The synthesis and biological evaluation of alkyl and benzyl naphthyridinium analogs of eupolauridine as potential antimicrobial and cytotoxic agents

Article abstract of DOI:10.1016/j.bmc.2016.02.028

Eupolauridine, an indenonaphthyridine alkaloid, has been previously reported by us to exhibit antifungal activity. This study describes the synthesis of new alkyl and benzyl naphthyridinium/pyridinium analogs of eupolauridine as potential antifungal agent

Full text of DOI:10.1016/j.bmc.2016.02.028

Accepted Manuscript
The Synthesis and Biological Evaluation of Alkyl and Benzyl Naphthyridinium
Analogs of Eupolauridine as Potential Antimicrobial and Cytotoxic Agents
Shahriyar Taghavi-Moghadam, Cecil D. Kwong, John A. Secrist III, Shabana
I. Khan, Alice M. Clark
PII:
S0968-0896(16)30118-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.028
BMC 12830
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 August 2015
15 February 2016
22 February 2016
Please cite this article as: Taghavi-Moghadam, S., Kwong, C.D., Secrist, J.A. III, Khan, S.I., Clark, A.M., The
Synthesis and Biological Evaluation of Alkyl and Benzyl Naphthyridinium Analogs of Eupolauridine as Potential
Antimicrobial and Cytotoxic Agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.02.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
The Synthesis and Biological Evaluation of Alkyl and Benzyl Naphthyridinium
Analogs of Eupolauridine as Potential Antimicrobial and Cytotoxic Agents
Shahriyar Taghavi-Moghadam,1 Cecil D. Kwong,1 John A. Secrist III,1* Shabana I.
Khan,2 and Alice M. Clark2
1Organic Chemistry Department, Southern Research Institute, 2000 Ninth Avenue South,
P.O. Box 55305, Birmingham, AL 35255 and 2National Center for Natural Products
Research, and Department of Pharmacognosy, School of Pharmacy, University of
Mississippi, University, MS 38677
*Corresponding author: secrist@southernresearch.org

2
Abstract
Eupolauridine, an indenonaphthyridine alkaloid, has been previously reported by
us to exhibit antifungal activity. This study describes the synthesis of new alkyl and
benzyl naphthyridinium/pyridinium analogs of the eupolauridine as potential antifungal
agents. A majority of the analogs exhibited antifungal activity against opportunistic
pathogens such as Candida albicans and Cryptococcus neoformans. Several of them
were also effective against bacteria (S. aureus, MRS, Pseudomnas and Mycobacterium)
and the malaria parasite (Plasmodium falciparum) to variable extents. A number of
analogs were also cytotoxic to human cancer cell lines.

3
Graphical Abstract
A series of alkyl and benzyl naphthyridinium analogs of the natural product
eupolauridine were prepared and evaluated for their antifungal, antibacterial and
antimalarial activities. Several compounds showed antifungal, antibacterial and
antimalarial activity to varying degrees. Some of them were cytotoxic to human cancer
cell lines.

4
1. Introduction
Eupolauridine is an indenonaphthyridine alkaloid that has been shown to have in
vitro efficacy against fungal pathogens namely Candida albicans, Cryptococcus
neoformans, and Aspergillus species.1-4 This natural product, isolated from Eupomatia
laurina and from Cleistopholis patens trees found in North Queensland and West Africa,
respectively,5,6 has also shown in vivo efficacy in a murine model of systemic
candidiasis.5 It is nontoxic towards mammalian cell lines and to mice,4,6 shows no
activity against mammalian DNA polymerase .7 It inhibits the catalytic activity of
Candida topoisomerase I (TopoI), completely inhibiting the DNA relaxation activity of
purified TopoI at 50 µg/mL.4 Additionally, it has also shown promising anti-toxoplasma
activity in human fibroblast host cells.8
Eupolauridine
Our interest in developing new eupolauridine-based antifungal and antibacterial
agents was inspired by these biological activities. Since there was little information
related to its mechanism of action and no SAR data was available for this compound, we
pursued a systematic SAR approach towards our goal of enhancing its biological activity.
We have prepared and evaluated a variety of substituents on the A, B. and D rings.
Herein we present our results on a series of alkyl and benzyl naphthyridinium derivatives,
compounds that should enhance aqueous solubility while maintain the main structural
features of eupolauridine. In this paper, we report the synthesis of a series of
eupolauridine analogs in which one of the naphthyridine/pyridine rings has been N-
alkylated with various alkyl and benzyl groups, producing the corresponding
eupolauridinium compounds. The synthesis of N-aminoeupolauridine is also reported.
All the newly synthesized analogs were screened for antifungal, antibacterial, and
antimalarial activities against a panel of representative pathogenic organisms. They were
also screened for anti-cellproliferative activity against four human solid tumor cell lines.
DNA topoisomerases are the enzymes that play important role in DNA replication by
regulating the topological state of DNA. These enzymes are considered as targets of a
number of anti-infective and anticancer agents, and we wished to determine if a
topoisomerase played any role in the mechanism of action of eupolauridine derivatives.
To further explore if the addition of alkyl and benzyl naphthyridinium moieties affected
the TopoI inhibitory activity of the parent compound, newly synthesized analogs were
also screened for inhibition of catalytic activity of both fungal and human TopoI.

5
2. Results and Discussion
2.1. Chemistry
The various alkyl- and benzyl- eupolauridinium targets described in this paper
were synthesized from eupolauridine (5a)9 or from other eupolauridine analogs from the
literature or prepared in our laboratories including A- and D-ring substituted analogs 5b-
f10 and benzoannulated analogs 10a-d,11 whose syntheses are shown in Schemes 1 and 2.
Scheme 1

6
Scheme 2
The initial targets within this series were analogs of the known compound, 1-N-
methyleupolauridinium iodide 11a12 but with different sized groups replacing methyl,
including 1-N-ethyl and 1-N-benzyl eupolauridinium analogs 11b and 11c, the
corresponding 2-methyl and 2-phenyleupolauridine-based analogs 12p and 12r, and
benzoeupolauridinium analogs 13a, 14f, and 13i. As shown in Schemes 3 and 4, these
compounds were prepared by treating eupolauridines 5a, 5e, and 5f and
benzoeupolauridines 10a, 10b, and 10d with the appropriate alkyl or benzyl halides.
After good antifungal and antibacterial activities were seen in the antifungal and
antibacterial assays of these analogs (see biological section for details), our study was
expanded to determine if substituent groups on the benzyls or on the D-ring could
further enhance these activities. Schemes 3 and 4 also show these eupolauridinium
targets, which were prepared by reacting 5a-f and benzoannulated analogs 10a-d with
the appropriate alkyl or benzyl halides or tosylate. We also prepared N-
aminoeupolauridine 11w, which was obtained in 15% overall yield by treating
eupolauridine with O-mesitylenesulfonyl-hydroxylamine,13-16 as is also shown in
Scheme 3.

7
Scheme 3
Precursor
5a
Product
11a
R1
H
R2
H
R3
CH3
X
I
5a
11b
H
H
CH3CH2
I
5a
5a
5a
5a
5a
5a
5a
5a
5a
5a
5d
5c
5b
5e
5e
5f
5f
5a
11c
11d
11e
11f
11g
11h
11i
11j
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C6H5CH2
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
I
p-F-C6H4CH2
m-Cl-C6H4CH2
p-Br-C6H4CH2
o,p-F2-C6H4CH2
p-NO2-C6H4CH2
p-CH3-C6H4CH2
p-CF3-C6H4CH2
3,5-(OCH3)2-C6H4CH2
C10H7CH2(naphth-2-yl)
C6H5CH2
11k
11l
H
H
11m, 12m*
11n, 12n*
11o, 12o*
12p
CH3
OCH3
F
C6H5CH2
C6H5CH2
CH3
C6H5CH2
CH3
C6H5CH2
H
H
H
H
H
H
H
H
H
CH3
CH3
C6H5
C6H5
H
H
H
H
12q
12r
12s
11t
11u
11v
11w
Br
I
Br
Br
Br
Br
Mes**
CH3CH2OCOCH2
C6H5COCH2
H2NCOCH2
H2N
5a
5a
5a
*)
**)
Mixture of both N(1)- and N(6)-isomers (11 and 12, respectively) in a ~1:1 ratio.
Mesitylene sulfonate salt. Prepared by the reaction of eupolauridine with freshly prepared O-
mesitylenesulfonylhydroxylamine. See experimental section for details.

8
Table 1:
Antimicrobial activity of alkyl and benzyl naphthyridinium analogs of
eupolauridine (IC50 and MIC values are in μg/ml)
S.
aureus
IC50
P.
auruginosa
IC50
A.
fumigatus
MIC
C.albicans
C.neoformans
MRS
IC50
M.intracellulare
MIC
IC50
MIC
12.5
IC50
4.8
10
MIC
25
eupo
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
12p
12q
12r
5.25
NA
5
2
50
35
50
13.33
0.43
0.87
0.18
0.3
0.58
1.19
0.33
0.57
0.92
0.3
0.48
0.7
0.52
8
0.82
0.4
0.95
NA
25
40
4.17
9.33
3
2.5
5.33
NA
3.17
6.5
20
0.67
6.17
16.67
1.5
6.25
12.5
6.25
6.25
6.25
1.56
3.12
0.78
0.78
1.56
6.25
0.78
3.12
3.12
0.39
1.56
1.56
0.78
50
50
50
40
50
50
50
50
50
50
50
50
6.25
12.5
25
0.78
12.5
25
35
30
20
27
30
9
22
3.12
NA
13.33
0.32
1.5
25
0.39
3.12
NA
1.56
0.78
3.12
NA
20
20
25
12s
11t
NA
11u
11v
11w
13a
13b
13c
13d
13e
14f
NA
NA
NA
23.3
16.67
NA
35
25
0.2
0.04
NA
16.1
5.17
NA
NA
NA
25
25
NA
NA
50
6.18
30
19.67
2
25
50
50
6.25
12.5
12.5
6.25
3.12
0.78
0.39
20
35
6
50
4
50
2.13
1.93
1.83
0.23
0.17
NA
5
10
25
5
2
0.39
0.098
4.5
2
4
2
50
10
14g
13h
13i
25
12.5
0.37
0.57
1.56
3.12
50
13j
Amphotericin B was used as control drug which showed an MIC of 0.156 µg/mL for C.albicans, 0.078 µg/mL
for C. neoformans and 1.25 µg/mL for A. fumigatus. IC50 is the concentration that causes a growth inhibition
of 50%. MIC (Minimum Inhibitory Concentration) is the concentration that inhibits 80% growth. NA = not
active at the highest concentration tested, 50 μg/mL.
Scheme 4

9
Precursor Product
R1
H
H
H
H
CH3
C6H5
C6H5
H
H
H
R2, R2
R4
CH3
CH3CH2
C6H5CH2
CH3
X
I
I
Br
TsO*
I
10a
10a
10a
10a
10c
10d
10d
10a
10b
10b
13a
13b
13c
13d
13e
14f
14g
13h
13i
H
H
H
H
H
H
H
CH3
CH3
I
CH3CH2
CH3CH2OCOCH2
CH3
Br
Br
I
H
-OCH2O-
-OCH2O-
13j
C6H5CH2
Br
*) p-Toluenesulfonate salt.
2.2. Biological Evaluation
Out of a total of 33 alkyl and benzyl naphthyridinium analogs of eupolauridine,
12 analogs (11c, 11e-g, 11i, 11l, 11o, 12r-s, 14f-g and 13j) exhibited antifungal activity
toward Candida albicans with IC50 values of <5 µg/ml (Table 1) and they were more
potent than eupolauridine (IC50 5.25 µg/mL) except 11c, 11g and 13j. Among them four
analogs (11l, 12r, 14f and 14g) showed the most potent activity with IC50 values of 0.67,
0.32, 0.2 and 0.04 µg/mL and MIC values in the range of 0.098 – 0.78 µg/mL,
respectively. The remaining analogs were either less active than eupolauridine or inactive
towards C. albicans. Though we did not perform any detailed solubility studies, the
quaternary salts did have improved solubility properties, as expected.
The alkyl and benzyl naphthyridinium analogs were also active against
Cryptococcus neoformans except 11t and 13h. Interestingly, nineteen of them (11c-g,
11i-o, 12q-s, 14f-g, and 13i-j) were very potent with IC50 values < 1 µg/ml and MIC
values in the range of 0.39 – 3.12 µg/mL in comparison to eupolauridine (IC50 4.8 and
MIC 25 µg/mL). Four others (11h, 13a, 13c-e) were also more effective (IC50 values 1-2
µg/mL) than eupolauridine.
Several analogs (11a, 11h, 12p, 11u-w, and 13c) were selective for Cryptococcus
with no activity towards Candida. The analog 14g was more active towards Candida (IC50
0.04 µg/mL, MIC 0.098 µg/mL) than Cryptococcus (IC50 0.17 µg/mL, MIC 0.39 µg/mL).
This compound was the most potent antifungal of the series with an over 100 fold
increase in activity for Candida and a 30 fold increase in activity for Cryptococcus
compared to the parent compound eupolauridine.
The activity data shows that most of the active analogs belong to the
benzyleupolauridinium bromide series, with the exception of the nitro-analog 11h.
Methyl- and ethyleupolauridinium analogs (11a and 11b) were not active against

10
Candida but moderately active against Cryptococcus. From the diazafluoranthenium salts
only 14f-g demonstrated antifungal activities towards both organisms.
Although eupolauridine showed activity against Aspergillus fumigatus (MIC 2
µg/mL) none of the synthesized analogs were effective against this fungal pathogen.
None of the analogs showed any significant antibacterial activities (data not shown)
except 13c, 14f-g (IC50 2-5 µg/mL).
Although eupolauridine did not exhibit any antimalarial activity (towards
Plasmodium falciparum), benzyleupolauridinium bromide and its derivatives (Table 2)
showed significant antimalarial activity. The IC50 values of 11d-g, and 11I-k were <20
ng/mL indicating a potent antimalarial activity. Among methyl-, ethyl-, and methoxy-
benzyleupolauridinium salts only 11l showed good antimalarial activity (IC50 19.5
ng/mL). Phenyleupolauridinium salts were also not very active against P. falciparum.
From the diazafluorenthenium salts, only dimethylbenzo analog 13e exhibited
antimalarial activity with an IC50 of 20 ng/mL.
The alkyl- and benzyl naphthyridinium analogs of eupolauridine reported in this
study were also tested for their cytotoxic properties against a panel of four human cancer
cell lines and one noncancerous mammalian kidney cell line in terms of their anti-cell
proliferative effects (Table 3). The compounds were not cytotoxic to mammalian kidney
(VERO) cells up to a concentration of 10 µg/ml except 11j and 14g, which showed mild
cytotoxicities (IC50 6.4 and 7.8 µg/ml). These concentrations are much higher than the
IC50 values of growth inhibition of Candida, Cryptococcus and Plasmodium indicating a
high selectivity index for antifungal and antimalarial activities.
Although eupolauridine was not cytotoxic to any of the cell lines, most of the
analogs have shown cytotoxicity (anti-cell proliferative activity) towards four cancer cell
lines included in this study, as shown in Table 3, indicating a possibility of anticancer
potential of these compounds. Based on these results it is clear that addition of alkyl- and
benzyl naphthyridinium moieties to eupolauridine resulted in molecules that were
cytotoxic to cancer cells. This could be explained in terms of topoisomeraseII-related
mechanism of action as described in an earlier study4. The involvement of other
mechanisms of course may well be important. Further evaluation of their cytotoxicities
and selectivity in animal models would be a logical next step for selecting potential
analogs that could serve as lead compounds.
Table 2: Antimalarial activity of alkyl and benzyl naphthyridinium analogs
of eupolauridine
P. falciparum
IC50 ng/ml
eupo
11a
11b
11c
11d
NA
60
47.5
22.5
17

11
11e
11f
4.2
13
11g
11h
11i
11j
11k
11l
15
35
9.5
8.5
11
19.5
62
32
33
110
95
460
40
500
170
90
3200
33
11m
11n
11o
12p
12q
12r
12s
11t
11u
11v
11w
13a
13b
13c
13d
13e
14f
33
41.5
38.5
20
NA
NA
560
28
14g
13h
13i
13j
9.4
Chloroquine was used as the control drug which showed IC50 <20 ng/mL. NA =
not active at the highest concentration tested, 4.76 μg/mL.
Table 3: Cytotoxicity of naphthyridinium analogs
cytotoxicity (IC50 μg/ml)
BT-549 SK-OV-3
SK-MEL
NA
KB
NA
VERO
>10
NA
NA
NA
NA
NA
NA
NA
eupo
11a
11b
11c
11d
11e
11f
11g
11h
11i
NA
NA
NA
NA
7.5
1.6
1.15
NA
4
>10
>10
NA
1.5
1.5
0.055
0.095
2.8
0.62
<1.1
NA
NA
5.4
6
1.15
0.65
6.2
NA
NA
NA
>10
0.68
0.18
NA
4.9
1.8
NA
NA
1.9
6.8
NA

12
11j
11k
11l
0.78
2
0.54
7
6
0.52
0.2
1.3
NA
>10
7
0.79
>10
>10
1.8
1.1
2.5
NA
3.5
6.2
1.2
NA
>10
NA
NA
7
>10
6
7.5
5.4
7.5
8
NA
1.2
1.3
1.35
0.08
<1.1
<1.1
0.58
0.15
0.53
NA
1.7
<1.1
0.25
7.6
6
>10
NA
1.3
1.3
1.2
1.3
4.5
4.5
4
6.4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>10
NA
NA
NA
NA
NA
NA
NA
7.8
NA
NA
NA
>10
<1.1
1.4
1.5
5.6
NA
4.2
9
0.7
NA
NA
6.5
>10
NA
NA
1.8
>10
>10
3.5
1.3
>10
5
11m
11n
11o
12p
12q
12r
12s
11t
11u
11v
11w
13a
13b
13c
13d
13e
14f
1
NA
>10
NA
NA
6.5
6.8
2.9
4.4
3.5
>10
6.5
NA
4.2
1
14g
13h
13i
5.9
1.2
0.6
1.3
13j
Doxorubicin
0.55
1.11
1.59
NA = not active (cytotoxic) at the highest concentration tested, 10 μg/mL.
Eupolauridine was initially found to inhibit the DNA relaxation activity of DNA
topoisomerase I and was more selective for fungal TopoI4. We also observed inhibition of
both human and candida TopoI enzymes in our study as shown in Table 4 with a little bit
higher selectivity towards the fungal enzyme in comparison to the human counterpart.
Based on this observation and to establish an SAR, we screened the synthesized analogs
for inhibition of TopoI isolated from Candida albicans4 and compared their activity with
human TopoI (commercially available). All the analogs have retained the activity of
topoisomeraseI inhibition except 11o and 11v (Table 4). The most active compounds
were 13a-e, 14f-g and 13i-j, with IC50s of < 1 µg/ml. Interestingly, only one compound
(12r) was much more selective for Candida TopoI inhibition over human TopoI with a
selectivity index of 2.92 as compared to eupolauridine with a selectivity of 1.55 (Table
4). Most of the compounds inhibited both human and fungal topoisomerase I to similar
extents. Inhibition of topoisomerase I might be responsible for the antifungal activities of
these compounds but certainly involvement of other mechanisms seems likely.
Compound 11o demonstrated no topoisomerase inhibition but showed antifungal activity;
similarly, compounds with no or mild antifungal activity have shown mild to moderate
inhibition of topoisomerase I. Hence a true correlation between the enzyme inhibition and
antifungal properties of these analogs could not be seen and this observation is in support
of previous study indicating that inhibition of catalytic activity of DNA TopoI does not
seem to be the principal mode of antifungal action of eupolauridine4 which seems to be

13
true for the alkyl and benzyl naphthyridinium analogs synthesized in this study.
Although, topoisomerase inhibition could be one of the possible contributing factors
towards their antifungal activities. We have therefore selected one compound, alkyl
naphthyridiniuim analog 14f, for further mechanistic studies, which will be reported in a
separate publication.16
Table 4: Topoisomerase I inhibition
Topol IC50 (μg/ml)
Topol
human
candida
4.2
10
Selectivity Index
eupo
11a
11b
11c
11d
11e
11f
11g
11h
11i
6.5
10
1.55
1
10
19
40
21
21
32
18
11
15
38
50
22
22
45
21
11
0.67
0.5
0.8
0.95
0.95
0.71
0.86
1
11j
11k
11l
30
12.5
21
31
15
30
0.97
0.83
0.7
11m
11n
11o
12p
12q
12r
15
17.5
5
NA
4.5
12
12
4.4
90
0.86
1.15
5.75
NA
5
11
35
4.3
75
50
NA
7.5
1.5
0.7
0.55
0.7
0.5
0.375
<6
1.11
0.92
2.92
0.98
0.83
0.91
12s
11t
11u
11v
11w
13a
13b
13c
13d
13e
14f
55
NA
15
0.5
0.83
1.00
0.96
1
1.8
0.7
0.57
0.7
0.7
0.475
<6
2.4
0.45
0.52
0.71
0.79
14g
13h
13i
2.3
0.425
0.52
0.96
0.94
1
13j
NA = not active at the highest concentration tested, 10 μg/mL.

14
3. Conclusion
As one phase of our systematic SAR studies on modified eupolauridine analogs,
we prepared a series of alkyl and benzyl naphthyridinium analogs and evaluated their
biological activities. We found a number of analogs that were significantly more potent
than the parent eupolauridine while also having enhanced solubilities. Compounds 14f
and 14g were the most effective antifungal agents against both Candida and
Cryptococcus, while several compounds (13i, 13j, 12q, and several of the 11 series) were
more effective against Crypotococcus than Candida. Though some antimalarial activity
was seen, it was relatively modest, though improved from the the phenyleupolauridinium
salts. The alkyl and benzyl naphthrydinium compounds were in some cases cytotoxic to
cancer cells. Evaluation of the topoisomerase I inhibitory activity of the various analogs
showed some inhibition, but it appears likely that one or more other mechanisms of
action are involved. We have selected 14f and studied its mechanism of action in more
detail, and those findings will be reported.16 These results and our other SAR studies on
eupolauridine derivatives modified in other ways in the A, B. and D rings demonstrate
that it is possible to maintain or improve antifungal activity while incorporating
modifications at a variety of sites on the eupolauridine ring system.
4. Experimental Section
4.1. General Methods
Melting points are uncorrected and were determined in open capillary tubes with a
Mel-Temp melting point apparatus. 1HNMR spectra were recorded on a Nicolet NT-300
NB spectrometer operating at 300.635 MHz (1H). Chemical shifts are expressed in parts
per million downfield from tetramethylsilane. Spectral assignments are supported by
proton decoupling. UV absorption spectra were determined on a Perkin-Elmer lambda 9
spectrometer by dissolving each compound in methanol. Numbers in parentheses are
extinction coefficients (lg()), [ ] = shoulder. Microanalyses were performed by the
Molecular Spectroscopy Section of Southern Research Institute. Mass spectra were
recorded on a Varian/MAT 311A double-focusing mass spectrometer in the fast atom
bombardment (FAB) mode. HPLC analyses were carried out on a Hewlett-Packard 1100
liquid chromatograph with a Bondapak C18 column (3.9 mm x 30 cm) or Phenomenex
Sphereclone 5µ ODS (1) column (250 X 4.6 mm) and diode-array detector for UV
monitoring (200-350 nm). All chromatographic separations were carried out by flash
chromatography using 230-400 mesh silica gel from E. Merck. TLC was carried out on
Merck precoated silica gel (60 F254) sheets or on Schleicher & Schuell cellulose (F
1440/LS 254) sheets with UV detection at 254 nm and 366 nm. Preparative TLC was
carried out on silica gel (60 F254, 2 mm) plates with appropriate solvents.
4.2. Synthesis
4.2.1. 1-N-Methyleupolauridinium iodide13 (11a) (1-N-Methyl-indeno[1,2,3-
ij]naphthyridinium iodide)

15
To a solution of eupolauridine1,13 (100 mg, 0.49 mmol) in acetone (5 ml,
anhydrous) was added methyl iodide (1 ml, 16.06 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature, the resulting orange precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (131 mg, 77%) that was chromatographically pure. m.p: 260-265 °C dec. (lit.13:
267-269 °C dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.65. UV (MeOH),

max[nm] (lg): 386 (3.92), 368 (3.89), [347 (3.54)], [310 (4.04)], 296 (4.23), 289 (4.23),
[240 (4.45)], 224 (4.61). FAB-MS (m/e): 219 (M)+. 1H-NMR (DMSO-d6): δ 4.66 (s, 3H,
CH3), 7.71 (t, J=7.9 Hz, 1H, Ph), 7.80 (t, J=7.4 Hz, 1H, Ph), 7.91 (d, J=5.9 Hz, 1H, Py),
8.10 (d, J=7.0 Hz, 1H, Ph), 8.29 (d, J=7.7 Hz, 1H, Ph), 8.33 (d, J=6.8 Hz, 1H, Py), 8.81
(d, J=6.8 Hz, 1H, Py), 8.99 (d, J=5.9 Hz, 1H, Py).
4.2.2. 1-N-Ethyleupolauridinium iodide (11b) (1-N-Ethyl-indeno[1,2,3-
ij][2,7]naphthyridinium iodide)
To a solution of eupolauridine (100 mg, 0.49 mmol) in acetone (5 ml, anhydrous)
was added ethyl iodide (1 ml, 12.50 mmol), and the mixture was heated at reflux for 24 h.
After cooling to room temperature, the resulting orange precipitate was collected, washed
with acetone and ether, and dried under high vacuum to give the product (62 mg, 35%) as
chromatographically pure material. A second fraction of the product (79 mg, 45%) was
isolated by additional quantity of ethyl iodide (1 ml, 12,50 mmol) to the filtrate and
further heating at reflux for 48 hours. m.p: 225 °C dec. (beginning of dec.); 246-250 °C
(completely dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.71. UV (MeOH),

max[nm] (lg): 387 (3.91), 368 (3.89), [349 (3.61)], [310 (4.06)], 296 (4.23), 289 (4.22),
[240 (4.47)], 225 (4.62). FAB-MS (m/e): 233 (M)+. 1H-NMR (DMSO-d6): δ 1.65 (t, J=7.3
Hz, 3H, CH3), 4.99 (q, J=7.3 Hz, 2H, CH2), 7.68-7.73 (dt, J=1.1, 7.7 Hz, 1H, Ph), 7.79 (t,
J=7.4 Hz, 1H, Ph), 7.91 (d, J=5.9 Hz, 1H, Py), 8.10 (d, J=7.3 Hz, 1H, Ph), 8.28 (d, J=7.5
Hz, 1H, Ph), 8.39 (d, J=6.8 Hz, 1H, Py), 8.87 (d, J=6.8 Hz, 1H, Py), 8.99 (d, J=6.2 Hz,
1H, Py). Anal. Calcd for C16H13IN2: C, 53.35; H, 3.64; N, 7.78. Found: C, 53.35; H, 3.71;
N, 7.76.
4.2.3. 1-N-Benzyleupolauridinium bromide (11c) (1-N-Benzylindeno[1,2,3-
ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (100 mg, 0.49 mmol) in acetone (5 ml, anhydrous)
was added benzyl bromide (1 ml, 8.41 mmol), and the mixture was heated at reflux for 48 h.
After cooling to room temperature, the resulting yellow precipitate was collected, washed
with acetone and ether, and dried under high vacuum to give the product (178 mg, 97%)
as chromatographically pure material. m.p: 244-246 °C (dec.). TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.75. UV (MeOH), max[nm] (lg): 389 (3.91), 370 (3.89), [352
(3.62)], [310 (4.10)], 297 (4.24), [288 (4.22)], [242 (4.49)], 228 (4.53). FAB-MS (m/e):
295 (M)+. 1H-NMR (DMSO-d6): δ 6.30 (s, 2H, CH2), 7.42-7.49 (m, 5H, Ph), 7.58-7.64
(dt, J=71.3, 7.7 Hz, 1H, Ph), 7.75 (t, J=7.7 Hz, 1H, Ph), 7.95 (d, J=6.1 Hz, 1H, Py), 8.08
(d, J=6.8 Hz, 1H, Ph), 8.18 (d, J=7.7 Hz, 1H, Ph), 8.43 (d, J=6.8 Hz, 1H, Py), 8.82 (d,
J=7.0 Hz, 1H, Py), 9.01 (d, J=5.93 Hz, 1H, Py). Anal. Calcd for C21H15BrN2 x 0.5 H2O :
C, 65.64; H, 4.20; N, 7.29. Found: C, 65.42; H, 4.01; N, 7.79.

16
4.2.4. 1-N-(4-Fluoro)benzyleupolauridinium bromide (11d) (1-N-(4-
Fluoro)benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 4-fluorobenzyl bromide (0.47, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (62 mg, 62%) as
chromatographically pure material. m.p: 180-184 °C (dec.). TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.75. FAB-MS (m/e): 313 (M)+. 1H-NMR (DMSO-d6): δ 6.27 (s,
2H, CH2), 7.27-7.32 (m, 2H, Ph), 7.49-7.57 (m, 2H, Ph), 7.62 (t, J=7.7 Hz, 1H, Ph), 7.75
(t, J=7.6 Hz, 1H, Ph), 7.93 (d, J=6.0 Hz, 1H, Py), 8.08 (d, J=7.3 Hz, 1H, Ph), 8.22 (d,
J=7.7 Hz, 1H, Ph), 8.40 (d, J=6.9 Hz, 1H, Py), 8.76 (d, J=6.9 Hz, 1H, Py), 9.00 (d, J=6.0
Hz, 1H, Py).
4.2.5. 1-N-(3-Chloro)benzyleupolauridinium bromide (11e) (1-N-(3-
Chloro)benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 3-chlorobenzyl bromide (0.51 g, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (73 mg, 73%) as
chromatographically pure material. m.p: 218-222 °C. TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.78. FAB-MS (m/e): 329, 331 (M)+. 1H-NMR (DMSO-d6): δ
(6.29 (s, 2H, CH2), 7.36 (d, J=6.8 Hz, 1H, Ph), 7.43-7.50 (m, 2H, Ph), 7.58-7.63 (m, 2H,
Ph), 7.75 (t, J=7.6 Hz, 1H, Ph), 7.94 (d, J=6.0 Hz, 1H, Py), 8.07 (d, J=7.3 Hz, 1H, Ph),
8.16 (d, J=7.7 Hz, 1H, Ph), 8.42 (d, J=6.9 Hz, 1H, Py), 8.78 (d, J=6.9 Hz, 1H, Py), 9.00
(d, J=6.0 Hz, 1H, Py).
4.2.6. 1-N-(4-Bromo)benzyleupolauridinium bromide (11f) (1-N-(4-
Bromo)benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 4-bromobenzyl bromide (0.62 g, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (82 mg, 74%) as
chromatographically pure material. m.p: >190 °C (beginning of dec.); 215-220 °C
(completely dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.78. FAB-MS (m/e):
373, 375 (M)+. 1H-NMR (DMSO-d6): δ 6.26 (s, 2H, CH2), 7.38 (d, J=8.4 Hz, 2H, Ph),
7.58-7.65 (m, 3H, Ph), 7.75 (t, J=7.5 Hz, 1H, Ph), 7.93 (d, J=6.1 Hz, 1H, Py), 8.07 (d,
J=7.3 Hz, 1H, Ph), 8.16 (d, J=7.7 Hz, 1H, Ph), 8.40 (d, J=6.9 Hz, 1H, Py), 8.77 (d, J=6.9
Hz, 1H, Py), 9.00 (d, J=6.0 Hz, 1H, Py).
4.2.7. 1-N-(2,4-Difluoro)benzyleupolauridinium bromide (11g) (1-N-(2,4-
Difluoro)benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 2,4-difluorobenzyl bromide (0.52 g, 2.5 mmol) in acetone (2 ml,

17
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (60 mg, 60%) as
chromatographically pure material. m.p: 192-196 °C (dec.). TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): FAB-MS (m/e): 331 (M)+. Rf: 0.75. 1H-NMR (DMSO-d6): δ 6.28 (s,
2H, CH2), 7.16-7.22 (m, 1H, Ph), 7.43-7.52 (m, 1H, Ph), 7.58-7.67 (m, 2H, Ph), 7.78 (t,
J=7.6 Hz, 1H, Ph), 7.92 (d, J=6.0 Hz, 1H, Py), 8.09 (d, J=7.2 Hz, 1H, Ph), 8.33 (d, J=7.7
Hz, 1H, Ph), 8.37 (d, J=6.8 Hz, 1H, Py), 8.69 (d, J=6.9 Hz, 1H, Py), 9.00 (d, J=6.0 Hz,
1H, Py).
4.2.8. 1-N-(4-Nitro)benzyleupolauridinium bromide (11h) (1-N-(4-
Nitro)benzylindeno [1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous) was
added a solution of 4-nitrobenzyl bromide (0.54, 2.5 mmol) in acetone (2 ml, anhydrous), and
the mixture was heated at reflux for 72 h. After cooling to room temperature, the resulting
yellow precipitate was collected, washed with acetone and ether, and dried under high
vacuum to give the product (68 mg, 66%) as chromatographically pure material. m.p:
223-226 °C. TLC, cellulose, (4%-Na-citrate: MeOH, 1:1): Rf: 0.74. FAB-MS (m/e): 340
(M)+. 1H-NMR (DMSO-d6):  6.46 (s, 2H, CH2), 7.57 (t, J=7.7 Hz, 1H, Ph), 7.67-7.76
(m, 3H, Ph), 7.95 (d, J=6.1 Hz, 1H, Py), 8.05-8.08 (m, 2H, Ph), 8.26 (d, J=8.7 Hz, 2H,
Ph), 8.45 (d, J=6.9 Hz, 1H, Py), 8.83 (d, J=6.9 Hz, 1H, Py), 9.02 (d, J=6.0 Hz, 1H, Py).
4.2.9. 1-N-(4-Methyl)benzyleupolauridinium bromide (11i) (1-N-(4-Methyl)benzyl-
indeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 4-methylbenzyl bromide (0.46, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give product (33 mg, 35%) containing ~20 %
starting material, according to 1H-NMR. The obtained mixture was screened without
purification. m.p: (of the mixture) >184 °C (beginning of dec.), 200-204 °C (completely
dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.78 (product), 0.39 (starting
material, eupolauridine). FAB-MS (m/e): 355 (M)+. 1H-NMR (DMSO-d6):  (target
compound only) 2.29 (s, 3H, CH3), 6.23 (s, 2H, CH2), 7.23 (d, J=8.0 Hz, 2H, Ph), 7.30 (d,
J=8.1 Hz, 2H, Ph), 7.60 (t, J=8.1 Hz, 1H, Ph), 7.74 (t, J=7.7 Hz, 1H, Ph), 7.91 (d, J=6.0
Hz, 1H, Py), 8.07 (d, J=7.3 Hz, 1H, Ph), 8.20 (d, J=7.7 Hz, 1H, Ph), 8.39 (d, J=6.9 Hz,
1H, Py), 8.76 (d, J=6.8 Hz, 1H, Py), 8.99 (d, J=5.9 Hz, 1H, Py).
4.2.10. 1-N-(4-Trifluoromethyl)benzyleupolauridinium bromide (11j) (1-N-(4-
Trifluoromethyl)-benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 4-trifluorobenzyl bromide (0.60 g, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (51 mg, 47%) as
chromatographically pure material. m.p:202-206 °C (dec.). TLC, cellulose, (4%-Na-

18
citrate:MeOH, 1:1):Rf: 0.83. FAB-MS (m/e): 363 (M)+. 1H-NMR (DMSO-d6):  6.41 (s,
2H, CH2), 7.59 (t, J=7.7 Hz, 1H, Ph), 7.63 (d, J=8.2 Hz, 2H, Ph), 7.74 (t, J=7.6 Hz, 1H,
Ph), 7.80 (d, J=6.1 Hz, 2H, Ph), 7.95 (d, J=6.0 Hz, 1H, Py), 8.07 (d, J=7.3 Hz, 1H, Ph),
8.12 (d, J=7.7 Hz, 1H, Ph), 8.43 (d, J=6.8 Hz, 1H, Py), 8.82 (d, J=6.8 Hz, 1H, Py), 9.01
(d, J=6.0 Hz, 1H, Py).
4.2.11. 1-N-(3,5-Dimethoxy)benzyleupolauridinium bromide (11k) (1-N-(3,5-
Dimethoxy)-benzylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 3,5-dimethoxyenzyl bromide (0.54, 2.5 mmol) in acetone (2 ml,
anhydrous), and the mixture was heated at reflux for 72 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the pure product (24 mg, 23%) as
chromatographically pure material. m.p: 200-205 °C. TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.78. FAB-MS (m/e): 355 (M)+. 1H-NMR (DMSO-d6):  3.72 (s,
6H, (OCH3)2), 6.18 (s, 2H, CH2), 6.55-6.58 (m, 3H, Ph), 7.63 (t, J=7.5 Hz, 1H, Ph), 7.75
(t, J=7.6 Hz, 1H, Ph), 7.92 (d, J=5.9 Hz, 1H, Py), 8.07 (d, J=7.3 Hz, 1H, Ph), 8.20 (d,
J=7.6 Hz, 1H, Ph), 8.39 (d, J=6.8 Hz, 1H, Py), 8.76 (d, J=6.7 Hz, 1H, Py), 8.99 (d, J=5.9
Hz, 1H, Py).
4.2.12. 1-N-(Naphth-2-yl)methyleupolauridinium bromide (11l) (1-N-(Naphth-2-yl)-
methylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added 2-(bromomethyl)naphthalene (0.54 g, 2.45 mmol), and the mixture was heated at
reflux for 48 h. After cooling to room temperature, the resulting orange precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (66 mg, 63%) as chromatographically pure material. m.p: 230-232 °C (dec.).
TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.76. FAB-MS (m/e): 345 (M)+. 1H-
NMR (DMSO-d6):  6.42 (s, 2H, CH2), 7.55-7.60 (m, 4H, Ph), 7.72-7.77 (m, 1H, Ph),
7.83-7.98 (m, 4H, 3 Ph, 1 Py), 8.03-8.12 (m, 3H, Ph), 8.37 (d, J=6.8 Hz, 1H, Py), 8.79 (d,
J=6.8 Hz, 1H, Py), 8.99 (d, J=6.2 Hz, 1H, Py).
4.2.13. 1-N-Benzyl-8(9)-methyleupolauridinium bromide (11m, 12m) (1-N-Benzyl-
8(9)-methylindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 8-methyl eupolauridine (50 mg, 0.23 mmol) in acetone (5 ml,
anhydrous) was added benzyl bromide (0.5 ml, 4.20 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature, the resulting orange precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the pure
product (75 mg, 84%) as a mixture of both 8-and 9-methyl –isomers in a tentative ratio
of ~ 1:1 according to 1H-NMR. The obtained mixture was screened without purification.
m.p. (mixture) : 234-238 °C (dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1):Rf: 0.83.
FAB-MS (m/e): 309 (M)+. 1H-NMR (DMSO-d6): (The 1H-NMR-spectrum shows both
the 1-N-benzyl-8-methyl - (the assignment with the index a) and 1-N-benzyl-9-methyl-
eupolauridinium bromide isomers (designated a and b, respectively) in tentative ratio of
1:1. The reported coupling constant values are approximated. Because of signal
overlapping, an exact calculation of the coupling constants is not possible)  2.41 (s, 3H,

19
CH3(a)), 2.47 (s, 3H, CH3(b)), 6.26 (s, 2H, CH2(a)), 6.29 (s, 2H, CH2(b)), 7.39-7.45 (m,
11H, 6 Ph(a), 5 Ph(b)), 7.54 (d, J=8.4 Hz, 1H, Ph(b)), 7.88-7.96 (m, 4H, 1 Py(a), 2 Ph(b),
1 Py(b)), 8.05 (d, J=8.1 Hz, 1H, Ph(a)), 8.11 (br s, 1H, Ph(a)), 8.36 (d, J=5.5 Hz, 1H,
Py(b)), 8.38 (d, J=5.5 Hz, 1H, Py(a)), 8.74 (d, J=7.0 Hz, 1H, Py(a)), 8.76 (d, J=7.0 Hz,
1H, Py(b)), 8.96 (d, J=6.2 Hz, 1H, Py(a)), 8.99 (d, J=5.9 Hz, 1H, Py(b)). Anal. Calcd for
C22H17BrN2: C, 67.88; H, 4.40; N, 7.20. Found: C, 67.71; H, 4.24; N, 7.06.
4.2.14. 1-N-Benzyl-8(9)-methoxyeupolauridinium bromide (11n, 12n) (1-N-Benzyl-
8(9)-methoxyindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 8-methoxy-eupolauridine (50 mg, 0.21 mmol) in acetone (5 ml,
anhydrous) was added benzyl bromide (0.5 ml, 4.20 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature, the resulting red precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (74 mg, 86%) as a mixture of both 8-and 9-methoxy –isomers in a tentative ratio
of ~ 3:2 according to 1H-NMR. The obtained mixture was screened without purification.
m.p (mixture): 216-220 °C (dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.78,
FAB-MS (m/e): 325 (M)+. 1H-NMR (DMSO-d6): (The 1H-NMR-spectrum of 11n shows
the 1-N-benzyl-8-methoxy and 1-N-benzyl-9-methoxy-eupolauridinium bromide isomers
(designated a and b, respectively) in tentative ratio of 3:2. The reported coupling constant
values are approximated. Because of signal overlapping, an exact calculation of the
coupling constants is not possible).  3.86 (s, 3H, OCH3(b)), 3.98 (s, 3H, OCH3(a)), 6.20
(s, 2H, CH2(a)), 6.31 (s, 2H, CH2(b)), 7.05-7.08(dd, J=2.5, 8.8 Hz, 1H, Ph(a)), 7.21 (d,
J=8.4 Hz, 1H, Ph(b)), 7.38-7.38 (m, 10H, 5 Ph(a), 5 Ph(b)), 7.59-7.62 (m, 2H, 1 Ph(a), 1
Ph(b)), 7.83 (d, J=5.9 Hz, 1H, Ph(b)), 7.91 (d, J=5.9 Hz, 1H, Ph(a)), 7.96 (d, J=8.4 Hz,
1H, Py(b)), 8.06 (d, J=8.6 Hz, 1H, Py(a)), 8.25 (d, J=6.8 Hz, 1H, Py(a)), 8.41 (d, J=7.0
Hz, 1H, Py(b)), 8.67 (d, J=6.8 Hz, 1H, Py(a)), 8.82 (d, J=6.8 Hz, 1H, Py(b)), 8.91 (d,
J=5.5 Hz, 1H, Py(b)), 8.99 (d, J=5.9 Hz, 1H, Py(a)).
4.2.15. 1-N-Benzyl-8(9)-fluoroeupolauridinium bromide (11o) (1-N-Benzyl-8(9)-
fluoroindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 8-fluoro eupolauridine (50 mg, 0.23 mmol) in acetone (5 ml,
anhydrous) was added benzyl bromide (0.5 ml, 4.20 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature, the resulting orange precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (45 mg, 51%) as a mixture of both 8-and 9-fluoro –isomers in a tentative ratio of
~ 1:1 according to 1H-NMR. A second fraction (21 mg, 23 %) was isolated by heating
the filtrate at reflux for another 24 hours. The obtained mixture was screened without
purification. m.p (mixture): 194-200 °C (dec.). TLC, cellulose, (4%-Na-citrate:MeOH,
1:1): Rf: 0.83. FAB-MS (m/e): 313 (M)+. 1H-NMR (DMSO-d6): (The 1H-NMR-spectrum
showed both the 1-N-benzyl-8-fluoro and 1-N-benzyl-9-fluoro-eupolauridinium bromide
isomers (11o and 12o, designated a and b, respectively) in tentative ratio of 1:1. The
reported coupling constant values are approximated. Because of signal overlapping an
exact calculation of the coupling constants is not possible).  6.25 (s, 2H, CH2(a)), 6.28
(s, 2H, CH2(b)), 7.38-7.50 (m, 11H, 6 Ph(a), 5 Ph(b)), 7.56-7.62 (m, 1H, Ph(b)), 7.91-
8.00 (m, 3H, 1 Py(a), 1 Ph(a), 1 Py(b)), 8.05-8.21 (m, 3H, 1 Ph(a), 2 Ph(b)), 8.36 (d,

20
J=6.8 Hz, 1H, Py(a)), 8.42 (d, J=6.4 Hz, 1H, Py(b)), 8.72-8.77 (m, 2H, Py(a), Py(b)), 8.98
(d, J=5.9 Hz, 1H, Py(b)), 9.02 (d, J=6.2 Hz, 1H, Py(a)).
4.2.16. 1,5-Dimethyleupolauridinium iodide (12p) (1,5-Dimethyl-indeno[1,2,3-ij][2,7]
naphthyridinium iodide)
To a solution of 2-methyleupolauridine (100 mg, 0.46 mmol) in acetone (7 ml,
anhydrous) was added methyl iodide (1 ml, 16.06 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature the resulting orange precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give product
(148 mg, 90%) that was chromatographically pure. m.p: 259-264 °C dec. TLC, cellulose,
(4%-Na-citrate:MeOH, 1:1): Rf: 0.70. FAB-MS (m/e): 233 (M)+. UV (MeOH), max[nm]
(lg): 387 (3.88), 368 (3.87), [350 (3.60)], [313 (4.05)], [300 (4.19)], 287 (4.25), [240
(4.53)], 229 (4.60). 1H-NMR (DMSO-d6)  2.80 (br s, 3H, C(5)-CH3), 4.64 (br s, 3H,
N(1)CH3), 7.66-7.82 (m, 2H, Ph & 1H, Py), 8.06 (d, J=7.9 Hz, 1H, Ph), 8.22-8.25 (m,
1H, Ph & 1H, Py), 8.74 (d, J=6.6 Hz, 1H, Py). Anal. Calcd for C16H13IN2: C, 53.35; H,
3.64; N, 7.78. Found: C, 53.19; H, 3.87; N, 7.67.
4.2.17. 1-N-Benzyl-5-methyleupolauridinium bromide (12q) (1-N-Benzyl-5-methyl
indeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 2-methyleupolauridine (50 mg, 0.23 mmol) in acetone (5 ml,
anhydrous) was added benzyl bromide (0.5 ml, 4.20 mmol), and the mixture was heated at
reflux for 48 h. After cooling to room temperature, the resulting yellow precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (66 mg, 74%) as chromatographically pure material. m.p: 218-222 °C. TLC,
cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.83. FAB-MS (m/e): 309 (M)+. 1H-NMR
(DMSO-d6):  2.81 (s, 3H, CH3), 6.28 (br s, 2H, CH2), 7.38-7.47 (m, 5H, Ph), 7.57-7.62
(dt, J=1.1, 7.7 Hz, 1H, Ph), 7.73 (t, J=7.5 Hz, 1H, Ph), 7.79 (br s, 1H, Py), 8.05 (d, J=6.8
Hz, 1H, Ph), 8.16 (d, J=7.7 Hz, 1H, Ph), 8.32 (d, J=6.8 Hz, 1H, Py), 8.77 (d, J=7.0 Hz,
1H, Py).
4.2.18. 1-N-Methyl-5-phenyleupolauridinium iodide (12r) (1-N-Methyl-5-
phenylindeno [1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2-phenyleupolauridine (100 mg, 0.46 mmol) in acetone (10 ml,
anhydrous) was added methyl iodide (1 ml, 16.06 mmol), and the mixture was heated at
reflux for 24 h. After cooling to room temperature, the resulting orange precipitate was
collected, washed with acetone (2x3 ml) and ether (3x10 ml), and dried under high
vacuum to give the product (126 mg, 84%) as a chromatographically pure material. m.p:
246-250 °C dec. TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.50. FAB-MS (m/e):
295 (M)+. UV (MeOH), max[nm] (lg), 439 (3.22), 392 (3.91), 372 (3.93), [352 (3.95)],
324 (4.51), 299 (4.38), [225 (4.49)]. 1H-NMR (DMSO-d6):  4.62 (br s, 3H, CH3), 7.63-
7.65 (m, 3H, Ph), 7.71-7.76 (m, 1H, Ph), 7.79-7.82 (m, 1H, Ph), 8.19-8.21 (d, J=7.0 Hz,
1H, Ph), 8.27-8.36 (m, 3H, Ph, 1H, Py), 8.47 (s, 1H, Ph), 8.76 (d, J=6.8 Hz, 1H, Py).
Anal. Calcd for C21H15IN2 x 0.2 H2O: C, 59.23; H, 3.64; N, 6.58. Found: C, 59.02; H,
3.83; N, 6.63.

21
4.2.19. 1-N-Benzyl-5-phenyleupolauridinium bromide (12s) (1-N-Benzyl-5-
phenylindeno [1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 2-phenyleupolauridine (50 mg, 0.18 mmol) in acetone (5 ml,
anhydrous) was added benzyl bromide (0.5 ml, 4.20 mmol), and the mixture was heated at
reflux for 48 h. After cooling to room temperature, the resulting yellow precipitate was
collected, washed with acetone and ether, and dried under high vacuum to give the
product (67 mg, 83%) as chromatographically pure material. m.p: 232-236 °C. TLC,
cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.62. FAB-MS (m/e): 371 (M)+. UV (MeOH),

max[nm] (lg): 442 (3.30), 394 (3.92), 374 (3.96), [356 (3.99)], 325 (4.51), [302 (4.38)],
276 (4.24), [250 (4.42)], 237 (4.47). 1H-NMR (DMSO-d6):  6.30 (s, 2H, CH2), 7.41-7.46
(m, 5H, Ph), 7.61-7.66 (m, 4H, Ph), 7.78 (t, J=7.7 Hz, 1H, Ph), 8.18 (d, J=7.3 Hz, 2H,
Ph), 8.34-8.37 (m, 3H, 2 Ph, Py), 8.52 (br s, 1H, Py), 8.77 (d, J=7.0 Hz, 1H, Py). Anal.
Calcd for C27H19BrN2: C, 71.85; H, 4.24; N, 6.21. Found: C, 71.59; H, 4.54; N, 6.12.
4.2.20. 1-N-Ethylacetatoeupolauridinium bromide (11t) (1-N-
Ethylacetatoindeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (100 mg, 0.49 mmol) in acetone (5 ml, anhydrous)
was added ethyl bromoacetate (1 ml, 9.02 mmol), and the mixture was stirred at room
temperature for 1 week.
The resulting yellow precipitate was collected, washed with acetone and ether,
and dried under high vacuum to chromatographically pure product (110 mg, 61%) as
material. In another experiment, the reaction mixture was heated at reflux for 72 h. Using
the same workup procedure mentioned above, the chromatographically pure product (73
mg, 40%) was again isolated. m.p: 207-209 °C (dec.). TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.58. FAB-MS (m/e): 292 (M)+. UV (MeOH), max[nm] (lg):
391 (3.90), 372 (3.88), [353 (3.64)], [311 (4.09)], 298 (4.22), [288 (4.20)], [256 (4.08)],
[242 (4.38)], 226 (4.43). 1H-NMR (DMSO-d6):  1.27 (t, J=7.0 Hz, 3H, CH3), 4.29 (q,
J=7.0 Hz, 2H, CH2), 6.15 (br s, 2H, CH2), 7.65-7.70 (m, 1H, Ph), 7.77 (t, J=7.7 Hz, 1H,
Ph), 7.96 (d, J=5.9 Hz, 1H, Py), 8.04 (d, J=7.5 Hz, 1H, Ph), 8.08 (d, J=6.8 Hz, 1H, Ph),
8.48 (d, J=6.6 Hz, 1H, Py), 8.86 (d, J=6.8 Hz, 1H, Py), 9.03 (d, J=6.2 Hz, 1H, Py). Anal.
Calcd for C18H15BrN2O2 x 0.4 H2O: C, 57.13; H, 4.21; N, 7.40. Found: C, 57.04; H, 4.13;
N, 7.42.
4.2.21. 1-N-Phenacyleupolauridinium bromide (11u) (1-N-Phenacylindeno[1,2,3-
ij][2,7] naphthyridinium bromide)
To a solution of eupolauridine (50 mg, 0.24 mmol) in acetone (5 ml, anhydrous)
was added a solution of 2-bromoacetophenone (0.48 g, 2.40 mmol) in acetone (1 ml,
anhydrous), and the mixture was heated at reflux overnight. After cooling to room
temperature, the resulting precipitate was collected, washed with acetone, ether, and dried
under high vacuum to give the pure product (32 mg, 32%) as a yellow solid. A second
fraction of the product (26 mg, 26%) was isolated by heating the filtrate at reflux for
another 48 h. m.p: 248-252 °C. TLC, cellulose, (4%-Na-citrate: MeOH, 1:1): Rf: 0.74.
FAB-MS (m/e): 323 (M)+. UV (MeOH), max[nm] (lg): 388 (3.87), 367 (3.86), [348
(3.61)], [320 (4.05)], [299 (4.25)], 288 (4.27), 236 (4.53), 227 (4.55). 1H-NMR (DMSO-
d6):  6.95 (s, 2H, CH2), 7.53-7.59 (m, 1H, Ph), 7.70-7.77 (m, 3H, Ph), 7.85 (d, J=7.5 Hz,
2H, Ph), 7.98 (d, J=5.9 Hz, 1H, Py), 8.09 (d, J=7.5 Hz, 1H, Ph), 8.19 (d, J=7.3 Hz, 2H,

22
Ph), 8.47 (d, J=6.8 Hz, 1H, Py), 8.78 (d, J=6.8 Hz, 1H, Py), 9.06 (d, J=5.9 Hz, 1H, Py).
Anal. Calcd for C22H15BrN2O: C, 65.52; H, 3.75; N, 6.95. Found: C, 65.34; H, 3.73; N,
7.06.
4.2.22. 1-N-Acetamidoeupolauridinium bromide (11v) (1-N-Acetamidoindeno[1,2,3-
ij][2,7]naphthyridinium bromide)
To a solution of eupolauridine (100 mg, 0.48 mmol) in acetone (10 ml,
anhydrous) was added a solution of bromoacetamide (0.70, 5.00 mmol) in acetone (6 ml,
anhydrous), and the mixture was heated at reflux for 48 h. After cooling to room
temperature, the resulting yellow precipitate was collected, washed with acetone and
ether, and dried under high vacuum to give the product (25 mg, 15%) as
chromatographycally pure material. A second fraction (26 mg, 16%) was isolated by
heating of the filtrate at reflux for another 48 h. m.p: 260 °C (beginning of dec.), 274-280
°C (completely dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.68. FAB-MS
(m/e): 262 (M)+. 1H-NMR (DMSO-d6):  5.80 (s, 2H, CH2), 7.67-7.80 (m, 2H, Ph), 7.93-
7.99 (m, 3H, 2 Ph, 1 NH), 8.08 (d, J=7.2 Hz, 1H, Py),8.22 (br s, 1H, NH), 8.42 (d, J=6.7
Hz, 1H, Py), 8.81 (d, J=6.7 Hz, 1H, Py), 9.02 (d, J=5.9 Hz, 1H, Py).
4.2.23. 1-N-Aminoeupolauridinium mesitylene sulfonate (11w) (1-N-Amino-1,6-
diazafluoranthenium mesitylene sulfonate, 1-N-Aminoindeno[1,2,3-
ij][2,7]naphthyridinium mesitylene sulfonate)
Freshly prepared O-mesitylenesulfonylhydroxylamine14-17 (300 mg, 1.39 mmol)
was added to a solution of eupolauridine (100 mg, 0.49 mmol) in methanol (15 ml), and
the mixture was stirred at room temperature for 30 min. The green reaction mixture was
added dropwise to ether (50 ml), and the resulting precipitate was collected, washed with
ether (2x 10 ml), dried under high vacuum to give the product (88 mg, 15%) as a green
solid. This material was submitted without any further purification for screening. FAB-
MS (m/e): 220 (M+H)+. 1H-NMR (DMSO-d6):  2.15 (s, 3H, CH3 (O-mesitylene)), 2.47
(s, 6H, CH3 (O-mesitylene)), 6.71 (s, 2H, Ph (O-mesitylene)), 7.70-7.76 (m, 2H, 2-Ph),
7.90 (d, J=5.9 Hz, 1H, Py), 8.09 (d, J=6.8 Hz, 1H, Ph), 8.29 (d, J=7.03 Hz, 1H, Py), 8.32-
8.35 (dd, J=1.1, 7.0 Hz, 1H, Ph), 8.64 (d, J=6.8 Hz, 1H, Py), 8.75 (br s, 2H, NH2), 8.93
(d, J=5.9 Hz, 1H, Py).
4.2.24. 6-Methyl-2,3-benzo-1,6-diazafluoranthenium iodide (13a) (1-N-Methyl-
benz[c] indeno[1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2,3-benzo-1,6-diazafluoranthene (300 mg, 1.18 mmol) in acetone
(30 ml, anhydrous) was added methyl iodide (3 ml, 48.18 mmol), and the mixture was
heated at reflux for 48 h. After cooling to room temperature, the resulting orange
precipitate was collected, washed with acetone and ether, and dried under high vacuum to
give product (403 mg, 86%) that was chromatographically pure. m.p: 256-260 °C dec.
TLC, cellulose, (4%-Na-citrat:MeOH, 1:1): Rf: 0.38. FAB-MS (m/e): 269 (M)+. UV
(MeOH), max[nm] (lg): 399 (3.77), 382 (3.75), 305 (4.66), 241 (4.52). 1H-NMR
(DMSO-d6): δ 4.72 (s, 3H, CH3), 7.76-7.88 (m, 2H, Ph), 7.94-7.99 (dt, J=1.3, 8.1 Hz, 1H,
Ph), 8.08-8.14 (dt, J=1.3, 7.3 Hz, 1H, Ph), 8.25 (d, J=7.5 Hz, 1H, Ph), 8.35 (d, J=8.4 Hz,
2H, Ph), 8.89-8.92 (dd, J=1.3, 7.9 Hz, 1H, Ph), 9.05 (d, J=6.8 Hz, 1H, Py), 9.10 (d, J=7.0

23
Hz, 1H, Py). Anal. Calcd for C19H13IN2: C, 57.60; H, 3.31; N, 7.07. Found: C, 57.14; H,
3.36; N, 6.97.
4.2.25. 6-Ethyl-2,3-benzo-1,6-diazafluoranthenium iodide (13b) (1-Ethylbenz[c]
indeno[1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2,3-benzo-1,6-diazafluoranthene (100 mg, 0.39 mmol) in acetone
(10 ml, anhydrous) was added ethyl iodide (1 ml, 48.18 mmol), and the mixture was heated
at reflux for 72 h. After cooling to room temperature, the resulting red-orange precipitate
was collected, washed with acetone and ether, and dried under high vacuum to give the
product (76 mg, 47%) as chromatographically pure material. m.p: >240 °C (beginning of
dec), 250-252 °C (completely dec.). TLC, cellulose, (4%-Na-citrat:MeOH, 1:1): Rf: 0.55.
FAB-MS (m/e): 283 (M)+. UV (MeOH), max[nm] (lg): 401 (3.76), 383 (3.74), [350
(3.74)], 307 (4.66), 242 (4.51). 1H-NMR (DMSO-d6):  1.69 (t, J=7.2 Hz, 3H, CH3) 5.06
(q, J=7.0 Hz, 2H, CH2), 7.76-7.88 (m, 2H, Ph), 7.93-7.98 (dt, J=1.1, 8.1 Hz, 1H, Ph),
8.08-8.13 (dt, J=1.3, 7.3 Hz, 1H, Ph), 8.24-8.26 (dd, J=1.3, 7.5 Hz, 1H, Ph), 8.34 (d,
J=7.9 Hz, 2H, Ph), 8.92 (d, J=7.0 Hz, 1H, Ph), 9.10 (d, J=6.6 Hz, 1H, Py), 9.16 (d, J=6.4
Hz, 1H, Py). Anal. Calcd for C20H15IN2 x 0.5 H2O: C, 57.30; H, 3.85; N, 6.68. Found: C,
57.67; H, 3.61; N, 6.99.
4.2.26. 6-N-Benzyl-2,3-benzo-1,6-diazafluoranthenium bromide (13c) (1-N-Benzyl-
benz[c]indeno[1,2,3-ij][2,7]naphthyridinium bromide)
To a solution of 2,3-benzo-1,6-diazafluoranthene (100 mg, 0.39 mmol) in acetone
(10 ml, anhydrous) was added benzyl bromide (1 ml, 8.41 mmol), and the mixture was
heated at reflux for 48 h. After cooling to room temperature, the resulting yellow
precipitate was collected, washed with acetone and ether, and dried under high vacuum to
give the product (128 mg, 77%) as chromatographically pure material. m.p: 248-252 °C.
TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.63. FAB-MS (m/e): 345 (M)+. UV
(MeOH), max[nm] (lg): 403 (3.77), 385 (3.75), [350 (3.77)], 307 (4.66), 243 (4.52). 1H-
NMR (DMSO-d6):  6.39 (s, 2H, CH2), 7.39-7.45 (m, 5H, Ph), 7.64-7.70 (dt,J=0.9, 7.7
Hz, 1H, Ph), 7.77-7.82 (dt, J=0.7, 7.7 Hz, 1H, Ph), 7.94-8.00 (dt, J=1.3, 8.4 Hz, 1H, Ph),
8.09-8.12 (dd, J=1.3, 8.1 Hz, 1H, Ph), 8.16 (d, J=7.7 Hz, 1H, Ph), 8.22 (d, J=7.3 Hz, 1H,
Ph), 8.34-8.37 (dd, J=1.1, 8.1 Hz, 1H, Ph), 8.92-8.95 (dd, J=1.3, 8.1 Hz, 1H, Ph), 9.17 (s,
2H, Py). Anal. Calcd for C25H17BrN2 x 0.3 H2O: C, 69.71; H, 4.12; N, 6.50. Found: C,
69.77; H, 4.08; N, 6.69.
4.2.27. 6-Methyl-2,3-benzo-1,6-diazafluoranthenium p-toluenesulfonate (13d) (1-
Methyl-benz[c]indeno[1,2,3-ij][2,7]naphthyridinium p-toluenesulfonate)
To a solution of 2,3-benzo-1,6-diazafluoranthene (100 mg, 0.39 mmol) in acetone
(10 ml, anhydrous) was added methyl p-toluenesulfonate (1 ml, 6.63 mmol), and the
mixture was heated at reflux for 48 h. After cooling to room temperature, the resulting
yellow precipitate was collected, washed with acetone and ether, and dried under high
vacuum to give the crude product (120 mg), contaminated with 13% (by 1H-NMR)
starting material. The crude product was suspended in acetone (10 ml, anhydrous), treated
with methyl p-toluensulfonate (1 ml, 6.63 mmol) and heated for an additional 72 hours at
reflux. The pure product (69 mg, 40%) was isolated as yellow solid. m.p: 238-242 °C.
TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.43. FAB-MS (m/e): 269 (M)+. UV

24
(MeOH), max[nm] (lg): 400 (3.77), 382 (3.75), [350 (3.71)], 305 (4.66), 241 (4.51). 1H-
NMR (DMSO-d6):  2.28 (s, 3H, CH3), 4.72 (s, 3H, CH3), 7.11 (d, J=7.9, 2H, Ph), 7.46
(d, J=8.1, 2H, Ph), 7.60-7.88 (m, 2H, Ph), 7.93-7.99 (dt, J=1.3, 8.1 Hz, 1H, Ph), 8.08-8.14
(dt, J=1.3, 7.3 Hz, 1H, Ph), , 8.24-8.26 (d, J=7.0 HZ, 1H, Ph), 8.34-8.37 (d, J=8.1 HZ,
2H, Ph), 8.89-8.92 (dd, J=0.9, 9.0 Hz, 1H, Ph), 9.05 (d, J=6.8 Hz 1H, Py), 9.09 (d, J=6.8
Hz 1H, Py). Anal. Calcd for C26H20N2O3S x 0.2 H2O: C, 70.60; H, 4.60; N, 6.33. Found:
C, 70.47; H, 4.60; N, 6.72.
4.2.28. 1,2-Dimethyl-benzo[e]-1,6-diazafluoranthenium iodide (13e) (1,2-Dimethyl
benz [f]indeno[1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2-methyl-4,5-benzo[e]-1,6-diazafluoranthene (100 mg, 0.37
mmol) in acetone (25 ml, anhydrous) was added methyl iodide (1 ml, 16.06 mmol), and
the mixture was heated at reflux for 3 days. The resulting yellow precipitate was filtered
from the hot mixture, washed with acetone, chloroform, and ether, and dried under high
vacuum to give the chromatographically pure product (60 mg, 40%). m.p: 238-244 °C
dec. TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.42. FAB-MS (m/e): 283 (M)+. UV
(MeOH), max[nm] (lg): 403 (3.82), 384 (3.81), [356 (3.72)], [322 (4.34)], 303 (4.64),
237 (4.50). 1H-NMR (DMSO-d6):  3.03 (s, 3H, CH3), 4.57 (s, 3H, CH3), 7.75-7.86 (m,
2H, Ph), 7.92-7.97 (dt, J=1.2, 7.3 Hz, 1H, Ph), 8.06-8.11 (dt, J=1.3, 8.3 Hz, 1H, Ph),
8.23-8.25 (dd, J=0.8, 7.3 Hz, 1H, Ph), 8.31 –8.34 (dd, J=0.8, 7.3 Hz, 1H, Ph), 8.48 (d,
J=7.8 Hz, 1H, Py), 8.78-8.81 (dd, J=1.1, 9.1 Hz, 1H, Py), 9.05 (br s, 1H, Py). Anal. Calcd
for C20H15IN2 x 0.4 HI: C, 52.06; H, 3.36; N, 6.07. Found: C, 52.03; H, 3.51; N, 5.95.
4.2.29. 1-N-Methyl-5-phenyl-2,3-benzo-1,6-diazafluoranthenium iodide (14f) (8-N-
Methyl-2-phenyl-benz[c]indeno[1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2-phenyl-2,3-benzo-1,6-diazafluoranthene (100 mg, 0.30 mmol)
in chloroform (10 ml, anhydrous) was added methyl iodide (1 ml, 16.06 mmol), and the
mixture was heated at reflux overnight. After cooling to room temperature, the resulting
red precipitate was collected, washed with acetone and ether, and dried under high
vacuum to give the product (14 mg, 10%) as a chromatographically pure material. A
second fraction (50 mg, 35%) of the product was obtained by adding methyl iodide (1 ml,
16.06 mmol) to the filtrate and further heating at reflux for 48 hours. m.p: 232-236 °C dec.
TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.28. FAB-MS (m/e): 345 (M)+. UV
(MeOH), max[nm] (lg): 450 (3.47), 402 (3.99), [380 (4.10)], [363 (4.16)], 324 (4.57),
[310 (4.41)], 274 (4.52), 236 (4.61). 1H-NMR (DMSO-d6):  4.82 (s, 3H, N(8)-CH3),
7.63-7.68 (m, 3H, Ph), 7.73-7.78 (dt, J=1.1, 7.9 Hz, 1H, Ph), 7.88 (t, J=7.4 Hz, 1H, Ph),
8.17-8.30 (m, 3H, Ph), , 8.49-8.52 (m, 2H, Ph), 8.60 (d, J=7.9, 1H, Ph), 8.78 (d, J=8.5,
1H, Ph), 9.13 (s, 1H, Py), 9.29 (dd, J=1.3, 7.9 Hz, 1H, Ph). Anal. Calcd for C25H17IN2 x
0.8 H2O: C, 61.69; H, 3.85; N, 5.76. Found: C, 61.72; H, 3.62; N, 5.53.
4.2.30. 1-N-Ethyl-5-phenyl-2,3-benzo-1,6-diazafluoranthenium iodide (14g) (8-N-
Ethyl-2-phenylbenz[c]indeno[1,2,3-ij][2,7]naphthyridinium iodide)
To a solution of 2-phenyl-2,3-benzo-1,6-diazafluoranthene (50 mg, 0.15 mmol) in
dioxane (10 ml, anhydrous) was added ethyl iodide (0.5 ml, 6.25 mmol), and the mixture
was heated at reflux 48h. After cooling to room temperature, the resulting red precipitate
was collected, washed with dioxane and ether, and dried under high vacuum to give the

25
product (11 mg, 15%) as chromatographically pure material. A second fraction (11 mg,
15%) of the product was isolated by adding of an excess of ethyl iodide (0.5 ml, 6.25
mmol) to the filtrate and further heating at reflux for 48 hours. m.p: >220 °C dec.
(beginning of dec.), 230-235 °C (completely dec.). TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.28, FAB-MS (m/e): 359 (M)+. 1H-NMR (DMSO-d6):  1.77 (t,
J=7.3 Hz, 3H, CH3), 5.34 (q, J=7.3 Hz, 2H, CH2), 7.62-7.71 (m, 3H, Ph), 7.75-7.80 (m,
1H, Ph), 7.90 (t, J=7.5 Hz, 1H, Ph), 8.17-8.31 (m, 3H, Ph), 8.46 (d, J=7.7, 1H, Ph), 8.52-
8.55 (m, 2H, Ph), 8.82 (d, J=8.6, 1H, Ph), 9.13 (s, 1H, Py), 9.29 (dd, J=1.5, 8.1 Hz, 1H,
Ph).
4.2.31. 1-N-Ethoxycarbonylmethyl-4,5-Benzo-1,6-diazafluoranthenium bromide
(13h) (1-N- Ethoxycarbonylmethylbenz[c]indeno[1,2,3-ij][2,7]naphthyridine)
To a solution of 2,3-benzo-1,6-diazafluoranthene (100 mg, 0.39 mmol) in acetone
(12 ml, anhydrous) was added ethyl bromoacetate (1 ml, 9.02 mmol) and the mixture was
heated at reflux overnight. The resulting yellow precipitate was collected, washed with
acetone and ether and dried under high vacuum to give the product (56 mg, 33%) as
chromatographically pure material. A second fraction (29 mg, 17%) was isolated by
adding excess ethyl bromoacetate (1 ml, 9.02 mmol) to the filtrate and heating the mixture
for 48 hours. m.p: 216-218 °C (dec.). TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf:
0.69. UV (MeOH), max[nm] (lg): 405 (3.78), 385 (3.76), [352 (3.78)], 309 (4.68), 243
(4.51). FAB-MS (m/e): 341 (M)+. 1H-NMR (DMSO-d6):  1.27 (t, J=7.0 Hz, 3H, CH3),
4.29 (q, J=7.0 Hz, 2H, CH2), 6.18 (s, 2H, CH2), 7.73-7.78 (m, 1H, Ph), 7.82-7.87 (m, 1H,
Ph), 7.95-8.00 (m, 1H, Ph), 8.05 (d, J=7.7 Hz, 1H, Ph), 8.11-8.16 (m, 1H, Ph), 8.24 (d,
J=7.3 Hz, 1H, Ph), 8.35 (d, J=8.1 Hz, 1H, Ph), 8.89 (d, J=7.0 Hz, 1H, Ph), 9.09 (d, J=7.0
Hz, 1H, Py), 9.18 (d, J=6.8 Hz, 1H, Py). Anal. Calcd for C22H17BrN2O2 x 0.1 H2O: C,
62.45; H, 4.10; N, 6.62. Found: C, 62.34; H, 4.08; N, 6.69.
4.2.32. 1-N-Methyl-5,6-dioxomethylenebenz[c]indeno[1,2,3-ij][2,7]naphthyridinium
bromide (13i)
To a solution of 5,6-dioxomethylenebenz[c]indeno[1,2,3-ij][2,7]naphthyridine
(100 mg, 0.34 mmol) in chloroform (35 ml, anhydrous) was added methyl iodide (1.0 ml,
16.06 mmol), and the mixture was heated at reflux for 7 days. After cooling to room
temperature, the resulting precipitate was collected, washed with chloroform, ether and
dried under high vacuum to give the product (85 mg, 58%), along with 10% unreacted
starting material as a impurity. This material was recrystallized from methanol (200 ml)
and dimethyl formamide (50 ml) to give the product (31 mg, 21%) as a red solid with a
purity of 96 %, according to 1H-NMR. m.p: 300-304 °C. TLC, cellulose, (4%-Na-
citrate:MeOH, 1:1): Rf: 0.22. FAB-MS (m/e): 313 (M)+. 1H-NMR (DMSO-d6):  4.67 (br
s, 3H, CH3), 6.41 (br s, 2H, CH2), 7.70-7.78 (m, 2H, Ph), 7.81 (s, 1H, Ph), 8.11 (d, J=7.0
Hz, 1H, Ph), 8.29 (d, J=3.5 Hz, 1H, Ph), 8.41 (s, 1H, Ph), 8.90-8.98 (m, 2H, Py).
4.2.33. 1-N-Benzyl-5,6-dioxomethylenebenz[c]indeno[1,2,3-ij][2,7]naphthyridinium
bromide (13j)
To a solution of 5,6-dioxomethylenebenz[c]indeno[1,2,3-ij][2,7]naphthyridine (50
mg, 0.17 mmol) in chloroform (10 ml, anhydrous) was added benzyl bromide (0.5 ml, 4.20
mmol), and the mixture was heated at reflux for 48 h. After cooling to room temperature,

26
the resulting precipitate was collected, washed with chloroform and ether, and dried
under high vacuum to give the pure product (35 mg, 44%) as an orange solid. A second
fraction of the product (27 mg, 34%) was isolated by heating the filtrate at reflux for
another 48 h. m.p: 280-284 °C. TLC, cellulose, (4%-Na-citrate:MeOH, 1:1): Rf: 0.43.
FAB-MS (m/e): 389 (M)+. UV (MeOH), max[nm] (lg): [411 (3.54)], [390 (3.75)], 336
(4.74), [296 (4.14)], 276 (4.28), 251 (4.37), [226 (4.46)]. 1H-NMR (DMSO-d6):  6.33 (s,
2H, CH2), 6.43 (s, 2H, CH2), 7.37-7.46 (m, 5H, Ph), 7.61 (t, J=7.7 Hz, 1H, Ph), 7.75 (t,
J=7.5 Hz, 1H, Ph), 7.82 (s, 1H, Ph), 8.11 (d, J=7.7 Hz, 2H, Ph), 8.45 (br s, 1H, Ph), 9.01
(d, J=6.8 Hz, 1H, Py), 9.06 (d, J=6.6 Hz, 1H, Py). Anal. Calcd for C26H17BrN2O2 x 0.7
HBr: C, 59.37; H, 3.39; N, 5.33. Found: C, 59.39; H, 3.58; N, 5.45.
4.3. Biology Methods
4.3.1. Antimicrobial Activity
All compounds were assessed for growth inhibitory activity against a panel of
microorganisms including Candida albicans B311 (Ca), Cryptococcus neoformans
(ATCC 52657, Cn), Mycobacterium intracellulare (ATCC 23068, Mi), Aspergillus
fumigatus (ATCC 26934, Afu), Pseudomonas aeruginosa (ATCC 15442, Pa),
Staphylococcus aureus (ATCC 6538, Sa) and methicillin-resistant Staphylococcus aureus
(MRS). Growth inhibition was determined in 96-well microtiter plates by incubating the
organism with diluted samples and assessing growth compared to controls.17 Ca was
cultured in Sabouraud’s dextrose broth, while mycophil broth was used to culture Cn and
the Aspergillus species. Trypticase soy broth was used for Pa and Eugon for Sa. Mi was
cultured in Middlebrook media with OADC supplement. Prior to the assay, the
appropriate media were inoculated with microorganisms and allowed to incubate for 24
(Ca, Pa, Sa, MRS) or 72 (Cn, Mi) hours. Yeasts were counted on a hemocytometer and
diluted in broth to 10,000 (Ca) or 200,000 (Cn) CFUs/mL. Pa, Sa and MRS were diluted
1:50 in broth while Mi was diluted 1:30. Inocula were prepared from the Aspergillus
species on the day of the assay by flooding a slant with a mat of fungus growth and
gently scraping with a pipet tip to release spores. On the day of the assay, inoculum was
added to diluted sample in the wells of a 96 well microtiter plate such that the greatest
final concentration tested was 50 g/mL (the highest DMSO concentration was 2.5%).
The plates were incubated at 37C (Ca, Mi, Pa, Sa, MRS) or at 30C (Cn, Afl, Afu).
Following incubation, the growth is determined as absorbance at 630 nm measured with
an EL-340 plate reader (Bio-Tek Instruments, Inc.).The inhibitory activity of the sample
was expressed as the Minimum Inhibitory Concentration (MIC), the lowest concentration
tested which inhibited 80% of growth compared to controls. IC50 values (the
concentration which inhibited 50% of growth) were also determined from graphs of
concentration versus % growth compared to the controls.18
4.3.2. Cytotoxicity
Compounds were assessed for inhibition of growth of human cancer cell lines:
SK-MEL, KB, BT-549 and SK-OV-3. Additionally, a cell line derived from monkey
kidney fibroblasts, VERO, was also used to represent non-cancerous cells. Cells were
cultured in RPMI-1640 media supplemented with 10% bovine serum and 60 g/mL

27
amikacin and incubated at 37C and 5% CO2. Cells were plated in 96-well tissue culture-
treated microtiter plates at 25,000 cells/well and allowed to adhere for 24 hours. Samples,
diluted in RPMI, were added to the wells such that highest final concentration was 10
g/mL. Following 48 hours of incubation with samples, growth was assessed by
measuring uptake of the supravital dye, neutral red.19
4.3.3. Antimalarial Activity
Compounds were tested for antimalarial activity in vitro against a chloroquine
sensitive strain of Plasmodium falciparum (D6). Parasites were cultured in A+ human red
blood cells suspended in RPMI 1640 supplemented with 10% normal type A+ human
serum, 25 mM HEPES, 27 mM bicarbonate and 60 ug/mL amikacin at 37°C and under a
gas mixture of 90% N2, 5% O2 and 5% CO2. Susceptibility assays were conducted in a
manner similar to Makler et al. (1993)20 in which infected red blood cells were incubated
for 48 hours with samples in wells of a microtiter plate. The highest concentration of
samples tested was 4.8 g/mL. Following incubation, the growth of parasite was assessed
with the Malstat reagent (Flow Inc., Portland, OR) followed by reduction of nitro blue
tetrazolium dye. Absorbance of the reduced dye was measured at 630 nm. The growth
assay is based on the specificity of 3-acetylpyridine adenine dinucleotide
(Malstatreagent) as a substrate of the parasite lactate dehydrogenase.20
4.3.4. Inhibition of TopoisomeraseI
The catalytic activity of TopoI was determined in terms of the conversion of supercoiled
DNA to relaxed DNA as described earlier.4 The assay for inhibition was performed using
the TopoI drug kit (TopoGen, Inc.) according to the procedure provided with the kit.The
IC50 values were calculated rom the dose response curves of %inhibition versus tested
concentration.
Acknowledgement
The authors wish to gratefully acknowledge support of this research by the National
Institute of Health grant R01 AI33865. We also thank Dr. James M. Riordan for
assistance with nmr spectral interpretation.

28
References
(1)
(2)
(3)
Hufford, C.D.; Liu, S.; Clark, A.M.; Oguntimein, B.O. Anticandidal Activity of
Eupolauridine and Onychine, Alkaloids from Cleistopholis patens. J. Nat. Prod.,
1987, 50, 961.
Clark, A.M.; Watson, E.S.; Khalid Ashfaq, M.; Hufford, C.D. In Vivo Efficacy of
Antifungal Oxaporphine Alkaloids in Experimental Candidiasis. Pharm. Res.,
1987, 4, 495-498.
Clark, A.M.; Hufford, C.D. Discovery and Development of Prototype Antifungal
Antibiotics from Higher Plants: Eupolauridine. 29th Interscience on
Antimicrobial Agents and Chemotherapy, September 17-20, 1989, Houston, TX,
Abstract No. 720.
(4)
(5)
Khan, S.I.; Nimrod A.C.; Mehrpooya M.; Nitiss J.L.; Wlker L.A.; Clark A.M.
Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases.
Antimicrob Agents Chemother,2002, 46, 1785-1792.
Read, R.W.; Taylor, W.C. Constituents of Eupomatia Species. V. The Isolation of
Eupomatenoid-13 (a New Neolignan), (±)-trans-Dehydrodiisoeugenol, and Other
Extractives from the Bark of Eupomatia laurina. Aust. J. Chem., 1979, 32, 2317-
2321.
(6)
Waterman, P.G.; Muhammad, I. Sesquiterpenes and Alkaloids from Cleistopholis
Patens. Phytochemistry, 1985, 24(1), 523-527.
(7)
(8)
(9)
Parker, W.B., unpublished results.
Clark, A.M.; Pfefferkorn, E., unpublished results.
Bracher, F. Polycyclic Aromatic Alkaloids. II. Synthesis of Onychine and
Eupolauridine. Arch. Pharm. (Weinheim), 1989, 322, 293-294.
(10) Taghavi-Moghadam, S.; Kwong, C.D.; Riordan, J.M.; Truss, J.W.; Secrist, J.A.
III; Khan, S.; Rogers, R.D; Clark, A.M. Manuscript In Preparation.
(11) Tong, T.H.; Wong, H.N.C. Synthesis of Eupolauridine and Its Benzo-Annulated
Derivative. Synth. Commun., 1992, 22(12), 1773-1782.
(12) Bowden, B.F.; Pickler, K.; Ritchie, E.; Taylor, W.C. Constituents of Eupomatia
species. IV. Structure and synthesis of eupolauridine (EL base-1) and some
observations on the structures of eupolauramine (EL base-2) and
hydroxyeupolauramine (EL base-3). Aust. J. Chem., 1975, 28, 2681-2701.

29
(13) Tamura, Y.; Minamikawa, J.; Miki, Y.; Matsugashita, S.; Ikeda, M. A novel
method for heteroaromatic N-imines. Tetrahedron Lett. 1972, 13, 4133-4135.
(14) Tamura, Y.; Miki, Y.; Minamikawa, J.; Ikeda, M. Mono-N-amino salts of
benzodiazepines and naphthyridines. J.Heterocycl. Chem. 1974, 11, 675-679.
(15) Wiemer, D.F.; Leonard, N.J. Site of N-amination of adenine and alkyladenines.
J.Org. Chem. 1974, 39, 3438-3440
(16) Kwong, C.D.; Jois, Y.H.R., unpublished results.
(17) Tripathi, S.K.; Xu, T.; Feng, Q.; Avula, B.; Shi, X.; Pan, X.; Mash, M.M.; Jacob,
M.R.; Ranga Rao, R.; Li, X.-C.; Baerson, S.R.; Khan, IlA.; Clark, A.M.; Agarwal,
A.K., manuscript in preparation.
(18) Chand, S.; Lusunzi, I.; Veal, D.A.; Williams, L.R.; Karuso, P. Rapid screening of
the antimicrobial activity of extracts and natural products. J. Antibiot., 1994, 47,
1295-1304
(19) Borenfreund E.; Babich H.; Martin-Alguacil N. Rapid Chemosensitivity Assay
with Human Normal and Tumoral cells in vitro. In vitro Dev. Cell. Biol. 1990, 26:
1030-1034.
(20) Makler, M. T.; Hinrichs, D. J. Measurement of the Lactate Dehydrogenase
Activity of Plasmodium falciparum as an assessment of Parasitemia. Am. J. Trop.
Med. Hyg. 1993, 48: 205-210.