Synthesis and antifungal activities in vitro of novel pyrazino [2,1-a] isoquinolin derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.12.050

A series of novel pyrazino[2,1-a]isoquinolin compounds were designed and synthesized, and their antifungal activities in vitro were evaluated. The results showed that all of the compounds exhibited antifungal activities. Some of them exhibited stronger an

Full text of DOI:10.1016/j.bmcl.2009.12.050

Bioorganic & Medicinal Chemistry Letters 20 (2010) 979–982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activities in vitro of novel pyrazino [2,1-a]
isoquinolin derivatives
Hui Tang a, Canhui Zheng a, Jiaguo Lv a, Juan Wu b, Yanan Li a, Hui Yang a, Bingyue Fu a, Chuntong Li a,
a,
Youjun Zhou a, , Ju Zhu
*
*
a Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Department of Pharmacy, Chengdu General Hospital, Chengdu Military Command Area, Chengdu 610083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrazino[2,1-a]isoquinolin compounds were designed and synthesized, and their anti-
fungal activities in vitro were evaluated. The results showed that all of the compounds exhibited anti-
fungal activities. Some of them exhibited stronger antifungal activities than that of lead compounds
and among them compound 11b was the most potent one, which showed more potent than that of
the active control fluconazole to the four of the five tested fungi. The studies presented here provide a
new structural type for the development of novel antifungal agents.
Received 4 October 2009
Revised 24 November 2009
Accepted 14 December 2009
Available online 21 December 2009
Keywords:
Antifungal
Ó 2009 Elsevier Ltd. All rights reserved.
Pyrazino[2,1-a]isoquinoline
Lanosterol 14
Synthesis
a-demethylase(CYP51)
Over the past three decades, the incidence of systemic fungal
infections has increased dramatically due to an increase in the
number of immunocompromised hosts.1–4 Although the arsenal
of antifungal drugs has expanded, currently available antifungal
drugs do not meet the increasing requirements of managing infec-
tion in the complex patient populations. The development of new
antifungal drugs has been constantly required in the clinical
therapy.
Lanosterol 14a-demethylase (CYP51) is one of the key enzymes
of sterol biosynthesis in fungi and also a prime target for the devel-
opment of antifungal drugs. Azole antifungals, such as fluconazole,
itraconzole, and voriconazole (Fig. 1), are the mainly CYP51 inhib-
itors used in the clinical antifungal therapy,5,6 which exert
antifungal activity through inhibiting the lanosterol 14a-demeth-
ylase (CYP51) of fungi by a mechanism in which the heterocyclic
nitrogen atom (N-3 of imidazole and N-4 of 1,2,4-triazole) binds
to the sixth coordination position of the heme iron atom of the
prophyrin in the substrate-binding site of the enzyme.7 Extensive
use and prolonged therapy with azole antifungals have led to resis-
tance. The search of new non-azole CYP51 inhibitors is meaningful.
We have focused on the search of non-azole CYP51 inhibitors
for several years. In the previous study,8–10 we reported the design
of non-azole lead molecules with a tetrahydroisoquinoline scaffold
based on the constructed three-dimensional model of Candida albi-
Figure 1. Chemical structures of fluconazole, voriconazole, lanosterol, and lead
compound L-6.
cans CYP51 by coupling structure-based de novo design. The bind-
ing study showed that tetrahydroisoquinoline scaffold was located
on the substrate-binding site of fungal CYP51, and affinity of the
lead molecule for CYP51 was mainly attributed to their nonbond-
ing interaction with the residues of apoprotein but without binding
with the heme, which was different from that of azole antifungals.
L-6 was the most potent compound discovered (Fig. 1). Lansterol is
the natural substrate for CYP51 with a skeleton of four rings in its
structure, and located on the same substrate-binding site of CYP51
with the tetrahydroisoquinoline compounds. Considering that the
* Corresponding authors. Tel.: +86 021 81871231 (Y.Z.).
E-mail addresses: zhuoyoujun2006@yahoo.com.cn (Y. Zhou), zhuju@smmu.e-
du.cn (J. Zhu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.050

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H. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 979–982
Table 1
In vitro antifungal activity MIC80 (l
g/ml)a
Compound
C. alb.
C. neo.
T. rub
M. gyp
A. fum
5a
5b
6a
6b
7a
8a
8b
9a
>64
>64
>64
64
>64
16
>64
32
16
16
32
>64
>64
4
8
16
8
>64
64
>64
>64
>64
32
16
16
8
>64
>64
>64
4
>64
8
16
4
4
>64
>64
>64
>64
>64
64
32
8
32
Figure 2. Chemical structures of the target compounds.
tetrahydroisoquinoline scaffold is smaller in the active site than
that of lansterol, the possible increase of affinity with CYP51 and
antifungal activities of target compounds is expected by expanding
the tetrahydroisoquinoline scaffold of lead molecules. On the basis
of the above hypothesis, the novel pyrazino [2,1-a] isoquinolin
derivatives were designed (Fig. 2), and their antifungal activities
in vitro were evaluated.
The chemical synthesis of pyrazino [2,1-a] isoquinolin deriva-
tives was outlined in Scheme 1. As a key intermediate of our de-
signed compounds, 4a–4b was synthesized respectively
according to the paper.11,12 Intermediate 4a–4b was allowed to re-
act with different alkyl halides in the presence of potassium car-
bonate in ethanol at 80 °C, and then the hydrogen chloride gas
was added to form compounds 5a–5b, 8a–8b, 11a–11b, and
14a–14b. Reduction of 5a–5b, 8a–8b, 11a–11b, and 14a–14b with
lithium aluminum hydride in THF yields the target compounds 6a–
6b, 9a–9b, 12a–12b, and 15a–15b. Cleavage of the methoxy groups
of 6a, 9a, 12a, 15a in HBr/CH3COOH provided N-substituted-
1,2,3,6,7,11b-hexahydro-4H-pyrazino [2,1-a] isoquinoline-9,10-
diol hydrobromide 7a, 10a, 13a, 16a. All the target compounds
were obtained as racemates.
The in vitro minimal inhibitory concentrations (MIC80) of the
compounds were determined by the micro-broth dilution method
in 96-well microtest plates according to the methods defined by
the National Committee for Clinical Laboratory Standards
(NCCLS).13 The tested fungi species included five pathogenic fungi,
which were found in dermatomycoses (Trichophyton rubrum, and
Microsporum gypseum) and systemic mycoses (Candida albicans,
Cryptococcus neoformans, and Aspergillus fumigatus). The in vitro
antifungal activities of all the title compounds were listed in
Table 1, in which fluconazole and L-6 were used as the controls.
In general, all the target compounds showed potent activities
9b
4
10a
11a
11b
12a
12b
13a
14a
14b
15a
15b
16a
L-6
FCZ
16
64
32
32
8
8
>64
>64
>64
>64
64
2
4
2
4
4
4
32
>64
16
64
16
16
4
16
32
4
8
8
32
2
0.5
2
2
8
64
64
32
4
16
16
2
64
32
16
8
16
64
>64
32
>64
>64
64
8
>64
64
>64
64
16
32
32
64
2
64
>64
a
Abbreviations: C. alb., Candida albicans; C. neo., Cryptococcus neoformans; T. rub.,
Trichophyton rubrum; M. gyp., Microsporum gypseum; A. fum., Aspergillus fumigatus;
FCZ: Fluconazole.
against most the test fungal pathogens. The MIC80 values indicated
that the compounds 8a, 9a, 9b, 11b, 12a–12b, and 13a showed
more excellent antifungal activities against four pathogenic fungi
than that of L-6. Noticeably, the MIC80 value of compounds 11b,
12a and 12b showed higher activities against nearly all the test
fungi except Candida albicans than that of fluconazole. Cryptococcus
neoformans has a worldwide distribution and is the most common
cause of life-threatening fungal infections. Compounds 7a, 9b, 10a,
11a–11b, and 13a exhibited comparable or stronger inhibitory
activities against Cryptococcus neoformans than fluconazole. Fluco-
nazole is not effective against Aspergillus fumigatus, while most of
our compounds showed potent activities. For example, the MIC80
values of compounds 9a and 12a against Aspergillus fumigatus are
8
lg/mL. On the Trichophyton rubrum strains, compound 9b, 11b,
12a–12b, and 13a showed better activities than fluconazole. On
Scheme 1. Synthesis of the target compounds 5a–16a. Reagents and conditions: (a) chloroacetyl chloride, NaHCO3, CH2Cl2; room temperature; (b) 2,2-dimethoxyethanamine
toluene, reflux; (c) H2SO4, CH2Cl2; (d) RX, K2CO3/KI; (e) LiAlH4, THF; (f) HBr/CH3COOH reflux.

H. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 979–982
981
Figure 3. The mode of action of compound L-6, lanosterol, and compound 13a with the active site of CYP51 of Candida albicans (Blue indicates the S1 subsite; Yellow indicates
the S2 subsite; green indicates the S3 subsite; Pink indicates the S4 subsite).
Table 2
groups on the benzene ring seemed to have little effect on the anti-
fungal activities. Compounds with hydrogen bonding donor sub-
stituents at positions 9 and 10, such as 7a, 10a, 13a, and 16a,
exhibited similar antifungal activities with that of those com-
pounds with hydrogen bonding acceptor substituents at positions
9 and 10 of pyrazino [2,1-a] isoquinolin ring, such as 6b, 9b, 12b,
and 15b. The interaction energies of title compounds 13a and lead
compound L-6 were in good qualitative agreement with their anti-
fungal activities in vitro (Table 2). All of the results gave us the sug-
gestion to further design and synthesize their derivatives.
In summary, a series of novel pyrazino [2,1-a] isoquinolin com-
pounds were successfully designed and synthesized. In vitro anti-
fungal activity assay indicated that the target compounds had
potent antifungal activity against both systemic pathogenic fungi
and dermatophytes. In particular, the most active compounds 8a,
9a–12b, and 13a exhibited broad antifungal spectrum, and showed
potent activities against Aspergillus fumigatus not inhibited effec-
tively by fluconazole, which are worthy of further evaluation.
Moreover, the mode of action of the pyrazino [2,1-a] isoquinolin
molecules showed that the affinity of the lead molecules for
CYP51 was mainly attributed to their nonbonding interaction with
the residues of apoprotein but without binding with the heme,
which was different from that of azoles. The studies presented here
provide a new structural type for the development of novel anti-
fungal agents.
A comparison of the calculated interaction energies of L-6 and 13a with CYP51 of
Candida albicans
Compounds
L-6
13a
Energy (kcal/mol)
MIC80
À9.91
À11.66
(l
g/ml)
64
8
the Microsporum gypseum strains, most of the compounds showed
inhibitory activity and compound 12a showed stronger inhibitory
activities than fluconazole with MIC80 values of 0.5 lg/mL. In par-
ticular, compounds 8a, 9a–12b, and 13a exhibited strong in vitro
antifungal activities with broad antifungal spectrum, which were
worthy of further evaluation.
The binding studies were carried out by the flexible docking
using the Affinity module within the Insight II software package.14
The mode of action of lead molecule 13a with the active site of
CYP51 of Candida albicans (Fig. 3) showed that the pyrazino [2,1-
a] isoquinolin ring interacted with the hydrophobic S2 subsite.
The hydroxy group in the benzene ring formed H-bonding interac-
tions with the residues HIS377 and Ser378 in the S4 subsite. The
lipophilic alkyl side chain at position 2 of the compound interacted
with the hydrophobic S3 subsite. The docking model showed that
the appropriate length of the substituents on the amino group of
target derivatives was important for antifungal activities. For
example, compounds 14a–16a were less potent because the alkyl
side chain was too long to be accommodated by the hydrophobic
S3 subsite. On the other hand, compounds 5a–7a were less potent
because the alkyl side chain was too short to interact firmly with
the hydrophobic S3 region, although it was able to enter the active
site. The substituents with hydrogen bond donor or acceptor
Acknowledgments
The work was supported by the National Natural Science Foun-
dation of China (Grant No. 20972187) and the National 863 Pro-
gram of China (No. 2008AA02Z302).

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H. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 979–982
7. Georgopapadakou, N. H.; Walsh, T. J. Antimicrob. Agents Chemother. 1996, 40,
Supplementary data
279.
8. Ji, H.; Zhang, W.; Zhang, M.; Kudo, M.; Aoyama, Y.; Yoshida, Y.; Sheng, C.; Song,
Y.; Yang, S.; Zhou, Y.; Lu, J.; Zhu, J. J. Med. Chem. 2003, 46, 474.
9. Ji, H.; Zhang, W.; Zhou, Y.; Zhang, M.; Zhu, J.; Song, Y.; Lu, J.; Zhu, J. J. Med. Chem.
2000, 43, 2493.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.050.
10. Zhu, J.; Lu, J.; Zhou, Y.; Li, Y.; Cheng, J.; Zheng, C. Bioorg. Med. Chem. Lett. 2006,
16, 5285.
References and notes
11. Kim, J. H.; Lee, Y. S.; Park, H.; Rim, C. S. Tetrahedron 1998, 54, 7395.
12. Laurent, S. A.-L.; Boissier, J.; Coslédan, F.; Gornitzka, H.; Robert, A.; Meunier, B.
Eur. J. Org. Chem. 2008, 5, 895.
1. Lupetti, A.; Danesi, R.; Campa, M.; Tacca, M. D.; Kelly, S. Trends Mol. Med. 2002,
8, 76.
13. NCCLS, reference method for broth dilution antifungal susceptibility testing of
yeasts; approved standard-second edition, M27-A2, National Committee for
Clinical Laboratory Standards, Villanova, PA, 2000.
14. InsightII 2000: Molecular Simulation Inc. 9685 Scranton Road, San Diego, CA
92121-3752, 1999.
2. Rogers, J. F.; Nafziger, A. N.; Bertino, J. S. Am. J. Med. 2002, 113, 746.
3. Su, F. W.; Perumalswami, P.; Grammer, L. C. Allergy 2003, 58, 1215.
4. Jeng, M. R.; Feusner, J. Pediatr. Hematl. Oncol. 2001, 18, 137.
5. Kale, P.; Johnson, L. B. Drugs Today (Barc) 2005, 41, 91.
6. Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M. Clin. Microbiol. Rev. 1999, 12, 40.