Identification of 3-phenylaminoquinolinium and 3-phenylaminopyridinium salts as new agents against opportunistic fungal pathogens

Article abstract of DOI:10.1016/j.bmc.2010.10.065

Previous studies on the indoloquinoline alkaloid, cryptolepine (2), revealed that it has antii-nfective properties among other activities. Using Structure-activity relationship (SAR) techniques, several ring-opened analogs of cryptolepine (3-phenylaminopyridinium and 3-phenylaminoquinolinium derivatives) were designed to improve the potency and lower the cytotoxicity shown by several of the precursor agents. Results indicate that these ring-opened analogs constitute new anti-infective agents with over a 100-fold potency and several fold lower cytotoxicity than cryptolepine from which they are derived.

Full text of DOI:10.1016/j.bmc.2010.10.065

Bioorganic & Medicinal Chemistry 19 (2011) 524–533
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 3-phenylaminoquinolinium and 3-phenylaminopyridinium
salts as new agents against opportunistic fungal pathogens
Tryphon K. Mazu a, Jagan R. Etukala a, Xue Y. Zhu a, Melissa R. Jacob b, Shabana I. Khan b, Larry A. Walker b,
Seth Y. Ablordeppey a,
⇑
a Florida A&M University, College of Pharmacy and Pharmaceutical Sciences, Tallahassee, FL 32307, United States
b The National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences, University of Mississippi, MS 38677, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies on the indoloquinoline alkaloid, cryptolepine (2), revealed that it has antii-nfective prop-
erties among other activities. Using Structure–activity relationship (SAR) techniques, several ring-opened
analogs of cryptolepine (3-phenylaminopyridinium and 3-phenylaminoquinolinium derivatives) were
designed to improve the potency and lower the cytotoxicity shown by several of the precursor agents.
Results indicate that these ring-opened analogs constitute new anti-infective agents with over a 100-fold
potency and several fold lower cytotoxicity than cryptolepine from which they are derived.
Published by Elsevier Ltd.
Received 21 September 2010
Revised 22 October 2010
Accepted 31 October 2010
Available online 5 November 2010
Keywords:
Cryptolepine analogs
Ring-opened analogs
Antifungal agents
Anti-opportunistic infection agents
Anti-bacterial agents
Agents against AIDS-related opportunistic
pathogens
1. Introduction
gillus fumigatus are three of the most common fungal pathogens
responsible for opportunistic infections in humans. The current
Opportunistic infections (OIs) may be caused by fungi, bacteria,
viruses, or parasites. Symptoms may vary according to the microor-
ganisms involved and the extent of their involvement. Conditions
leading to immunodeficiency, including malnutrition, recurrent
infections, immunosuppressive agents for organ transplant
recipients, chemotherapy for cancer, acquired immunodeficiency
syndrome (AIDS), genetic predisposition, antibiotic treatment, med-
ical procedures and pregnancy among others expose these individ-
uals to the risk of OIs.1–3 The most prevalent of these conditions is
AIDS and since the discovery of the virus that causes AIDs (HIV) in
1981, a steady increase in the number of people worldwide living
with HIV has been reported. This number, according to the WHO,
has risen from around 8 million in 1990 to about 33 million and is
still growing.3 Since 1981, more than 25 million people have died
of AIDS and a large percentage of these individuals mostly die from
opportunistic infections or malignancies associated with the pro-
gressive failure of the immune system.3 In addition to immune-sup-
pressed patients, there are indications in the literature that even
immunocompetent individuals may be at risk for some opportunis-
tic infections.4 Candida albicans, Cryptococcus neoformans and Asper-
treatment options for the cure of these fungal pathogens are limited
by toxicity, resistance development and drug interactions among
others.5,6 Consequently, new drug entities especially those with
new mechanisms of action are of great need.
Indolo[3,2-b]quinoline ring or quindoline (1) constitutes an
important lead scaffold in the quest to find new agents against
cancer, opportunistic infections, malaria, diabetes, and several
other diseases.6 While the natural product cryptolepine (2) pro-
vided the impetus for the interest in this lead scaffold, several
lines of evidence suggest caution about the long-term prospects
of using cryptolepine.7 Armed with this information, we have ex-
plored the possibility of modifying the structure to limit the cyto-
toxicity associated with the pharmacology of cryptolepine. Earlier
on, we reported that the anti-infective properties of cryptolepine
can be modified by substituting at various positions on the quind-
oline structure including isosteric replacement of the N-10 atom
with sulfur.8 Furthermore, it was observed that opening the ring
at the benzothiophene moiety produced analogs of N-substituted
benzothieno[3,2-b]quinolines with potent antimicrobial activity.
As a result, we opine that substitution could be used to obtain
selective agents and curtail the cytotoxicity associated with cry-
ptolepine. We further surmised that since cryptolepine interca-
lates into DNA in a unique fashion as a result of its flat and
rigid tetracyclic structure10,11 and since some of its toxicity may
⇑
Corresponding author. Tel.: +1 850 599 3834; fax: +1 850 599 3934.
E-mail address: seth.ablordeppey@famu.edu (S.Y. Ablordeppey).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.10.065

T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
525
be related to this intercalation, opening the ring to allow for flex-
2.3. N-5 and N-10 alkylations
ibility may decrease intercalation into DNA and hence reduce
cytotoxicity.
Evidence suggests that the active form of cryptolepine consists
of the flat tetracyclic aromatic ring with a positive charge on the N-
R2
CH3
CH3
I
I
I
6
9
4
N
N
N
N
R
N
S
N
N
11
1
H
H
R1
5-Methyl-Benzo[b]thieno[3,2-b]
Ring-opened Analogs (4)
Quindoline (1)
Cryptolepine Hydroiodide (2)
quinolin-5-ium Iodide(3)
Hence, in this paper, we report the synthesis and evaluation of
cryptolepine analogs substituted at all synthetically accessible
positions on the indole and quinoline moieties with a chlorine
atom previously shown to enhance potency and the exploration
of the possibility that ring-opened analogs might offer opportunity
for increased potency and lower cytotoxicity.
5 atom brought about by the N-methyl substituent.16 It was also
observed that changes in the electron density around the N-5 atom
do not appear to affect activity significantly.17 Through systematic
modification of the N-5 alkyl groups,
x-cyclohexylpentyl group
has been identified with an enhanced anti-infective potency. Thus,
compounds proposed in this manuscript are adorned with the
cyclohexylpentyl group on the N-atom.
x-
2. Design of target compounds
2.4. Synthesis
2.1. Substitution around the quindoline ring
Literature methods were employed in the synthesis of these
analogs.18 As previously reported, substituted cryptolepine deriva-
tives 11a–d were obtained by a series of reactions beginning with
treating the commercially available 3-aminoquinoline, 8 with
substituted phenyl boronic acid 7 in the presence of copper acetate
and triethylamine. This reaction gave N-phenyl quinoline-3-
amines 9 in good yield (80–90%). The intermediate 9 was then
heated in trifluoroacetic acid in the presence of palladium acetate
to obtain the cyclized product, 10. Methylation of the N-5 (10a–d)
yielded the target compounds, 11a–d (Scheme 1).
In a previous publication8 we have identified the electron with-
drawing and hydrophobic chlorine atom to enhance antifungal po-
tency when substituted at the 2-position of cryptolepine. To obtain
the optimal position for substitution on the indoloquinoline ring,
we proposed substitution of chlorine at all synthetically accessible
positions on the quindoline ring. Subsequent evaluation of the
antifungal properties of the synthetic compounds would lead to
the identification of the optimal position with respect to electron
withdrawing and hydrophobic groups.
2-Substituted cryptolepine derivatives, 12 were obtained as
previously reported8 and compounds 16a–d were obtained as de-
tailed in Scheme 2 below.17
For the ring-opened systems (see below), substituent selections
will be guided by a 2D Craig plot9 in order to use the minimum
CH3
N
C
CH3
N
R2
N
R2
N
Cl
X
X
X
D
A
B
R1
R
R
N
H
N
N
R1
N
R1
H
3-Phenylaminopyridinium Salts
3-Phenylaminoquinolinium Salts
Cryptolepine HCl
Substituted Cryptolepines
(4)
(2)
(6)
(5)
number of compounds to explore the electronic and hydrophobic
space around the ring systems.
Compound 19, 5-iodopentylcyclohexane was obtained by the
reaction of cyclohexyl magnesium chloride, 17 with 1,5-dibromo-
pentane in the presence of lithium tetrachloro cuprate in THF to
yield the intermediate 5-bromopentylcyclohexane 18, which was
then converted to the more reactive 5-iodopentylcyclohexane, 19
by the reaction of 18 with sodium iodide in acetone (Scheme 3).
To prepare 3-phenylaminoquinolinium analogs 21a–m, inter-
mediate 9 was converted to 20 by deprotonation of the secondary
aromatic amine with sodium hydride and subsequently alkylated
with 5-iodopentylcyclohexane to obtain 19. The target compounds,
21a–m, were obtained by treating 20 with iodomethane, 5-phenyl-
pentyl iodide or 19 in toluene (Scheme 4).
Similarly, the first step in the synthesis of 3-phenylaminopyrid-
inium analogs, 26a–t, involved coupling the available substituted
phenyl boronic acid or halide 22, with 3-aminopyridine 23 to give
substituted phenylaminopyridine, 24. The conversion of 24 to 25
required selective alkylation of the secondary diarylamine which
was accomplished by deprotonation with sodium hydride followed
by alkylation with the appropriate alkyl halide. The target
2.2. Opening of ring B and excision of ring D
Exploitation of a new agent’s conformational flexibility (such as
torsional flexibility about strategically located chemical bonds) is a
powerful element of drug design.12 The flat tetracyclic structure of
cryptolepine enables it to intercalate into DNA in a previously un-
known fashion.10b,13 In addition to DNA intercalation, cryptolepine
is also known to interfere with topoisomerase II.14 Thus, we
hypothesize that by opening ring B in cryptolepine, the flat topog-
raphy of cryptolepine would be lost and consequently its unique
binding to DNA would be curtailed and thus the potential to de-
crease toxicity brought on by intercalation into DNA. This hypoth-
esis will be tested by synthesizing ring-opened analogs, 4. In
addition, the corresponding ring-opened analogs of d-carbolinium
compounds would also be synthesized as the d-carboline moiety
was proposed as a pharmacophoric group in cryptolepine.15

526
T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
N
N
i
R
R
B(OH)2
+
H2N
N
H
8
7
9
ii
CH3
N
N
6
I
11a R = 6-Cl
11b R = 7-Cl
11c R = 7-Br
iii
R
7
R
8
N
H
N
10
H
9
11d R = 9-Cl
11(a-d)
Scheme 1. Reagents and conditions: (i) CH2Cl2, Cu(OAc)2, TEA, MS-powder, rt, 12–24 h; (ii) Pd(OAc)2, CF3COOH, 80 °C, 6 h; (iii) CH3I, toluene 110 °C, 12–24 h, sealed tube.
H
OAc
R
O
O
N
N
N
i
ii
+
R
R
N
N
H
N
H
H
14
iii
13
COOH
iv
R1 = H; X = OTf
R1 = H; X = OTf
16a-c
16a R = 1-Cl;
16b R= 3-Cl;
CH3
N
for
X
N
iv
16c R = 3-Cl, 11-Cl;
R1 = H; X = I
R
R
for 16d
N
N
R
= C11H21; X = I
16d R =2-Cl;
1
R1
15
R1
16a-d
Scheme 2. Reagents and conditions: (i) aqueous KOH, 10 days; (ii) paraffin oil, 300 °C, 3 h; (iii) NaH, R1-I, DME, rt, 12 h; (iv) CH3I, toluene, 110 °C, 12–24 h or CH3OTf, toluene,
rt, 24 h.
ii
all chloro-substituents when compared to the unsubstituted cry-
ptolepine except for the 6- and 9-chloro substituents which were
inactive. Position 4 was not investigated because all attempts to
alkylate the N-5 atom of the quinoline with chlorine at the 4-posi-
tion were unsuccessful. Apparently, the chlorine atom sterically
and electronically hindered the introduction of the methyl group
onto the N-atom. Replacement of the iodide with trifluorometh-
anesulfonate (triflate) co-ion in 16a and 16b did not have any sig-
nificant change on antifungal activity of these analogs. Overall, all
chloro-substituted cryptolepine analogs were inactive against A.
fumigatus and showed considerable cytotoxicity against Vero cell
lines, an indication of their possible toxicity in humans (Table 1).
The exception to this observation was when N-10 is alkylated with
i
(CH2)5I
19
MgCl
(CH2)5Br
18
17
Scheme 3. Reagents and conditions: (i) 1,5-dibromopentane, Li2CuCl4, THF, 0 °C,
12 h; (ii) NaI, acetone, reflux, 12 h.
compounds were then obtained by methylation of the pyridine
nitrogen, 25 to form the desired target compounds, 26a–t (Scheme
5).
3. Result and discussion
x
-cyclohexylpentyl moiety (16d).
We have previously shown that substitution of
Biological evaluation of all analogs was performed against three
common opportunistic fungal pathogens C. albicans (Ca), C. neofor-
mans (Cn), and A. fumigatus (Af). Evaluation of chlorine-substituted
cryptolepine analogs (11a–d and 16a–d) was intended to identify
the optimum location on the indoloquinoline moiety. There was
an increase in potency against C. albicans and C. neoformans for
x-cyclohexyl-
pentyl group on the N-5 atom of ring C enhanced potency against
fungi.17 However, cytotoxicity against Vero cell lines was increased
as well. Alkylation of 2-chlorocryptolepine’s N-10 atom with
cyclohexylpentyl (16d) was investigated to find if potency would
x-
R2
I
4
N
N
N
i
ii
R
R
R
3
N
N
H
N
2
R1
R1
21a - m
20
9
21a
R1 = H, R2 = CH3
R = H;
21b R = 2-Cl; R1 = H, R2 = CH3
R1 = C11 H21, R2 = CH3
R1 = C11 H21, R2 = CH3
R1 = C11 H21, R2 = CH3
R1 = C11 H21, R2 = CH3
R1 = H, R2 = C11H15
R1 = H, R2 = C11H21
21f R = 4-Cl;
21g R = H;
21j
R = 2-Cl;
21k R = 3-Cl;
R1 = H, R2 = CH3
R1 = H, R2 = CH3
R1 = H, R2 = C11H15
21c R = 3-Cl;
21d
R = 4-Cl;
21e R = H;
21h R =3-Cl; R1 = H, R2 = C11H21
21i
21l R = 4-Cl;
21m R =H;
R = 4-Cl; R1 = H, R2 = C11H21
Scheme 4. Reagents and conditions: (i) NaH, cyclohexylpentyl iodide, DME, rt, 12 h; (ii) CH3I, toluene 110 °C, 12–24 h.

T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
527
N
N
N
R
ii
i
R
R
+
N
N
H2N
(Y)
H
25
R1
23
22
24
Y = B(OH)2, Br, or I
iii
26a R = H;
26k R = 3-Cl;
R1 = H
R1 =C11 H21
26b R =4-Cl;
26l R = 3-CF3; R1 = C11 H21
R1 = C11H21
26c R = 4-CF3;
26d R =4-F;
26e R = 4-H;
26m R =3-CH3; R1 = C11 H21
26n R =3-OCH3; R1 = C11 H21
R1 = C11H21
R1 = C11H21
CH3
I
4
N
R1 =C11H21
R1 = C11 H21
R1 = C11 H21
26o R =3-CN;
26p R =2-Cl;
26q R =2-CH3;
R
26f R =4-CH3; R1 = C11H21
N
3
26g R =4-C2H5;
26h R = 4-OCH3;
26i R = 4-SCH3;
26j R = 4-CN;
R1 = C11H21
R1 = C11H21
R1 = C11H21
R1 = C11H21
R1 = C11 H21
R1 = C11 H21
R1 = C11 H21
R1 = C11 H21
2
R1
26r R =2-CF3;
26s R = 2-OCH3;
26t R =2-CN;
26a-t
Scheme 5. Reagents and conditions: (i) (Y = B(OH)2), CH2Cl2, Cu(OAc)2, TEA, molecular sieve, rt, 24 h, (Y = Br, I) dioxane, Pd(OAc)2, 80 °C, 24 h; (ii) DME, 5-iodo-
pentylcyclohexane, NaH, 0 °C, rt, 12 h; (iii) CH3I, toluene, 110 °C, 12 h.
Table 1
Antifungal activity and cytotoxicity of substituted cryptolepine analogs
CH3
X
6
4
N
3
7
R
8
2
N
11
9
1
R1
Compoundsa
R
R1
X
IC50/MIC/MFC (lg/mL)
TC50 (lg/mL)
Ca
0.6/>20
0.5/5.5/>20
3.0/>20
1.0/10/>20
>20
4.5/>20
2.5/>20
>20
0.6/1.3/>20
20/>20
Cn
Afu
Vero cells
16a
1-Cl
2-Cl
3-Cl
3-Cl, 11-Cl
6-Cl
7-Cl
7-Br
9-Cl
2-Cl
H
H
H
H
H
H
H
H
OTf
I
OTf
I
I
I
I
I
I
7.0/>20
2.0/5.0/>20
15/>20
6.0/10/>20
>20
15/>20
2.5/20
>20
0.9/2.5/10
12.5b/20
1.3/0.5/1.0
>20
10/>20/>20
>20
>20
>20
>20
10 (MIC)
>20
0.8/1.3/>20
NT
0.7/1.3/5.0
5
NT
2.1
10
>4.7
1.5
0.93
3.2
1.4
3.2c
7.6c
12c
16b
16c
11a
11b
11c
11d
16d
C
11H21
Cryptolepine
Amphotericin B
0.2/0.6/1.3
Abbreviations: Ca = Candida albicans, Cn = Cryptococcus neoformans, Afu = Aspergillus fumigatus. OTf = triflate; NT = not tested.
a
All compounds were subjected to CHN analysis and each passed within 0.4% of the theoretical value.
b
MIC value; previously published.18
c
Data previously reported.8 IC50 = the concentration that affords 50% inhibition of growth, MIC = minimum inhibitory concentration is the lowest test concentration that
allows no detectable growth; MFC (minimum fungicidal concentration) is the lowest test concentration that kills the organism; TC50 = the concentration, that is, toxic to 50%
of cells.
be retained and if cytotoxicity would be attenuated. While potency
was enhanced against all three fungal pathogens, cytotoxicity was
the diarylamino group while retaining the necessary N-methyl
group on the quinoline, produced the most potent analogs among
increased as well (TC50 = 1.4
lg/mL). It became clear that the tetra-
the series with no cytotoxicity at 10 lg/mL. While the presence of
cyclic structure per se may be responsible for the cytotoxicity and
we needed to deviate from the linear and flat tetracycle.
the chlorine atom improved activity against Ca and Af, it appears to
have no effect in the case of Cn (cf. 21i–k vs 21l). Substitution of
the chlorine atom at the para position of the phenyl A ring appears
to provide the optimum effect for activity, Thus, 3-phenylamino-
One approach to modify the linear flat topography of cryptole-
pine is to open the ring system. The pyrrole ring of the indole moi-
ety offers a synthetically attractive target for opening and was thus
selected for investigation, while maintaining the chlorine atom at
the o, m- and p-positions of the phenyl ring. We report the anti-
fungal activity and cytotoxicity evaluation of the resulting com-
pounds (21a–l) in Table 2. Compounds 21a–d, the N-methylated
quinolinium derivatives, with an appropriately substituted
x-
cyclohexylpentyl group, 21f–l, offer a new promising scaffold for
further investigation.
We have also previously reported that alkylating cryptolepine
with an
x-cyclohexylpentyl group on the diarylamino function
analogs showed no activity at 20
tuted -phenylpentyl analog, 21e were synthesized and evaluated.
These showed improved activity and in addition produced no cyto-
toxicity at 10 g/mL. Replacing the -phenylpentyl group with
cyclohexylpentyl group, 21f–h, led to further improvements in
activity. Finally, substituting the -cyclohexylpentyl group on
lg/mL. Next, quinoline N-substi-
showed potent antifungal activity but significant cytotoxicity.17
Since ring opening resulted in attenuating cytotoxicity in cryptole-
pine, we hypothesized that using a similar strategy for the d-carb-
oline system might lead to improvements in cytotoxicity. The
antifungal activity and cytotoxicity evaluation of the compounds
(26a–t) are reported in Table 3. With no substitution on the
x
l
x
x-
x

528
T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
Table 2
Antifungal activity and cytotoxicity of 3-phenylaminoquinolinium analogs
R2
N
I
4
R
3
N
R1
2
Compoundsa
R
R1
R2
IC50/MIC/MFC (
Cn
lg/mL)
TC50 (lg/mL)
Ca
Afu
Vero cells
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
NA
NA
NA
NA
15/>20/>20
3.5/10/>20
2.0/6.3/13
2.0/>20
0.63/1.3/1.3
0.9/2.5/10
0.5/1.3/5.0
1.2/2.5/2.5
20/250/NT
0.2/0.6/1.3
NA
NA
NA
NA
NA
NA
NA
NA
10/>20/>20
1.0/2.5/10
3.5/6.3/>20
NA
3.1/5.0/20
1.0/2.5/10
0.8/1.3/10
4.6/10/10
NT
NA
NA
NA
NA
>25
NT
>10
>10
NT
>10
10
9.8
3.2c
7.6c
2-Cl
3-Cl
4-Cl
4-Cl
H
3-Cl
4-Cl
2-Cl
3-Cl
4-Cl
H
C11H15
4.0/6.3/12.5
1.5/2.5/10
2.0/3.1/50
1.0/1.6/>20
0.2/0.3/0.6
0.9/1.3/5.0
0.4/0.6/2.5
0.2/0.3/0.6
12.5b/NT/NT
1.3/0.5/1.0
C11H21
C11H21
C11H21
C
C
C
11H21
11H21
11H21
CH3
CH3
CH3
CH3
C11H21
Cryptolepine
Amphotericin B
0.7/1.3/5.0
Abbreviations: Ca = Candida albicans, Cn = Cryptococcus neoformans, Afu = Aspergillus fumigatus. Otf = triflate; Crypto = cryptolepine. Amph B = amphotericin B; NT = not tested.
NA = not active at 20 g/mL.
l
a
All compounds were subjected to CHN analysis and each passed within 0.4% of the theoretical value.
MIC value; previously published.18
b
c
Data previously reported2,11,19 IC50 = the concentration that affords 50% inhibition of growth, MIC = minimum inhibitory concentration is the lowest test concentration
that allows no detectable growth; MFC (minimum fungicidal concentration) is the lowest test concentration that kills the organism; TC50 = the concentration, that is, toxic to
50% of cells.
diarylamino function (26a), there was no activity against any of the
fungi. Introduction of the -cyclohexylpentyl group on the diaryla-
mino N-atom with the pyridine methyl group in place (26e) led to
significant improvement in antifungal activity and in particular no
cytotoxicity was observed at 10 lg/mL. It was of interest at this
point to probe the electronic effect and lipophilicity on the phenyl
ring as this would affect the electron density at the diaryl nitrogen.
The para position was first selected for exploration as it was the
optimum position for substitution in the quinolinium series (Table
2). The presence of a fluorine atom (26d) at the para position has
no effect on activity but a 4-chloro (26b) and the more potent elec-
tron withdrawing trifluoromethyl group (26c) both increased
activity significantly against all three pathogens and showed no
constitute novel anti-infective agents with high potency and low
cytotoxicity. On average, these analogs are over 100-fold (Cn) and
over 250-fold (Ca) more potent than cryptolepine, the original natu-
ral product. Substitution on the phenyl ring of ring-opened analogs
with electron withdrawing and hydrophobic groups (such as Cl, CF3)
tends to enhance activity. The in vitro cytotoxicity assay against
mammalian kidney fibroblast (Vero) cells indicates that opening
x
cryptolepine’s indole ring and introducing the
x-cyclohexylpentyl
group on N-atoms resulted in a decrease in toxicity. Finally, 3-phe-
nylaminopyridinium and 3-phenylamino-quinolinium derivatives
are not only very potent against the selected microorganisms but
they also have lower toxicity than cryptolepine.
cytotoxicity at 10 lg/mL against Vero cells. Similarly, increase in
5. Experimental
potency was observed for the electron donating and lipophilic alkyl
substituents (26fand 26g) and the thiomethyl group (26i) at the 4-
position. However, activity decreased somewhat for the weakly
hydrophilic methoxy (26h) along with the even more hydrophilic
cyano substituent (26j).
These results would appear to point to the importance of lipo-
philicity and not electronic effects on activity. The 3-position of
the phenyl ring was also evaluated using the chloro (26k), trifluo-
romethyl (26l) and methyl (26m) groups. These analogs also
showed significant potency albeit generally lower than the substit-
uents at the 4-position. Finally, the same substituents were evalu-
ated at the 2-position (26p–t). Overall, activities of the 2-
substituents were similar to those of the 3-substituents.
Reagents and solvents were obtained from commercial suppli-
ers (Sigma–Aldrich, Alfa Aesar and VWR) and except for tetrahy-
drofuran (THF) which was dried over sodium metal, all other
reagents and solvents were used without further purification. Flash
Chromatography was performed with Selecto Scientific silica gel
(63–200 mesh). Analytical TLC was performed on pre-coated
Whatman Ltd aluminum plates (Silica gel 60 Å). NMR spectra were
obtained on Varian 300-MHZ NMR spectrometer at FAMU. Melting
points were determined on Gallenkamp (UK) apparatus. CHN ana-
lyzes, performed with a Perkin Elmer 2400 CHN elemental ana-
lyzer, were carried out by Atlantic Microlab, Inc., Norcross, GA
and are within 0.4% of theoretical values unless otherwise noted.
4. Conclusion
5.1. General procedure for the synthesis of N-phenylquinolin-3-
amine derivatives (9) and N-phenylpyridin-3-amine derivatives
(24)
Using the method previously reported by our labs, we were able
to synthesize the target compounds in relatively good yield. Biolog-
ical results indicated that substituted cryptolepine analogs, 11a–d
and 16a–d are active but continue to show significant toxicity. How-
ever, the corresponding ring-opened analogs, 3-phenylaminoquino-
linium 21a–l and 3-phenylaminopyridinium derivatives 26a–t now
To a solution of 3-aminoquinoline or 3-aminopyridine (1 g) in
dichloromethane (30 mL) was added portion-wise phenyl boronic
acid (1.63 g, 13.36 mmol), triethylamine (1.5 g, 10.41 mmol), cop-
per acetate (1.90 g, 10.41 mmol), and molecular sieves (2 g) under

T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
529
Table 3
Antifungal activity and cytotoxicity data of 3-phenylaminopyridinium analogs
CH3
N
I
4
R
3
N
2
R1
Compoundsa
R
R1
H
IC50/MIC/MFC (
Cn
lg/mL)
TC50 (lg/mL)
Ca
Afu
Vero cells
26a
H
NA
NA
NA
NA
26b
26c
26d
26e
26f
26g
26h
26i
26j
26k
26l
26m
26n
26o
26p
26q
4-Cl
4-CF3
4-F
C
11H21
1.5/3.1/NT
1.0/1.6/NT
1.5/2.5/5.0
1.4/2.5/2.5
1.5/2.5/2.5
1.5/2.5/5.0
0.4/0.6/5.0
1.4/2.5/2.5
1.2/1.3/2.5
2.6/5.0/5.0
0.7/1.3/1.3
1.5/2.5/5.0
1.4/2.5/2.5
3.6/5.0/5.0
3.5/5.0/5.0
2.0/2.5/2.5
2.3/5.0/5.0
1.5/2.5/2.5
3.0/5.0/5.0
6.6/10/20
1.0/1.6/NT
1.5/2.5/5.0
NA/NA/NA
19.7/NA/NA
1.5/5.0/5.0
2.7/5.0/ND
NA
6.4/10.0/10.0
NA
12.4/20.0/20.0
1.5/2.5/10.0
8.7/10.0/20.0
NA
NA
18/>20/>20
NA
19>20>20
NA
NA
>10
>10
>10
>10
>10
10
>10
>10
>10
>10
>10
>10
>10
>10
NT
C11H21
C
C
C11H21
C11H21
C11H21
C11H21
C
C
C11H21
C11H21
C11H21
C
C
C11H21
C11H21
C11H21
1.0/2.5/10.0
9.6/20.0/NA
8.3/20.0/NA
1.5/2.5/10.0
1.3/2.5/5.0
5.1/10/20
2.0/5.0/10
19/20/>20
3.0/5.0/10
3.0/5.0/20
3.0/5.0/10
8.9/20/>20
NA
11H21
11H21
H
4-CH3
4-C2H5
4-OCH3
4-SCH3
4-CN
3-Cl
3-CF3
3-CH3
3-OCH3
3-CN
2-Cl
11H21
11H21
11H21
11H21
2.8/5.0/5.0
2.6/5.0/5.0
2.6/5.0/5.0
5.0/10.0/10
NA
2-CF3
2-CH3
2-OCH3
2-CN
NT
NT
NT
>10
3.2b
7.6
26r
26s
26t
C
11H21
Cryptolepine
Amphotericin B
20/250/NTb
0.2/0.6/1.3
12.5 (MIC)b
1.3/0.5/1.0
NT
0.7/1.3/5.0
Abbreviations: Ca = Candida albicans, Cn = Cryptococcus neoformans, Afu = Aspergillus fumigatus. NT = not tested. NA = not active at 20
l
g/mL.
a
All compounds were subjected to CHN analysis and each passed within 0.4% of the theoretical value.
b
Data previously reported2,11,19 IC50 = the concentration that affords 50% inhibition of growth, MIC = minimum inhibitory concentration is the lowest test concentration
that allows no detectable growth; MFC (minimum fungicidal concentration) is the lowest test concentration that kills the organism; TC50 = the concentration, that is, toxic to
50% of cells.
stirring. The reaction mixture was stirred at room temperature for
12–24 h and monitored by TLC. Once reaction is completed, the
reaction was quenched by the drop wise addition of aqueous
ammonia (15 mL). The resulting mixture was extracted with
dichloromethane, washed with brine then with water, and dried
over anhydrous Na2SO4. The solvent was removed under reduced
pressure and the residue purified by column chromatography
using ethyl acetate and hexanes as eluent.
5.4. General procedure for the synthesis of 10H-indolo[3,2-
b]quinoline derivatives (14)
A mixture of quindoline-11-carboxylic acid derivative 13 (3.2 g)
and parafin oil (30 mL) was heated at 300 °C for 3 h, allowed to
cool to rt and diluted in hexanes (100 mL), filtered and washed
with hexanes (3 Â 50 mL). The crude product was purified by col-
umn chromatography (hexane/EtOAc 1:1) to yield 14.
5.2. General procedure for the synthesis of 10H-indolo[3,2-
b]quinoline derivatives (10)
5.5. Synthesis of 5-bromopentylcyclohexane (18)
To a solution of 1,5-dibromopentane (16 g, 70 mmol) in THF
(20 mL) was added Li2CuCl4 (12.7 g, 62 mmol) under N2 at 5–
10 °C. The resulting mixture was stirred for 25 min and then cyclo-
hexyl magnesium bromide (10 g, 70 mmol) was added drop-wise
over 30 min. The reaction continued to stir at 0 °C for another 1 h
and then at rt for 12 h. The reaction mixture was cooled to 0 °C,
saturated NH4Cl solution (20 mL) added and then EtOAc
(100 mL). The organic layer was separated, pooled, washed with
brine and then dried over anhydrous Na2SO4. The filtrate was sub-
jected to rotary evaporation and the crude product was purified by
column chromatography using hexanes as eluent to yield 18 as an
oily liquid (75% yield). 1H NMR (300 MHz, CDCl3): d 0.8 (t, 2H,
J = 10.2 Hz), 1.00–1.38 (m, 9H), 1.52–168 (m, 6H), 1.70–1.80 (m,
2H), 3.38 (t, 2H, J = 7.2 Hz).
To
a solution of phenyl-quinolin-3-yl-amine derivatives 9
(400 mg) in CF3COOH (8 mL), was added Pd(OAc)2 (300 mg,
1.34 mmol) and the reaction mixture was refluxed for 6 h at
72 °C. The reaction mixture was allowed to cool to room tempera-
ture, poured on ice cold water (15 mL), neutralized with aqueous
NH3 and extracted with EtOAc (3 Â 50 mL), washed with brine
and dried over anhydrous sodium sulfate. The solvent was re-
moved under reduced pressure and the crude product was purified
on column chromatography using hexane/EtOAc, 2:3.
5.3. General procedure for the synthesis of 10H-indolo[3,2-
b]quinoline-11-carboxylic acid derivatives (13)
A mixture of 1H-indol-3-yl acetate (5 g, 29 mmol), isatin deriv-
ative (5.2 g), and KOH (22 g) in water (200 mL) was stirred for
10 days at rt, filtered and the filtrate was acidified to pH 1 with
concentrated HCl. The precipitate of quindoline-11-carboxylic acid
derivative 13 was collected, washed with water, dried, and used for
the next step without further purification.
5.6. Synthesis of 5-iodopentylcyclohexane (19)
A mixture of (5-bromopentyl)cyclohexane 18 (2 g, 8.6 mmol) in
acetone (20 mL) and NaI (2.57 g, 17.2 mmol) was refluxed for 12 h
and then allowed to cool to rt. The solvent was removed in

530
T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
vacuum; the residue was diluted with H2O (50 mL) and extracted
with EtOAc (30 mL). The organic layer was washed with brine
(30 mL), dried over anhydrous Na2SO4, solvent removed under vac-
uum and the crude product purified on column chromatography
using hexanes as eluent to yield the pure product as an oily liquid
(90% yield, boiling point 291 °C). 1H NMR (300 MHz, CDCl3): d 0.8
(t, 2H, J = 10.2 Hz), 1.00–1.38 (m, 9H), 1.52–168 (m, 6H), 1.70–
1.80 (m, 2H), 3.10 (t, 2H, J = 6.9 Hz).
5.9.2. 7-Chloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
iodide (11b)
Yellow solid (45% yield), mp: 206–207 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.22 (s, 1H), 8.78 (d, 1H, J = 1.8 Hz), 8.68 (d, 1H,
J = 9.0 Hz), 8.50 (d, 1H, J = 7.5 Hz), 8.24–8.18 (m, 1H), 7.96 (t, 1H,
J = 6.3 Hz), 7.90 (d, 1H, J = 2.1 Hz), 7.82 (d, 1H, J = 9.0 Hz), 5.08 (s,
3H). Anal. Calcd for C16H12ClIN2: C, 48.70; H, 3.06; N, 7.10. Found:
C, 48.70; H, 3.06; N, 6.99.
5.9.3. 7-Bromo-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
Iodide (11c)
5.7. General procedure for the synthesis of 20 and 25 derivatives
Yellow solid (74% yield), mp: 260–261 °C; 1H NMR (300 MHz,
DMSO-d6): 13.04 (s, 1H), 9.36 (s, 1H), 9.00 (s, 1H), 8.10 (d, 1H,
J = 8.8 Hz), 8.59 (d, 1H, J = 8.1 Hz), 8.19 (t, 1H, J = 8.8 Hz), 8.06 (d,
1H, J = 8.4 Hz), 7.98 (t, 1H, J = 7.2 Hz), 7.83 (d, 1H, J = 8.8 Hz), 5.03
(s, 3H). Anal. Calcd for C16H12BrIN2: C, 43.77; H, 2.75; N, 6.38.
Found: C, 44.05; H, 2.81; N, 6.20.
To a solution of 9 or 24 (100 mg) in 1,2-dimethoxyethane
(5 mL), NaH (20 mg, 0.83 mmol), and 5-iodo-pentylcyclohexane
(636 mg, 2.3 mmol) were added in that order. The mixture was
stirred at rt for 12 h, solvent was evaporated and the residue par-
titioned between H2O (10 mL) and EtOAc (2 Â 30 mL). The pooled
organic phase was washed with brine and dried over anhydrous
Na2SO4 and solvent was removed under reduced pressure. The
crude product was purified on column chromatography (hexane/
EtOAc, 4:1).
5.9.4. 9-Chloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
iodide (11d)
Yellow solid (57% yield), mp: 251–253 °C; 1H NMR (300 MHz,
DMSO-d6): d 13.70 (s, 1H), 9.80 (s, 1H), 9.26–9.18 (m, 1H), 8.90
(d, 1H, J = 8.4 Hz), 8.70–8.60 (m, 1H), 8.20–8.10 (m, 2H), 8.00 (d,
1H, J = 7.5 Hz), 7.55 (t, 1H, J = 7.5 Hz), 4.80 (s, 3H). Anal. Calcd for
5.8. General procedure for synthesis of 5-methyl-10H-
indolo[3,2-b] quinolin-5-ium triflate derivatives 16a–b
C
16H12ClIN2Á0.73H2O: C, 45.65; H, 3.22; N, 6.65. Found: C, 45.64;
To a solution of 14 (100 mg) in toluene (3 mL), in a sealed pres-
sure tube, methyl triflate (0.3 g, 1.8 mmol) was added. The reaction
mixture was stirred at rt for 24 h, then diluted with Et2O (15 mL),
the precipitate was filtered and washed with Et2O (3 Â 20 mL). The
crude product was recrystallized from MeOH.
H, 3.18; N, 6.62.
5.9.5. 3,11-Dichloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
iodide (16c)
Yellow solid (33% yield), mp: 236–238 °C; 1H NMR (300 MHz,
CD3OD): d 5.00 (s, 3H), 7.55–7.61 (m, 1H), 7.89–8.01 (m, 3H),
8.62 (d, 1H, J = 9 Hz), 8.71 (d, 1H, J = 9 Hz), 8.763 (d, 1H,
J = 1.5 Hz). Anal. Calcd for C16H11Cl2IN2Á5.5H2O: C, 36.39; H, 2.10;
N, 5.30. Found: C, 36.32; H, 2.48; N, 5.21.
5.8.1. 1-Chloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
triflate (16a)
Yellow solid (71% yield), mp: 289–291 °C; 1H NMR (300 MHz,
CD3OD): d 5.14 (s, 3H), 7.57–7.63 (m, 1H), 7.85 (d, 1H, J = 8.4 Hz),
7.97–8.02 (m, 1H), 8.08–8.14 (m, 2H), 8.64–8.68 (m, 1H), 8.76 (d,
5.9.6. 2-Chloro-10-(5-cyclohexylpentyl)-5-methyl-10H-
indolo[3,2-b]quinolin-5-ium iodide (16d)
1H, J = 8.7 Hz), 9.41 (s, 1H). Anal. Calcd for
C17H12ClF3I-
Yellow solid (72% yield), mp: 215–215 °C; 1H NMR (300 MHz,
CD3OD): d 9.36 (s, 1H), 8.81 (d, 1H, J = 8.4 Hz), 8.71 (d, 1H,
J = 9.6 Hz), 8.58 (d, 1H, J = 2.4 Hz), 8.13 (dd, 1H, J = 2.4, 2.1 Hz),
8.02–7.96 (m, 2H), 7.63 (t, 1H, J = 7.2), 5.12 (s, 3H), 4.72 (t, 2H,
J = 7.2 Hz), 2.02–1.97 (m, 2H), 1.68–1.38 (m, 9H), 1.22–1.14 (m,
6H), 0.82 (t, 2H, J = 6 Hz). Anal. Calcd for C27H32ClIN2: C, 59.29; H,
5.90; N, 5.12. Found: C, 59.15; H, 6.07; N, 5.08.
N2O3SÁ0.5H2O: C, 47.95; H, 2.84; N, 6.58. Found: C, 47.93; H,
2.81; N, 6.58.
5.8.2. 3-Chloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
triflate (16b)
Yellow solid (82% yield), mp: 226–228 °C; 1H NMR (300 MHz,
CD3OD): d 5.06 (s, 3H), 7.54–7.59 (m, 1H), 7.81 (d, 1H, J = 8.7 Hz),
7.90–7.98 (m, 2H), 8.46 (d, 1H, J = 8.7 Hz), 8.72–8.76 (m, 2H),
9.13 (s, 1H). Anal. Calcd for C17H12ClF3IN2O3SÁ0.5H2O: C, 47.95; H,
2.84; N, 6.58. Found: C, 47.88; H, 2.84; N, 6.54.
5.9.7. 1-Methyl-3-(phenylamino)quinolinium iodide (21a)
Yellow solid (86% yield), mp: 182–183 °C; 1H NMR (300 MHz,
CD3OD): d 4.6 (s, 3H), 7.18 (m, 1H), 7.36 (dd, 2H, J = 0.9, 7.5 Hz),
7.44 (m, 2H), 7.82 (t, 2H, J = 6.0 Hz), 7.94 (m, 2H), 8.10 (d, 1H,
J = 8.4 Hz), 8.26 (d, 1H, J = 8.6 Hz), 8.46 (d, 1H, J = 2.4 Hz), 9.04 (d,
1H, J = 2.7 Hz). Anal. Calcd for C16H15IN2Á1.3H2O: C, 46.98; H,
4.34; N, 6.85. Found: C, 46.81; H, 3.95; N, 6.54.
5.9. General procedure for the synthesis of 11a–d, 16c–d, 21a–l,
and 26a–t derivatives
To a solution of10, 15, 20, or 25 (100 mg) in toluene (3 mL), in a
sealed pressure tube, CH3I (0.3 g, 2.1 mmol) was added. The mix-
ture was stirred at 110 °C for 24 h, allowed to cool to rt, diluted
with Et2O (15 mL), the precipitate filtered and washed with Et2O
(3 Â 20 mL). The crude product was recrystallized from MeOH.
5.9.8. 3-(2-Chlorophenylamino)-1-methylquinolinium iodide
(21b)
Yellow solid (55% yield), mp: 208–210 °C; 1H NMR (300 MHz,
CD3OD): d 9.10 (d, 1H, J = 2.4 Hz), 8.34 (d, 2H, J = 9.0 Hz), 8.10 (d,
1H, J = 8.4 Hz), 7.96 (t, 1H, J = 9.0 Hz), 7.84 (t, 1H, J = 7.5 Hz), 7.58
(dd, 2H, J = 8.1, 1.5 Hz), 7.40 (t, 1H, J = 8.1 Hz), 7.24 (t, 1H,
J = 7.2 Hz), 4.90 (s, 3H). Anal. Calcd for C16H14ClIN2Á1.8CH3OH: C,
42.30; H, 3.11; N, 6.17. Found: C, 42.49; H, 3.23; N, 6.09.
5.9.1. 6-Chloro-5-methyl-10H-indolo[3,2-b]quinolin-5-ium
Iodide (11a)
Yellow solid (67% yield), mp: 211–212 °C; 1H NMR (300 MHz,
DMSO-d6): 13.28 (s, 1H), 9.37 (s, 1H), 8.63 (d, 1H, J = 9.0 Hz), 8.59
(d, 1H, J = 9.0 Hz), 8.21 (t, 1H, J = 8.1 Hz), 7.97 (t, 1H, J = 7.5 Hz),
7.88 (m, 2H), 7.60 (d, 1H, J = 7.1 Hz), 5.08 (s, 3H). Anal. Calcd for
5.9.9. 3-(3-Chlorophenylamino)-1-methylquinolinium iodide
(21c)
Yellow solid (42% yield), mp: 211–213 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.42 (s, 1H), 9.24 (d, 1H, J = 2.4 Hz), 8.64 (d, 1H,
C16H12ClIN2: C, 48.70; H, 3.06; N, 7.10. Found: C, 48.49; H, 3.05;
N, 6.97.

T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
531
J = 2.4 Hz), 8.34 (d, 1H, J = 9.0 Hz), 8.26 (d, 1H, J = 8.1 Hz), 7.98–7.92
(t, 1H, J = 7.5 Hz), 7.87 (t, 1H, J = 7.8 Hz), 7.39 (t, 1H, J = 8.1 Hz), 7.32
(t, 1H, J = 2.1 Hz), 7.24 (dd, 1H, J = 6.3, 1.2 Hz), 7.08 (dd, 1H, J = 6.0,
1.2 Hz), 4.60 (s, 3H). Anal. Calcd for C16H14ClIN2Á0.39H2O: C, 47.61;
H, 3.50; N, 6.94. Found: C, 47.75; H, 3.52; N, 6.89.
(dd, 1H, J = 1.5, 7.2 Hz), 8.04 (d, 1H, J = 2.4 Hz), 7.90–7.78 (m, 2H),
7.57–7.748 (m, 3H), 4.57 (s, 3H), 3.81 (t, 2H, J = 7.2 Hz), 1.63–
1.59 (m, 7H), 1.35–1.28 (m, 4H), 1.21–1.05 (m, 6H), 0.81 (t, 2H,
J = 9.0 Hz). Anal. Calcd for C27H34ClIN2Á0.8H2O: C, 57.57; H, 6.08;
N, 4.97. Found: C, 57.56; H, 6.27; N, 4.96.
5.9.10. 3-(4-Chlorophenylamino)-1-methylquinolinium iodide
(21d)
5.9.16. 3-((3-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-
methylquinolinium iodide (21j)
Yellow solid (45% yield), mp: 153–155 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.03 (d, 1H, J = 2.4 Hz), 8.50 (d, 1H, J = 2.4 Hz), 8.29
(d, 1H, J = 8.7 Hz), 8.11 (d, 1H, J = 7.2 Hz), 7.96–7.91 (m, 1H),
7.87–7.81 (m, 1H), 7.43–7.41 (m, 2H), 7.35–7.31 (m, 2H), 4.63 (s,
3H). Anal. Calcd for C16H14ClIN2: C, 48.45; H, 3.56; N, 7.06. Found:
C, 49.00; H, 3.88; N, 6.70.
Yellow solid (74% yield), mp: 85–87 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.21 (d, 1H, J = 2.7 Hz), 8.57 (d, 1H, J = 2.4 Hz), 8.30
(d, 1H, J = 8.7 Hz), 8.23 (d, 1H, J = 9.3 Hz), 7.99–7.93 (m, 1H), 7.87
(t, 1H, J = 7.5 Hz), 7.43 (t, 1H, J = 8.4 Hz), 7.38 (t, 1H, J = 2.1 Hz),
7.27–7.22 (m, 2H), 4.53 (s, 3H), 3.88 (t, 2H, J = 7.2 Hz), 1.63–1.59
(m, 6H), 1.35–1.26 (m, 5H), 1.15–1.08 (m, 6H), 0.81 (t, 2H,
J = 9.6 Hz). Anal. Calcd for C27H34ClIN2: C, 59.08; H, 6.24; N, 5.10.
Found: C, 59.25; H, 6.26; N, 5.21.
5.9.11. 3-(4-Chlorophenylamino)-1-(5-phenylpentyl)quinolin-
ium iodide (21e)
Yellow solid (36% yield), mp: 182–183 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.36 (s, 1H), 9.18 (d, 1H, J = 2.4 Hz), 8.58 (d, 1H,
J = 2.4 Hz), 8.38 (d, 1H, J = 8.7 Hz), 8.22 (d, 1H, J = 8.1 Hz), 7.91 (t,
1H, J = 7.2 Hz), 7.83 (t, 1H, J = 7.2 Hz), 7.42 (d, 2H, J = 9.0 Hz), 7.30
(d, 2H, J = 9.0 Hz), 7.23 (t, 2H, J = 6.9 Hz), 7.16–7.10 (m, 3H), 4.98
(t, 2H, J = 7.5 Hz), 2.56 (t, 2H, J = 7.5 Hz), 2.02–1.90 (m, 2H), 1.66–
1.56 (m, 2H), 1.46–1.36 (m, 2H). Anal. Calcd for C26H26IClN2: C,
59.05; H, 4.96; N, 5.30. Found: C, 58.99; H, 4.99; N, 5.22.
5.9.17. 3-((4-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-
methylquinolinium iodide (21k)
Yellow solid (79% yield), mp: 176–177 °C; 1H NMR (300 MHz,
(DMSO-d6): d 9.10 (d, 1H, J = 2.7 Hz), 8.46 (d, 1H, J = 2.4 Hz), 8.26
(d, 1H, J = 8.7 Hz), 8.20–8.18 (dd, 1H, J = 7.2, 1.2 Hz), 7.96–7.90
(m, 1H), 7.84 (t, 1H, J = 7.8 Hz), 7.50 (d, 2H, J = 9.0 Hz), 7.36 (d,
2H, J = 9.0 Hz), 4.54 (s, 3H), 3.86 (t, 2H, J = 7.5 Hz), 1.70–1.50 (m,
7H), 1.40–1.24 (m, 4H), 1.20–1.04 (m, 6H), 0.79 (t, 2H,
J = 10.2 Hz). Anal. Calcd for C27H34ClIN2: C, 59.08; H, 6.24; N,
5.10. Found: C, 59.25; H, 6.26; N, 5.21.
5.9.12. 1-(5-Cyclohexylpentyl)-3-(phenylamino)quinolinium
iodide (21f)
Yellow solid (13% yield), 156–158 °C; 1H NMR (300 MHz,
CD3OD): d 0.80–1.00 (m, 2H), 1.00–1.38 (m, 7H), 1.40–1.58 (m,
4H), 1.60–1.80 (m, 4H), 2.00–2.20 (m, 2H), 5.00 (t, 2H, J = 7.2 Hz),
7.20 (t, 1H, J = 6.8 Hz), 7.35 (d, 2H, J = 8.6 Hz), 7.46 (t, 2H,
J = 7.0 Hz), 7.80 (t, 1H, J = 7.6 Hz), 7.82–8.00 (m, 1H), 8.10 (d, 1H,
J = 8.4 Hz), 8.38 (d, 1H, J = 9.0 Hz), 8.45 (d, 1H, J = 2.4 Hz), 9.0 (d,
1H, J = 2.7 Hz). Anal. Calcd for C26H33IN2Á1.4H2O: C, 56.99; H,
6.55; N, 5.08. Found: C, 56.63; H, 6.25; N, 4.77.
5.9.18. 3-((5-Cyclohexylpentyl)(phenyl)amino)-1-methylquin-
olinium iodide (21l)
Yellow solid (74% yield), mp: 156–158 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.06 (d, 1H, J = 2.4 Hz), 8.36 (d, 1H, J = 2.4 Hz), 8.24
(d, 1H, J = 9.0 Hz), 8.16 (d, 1H, J = 8.4 Hz), 7.90–7.78 (m, 2H), 7.46
(t, 2H, J = 7.8 Hz), 7.34 (d, 2H, J = 7.8 Hz), 7.26 (t, 1H, J = 7.5 Hz),
4.50 (s, 3H), 3.86 (t, 2H, J = 7.5 Hz), 1.70–1.50 (m, 7H), 1.40–1.20
(m, 4H), 1.20–1.00 (m, 6H), 0.79 (t, 2H, J = 10.2 Hz). Anal. Calcd
for C27H35IN2: C, 63.03; H, 6.86; N, 5.44. Found: C, 62.86; H, 6.88;
N, 5.28.
5.9.13. 3-(3-Chlorophenylamino)-1-(5-cyclohexylpentyl)quino-
linium iodide (21g)
Yellow solid (74% yield), mp: 128–129 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.40 (s, 1H), 9.28 (d, 1H, J = 2.4 Hz), 8.66 (d, 1H,
J = 1.8 Hz), 8.42 (d, 1H, J = 9.0 Hz), 8.28 (d, 1H, J = 8.1 Hz), 7.93 (t,
1H, J = 7.2 Hz), 7.84 (t, 1H, J = 7.2 Hz), 7.37 (t, 1H, J = 8.1 Hz), 7.32
(d, 1H, J = 1.5 Hz), 7.24–7.20 (dd, 1H, J = 6.9, 1.2 Hz), 7.08 (d, 1H,
J = 8.1 Hz), 4.98 (t, 2H, J = 7.2 Hz), 1.92 (t, 2H, J = 6.6 Hz), 1.70–
1.50 (m, 5H), 1.40–1.26 (m, 4H), 1.20–1.00 (m, 6H), 0.81 (t, 2H,
J = 9.9 Hz). Anal. Calcd for C26H32ClIN2: C, 58.38; H, 6.03; N, 5.24.
Found: C, 58.36; H, 5.99; N, 5.17.
5.9.19. 1-Methyl-3-(phenylamino)pyridinium iodide (26a)
Yellow solid (55% yield), mp: 161–162 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.30 (s, 1H), 8.42 (s, 1H), 8.28 (d, 1H, J = 6.0), 7.96–
7.90 (dd, 1H, J = 6.6, 2.1 Hz), 7.81 (dd, 1H, J = 8.7, 3.3 Hz), 7.42–
7.38 (m, 2H), 7.28–7.24 (m, 2H), 7.10 (t, 1H, J = 7.2 Hz), 4.20 (s,
3H). Anal. Calcd for C27H35IN2Á1.5H2O: C, 42.50; H, 3.86; N, 8.26.
Found: C, 42.66; H, 3.79; N, 8.18.
5.9.20. 3-((4-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-methyl-
pyridinium iodide (26b)
5.9.14. 3-(4-Chlorophenylamino)-1-(5-cyclohexylpentyl)quino-
linium iodide (21h)
Yellow solid (75% yield), mp: 134–135 °C; 1H NMR (300 MHz,
CDCl3): d 8.09 (d, 2H, J = 1.8 Hz), 7.67 (t, 2H, J = 6.3 Hz), 7.57–7.54
(m, 2H), 7.34–7.31 (m, 2H), 4.3 (s, 3H), 3.78 (t, 2H, J = 7.8 Hz),
3.04–2.99 (m, 2H), 2.22–2.17 (m, 2H), 1.70–1.62 (m, 6H), 1.35–
1.16 (m, 9H). Anal. Calcd for C23H32ClIN2Á0.05H2O: C, 55.37; H,
6.47; N, 5.62. Found: C, 55.28; H, 6.45; N, 5.61.
Yellow solid (40% yield), mp: 177–178 °C; 1H NMR (300 MHz,
DMSO-d6): d 9.38 (s, 1H), 9.18 (d, 1H, J = 2.4 Hz), 8.58 (d, 1H,
J = 2.1 Hz), 8.40 (d, 1H, J = 8.7 Hz), 8.22 (d, 1H, J = 8.1 Hz), 7.90 (t,
1H, J = 7.2 Hz), 7.82 (t, 1H, J = 6.9 Hz), 7.42 (d, 2H, J = 9.0 Hz), 7.30
(d, 2H, J = 9.0 Hz), 4.96 (t, 2H, J = 7.5 Hz), 1.9 (t, 2H, J = 6.9 Hz),
1.70–1.50 (m, 5H), 1.40–1.28 (m, 4H), 1.20–1.00 (m, 6H), 0.80 (t,
2H, J = 9.6 Hz). Anal. Calcd for C26H32ClIN2Á0.6H2O: C, 57.22; H,
5.91; N, 5.13. Found: C, 57.26; H, 5.86; N, 5.14.
5.9.21. 3-((5-Cyclohexylpentyl)(4-(trifluoromethyl)phenyl)-
amino)-1-methylpyridinium iodide (26c)
Yellow solid (48% yield), mp: 130–131 °C; 1H NMR (300 MHz,
CDCl3) d 8.51 (s, 1H), 8.23 (d, 1H, J = 5.7 Hz), 7.79 (d, 2H,
J = 8.4 Hz), 7.65–7.60 (m, 1H), 7.45 (d, 3H, J = 8.1), 4.60 (s, 3H),
3.95 (t, 2H, J = 7.2 Hz), 1.69–1.61 (m, 10H), 1.17–1.13 (m, 9H). Anal.
Calcd for C24H32F3IN2: C, 54.14; H, 6.06; N, 5.26. Found: C, 53.91; H,
6.04; N, 5.17.
5.9.15. 3-((2-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-
methylquinolinium iodide (21i)
Yellow solid (29% yield), mp: 131–132 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.87 (d, 1H, J = 2.7 Hz), 8.25 (d, 1H, J = 9.0 Hz), 8.16

532
T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
5.9.22. 3-((5-Cyclohexylpentyl)(4-fluorophenyl)amino)-1-
methylpyridinium iodide (26d)
1H), 7.52–7.46 (m, 1H), 7.41–7.35 (m, 2H), 7.27–7.21 (m, 2H),
4.60 (s, 3H), 3.91 (d, 2H, J = 7.8 Hz), 1.67–1.59 (m, 8H), 1.39–1.16
(m, 10H), 0.86 (d, 1H, J = 9.6 Hz). Anal. Calcd for
Yellow solid (66% yield), mp: 129–130 °C; 1H NMR (300 MHz,
CDCl3): d 8.46 (s, 1H), 8.23 (d, 1H, J = 6 Hz), 7.63–7.48 (m, 2H),
7.40–7.36 (m, 1H), 7.15–7.09 (m, 2H), 6.99–6.95 (m, 1H), 4.59 (s,
3H), 3.91 (t, 2H, J = 7.8 Hz), 1.68–1.64 (m, 11H), 1.30–1.14 (m,
8H). Anal. Calcd for C23H32FIN2: C, 57.26; H, 6.69; N, 5.81. Found:
C, 57.28; H, 6.61; N, 5.73.
C
23H32ClIN2Á0.05H2O: C, 55.37; H, 6.47; N, 5.62. Found: C, 55.28;
H, 6.45; N, 5.61.
5.9.30. 3-((5-Cyclohexylpentyl)(3-(trifluoromethyl)phenyl)-
amino)-1-methylpyridinium iodide (26l)
Yellow solid (35% yield), mp: 124–125 °C; 1H NMR (300 MHz,
CDCl3): d 8.44 (s, 1H), 8.22 (d, 1H, J = 5.1 Hz), 7.62–7.51 (m, 1H),
7.49–7.38 (m, 3H), 7.27–7.20 (m, 2H), 4.6 (s, 3H), 3.89 (t, 2H,
J = 7.8 Hz), 1.67–1.61 (m, 9H), 1.39–1.15 (m, 10H). Anal. Calcd for
5.9.23. 3-((5-Cyclohexylpentyl)(phenyl)amino)-1-methylpyrid-
inium iodide (26e)
Yellow solid (62% yield), mp: 125–126 °C; 1H NMR (300 MHz,
CDCl3): d 8.42 (s, 1H), 8.17 (s, 1H), 7.63–7.26 (m, 7H), 4.62 (s,
3H), 3.95 (t, 2H, J = 7.5 Hz), 1.73–1.66 (m, 12H), 1.59–1.22 (m,
9H). Anal. Calcd for C23H33IN2: C, 59.48; H, 7.16; N, 6.03. Found:
C, 59.35; H, 7.11; N, 5.90.
C
24H32F3IN2Á0.05H2O: C, 54.14; H, 6.06, N, 5.26. Found: C, 54.05;
H, 6.05; N, 5.25.
5.9.31. 3-((5-Cyclohexylpentyl)(m-tolyl)amino)-1-methylpyrid-
inium iodide (26m)
5.9.24. 3-((5-Cyclohexylpentyl)(p-tolyl)amino)-1-methylpyrid-
inium iodide (26f)
Yellow solid (55% yield), mp: 130–131 °C; 1H NMR (300 MHz,
CDCl3): d 8.26 (s, 1H), 8.22 (d, 1H, J = 5.7 Hz), 7.58–7.53 (m, 1H),
7.43–7.27 (m, 3H), 7.03 (d, 2H, J = 7.8 Hz), 4.55 (s, 3H), 3.85 (t,
2H, J = 7.8 Hz), 2.41 (s, 3H), 1.72–1.57 (m, 10H), 1.43–1.14 (m,
7H), 0.87–0.80 (t, 2H). Anal. Calcd for C24H35IN2: C, 60.25; H,
7.37; N, 5.86. Found: C, 60.14; H, 7.37; N: 5.86.
Yellow solid (60% yield), mp: 150–151 °C; 1H NMR (300 MHz,
CDCl3): d 8.26 (d, 1H, J = 5.4 Hz), 8.19 (s, 1H), 7.60–7.55 (m, 1H),
7.32 (d, 3H, J = 7.8 Hz), 7.10 (d, 2H, J = 8.7 Hz), 4.53 (s, 3H), 3.84–
3.79 (m, 2H), 2.42 (s, 3H), 1.66–1.63 (m, 12H), 1.15–1.12 (m, 7H).
Anal. Calcd for C24H35IN2Á0.05H2O: C, 60.14; H, 7.36; N, 5.84.
Found: C, 60.09; H, 7.31; N, 5.79.
5.9.32. 3-((5-Cyclohexylpentyl)(3-methoxyphenyl)amino)-1-
methylpyridinium iodide (26n)
5.9.25. 3-((5-Cyclohexylpentyl)(4-ethylphenyl)amino)-1-
methylpyridinium iodide (26g)
Yellow solid (45% yield), mp: 158–159 °C; 1H NMR (300 MHz,
CDCl3) d 8.29 (s, 1H), 8.10 (s, 1H), 7.58 (s, 1H), 7.46 (s, 1H), 7.30–
7.23 (m, 2H), 7.15–7.08 (m, 2H), 4.56 (s, 3H), 3.82–3.79 (m, 3H),
1.62–1.60 (m, 15H), 1.34–1.16 (m, 6H). Anal. Calcd for C24H35I-
N2OÁ0.1H2O: C, 58.30; H, 7.13; N, 5.67. Found: C, 58.09; H, 7.11;
N, 5.64.
Yellow solid (65% yield), mp: 159–160 °C; 1H NMR (300 MHz,
CDCl3): d 8.29 (s, 1H), 8.14 (d, 1H, J = 2.1 Hz), 7.55–7.50 (m, 1H),
7.36–7.12 (m, 5H), 4.54 (s, 3H), 3.84 (t, 2H, J = 7.8 Hz), 2.75–2.68
(m, 2H), 1.65 (d, 4H, J = 9.3 Hz), 1.52–1.29 (m, 12H), 1.27–1.09
(m, 6H). Anal. Calcd for C25H37IN2Á0.05H2O: C, 60.97; H, 7.57; N,
5.69. Found: C, 60.86; H, 7.56; N, 5.68.
5.9.33. 3-((3-Cyanophenyl)(5-cyclohexylpentyl)amino)-1-
methylpyridinium iodide (26o)
5.9.26. 3-((5-Cyclohexylpentyl)(4-methoxyphenyl)amino)-1-
methylpyridinium iodide (26h)
Yellow solid (46% yield), mp: 112–113 °C; 1H NMR (300 MHz,
CDCl3) d 8.42 (d, 2H, J = 4.8 Hz), 7.77–7.70 (m, 4H), 7.46 (d, 2H,
8.1 Hz), 4.60 (s, 3H), 3.91 (t, 2H, J = 7.5 Hz), 1.65 (d, 7H,
J = 8.7 Hz), 1.42–1.15 (m, 10H), 0.87–0.80 (m, 2H). Anal. Calcd for
Yellow solid (50% yield), mp: 115–116 °C; 1H NMR (300 MHz,
CDCl3): d 8.18 (d, 2H, J = 5.4 Hz), 7.56–7.54 (m, 1H), 7.32–7.01
(m, 5H), 4.6 (s, 3H), 3.86–3.77 (m, 5H), 3.51–3.44 (m, 9H), 1.67–
C24H32IN3: C, 58.90; H, 6.59; N, 8.59. Found: C, 59.03; H, 6.54; N,
1.61 (m, 5H), 1.23–1.14 (m, 5H). Anal. Calcd for
C24H35I-
8.56.
N2OÁ0.05H2O: C, 58.30; H, 7.13; N, 5.67. Found: C, 58.19; H, 7.12;
N, 5.66.
5.9.34. 3-((2-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-
methylpyridinium iodide (26p)
5.9.27. 3-((5-Cyclohexylpentyl)(4-(methylthio)phenyl)amino)-
1-methylpyridinium iodide(26i)
Yellow solid (45% yield), mp: 125–126 °C; 1H NMR (300 MHz,
CDCl3) d 8.29 (s, 2H), 7.64–7.60 (m, 2H), 7.49–7.43 (m, 3H), 7.27
(s, 1H), 4.60 (s, 3H), 3.87 (t, 2H, J = 7.8 Hz), 1.67–1.61 (m, 12H),
1.39–1.15 (m, 10H). Anal. Calcd for C23H32ClIN2: C, 55.37; H,
6.47; N, 5.62. Found: C, 55.29; H, 6.48; N, 5.58.
Yellow solid (55% yield), mp: 134–135 °C; 1H NMR (300 MHz,
CDCl3): d 8.34 (s, 1H), 8.09 (d, 1H, J = 6 Hz), 7.56–7.51 (m, 1H),
7.39–7.31 (m, 1H), 7.27–7.16 (m, 5H), 4.55 (s, 3H), 3.85 (t, 2H,
J = 7.8 Hz), 2.54 (s, 3H), 1.67–1.54 (m, 10H). Anal. Calcd for
C24H35IN2S: C, 56.46; H, 6.91; N, 5.49. Found: C, 56.59; H, 7.01;
5.9.35. 3-((5-Cyclohexylpentyl)(2-(trifluoromethyl)phenyl)-
amino)-1-methylpyridinium iodide (26q)
N, 5.61.
Yellow solid (40% yield), mp: 137–138 °C; 1H NMR (300 MHz,
CDCl3) d 8.26 (d, 1H, J = 5.7), 8.06 (s, 1H), 7.90–7.81 (m, 2H),
7.67–7.61 (m, 2H), 7.53 (d, 1H, J = 7.8 Hz), 7.17 (d, 1H, J = 8.4 Hz),
4.56 (s, 3H), 1.78–1.60 (m, 12H), 1.32–1.16 (m, 4H), 0.85 (t, 1H).
Anal. Calcd for C24H32F3IN2: C, 54.14; H, 6.06; N, 5.26. Found: C,
53.96; H, 6.00; N, 5.26.
5.9.28. 3-((4-Cyanophenyl)(5-cyclohexylpentyl)amino)-1-
methylpyridinium iodide (26j)
Yellow solid (50% yield), mp: 50–51 °C; 1H NMR (300 MHz,
CDCl3): d 8.55 (s, 1H), 8.41 (d, 1H, J = 6 Hz), 7.80–7.70 (m, 3H),
7.60–7.57 (m, 1H), 7.46 (d, 2H, J = 8.4 Hz), 7.27 (s, 1H), 4.60 (s,
3H), 3.95 (t, 2H, J = 7.5 Hz), 1.68–1.65 (m, 8H), 1.29–1.13 (m, 9H).
Anal. Calcd for C24H32IN3Á0.05H2O: C, 58.90; H, 6.59; N, 8.59.
Found: C, 58.79; H, 6.58; N, 8.57.
5.9.36. 3-((5-Cyclohexylpentyl)(o-tolyl)amino)-1-methylpyrid-
inium iodide (26r)
Yellow solid (60% yield), mp: 160–161 °C; 1H NMR (300 MHz,
CDCl3) d 8.24 (d, 2H, J = 5.7 Hz), 7.59–7.38 (m, 1H), 7.42–7.38 (m,
3H), 7.13–7.07 (m, 2H), 4.59 (s, 3H), 2.15 (s, 3H), 1.67–160 (m,
10H), 1.59–1.32 (m, 4H), 1.39–1.16 (m, 10H), 0.86 (d, 1H,
5.9.29. 3-((3-Chlorophenyl)(5-cyclohexylpentyl)amino)-1-
methylpyridinium iodide (26k)
Yellow solid (55% yield), mp: 115–116 °C; 1H NMR (300 MHz,
CDCl3): d 8.45 (s, 1H), 8.27 (d, 1H, J = 5.7 Hz), 7.63–7.7.59 (m,

T. K. Mazu et al. / Bioorg. Med. Chem. 19 (2011) 524–533
533
J = 9.6 Hz). Anal. Calcd for C24H35IN2: C, 60.25; H, 7.37; N, 5.86.
Acknowledgement
Found: C, 60.29; H, 7.46; N, 5.82.
We are grateful for financial support from the National Insti-
tutes of Health, National Institute of Allergy and Infectious Dis-
eases, through AREA grant number R15 Al37976-01, Research
Centers at Minority Institutions (RCMI) grant number G12 RR
03020, and a Title III grant to Florida A&M University. This re-
search was also supported in part by the Pharmaceutical Re-
5.9.37. 3-((5-Cyclohexylpentyl)(2-methoxyphenyl)amino)-1-
methylpyridinium iodide (26s)
Yellow solid (40% yield), mp: 147–148 °C; 1H NMR (300 MHz,
CDCl3) d 8.30 (d, 1H, J = 5.7 Hz), 8.10 (s, 1H), 7.59–7.54 (m, 1H),
7.44 (t, 1H, J = 8.4 Hz), 7.22–7.10 (m, 4H), 4.57 (s, 3H), 3.79–3.73
(m, 5H), 1.66–1.60 (m, 15H), 1.35–0.83 (m, 4H). Anal. Calcd for
search Center NIH/NCRR
1
C06-RR12512-01 Grant. We
C
24H35IN2OÁ0.01H2O: C, 58.30; H, 7.13; N, 5.67. Found: C, 58.28;
appreciate the generous contributions of Dr. Wang Zhang, Senior
Technician responsible for instrumentation for his contribution
to the generation of NMR spectra of compounds in this paper
and Ms. Marsha Wright for biological evaluation. Antifungal
and antibacterial testing were supported by the NIH, NIAID, Divi-
sion of AIDS, Grant No. AI 27094 and the USDA Agricultural Re-
search Service Specific Cooperative Agreement No. 58-6408-2-
0009, respectively.
H, 7.13; N, 5.66.
5.9.38. 3-((2-Cyanophenyl)(5-cyclohexylpentyl)amino)-1-
methylpyridinium iodide (26t)
Yellow solid (48% yield), mp: 117–118 °C; 1H NMR (300 MHz,
CDCl3) d 8.34 (d, 1H, J = 1.8 Hz), 7.91–7.82 (m, 2H), 7.70 (t, 2H,
J = 6.9 Hz), 7.61–7.56 (m, 1H), 7.34–7.28 (m, 1H), 4.58 (s, 3H),
3.96 (t, 2H, J = 7.8 Hz), 1.76–1.63 (m, 8H), 1.44–1.13 (m, 10H),
0.86–0.80 (m, 2H). Anal. Calcd for C24H32IN3: C, 58.90; H, 6.59; N,
8.59. Found: C, 58.90; H, 6.58; N, 8.46.
References and notes
1. www.cdc.gov.
5.10. Antifungal testing
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All organisms were obtained from the American Type Culture
Collection (Manassas, VA) and includeC. albicans ATCC 90028,C. neo-
formans ATCC 90113, and A. fumigatus ATCC 90906. Susceptibility
testing was performed using a modified version of the NCCLS meth-
ods.19 DMSO solutions of samples were serially diluted in saline, and
transferred in duplicate to 96 well microplates. Microbial suspen-
sions were diluted in broth to afford desired colony forming units/
mL according to the 0.5 McFarland Standard [C. albicans: either Sab-
oraud Dextrose broth (SDB) or RPMI 1640, C. neoformans: SDB, A.
fumigatus: YM broth (for MICs) or RPMI-1640 + 5% Alamar Blue
(for IC50 determination)]. Middlebrook 7H9 broth with OADC (Oleic
Acid-Albumin-Dextrose-Catalase.) enrichment + 5% Alamar Blue.
After adding microbial cultures to the samples affording a final vol-
7. (a) Ansah, C.; Khan, A.; Gooderham, N. J. Toxicology 2005, 208, 141; (b) Ansah,
C.; Gooderham, N. J. Toxicol. Sci. 2002, 70, 245.
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Chem. 2002, 10, 1337; (b) Zhu, X. Y.; Mardenborough, L. G.; Li, S.; Khan, A.;
Zhang, W.; Fan, P.; Jacob, M. R.; Khan, S. I.; Walker, L. A.; Ablordeppey, S. Y.
Bioorg. Med. Chem. 2007, 15, 686; (c) Eyunni, S. V. K.; Boateng, C. A.; Zhu, X. Y.;
Etukala, J. R.; Bricker, B.; Ashfaq, M. K.; Jacob, M. R.; Khan, S. I.; Walker, L. A.;
Ablordeppey, S. Y. Bioorg. Med. Chem. (under review).
ume of 200 lL and final test concentration starting with 20 lg/mL,
plates were read prior to and after incubation using either fluores-
cenceat544ex/590em (A. fumigatus)usingthePolarstarGalaxyPlate
Reader (BMG Lab Technologies, Germany) or optical density at
630 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments,
Vermont). Growth (saline only), solvent and blank (media only) con-
trols were included on each test plate. Drug control [Amphotericin B
(ICN Biomedicals, Ohio)] is included in each assay. Percent growth is
calculated and plotted versus test concentration to afford the IC50
(sample concentration that affords 50% growth of the organism).
The minimum inhibitory concentration (MIC) was determined by
visually inspecting the plate, and is defined as the lowest test con-
centration that allows no detectable growth (for Alamar Blue assays,
no color change from blue to pink).20
9. Van de Waterbeemd, H.; Rose, S. In The Practice of Medicinal Chemistry;
Wermuth, C. G., Ed., 2nd ed.; Elsevier Academic Press, 2004; p 351.
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5.11. Toxicity assay
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19. National Committee for Clinical Laboratory Standards. Reference method for
broth dilution antifungal susceptibility testing of yeasts. Approved standard
M27-A. National Committee for Clinical Laboratory Standards, Wayne, PA,
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20. Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; Madico, G.;
Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.;
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The in vitro cytotoxicity was determined against mammalian
kidney fibroblast (VERO) cells. The assay is performed in 96-well
tissue culture-treated microplates and compounds were tested
up to a highest concentration of 20 l
g/mL as described earlier.21
In brief, cells (25,000 cells/well) were seeded to the wells of the
plate and incubated for 24 h. Samples were added and plates
were again incubated for 48 h. The number of viable cells was
determined according to neutral red assay as previously de-
scribed.22,23 IC50 values were determined from dose curves of
growth inhibition versus concentration. Doxorubicin was used
as a positive control, while DMSO was used as the negative
(vehicle) control.
21. Jain, M.; Khan, S. I.; Tekwani, B. L.; Jacob, M. R.; Singh, S.; Singh, P. P.; Jain, R.
Bioorg. Med. Chem. 2005, 13, 4458.
22. Meenakshi, J.; Khan, S. I.; Babu, T. L.; Jacob, M. R.; Savita, S.; Prati, P. S.; Rahul, J.
Bioorg. Med. Chem. 2005, 13, 4458.
23. Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, A. Lipids 2005, 40, 375.