Benzothieno[3,2-b]quinolinium and 3-(phenylthio)quinolinium compounds: Synthesis and evaluation against opportunistic fungal pathogens

Article abstract of DOI:10.1016/j.bmc.2010.11.008

Substitution around 5-methyl benzothieno[3,2-b]quinolinium (2) ring system was explored in order to identify positions of substitution that could improve its antifungal profile. The 3-methoxy (10b) was active against C. albicans, C. neoformans, and A. fum

Full text of DOI:10.1016/j.bmc.2010.11.008

Bioorganic & Medicinal Chemistry 19 (2011) 458–470
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzothieno[3,2-b]quinolinium and 3-(phenylthio)quinolinium compounds:
Synthesis and evaluation against opportunistic fungal pathogens
Comfort A. Boateng a, , Suresh V. K. Eyunni a, , Xue Y. Zhu a, Jagan R. Etukala a, Barbara A. Bricker a,
M. K. Ashfaq b, Melissa R. Jacob b, Shabana I. Khan b, Larry A. Walker b, Seth Y. Ablordeppey a,
⇑
a College of Pharmacy and Pharmaceutical Sciences, Florida A & M University, Tallahassee, FL 32307, USA
b The National Center for the Development of Natural Products, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Substitution around 5-methyl benzothieno[3,2-b]quinolinium (2) ring system was explored in order to
identify positions of substitution that could improve its antifungal profile. The 3-methoxy (10b) was
active against C. albicans, C. neoformans, and A. fumigatus and the 4-chloro (10f) analog showed moderate
increases in anti-cryptococcal and anti-aspergillus activities. The effectiveness of 10b and 10f were val-
idated in murine models of candidiasis and cryptococcosis, respectively. The efficacy of 10f in reducing
brain cryptococcal infection and its observation in the brain of mice injected with this quaternary com-
pound confirm the capacity of these compounds to cross the blood–brain barrier of mice. Overall, several
of the chloro and methoxy substituted compounds showed significant improvements in activity against
A. fumigatus, the fungal pathogen prevalent in patients receiving organ transplant. Opening the benzo-
thiophene ring of 2 to form 1-(5-cyclohexylpentyl)-3-(phenylthio)quinolinium compound (3) resulted
in the identification of several novel compounds with over 50-fold increases in potency (cf. 2) while
retaining low cytotoxicities. Thus, compound 3 constitutes a new scaffold for development of drugs
against opportunistic infections.
Received 2 September 2010
Revised 25 October 2010
Accepted 3 November 2010
Available online 10 November 2010
Keywords:
Benzothienoquinoline
Antifungal agents
Opportunistic infection agents
Cryptolepine isostere
Quinolinium salts
Benzothienoquinolinium salts
Sulfur isostere
Published by Elsevier Ltd.
1. Introduction
with improved antifungal efficacy and low toxicity. We recently
reported that analogs of sulfur isosteres of cryptolepine,
Infections caused by opportunistic pathogens continue to be of
public health concern.1,2 Candida albicans, Cryptococcus neoformans,
and Aspergillus fumigatus are three of the most common fungal
pathogens responsible for opportunistic infections in humans.
Patients with AIDS, lymphoma, those on long-term corticosteroids,
organ transplant patients and even diabetes patients are at risk for
opportunistic infections.3–6 Antifungal medications are used to
treat opportunistic infections and intravenous therapy with
amphotericin B has been and is one of the most common treatment
options.6–8 Amphotericin B is not available orally and hence is
often combined with an oral medication, such as flucytosine or
one of the azoles.9–16 The unavailability of oral forms of amphoter-
icin B, its severe nephrotoxic side effects along with other side
effects, resistance development, drug interactions associated with
the azoles in particular and the paucity of biological targets
support the need for newer agents.17–21
benzothieno[3,2-b]quinolinium salts (2), have the potential to
become useful agents with broad antifungal properties and low
cytotoxicity.30 It was also found that while the unsubstituted
5-methyl benzothieno[3,2-b]quinolinium salt showed antifungal
properties, the few 2-substituted analogs evaluated were disap-
pointingly less potent. However, substitution at other positions
to enhance the antifungal profile of the benzothienoquinolinium
moiety has not been explored. In addition, it was of interest to ex-
plore flexible ring-opened analogs of the benzothienoquinolines
(Chart 1) in order to confirm previous observations that the tetra-
cyclic ring might not be required for its activity. Although the qua-
ternary form of the tetracyclic structure appears to be the active
form,31 we were concerned that the presence of a permanent pos-
itively charged atom in the N-alkylated benzothienoquinolinium
salts might limit bioavailability and prevent effectiveness in vivo.
This would be of primary concern especially when the compounds
are designed to target infections of the CNS.
Towards this end, we embarked on structural modifications of
the natural product cryptolepine22–29 (Chart 1) to obtain agents
Hence, in this paper, we sought to synthesize and evaluate
substitution at all synthetically accessible positions on the benzo[b]-
thiophene and quinoline moieties, to explore whether ring-opened
analogs might offer opportunity for increased potency and selectiv-
ity and to validate the efficacy of the benzothieno[3,2-b]quinolini-
um salts in murine models of candidiasis and brain cryptococcosis,
⇑
Corresponding author. Tel.: +1 850 599 3834; fax: +1 850 599 3934.
E-mail address: seth.ablordeppey@famu.edu (S.Y. Ablordeppey).
Designated first authors.
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.11.008

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
459
Chart 1.
two common opportunistic infections associated particularly with
AIDS.32–35
pounds 10c and 10e in moderate yields. Similarly, 5-methyl triflate
derivatives of 1- and 3-substituted benzothienoquinolines were
prepared in good yields. The 1-methoxy analog 11a was synthe-
sized to evaluate the effect of the counter ion (IÀ and ÀOTf) on
biological activity. Finally, N-5 methyl iodides of benzothienoquin-
olines, 18a–f, were synthesized in good yields using methyl iodide
at elevated temperatures. Attempts to substitute the 5-methyl
2. Results and discussion
2.1. Synthesis
group with the more hydrophobic
x-phenyl- or x-cyclohexyl-pen-
Substituted 5-methylbenzothieno[3,2-b]quinolinium salts were
synthesized using modifications of previously published proce-
dures,30,36 as outlined in Schemes 1 and 2. Briefly, suitably substi-
tuted anthranilic acids from commercial sources were acylated
with (phenylthio)acetyl chloride (5) to obtain 1-, 3-, and 4-substi-
tuted 6-(2-phenylsulfanylacetylamino)benzoic acids, 6a–f. To
obtain the corresponding 7-, 8-, and 9-substituted compounds
(14a–f), 2-(2-bromoacetamido)benzoic acid (13), (prepared by
condensing 2-aminobenzoic acid with bromoacetyl bromide) was
reacted with suitably substituted thiophenols. In a double intramo-
lecular cyclization reaction with polyphosphoric acid (PPA), the
acylated intermediates (6a–f and 14a–f) were converted to substi-
tuted-11-quinolones, which were subsequently treated with POCl3
to form 11-chlorinated benzothieno[3,2-b]quinolines. Hydrogena-
tion on Pd/C to remove the 11-substituted chlorine selectively
was achieved as previously described.30,37 Attempts to prepare
5-methyl iodides of 4-substituted benzothieno[3,2-b]quinolines
were unsuccessful. This may be due to the bulkiness of the groups
at the 4-position, their proximity to the N-5 atom of the rigid tet-
racyclic ring and deactivation of the N atom. However, 5-methyl
triflates of the quinolines 9a–c were successfully synthesized albeit
in poor to moderate yields depending on the reaction times and the
amount of the stronger methylating MeOTf added. Addition of
excess of MeOTf to 9c and 9e at room temperature yielded com-
tyl groups were unsuccessful as very low yields and difficult
solubility problems prevented successful delivery of the products.
Synthesis of 1-substituted-3-(phenylthio)quinolinium iodides
(3) required 3-iodoquinoline (20) as the starting material (Scheme
3). A Finkelstein reaction was applied to 3-bromoquinoline to ex-
change the bromide for the desired iodide.38 Using a copper-cata-
lyzed carbon–sulfur bond formation approach, benzenethiols was
reacted with 3-iodoquinoline to obtain 3-(phenylthio)quinoline
(21) in good yield.39 Subsequent alkylation of 21 with appropriate
alkyl halides provided the desired target compounds as shown in
Scheme 3.
2.2. Biological evaluation
Previous studies in our labs have revealed that substitution on
the ring of cryptolepine (1) enhanced its potency and broadened
its anti-opportunistic activity profile. Further optimization led to
the identification of the sulfur isostere (2) of cryptolepine as a
potential anti-opportunistic agent.30 Thus, it was of interest to
explore substitution on the 5-methyl benzothienoquinolinium
compound in order to optimize its anti-opportunistic profile. The
observation that introduction of a halogen on the core structure
of cryptolepine enhanced potency motivated the initial decision
Scheme 1. Synthetic procedure for quinoline-substituted benzothienoquinolinium compounds. Reagents and conditions: (a) NaOH, H2O, rt; (b) PPA, 130 °C, 3 h; (c) POCl3,
110 °C, 3 h; (d) 10% Pd/C, EtOAc/AcOH, NaOAc, H2 gas; (e) Py. HCl, 175 °C, 2 h; (f) MeOTf, toluene, rt, 24 h; (g) CH3I, sulfone, 110 °C. Reflux for 12 h.

460
C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
Scheme 2. Synthetic procedure for benzothiophene-substituted benzothienoquinolines. Reagents and conditions: (a) DMF/1,4-dioxane (1:1), rt, 24 h; (b) acetone, K2CO3,
KI, reflux, 24 h; (c) PPA, 130 °C, 3 h; (d) POCl3, 110 °C, 3 h; (e) 10% Pd/C, EtOAc/AcOH, NaOAc, H2 gas; (f) CH3I, sulfone, 110 °C. Reflux for 24 h.
Scheme 3. Synthesis of substituted 3-(phenylthio)quinolinium salts. Reagents and conditions: (i) CuI, CH3NHCH2CH2NHCH3, NaI, dioxane, reflux under N2, 110 °C, 48 h;
(ii) benzenethiol, CuI, HOCH2CH2OH, K2CO3, iPrOH, reflux under N2, 110 °C, 48 h; (iii) C6H5(CH2)4CH2-I (PP-I) or C6H11(CH2)4CH2-I (CP-I) or CH3I, sulfone, 110 °C, 12–16 h.
to use a chlorine atom to probe all synthetically accessible posi-
tions on 2. Thus, compounds 10d–f and 18d–f were synthesized
and evaluated against C. neoformans, C. albicans, and A. fumigatus.
Substitution at position 6 was unsuccessful as ring closure consis-
tently occurred on the side away from the 3-chloro position of the
benzenethiol to form the 9-substituted product. Methoxy-substi-
tuted analogs (10a–c, i, and 18a–c) were also synthesized to pro-
vide an avenue for comparing the presence of an electron
donating substituent (with the electron withdrawing chlorine) on
the benzothienoquinoline structure and its effect on anti-opportu-
nistic activities.
against Ca). Several of the same compounds however showed mod-
erate to very potent activities against A. fumigatus, the opportunis-
tic fungus that causes infections in organ transplant patients.40
Clearly, the OMe-substituted benzothienoquinolinium salts show
a higher level of potency against A. fumigatus than the Cl-substi-
tuted analogs. This observation together with the loss of activity
observed for the 4-phenolic substitution (10g) may suggest that
substituents with negative sigma and positive pi values may be
associated with enhancing potency and need to be further ex-
plored. The potential of these compounds to induce toxicity in Vero
cells was also investigated. None of the compounds showed cyto-
toxicity in Vero cells up to 4.7
lg/mL and in fact most of them
2.3. In vitro evaluation
showed no cytotoxicity at 10 g/mL. These results are very encour-
l
aging and warrant further investigation of the potential of these
compounds as new anti-opportunistic infection agents. Com-
pounds 10f and 10b were selected as representatives of the benzo-
thienoquinolines for further evaluation of their in vivo efficacy
against C. neoformans and C. albicans.
In an earlier study, it was observed that opening the benzothi-
enoquinoline ring to form 3-phenylthio-quinolinium salt which
demonstrated activity against opportunistic pathogens. Compound
22a, the unsubstituted N-methyl-3-phenylthioquinolinium salt,
The results of the in vitro antifungal screening of the Cl and
OMe-substituted benzothienoquinolines are presented in Table 1.
When both IC50 and MIC values are between 1 and 10
compounds are considered active, moderately active between
10–15 g/mL and weak above 15 up to 20 g/mL. Most of the
lg/mL, the
l
l
compounds show only weak activities against C. neoformans (Cn)
and C. albicans (Ca) except for compound 10f (IC50 = 4.0 and
MIC = 10 lg/mL against Cn) and 10b (IC50 = 1.5, MIC = 10 lg/mL

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
461
Table 1
Physicochemical data and antifungal activities of 5-methylbenzothieno[3,2-b]quinolinium analogs
Compd
R
Y
Mpb (°C)
Empirical formulac
IC50/MIC
lg/mL
TC50 lg/mL
C. neoformans
C. albicans
A. fumigatus
Vero cells
2aa
H
H
1-Cl
2-Cl
3-Cl
4-Cl
7-Cl
8-Cl
OTf
237–238
215–216
285–288
189.3
C17H12F3NO3S2Á0.2H2O
>15/>20
NT/>20/(2.5)d
NA
15/>20
10/>20
4.0/10
NA
NA
15/>20
NA
10/>20
NT/7.8/(1.0)d
NA
2.0/>20
NA
6.5/>20
NT/>20
3/10
NT/2.5
NA
>23.8
>23.8
NT
>23.8
>10
>10
>10
>10
NT
2ba
C16H12INS
10d
IOTf
I
OTf
OTf
I
I
I
I
OTf
I
OTf
OTf
C17H11NO3S2Cl
10ha
10e
C16H11ClINSÁ0.2H2O
220–221
225–226
203–204
218–219
192–193
216–217
225–227
216–217
222–223
180–181
208–209
199–200
228–229
200–201
C
C
17H11NO3S2Cl
17H11NO3S2Cl
10f
18d
18e
18f
NA
6.0/10.0
NA
C16H11ClINS
20/>20
2.5/>20
>20
15/>20
15/>20
>20
1.5/10
15/>20
NA
C
C
C
C
16H11ClINSÁ0.4H2O
9.5/>20
6.5/10
1.5/2.5
0.8/1.3
20/ND
0.4/0.6
3.0/5.0
NA
8.0/20
1.0/2.5
3.0/5.0
1.5/2.5
9-Cl
16H11ClINSÁ0.35CH3OH
17H14NOSIÁ0.1H2O
11a
1-OMe
1-OMe
2-OMe
3-OMe
4-OMe
1-OH
7-OMe
8-OMe
9-OMe
NT
10a
18H14NO4S2F3Á0.65H2O
NA
>4.7
>23.8
>10
NT
NT
NT
NT
>10
6.5
10ia
10b
C17H14INOSÁ0.5H2O
NT/8–15
6.0/>20
15/>20
NA
NA
NA
C
C
C
C
C
C
18H14NO4S2F3Á0.25H2O
18H14NO4S2F3Á0.1H2O
17H12NO4S2F3Á0.25EtOAc
17H14NOSIÁ0.2H2O
17H14NOSI
10c
10g
18a
18b
OTf
I
I
I
NA
6.0/20
7.0/>20
0.3/0.6
18c
17H14NOSI
10/>20
1.5/2.5
Amphotericin B
Abbreviations: NT = not tested. NA = not active at 20 lg/mL; IC50 = the concentration that affords 50% inhibition of growth; MIC = minimum inhibitory concentration, is the
lowest test concentration that allows no detectable growth.
a
Previously reported.30
Melting points were uncorrected.
b
c
All compounds were subjected to CHN analysis and each passed within 0.4% of the theoretical value.
d
Data in brackets previously published.30a
was synthesized and evaluated against four opportunistic patho-
gens as shown in Table 2 to probe this observation. The results
show that ring-opening at the benzo[b]thiophene moiety did not
significantly impact activity. However, replacing the methyl group
of the former (22b) with the later (22c), showed the later to be
the more potent against all pathogens tested except for A. fumiga-
tus where the reverse is the case. With potencies and cytotoxicities
comparable to that of amphotericin B, and in particular having
fungicidal activity, the compounds with the N-cyclohexylpentyl
3-phenylthio-quinolinium scaffold have the potential of serving
as new anti-opportunistic infection agents warranting further
investigation and development. These are currently on-going.
with an
x-phenylpentyl group, previously reported to enhance
activity,28 led to compound 22b with a much enhanced activity
against all the fungal pathogens including C. krusei. It has been re-
ported that C. krusei is prevalent in bone marrow transplantation
and accounts for 2–3% of all Candida bloodstream infections.41
Replacement of the
x
-phenylpentyl group (22b) with the more
2.4. In vivo evaluation
hydrophobic
x-cyclohexylpentyl group in line with previous
observations that the later produced improvement in potency28
Prior to in vivo efficacy studies, maximum tolerated dose (MTD)
studies were carried out to determine the highest dose that could
led to compound 22c. Comparing the two unsubstituted analogs
Table 2
Antifungal activities of N-alkyl 3-substituted phenylthioquinolinium analogs
Compd
R
R1
IC50/MIC/MFC (
C. albicans
l
g/mL)
TC50 (lg/mL)
C. neoformans
C. krusei
A. fumigatus
Vero cells
22a
22b
22c
Cryp
2b
H
H
H
Me
PP
CP
15/20/>20
1.5/5.0/10
0.5/1.3/2.5
12.5 (MIC)
NT/>20/>20
1.5/2.5/2.5
6.0/20/>20
5.5/10/>20
2.7/5.0/10
250 (MIC)
NT/7.8/>20
0.3/0.6/1.3
NT
NT/>2.5/20
NT/1.3/>20
8.6/10/>20
NT
NA
1.5/2.5/5.0
NT
NT
4.6
3.2
>23.8
6.5
1.5/5.0/20
0.7/1.3/2.5
NT
NT
0.5/1.3/1.3
AmB
Abbreviations: AmB = amphotericin B; Cryp = cryptolepine; NT = not tested. NA = not active at 20 lg/mL; PP = 5-phenylpentyl-; CP = 5-cyclohexylpentyl-; 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 microorganism. TC50 = the concentration that is toxic to 50% of cells.

462
C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
Drug dose
CFU/gm Brain
10 mg/kg
7.9x107 1.9x107
5 mg/kg
6.58x107 3.2 x107
4.47x107 1.0 x107
1.0 mg/kg
Amphotericin B
1.2 mg/kg
6.78x107 2.9 x107
Vehicle
8.88x107 3.9 x107
Figure 1. Effect of 10f on C. neoformans infection in mice.
be delivered without any apparent adverse effects. The test com-
pounds were dissolved in an appropriate vehicle and daily doses
were administered for 3 (10b) or 5 (10f) days. Body weights and
general appearances of the animals were observed daily and signs
of distress were recorded. Animals were sacrificed at the termina-
tion of the scheduled experiment or earlier if signs of increased
morbidity after administration of the compounds were observed.
Necropsy was performed and gross lesions were recorded.
To have in vivo efficacy, the effect of compounds in inhibiting
the growth of opportunistic pathogens must be demonstrated. To
this end, cryptococcosis was induced in mice by infecting them
with C. neoformans ATCC 52657 (5 Â 105 cells) via the lateral tail
vein. The mice were divided in five groups of five each and begin-
ning 24 h post-infection, they were treated daily with increasing
doses of the test compound, amphotericin B (1.2 mg/kg) or the
vehicle intraperitoneally (ip) for the next 5 days. On day 6 post-
infection, all mice were sacrificed, their brains aseptically removed,
and CFU of C. neoformans per gram brain was determined.
tested. Maximum activity was observed at 1 mg/kg where half of
the organisms were killed in the brain tissue compared to negative
control, whereas amphotericin B killed approximately 24%. Thus, at
this dose (1 mg/kg), 10f was about twice as effective as amphoter-
icin B. At 5 mg/kg, the anti-cryptococcus activity was comparable
to that of amphotericin B. Increasing the dose to 10 mg/kg did
not result in additional improvement but instead reduced activity.
The mechanism by which the activities at higher concentrations of
10f are attenuated is not known at this time but is not unique. This
observation parallels the behavior reported for caspofungin42 (an
echinocandin) and may reflect several possible events including
an adverse effect on the immune system at higher doses or solubil-
ity problems leading to precipitation of the compound on injection.
Thus, the results clearly show that despite their quaternary
nature, these compounds are capable of entering cells and more
importantly, in crossing the blood–brain barrier (BBB) to elicit their
anti-infective actions and hence providing us with the first
evidence that these compounds are capable of crossing the BBB.
Compound 10f was dissolved in DMSO/PEG/PBS (1:6:7) and in-
jected ip once a day for 5 days using doses ranging from 1 mg/kg to
20 mg/kg. At 10 and 20 mg/kg, mice showed decreased mobility
and signs of discomfort. However, these signs were not noticeable
after 2 h post injection. The body weights of mice showed no
noticeable changes up to 10 mg/kg but at 20 mg/kg, loss of weight
was observed. At necropsy, no significant pathological lesions were
observed at doses less than 10 mg/kg. Slight distension of the duo-
denum, jejunum and cecum was seen at 10 mg/kg. However, at
20 mg/kg, enlargement of the liver with fusion of the hepatic lobes
was observed. The intestines were moderately distended, espe-
cially the duodenum, jejunum, and cecum. The intestinal content
was dark colored. No gross pathology was observed in the kidneys,
spleen and other visceral organs.
Figures 1 and 2 show the effect of various doses of 10f against
C. neoformans infection in mice. In Figure 1, the CFU per gram of
brain sample is displayed for each dose while Figure 2 shows
the% reduction in CFU per dose of 10f. Amphotericin B is included
as the positive control while the vehicle served as a negative con-
trol. Compound 10f showed anti-cryptococcus activity at all doses
2.5. Murine model of systemic candidiasis
2.5.1. Tissue burden
Mice were infected with C. albicans strain B311 ATCC 32354
(7.5 Â 105 yeast cells) via the lateral tail vein and divided in five
groups of five mice each. Seven, 24, and 48 h post-infection, mice
were injected ip with various doses of the test compound, ampho-
tericin B (1.2 mg/kg) or the vehicle. At 72 h post-infection all mice
were sacrificed, their kidneys were collected aseptically and CFU of
C. albicans per gram of kidney was determined.
2.5.2. Maximum tolerated dose (MTD) of 10b
The compound was dissolved in DMSO/PEG/PBS (1:3:3) and in-
jected ip once a day for 3 days. The compound was well tolerated
by the mice and the MTD was 10 mg/kg. No significant changes
in body weights were observed. At necropsy, no gross pathological
lesions were observed in the visceral tissues at 1–10 mg/kg doses.
However, mice injected with 20 mg/kg for 3 days showed white
deposits of unknown origin in the peritoneal cavity.
% Reduction
Drug dose
CFU/gm Brain
10 mg/kg
5 mg/kg
10
26
50
1.0 mg/kg
Amphotericin B
1.2 mg/kg
24
0
Vehicle
Figure 2. Reduction of C. neoformans infection by 10f in mouse brain.

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
463
Drug dose
CFU/gm Kidney
10 mg/kg
5 mg/kg
1 mg/kg
5.39x104 1.7 x104
2.83x105 5.7x104
2.50 x105 1.1 x105
Amphotericin B
1.2 mg/kg
4.76x103 7.5x102
3.81x105 1.1 x105
Vehicle
Figure 3. Effect of 10b on C. albicans infection in mice.
% Reduction
CFU/gm Kidney
Drug dose
10 mg/kg
86
5 mg/kg
1 mg/kg
26
34
99
Amphotericin B
1.2 mg/kg
Vehicle
0
Figure 4. Reduction of C. albicans infection by 10b in the kidney.
2.5.3. Candidiasis tissue burden
Compound 10f was identified in this extract (Fig. 6A). Subse-
quently, the extract was spiked with a standard solution of 10f
and injected into the HPLC. The identified peak and the standard
peak co-eluted providing further confirmation (Fig. 6B) of the pres-
ence of 10f.
Figures 3 and 4 show the effect of 10b against candidiasis in
vivo. In Figure 3, the CFU per gram of kidney sample is displayed
for each dose while Figure 4 shows the% reduction in CFU per dose
of 10b. Amphotericin B is included as the positive control while the
vehicle serves as a negative control. Amphotericin B, at a dose of
1.2 mg/kg, eliminated 99% of the infection from the kidneys
compared to the vehicle while a dose of 10 mg/kg of 10b produced
86% reduction in C. albicans tissue burden. At 1 mg/kg and 5 mg/kg
the activity of 10b against C. albicans was limited to a 26% and 34%
reduction, respectively.
3. Conclusion
The tetracyclic benzothieno[3,2-b]quinolinium compounds as a
group have weak to good in vitro potency against C. neoformans,
C. albicans, and A. fumigatus. These activities were generally fungi-
The efficacy of these quaternary compounds especially in the
brain suggests that they have the capacity to cross the blood–brain
barrier. To provide further proof that this is the case, mice were
dosed ip with 10f in an identical manner as in the in vivo efficacy
studies. After 15–60 min post injection, mice were anesthesized,
sacrificed, the brain perfused with phosphate buffered saline
(PBS), removed and frozen until the day of extraction. An HPLC
chromatogram of a perfused brain of a mouse dosed with 10f is
shown in Figure 5.
The results show the retention time and UV spectra match the
10f standard. No peak above detection limit at the retention time
of 10f was observed in a blank brain extract treated by the same
procedure. A mass spectrum of the same extract confirms 10f with
a parent peak m/e of 284.8 (m/e of a MeOH solution of 10f was
284.4), within the resolution of the instrument (Fig. 5).Another
perfused brain from a rat dosed with 40 mg/kg of 10f was macer-
ated, extracted with 1.5 mL of 30:70 (2.3 M HClO4/MeOH), treated
with a sonic dismembrator, refrigerated, centrifuged at 10,000 g for
static below 20 lg/mL concentrations. However, selected agents
(10b and 10f) demonstrated significant in vivo efficacy against
C. albicans and C. neoformans comparable to that of amphotericin
B. It was further demonstrated that these compounds are able to
cross not only cell membranes but also the blood–brain barrier
to produce their anti-infective activities. Interestingly, opening
the benzo[b]thiophene ring and replacing the N-methyl group with
N-5-phenylpentyl or 5-cyclohexylpentyl groups, to form substi-
tuted 3-(phenylthio)quinolinium compounds, produced as high
as 50-fold increases in potency without increasing cytotoxicity.
These analogs also demonstrated significantly higher activity
against C. krusei than the other pathogens tested. In addition,
unlike their tetracyclic counterparts, these ring-opened analogs
demonstrated fungicidal activity as well.
4. Experimental section
4.1. Chemistry
5 min, and the supernatant concentrated to 300 lL. This extract
was injected into the HPLC system as described in the biological
testing section, with an isocratic mobile phase of 70C:30D (where
C is 90% (7.5 mM sodium heptanesulfonate + 0.1 M phosphoric acid
adjusted to pH 3.00 with triethylamine) + 10% acetonitrile and D is
100% acetonitrile.).
All the reagents were purchased from Sigma–Aldrich, Fisher
Scientific or Alfa Aesar and used without further purification. Flash
chromatography was performed with Davisil grade 634 silica gel.
Analytical thin layer chromatography (TLC) was carried out on

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C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
Figure 5. HPLC chromatograms of extracts from a perfused brain of a 10f-dosed mouse (above), a blank brain from an untreated mouse (below, left) and a mass spectrum of
the extract (below, right).
Figure 6. HPLC chromatograms of perfused brain extracts of 10f-dosed mouse [6A] and a spiked extract [6B].
Merck TLC plates coated with Silica Gel 60 F254 (0.25 mm layer
thickness); visualization was carried out using UV lamp
over Na2SO4, filtered, and solvent was evaporated in vacuo. The
residue obtained was recrystallized using EtOAc/hexane (3:1) to
give compounds 6a–f as white solids.
a
(254 nm). Melting points were determined on an Electrothermal
MEL-TEMPÒ 3.0 device and are uncorrected. Nuclear Magnetic
Resonance (1H NMR) spectra were obtained on a Varian 300 MHz
Mercury NMR Spectrometer. Elemental analyses were carried out
by Atlantic Microlab, Inc., Norcross, GA, and were within 0.4% of
the theory unless otherwise noted.
4.2.1. 1-Methoxy-6-(2-phenylsulfanyl-acetylamino)-benzoic
acid (6a)
Yield 67%. Mp 97–98 °C. 1H NMR (CDCl3): d 11.56 (br s, 1H, NH),
8.53–8.50 (dd, 1H, J = 8.7, 0.9 Hz), 7.52–7.46 (t, 1H, J = 8.4 Hz),
7.44–7.41 (m, 2H), 7.29–7.24 (m, 2H), 7.20–7.17 (m, 1H), 6.76 (d,
1H, J = 8.7 Hz), 4.07 (s, 3H), 3.81 (s, 2H).
4.2. General procedure for the synthesis of substituted 6-(2-
phenylsulfanyl-acetylamino)-benzoic acids 6a–f, (Method A)
4.2.2. 3-Methoxy-6-(2-phenylsulfanyl-acetylamino)benzoic acid
(6b)
A mixture of substituted anthranilic acids 4a–f (30 mmol),
NaOH (60 mmol) and phenylthioacetyl chloride (30 mmol) in
H2O (20 mL) was stirred at 0 °C for 1 h, and then at rt for 2 h. The
mixture was acidified with 5% HCl (pH 4–5) and then extracted
with EtOAc (2 Â 500 mL). The combined organic layer was dried
Yield 82%. Mp 188–189 °C. 1H NMR (CDCl3): d 11.92 (br s, 1H,
NH), 8.41 (d, 1H, J = 2.4 Hz), 8.06 (d, 1H, J = 8.70 Hz), 7.29–7.24
(m, 2H), 7.40 (m, 2H), 20 (d, 1H, J = 7.5 Hz), 6.68 (dd, 1H, J = 9.0,
2.2 Hz), 3.88 (s, 3H), 3.82 (s, 2H).

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
465
4.2.3. 4-Methoxy-6-(2-phenylsulfanyl-acetylamino)benzoic acid
4.4. General procedure for the synthesis of substituted 11-chlo-
(6c)
robenzothieno[3,2-b]quinolines 8a–f, (Method C)
Yield 85%. Mp 167–168 °C. 1H NMR (CD3OD): d 7.47–7.42 (m,
3H), 7.34–7.27 (m, 3H), 7.24–7.18 (m, 2H), 3.81 (s, 3H), 3.79
(s, 2H).
A suspension of compound 7 (28 mmol) in POCl3 (20 mL) was re-
fluxed with stirring at 110 °C for 3 h. After evaporating the POCl3, the
reaction mixture was poured into ice/water (50 mL) and then neu-
tralized with 10% NaOH. The aqueous solution was extracted with
CHCl3 (3 Â 200 mL) and the combined organic layer was washed
with brine, dried over sodium sulfate, filtered, and solvent evapo-
rated. The residue obtained was column chromatographed over
silica gel with hex/EtOAc (3:2) as eluent to give compounds 8a–f.
4.2.4. 1-Chloro-6-(2-phenylsulfanyl-acetylamino)benzoic acid
(6d)
Yield 82%. Mp 153–154 °C. 1H NMR (CDCl3): d 10.25 (br s, 1H,
NH), 8.22 (d, 1H, J = 8.4), 7.42–7.36 (dd, 1H, J = 8.4, 8.1 Hz), 7.35–
7.16 (m, 6H), 3.80 (s, 2H).
4.2.5. 3-Chloro-6-(2-phenylsulfanyl-acetylamino)benzoic acid
(6e)
4.4.1. 1-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (8a)
Yield 92%. Mp 192–193 °C. 1H NMR (DMSO-d6): d 8.54 (d, 1H,
J = 7.2 Hz), 7.86 (d, 1H, J = 8.4 Hz), 7.80 (d, 1H, J = 8.4 Hz), 7.62–
7.48 (m, 3H), 6.92 (d, 1H, J = 7.8 Hz), 3.96 (s, 3H).
Yield 82%. Mp 161–163 °C.1H NMR (CDCl3): d 8.54 (d, 1H,
J = 2.1), 8.02 (d, 1H, J = 8.4), 7.39–7.36 (m, 2H), 7.29–7.25 (m, 3H),
7.21–7.10 (m, 2H), 3.82 (s, 2H).
4.4.2. 3-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (8b)
Yield 78%. Mp 209–210 °C. 1H NMR (CDCl3): d 8.58 (d, 1H,
J = 8.10 Hz), 8.18 (d, 1H, J = 9.3 Hz), 7.87 (d, 1H, J = 7.5 Hz), 7.64–
7.53 (m, 3H), 7.34 (dd, 1H, J = 9.3, 2.1 Hz), 4.016 (s, 3H).
4.2.6. 4-Chloro-6-(2-phenylsulfanyl-acetylamino)benzoic acid
(6f)
Yield 86%. Mp 178–179 °C. 1H NMR (CDCl3): d 9.39 (s, 1H, NH),
7.87 (dd, 1H, J = 7.8, 1.5), 7.61 (dd, 1H, J = 8.1, 1.5 Hz), 7.41 (dd, 2H,
J = 7.8, 1.5 Hz), 7.32–7.20 (m, 4H), 3.85 (s, 2H).
4.4.3. 4-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (8c)
Yield 94%. Mp 201–202 °C. 1H NMR (DMSO-d6): d 8.74 (d, 1H,
J = 7.5 Hz), 7.8–7.83 (m, 2H), 7.65–7.53 (m, 3H), 7.16 (d, 1H,
J = 7.8 Hz), 4.19 (s, 3H).
4.3. General procedure for the synthesis of substituted benzo-
thieno[3,2-b]quinolin-11(5H)-ones, 7a–f, (Method B)
Compound 6 (20 mmol) was mixed with polyphosphoric acid
(PPA, 125 g) and heated at 130–135 °C for 3 h. After cooling to rt,
the mixture was poured into ice/water (200 mL), neutralized with
50% NaOH solution to give a highly insoluble dark brown solid
which was washed with H2O, EtOAc, and Et2O, dried over P2O5
and used as such for the next step without further purification.
4.4.4. 1,11-Dichlorobenzothieno[3,2-b]quinoline (8d)
Yield 64%. Mp 200–201 °C. 1H NMR (CDCl3): d 8.58 (d, 1H,
J = 7.8 Hz), 8.26 (dd, 1H, J = 8.7, 1.5 Hz), 7.88 (d, 1H, J = 7.8 Hz),
7.72 (m, 1H), 7.66–7.55 (m, 3H).
4.4.5. 3,11-Dichlorobenzothieno[3,2-b]quinoline (8e)
Yield 71%. 1H NMR (CDCl3): d 8.58 (d, 1H, J = 8.1 Hz), 8.30 (d, 1H,
J = 2.1 Hz), 8.23 (d, 1H, J = 9.0 Hz), 7.86 (d, 1H, J = 7.8 Hz), 7.69–7.56
(m, 3H).
4.3.1. 1-Methoxybenzothieno[3,2-b]quinolin-11(5H)-one (7a)
Yield 95% (crude). 1H NMR (DMSO-d6): d 12.53 (br s, 1H, NH),
8.50–8.47 (dd, 1H, J = 7.2, 1.8), 8.07–8.04 (dd, 1H, J = 7.2, 1.5),
7.61–7.52 (m, 3H), 7.33 (d, 1H, J = 8.4), 6.81 (d, 1H, J = 8.10), 3.83
(s, 3H).
4.4.6. 4,11-Dichlorobenzothieno[3,2-b]quinoline (8f)
Yield 72%. Mp 182 °C. 1H NMR (CDCl3): d 8.73 (dd, 1H, J = 7.8,
1.8 Hz), 8.26 (dd, 1H, J = 8.7. 1.2 Hz), 7.94 (dd, 1H, J = 7.5, 1.2 Hz),
7.88 (d, 1H, J = 8.4 Hz), 7.69–7.64 (ddd, 1H, J = 8.10, 7.20, 1.5 Hz),
7.62–7.55 (m, 2H).
4.3.2. 3-Methoxybenzothieno[3,2-b]quinolin-11(5H)-one (7b)
Yield 95% (crude). 1H NMR (DMSO-d6): d 8.49 (d, 1H, J = 7.20),
8.13 (d, 1H, J = 9.0 Hz), 8.08 (d, 1H, J = 7.5 Hz), 7.53 (m, 2H), 7.17
(d, 1H, J = 1.80 Hz), 6.98 (dd, 1H, J = 8.7, 1.8), 3.89 (s, 3H).
4.5. General procedure for the synthesis of methoxy-substituted
benzothieno[3,2-b]quinolines 9a–c, (Method D)
4.3.3. 4-Methoxybenzothieno[3,2-b]quinolin-11(5H)-one (7c)
Yield 84% (crude). 1H NMR (DMSO-d6): d 11.9 (br s, 1H, NH), 9.0
(d, 1H, J = 8.10), 8.1 (d, 1H, J = 7.7 Hz), 7.81 (d, 1H, J = 7.14 Hz),
7.65–7.53 (m, 2H), 7.38–7.30 (m, 2H), 4.08 (s, 3H).
Methoxy-substituted compounds, 8a–c (10 mmol) were each
dissolved in a mixture of AcOH and NaOAc (3 g) and Pd/C (10%,
1.5 g) was added. The resulting suspension was hydrogenated
(60–65 psi) at rt for 2–4 h. The mixture was filtered to remove
the Pd/C and the filtrate was concentrated to a small volume, basi-
fied with saturated aqueous NaHCO3, and extracted with EtOAc
(2 Â 100 mL). The combined organic layers was dried (Na2SO4),
and filtered. The filtrate was concentrated at reduced pressure to
afford the corresponding substituted benzothieno[3,2-b]quinolines
9a–c as pale yellow to white solids. These intermediates were used
as such for the next step without further purification.
4.3.4. 1-Chlorobenzothieno[3,2-b]quinolin-11(5H)-one (7d)
Yield 92% (crude). 1H NMR (DMSO-d6): d 8.51 (dd, 1H, J = 7.8,
0.9 Hz), 8.06 (dd, 1H, J = 7.2, 1.2 Hz), 7.77 (dd, 1H, J = 8.4, 1.2 Hz),
7.63–7.52 (m, 3H), 7.31 (dd, 1H, J = 7.5, 0.9 Hz).
4.3.5. 3-Chlorobenzothieno[3,2-b]quinolin-11(5H)-one (7e)
Yield 92% (crude). 1H NMR (DMSO-d6): d 8.50 (d, 1H, J = 7.5 Hz),
8.21 (d, 1H, J = 8.7 Hz), 7.86 (s, 1H), 8.12–8.04 (m, 1H), 7.68–7.58
(m, 2H), 7.86 (s, 1H), 7.38 (dd, 1H, J = 8.7, 2.1 Hz).
4.5.1. 1-Methoxybenzothieno[3,2-b]quinolines (9a)
Yield 68%. Mp 190–191 °C. 1H NMR (CDCl3): d 8.94 (s, 1H), 8.59
(d, 1H, J = 7.5 Hz), 7.81 (dd, 2H, J = 8.10, 9.0 Hz), 7.59–7.44 (m, 3H),
6.80 (d, 1H, J = 7.5), 3.96 (s, 3H).
4.3.6. 4-Chlorobenzothieno[3,2-b]quinolin-11(5H)-one (7f)
Yield 94% (crude). 1H NMR (DMSO-d6): d 11.48 (s, 1H, NH), 8.91
(d, 1H, J = 7.8 Hz), 8.25 (d, 1H, J = 8.10 Hz), 8.11 (d, 1H, J = 7.8 Hz),
7.94 (d, 1H, J = 7.2 Hz), 7.67–7.55 (m, 2H), 7.42 (dd, 1H, J = 8.10,
7.80 Hz).
4.5.2. 3-Methoxybenzothieno[3,2-b]quinolines (9b)43
Yield 88%. Mp 148–149 °C. 1H NMR (CDCl3): d 8.65 (dd, 1H,
J = 7.0, 1.5), 8.51 (s, 1H), 7.87 (dd, 1H, J = 8.10, 1.5 Hz), 7.80

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C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
(d, 1H, J = 9.0 Hz), 7.63–7.51 (m, 3H), 7.27–7.24 (m, 1H), 4.02
(s, 3H).
4.7.2. 3-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10b)
Yield 68%. Mp 221–223 °C. 1H NMR (CD3OD): d 9.59 (s, 1H), 9.0
(d, 1H, J = 8.4 Hz), 8.35 (d, 1H, J = 9.0 Hz), 8.29 (d, 1H, J = 8.4 Hz),
7.98–7.93 (m, 1H), 7.88–7.82 (m, 2H), 7.69–7.65 (dd, 1H, J = 9.0,
2.4 Hz), 5.08 (s, 3H), 4.22 (s, 3H). Anal. Calcd for C18H14F3NO4S2Á
0.25H2O: C, 49.82; H, 3.25; N, 3.23. Found: C, 49.82; H, 3.27; N,
3.11.
4.5.3. 4-Methoxybenzothieno[3,2-b]quinolines (9c)
Yield 78%. Mp 164–165 °C. 1H NMR (CDCl3): d 8.78 (dd, 1H,
J = 7.5, 1.5 Hz), 8.58 (s, 1H), 7.86 (dd, 1H, J = 8.4, 1.2 Hz),
7.64–7.55 (m, 2H), 7.55–7.50 (m, 2H), 7.12–7.09 (m, 1H), 4.17
(s, 3H).
4.7.3. 4-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10c)
4.6. General procedure for the synthesis of chloro-substituted
benzothieno[3,2-b]quinolines 9d–f, (Method E)
Yield 28%. Mp 180–181 °C. 1H NMR (CD3OD): d 9.62 (s, 1H), 8.95
(d, 1H, J = 8.4 Hz), 8.26 (d, 1H, J = 8.4 Hz), 7.96–7.91 (m, 3H), 7.87–
7.79 (m, 2H), 5.15 (s, 3H), 4.24 (s, 3H). Anal. Calcd for
Chloro-substituted compounds 8d–f (10 mmol) were dissolved
in EtOAc. Pd/C (10%, 1.5 g) was added to the solution. The resulting
suspension was hydrogenated (60–65 psi) at rt for 6–8 h. The mix-
ture was filtered, the filtrate was concentrated to dryness, and the
residue was purified by column chromatography on silica gel
(eluted with EtOAc–hexane 10–15%) to afford the corresponding
substituted benzothieno[3,2-b]quinolines 9d–f as pale yellow to
white solids.
C
18H14F3NO4S2Á0.1H2O: C, 50.13; H, 3.32; N, 3.25. Found: C,
49.85; H, 3.22; N, 3.10.
4.7.4. 1-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10d)
Yield 62%. Mp 285–287 °C. 1H NMR (CD3OD): d 10.16 (s, 1H),
9.04 (d, 1H, J = 8.4 Hz), 8.72 (d, 1H, J = 9.0 Hz), 8.35 (d, 1H,
J = 8.4 Hz), 8.28–8.17 (m, 2H), 8.06–8.01 (m, 1H), 7.93–7.87 (m,
1H), 5.19 (s, 3H). Anal. Calcd for C17H11ClF3NO3S2: C, 47.06; H,
2.56; N, 3.23. Found: C, 46.96; H, 2.47; N, 3.21.
4.6.1. 1-Chlorobenzothieno[3,2-b]quinolines (9d)
Yield 72%. Mp 173–174 °C. 1H NMR (CDCl3): d 9.03 (s, 1H), 8.68
(d, 1H, J = 7.80 Hz), 8.24 (m, 1H), 7.89 (d, 1H, J = 7.2 Hz), 7.68–7.65
(m, 2H), 7.63–7.58 (m, 2H).
4.7.5. 3-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10e)
4.6.2. 3-Chlorobenzothieno[3,2-b]quinolines (9e)
Yield 65%. Mp 220–221 °C. 1H NMR (CD3OD): d 9.76 (s, 1H), 9.03
(d, 1H, J = 8.4 Hz), 8.88 (s, 1H), 8.46 (d, 1H, J = 8.7 Hz), 8.32 (d, 1H,
J = 8.1 Hz), 8.07–8.00 (m, 2H), 7.92–7.86 (m, 1H), 5.13 (s, 3H). Anal.
Calcd for C17H11ClF3NO3S2Á0.2H2O: C, 46.68; H, 2.53; N, 3.20.
Found: C, 46.58; H, 2.52; N, 3.12.
Yield 42%. 1H NMR (CDCl3): d 8.63 (s, 1H), 8.57 (d, 1H,
J = 7.2 Hz), 8.29 (s, 1H), 7.84 (d, 1H, J = 8.1 Hz), 7.66–7.52 (m, 3H).
4.6.3. 4-Chlorobenzothieno[3,2-b]quinolines (9f)
Yield 52%. Mp 146.0–146.5 °C. 1H NMR (CDCl3): d 8.73 (dd, 1H,
J = 7.8, 0.9 Hz), 8.27 (dd, 1H, J = 8.7, 1.2 Hz), 7.94 (dd, 1H, J = 7.2,
1.2 Hz), 7.86 (d, 1H, J = 7.8 Hz), 7.69–7.55 (m, 4H).
4.7.6. 4-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10f)
Yield 46%. Mp 225–226 °C. 1H NMR (CD3OD): d 9.77 (s, 1H), 9.01
(d, 1H, J = 8.4 Hz), 8.44–8.39 (m, 2H), 8.33 (d, 1H), J = 8.10 Hz),
8.07–7.87 (m, 3H), 5.21 (s, 3H). 13C NMR (DMSO-d6): 152.99,
146.46, 142.31, 138.89, 138.79, 135.47, 134.81, 131.07, 130.24,
4.6.4. 1-Hydroxybenzothieno[3,2-b]quinoline (9g)
Compound 9a (0.53 g, 2 mmol) was added to preheated (150 °C)
pyridinium hydrochloride (10 g) and the mixture was further
heated at 175 °C for 6 h. The reaction mixture was allowed to cool
to rt and H2O (100 mL) was added. The solid obtained was filtered,
washed with H2O (50 mL) and recrystallized using EtOAc/hexane
(4:1) to give a yellow solid. Yield 96%. Mp 164–165 °C. 1H NMR
(DMSO-d6): d 9.86 (br s, 1H, OH), 8.97 (s, 1H), 8.75 (dd, 1H,
J = 7.8, 1.2 Hz), 8.09 (d, 1H, J = 7.5 Hz), 7.71–7.59 (m, 2H), 7.50
(m, 2H), 7.18–7.13 (t, 1H, J = 4.2 Hz).
129.91, 129.33, 127.92, 127.26, 125.73, 124.54, 121.40 (q,
(C–F) = 322.36 Hz), 51.08. Anal. Calcd for C17H11ClF3NO3S2: C,
47.06; H, 2.56; N, 3.23. Found: C, 46.90; H, 2.59; N, 3.25.
J
4.7.7. 4-Hydroxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10g)
Yield 68%. Mp 208–209 °C. 1H NMR (CD3OD): d 9.46 (s, 1H), 8.92
(d, 1H, J = 8.7 Hz), 8.22 (d, 1H, J = 8.10 Hz), 7.95–7.90 (m, 1H), 7.83–
7.71 (m, 2H), 7.64–7.62 (m, 1H), 7.46 (d, 1H, J = 7.8 Hz), 5.27 (s,
3H). Anal. Calcd for C17H12F3NO4S2Á0.25EtOAc: C, 49.42; H, 3.23;
N, 3.20. Found: C, 49.72; H, 3.29; N, 3.37.
4.7. General procedure for the synthesis of substituted 5-
methylbenzothieno[3,2-b]quinolin-5-ium triflates 10a–g,
(Method F)
MeOTf (0.5 mL) was added to a suspension of compound 9
(100 mg) in toluene (3 mL) and stirred for 24 h at rt. The precipi-
tate thus obtained was filtered, washed with Et2O and recrystal-
lized from either MeOH-Et2O or MeOH/EtOAc to afford the
desired substituted 5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate derivatives as yellow solids in moderate to good yields.
4.8. General procedure for the synthesis of 1-methoxy-5-meth-
ylbenzothieno[3,2-b]quinolin-5-ium iodide (11a), (Method G)
CH3I (0.5 mL) was added to a solution of compound 9a (100 mg)
in tetramethyl sulfone (0.2 mL). The mixture was heated at 110 °C
for 12 h in a sealed pressure tube. After cooling to rt, Et2O was
added to form a precipitate. The precipitate was collected, washed
with Et2O (25 mL) and recrystallized from MeOH–Et2O as a yellow
solid. Yield 82%. Mp 216–217 °C. 1H NMR (CD3OD): d 10.01 (s, 1H),
9.0 (d, 1H, J = 8.4 Hz), 8.31–8.22 (m, 3H), 7.97 (t, 1H, J = 8.1, 6.9 Hz),
7.86 (t, 1H, J = 7.5 Hz), 7.46 (d, 1H, J = 7.2 Hz), 5.11 (s, 3H), 4.22 (s,
3H). Anal. Calcd for C17H14INOSÁ0.1H2O: C, 49.70; H, 3.48; N, 3.41.
Found: C, 49.69; H, 3.50; N, 3.40.
4.7.1. 1-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
triflate (10a)
Yield 72%. Mp 225–227 °C. 1H NMR (CD3OD): d 9.98 (s, 1H), 9.0
(d, 1H, J = 8.4 Hz), 8.28–8.17 (m, 3H), 7.99–7.93 (m, 2H), 7.88–7.82
(m, 1H), 7.45 (d, 1H, J = 7.8 Hz), 5.09 (s, 3H), 4.21 (s, 3H). Anal.
Calcd for C18H14F3NO4S2Á0.65H2O: C, 49.01; H, 3.20; N, 3.18. Found:
C, 49.03; H, 3.21; N, 3.06.

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
467
4.9. General procedure for the synthesis of 2-(2-bromoacetyl-
4.10.6. 2-(2-(4-Chlorophenylsulfanyl)acetylamino)benzoic acid
amido)benzoic acid (13). (Method H)
(14f)
Yield 95%. Mp 183–184 °C. 1H NMR (CD3OD): d 8.56–8.50 (dd,
1H, J = 0.9, 7.8 Hz), 8.04–8.02 (dd, 1H, J = 1.5, 6.6 Hz), 7.48 (m,
1H), 7.44–7.38 (m, 2H), 7.30–7.24 (m, 2H), 7.16–7.10 (m, 1H),
3.90 (s, 2H).
A mixture of anthranilic acid (15 g, 109 mmol), DMF (35 mL), and
1,4-dioxane (35 mL) was cooled to 0 °C in a round bottom flask and
bromoacetyl bromide (22 g, 109 mmol) slowly added so that the
temperature did not rise above 1 °C. At the end of the addition, the
temperature was maintained at 0 °C for 15 min longer and then
the mixture was stirred overnight at room temperature. The con-
tents of the flask was poured into water (400 mL), and the resulting
precipitate 13 was filtered, washed with water (3 Â 100 mL), and
dried to yield a colorless crystalline compound 13 (20 g). No
further purification was attempted. 1H NMR: (CD3OD): d 8.58–
8.54 (dd, 1H, J = 0.6, 7.8 Hz), 8.12–8.08 (dd, 1H, J = 1.5, 6.3 Hz),
7.60–7.54 (dtd, 1H, J = 1.2, 1.5 and 5.7 Hz), 7.20 (dtd, 1H, J = 1.2,
1.2 and 5.7 Hz), 4.10 (s, 2H).
4.11. Substituted benzothieno[3,2-b]quinoline-11(5H)-ones
15ª–15f using General Method B
Compound 6 (20 mmol) was mixed with PPA (125 g) and heated
at 130–135 °C for 3 h. After cooling to rt, the mixture was poured
into ice/water (200 mL), neutralized with 50% NaOH solution to
give a highly insoluble dark brown solid which was washed with
H2O, EtOAc, and Et2O, dried over P2O5 and used as such for the next
step without further purification.
4.10. General procedure for the synthesis of substituted 2-(2-
phenylsulfanylacetylamido)benzoic acids 14a–f, (Method I)
4.11.1. 9-Methoxybenzothieno[3,2-b]quinoline-11(5H)-one
(15a)
Yield 52%. Mp 225–227 °C. 1H NMR (DMSO-d6): d 8.32 (d, 1H,
J = 7.8 Hz), 8.21 (d, 1H, J = 8.1 Hz), 7.90–7.83 (m, 2H), 7.64
(t, 1H, J = 7.8 Hz), 7.48–7.43 (m, 1H), 7.31 (d, 1H, J = 8.1 Hz), 4.07
(s, 3H).
A mixture of substituted benzenethiol (38 mmol), K2CO3 (2.0 g)
and KI (200 mg) in acetone (100 mL) was stirred for 15 min. 2-(2-
Bromoacetylamido)-benzoic acid (13) (38 mmol) was added to the
reaction mixture and refluxed for 24 h. The reaction was allowed to
cool to rt and acetone was removed on rotary evaporator. The res-
idue obtained was dissolved in water (200 mL) and 10% HCl
(100 mL) was added. The resulting white precipitate was filtered,
washed with H2O (2 Â 100 mL) and dried to give the desired com-
pounds 14a–f in good to excellent yields. These intermediates
were used as such for the next step without further purification.
4.11.2. 8-Chlorobenzothieno[3,2-b]quinoline-11(5H)-one (15e)
Yield 48%. Mp 217–219 °C. 1H NMR (DMSO-d6): d 12.80 (s, 1H),
8.54–8.48 (d, 1H, J = 8.4 Hz), 8.28–8.26 (d, 1H, J = 2.1 Hz), 8.24–8.18
(d, 1H, J = 8.1 Hz), 7.80–7.70 (m, 2H), 7.64–7.60 (dd, 1H, J = 2.1,
6.9 Hz), 7.34–7.32 (m, 1H).
1H NMR spectra of compounds 15b–d and f could not be ob-
tained because they were insoluble in DMSO/MeOH.
4.10.1. 2-(2-(2-Methoxyphenylsulfanyl)acetylamido)benzoic
acid (14a)
4.12. Substituted 11-chlorobenzothieno[3,2-b]quinolines
16a–e, using General Method C
Yield 86%. Mp 172–173 °C. 1H NMR (CD3OD): d 8.53 (d, 1H,
J = 8.1 Hz), 8.04–8.01 (m, 1H), 7.52–7.47 (m, 1H), 7.37–7.34 (m,
1H), 7.24–7.18 (m, 1H), 7.13 (t, 1H, J = 7.5 Hz), 6.94 (d, 1H,
J = 8.1 Hz), 6.82 (t, 1H, J = 7.8 Hz), 3.83 (s, 3H), 3.75 (s, 2H).
4.12.1. 9-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (16a)
Yield 68%. Mp 200–201 °C. 1H NMR (CDCl3): d 8.33–8.28 (m,
2H), 8.22 (d, 1H, J = 8.1 Hz), 7.80 (t, 1H, J = 8.1 Hz), 7.70–7.65 (m,
1H), 7.53 (t, 1H, J = 7.8 Hz), 7.10 (d, 1H, J = 7.8 Hz), 4.06 (s, 3H).
4.10.2. 2-(2-(3-Methoxyphenylsulfanyl)acetylamido)benzoic
acid (14b)
Yield 78%. Mp 167–169 °C. 1H NMR (CD3OD): d 8.55 (d, 1H,
8.7 Hz), 8.04 (dd, 1H, J = 8.1, 1.8 Hz), 7.08 (m, 1H), 7.18–7.12 (m,
2H), 6.97–6.94 (m, 2H), 6.75 (dd, 1H, J = 9.6, 2.7 Hz), 3.82 (s, 2H),
3.69 (s, 3H).
4.12.2. 8-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (16b)
Yield 54%. Mp 199–200 °C. 1H NMR (DMSO-d6): d 8.25 (d, 1H,
J = 8.7 Hz), 8.20 (d, 1H, J = 8.1 Hz), 7.69 (d, 1H, J = 8.4 Hz), 7.47–
7.40 (m, 2H), 7.12 (dd, 1H, J = 6.9, 7.2 Hz), 7.03 (dd, 1H, J = 8.4,
1.1 Hz), 3.84 (s, 3H).
4.10.3. 2-(2-(4-Methoxyphenylsulfanyl)acetylamido)benzoic
acid (14c)
4.12.3. 7-Methoxy-11-chlorobenzothieno[3,2-b]quinoline (16c)
Yield 56%. Mp 207–209 °C. 1H NMR (CDCl3): d 8.31 (m, 2H), 8.09
(s, 1H), 7.83 (dd, 1H, J = 8.4, 7.2 Hz), 7.74 (m, 1H), 7.25 (m, 2H), 4.01
(s, 3H).
Yield 72%. Mp 175–176 °C; 1H NMR (CD3OD): d 8.53 (d, 1H,
J = 8.4 Hz), 8.07 (dd, 1H, J = 7.8, 1.5 Hz), 7.51 (dd, 1H, J = 7.20,
8.4 Hz), 7.43 (dd, 2H, J = 9.0, 2.1 Hz), 7.14 (dd, 1H, J = 7.2, 7.8 Hz),
6.84 (dd, 2H, J = 9.0, 2.1 Hz), 3.73 (s, 3H), 3.68 (s, 2H).
4.12.4. 9,11-Dichlorobenzothieno[3,2-b]quinoline (16d)
Yield 63%. Mp 221–222 °C. 1H NMR (CDCl3): d 8.60–8.54 (d, 1H,
J = 7.8 Hz), 8.38–8.30 (d, 2H, J = 8.4 Hz), 7.88–7.82 (m, 1H), 7.76–
7.70 (m, 1H), 7.68–7.62 (dd, 1H, J = 0.9, 6.9 Hz), 7.58–7.52 (m, 1H).
4.10.4. 2-(2-(2-Chlorophenylsulfanyl)acetylamido)benzoic acid
(14d)
Yield 95%. Mp 171–173 °C. 1H NMR (CD3OD): d8.44–8.38 (d, 1H,
J = 8.1 Hz), 8.04–8.00 (dd, 1H, J = 1.5, 6.3 Hz), 7.52–7.46 (dd, 1H,
J = 1.5, 6.3 Hz), 7.40–7.33 (m, 1H), 7.32–7.30 (m, 1H), 7.23–7.20
(m, 1H), 7.18–7.12 (m, 1H), 7.08–7.00 (m, 1H), 3.90 (s, 2H).
4.12.5. 8, 11-Dichlorobenzothieno[3,2-b]quinoline (16e)
Yield 59%. Mp 252–253 °C. 1H NMR (CDCl3): d 8.56–8.50 (d, 1H,
J = 8.4 Hz), 8.36–8.26 (m, 2H), 7.88–7.80 (m, 2H), 7.74–7.68 (m,
1H), 7.58–7.52 (dd, 1H, J = 1.8, 6.6 Hz).
4.10.5. 2-(2-(3-Chlorophenylsulfanyl)acetylamido)benzoic acid
(14e)
Yield 96%. Mp 167–168 °C. 1H NMR (CD3OD): d 8.44–8.40 (dd,
1H, J = 0.9, 7.5 Hz), 8.04–7.88 (dd, 1H, J = 1.5, 6.0 Hz), 7.44 (t, 1H,
J = 2.1 Hz), 7.38–7.30 (m, 2H), 7.28–7.22 (t, 1H, J = 7.8 Hz), 7.18–
7.14 (m, 1H), 7.08–7.00 (m, 1H), 3.80 (s, 2H).
4.12.6. 7,11-Dichlorobenzothieno[3,2-b]quinoline (16f)
Yield 45%. Mp 69–71 °C. 1H NMR (CDCl3): d 8.60 (d, 1H,
J = 1.5 Hz), 8.38–8.26 (t, 2H, J = 7.8 Hz), 7.88–7.76 (m, 2H), 7.74–
7.68 (m, 1H), 7.62–7.58 (dd, 1H, J = 2.1, 6.3 Hz).

468
C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
4.13. General procedure for the synthesis of methoxy-substi-
tuted benzothieno[3,2-b]quinolines 17a–c, (Method D)
C
17H14INOSÁ0.2H2O: C, 49.26; H, 3.50; N, 3.38. Found: C, 49.27;
H, 3.41; N, 3.33.
4.13.1. 9-Methoxybenzothieno[3,2-b]quinolines (17a)
Yield 62%. Mp 168–169 °C. 1H NMR (CDCl3): d 8.63 (s, 1H), 8.27
(t, 2H, J = 7.2 Hz), 7.92 (d, 1H, J = 8.4 Hz), 7.75 (t, 1H, J = 8.4 Hz),
7.60–7.50 (m, 2H), 7.09 (d, 1H, J = 8.1 Hz), 4.05 (s, 3H).
4.15.4. 7-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18f)
Yield 56%. Mp 203–204 °C. 1H NMR (CD3OD): d 9.84 (s, 1H), 9.04
(d, 1H, J = 1.5 Hz), 8.82–8.76 (d, 1H, J = 9.0 Hz), 8.54–8.48 (d, 1H,
J = 8.4 Hz), 8.40–8.32 (m, 2H), 8.12–8.06 (t, 1H, J = 7.8 Hz), 8.04–
7.98 (dd, 1H, J = 2.1, 6.6 Hz), 5.20 (s, 3H). Anal. Calcd for
4.13.2. 8-Methoxybenzothieno[3,2-b]quinolines (17b)43
Yield 54%. Mp 185–186 °C. 1H NMR (CDCl3): d 8.52 (d, 1H,
J = 8.4 Hz), 8.30 (d, 1H, J = 9.0 Hz), 7.81 (dd, 1H, J = 6.6, 8.4 Hz),
7.68 (dd, 1H, J = 7.8, 7.8 Hz), 7.32 (d, 1H, J = 2.4 Hz), 7.26 (s, 1H),
7.16 (dd, 1H, J = 8.7, 2.1 Hz), 3.95 (s, 3H).
C16H11ClINS: C, 46.68; H, 2.69; N, 3.40. Found: C, 46.39; H, 2.55;
N, 3.30.
4.15.5. 8-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18e)
4.13.3. 7-Methoxybenzothieno[3,2-b]quinolines (17c)
Yield 56%. Mp 176–178 °C. 1H NMR (CDCl3): d 8.55 (s, 1H), 8.30
(d, 1H, J = 8.70 Hz), 8.14 (s, 1H), 7.90 (d, 1H, J = 8.10 Hz), 7.78–7.68
(m, 2H), 7.59 (dd, 1H, J = 8.1, 6.9 Hz), 7.26 (dd, 1H, J = 8.7, 1.8 Hz),
4.01 (s, 3H).
Yield 61%. Mp 218–219 °C. 1H NMR: (CD3OD): d 9.80 (s, 1H),
9.06–9.00 (d, 1H, J = 9.6 Hz), 8.80–8.72 (d, 1H, J = 9.0 Hz), 8.52–
8.48 (d, 1H, J = 8.7 Hz), 8.44 (d, 1H, J = 2.1 Hz), 8.40–8.30 (m, 1H),
8.10–8.04 (t, 1H, J = 7.5 Hz), 7.94–7.86 (dd, 1H, J = 1.8, 7.2 Hz),
5.20 (s, 3H). Anal. Calcd for C16H11ClINSÁ0.4H2O: C, 45.88; H,
2.84; N, 3.34. Found: C, 45.81; H, 2.60; N, 3.38.
4.14. General procedure for the synthesis of chloro-substituted
benzothieno[3,2-b]quinolines 17d–f, (Method E)
4.15.6. 9-Chloro-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18d)
4.14.1. 9-Chlorobenzothieno[3,2-b]quinoline (17d)
Yield 26%. Mp 192–193 °C. 1H NMR: (CD3OD): d 9.88 (s, 1H).
9.04–9.02 (d, 1H, J = 8.4 Hz), 8.80–8.78 (d, 1H, J = 9.3 Hz),
8.54–8.50 (d, 1H, J = 8.1 Hz), 8.42–8.34 (m, 1H), 8.14–8.04 (m, 2H),
7.94–7.88 (t, 1H, J = 7.8 Hz), 5.20 (s, 3H). Anal. Calcd for C16H11ClNSÁ
0.35CH3OH: C, 45.24; H, 2.88; N, 3.23. Found: C, 44.90; H, 2.56; N,
3.22.
Yield 58%. Mp 179–180 °C. 1H NMR (CDCl3): d 8.64 (s, 1H), 8.62–
8.56 (d, 1H, J = 7.8 Hz), 7.96–7.90 (d, 1H, J = 8.4 Hz), 7.82–7.76 (m,
1H), 7.64–7.58 (m, 2H), 7.56–7.50 (t, 1H, J = 7.5 Hz).
4.14.2. 8-Chlorobenzothieno[3,2-b]quinoline (17e)
Yield 52%. Mp 252–253 °C. 1H NMR (CD3OD): d 8.60 (s, 1H).
8.58–8.56 (d, 1H, J = 8.7 Hz), 8.30–8.26 (d, 1H, J = 8.7 Hz), 7.94–
7.90 (d, 1H, J = 8.1 Hz), 7.82–7.74 (m, 1H), 7.64–7.58 (m, 1H),
7.56–7.52 (dd, 1H, J = 1.8, 6.6 Hz).
4.16. General procedure for the synthesis of 3-iodoquinoline
(20), (Method F)
A mixture of 3-bromoquinoline (19) (9.83 g, 47.3 mmol), CuI
(450 mg, 2.4 mmol), NaI (14.15 g, 94.52 mmol), N,N-dimethyleth-
ylenediamine (0.5 mL, 415 mg, 4.7 mmol) in dioxane (47 mL) was
heated, stirred, and refluxed under N2 for 44–48 h at 110 °C. The
reaction was monitored by GC/MS till the conversion reached
100%. After cooling to room temperature, the mixture was diluted
with 30% aqueous ammonia (20 mL), followed by extraction with
EtOAc (3 Â 30 mL). The combined organic layers was washed with
brine (100 mL), dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated by rotary evaporation under reduced pres-
sure to yield the pure product as a pale yellow solid in almost
quantitative yield. The product was used as such for the next step
without further purification. 1H NMR (CDCl3): d 9.03 (d, 1H,
J = 2.4 Hz), 8.54–8.53 (m, 1H), 8.08–8.04 (m, 1H), 7.76–7.69 (m,
2H), 7.59–7.53 (m, 1H).
4.14.3. 7-Chlorobenzothieno[3,2-b]quinoline (17f)
Yield 46%. Mp 190–192 °C. 1H NMR (CD3OD): d 8.86 (s, 1H), 8.68
(s, 1H), 8.44–8.50 (d, 1H, J = 8.7 Hz), 7.98–7.92 (d, 1H, J = 8.4 Hz),
7.86–7.76 (m, 2H), 7.70–7.58 (m, 2H).
4.15. Substituted 5-methylbenzothieno[3,2-b]quinolin-5-ium
iodides 18–f were synthesized using General Method E
4.15.1. 9-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18a)
Yield 68%. Mp 200–201 °C. 1H NMR (CD3OD): d 9.77 (s, 1H), 8.76
(d, 1H, J = 9), 8.60 (d, 1H, J = 8.4 Hz), 8.47 (d, 1H, J = 8.4 Hz), 8.33
(t, 1H, J = 7.5 Hz), 8.04 (t, 1H, J = 7.5 Hz), 7.83 (t, 1H, J = 8.1 Hz),
7.55 (d, 1H, J = 8.4 Hz), 5.14 (s, 3H), 4.12 (s, 3H). Anal. Calcd for
C17H14INOS: C, 50.13; H, 3.46; N, 3.44. Found: C, 50.10; H, 3.46;
N, 3.42.
4.17. Synthesis of 3-(phenylthio)quinolines (21)
4.15.2. 8-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18b)
A mixture of Cu (I) iodide (20 mg, 0.10 mmol), potassium
carbonate (540 mg, 3.92 mmol), 3-iodoquinoline (500 mg,
1.96 mmol), substituted benzenethiol (0.21 mL, 1.96 mmol), ethyl-
ene glycol (0.22 mL, 3.92 mmol), and 2 mL of 2-propanol was
heated at 110 °C and allowed to reflux under N2 for 30 h. After
cooling to room temperature, the mixture was diluted with
distilled water (20 mL) and the extracted EtOAc (3 Â 15 mL). The
organic layer was washed with brine (50 mL), dried over anhy-
drous Na2SO4 and concentrated by rotary evaporation at reduced
pressure to provide crude product. The crude product was purified
by column chromatography on silica gel using hexane/EtOAc
(9.8:0.2) to afford a pure 3-(phenylthio)quinoline.44 Yield 92%.
Mp 78–79 °C. 1H NMR (CDCl3): d 8.81 (s, 1H), 8.08–8.06 (m, 2H),
7.71–7.25 (m, 8H).
Yield 62%. Mp 228–229 °C. 1H NMR (CD3OD): d 4.07 (s, 3H), 5.05
(s, 3H), 7.85 (s, 1H), 8.01–7.96 (t, 1H, J = 7.5 Hz), 8.30–8.24 (t, 1H,
J = 7.2 Hz), 8.41 (d, 1H, J = 8.10 Hz), 8.68 (d, 1H, J = 9.30 Hz), 8.92
(d, 1H, J = 9.6 Hz), 9.56 (s, 1H). Anal. Calcd for C17H14INOS: C,
50.13; H, 3.46; N, 3.44. Found: C, 50.01; H, 3.39; N, 3.43.
4.15.3. 7-Methoxy-5-methylbenzothieno[3,2-b]quinolin-5-ium
iodide (18c)
Yield 78%. Mp 199–200 °C. 1H NMR (CD3OD): d 9.73 (s, 1H),
8.74 (d, 1H, J = 9.3 Hz), 8.47 (d, 1H, J = 8.4 Hz), 8.37–8.30 (m, 2H),
8.2 (d, 1H, J = 9.0 Hz), 8.06–8.01 (t, 1H, J = 7.8 Hz), 7.68–7.64
(dd, 1H, J = 9.0, 1.8 Hz), 5.18 (s, 3H), 4.06 (s, 3H). Anal. Calcd for

C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
469
4.18. Synthesis of 1-(alkylsubstituted)-3-(phenylthio)quino-
linium iodides (22a–c)
EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont). Growth
(saline only), solvent, and blank (media only) controls were
included on each test plate. Drug control amphotericin B (ICN
Biomedicals, Ohio) for fungi was included in each assay. Percent
growth was calculated and plotted versus test concentration to af-
ford 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 concentration that allows no detectable growth (for
Alamar Blue assays, no color change from blue to pink).
3-(Phenylthio)quinoline (100 mg, 0.39 mmol) was added to the
appropriate alkyl iodide (130 mg, 0.470 mmol) in tetramethylene
sulfone (0.5 mL). The mixture was heated at 110 °C for 16 h in a
sealed pressure tube and then allowed to cool to rt after which
EtOAc (15 mL) was added to precipitate the solid. The precipitate
was collected, washed with additional EtOAc and dried to form
the pure solid powder and no further purification was necessary
4.18.1. 1-Methyl-3-(phenylthio)quinolinium iodide (22a)
Yield 81%. Mp 145–146. 1H NMR (DMSO): 9.69 (1H, s), 9.19 (1H,
s), 8.48 (1H, d, J = 7.5 Hz), 8.37 (1H, d, J = 8.4 Hz), 8.25 (1H, t,
J = 8.1 Hz), 8.03 (1H, t, J = 8.9 Hz), 7.50 (5H, m), 4.60 (3H, s). Anal.
Calcd for C16H14INS: C 50.67, H 3.72, N 3.69; Found: 50.63,
H 3.66, N 3.51.
4.19.2. Cytotoxicity assay
The in vitro cytotoxicity was determined against mammalian
kidney fibroblast (VERO) cells. The assay was performed in 96-well
tissue culture-treated microplates and compounds were tested up
to a highest concentration of 23.8 l
g/mL as described earlier.47 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 by
the neutral red assay as previously described.47 IC50 values were
determined from dose curves of growth inhibition versus concen-
tration. Doxorubicin was used as a positive control, while DMSO
was used as the negative (vehicle) control.
4.18.2. 1-(Phenylpentyl)-3-(phenylthio)quinolinium iodide
(22b)
Yield 75%. Mp 166–167 °C. 1H NMR (DMSO): 9.69 (1H, s), 9.17
(1H, s), 9.57 (1H, d, J = 9.0 Hz), 8.39 (1H, d, J = 8.0 Hz), 8.23 (1H, t,
J = 7.5 Hz), 8.02 (1H, t, J = 7.5 Hz), 7.50 (5H, m), 7.24 (2H, t, J =
7.0 Hz), 7.15 (3H, m), 5.02 (2H, t, J = 7.4 Hz), 2.54 (2H, t, J =
7.4 Hz), 1.98 (2H, m), 1.60 (2H, m), 1.39 (2H, m). 13C NMR
(DMSO-d6): 151.64, 148.23, 142.58, 136.99, 136.29, 132.90,
131.98, 131.39, 131.13, 130.93, 130.84, 130.56, 129.47, 128.95,
128.92, 126.37, 119.76, 58.17, 35.56, 31.03, 29.97, 25.99. Anal.
Calcd for C26H26INSÁ0.2H2O: C, 60.63; H, 5.09; N, 2.72. Found:
60.60; H, 5.02; N, 2.73.
4.19.3. Identification of 10f in brain samples
Under general anesthesia, mice brains were perfused with
phosphate buffered saline (PBS) until >99.5% white. Mice were
euthanized, brains were harvested and frozen, weighed, thawed
at room temperature and then macerated in a silanized glass vial
with a spatula before adding 0.5 M HClO4 (2 mL) and vortex mix-
ing. Additional 0.5 M HClO4 was added if needed to obtain a pH
of 1–1.5 on a pH paper. The mixture was then homogenized on
ice with a Kinematica Polytron 3100 homogenizer at the highest
setting for 2 min, followed by two 45 s treatments on ice with a
Fisher 550 Sonic Dismembrator. Four mL MeOH (HPLC grade)
was added and the mixture vortexed and sonified for 5 min. The
supernatant was removed after centrifugation and filtered if
needed into a clean silanized glass vial. The remaining brain tissue
was extracted again with 2 mL of 0.5 M HClO4 plus 4 mL MeOH at a
pH of 1–1.5. The supernatants were combined and the pH was then
adjusted to ꢀ7 with concd NH4OH.48,49
4.18.3. 1-(Cyclohexylpentyl)-3-(phenylthio)quinolinium iodide
(22c)
Yield: 73%. Mp 174–176 °C. 1H NMR (DMSO-d6): d 9.66 (d, 1H,
J = 2.1 Hz), 9.16 (d, 1H, J = 1.5 Hz), 8.58 (d, 1H, J = 9 Hz), 8.38 (t,
1H, J = 7.2 Hz), 8.25–8.19 (m, 1H), 8.01 (t, 1H, J = 7.5 Hz), 7.56–
7.41 (m, 5H), 5.01 (t, 2H, J = 7.2 Hz), 1.93–1.91 (m, 2H), 1.64–1.61
(m, 5H), 1.30–1.11 (m, 10H), 0.86–0.79 (m, 2H). 13C NMR (DMSO-
d6): 151.60, 148.26, 137.04, 136.29, 132.88, 132.07, 131.47,
131.12, 130.95, 130.81, 130.59, 129.49, 119.71, 58.32, 37.57,
37.38, 33.52, 30.21, 26.88, 26.72, 26.52, 26.45. Anal. Calcd for
C26H32INS: C, 60.34; H, 6.23; N, 2.71. Found: C, 60.48; H, 6.27; N,
After centrifugation if necessary, the extract was cleaned up
using a 225 mg Waters Oasis WCX plus SPE cartridge as follows:
The cartridge was rinsed (5 mL MeOH) and conditioned (with
5 mL of 70:30 MeOH/H2O at pH 7). The extract (12 mL) was slowly
loaded, washed with 5% NH4OH (4 mL), then with 20% MeOH
(5 mL) and another aliquot of MeOH (4 mL). The column was
eluted slowly with 5% HCOOH in MeOH. Eluents were collected
in 2 mL fractions (16 mL total) and analyzed by HPLC separately.
Eluents were combined and then evaporated under nitrogen in a
40 °C water bath. The residue was reconstituted in MeOH
2.75.
4.19. Biological testing
4.19.1. Antifungal testing
All organisms were obtained from the American Type Culture
Collection (Manassas, VA) and include C. albicans ATCC 90028,
C. krusei ATCC 6258, C. neoformans ATCC 90113, and A. fumigatus
ATCC 90906. Susceptibility testing was performed using a modified
version of the CLSI methods45 as described by Samoylenko et al.46
Amphotericin B (ICN Biomedicals, Ohio) was used as a positive
control in each assay.
(100 lL) and injected into the HPLC (Waters 2695 Alliance system
with a 2996 PDA detector). The HPLC conditions were: Analytical
wavelength—294 nm, Flowrate 1.00 mL/min, Column–Alltech
Briefly, DMSO solutions of samples were serially diluted in sal-
ine and transferred in duplicate to 96-well microplates. Microbial
suspensions were diluted in broth to afford desired colony forming
units/mL according to the 0.5 McFarland Standard [C. albicans:
either Saboraud Dextrose broth (SDB) or RPMI 1640, C. neoformans:
SDB, A. fumigatus: either YM broth (for MICs) or RPMI-1640 + 5%
Alamar Blue (for IC50 determination). After adding microbial
Nucleosil 100, C-18, 15 cm  4.6 cm, 5
l with C-18 guard cartridge,
injection volume—60 L, Gradient elution was employed using
l
mobile phases C, (90% 7.5 mM sodium heptanesulfonate, 0.1 M
H2PO4, adjusted to pH 3.00 with NEt3) + 10% CH3CN and D (100%
acetonitrile).50
Acknowledgments
cultures to the samples affording a final volume of 200
lL and final
test concentration starting with 20 g/mL, plates were read prior
l
We gratefully acknowledge research support from the National
Institutes of Health, National Institute of Allergy and Infectious
Diseases, through AREA Grant Number R15 Al37976-01, Research
Centers at Minority Institutions (RCMI) Grant Number G12 RR
to and after incubation using either fluorescence at 544ex/590em
(A. fumigatus) using the Polarstar Galaxy Plate Reader (BMG Lab-
Technologies, Germany) or optical density at 630 nm using the

470
C. A. Boateng et al. / Bioorg. Med. Chem. 19 (2011) 458–470
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03020, and Title III grant to Florida A&M University. This research
was supported in part by the Pharmaceutical Research Center NIH/
NCRR 1 C06-RR12512-01 grant. We also appreciate the generous
contributions of Dr. Wang Zhang, Senior Technician responsible
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NMR spectra of compounds in this paper and Ms. Marsha Wright
for antifungal testing. Antifungal testing was supported by the
NIH, NIAID, Division of AIDS, Grant No. AI 27094.
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