Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4-N-arylamine-1- butenes and related compounds with inhibitory activities on β(1-3) glucan and chitin synthases

Article abstract of DOI:10.1016/S0968-0896(00)00003-1

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4- aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2- substituted 4-methyl-tetrahydroquin

Full text of DOI:10.1016/S0968-0896(00)00003-1

Bioorganic & Medicinal Chemistry 8 (2000) 691±698
Inhibitors of the Fungal Cell Wall. Synthesis of
-Aryl-4-N-arylamine-1-butenes and Related Compounds with
Inhibitory Activities on ꢀ(1±3) Glucan and Chitin Synthases
4
Juan M. Urbina, Juan C. G. Cortes, Alirio Palma, Silvia N. Lo
 Â
pez, c
a
b
a
c,
d
b,y
Susana A. Zacchino, * Ricardo D. Enriz, Juan C. Ribas
a,y
and Vladimir V. Kouznetzov
aLaboratory of Fine Organic Chemistry, School of Chemistry, Industrial University of Santander, A.A. 678,
Bucaramanga, Colombia
b
Instituto de Microbiologõ Âa Bioquõ Âm ica, Campus ``Miguel de Unamuno'', Edi®cio Departamental (37007)-Salamanca, Spain
cFarmacognosia, Facultad de Ciencias Bioquõ Âm icas y Farmac e uticas, Universidad Nacional de Rosario, Suipacha 531,
(
2000)-Rosario, Argentina
d
Quõ Âm ica General, Facultad de Quõ Âm ica, Bioquõ Âm ica y Farmacia, Chacabuco y Pedernera, (5700)-San Luis, Argentina
Received 10 June 1999; accepted 12 October 1999
AbstractÐAs part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthe-
sized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetra-
hydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution assays have shown that 4-
aryl homoallylamines not possessing halogen in their structures, tetrahydroquinolines and quinolines, display a range of antifungal
properties in particular against Epidermophyton ¯occosum and Microsporum canis. Regarding the mode of action, all active com-
pounds showed in vitro inhibitory activities against b(1±3) glucan-synthase and mainly against chitin-synthase. These enzymes
catalyze the synthesis of b(1±3) glucan and chitin, respectively, major polymers of the fungal cell wall. Since fungal but not mam-
malian cells are encased in a cell wall, its inhibition may represent a useful mode of action for these antifungal compounds. # 2000
Elsevier Science Ltd. All rights reserved.
3
Introduction
for them and new and safe antifungal agents are still
needed.
The incidence of fungal infections especially invol-
ving immunocompromised patients has dramatically
increased during the last years. Systemic mycoses and
Since fungal, but not mammalian cells, are encased in a
carbohydrate-containing wall, required for the growth
and viability of fungi, the fungal cell wall has emerged
as a major target for antifungal drugs. As part of a
program aimed to identify novel antifungal agents, we
focused on developing antifungal compounds that
would selectively inhibit the synthesis of the fungal cell
wall.4
1
some forms of dermatomycoses are very dicult to
eradicate and they are the cause of a great mortality in
patients receiving antineoplasic chemotherapy, organ
2
transplants or su€ering AIDS.
±7
The mode of action of most known antifungal drugs in
use today, is the inhibition of some of the steps of
ergosterol biosynthesis, which are common to human
cholesterol and sexual hormones biosynthesis. As a
consequence, many adverse e€ects have been reported
Considering that certain 4-arylsubstituted 4-N-aryl-
amine-1-butenes (homoallylamines) have showed in vitro
antifungal properties against a panel of phytopatho-
8
genic fungi and on the basis of our previous experience
in synthesizing this type of compound,8 we prepared
a series of homoallylamines from the aldimines 1±11
(Table 1) in order to evaluate their antifungal properties
±11
*
Corresponding author. E-mail: szaabgil@ citynet.net.ar
The authors contributed equally to the direction of this work.
y
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00003-1

6
92
J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
Table 1. Synthetic aldimines used as starter compounds for preparing
homoallylamines
of fungal cell wall, these compounds were tested for
their inhibitory activity on b(1±3) glucan-synthase and
chitin-synthase, enzymes that catalyze the synthesis of
b(1±3) glucan and chitin, two major polymers of the
fungal cell wall.1
6,17
Results and Discussion
Chemistry
Compd
Type
R
1
R
2
R
3
1
2
3
4
5
6
7
8
9
A
A
A
A
A
A
A
A
A
B
H
CH
OCH
F
Cl
Br
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
The synthesis of 4-methyl-(4,6-dimethyl)-2-phenyl-quin-
olines was performed from readily accesible aldi-
3
3
18,19
mines,
(
following Scheme 1. The starting compounds
1±11; Table 1) were readily prepared in high yield from
H
OCH
N(CH
H
p-substituted anilines and benzaldehydes in following a
known procedure.11 The 4-aryl- or 4-alkyl-4-N-aryl-
amino-1-butenes 12±22 (Table 2) were obtained (Scheme 1)
by addition of allylmagnesium bromide to aromatic
aldimines 1±11 using the Grignard procedure. Allyl-
magnesium bromide was prepared from allyl bromide
3
3
)
2
CH
Ð
Ð
3
10
1
H
CH
Ð
Ð
1
B
3
and magnesium in anhydrous Et O. To the best of our
2
knowledge, homoallylamines 12±22 have not been
reported previously. They were isolated as stable yellow
oils in 53±98% after distillation under reduced pressure
followed by chromatographic technics. Their structure
was established using IR, NMR spectroscopy and mass
spectrometry. Their IR spectra showed the bands of
against human pathogenic fungi and to study the struc-
tural requirements for the antifungal activity.
In addition, we demonstrate here, by using a novel syn-
thetic pathway, that homoallylamines could serve as
versatile precursors to obtain 2-substituted 4-methyl
quinolines via tetrahydroquinolines; both type of com-
�
1
the amine group in the region of 3401±3416 cm . In the
1
H NMR spectra of these compounds the allyl radical
protons generated three groups of signals: the multiplet
8,12±15
pounds with important biological properties.
The
possibility of being useful intermediates for synthesizing
tetrahydroquinolines and quinolines add interest to
homoallylamines which, in contrast with homoallylic
alcohols, have not practically been explored in hetero-
cycle synthesis.
signal of HCˆ appeared in a zone 5.77 ppm, the protons
of ˆCH groups observed in 5.18 ppm as triple-
2
doublets, and the protons of CH -group resonated in
2
the region 2.61 ppm as two multiplets (HA and H are
B
diastereotopic).
Homoallylamines 12±22, tetrahydroquinolines 23 and
24 and quinolines 25 and 26, were tested for antifungal
properties with the agar dilution method against a panel
of standardized dermatophytes and other yeasts and
®lamentous fungi. Then, to gain insight into the capacity
of active compounds to interfere with the biosynthesis
Then, we performed electrophilic cyclization of selected
homoallylamines (12 and 13) under acidic conditions
(H SO ) and obtained the 4-methyl-(4,6-dimethyl)-2-
2
4
phenyl-1,2,3,4-tetrahydroquinolines (23 and 24). These
tetrahydroquinolines were prepared in 58±66% yields
9
as a mixture of cis/trans isomers and then oxidized
ꢀ
Scheme 1. Preparation of homoallylamines, 4-methyl-1,2,3,4-tetrahydroquinolines and 4-methylquinolines. (a) Allylbromide+Mg/Et
ꢀ
2 4
H SO 75% W/V; (c) DDQ/Bz, 35 C.
2
O, 10 C; (b)

J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
693
Table 2. MIC values (mg/mL) of homoallylamines, tetrahydroquinolines and quinolines acting against dermatophytes
Compd
Type
R
1
R
2
R
3
M. ca
M. gb
T. mc
T. rd
E. f e
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
C
C
C
C
C
C
C
C
C
D
D
E
E
F
F
H
CH
OCH
F
Cl
Br
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
Ð
Ð
H
H
H
30
30
30
>50
>50
>50
30
>50
30
>5 0
>5 0
50
50
25
0.75
>50
15
30
>50
>50
>50
>50
>50
>50
>50
>50
>5 0
>5 0
25
30
>50
30
30
>50
30
12.5
3.12
3.12
30
30
30
3.12
>50
3.12
>5 0
>5 0
3
3
>50
>50
>50
>50
>50
>50
>5 0
>5 0
25
25
12.5
25
6.25
12.5
>50
>50
>50
>50
>50
>50
>5 0
>5 0
25
25
25
12.5
25
H
OCH
N(CH
H
3
3
)
2
3
H
Ð
Ð
H
H
H
CH
H
CH
H
3
3
3
12.5
25
12.5
12.5
12.5
0.3
12.5
12.5
6.25
6.25
CH
H
H
Amphotericin B
Ketoconazole
15
25
a
Microsporum canis C 112.
Microsporum gypseum C 115.
Trichophyton mentagrophytes ATCC 9972.
b
c
d
Trichophyton rubrum C 113.
Epidermophyton ¯occosum C 114.
e
ꢀ
6) as outlined in Scheme 1.
(
2
DDQ, benzene, 35 C) to known quinolines (25 and
2
tested dermatophytes. Compound 14, with an OCH on
3
0
ring A, showed broad activity too, inhibiting all
fungi except Microsporum gypseum. Nevertheless, it is
interesting to note that when OCH3 changes from
ring A to B (Compounds 14!18) the spectrum of
action narrows down, being compound 18 active only
against M. canis and E. ¯occossum. This same range of
inhibition was observed with compounds 13 and 20,
both having methyl groups on di€erent positions of
ring A.
Antifungal assays
Homoallylamines 12±22, tetrahydroquinolines 23 and
24, and quinolines 25 and 26 were evaluated for anti-
fungal properties with the agar dilution method
against a panel of human pathogenic dermatophytes
and other fungi and yeasts.
21
Concentrations up to 50 mg/mL of each compound,
were incorporated into growth media according to
reported procedures.4
Within the activity shown by homoallylamines against
the most sensitive fungus E. ¯occosum, variations in the
electronic properties appear to a€ect the antifungal
activities. Structures containing halogen atoms (com-
pounds 15±17 and 19) were drastically less active than
the non-substituted analogue (compound 12). The pre-
sence of methyl or methoxyl groups bound to benzene
rings (A or B) enhance up to four times the activity of
these compounds relative to the non-substituted analo-
gue (compare activities of compounds 13, 14, 18, 20
(MICs=3.12 mg/mL) and 12 (MIC=12.5 mg/mL). Posi-
tion changes of the methyl substituent within the same
ring (compounds 13 and 20), do not produce changes in
the fungistatic action (MICs=3.12 mg/mL). In addition,
structures containing an aliphatic chain instead of the
benzene ring B, do not show activity (compounds 21
and 22) suggesting that the phenyl group in C-4 is
necessary for activity in homoallylamines.
Results showed that none of the compounds tested dis-
played any activity against the yeasts Candida albicans,
Candida tropicalis, Saccharomyces cerevisiae or Crypto-
coccocus neoformans nor against the ®lamentous fungi
Aspergillus niger, Aspergillus fumigatus or Aspergillus
¯
avus (results not shown). In contrast, di€erent results
were obtained for the compounds of the series against
dermatophytes. These results are shown in Table 2.
As main observations, it can be stated that all the tested
dermatophytes were inhibited at 50 mg/mL, and some of
them at lower concentrations. The most sensitive species
was E. ¯occosum.
Concerning the spectra of action of homoallylamines
tested, only the non-substituted structure 12 displayed
signi®cant activity (MICs <50 mg/mL) against all the
In order to add more information about activities of
homoallylamines, we cycled compounds 12 and 13 into

6
94
J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
tetrahydroquinolines 23 and 24 and then oxidized them
to quinolines 25 and 26.
Results obtained in the agar dilution assays showed that
cyclic compounds 23 and 25 displayed the same activity
as non-substituted compound 12 against E. ¯occosum.
However, the cyclization of the methyl derivative 13
(
MIC=3.12 mg/mL) diminished four times the inhibi-
tory activity (MICs=12.5 mg/mL for compounds 24 and
6). Analyzing the results obtained in the remaining
2
fungi tested, the most striking result was that with
compound 26. It displays an activity 66-fold higher
than its non-aromatic analogue 24 (MICs=0.75 and
50 mg/mL, respectively) and 40-fold higher than homo-
allylamine 13. In addition, compound 26 was active
only against M. canis, which was scarcely inhibited by
all the other compounds tested.
Figure 1. E€ect of di€erent concentrations of antifungal homoallyl-
amines, 4-methyl 1,2,3,4-tetrahydroquinolines and 4-methylquinolines
on in vitro incorporation of [14C]-glucose into insoluble b(1Ð3) glucan
expressed as residual activity of the enzyme (% incorporation com-
pared to the incorporation in the absence of antifungal compounds).
Points are the mean of duplicate tests.
To gain insight into the mode of action of the most
active antifungal compounds, we tested them for their
capacity of inhibiting in vitro either b(1±3) glucan syn-
5
,17
or chitin synthase 1 activities.22 Results of the
thase
in vitro assays are listed in Table 3. Regarding activity
against b(1±3) glucan synthase, all homoallylamines
tested (compounds 12±14, 18 and 20) exhibited a mod-
erate inhibitory activity at 20 mg/assay (% of inhibition
ranging from 25 to 50%). Their cyclic derivatives, tet-
rahydroquinoline 23 and quinoline 25 showed inhibi-
tory activities between 42 and 60%.
Results obtained in the chitin synthase-1 assay, showed
that 20 mg of antifungal compounds 12±14, 18, 20, 23±
26 possess a strong inhibitory e€ect on enzyme activity
(% of inhibition from 50 to 88% (Table 3)). All homo-
allylamines tested displayed IC <0.20 mg/mL, with qui-
50
nolines 25 and 26 as the most active inhibitors
(IC =0.02 and 0.04 mg/mL, respectively (Fig. 2)).
50
Since compounds 12, 13, 20, 23 and 25 displayed a b(1±3)
glucan synthase inhibition 550%, ®ve serial dilutions
of them (1±20 mg) were tested for enzyme inhibition
and their average inhibitory concentration calculated
The results here reported allow us to infer that the
assayed homoallylamines, tetrahydroquinolines and
quinolines would act by inhibiting the biosynthesis of
the two major polymers of fungal wall as one of their
possible mode of action.
(
Fig. 1). Among them, homoallylamine 12 and its
derivatives 23 and 25 showed the most potent e€ects
with similar IC50 values (0.25, 0.23 and 0.23 mg/mL,
respectively). These values are lower than the well-
known, b(1±3) glucan synthase inhibitor Aculeacin A
Alternatively, since the crude enzymes were prepared
from S. cerevisiae, the genus di€erence would account
for the lack of a strict correlation between activities
shown by compounds in cellular and enzymatic assays.
(
IC 50.5 mg/mL).
5
0
Table 3. Capacity of inhibiting b(1±3) glucan synthase and chitin
synthase expressed in % of inhibition and IC50 values (mg/mL)
Glucan synthase assay
Chitin synthase assay
Compd
% Ia
IC50b
% Ic
IC50b
1
1
1
1
2
2
2
2
2
2
3
4
8
0
3
4
5
6
60.10‹4.36
53.35‹3.27
25.34‹5.78
36.02‹4.63
51.67‹3.94
53.48‹4.90
41.64‹7.16
59.46‹9.20
34.32‹3.08
0.25
0.32
>0.50
>0.50
0.33
0.23
>0.50
0.23
>0.50
0.10
>0.50
87.07‹1.04
56.36‹2.57
68.42‹1.02
75.26‹0.65
70.01‹1.77
73.41‹0.10
49.75‹0.28
87.90‹0.88
84.11‹0.50
0.09
0.19
0.15
0.07
0.17
0.10
0.40
0.02
0.04
Papulacandin B
Aculeacin A
Nikkomicin
0.0006d
a
%
of inhibition at 20 mg/assay (total volume: 40 mL): mean‹SEM.
Figure 2. E€ect of di€erent concentrations of antifungal homoallyl-
amines, 4-methyl-1,2,3,4-tetrahydroquinolines and 4-methylquinolines
on the in vitro chitin synthase 1 activity, expressed as residual activity
of the enzyme (see legend of Fig. 1 for details).
b
Concentration (mg/mL) that produces 50% of inhibition.
c
% of inhibition at 20 mg/assay (total volume: 50 mL): mean‹SEM.
Value obtained from ref 29.
d

J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
695
Conclusions
4-Aryl or 4-alkyl-4-N-arylamino-1-butenes (12±22). Gen-
eral procedure. The aldimines (1±11) (32 mmol) dis-
solved in 25 mL of Et O were added slowly to a
In an attempt to generate lead structures that might
have selective antifungal activities, we have synthesized
a series of aryl and alkyl homoallylamines from aldi-
mines 1±11. Then, we transformed some of them into 4-
methylquinolines 25±26 via the 4-methyl-1,2,3,4-tetra-
hydroquinolines 23 and 24 by using a novel synthetic
pathway, showing that homoallylamines could serve as
useful precursors for this type of biologically important
compounds. Agar dilution assays with human patho-
genic fungi have shown that structures 12±14, 18 and 20
possess potent antifungal properties in particular
against E. ¯occosum. Tetrahydroquinolines 23 and 24
and quinolines 25 and 26 also display antifungal activ-
ities; compound 26 being particularly active against M.
canis. Since dermatophytes are a group of highly spe-
cialized fungi which characteristically infect the kerati-
nized areas of the body and dermatophytoses are
extremely common and very dicult to eradicate, it is
interesting to note that 4-aryl homoallylamines and
their derivatives, display similar or stronger antifungal
activities than Amphotericin B or Ketoconazole on the
species tested in our experiments.
2
magnetically stirred suspension of allyl magnesium bro-
mide prepared from allyl bromide (95 mmol, 11.45 g)
and magnesium (158 mmol, 3.84 g) in 100 mL Et O, at
2
ꢀ
ꢀ
10 C. The mixture was heated to 30±35 C during 4 h,
cooled to 0 C and treated with a NH Cl solution (pH
4
ꢀ
8±9). Two liquid±liquid extractions with Et O (50 mL
2
each) were performed. The organic layers were com-
bined and dried over anhydrous MgSO . The residue
4
from ether evaporation was puri®ed by column chro-
matography over silica to give products 12±22, which
were obtained as yellow oils as follows:
�
1
(s, NH); 1
H
12. Yield 58%. IR (®lm): n 3413 cm
NMR (CDCl ): d 2.51 (1H, m, H C±Cˆ), 2.63 (1H, m,
3
2
H C±Cˆ), 4.16 (1H, br. s, H±N), 4.42 (1H, dd, H±C±
2
Ar), 5.18 (2H, td, H Cˆ), 5.77 (1H, m, H±Cˆ),
2
6.52 (2H, d, o-Ph±N), 6.67 (1H, t, p-Ph-N), 7.10 (2H,
t, m-Ph±N), 7.26 (1H, t, p-Ph±C), 7.35 (2H, t, m-Ph±C),
7.36 (2H, d, o-Ph-C); 13C NMR (CDCl ): d 43.40,
57.23, 113.57, 117.46, 118.39, 126.39, 126.92, 128.67,
129.16, 134.74, 143.64, 147.43; MS m/z (EI): 223
3
(
H, 7.81; N, 6.19; calcd for C H N: C, 86.05; H, 7.67;
M+, 3%), 182 (M� C H , 100%). Found: C, 86.00;
3
5
Regarding the mode of action, active compounds 12±14,
16 17
18, 20 and 23±26 showed inhibition against b(1±3) glu-
N, 6.27.
can synthase and chitin synthase activities. The fact that
these compounds inhibit the synthesis of both, glucan
and chitin at the same time is particularly attractive.
We should keep in mind that combinations of chitin
synthase and glucan synthase inhibitors are usually
�
1
(s, NH); 1
H
13. Yield 53%. IR (®lm): n 3411 cm
NMR (CDCl ): d 2.55 (1H, m, H C±C), 2.62 (1H, m,
3
2
H C±Cˆ), 3.95 (1H, br. s, H±N), 4.36 (1H, dd, H±C±
2
Ar), 5.17 (2H, td, H C=), 5.76 (1H, m, H±Cˆ), 6.42
2
2
3
required.
(2H, d, o-Ph±N), 6.90 (2H, d, m-Ph±N), 7.23 (1H, t, p-
Ph±C), 7.35 (2H, t, m-Ph±C), 7.36 (2H, d, o-Ph±C);
1
3
C
The antifungal activity of homoallylamines, tetra-
hydroquinolines and quinolines against some dermato-
phytes, combined with their selective mode of action,
make these compounds attractive leaders for the devel-
opment of most potent and mainly safer antifungal
drugs.
NMR (CDCl ): d 20.44, 43.44, 57.45, 113.66, 118.32,
3
126.37, 126.58, 126.99, 128.64, 129.86, 134.85, 143.84,
145.0; MS m/z (EI): 237 (M+, 4%), 196 (M� C H ,
3
5
100%). Found: C, 85.90; H, 8.18; N, 5.82; calcd for
C H N: C, 86.03; H, 8.07; N, 5.90.
17
19
�
1
(s, NH); 1
H
1
4. Yield 78%. IR (®lm): n 3401 cm
NMR (CDCl ): d 2.48 (1H, m, H C±Cˆ), 2.60 (1H, m,
3
2
H C±Cˆ), 3.94 (1H, br. s, H±N), 4.31 (1H, dd, H±C±
2
Experimental
Chemistry
Ar), 5.17 (2H, td, H Cˆ), 5.78 (1H, m, H±Cˆ), 6.46
2
(2H, d, o-Ph±N), 6.46 (2H, d, m-Ph±N), 7.25 (1H, t, p-
Ph±C), 7.35 (2H, t, m-Ph±C), 7.35 (2H, d, o-Ph±C);
1
3
C
All reagents were purchased from Aldrich, commercial
grade. IR spectra were recorded on a Perkin±Elmer
NMR (CDCl ): d 43.54, 55.81, 57.99, 114.72, 114.79,
3
118.36, 126.43, 127.03, 128.66, 134.90, 141.71, 143.93,
152.03; MS m/z (EI): 253 (M+, 7%), 212 (M� C H ,
1
599B-FT spectrometer. H NMR spectra were deter-
mined on a Jeol 400 MHz in CDCl3 with tetra-
3
5
100%). Found: C, 80.53; H, 7.80; N, 5.25; calcd for
C H NO: C, 80.60; H, 7.56; N, 5.53.
13
methylsilane (TMS, d 0 ppm) as internal standard.
C
NMR spectra were recorded with CDCl as an internal
17
19
3
�
1
(s, NH); 1
H
standard at d=77.0 at 100 MHz on a Jeol spectrometer.
MS spectra were obtained with an HP 5890 series II gas
chromatograph interfaced to an HP 5972 mass selective
detector that used electron impact ionization (70 eV).
Elemental analyses were performed on a Leco CHN-600
analyzer. Column chromatography was carried out on
columns packed with SiO2.
15. Yield 74%. IR (®lm): n 3414 cm
NMR (CDCl ): d 2.50 (1H, m, H C±Cˆ), 2.63 (1H, m,
3
2
H C±Cˆ), 4.12 (1H, br. s, H±N), 4.35 (1H, dd, H±C±
2
Ar), 5.22 (2H, td, H Cˆ), 5.80 (1H, m, H±Cˆ), 6.46
2
(2H, dd, o-Ph±N), 6.82 (2H, t, m-Ph±N), 7.24 (1H, dd,
p-Ph±C), 7.36 (2H, t, m-Ph±C), 7.38 (2H, d, o-Ph-C);
13
C NMR (CDCl ): d 43.51, 57.77, 114.4, 115.5, 118.57,
3
1
26.41, 127.22, 128.95, 134.73, 144.0, 148.0, 160.3; MS
The aldimines were prepared according to the litera-
1
m/z (EI): 241 (M+, 4%), 200 (M� C H , 100%).
3
5
8
ture. The solid products were obtained with nearly
quantitative yield and con®rmed by IR spectra.
Found: C, 79.50; H, 6.95; N, 5.65; calcd for C H FN:
16 16
C, 79.64; H, 6.68; N, 5.80.

6
96
J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
�
1
(s, NH); 1
1
6. Yield 72%. IR (®lm): n 3416 cm
H
H±Cˆ), 6.63 (2H, d, o-Ph±N), 6.72 (1H, t, p-Ph±N),
NMR (CDCl ): d 2.50 (1H, m, H C±Cˆ), 2.63 (1H, m,
7.21 (2H, t, m-Ph±N); 13C NMR (CDCl ): d 14.32,
3
2
3
H C±Cˆ), 4.19 (1H, br. s, H±N), 4.35 (1H, dd, H±C±
19.40, 36.79, 38.71, 52.20, 113.26, 116.95, 117.63,
129.42, 135.12, 147.92; MS m/z (EI): 189 (M+, 7%),
148 (M� C H , 100%). Found: C, 82.38; H, 10.20; N,
2
Ar), 5.20 (2H, td, H Cˆ), 5.76 (1H, m, H±Cˆ), 6.45
2
(2H, d, o-Ph±N), 7.05 (2H, d, m-Ph±N), 7.20 (1H, d, p-
Ph±C), 7.38 (2H, dd, m-Ph±C), 7.52 (2H, d, o-Ph±C);
3
5
7.52; calcd for C H N: C, 82.48; H, 10.12; N, 7.40.
13 19
1
3
C NMR (CDCl ): d 43.15, 57.05, 114.48, 118.47,
3
�
1
(s, NH); 1
H
1
1
1
26.15, 126.3, 127.17, 128.60, 128.77, 134.33, 142.91,
45.74; MS m/z (EI): 257 (M+, 4%), 216 (M� C H ,
22. Yield 43%. IR (®lm): n 3411 cm
NMR (CDCl ): d 0.97 (3H, t, CH ), 1.49 (4H, m, -
3
5
3
3
00%). Found: C, 74.80; H, 6.10; N, 5.30; calcd for
(CH ) -), 2.07 (2H, m, H C±Cˆ), 2.26 (3H, s, CH ±Ph),
2
2
2
3
C H ClN: C, 74.55; H, 6.26; N, 5.43.
1
3.43 (1H, t, H±C±N), 3.67 (1H, br. s, H±N), 5.13 (2H, d,
H Cˆ), 5.82 (1H, m, H±Cˆ), 6.55 (2H, d, o-Ph±N), 7.02
6
16
2
�
1
1
13
1
7. Yield 60%. IR (®lm): n 3414 cm (s, NH); H NMR
(2H, d, m-Ph±N); C NMR (CDCl ): d 14.28, 19.38,
3
(
4
CDCl ): d 2.55 (1H, m, H C±Cˆ), 2.69 (1H, m, H C±Cˆ),
20.50, 36.81, 38.69, 52.54, 113.48, 117.45, 126.7, 129.89,
135.72, 145.64; MS m/z (EI): 203 (M+, 8%), 162
(M� C H , 100%). Found: C, 82.64; H, 10.46; N, 6.95;
3
2
2
.29 (1H, br. s, H±N), 4.42 (1H, dd, H±C±Ar), 5.25 (2H, td,
H Cˆ), 5.83 (1H, m, H±Cˆ), 6.45 (2H, d, o-Ph±N), 7.23
2
3
5
(
2H, d, m-Ph±N), 7.32 (1H, t, p-Ph±C), 7.33 (2H, t, m-Ph±
calcd for C H N: C, 82.70; H, 10.41; N, 6.89.
14 21
13
C), 7.41 (2H, d, o-Ph±C); C NMR (CDCl ): d=43.38,
3
5
1
5
4
7.22, 109.19, 115.29, 118.77, 126.43, 127.37, 128.89,
31.94, 134.57, 143.08, 146.43; MS m/z (EI): 301 (M+,
%), 260 (M� C H , 100%). Found: C, 63.08; H, 5.60; N,
Synthesis of tetrahydroquinolines (23 and 24). General
method. Sulfuric acid 75% w/v (2.0 mL) was added to
1.0 g of the allylamines (12 and 13) and the mixture was
3
5
ꢀ
.51; calcd for C H BrN: C, 63.59; H, 5.34; N, 4.63.
heated to 60 C for 8±12 h while stirring vigorously. The
1
6
16
reaction progress was monitored via TLC (heptane,
chromatoplates of Silufol UV254). After the reaction
was completed, the mixture was cooled down to room
temperature and a concentrated ammonium hydroxide
solution was added (pH 8±9). Two 30 mL extractions
�
1
(s, NH); 1
H
1
8. Yield 85%. IR (®lm): n 3411 cm
NMR (CDCl ): d 2.54 (1H, m, H C±Cˆ), 2.64 (1H, m,
3
2
H C±Cˆ), 4.19 (1H, br. s, H±N), 4.41 (1H, dd, H±C±
2
Ar), 5.23 (2H, td, H Cˆ), 5.83 (1H, m, H±Cˆ), 6.56
2
(
Ph±C), 7.15 (2H, t, m-Ph±N), 7.35 (2H, d, o-Ph±C);
2H, d, o-Ph±N), 6.71 (1H, t, p-Ph±N), 6.92 (2H, d, m-
C
with Et O were performed. The organic layers were
2
13
combined and dried with anhydrous Na SO . The resi-
2
4
NMR (CDCl ): d 43.54, 55.35, 56.69, 113.64, 114.12,
due after ether evaporation was puri®ed by column
chromatography over silica to give products 23 and 24
as yellow oils as follows.
3
1
1
1
17.45, 118.31, 127.48, 129.21, 134.93, 135.66, 147.56,
58.69; MS m/z (EI): 253 (M+, 2%), 212 (M� C H ,
3
5
00%). Found: C, 80.52; H, 7.70; N, 5.28; calcd for
�
1
C H NO: C, 80.60; H, 7.56; N, 5.53.
1
23. Yield 58%. IR (®lm): n 3383 cm (s, NH); MS m/z
7
19
+
2
reported.
23 (M , 100%), having similar properties to those
9
�
1
(s, NH); 1
19. Yield 77%. IR (®lm): n 3415 cm
H
NMR (CDCl ): d 2.60 (1H, m, H C±Cˆ), 2.70 (1H, m,
3
2
�
1
(s, NH); 1
H
H C±Cˆ), 4.26 (1H, br. s, H-N), 4.40 (1H, t, H±C±Ar),
24. Yield 66%. IR (®lm): n 3373 cm
2
5
.28 (2H, t, H Cˆ), 5.90 (1H, m, H±Cˆ), 6.54 (2H, d,
NMR: d 1.36 (3H, d, 6.95 Hz, 4-CH ), 1.78 (1H, dd, 3-
2
3
m-Ph±C), 6.83 (2H, d, o-Ph±N), 7.15 (2H, d, m-Ph±N),
H ), 2.12 (1H, ddd, 3-H ), 2.28 (3H, s, 6-CH ), 3.13
(1H, m, 4-H), 3.85 (1H, s, N-H), 4.46 (1H, d, 2-H), 6.46
(1H, d, 8-H), 6.84 (1H, d, 7-H), 7.32 (1H, t, p-Ph), 7.38
(2H, t, m-Ph), 7.44 (2H, t, o-Ph); 13C NMR (CDCl ): d
20.35, 20.78, 31.39, 41.94, 57.19, 114.37, 126.37, 127.52,
128.69, 142.52, 144.78. MS m/z (EI) 237 (M , 100%).
Found: C, 85.85 H, 8.22; N, 5.85; calcd for C H N: C,
86.03; H, 8.07; N, 5.90.
ax
eq
3
7
4
1
.32 (2H, d, o-Ph±C); 13C NMR (CDCl ): d 40.79,
3.48, 56.88, 112.95, 114.84, 118.25, 121.82, 127.21,
29.06, 130.76, 135.15, 146.96, 149.96; MS m/z (EI): 259
3
3
(
calcd for C H ClN : C, 71.87; H, 7.04; N, 9.31.
M� C H , 100%). Found: C, 71.60; H, 7.25; N, 9.44;
3
5
+
1
8
21
2
17
19
�
1
(s, NH); 1
20. Yield 98%. IR (®lm): n 3431 cm
H
NMR (CDCl ): d 2.49 (1H, m, H C±Cˆ), 2.61 (1H, m,
3
2
H C±Cˆ), 4.07 (1H, br. s, H±N), 4.39 (1H, dd, H±C±
Synthesis of quinolines (25 and 26). General method. To
a solution of 1.0 mmol of the tetrahydroquinolines 23 or
24 dissolved in 30 mL of benzene, 2.1 mmol of DDQ
dissolved in benzene were added slowly. The reaction
mixture was stirred at room temperature for 4 h, mon-
itoring progress via TLC (heptane:ethylacetate (50:1),
chromatoplates of Silufol UV254). At the end of the
reaction the mixture was ®ltered (solid product is
DDHQ). The benzene was distilled; the residue was
puri®ed by column chromatography over silica (with
heptane:ethyl acetate, 50:1) to yield quinolines 25 and
26 as yellow oils as follows.
2
Ar), 5.18 (2H, td, H Cˆ), 5.77 (1H, m, H±Cˆ), 6.28
2
(2H, d, o-Ph±N), 6.58 (1H, t, m-Ph±N), 6.91 (1H, t, p-
Ph±N), 7.02 (1H, d, m-Ph±N), 7.20 (2H, t, m-Ph±C),
1
3
7
.29 (1H, t, p-Ph±C), 7.34 (2H, d, o-Ph±C); C NMR
(
CDCl ): d 17.77, 43.93, 57.12, 117.15, 118.62, 122.18,
3
126.43, 127.19, 128.84, 130.15, 135.21, 143.92, 145.44;
MS m/z (EI): 237 (M+, 5%), 196 (M� C H , 100%).
3
5
Found: C, 85.85; H, 8.10; N, 5.82; calcd for C H N:
19
1
7
C, 86.03; H, 8.07; N, 5.90.
�
1
(s, NH); 1
H
21. Yield 66%. IR (®lm): n 3407 cm
NMR (CDCl ): d 0.98 (3H, t, -CH ), 1.49 (4H, m,
3
3
1
-
(CH ) -), 2.30 (2H, m, H C±Cˆ), 3.49 (1H, t, HC±N),
25. Yield 59%. H NMR: d 2.74 (3H, s, 4-CH ), 7.47
2
2
2
3
3
.57 (1H, br. s, H±N), 5.14 (2H, d, H Cˆ), 5.87 (1H, m,
(1H, t, p-Ph), 7.53 (2H, t, m-Ph), 7.69 (1H, s, 3-H), 7.73
2

J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
697
(
2H, d, o-Ph), 7.97 (1H, d, 8-H), 8.14±8.23 (3H, m, 5±7-
1
Enzymatic assays
3
H); C NMR (CDCl ): d 19.13, 119.87, 123.73, 126.12,
3
1
1
1
27.34, 127.65, 128.68, 128.89, 129.31, 129.45, 130.35,
+
39.91, 144.93, 148.20, 157.17. MS m/z (EI) 219 (M ,
00%). Found: C, 87.57; H, 6.10; N, 6.27; calcd for
Strains and culture conditions. The S. cerevisiae strain
used was MATa ura3 leu2 his3 pep4::HIS3 nuc1::LEU2.
Routine yeast growth (YES)25 was as described.
C H N: C, 87.64; H, 5.98; N, 6.39.
1
6
13
Enzyme preparation. Cell extracts were obtained essen-
26
tially as described previously.
1
2
6. Yield 34%. H NMR: d 2.57 (3H, s, 6-CH ), 2.74
3
(
3H, s, 4-CH ), 7.43 (1H, t, p-Ph), 7.51 (2H, t, m-Ph),
3
7
8
.52 (2H, d, o-Ph), 7.55 (1H, s, 5-H), 7.68 (1H, s, 3-H),
.05 (1H, d, 7-H), 8.12 (1H, d, 8-H); MS m/z (EI) 233
Early logarithmic phase cells grown in 100 mL YES
medium were collected, washed once with 50 mM Tris±
HCl pH 7.5, suspended in 100 mL of the same bu€er and
broken with glass beads in a FastPrep FP120 apparatus
+
(M , 100%). Found: C, 87.35; H, 6.70; N, 5.90; calcd
for C H N: C, 87.52; H, 6.48; N, 6.00.
1
7
15
(Savant, BIO 101, Inc.) (once a 15 s pulse at speed of 6).
Broken material was collected and cell debris was
removed by low speed centrifugation (5000 g, 5 min at
ꢀ
Biological Evaluation
4
C). The supernatant was centrifuged at 48,000 g for
ꢀ
Microorganisms and media
30 min at 4 C and the pellet was resuspended in 50 mM
Tris±HCl pH 7.5 containing 33% glycerol (at a con-
centration of 3 mg protein per mL) and stored at
The following microorganisms used for the fungistatic
evaluation were purchased from American Type Cul-
ture Collection (Rockville, MD): C. albicans ATCC
0231, S. cerevisiae ATCC 9763, C. neoformans ATCC
2264, A. ¯avus ATCC 9170, A. fumigatus ATCC
6934, A. niger ATCC 9029 and Trichophyton menta-
ꢀ
� 70 C. Protein was quanti®ed by the Bradford dye-
binding procedure27 using the Bio-rad Protein Assay
Dye Reagent and bovine serum albumin as standard.
1
3
2
ꢀ(1±3)-Glucan synthase assay. b(1±3)-Glucan synthase
2
6
grophytes ATCC 9972. Strains were grown on Sabour-
aud-chloramphenicol agar slants for 48 h at 30 C. Cell
assay was essentially as described previously. The
standard assay mixture contained 5 mL enzyme (15 mg
protein), in a total volume of 40 mL. Two mL of ethanol
or the corresponding compounds (kept in stock solution,
ꢀ
suspensions in sterile distilled water were adjusted to
6
24
give a ®nal concentration of 10 viable yeast cells/mL.
Dermatophytes: M. canis C 112, Trichophyton rubrum C
13, E. ¯occosum C 114 and M. gypseum C 115 are
clinical isolates and were kindly provided by CERE-
MIC, Centro de Referencia Micologica, Facultad de
Ciencias Bioquõmicas y Farmaceuticas, Suipacha 531-
2000)-Rosario, Argentina.
ꢀ
10 mg/mL in ethanol at � 20 C) were added to each
ꢀ
1
reaction. The reaction was incubated for 1 h at 30 C
and stopped by addition of 1 mL 10% trichloroacetic
acid. All reactions were carried out in duplicate.
Â
Â
Â
(
The drugs Papulacandin B and Aculeacin A were gen-
erous gifts from K. Scheibli and P. Traxler (Ciba-Geigy,
Basel, Switzerland) and K. Mizuno (Tokyo Jozo Co.
Ltd., Tagatagun, Shizuoka-ken, Japan), respectively.
The antibiotics were kept in stock solution (10 mg/mL
Organisms were maintained on slopes of Sabouraud-
dextrose agar (SDA, Oxoid) and subcultured every 15 days
to prevent pleomorphic transformations. Spore suspen-
24
ꢀ
sions were obtained according to reported procedures
6
in methanol) at � 20 C.
and adjusted to 10 spores with colony forming ability/mL.
Chitin synthase 1 assay. Chitin synthase 1 assay was
performed as described previously.28 The standard
assay mixture contained 10 mL enzyme (30 mg protein),
in a total volume of 50 mL. Two mL of ethanol or the
corresponding compounds (kept in stock solution,
Antifungal assays
The antifungal activity of homoallylamines was eval-
uated with the agar dilution method by using Sabouraud-
chloramphenicol agar for both yeast and dermatophyte
species. The assay was carried out in 96-well microtiter
plates. Stock solutions of compounds in DMSO were
diluted to give serial 2-fold dilutions that were added to
each medium resulting in concentrations ranging from
ꢀ
10 mg/mL in ethanol at � 20 C) were added to each
reaction. Enzyme activation was performed by partial
proteolysis of the reaction mixture with 2 mL trypsin
ꢀ
(0.166 m g/mL) during 15 min at 30 C and stoped by the
addition of 2 mL tripsin inhibitor (0.25 mg/mL). The
reaction was incubated for 1 or 2 h at 30 C and stopped
ꢀ
0.10 to 50 mg/mL. The ®nal concentration of DMSO in
the assay did not exceed 2%. Five mL of yeast cell or
spore suspensions were added to each Sabouraud-
chloramphenicol agar well. The antifungal agents
Ketoconazole (Janssen Pharmaceutica) and Amphoter-
icin B (Sigma Chemical Co.) were included in the assay
as positive controls. Drug-free solution was also used as
blank control. The plates were incubated 24, 48, or 72 h
by addition of 1 mL 10% trichloroacetic acid. All reac-
tions were carried out in duplicate.
Acknowledgements
This work was supported by respective grants No.115-
05-353-96 from Colciencias (Colombia), PEI 239/97
from CONICET and Project B050 from Universidad
Nacional de Rosario (Argentina), grant BIO98-0814-
ꢀ
at 30 C (according to the control fungus growth) and
up to 15 days for dermatophyte strains. MIC was
de®ned as the lowest compound concentration, showing
no visible fungal growth after incubation time.
C02-01 from ``Comision Interministerial de Ciencia y
Â

6
98
J. M. Urbina et al. / Bioorg. Med. Chem. 8 (2000) 691±698
Tecnologõ
Â
Spain). J.M.U., A.P. and V.V.K are indebted to Dr.
Leticia Quintero and Miss Sara Montiel (Universidad
Autonoma de Puebla, Mexico) for the NMR measure-
a'' (Spain) and a contract with Lilly S.A.
È
11. Kouznetsov, V. V.; Ocal, N.; Turgut, Z.; Zubkov, F.;
Kaban, S.; Varlamov, A. V. Mon. Chem. 1998, 129, 671.
(
1
2. Fournet, A.; Hocquemiller, R.; Roblot, F.; Cave
Richomme, P.; Bruneton, J. J. Nat. Prod. 1993, 56, 1547.
3. Fournet, A.; Barrios, A. A.; Munoz, V.; Hocquemiller, R.;
Â
, A.;
1
Ä
ments. J.C.C. and J.C.R. wish to thank A. Dura
Â
n
Bruneton, J.; Richomme, P.; Gantier, J. Antimicrob. Agents
Chemother. 1993, 37, 859.
(
CSIC/Univ. de Salamanca) for helping and supporting
this work. Collaboration from international project X-2
of RIPRONAMED (Iberoamerican Network of Med-
icinal Natural Products), part of the Iberoamerican
Program of Science and Technology for the Develop-
ment (CYTED), is gratefully acknowledged.
1
4. Yates, F. In Comprehensive Heterocyclic Chemistry; Boul-
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