Identification of bis-quindolines as new antiinfective agents

Article abstract of DOI:10.1016/j.bmc.2005.04.008

Several N-substituted quindolines were made to further evaluate the role of N-alkylation on the activity of indoloquinolines as antifungal agents. While N-5 substitution is required for these activities, N-10 alkylation alone leads to inactive products bu

Full text of DOI:10.1016/j.bmc.2005.04.008

Bioorganic & Medicinal Chemistry 13 (2005) 3955–3963
Identification of bis-quindolines as new antiinfective agents
Leroy G. Mardenborough,a Xue Y. Zhu,a Pincheng Fan,a Melissa R. Jacob,b
Shabana I. Khan,b Larry A. Walkerb and Seth Y. Ablordeppeya,*
aCollege of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA
bThe National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences,
University of Mississippi, University, MS 38677, USA
Received 26 March 2005; revised 5 April 2005; accepted 5 April 2005
Available online 28 April 2005
Abstract—Several N-substituted quindolines were made to further evaluate the role of N-alkylation on the activity of indoloquin-
olines as antifungal agents. While N-5 substitution is required for these activities, N-10 alkylation alone leads to inactive products
but is tolerated in the presence of N-5 alkyl groups. It was also discovered that bis-quindolines appear to have a more expanded
antimicrobial spectrum and lower cytotoxicity than their monomeric counterparts.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The unique structural arrangement of the quindolines
has inspired our curiosity in seeking to understand the
contributions of the various components. Consequently,
we have shown that alkylation of the N-5 atom in
quindoline is necessary for several of the activities.12,13
In particular, we have reported that x-phenylpentyl
and x-cyclohexylpentyl moieties on the N-5 atom of
the quindoline ring produce a high antifungal potency
and broaden the spectrum of activities.12 It is interesting
to note that N-5 alkylation produces an anhydronium
base in which the N-5 nitrogen becomes positively
charged under acidic conditions, that is, aromatic qua-
ternary nitrogen (2b), but reverts to sp3 type nitrogen
under basic conditions (2a). This physical characteristic
of cryptolepine is also accompanied by a color change
from pink in a basic to orange in an acidic environment;
suggesting that a significant change in electron distribu-
tion has occurred. This unique behavior may allow for
The tetracyclic structure of indolo[3,2-b]quinoline, also
referred to as quindoline (1) constitutes an important
structural moiety in the literature because of its effect
on numerous biological functions.1 For example, cry-
ptolepine (2a and b) and several of its analogs display
antiaggregatory,2 antifungal,3 antihypertensive,4 antihy-
perglycemic,5 antibacterial,6 anticancer,7 antimalarial,8
activities among others. A unified mechanism by which
the drug produces the different biological activities has
not been elucidated. However, cryptolepine has been
shown to bind to DNA fragments9 in a rather unique
fashion. Furthermore, it intercalates DNA and stimu-
lates topoisomerase II mediated cutting of DNA.7,10
More recently, cryptolepine has been identified as a po-
tential inhibitor of telomerase and a G-quadruplex
DNA stabilizing agent.11
Keywords: Cryptolepine; Indoloquinoline; Quindoline; Quaternary amines; Indole bis-quindolines; Antifungal; Antiinfective; Antiparasitic;
Antimalarial.
*
Corresponding author. Tel.: +1 8505 993834; fax: +1 8505 993934; e-mail: seth.ablordeppey@famu.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.008

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L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
Chart 1. Compounds synthesized and tested for biological activity.
easy entry into cells in the basic form and also for
the production of its pharmacological effect in the salt
form.
2. Chemistry
The construction of the tetracyclic structure of quindo-
line has been widely reported.15–21 In this paper, we used
a previously reported method15 to construct the quindo-
line unit. Briefly, a substituted or unsubstituted anthra-
nilic acid was acylated with 2-bromoacetyl bromide and
the resulting alkyl halide was used to alkylate aniline.
The alkylated aniline underwent a double cyclization
reaction in the presence of polyphosphoric acid (PPA)
to yield a quindolone, which was chlorinated with phos-
phorous oxychloride (POCl3). The resulting chloride
was dechlorinated with hydrogen on palladium to ob-
tain the desired quindoline (Scheme 1). For compound
6f, 2-iodoquindoline was converted to 2-thiophenylqu-
indoline as previously reported.14
The hypothesis that a charged N-5 atom is necessary for
biological activity was tested by alkylating the N-10
atom of cryptolepine to prevent anhydronium base form-
ation and to produce a compound with a permanently
charged N-5 atom (3a). Because compound 3a showed
antifungal activity, we reported that the active form of
the indoloquinoline ring system is the salt form in which
N-5 is positively charged.14 This view is consistent with
the binding mode of cryptolepine to DNA fragments re-
ported by Aymami and co-workers.9 on the basis of
their X-ray crystallographic work. Thus, the purpose
of this study was to investigate the effect of various com-
binations of arylalkyl substituents (on N-5 and N-10
atoms) on the antiinfective properties of these agents
(Chart 1).
Specific alkylation of either the N-5 or N-10 atoms
was accomplished using the methods we previously
Scheme 1. The general synthetic method for substituted quindolines. Reagents: (i) BrCH2COBr, NaOH; (ii) DMF, PhNH2; (iii) (a) PPA, 130 °C; (b)
POCl3; (c) H2/10% Pd–C.

L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
3957
Scheme 2. Synthesis of bis-alkylated quindolines. Reagents: (i) trimethylene sulfone (TMS), I(CH2)5I; (ii) NaH, I(CH2)5I; (iii) CH3I, TMS.
reported.16 The synthesis of bis-quindolines linked
through their N-5 atoms was accomplished by heating
quindoline with 1,4-diiodobutane (9) or the correspond-
ing 1,5-diiodopentane (10). The formation of bis-quind-
olines, joined through the N-10 atoms required a
strongly basic medium and was achieved by the intro-
duction of sodium hydride. Bis-alkylation at N-10 was
then followed by bis-methylation of N-5 (Scheme 2).
electron withdrawing bromine while enhancing anti-
cryptococcal activity, had little or no effect on antican-
dida action.14 Similarly, the electron-donating methoxy
group has little or no effect on either activity when com-
pared to 3a. We have previously shown that alkylation
of N-5 in quindoline with x-phenylpentyl or x-cyclo-
hexylpentyl moieties as in 4a and 4b enhances antifungal
activity (Table 1). In this form, the positively charged N-
5 atom is retained. Thus, it was of interest to investigate
the contribution of the positively charged N-5 atom.
3. Results
One way a non-N-5 alkylated quindoline can produce a
positively charged N-5 atom is to form the salt. Hence,
we synthesized two N-10 methylated quindolines with
Re-evaluation of N-10 methylated cryptolepines (3a–c),
confirmed that substitution at the 2-position with an
Table 1. Physicochemical data and antifungal activities of synthetic compounds
Comp
Solva
% Yieldb
MP (°C)c
Empiricald formula
IC50(lg/mL)
Ca
Cn
2be
A
A
A
A
A
A
B
73
100
57
27
67
34
94
62
54
54
73
64
71
88
43
67
30
100
22
65
45
78
76
87
75
265–268
304–306
285–288
262–264
218–219
262–264
110–112
160–162
223–224
223–224
229–231
231–233
232–234
231–233
221–223
215–217
203–204
217–220
196–198
79–83
C16H13N2I
C17H15N2I
15.6
6.3
0.4
4.3
1.3
0.3
62.5f
11
180
3.1f
3.1f
2.1
3ae
3be
C17H14N2BrI
C18H17N2OIÆ1.0MeOH
C26H25N2Br
3ce
4ae
80
4be
5a
C26H31N2Br
61.3
125f
43
C
16H12N2Æ0.3H2O
C16H11N2Br
26H25N2ClÆHCl
C26H29N2BrÆHCl
27H29N3ÆHCl
5b
5c
B
A
A
A
A
A
A
A
A
A
A
C
C
A
D
A
A
A
C
9.0
>50
>50
>50
>50
>50
>50
15
18.0
>50
>50
>50
>50
>50
>50
>20
4.3
6a
6b
C
6c
6d
C26H29N2FÆHCl
C27H32N2OÆHCl
C27H32N2ÆHCl
C32H34N2SÆHCl
C27H27N2I
6e
6f
7a
7be
7ce,g
8a
C27H27N2BrÆ1.4MeOH
C27H26N2Br2
4.3
1.3
43
43
C
16H14NI
C20H28NBrÆ0.8H2O
16H26NBrÆ1.4H2O
C34H30N4I2Æ0.5H2O
35H30N4I2Æ3.5H2O
C35H28N4Æ0.6H2O
37H34N4I2Æ0.5H2O
180
80
8b
8c
20
86–89
C
3.5
4.0
1.5
NA
2.0
0.6
20
9
10
252–254
256–258
Oily
>50
>50
NA
2.0
C
11
12
238–240
C
Amphotericin B
0.1
Abbreviations in the table are as follows: Ca = Candida albicans, Cn = Cryptococcus neoformans. NA = not active at 180 lg/mL.
a Recrystallization solvents are: A = MeOH–Et2O, B = hexane–EtOAc, C = EtOH–Et2O, D = MeOH–CH2Cl2.
b Yields were not optimized.
c Melting points were uncorrected.
d All compounds were subjected to CHN analysis and each passed within 0.4% of the theoretical value.
e Compounds were previously reported.12,14
f MIC values in microgram per milliliter.
g Compound crystallized with 0.5H2O and 1.0MeOH.

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L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
N-5 atoms, which should be converted to their salt form
in aqueous solution (5a and 5b), for evaluation. The re-
sults showed that these compounds were only weakly ac-
tive against Cryptococcus neoformans and Candida
albicans. Compound 5c was synthesized to explore the
possibility that antifungal potency might be enhanced
in the same way that x-phenylpentyl groups at N-5 en-
hanced the potency of quindoline. Indeed, a moderate
increase in potency (ꢀ10-fold) over 5a was observed.
The enhanced potency of compound 4b over cryptole-
pine and the increased potency of 2-substituted ana-
logs14 also led to the synthesis of several analogs (6a–
f) with x-cyclohexylpentyl substituent at N-10. The N-
10 alkylated analogs were also of interest because N-
10 alkylation enables both the free base form and the
salt form to co-exist in an aqueous medium, allowing
the free base form of the drug to penetrate fungal cell
membranes while the presumed active protonated form
interferes with cell reproduction. The results indicate
that N-10 alkylated analogs of quindoline have little
or no activity as antifungal agents. Since quindoline
salts have no antifungal activity, but N-5 alkylated
quindolines do have activity, it appears that a positive
N-5 atom along with a hydrophobic interaction with
the N-5 alkyl groups may be important for the actions
of these alkylated indoloquinolines.
constitute important structural features for the activities
observed in N-alkyl substituted quindolines.
Since quindolines with phenyl and cyclohexyl moieties
placed five carbon atoms away from N+-5 display high
antifungal potency, we investigated the possibility that
bis-quindolines five carbons from each other might simi-
larly show enhanced antifungal potency perhaps by
interacting with two adjoining DNA molecules. Com-
pounds 10, in which the pyridine N-atoms are con-
nected, and 11 and 12, where the tetracycles are joined
by the indole N-atoms, were thus synthesized and evalu-
ated for biological activity. The results show that the
positively charged quaternary atom is required for acti-
vity even in the bis-quindolines; compounds 10 and 12
possess potent activity while compound 11 (the non-
alkylated compound) displays no activity. In addition,
consistent with our previous observation that an x-
cycloalkylpentyl substituent on N-5 displays an en-
hanced potency, a bis-quindoline separated by a 5-car-
bon chain (compound 10) was more potent than that
separated by a 4-carbon chain (compound 9). Although
alkylation on the indole N-atom did not result in activ-
ity (compound 11), methylation of the N-5 atoms (com-
pound 12) resulted in potent activity and there was little
difference in the antifungal activities of 10 and 12 when
compared together.
Since alkylation of both N-5 and N-10 appears to be
beneficial (3a–c), it became of interest to examine the
position for optimum placement of the alkyl groups.
Thus, compounds 7a–c were prepared and evaluated
for their antifungal properties and the results are re-
corded in Table 1. These results indicate that when N-
5 is substituted with the longer x-phenylpentyl moiety
and N-10 is methylated (7b), this combination is more
potent than the reverse (7a) in C. neoformans and C.
albicans. However, there appears to be no significant
overall improvement in potency compared to the doubly
methylated compounds, 3a–c.
A selected number of the compounds (7a, 9, 10, and 12)
based on their novelty, was further evaluated in addi-
tional assays available in our laboratories with cryptole-
pine as the benchmark, to explore their antimicrobial
spectrum. Their cytotoxicities to a mammalian cell were
also determined. The results are reported in Tables 2
and 3. Evaluation of these results shows that com-
pounds 7a, 10, and 12 have more expansive antimicro-
bial/antiparasitic spectra than cryptolepine. All three
compounds also display activity against methicillin-
resistant Staphylococcus aureus (MRSA). Similarly, all
four compounds displayed significant activity against
Mycobacterium intracellulare (Mi). Among the four
compounds tested however, only 7a showed significant
potency against Aspergillus fumigatus (Af), Candida kru-
sei (Ck), and Plasmodium falciparum (Pf) and only com-
pound 10 displayed activity against Pseudomonas
aeruginosa (Pa). Thus, among the bis-quindolines, join-
ing the pyridine nitrogens (compounds 9 and 10) by
an alkyl chain appears to be more effective than through
The importance of the quaternary N-5 atom in these
indoloquinolines also led us to investigate compounds
8a–c in order to dispel the notion that the antifungal
activity of these compounds was intricately associated
with any functionality with a quaternary N+ atom and
an alkyl function such as the x-cyclohexylpentyl moiety.
As shown in Table 1, there was little antifungal activity
associated with 8a–c suggesting that the A and B rings
Table 2. The effect of N-alkylation on the antimicrobial activity of selected quindolines
Compound
IC50 (lg/mL)
Af
Ck
20
2.0
MRSA
Mi
Pa
Sa
10.0
0.60
>20
3.0
4.0
NT
0.10
Cryptolepine
7a
>20
20
2.0
15.0
1.0
>20
>20
>20
2.5
9
10
>20
20
>20
>20
15
>20
3.0
6.0
5.5
7.0
1.5
12
Amphotericin B
Ciprofloxacin
20
0.31
NT
15.0
NT
>20
NT
0.60
NT
NT
0.15
0.20
0.06
Abbreviations: NT = not tested. Aspergillus fumigatus (Af), Candida krusei (Ck), methicillin-resistant Staphylococcus aureus (MRSA), Mycobacte-
rium intracellulare (Mi); Pseudomonas aeruginosa (Pa); Staphylococcus aureus (Sa).

L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
Table 3. Antimalarial activity and cytotoxicity of selected quindolines
3959
Compound
Plasmodium falciparum (IC50, ng/mL)
Cytotoxicity (Vero) (TC50, ng/mL)
Pf (D6)
SI
Pf (W2)
SI
Cryptolepine
7a
180
62
23.3
145
420
28
10
321
4200
9000
9
10
>528
120
260
14.5
11
NA
88
NC
>198
>91.5
>1400
>2800
>270
>170
>317
>23,800
>23,800
>23,800
>23,800
6500
12
140
75
Chloroquine
Artemisinin
Amphotericin B
6
NT
>7677
NT
Abbreviations: NC = no cytotoxicity observed up to 5 lg/mL; NT = not tested. NA = not active up to 20 lg/mL; SI = IC50 (vero cells)/IC50 (Pf).
D6 = D6 Clone; W2 = W2 Clone.
the indole nitrogen (compound 11). In addition, the
5-chain compound (10) was more potent than the 4-
chain structure (9). Interestingly, cryptolepine was more
toxic than any of the four selected compounds. It is
important to note that these compounds may not be act-
ing through their monomeric units since that would
have resulted in similar or higher toxicity and decreased
potency per unit weight of compound. The fact that
compounds 10 and 12 displayed no cytotoxicity up to
23.8 lg/mL and are potent against a wide spectrum of
microorganisms, suggest that these bis-quindolines
may have therapeutic advantage over their monomeric
counterparts as new potential antiinfectives.
and are within 0.4% of theory unless otherwise noted.
Flash chromatography was performed with Davisil grade
634 silica gel. N,N-Dimethylformamide was distilled from
CaSO4 and stored over 4 A molecular sieves. Sulfolane
˚
was dried over 4 A molecular sieves. 5-Cyclohexylpentyl
bromide and 5-phenylpentyl bromide were prepared by
treatment of the corresponding alcohols with PBr3. The
remaining alkyl halides were obtained from either Sig-
ma–Aldrich Chemicals or Fisher Scientific and were used
without further purification. Quindoline (10H-indolo[3,2-
b]quinoline), the starting material in several of the synthe-
ses below was obtained as previously reported.12
˚
5.1. General method for the synthesis of N-10 alkylated
quindolines
4. Conclusion
5.1.1. 10-Methylquindoline (5a). A mixture of quindoline
(400 mg, 1.83 mmol), KOH (0.2 g), acetone (20 mL),
and iodomethane (0.5 mL) was stirred at room temper-
ature for 30 min. A second portion of an equal amount
of KOH and iodomethane was added. The mixture was
stirred for another 2 h, acetone was removed by rotary
evaporation and the residue chromatographed using
20:30% EtOAc–hexanes (gradient elution) to yield a yel-
low solid (0.42 g, 98%). Recrystallization using EtOAc–
hexanes afforded the desired product, mp 110–112 °C.
1H NMR (CDCl3): d 3.85 (s, 3H), 7.33 (dd, 1H,
J = 7.7, 7.7 Hz), 7.40 (d, 1H, J = 8.1 Hz), 7.52 (ddd,
1H, J = 7.1, 7.1, 1.3 Hz), 7.65 (m, 2H), 7.90 (s, 1H),
7.95 (dd, 1H, J = 8.5, 0.9 Hz), 8.33 (d, 1H, J = 8.2 Hz),
8.55 (dd, 1H, J = 7.8, 0.9 Hz). Anal. Calcd for:
C16H12N2Æ0.3H2O, C, 80.85; H, 5.34; N, 11.79. Found:
C, 80.60; H, 5.52; N, 11.64.
Evaluation of the indoloquinolines prepared in this arti-
cle confirms the importance of N-5 alkylation. The basi-
city of this nitrogen and consequently the formation of
the positive charge appear to be important. On the other
hand, alkylation of the non-basic indole nitrogen does
not lead to activity. Despite this observation, simulta-
neous alkylation of both nitrogen atoms results in im-
proved potency and extension in antiinfective
spectrum. Similarly, bis-quindolines obtained by double
alkylation of the pyridine N-5 atoms produce a broad
spectrum antimicrobial activity while connection
through the indole N-10 atoms led to compounds with-
out activity. Subsequent alkylation of the N-5 in this
type of bis-quindolines however, resulted in similar anti-
infective properties. The broad spectrum of antimicro-
bial actions and the lower cytotoxicity displayed by
the bis-quindolines indicate this group may be acting
through a different mechanism of action from that of
their monomeric counterparts. These results suggest a
more systematic study of the doubly alkylated com-
pounds, including the bis-quindolines, in these and other
antiinfective assays, is warranted.
5.1.2. 2-Bromo-10-methylquindoline (5b). Compound 5b
was similarly obtained and recrystallized from hexane–
EtOAc, mp 160–162 °C. 1H NMR (CDCl3): d 4.82
(3H, s), 7.38 (2H, m), 7.65 (2H, m), 7.74 (1H, s), 8.05
(1H, d, J = 2.2 Hz), 8.18 (1H, d, J = 8.6 Hz), 8.51 (1H,
d, J = 7.4 Hz). Anal. Calcd for C16H11BrN2: C, 61.76;
H, 3.56; N, 9.00. Found: C, 61.71; H, 3.64; N, 8.90.
5. Experimental
5.1.3. 10-(5-Phenylpentyl)quindoline HCl (5c). Com-
pound 5c was similarly obtained and recrystallized from
MeOH–Et2O, mp 223–224 °C. H NMR (DMSO-d6): d
1.36 (2H, m), 1.57 (2H, m), 1.90 (2H, m), 2.51 (2H, m),
4.60 (2H, m), 7.13 (5H, m), 7.46 (1H, m), 7.88 (4H, m),
8.00 (1H, t, J = 7.2 Hz), 8.35 (1H, d, J = 8.1 Hz), 8.53
Melting points were determined on a Gallenkamp (UK)
apparatus and are uncorrected. NMR spectra were ob-
tained on a Varian 300 MHz Mercury or a Bruker
270 MHz NMR Spectrometer. Elemental analyses were
carried out by Atlantic Microlab, Inc., Norcross, GA
1

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L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
(1H, d, J = 8.8 Hz), 8.84 (1H, d, J = 7.8 Hz), 9.02 (1H,
s). Anal. Calcd for C26H25ClN2: C, 77.89; H, 6.28; N,
6.99. Found: C, 77.87; H, 6.34; N, 6.95.
5.1.8. 2-Methyl-10-cyclohexylpentyl quindoline HCl (6e).
This compound was prepared from 2-methylquindo-
line14 and cyclohexylpentyl bromide according to the
procedure described above. Recrystallization in
MeOH–Et2O afforded 2-methyl-10-cyclohexylpentyl
quindoline HCl (0.15 g, 88%) mp: 231–233 °C. 1H
NMR (CDCl3): d 0.80 (m, 2H), 1.1 (m, 6H), 1.29 (s,
br, 3H), 1.62 (m, 6H), 1.84 (s, br, 2H), 2.47 (s, 3H),
4.46 (s, br, 2H), 7.30 (m, 2H), 7.47 (d, 1H, J = 8.8 Hz),
7.61 (t, 1H, J = 7.3 Hz), 7.76 (s, 1H), 8.45 (s, 1H), 8.70
(d, 1H, J = 8.8 Hz), 9.10 (d, 1H, J = 7.8 Hz). Anal.
Calcd for C27H33ClN2: C, 77.03; H, 7.90; N, 6.65.
Found: C, 76.86; H, 7.76; N, 6.56.
5.1.4. 2-Bromo-10-cyclohexylpentyl quindoline HCl (6a).
A mixture of KOH (0.115 g), acetone (4 mL), 2-bro-
moquindoline14 (0.12 g, 0.4 mmol), and cyclohexylpen-
tyl bromide (1.5 mL) was refluxed with stirring at
50 °C for 1 h, after which the acetone was evaporated
in vacuo. The crude product was chromatographed on
a column of silica gel and eluted with gradient ethyl ace-
tate–hexane to yield the pure free base product. The HCl
salt was obtained by dissolving the product in MeOH
and adding excess ethereal HCl. Recrystallization from
MeOH–Et2O afforded 2-bromo-10-cyclohexylpentyl
quindoline HCl (0.105 g, 54%) mp: 223–224 °C. 1H
NMR (CDCl3): d 0.79 (m, 2H), 1.11 (m, 6H), 1.29 (s,
br, 6H), 1.60 (m, 6H), 1.83 (s, br, 2H), 4.51 (t, 1H,
J = 5.9 Hz), 7.24 (s, 1H), 7.31 (t, 2H, J = 8.8 Hz), 7.68
(t, 2H, J = 8.8 Hz), 8.18 (s, 1H), 8.53 (s, 1H), 8.68 (d,
1H, J = 8.8 Hz), 9.13 (d, 1H, J = 7.8 Hz). Anal. Calcd
for C26H30BrClN2: C, 64.27; H, 6.22; N, 5.77. Found:
C, 64.19; H, 6.25; N, 5.64.
5.1.9. 2-Thiophenyl-10-cyclohexylpentyl quindoline HCl
(6f). This compound was prepared from 2-thio-
phenylquindoline14 and cyclohexylpentyl bromide
according to the procedure described above. Recrystalli-
zation in MeOH–Et2O afforded 2-thiophenyl-10-cyclo-
hexylpentyl quindoline HCl (0.1 g, 43%) mp: 221–
1
223 °C. H NMR (CDCl3): d 0.80 (m, 2H), 1.10 (s, br,
6H), 1.52 (s, br, 3H), 1.60 (s, br, 6H), 1.82 (s, br, 2H),
4.42 (t, 2H, J = 7.3 Hz), 7.32 (m, 2H), 7.43 (m, 3H),
7.59 (m, 4H), 7.70 (s, 1H), 8.3 (s, 1H), 8.82 (d, 1H,
J = 9.3 Hz), 9.20 (d, 1H, J = 7.8 Hz). Anal. Calcd for
C32H35ClN2S: C, 71.52; H, 6.56; N, 5.21. Found: C,
71.63; H, 6.72; N, 5.24.
5.1.5. 2-Cyano-10-cyclohexylpentyl quindoline HCl (6b).
This compound was prepared from 2-cyanoquindoline14
and cyclohexylpentyl bromide according to the proce-
dure described above. Recrystallization in MeOH–
Et2O afforded 2-cyano-10-cyclohexylpentyl quindoline
5.1.10. Synthesis of 5-methyl-10-(5-phenylpentyl)quindo-
linium iodide (7a). A mixture of quindoline (420 mg,
1.93 mmol) and 5-phenyl-1-iodopentane (600 mg,
2.19 mmol) in DME (15 mL) was added with stirring
at 0 °C, to NaH (100 mg, 4.15 mmol). After the addition
was complete, the mixture was refluxed under N2 for
10 h and allowed to cool to room temperature. The
resulting mixture was filtered through a short pad of sil-
ica gel and the filtrate was concentrated in vacuo to dry-
ness. The residue was purified by column
chromatography to give the desired product (610 mg,
1
HCl (0.13 g, 73%) mp: 229–231 °C. H NMR (CDCl3):
d 0.85 (m, 2H), 1.35 (m, 11H), 1.65 (m, 4H), 2.0 (m,
2H), 4.68 (t, 2H, J = 7.4 Hz), 7.58 (t, 1H, J = 7.8 Hz),
7.93 (d, 1H, J = 8.8 Hz), 8.02 (m, 1H), 8.22 (dd, 1H,
J = 1.4, 7.3 Hz), 8.50 (d, 1H, J = 8.8 Hz), 8.65 (d, 1H,
J = 8.3 Hz), 8.9 (s, 1H), 9.3 (s, 1H). Anal. Calcd for
C27H30ClN3: C, 75.07; H, 7.00; N, 9.73. Found: C,
75.20; H, 7.18; N, 9.72.
5.1.6. 2-Fluoro-10-cyclohexylpentyl quindoline HCl (6c).
This compound was prepared from 2-fluoroquindoline14
and cyclohexylpentyl bromide according to the proce-
dure described above. Recrystallization in MeOH–
Et2O afforded 2-fluoro-10-cyclohexylpentyl quindoline
87%).
A mixture of the N-5 alkylated product
(320 mg, 0.88 mmol), MeI (0.2 mL), and sulfolane
(2 mL) was sealed in a tube and then heated at 100 °C
for 16 h. After cooling to room temperature, Et2O
(20 mL) was added to precipitate a yellow solid, which
was collected by filtration. The solid was recrystallized
from CH2Cl2–MeOH and Et2O to give compound 7a
1
HCl (0.11 g, 64%) mp: 231–233 °C. H NMR (CDCl3):
d 0.80 (s, br, 2H), 1.1 (s, br, 6H), 1.31 (s, br, 3H), 1.62
(s, br, 6H), 1.86 (s, br, 1H), 4.48 (t, 2H J = 6.8 Hz),
7.34 (s, 1H), 7.37 (s, 1H), 7.5 (m, 1H), 7.7 (m, 2H), 8.5
(s, 1H), 8.95 (dd, 1H, J = 4.9, 9.2 Hz), 9.2 (d, 1H,
J = 7.8 Hz). Anal. Calcd for C26H30ClFN2: C, 73.48;
H, 7.12; N, 6.59. Found: C, 73.38; H, 7.03; N, 6.49.
1
(360 mg, 80%); mp: 215–217 °C. H NMR (DMSO-d6)
1.38 (m, 2H), 1.59 (m, 2H), 1.87 (m, 2H), 2.49 (t, 2H,
J = 3.0 Hz), 4.70 (t, 2H, J = 7.1 Hz), 5.05 (s, 6H), 7.12
(m, 5H), 7.57 (t, 1H, J = 8.0 Hz), 8.02 (m, 3H), 8.19 (t,
1H, J = 7.5 Hz), 8.52 (d, 1H, J = 7.6 Hz), 8.75 (d, 1H,
J = 9.0 Hz), 8.85 (d, 1H, J = 8.4 Hz), 9.60 (s, 1H). Anal.
Calcd for C27H27IN2: C, 64.04; H, 5.37; N, 5.53. Found:
C, 63.77; H, 5.40; N, 5.56.
5.1.7. 2-Methoxy-10-cyclohexylpentyl quindoline HCl
(6d). This compound was prepared from 2-methoxyqu-
indoline14 and cyclohexylpentyl bromide according to
the procedure described above. Recrystallization in
MeOH–Et2O afforded 2-methoxy-10-cyclohexylpentyl
quindoline HCl (0.13 g, 71%) mp: 232–234 °C. 1H
NMR (CDCl3): d 0.8 (m, 2H), 1.10 (m, 6H), 1.28 (m,
3H), 1.62 (m, 6H), 1.81 (m, 2H), 3.98 (s, 3H), 4.42 (t,
2H, J = 6.8 Hz), 7.18 (s, 1H), 7.27 (m, 3H), 7.64 (t, 1H,
J = 8.3 Hz), 8.39 (s, 1H), 8.65 (d, 1H, J = 9.8 Hz), 9.15
(d, 1H, J = 7.8 Hz). Anal. Calcd for C27H33ClN2O: C,
70.05; H, 7.18; N, 6.05. Found: C, 70.18; H, 7.36; N, 6.04.
5.2. General method for alkylation (compounds 8a–c)
Alkyl halide (0.8 mL) was added to a 10-mL round-bot-
tomed flask containing quinoline (100 mg, 0.46 mmol)
and sulfolane (2.0 mL). The mixture was heated at
about 100 °C in a sealed flask overnight and allowed
to cool to room temperature. The mixture was directly
chromatographed with 5:20% MeOH–CH2Cl2 (gradient

L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
3961
elution) to give a solid, which was recrystallized from an
appropriate solvent system as shown below.
J = 7.5, 9.0 Hz), 7.88 (d, 2H, J = 9.0 Hz), 7.93 (d, 2H,
J = 9.5 Hz), 7.98 (d, 2H, J = 8.0 Hz), 8.10 (t, 2H,
J = 8.0 Hz), 8.54 (d, 2H, J = 8.5 Hz), 8.59 (d, 2H,
J = 8.5 Hz), 8.71 (d, 2H, J = 9.5 Hz), 9.31 (s, 2H), 12.92
(s, 2H). Anal. Calcd for C35H30I2N4Æ3.5H2O: C, 51.05;
H, 3.67; N, 6.80. Found: C, 51.06; H, 3.69; N, 6.80.
5.2.1. Synthesis of 1-methyl-2-phenylquinolinium iodide
(8a). Precipitated solid was recrystallized from EtOH–
1
Et2O to produce 8a (65 mg, 4%); mp: 196–198 °C. H
NMR (DMSO-d6) 4.37 (3H, s), 7.72 (3H, m), 7.80
(2H, m), 8.08 (1H, t, J = 7.8 Hz), 8.16 (1H, d, J =
8.4 Hz), 8.32 (1H, t, J = 8.7 Hz), 8.52 (1H, d, J = 8.1
Hz), 8.62 (1H, d, J = 9.0 Hz), 9.27 (1H, d, J = 8.7 Hz),
Anal. Calcd for C16H14IN: C, 55.35; H, 4.06; N, 4.03.
Found: C, 55.23; H, 4.09; N, 4.02.
5.2.6. Synthesis of 1,5-bis(indolo[3,2-b]quindolin-10-yl)-
pentane (11). A mixture of 10H-indolo[3,2-b]quinoline
(860 mg, 3.9 mmol), and 1,5-diiodopentane (640 mg,
1.95 mmol) was dissolved in DME (15 mL) and NaH
(200 mg, 8.3 mmol) was added with stirring at 0 °C.
After the addition was completed, the mixture was re-
fluxed under N2 for 10 h and allowed to cool to room
temperature. The mixture was then filtered through a
short pad of silica gel and the filtrate was concentrated
in vacuo to dryness. The residue was purified by column
chromatography to give a soft solid, compound 11
(860 mg, 87.5%). 1H NMR (CDCl3) 1.43 (m, 2H),
1.89 (m, 4 H), 4.20 (t, 4H, J = 6.8 Hz), 7.25 (m, 4H),
7.49 (m, 4H), 7.61 (m, 2H), 7.77 (s, 2H), 7.81 (d, 2H,
J = 7.6 Hz), 8.28 (d, 2H, J = 8.4 Hz), 8.50 (d, 2H,
J = 7.6 Hz). Anal. Calcd for C35H28N4Æ0.6H2O: C,
81.56; H, 5.48; N, 10.87. Found: C, 81.60; H, 5.35; N,
10.76.
5.2.2. Synthesis of 5-cyclohexylpentylquinolinium bro-
mide (8b). Precipitated solid was recrystallized from
EtOH–Et2O to give the desired product (yield 65%),
mp: 79–83 °C. 1H NMR (DMSO-d6) 0.83 (2H, m),
1.22 (10H, m), 1.63 (5H, m), 1.96 (2H, m), 5.05 (2H, t,
J = 7.8 Hz), 8.05 (1H, t, J = 7.3 Hz), 8.19 (1H, dd,
J = 5.8 Hz, J = 8.8 Hz), 8.27 (1H, m), 8.49 (1H, d,
J = 8.3 Hz), 8.62 (1H, d, J = 8.8 Hz), 9.30 (1H, d,
J = 8.8 Hz), 9.57 (1H, d, J = 5.8 Hz). Anal. Calcd for
C20H28NBrÆ0.8H2O: C, 63.76; H, 7.49; N, 3.72. Found:
C, 63.89; H, 7.71; N, 3.64.
5.2.3. Synthesis of 5-cyclohexylpentylpyridinium bromide
(8c). Precipitated solid was recrystallized from EtOH–
Et2O to give the desired product (yield 45%), mp: 86–
89 °C. 1H NMR (DMSO-d6) d (ppm) 0.83 (2H, m),
1.20 (10H, m), 1.62 (5H, m), 1.89 (2H, m), 4.61 (2H, t,
J = 7.5 Hz), 8.16 (2H, m), 8.61 (1H, t, J = 7.8 Hz),
9.15 (2H, d, J = 5.4 Hz). Anal. Calcd for C16H26NBrÆ
1.4H2O: C, 56.94; H, 8.60; N, 4.15. Found: C, 56.60;
H, 8.15; N, 4.02.
5.2.7. Synthesis of 1,5-bis(indolo[3,2-b]quindolin-10-ium)-
pentane dioodide (12). A mixture of bis-quindoline, 11
(300 mg, 0.69 mmol), MeI (0.2 mL), and sulfolane
(2 mL) was sealed in a tube, and the solution was heated
at 100 °C for 16 h and allowed to cool to room temper-
ature. Et2O (20 mL) was added to produce a yellow pre-
cipitate, which was collected by filtration. The resulting
solid was recrystallized from CH2Cl2–MeOH and Et2O
to give compound 12 (410 mg, 75%); mp: 238–240 °C.
1H NMR (DMSO-d6) 1.43 (br s, 2H), 1.91 (br s, 4H),
4.63 (t, 4H, J = 6.7 Hz), 5.03 (s, 6H), 7.51 (t, 2H,
J = 7.9 Hz), 7.91 (m, 6H), 8.18 (m, 2H), 8.36 (d, 2H,
J = 7.5 Hz), 8.76 (d, 2H, J = 9.3 Hz), 8.81 (d, 2H,
J = 8.5 Hz), 9.48 (s, 2H). Anal. Calcd for C37H34I2-
N4Æ0.5H2O: C, 55.72; H, 4.30; N, 7.03. Found: C,
55.54; H, 4.36; N, 6.94.
5.2.4. Synthesis of 1,4-bis(10-indolo[3,2-b]quinolin-5-ium)-
butane diiodide (9). A mixture of 10H-indolo[3,2-b]quin-
oline (700 mg, 3.2 mmol), 1,4-diiodobutane (400 mg,
1.3 mmol), and sulfolane (4 mL) was sealed in a tube,
and the solution was heated at 100 °C for 16 h and
allowed to cool to room temperature. Et2O (20 mL)
was added to form a yellow precipitate and was
collected by filtration. The solid was recrystallized from
CH2Cl2–MeOH and Et2O to give compound 9 (850 mg,
78%); mp: 252–254 °C. 1H NMR (DMSO-d6) 2.58 (br s,
4 H), 5.58 (br s, 4H), 7.42 (t, 2H, J = 7.4 Hz), 7.95 (m,
6H), 8.15 (t, 2H, J = 7.6 Hz), 8.50 (d, 2H, J = 8.5 Hz),
8.59 (d, 2H, J = 7.8 Hz), 8.80 (d, 2H, J = 9.1 Hz), 9.32
(s, 2H), 12.99 (br s, 2H). Anal. Calcd for C34H30I2-
N4Æ0.5H2O: C, 53.91; H, 3.99; N, 7.40. Found: C,
54.07; H, 3.64; N, 7.40.
6. Biological testing
Compounds were evaluated in vitro against a panel of
microorganisms, including C. albicans ATCC 90028
(Ca), C. krusei ATCC 6258 (Ck), C. neoformans ATCC
90113 (Cn), Staphylococcus aureus ATCC 29213 (Sa),
methicillin-resistant S. aureus ATCC 43300 (MRSA),
P. aeruginosa ATCC 27853 (Pa), A. fumigatus ATCC
90906 (Af), and M. intracellulare ATCC 23068 (Mi) as
previously reported.22 All organisms were obtained from
the American Type Culture Collection (Manassas, Va.).
Susceptibility testing was performed using a modified
version of the NCCLS methods23–25 for all organisms
except for M. intracellulare, for which the modified Ala-
mar blue procedure described by Franzblau et al.26 was
followed. Briefly, samples (dissolved in DMSO) were
serially diluted by using 0.9% saline and transferred in
duplicate to 96-well microplates. Microbial inocula were
prepared after comparison of the absorbance (at
5.2.5. Synthesis of 1,5-bis(10-indolo[3,2-b]quinolin-5-ium)-
pentane diiodide (10). A mixture of 10H-Indolo[3,2-b]-
quinoline (400 mg, 1.84 mmol), 1,5-diiodopentane (280
mg, 0.87 mmol), and sulfolane (4 mL) was sealed in a
tube, and the solution was heated at 100 °C for 16 h
and allowed to cool to room temperature. Et2O
(20 mL) was added to precipitate a yellow solid, which
was collected by filtration. The solid was recrystallized
from CH2Cl2–MeOH and Et2O to give compound 10
1
(520 mg, 76%); mp: 256–258 °C. H NMR (DMSO-d6)
2.00 (m, 2H), 2.23 (m, 4H), 5.52 (m, 4H), 7.50 (dd, 2H,

3962
L. G. Mardenborough et al. / Bioorg. Med. Chem. 13 (2005) 3955–3963
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Acknowledgements
11. Guyen, B.; Schultes, C. M.; Hazel, P.; Mann, J.; Neidle, S.
Org. Biomol. Chem. 2004, 2, 981–988.
We gratefully acknowledge research support from the
National Institutes of Health, National Institute of Al-
lergy and Infectious Diseases, through AREA grant
number R15 Al37976-01, Research Centers at Minority
Institutions (RCMI) grant number G12 RR 03020,
MBRS grant # GM 08111 and Title III grant to Florida
A&M University. We also appreciate the generous con-
tributions of Dr. Tom Gedric of Florida State Univer-
sity Chemistry Department who obtained the NMR
spectra of compounds in this paper.
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