Design and synthesis of antifungal benzoheterocyclic derivatives by scaffold hopping

Article abstract of DOI:10.1016/j.ejmech.2011.01.075

The incidence of invasive fungal infections and associated mortality is increasing dramatically. Although azoles are first-line antifungal agents, cross-resistance and hepatic toxicity are their two major limitations. The discovery of novel non-azole lead compounds will be helpful to overcome these problems. On the basis of our previously reported benzopyran non-azole CYP51 inhibitor, scaffold hopping was used to design structurally diverse new compounds and expand the structure-activity relationships of the lead structure. Five kinds of scaffolds, namely benzimidazole, benzoxazole, benzothiazole, quinazolin-4-one and carboline, were chosen for synthesis. In vitro antifungal activity data and results from molecular docking revealed that the scaffold was important for the antifungal activity. Several compounds showed potent activity against both standard and clinically resistant fungal pathogens, suggesting that they can serve as a good starting point for the discovery of novel antifungal agents.

Full text of DOI:10.1016/j.ejmech.2011.01.075

European Journal of Medicinal Chemistry 46 (2011) 1706e1712
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of antifungal benzoheterocyclic derivatives
by scaffold hopping
1
1
Chunquan Sheng , Xiaoying Che , Wenya Wang, Shengzheng Wang, Yongbing Cao, Jianzhong Yao,
*
*
*
Zhenyuan Miao , Wannian Zhang
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 October 2010
Received in revised form
The incidence of invasive fungal infections and associated mortality is increasing dramatically. Although
azoles are first-line antifungal agents, cross-resistance and hepatic toxicity are their two major limita-
tions. The discovery of novel non-azole lead compounds will be helpful to overcome these problems. On
the basis of our previously reported benzopyran non-azole CYP51 inhibitor, scaffold hopping was used to
design structurally diverse new compounds and expand the structureeactivity relationships of the lead
structure. Five kinds of scaffolds, namely benzimidazole, benzoxazole, benzothiazole, quinazolin-4-one
and carboline, were chosen for synthesis. In vitro antifungal activity data and results from molecular
docking revealed that the scaffold was important for the antifungal activity. Several compounds showed
potent activity against both standard and clinically resistant fungal pathogens, suggesting that they can
serve as a good starting point for the discovery of novel antifungal agents.
13 January 2011
Accepted 17 January 2011
Available online 22 February 2011
Keywords:
Non-azole lead
CYP51
Scaffold hopping
Benzoheterocyclic derivatives antifungal
activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Molecular docking
1. Introduction
extensive use of azole antifungal agents has led to severe resistance,
which significantly limited the efficacy of them [6]. This situation
Systemic and invasive fungal infections are often life-threat-
ening and have become increasingly common in the immuno-
compromised hosts, such as patients undergoing anticancer
chemotherapy or organ transplants and patients with AIDS [1,2].
Azole antifungal agents were broadly used as the first-line drugs in
clinic because of their high therapeutic index. They act by
prompted us to search novel non-azole lead compounds with more
structural specificity for the fungal CYP51 enzymes and separate
their activity from toxicity.
In our previous studies, three-dimensional (3D) models of
CYP51was constructed using homology modeling [7e10]. Further-
more, we reported the first example of structure-based de novo
design of novel non-azole CYP51 inhibitors [11]. The benzopyran
inhibitors (Fig. 1) were bound to CYP51 mainly through non-bond
interactions without coordinating with the heme [11], which rep-
resenting a promising class of lead structures for the development
of selective antifungal agents without cross inhibiting other cyto-
chrome P450 proteins. Therefore, the extension of structur-
eeactivity relationships (SARs) of these CYP51 inhibitors is of great
importance. Herein, a series of benzoheterocyclic derivatives were
designed and synthesized by scaffold hopping. In vitro antifungal
activity assay indicated that several compounds showed improved
antifungal activity.
competitive inhibition of the lanosterol 14a-demethylase (CYP51),
a key enzyme in ergosterol biosynthesis pathway of fungal cell
membrane [3]. Azole antifungal agents inhibit CYP51 through the
formation of a coordination bond between the heterocyclic
nitrogen atom (N-3 of imidazole and N-4 of triazole) and the heme
iron atom in the active site of the enzyme [4]. However, the azole
pharmacophore is also responsible for the hepatotoxicity of azole
antifungal agents, because of the coordination binding of its
nitrogen atom to the heme of a lot of host cytochrome P450
enzymes (particularly mammalian CYP3A4) [5]. Moreover, the
*
Corresponding author. Tel./fax: þ86 21 81870243.
2. Chemistry
*
* Corresponding author. Tel./fax: þ86 21 81871233.
E-mail addresses: miaozhenyuan@hotmail.com (Z. Miao), zhangwnk@hotmail.
The synthetic route of target compounds was outlined in
Scheme 1 and Scheme 2. In general, the target compounds were
com (W. Zhang).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.075

C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
1707
OH
F
14aec were obtained by reacting 13 with various 1-bromoalkanes
N
N
ꢀ
in the presence of NaH and DMF at 80 C.
g
-Carboline derivatives
N
N
F
CH3
N
N
HO
O
17aee were prepared following the standard Fischer synthesis
from substituted phenylhydrazines 16aee and N-carbethoxy-
4-piperidinone [12,13]. Free NH of 16aee was treated with excess
iodomethane in the presence of KOH and DMF to give 18aee. Then,
the N-carbethoxy group of 18aee was removed by hydrolysis
reaction using KOH/EtOH condition. Finally, carbolines 19aee were
OH
Fluconazole
Non-azole CYP51 Inhibitor
substituted with various 1-bromoalkanes in the presence of K
and EtOH at 80 C to afford the title compounds 20aeo.
2
CO
3
ꢀ
Fig. 1. Chemical structures of representative CYP51 inhibitors.
obtained via a two-step strategy. First, the benzoheterocyclic scaf-
fold was constructed by various ring closure reactions. Then, the
alkyl side chains were substituted on the scaffold to afford the
target compounds. Benzene-1,2-diamine and ethyl chloroacetate
were condensed in dilute HCl solution to afford 2-(chloromethyl)-
3. Pharmacology
In vitro antifungal activity was measured according to the
National Committee for Clinical Laboratory Standards (NCCLS)
recommendations. The antifungal activity of the target compounds
was expressed as the minimal inhibitory concentration (MIC) that
achieved 80% inhibition of the tested fungi using fluconazole as
a reference drug. The MIC determination was performed by means
of the serial dilution method in 96-well microtest plates with RPMI
1640 (Sigma) buffered with 0.165 M MOPS (Sigma) as the test
medium. Fluconazole was used as the reference drug. Tested fungal
strains were obtained from the ATCC or were clinical isolates. The
MIC value was defined as the lowest concentration of test
compounds that resulted in a culture with turbidity less than or
equal to 80% inhibition when compared to the growth of the
control. Test compounds were dissolved in DMSO serially diluted in
1
2 3
H-benzimidazole (2). In the presence of K CO , scaffold 2 was
substituted by various 1-bromide alkylamines in MeOH at room
temperature to give compounds 3aec. 2-Chloroacetonitrile (4) was
transformed to ethyl 2-chloroacetimidate (5) by reacting with
ꢀ
diethyl ether in absolute EtOH at 0 C. Compound 5 was condensed
with 2-aminophenol and 2-aminobenzenethiol in CH
2 2
Cl to afford
2
-(chloromethyl)benzoxazole (6) and 2-(chloromethyl)benzothia-
zole (9), respectively. Target compounds 7aec and 10aec were
obtained by a similar procedure with 3aec. 2-amino-5-methyl-
benzoic acid (11) was treated with formic anhydride to afford
benzoxazin-4-one 12, which was subsequently reacted with
ꢀ
ꢀ
formamide at 150 C to give quinazolin-4-one 13. Compounds
growth medium. The yeasts were incubated at 35 C, and the
H
H
NH2
NH2
HN (CH ) CH3
2 n
a
N
b
N
CH Cl
2
N
N
3a n = 5
3b n = 6
3c n = 7
2
1
d
c
O
O
N
O
HN (CH2)nCH3
Cl
e
ClCH CN
2
CH Cl
2
NHHCl
N
4
5
6
7a n = 5
7
b n = 6
c n = 7
7
NH2
S
N
HN (CH ) CH3
2 n
f
S
g
CH Cl
2
SH
N
8
9
10a n = 5
1
0b n = 6
0c n = 7
1
O
O
O
H C
COOH
NH2
3
H C
3
h
H C
i
3
H C
3
j
(CH )nCH3
2
N
O
NH
N
N
13
N
1
1
12
14a n = 5
1
4b n = 6
4c n = 7
1
ꢀ
Scheme 1. Reagents and conditions: a. ethyl chloroacetate, dilute HCl solution, rtw110 C, 8h, 79.16%; b. 1-bromide alkylamine, K
2
3
CO , MeOH, rt, 10e30min, 10e20%; c. absolute
ꢀ
ꢀ
EtOH, HCl (g), diethyl ether, 0 C, 5.5h, 81.47%; d. 2-aminophenol, CH
2
Cl
2
, 0 Cwrt, 99.5%; e. 1-bromide alkylamine, K
, MeOH, rt, 10e30min, 15e20%; h. formic anhydride, reflux, 5h, 68.2%; i. formamide, 150 C, 7h, 75.5%; j. 1-bromoalkane, NaH,
2
CO
3
, MeOH, rt, 10e30min 1 0e25%; f. 2-aminobenzenethiol,
ꢀ
ꢀ
CH
2
Cl
2
, 0 Cw rt, 67.8%; g. 1-bromide alkylamine, K
2
CO
3
ꢀ
DMF, 80 C, 8h, 85%.

1708
C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
NH2
NHNH2
H
N
a
b
c
.
HCl
O
N
Et
R
R
R
O
15a-e
16a-e
17a-e
CH3
N
CH3
N
CH3
N
d
e
O
N
HN
N
Et
R
R
R
(
CH )nCH
3 2
O
1
1
1
1
1
9a R = F
9b R = Cl
9c R = Br
9d R = I
18a-e
20a-o
2
0a R = F, n = 5
0b R = F, n = 6
20c R = F, n = 7
20i R = Br, n = 7
2
20j R = I,
20k R = I,
20l R = I,
n = 5
n = 6
n = 7
9e R = CH3
2
2
2
2
2
0d R = Cl, n = 5
0e R = Cl, n = 6
0f R = Cl, n = 7
0g R = Br, n = 5
0h R = Br, n = 6
20m R = CH , n = 5
3
20n R = CH , n = 6
3
20o R = CH , n = 7
3
O, ꢁ5eꢁ20 ꢀC, 45 min, 50.5%e75.2%; b. ethyl 4-oxopiperidine-1-carboxylate, alchol, 80 C,
ꢀ
Scheme 2. Reagents and conditions: a. sodium nitrite, hydrochloric acid, water, SnCl
2
$2H
2
ꢀ
ꢀ
ꢀ
4h, 45%e50%; c. CH
3
I, KOH, DMF, 30 C, 10h, 13.3%e62.6%; d. KOH, alchol, water, 80 C, 10h, 49.4e81.8%; e. 1-bromoalkane, K
2
CO
3
, alcohol, 80 C, 14.2%e25%.
growth MIC was determined at 24 h for Candida species, at 72 h for
C. neoformans, and at 7 days for filamentous fungi.
Compounds 3b and 3c have longer alkyl side chain and their
activity against C. albicans was lower than that of 3a. Interestingly,
they showed improved activity against a fluconazole-resistant
4
. Results and discussion
strain with their MIC80 values of 32 mg/mL and 16 mg/mL, respec-
tively. Moreover, compound 3c showed broad antifungal spectrum.
It’s MIC80 value against C. neoformans, C. tropicalis, T. rubrum,
4.1. Design rationale
M. gypseum and A. fumigatus was 16
mg/mL. Especially, compound
Previous modeling studies revealed that the active site of
3c showed moderate activity against A. fumigatus (MIC80 ¼ 32
m
g/
CACYP51 could be divided into four pockets (S1-S4) [11]. S1
subsite is a hydrophilic hydrogen bond binding pocket, while S2
subsite is above the heme ring representing the core hydrophobic
area. S3 and S4 subsite represent a narrow and hydrophobic cleft
facing BC loop) and a hydrophobic hydrogen bond binding site
facing FG loop), respectively. The binding mode of the benzo-
pyran lead compound indicated that the core scaffold was located
in S2 subsite with its long alkyl side chain oriented into the
hydrophobic S3 subsite. The SARs of the substitutions on the
benzopyran ring and the length of alkyl side chain have been
investigated [11]. Interestingly, the replacement of the benzopyran
scaffold by the tetrahydroisoquinoline ring led to the increase of
the antifungal activity [14], which encouraged us to expand the
SARs of the scaffold. Scaffold hopping is a useful method to
discover structurally novel compounds by modifying the central
core structure of the known active compounds [15]. In consider-
ation of synthetic accessibility, we designed five kinds of scaffolds
mL), while fluconazole was inactive. Benzoxazole derivatives 7aec
revealed moderate to good activities against most of the tested
fungi with their MIC80 values in the range of 1
The activity of compound 7c against C. neoformans
(MIC80 ¼ 1 g/mL) and T. rubrum (MIC80 ¼ 4 g/mL) was compa-
rable to that of fluconazole. Moreover, it showed higher activity
g/mL) than fluconazole. When the
mg/mL to 64 mg/mL.
(
(
m
m
against M. gypseum (MIC80 ¼ 8
m
benzoxazole scaffold of 7aec was replaced by benzothiazole, the
antifungal activity was slightly decreased. The MIC80 range of
compounds 10aec is 16 mg/mL to 32 mg/mL. The loss of antifungal
activity was observed for quinazolinone derivatives 14aec. Only
compound 14b showed moderate activity against A. fumigatus
(MIC80 ¼ 64
mg/mL). For carboline derivatives 20aeo, 8-F (20aec),
8-Cl (20def), 8-Br (20gei) and 8-I (20jel) analogs only showed
marginal antifungal activities. On the other hand, the antifungal
activity of 8-methyl derivatives 20meo was improved. They
showed broad spectrum with MIC80 values in the range of 16
to 64 g/mL. Especially, compounds 8n and 8o revealed the highest
activity against A. fumigatus (MIC80 ¼ 16 g/mL).
mg/mL
(
i.e. benzimidazole, benzoxazole, benzothiazole, quinazolin-4-one
m
and carboline) for synthesis (Fig. 2). Based on our previous SAR
results, the length of side chains was set to 6 to 8 carbon atoms. As
a result, a series of benzoheterocyclic derivatives with long alkyl
side chains were synthesized.
m
From the antifungal activity data, preliminary SARs of the
synthesized compounds are obtained as follows. In general, the
chemical scaffold of the designed compounds is important for their
antifungal activity. Benzimidazole and benzoxazole are found to be
favorable for the antifungal activity. Benzimidazole derivative 3a
showed the highest activity against C. albicans. On the C. neoformans
strain, benzoxazole derivative 7c is the most active. The effect of the
length of alkyl side chain on the antifungal activity is not obvious.
The introduction of quinazolinone scaffold leads to the loss of anti-
fungal activity. For the carboline derivatives, the substitutions on the
C-8 position phenyl ring play an important role for their antifungal
4
.2. In vitro antifungal activities and SARs
The antifungal activity of the target compounds was shown in
Table 1 and Table 2. Benzimidazole derivative 3a showed potent
g/mL), which was compa-
activity against C. albicans (MIC80 ¼ 1
m
rable to fluconazole. However, its antifungal spectrum was narrow
because it was inactive against other tested pathogenic fungi.

C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
1709
Fig. 2. Scaffold hopping of benzopyran non-azole CYP51 inhibitors.
activity. Most of the halogen substituted compounds are found
to be inactive. Among them, only iodine derivative 20l showed
marginal activity. The methyl group seems to be the most favorable.
Compounds 20meo show improved antifungal activity and
spectrum.
interactions with Phe228, Met306, Leu376 and Val509. Among the
designed scaffolds, only benzimidazole has a free NH group to form
hydrogen-bonding interaction with Met306. The replacement of
benzimidazole by other benzoheterocyclic led to the decreased
activity against C. albicans. Therefore, the hydrogen bonding
interaction with CACYP51 might important for the antifungal
activity. On the other hand, the scaffold of the designed compounds
was located in a hydrophobic environment. Hydrophobic substi-
tutions on the scaffold could improve the binding affinity with
CACYP51. The hypothesis was supported by the SAR of the carboline
4.3. Binding mode of the compounds
In an attempt to understand the binding mode of the designed
compounds, representative active derivative 3a was docked into
the active site of CACYP51 using the Affinity module within InsightII
2
000 software package [16]. As shown in Fig. 3, the benzimidazole
core of compound 3a was located above the heme ring and formed
hydrophobic interactions with the surrounding residues lined with
Phe145, Leu139, Ile304 and Phe145. The imidazole NH formed
a hydrogen bond with backbone carbonyl oxygen atom of Met306.
The long alkyl side chain formed hydrophobic and van der Waals
Table 2
g mL 1).a
ꢁ
In vitro antifungal activities of the compounds (MIC80
,
m
Compd.
C. alb
C. par
C. kru
C. tro
C. neo
A. fum
14a
14b
14c
20a
>64
>64
>64
>64
64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
64
>64
>64
64
>64
64
>64
>64
64
>64
>64
>64
>64
>64
64
Table 1
20b
20c
ꢁ
In vitro antifungal activities of the compounds (MIC80
,
m
g$mL 1).a
64
64
2
2
0d
0e
>64
>64
>64
>64
>64
64
>64
>64
64
64
16
16
1
>64
>64
64
>64
64
16
64
64
16
64
16
16
1
Compd.
C. alb
C. neo
C. tro
T. rub
A. fum
M. gyp
C. albb
>64
64
3
3
3
7
7
7
1
1
1
a
b
c
a
b
c
0a
0b
0c
1
>64
>64
64
64
64
64
64
64
1
>64
64
16
64
16
1
64
64
16
1
>64
>64
16
64
64
16
64
64
64
1
64
64
16
16
16
4
64
16
16
4
>64
>64
32
>64
64
>64
>64
16
16
16
8
64
16
64
16
>64
32
16
>64
64
64
64
32
>64
e
20f
20g
20h
20i
20j
20k
20l
20m
20n
20o
FLZ
>64
>64
>64
>64
>64
64
16
64
64
0.5
64
>64
>64
>64
64
>64
>64
1
32
>64
>64
>64
>64
64
64
16
16
>64
FLZ
1
a
Abbreviations: C. alb. Candida albicans; C. neo. Cryptococcus neoformans; C. tro.
Candida tropicalis; T. rub. Trichophyton rubrum; A. fum. Aspergillus fumigatus; M. gyp.
Microsporum gypseum; FLZ: Fluconazole.
a
Abbreviations: C. alb. Candida albicans; C. neo. Cryptococcus neoformans; C. tro.
Candida tropicalis; T. rub. Trichophyton rubrum; A. fum. Aspergillus fumigatus;
M. gyp. Microsporum gypseum; FLZ: Fluconazole.
b
Clinical isoate resistant to FLZ.

1710
C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
Fig. 3. The binding mode of compound 3a with the active site of CACYP51. Important residues involved in inhibitor binding are shown in stick mode and the hydrogen bond was
depicted as red dotted line. The heme atoms are colored purple, the nitrogen atoms are colored blue, the oxygen atoms are colored red, and the carbon atoms are colored gray
(
CYP51) and cyan (inhibitor). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compounds 20aeo. The methyl substituted derivatives showed
better activity against C. albicans than the corresponding halogen
substituted compounds.
a Kratos-concept mass spectrometer under electron impact ioni-
zation (EI) conditions. TLC analysis was carried out on silica gel
plates GF254 (Qindao Haiyang Chemical, China). Silica gel column
chromatography was performed with Silica gel 60 G (Qindao
Haiyang Chemical, China). Commercial solvents were used without
any pretreatment.
5
. Conclusion
Cross-resistance and hepatic toxicity are two major limitations
for azole antifungal agents. The discovery of novel non-azole CYP51
inhibitors is great significance to overcome these problems. Staring
from our previously designed non-azole lead structure, scaffold
hopping was used to design structurally diverse antifungal benzo-
heterocyclic derivatives. SAR of the lead structure was further
extended. Moreover, the synthesized non-azole antifungal
compounds have several advantages. (1) They bound with CYP51
through non-bond interactions without coordination to the iron
atom, which is helpful to reduce the toxicity of azoles due to cross
inhibition of other cytochrome P450 proteins. (2) As compared with
the lead structure, several compounds (e.g. 3a and 7c) showed
improved antifungal activity, which can serve as a good starting
point for further optimization. (3) Several compounds were active
against fluconazole-resistant clinical isolates, which affords an
opportunity to overcome the emerging severe resistance of azoles.
Further structural optimization studies are in progress.
6.1.1. Chemical synthesis of 2-(chloromethyl)-1H-benzimidazole (2)
A solution of benzene-1,2-diamine (10.8 g, 0.10 mol) and ethyl
chloroacetate (15.9 g, 0.13 mol) in dilute HCl solution (4 M, 90 mL)
was stirred at room temperature for 4 h. Subsequently, the reaction
ꢀ
mixture was heated to 110 C and refluxed for 4 h. Then, the
resulting solution was poured into ice water (250 mL), and the pH
value was adjusted to 9 by ammonia. The solid was filtrated and
washed by water to afford intermediate 2 as pale yellow solid
(13.22 g, yield 79.16%), which can be used directly in the next step
1
without purification. H NMR (DMSO, 500 MHz, ppm):
d 7.56 (m,
2H), 7.21 (m, 2H), 4.94 (s, 2H).
6.1.2. Chemical synthesis of N-((1H-benzoimidazol-2-yl)methyl)
hexan-1-amine (3a)
A solution of compound 2 (1.66 g, 0.01 mol) in MeOH (25 mL)
was added dropwise to the suspension of hexan-1-amine (3.04 g,
2 3
0.03 mol) and anhydrous K CO (2.76 g, 0.02 mol) in MeOH (10 mL).
6
. Experimental protocols
The mixture was stirred at room temperature for 0.5 h. The solvent
was evaporated under reduced pressure. The residue was purified
6
.1. General procedure for the synthesis of compounds
by column chromatography (CH
2
Cl
yellow oil (0.28 g, 12.1%). H NMR (500 MHz, DMSO, TMS):
(w, 1H), 7.10e7.50 (m, 4H), 3.90 (s, 2H), 2.52 (m, 2H), 1.43 (m, 2H),
2
: MeOH 100:2, v/v) to give 3a as
1
d11.82
Nuclear magnetic resonance (NMR) spectra were recorded on
13
a Bruker 500 spectrometer with TMS as an internal standard and
CDCl
as solvent. Chemical shifts ( values) and coupling constants
J values) are given in ppm and Hz, respectively. ESI mass spectra
1.24 (m, 6H), 0.94 (t, 3H, J ¼ 7.0 Hz). C NMR (500 MHz, CDCl
3
,
3
d
TMS): 147.32, 138.22, 122.96, 115.19, 47.97, 45.35, 31.17, 27.20,
d
(
26.37, 22.40, 13.83. MS (ESI) m/z: 232 (M þ 1).
were performed on an API-3000 LCeMS spectrometer. High-reso-
lution mass spectrometry measurements were performed on
The synthetic procedure for the target compounds 3bec, 7aec
and 10aec were similar to the synthesis of compound 3a.

C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
1711
6
.1.3. Chemical synthesis of ethyl 8-bromo-3,4-dihydro-1H-pyrido
6.1.9. N-(benzooxazol-2-ylmethyl)hexan-1-amine (7a)
1
[4,3-b]indole ꢁ2(5H)-carboxylate (17c)
H NMR (500 MHz, DMSO, TMS):
d6.76e6.91 (m, 4H), 4.38 (s,
A solution of 4-bromophenylhydrazine hydrochloride (4.47 g,
.02 mol) and 1-carbethoxy-4-piperidone (3.42 g, 0.02 mol) in
2H), 3.28 (m, 2H), 1.52 (m, 2H), 1.29 (m, 6H), 0.87 (t, 3H,
J ¼ 6.9 Hz).
0
EtOH (25 mL) was refluxed for 3 h. The reaction mixture was
allowed to stand at room temperature overnight, and the solid
product was collected by filtration, washed with 50% aqueous EtOH,
6.1.10. N-(benzooxazol-2-ylmethyl)heptan-1-amine (7b)
1
H NMR (500 MHz, DMSO, TMS): d6.76e6.91 (m, 4H), 4.38 (s,
and recrystallized from 95% EtOH to give 17c as pale yellow solid
2H), 3.28 (m, 2H), 1.52 (m, 2H), 1.29 (m, 8H), 0.87 (t, 3H, J ¼ 6.9 Hz).
1
(
(
2
3.09 g, yield 47.5%). H NMR (300 MHz, CDCl
3
, TMS):
d
7.27e7.88
MS (ESI) m/z: 247 (M þ 1).
m, 3H), 4.66 (s, 2H), 4.21 (q, J ¼ 7.2 Hz, 2H), 3.87 (br, 2H), 2.86 (br,
H), 1.31 (t, J ¼ 7.2 Hz, 3H). MS (ESI) m/z: 323 (M).
6.1.11. N-(benzooxazol-2-ylmethyl)octan-1-amine (7c)
1
H NMR (500 MHz, DMSO, TMS): d6.76e6.91 (m, 4H), 4.38 (s,
6
1
.1.4. Chemical synthesis of ethyl 8-bromo-5-methyl-3,4-dihydro-
H-pyrido[4,3-b] indole-2(5H)-carboxylate (18c)
2
H), 3.28 (m, 2H), 1.52 (m, 2H), 1.29 (m, 10H), 0.86 (t, 3H, J ¼ 6.9 Hz).
13
C NMR (500 MHz, CDCl
3
, TMS): d154.06, 145.45, 135.43, 124.17,
3
KOH (2.02 g, 0.036 mol) and CH I (7.10 g, 0.09 mol) was added to
a solution of compound 11c (2.96 g, 0.009 mol) in DMF (50 mL). The
reaction mixture was stirred at room temperature for 12 h, then
123.20, 122.65, 115.19, 67.33, 63.06, 40.82, 31.77, 29.78, 29.25, 26.97,
2
6
2
2.61, 14.05. MS (ESI) m/z: 261 (M þ 1).
diluted with H
2
O (100 mL) and extracted with ethyl acetate
50 mL ꢂ 3). The combined organic layers were washed with H
SO , and filtrated, and the
.1.12. N-(benzothiazol-2-ylmethyl)hexan-1-amine (10a)
(
(
2
O
1
H NMR (500 MHz, DMSO, TMS): d7.36e8.05 (m, 4H), 4.08 (s,
100 mL ꢂ 3), dried over anhydrous Na
2
4
H), 2.88 (s, 1H), 2.59 (t, 2H), 1.45 (m, 2H), 1.29 (m, 6H), 0.86 (t, 2H,
solvent was evaporated under reduced pressure. The residue was
J ¼ 7.0 Hz). MS (ESI) m/z: 249 (M þ 1).
purified by silica gel column chromatography (hexane: EtOAc ¼ 3:1,
1
v/v) to give 18c as pale yellow solid (0.41 g, yield 13.3%). H NMR
6
.1.13. N-(benzothiazol-2-ylmethyl)heptan-1-amine (10b)
(
2
1
300 MHz, DMSO, TMS):
H, J ¼ 6.9 Hz), 3.89 (br, 2H), 3.63 (s, 3H), 2.83 (m, 2H),
¼ 7.2 Hz, J
.31 (t, 3H, J ¼ 7.0 Hz).
d
7.14e7.59 (m, 3H), 4.65 (s, 2H), 4.20 (dd,
1
H NMR (500 MHz, DMSO, TMS): d7.37e8.05 (m, 4H), 4.08 (s,
1
2
2
0
H), 2.76 (s, 1H), 2.59 (t, 2H, J ¼ 7.0 Hz), 1.46 (m, 2H), 1.29 (m, 8H),
.85 (t, 3H, J ¼ 6.8 Hz). MS (ESI) m/z: 263 (M þ 1).
6.1.5. Chemical synthesis of 8-bromo-2-hexyl-5-methyl-2,3,4,5-
6
.1.14. N-(benzothiazol-2-ylmethyl)octan-1-amine (10c)
tetrahydro-1H- pyrido[4,3-b]indole (20g)
1
H NMR (500 MHz, DMSO, TMS): d7.37e8.05 (m, 4H), 4.08 (s,
A solution of compound 18c (0.07 g, 0.26 mmol) in EtOH (10 mL)
2
H), 2.59 (t, 2H, J ¼ 7.0 Hz), 1.44 (m, 2H), 1.29 (m, 10H), 0.85 (t, 3H,
was added 1-bromohexane (0.17 g, 1.06 mmol) and anhydrous
ꢀ
J ¼ 6.9 Hz). MS (ESI) m/z: 277 (M þ 1).
K
2
CO
3
(0.15 g, 1.05 mmol). The reaction mixture was fluxed at 80 C
for 8 h. After filtration, the solvent was evaporated under reduced
pressure. The residue was purified by column chromatography
6
.1.15. 3-Heptyl-6-methylquinazolin-4(3H)-one (14b)
1
3
H NMR (500 MHz, CDCl , TMS): d8.11 (s, 1H), 7.56e7.98 (m, 3H),
(
2
2
2 2
CH Cl : MeOH 100:2, v/v) to give 20g as brown oil (0.02 g, yield
1
3.99 (t, 2H, J ¼ 7.4 Hz), 2.50 (s, 3H),1.78 (m, 2H),1.35 (m, 8H), 0.89 (t,
2.2%). H NMR (500 MHz, CDCl
3
, TMS): d7.09e7.52 (m, 3H), 3.64 (s,
3
6
3
H, J ¼ 7.0 Hz). MS (ESI) m/z: 259 (M þ 1).
H), 3.58 (s, 3H), 2.87 (m, 2H), 2.83 (m, 2H), 2.59 (t, 2H, J ¼ 7.7 Hz),
13
1
(
1
.62 (m, 2H), 1.34 (m, 6H), 0.90 (t, 3H, J ¼ 6.8 Hz). C NMR
500 MHz, CDCl , TMS): 135.8, 135.4, 127.4, 123.2, 120.2, 112.1,
10.0, 107.6, 58.1, 50.6, 49.5, 31.8, 29.2, 27.6, 27.3, 22.9, 22.6, 14.0.
The synthetic methods for the target compounds 20aeo were
similar to the above procedure.
.1.16. 3-Octyl-6-methylquinazolin-4(3H)-one (14c)
3
d
1
3
H NMR (500 MHz, CDCl , TMS): d8.10 (s, 1H), 7.56e7.98 (m,
H), 3.99 (t, 2H, J ¼ 7.4 Hz), 2.50 (s, 3H), 1.78 (m, 2H), 1.34 (m,
13
1
d
0H), 0.89 (t, 3H, J ¼ 7.0 Hz). C NMR (500 MHz, CDCl
161.03, 146.13, 145.79, 137.39, 135.50, 127.18, 126.08, 121.91,
46.99, 31.69, 29.38, 29.09, 26.64, 22.56, 21.08, 14.00. MS (ESI) m/z:
73 (Mþ1).
3
, TMS):
6
(
.1.6. Chemical synthesis of 3-hexyl-6-methylquinazolin-4(3H)-one
14a)
NaH (60% oil, 0.8 g, 0.02 mol) was suspended to a solution of
2
intermediate 13 (0.16 g, 0.01 mol) in DMF (10 mL), and the mixture
was stirred at room temperature for 2 h. Then, 1-bromohexane
6.1.17. 8-Fluoro-2-hexyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido
[4,3-b]indole (20a)
1
(
8
0.17 g, 0.01 mol) was added, the resulting mixture was heated to
0 C and stirred for 9 h. After filtration, the solvent was evaporated
3
H NMR (500 MHz, CDCl , TMS): d6.86e7.25 (m, 3H), 3.74 (s,
ꢀ
2H), 3.60 (s, 3H), 2.95 (m, 2H), 2.87 (m, 2H), 2.65 (t, 2H, J ¼ 7.6 Hz),
1.66 (m, 2H), 1.34 (m, 6H), 0.89 (t, 3H, J ¼ 7.0 Hz). MS (ESI) m/z: 290
(M).
under reduced pressure and the residue was purified by column
chromatography (hexane: EtOAc ¼ 4:1, v/v) to give 14a as white
1
solid (0.18 g, yield 75.0%). H NMR (500 MHz, CDCl
3
, TMS):
d8.11 (s,
1
2
H), 7.56e8.02 (m, 3H), 4.00 (t, 2H, J ¼ 7.4 Hz), 2.50 (s, 3H), 1.78 (m,
6.1.18. 8-Fluoro-2-heptyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido
H), 1.35 (m, 6H), 0.89 (t, 3H, J ¼ 7.0 Hz). MS (ESI) m/z: 245(M þ 1).
[4,3-b]indole (20b)
1
The synthetic procedure for the target compounds 14bec was
H NMR (500 MHz, CDCl , TMS): d6.87e7.15 (m, 3H), 3.64 (s,
3
similar to the synthesis of compound 14a.
2H), 3.60 (s, 3H), 2.88 (m, 2H), 2.85 (m, 2H), 2.60 (t, 2H, J ¼ 7.7 Hz),
.63 (m, 2H),1.29e1.35 (m, 8H), 0.89 (t, 3H, J ¼ 7.0 Hz). MS (ESI) m/z:
303 (M þ 1).
1
6.1.7. N-((1H-benzoimidazol-2-yl)methyl)heptan-1-amine (3b)
1
H NMR (500 MHz, DMSO, TMS):
d6.75e6.91 (m, 4H), 4.38 (s,
2
H), 3.28 (m, 2H), 1.51 (m, 2H), 1.30 (m, 8H), 0.87 (t, 3H, J ¼ 6.90 Hz).
6
.1.19. 8-Fluoro-5-methyl-2-octyl-2,3,4,5-tetrahydro-1H-pyrido
MS (ESI) m/z: 246 (M þ 1).
[4,3-b]indole (20c)
1
6.1.8. N-((1H-benzoimidazol-2-yl)methyl)octan-1-amine (3c)
3
H NMR (500 MHz, CDCl , TMS): d6.86e7.14 (m, 3H), 3.64 (s,
1
H NMR (500 MHz, DMSO, TMS):
d
12.22 (w, 1H), 7.10e7.48 (m,
2H), 3.59 (s, 3H), 2.92 (m, 2H), 2.83 (m, 2H), 2.59 (t, 2H, J ¼ 7.7 Hz),
1.62 (m, 2H), 1.27e1.34 (m, 10H), 0.89 (t, 3H, J ¼ 7.0 Hz). MS (ESI)
m/z: 319 (M þ 1).
4
3
H), 3.88 (s, 2H), 2.50 (m, 2H), 1.43 (m, 2H), 1.26 (m, 10H,), 0.85 (t,
H, J ¼ 6.9 Hz). MS (ESI) m/z: 260 (M þ 1).

1712
C. Sheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1706e1712
6
.1.20. 8-Chloro-2-hexyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido
6.1.29. 2-Heptyl-5,8-dimethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]
indole (20n)
1H NMR (500 MHz, CDCl
, TMS): d6.96e7.19 (m, 3H), 3.66 (s,
3
[4,3-b]indole (20d)
1
H NMR (500 MHz, CDCl
3
, TMS):
d
7.07e7.36 (m, 3H), 3.65
(
s, 2H), 3.59 (s, 3H), 2.89 (m, 2H), 2.84 (m, 2H), 2.60 (t, 2H,
2H), 3.58 (s, 3H), 2.90 (m, 2H), 2.83 (m, 2H), 2.61 (t, 2H, J ¼ 7.8 Hz),
J ¼ 7.7 Hz), 1.63 (m, 2H), 1.35 (m, 6H), 0.90 (t, 3H, J ¼ 6.7 Hz).
2.43 (s, 3H), 1.63 (m, 2H), 1.29e1.35 (m, 8H), 0.89 (t, 3H, J ¼ 7.0 Hz).
6.1.21. 8-Chloro-2-heptyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido
6.1.30. 2-Octyl-5,8-dimethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]
[
4,3-b]indole (20e)
indole (20o)
1
1H NMR (500 MHz, CDCl
H NMR (500 MHz, CDCl
3
, TMS):
d
7.06e7.36 (m, 3H), 3.65
3
, TMS):
d6.95e7.19 (m, 3H), 3.68 (s, 2H),
(
s, 2H), 3.58 (s, 3H), 2.88 (m, 2H), 2.83 (m, 2H), 2.60 (t, 2H,
3.57 (s, 3H), 2.88 (m, 2H), 2.82 (m, 2H), 2.60 (t, 2H, J ¼ 7.7 Hz), 2.43 (s,
13
J ¼ 7.7 Hz), 1.62 (m, 2H), 1.30e1.35 (m, 8H), 0.89 (t, 3H, J ¼ 6.8 Hz).
3H, CH
NMR (500 MHz, CDCl
108.3,107.2, 58.1, 50.9, 49.7, 31.8, 29.6, 29.3, 29.0, 27.7, 22.8, 22.6,14.1.
3
),1.63 (m, 2H),1.28e1.34 (m,10H), 0.89 (t, 3H, J ¼ 6.9 Hz).
C
3
, TMS): 135.5,133.9,127.8,125.9, 122.0, 117.3,
d
6
.1.22. 8-Chloro-5-methyl-2-octyl-2,3,4,5-tetrahydro-1H-pyrido
[
4,3-b]indole (20f)
1
3
H NMR (500 MHz, CDCl , TMS): d7.07e7.36 (m, 3H), 3.65
6.2. Flexible molecular docking
(
s, 2H), 3.59 (s, 3H), 2.88 (m, 2H), 2.83 (m, 2H), 2.60 (t, 2H,
J ¼ 7.7 Hz), 1.62 (m, 2H), 1.29e1.34 (m, 10H), 0.89 (t, 3H, J ¼ 6.9 Hz).
The 3D structure of CACYP51 was obtained by homology
modeling [9]. Flexible ligand docking procedure in the Affinity
module within InsightII was used to define the lowest energy
position for the substrate using a Monte Carlo docking protocol. The
detailed docking parameters were from our previous studies [17].
6.1.23. 8-Bromo-2-heptyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido
[
4,3-b]indole (20h)
1
3
H NMR (500 MHz, CDCl , TMS): d7.09e7.52 (m, 3H), 3.64
(
s, 2H), 3.58 (s, 3H), 2.83 (m, 2H), 2.88 (m, 2H), 2.59 (t, 2H,
J ¼ 7.8 Hz), 1.63 (m, 2H), 1.26e1.35 (m, 8H), 0.89 (t, 3H, J ¼ 6.9 Hz).
Acknowledgment
6
.1.24. 8-Bromo-5-methyl-2-octyl-2,3,4,5-tetrahydro-1H-pyrido
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 30930107, 30973640), Shanghai
Rising-Star Program (Grant Nos. 09QA1407000) and Shanghai
Leading Academic Discipline Project (Project Nos. B906).
[4,3-b]indole (20i)
1
3
H NMR (500 MHz, CDCl , TMS): d7.09e7.52 (m, 3H), 3.64
(
s, 2H), 3.58 (s, 3H), 2.88 (m, 2H), 2.83 (m, 2H), 2.59 (t, 2H,
J ¼ 7.7 Hz), 1.62 (m, 2H), 1.29e1.34 (m, 10H), 0.89 (t, 3H, J ¼ 6.8 Hz).
References
6.1.25. 8-Iodo-2-hexyl-5-methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-
b]indole (20j)
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1
3
H NMR (500 MHz, CDCl , TMS): d7.01e7.72 (m, 3H), 3.65 (s, 2H),
3
.58 (s, 3H), 2.89 (m, 2H), 2.84 (m, 2H), 2.60 (t, 2H, J ¼ 7.7 Hz), 1.63
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6.1.27. 8-Iodo-5-methyl-2-octyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-
b]indole (20l)
1H NMR (500 MHz, CDCl
3
, TMS): d7.00e7.72 (m, 3H), 3.63
[
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(
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J ¼ 7.7 Hz), 1.62 (m, 2H), 1.28e1.33 (m, 10H), 0.89 (t, 3H,
13
J ¼ 7.0 Hz). C NMR (500 MHz, CDCl
3
, TMS): d136.2, 135.0, 128.7,
[
128.2, 126.4, 110.6, 107.4, 82.1, 58.1, 50.7, 49.4, 31.8, 29.6, 29.2, 27.6,
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2.8, 22.6, 14.1.
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indole (20m)
[15] H. Zhao, Drug Discov. Today 12 (2007) 149e155.
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2121-3752, 1999.
1H NMR (500 MHz, CDCl
3
, TMS): d6.96e7.19 (m, 3H), 3.95 (s, 2H),
9
3
.58 (s, 3H), 2.88 (m, 2H), 2.83 (m, 2H), 2.60 (t, 2H, J ¼ 7.8 Hz), 2.43
[17] C. Sheng, W. Zhang, H. Ji, M. Zhang, Y. Song, H. Xu, J. Zhu, Z. Miao, Q. Jiang,
(
s, 3H), 1.63 (m, 2H), 1.32 (m, 6H), 0.90 (t, 3H, J ¼ 6.9 Hz).
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