Structure-activity relationship study of homoallylamines and related derivatives acting as antifungal agents

Article abstract of DOI:10.1016/j.bmc.2005.10.036

The synthesis, in vitro evaluation, and structure-activity relationship studies of homoallylamines and related derivatives acting as antifungal agents are reported. Among them, compounds N-(4-bromophenyl)-N-(2-furylmethyl)amine and N-(4-chlorophenyl)-N-(2

Full text of DOI:10.1016/j.bmc.2005.10.036

Bioorganic & Medicinal Chemistry 14 (2006) 1851–1862
Structure–activity relationship study of homoallylamines
and related derivatives acting as antifungal agents
Fernando D. Suvire,a Maximiliano Sortino,c Vladimir V. Kouznetsov,b
Leonor Y. Vargas M,b Susana A. Zacchino,c Uriel Mora Cruzb and Ricardo D. Enriza,*
aFacultad de Quı´mica, Bioquı´mica y Farmacia, Universidad Nacional de San Luis (U.N.S.L.),
Chacabuco 917, 5700 San Luis, Argentina
bLaboratorio de Quı´mica Orga´nica y Biomolecular, Escuela de Quı´mica, Universidad Industrial de Santander,
A.A. 678 Bucaramanga, Colombia
cFarmacognosia, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario,
Suipacha 531, 2000 Rosario, Argentina
Received 9 September 2005; revised 19 October 2005; accepted 20 October 2005
Available online 10 November 2005
Abstract—The synthesis, in vitro evaluation, and structure–activity relationship studies of homoallylamines and related derivatives
acting as antifungal agents are reported. Among them, compounds N-(4-bromophenyl)-N-(2-furylmethyl)amine and N-(4-chlor-
ophenyl)-N-(2-furylmethyl)amine reported here exhibited remarkable antifungal activity against dermatophytes. Theoretical calcu-
lations allow us to determine the minimal structural requirements to produce the antifungal response and can provide a guide for the
design of compounds with these properties.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
shell that surrounds and protects the cell against envi-
ronment and mechanical stress.7 With considerable var-
iation among different species, the main components of
cell wall of most fungi include chitin, b- or a-linked
glucans, and a variety of mannoproteins.8
The current interest in the development of new antifungal
agents can partially be due to the dramatic rise in the num-
ber of AIDS cases and the subsequent suppression of the
immune system in patients with the disease. Other condi-
tions that have spurred the development of new anti-
fungal agents include the increase in the frequency of
bone marrow and organ transplants, the use of antineo-
plastic agents, long-term use of corticoids, and even the
indiscriminate use of antibiotics.1–3 On the other hand,
the emergence of fungal resistance to currently available
antifungal agents, specially azoles, leads to an increasing
need for new and effective antifungal agents.4
In the course of our ongoing screening program for new
and selective antifungal compounds, we have previously
reported the antifungal activity of different compounds
obtained from natural9–12 and synthetic13–18 sources.
Among them, a series of 4-aryl- or 4-alkyl-N-arylamino-
1-butenes (ÔhomoallylaminesÕ) and related tetrahydro-
quinolines and quinolines16 display a range of antifungal
properties against dermatophytes, which are responsible
for most dermatomycoses in humans. More recently, we
extended our study introducing a new series of 4-N-aryla-
mino-1-butenes containing the pyridinyl or quinolinyl
moieties at C4 and other structurally related com-
pounds.17 Regarding their mode of action, active com-
pounds showed in vitro inhibitory activities against
(1,3)b-D-glucan-synthase and mainly against chitin-
synthase, enzymes that catalyze the synthesis of major
fungal cell wall polymers.7,8 Since fungal but not mamma-
lian cells possess a cell wall, these structures appeared
as promising leads for the development of selective
antifungal compounds.
Owing to their eukaryotic nature, fungal cells have only
a restricted set of specific targets that do not overlap
with their mammalian counterparts.5 The cell wall of
most fungi offers a selective target for drug design
because several enzymes involved in the synthesis of
specific components of the fungal cell wall are not found
in human cells.5,6 The fungal cell wall consists of a rigid
Keywords: Homoallylamines; Antifungal activity; SAR; Synthesis.
*
Corresponding author. E-mail: denriz@unsl.edu.ar
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.036

1852
F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
In an attempt to improve the biological profile of homo-
allylamines, we present here the synthesis and antifungal
activity of 15 additional heterocyclic analogues not
reported previously with or without the allylamine
group.
(a) Compounds containing a flexible connecting chain
(homoallylamines).
(b) Compounds containing a conformationally restricted
non-aromatic connecting ring (tetrahydroquinolines).
(c) Compounds containing a conformationally restricted
aromatic connecting ring (quinolines).
With both, the results reported here, and the previously
published data,16,17 we have performed a comprehensive
structure–activity relationship (SAR) study on 43 mole-
cules (20 homoallylamines and 23 structural analogues),
in order to determine the minimal structural requirements
for these compounds to produce the antifungal effect.
How can theoretical calculations contribute to the
understanding of the activity of these molecules? One
way is to study a series of chemically related compounds
and to obtain more precise information about how the
different members of a series resemble each other in
terms of spatial orientations for the recognition of the
receptor. These differences may be correlated with the
antifungal activities.
We also discuss a possible pharmacophore model for
these compounds and the stereoelectronic requirements
necessary to elicit the activity.
We conducted a computer-assisted conformational and
electronic study on the most representative molecules
of each series.
2. Results and discussion
2.1. Chemistry
Regarding conformational analysis RHF/6-31G(d) cal-
culations on compounds 3 and 15 were carried out
and the low-energy conformations obtained for these
homoallylamines were compared with those previously
reported for tetrahydroquinolines and quinolines.18 Tet-
rahydroquinoline molecules were shown to possess eight
distinct forms (without considering enantiomeric
forms), varying in the orientation of both substituents,
CH3 group and pyridinyl ring.18 The preferred confor-
mation adopted by homoallylamines is closely related
to those previously reported for tetrahydroquinolines18
(Fig. 1). In contrast, quinoline derivatives display a qua-
si-planar conformation as the preferred form, which is
not viable for tetrahydroquinoline (and homoallylam-
ines) structures due to the sp3 form of their chiral
carbon.
The N-arylaldimines, principal starting materials in this
investigation, were prepared from commercially avail-
able aromatic aldehydes (benzaldehydes, 2-furaldehy-
des, and 2-thiophenecarboxyaldehyde) and substituted
anilines according to literature procedures.16,19 New ser-
ies of obtained aldimines were converted into secondary
N-benzylanilines 12–14 (structure I in Table 1), N-(2-
furylmethyl)anilines 34–40, N-(2-thienylmethyl)anilines
41–43 (Table 2) or N-aryl-N-[1-(thien-2-yl)but-3-enyl]-
amines 44, 45 (Ôhomoallylamines20Õ) (structure III). Be-
cause the reduction of aldimines with an excess of
NaBH4 in the methanol is still the reaction of choice
to produce secondary amines in a reasonably good yield,
we employed this method in our work. Thus, first series
was easily prepared obtaining these secondary amines as
a colored solid or liquid in 75–98% yields after purifica-
tion using a SiO2 chromatography column. Using the
nucleophilic addition of allylmagnesium bromide to
the C@N bond of respective aldimines, we prepared
new homoallylamines, which were isolated as stable oils
in 70–77% yields (Scheme 1). N-Substituted anilines 12–
14, 34–40, and 41–43 were synthesized according to
reported methodologies.21,22
Taking into account this difference, we here focused this
study on tetrahydroquinolines and homoallylamines.
Quinoline derivative SAR studies will be reported in a
separate paper.
The antifungal activities obtained for homoallylamines
and tetrahydroquinolines are summarized in Tables 1
and 2.
Compounds 1 and 2 were easily prepared from the
respective aldimines using the Grignard reaction with
allylmagnesium bromide. Compound 22 was obtained
To evaluate the SARs, the effects of structural changes
in three regions of the molecules were considered: varia-
tion in the A and B rings as well as in the connecting
chain (see a general structure in Scheme 2).
from compound
cyclization.16,17
1
under acidic intramolecular
2.2.1. Role of ring B. We have previously reported
that the change of the phenyl ring B by heterocyclic
rings such as a-, b- or c-pyridine (compounds 15–21,
23–31 in Table 1) gives very active compounds. In
the present paper, we extend the range of heterocyclic
rings replacing phenyl ring by other heterocycles such
as furan (compounds 34–40) and thiophene (41–45).
Interesting enough, some of them displayed remark-
able activity (compounds 38, 39, 41–43, and 45
MIC = 3.25–50 lg/ml) (Table 2), giving further sup-
port for the bio-isosteric nature of these alternative
aromatic rings.
2.2. Structure–antifungal activity relationship study
Our previous works16,17 provided a considerable diversi-
ty of chemical structures of antifungal homoallylamines
and their derivatives, most compounds possessing two
aromatic rings with a flexible or a rigid connecting unit.
With the aim of understanding the experimental results,
we grouped these compounds into three main classes,
according to the type of connection between both
aromatic rings:

F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
1853
Table 1. MIC values (lg/ml) of homoallylamines and related compounds acting against dermatophytes
Compound
Ref.
Type
Substituents
Activity
Tm
R
R1
R2
R3
R4
X
Mc
Mg
Tr
Ef
1
2
16
17
16
16
16
16
16
17
16
16
16
III
IV
I
—
—
—
—
—
—
H
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
H
H
H
H
H
H
H
H
H
H
H
—
—
H
—
—
>50
>50
30
>50
>50
30
>50
>50
30
>50
>50
30
>50
>50
3
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
Nb
Na
Nb
Nc
Nb
Nb
Nb
—
12.5
3.12
4
I
CH3
H
H
H
30
30
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
30
>50
>50
30
5
I
CH3
H
H
3.12
3.12
3.12
6
I
OCH3
H
H
30
30
7
I
H
OCH3
H
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
>50
>50
50
8
I
H
OCH3
H
>50
>50
>50
>50
—
50
9
I
F
Cl
H
30
30
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Ket.
Amp.
Terb.
I
H
H
I
Br
H
H
H
I
H
H
—
I
H
C2H5
H
H
H
—
—
50
25
—
—
I
H
H
OCH3
H
17
17
17
17
17
17
17
17
16
16
17
17
17
17
17
17
17
17
17
I
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
Allyl
—
H
H
12.5
>50
6.25
50
6.25
50
50
12.5
>50
6.25
50
6.25
12.5
6.25
>50
12.5
>50
6.25
50
1.50
I
CH3
CH3
CH3
F
H
H
>50
50
>50
50
I
H
H
I
H
H
25
25
50
I
H
H
12.5
25
I
Cl
Br
H
H
6.25
3.12
12.5
12.5
>50
3.12
1.50
I
H
H
12.5
>50
25
V
II
II
II
II
II
II
II
II
II
VI
VI
—
H
—
H
—
H
>50
>50
12.5
—
—
CH
CH
Nb
Na
Nb
Nc
Nb
Nb
Nb
Nb
Nc
50
50
25
25
25
25
CH3
H
H
H
25
12.5
50
—
—
H
H
>50
>50
25
50
>50
>50
12.5
25
>50
>50
50
CH3
CH3
CH3
F
H
H
>50
25
25
50
—
—
H
H
H
H
50
25
25
50
50
50
50
25
—
—
H
H
25
Cl
Br
H
H
25
25
50
12.5
>50
>50
>50
6.25
12.5
0.006
12.5
6.25
—
H
H
>50
>50
>50
15
25
>50
>50
25
>50
>50
25
Allyl
Allyl
H
H
H
>50
>50
H
H
H
6.25
12.5
0.006
6.25
12.5
0.003
50
0.3
0.004
0.01
Mc, Microsporum canis C 112; Mg, Microsporum gypseum C 115; Tr, Trichophyton rubrum C 110; Tm, Trichophyton mentagrophytes ATCC 9972; Ef,
Epidermophyton floccosum C 114. Amp., Amphotericin B; Ket., Ketoconazole; Terb., Terbinafine.
From the calculated electron distribution in the mole-
cule, some properties such as the net atomic charges
and bond polarities can be predicted, which help us to
characterize the nature of interactions at specific recep-
tor sites. The electron distribution can also be used to
quantitatively map the electrostatic potential generated
by a molecule in all regions surrounding it.23
The molecular electrostatic potentials (MEPs) for com-
pounds 7 and 15 (Fig. 2) show a closely related stereo-
electronic behavior for both compounds. The 0.002
el/au3 MEP maps of molecules 7 and 15 show three clear
minima V(r)min, being the lowest-energy minima the zone
located in the in-plane lone pair region of N in com-
pound 15, and the minimum located in the proximity

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F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
Table 2. MIC values (lg/ml) of related compounds acting against dermatophytes
ID
Substituents
R3
Activity
Tm
R1
R2
R4
X
Mc
Mg
Tr
Ef
34
35
H
H
H
H
O
O
O
O
O
O
O
S
>50
50
>50
50
>50
25
>50
50
>50
50
H
CH3
H
H
H
36
37
OCH3
OCH3
Br
H
H
>50
>50
>50
50
50
50
>50
25
50
50
CH3
H
H
H
38
39
H
H
6.25
12.5
6.25
12.5
12.5
12.5
>50
25
3.125
6.25
25
3.125
12.5
25
Cl
F
H
H
H
40
41
H
H
H
62.5
25
>50
25
H
H
H
H
12.5
12.5
25
12.5
12.5
12.5
42
43
H
H
H
CH3
H
S
12.5
25
12.5
12.5
125
50
6.25
12.5
0.006
25
50
CH3
H
H
H
S
44
45
H
Allyl
Allyl
H
S
125
6.25
125
6.25
6.25
12.5
0.006
125
12.5
6.25
12.5
0.003
125
6.25
25
0.3
0.004
CH3
H
H
S
Ket.
Amp.
Terb.
15
50
0.01
Mc, Microsporum canis C 112; Mg, Microsporum gypseum C 115; Tr, Trichophyton rubrum C 110; Tm, Trichophyton mentagrophytes ATCC 9972; Ef,
Epidermophyton floccosum C 114. Amp., Amphotericin B; Ket., Ketoconazole; Terb, Terbinafine.
R1
5
4
6
3
1
2
R2
N
H
R1
R2
R3
R4
12-14
NH2
R1
R2
a
R3
b
b
O
1
N
H
2
3
5
R1
R2
R4
4
R3
N
c
Aryl
34-40
R3
c
b
R1
R2
R1
R2
1
2
N
S
N
H
1
4
2
H
5
R4
R1
3
R3
R3
I
3
X
R4
4
Ref.16,17
S
41-43
R2
N
H
R3
R1
44,45
R2
N
H
R3
X
R4
II
Scheme 1. Preparation of N-substituted anilines and homoallylamines. Reagents and conditions: (a) ArCHO, EtOH (benzene or toluene), rt to
reflux; (b) NaBH4, MeOH, rt to reflux; (c) allylmagnesium bromide/Et2O, 15–34 ꢁC, then H2O/NH4Cl/ice.
of the CH3O group in compound 7. The other two min-
ima correspond to the zones located in the vicinity of the
N atom in the connecting chain and in ring A. It should
be noted that the V(r)min, values, in the in-plane lone pair
regions of N, are in accordance with previous findings,24
and indicate that these heteroatoms are H-bond
acceptors.
It was particularly noteworthy that compounds 6 and 7
displayed almost the same antifungal activity. This

F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
1855
Based on these results we postulate a short-range elec-
trostatic interaction for ring B, which may result in
Bro¨nsted (H-bonded) complex formation. Thus, in
an exploratory theoretical study, we simulate the puta-
tive electrostatic interactions between the homoallyl-
amine molecule and its molecular receptor in terms
of representative smaller molecules. For instance,
methyl alcohol was used to mimic the side chain of
a serine residue which is a very good candidate to per-
form H-bonding interactions.25 Alternative moieties
present on ring B of homoallylamines were used as
interacting counterparts (Scheme 2). The use of model
compounds to calculate and simulate molecular inter-
actions (MI) is necessary since homoallylamines are
too large for accurate quantum mechanical MI calcu-
lations and the number of ligand models to be
screened is large. We include electron correlation as
well as solvation energies for studying the relative
binding free energies of ligand–receptor interactions,
which were demonstrated to be necessary in this type
of calculations.25,26
The energies of interaction (EI) were calculated by
approximation neglecting the superimposition of error
due to the difference between the total energies of the
complex with the sum of the total energies of the
components:
EI ¼ ECx ꢀ ðEBC þ EACÞ;
where EI is the energy interaction, ECx is the complex
energy, EBC is the energy of proton-donor component
(i.e., Bro¨nsted acid), and EAC is the energy of proton
acceptor component (i.e., Bro¨nsted base).
Figure 1. Spatial view of the preferred conformations obtained for
homoallylamines, tetrahydroquinolines, and quinolines using RHF/6-
31G(d) calculations.
The energies of all the complexes obtained and their
components are summarized in Table 3. The interaction
energies obtained for the different complexes are also
shown in this table.
result can be explained by the ability of compound 6 to
adopt a conformation very similar to that of compound
7. In this spatial ordering, the CH3O group of ring B in
compound 7 and the CH3O group of ring A in com-
pound 6 overlap, whereas the benzene ring A of 7 eclips-
es the benzene ring B of 6. The similarity between the
spatial ordering adopted by both compounds 6 and 7
is shown in Figure 3 where it is also possible to appreci-
ate the similar spatial position for the lone pairs of their
respective N atoms. In short, ring A of 7 is acting simi-
larly to ring B in the whole structure of compound 6.
The antifungal activity shown by 6, 7, and 8 provides
additional support for this hypothesis: whereas com-
pounds 6 and 7 were active, compound 8 was inactive.
It should be noted that the only structural difference
between compounds 6 and 8 is the spatial position of
the OCH3 group in ring A (in fact, they are simple
positional isomers). It appears that the spatial orienta-
tion of the substituents in the general structural scaffold
confers high affinity to the ligands. These results suggest
that a spatially oriented molecular interaction (i.e., type
hydrogen bond or dipole–dipole) could be operative in
this case. Additional evidence for this hypothesis comes
from the different activities of compounds 16, 17, and 18
as well as those displayed by 26, 27, and 28. Note the
different antifungal activity of these compounds as a
function of the different positions of the nitrogen atom
at ring B.
Figure 4 gives the different geometries obtained for com-
plexes I–IV. The starting geometries of complexes I–III
converged to the same kind of hydrogen-bonding inter-
actions for such complexes, independent of the level of
theory used. In contrast, complex IV displayed a clear
electrostatic interaction but not hydrogen bonding be-
cause of an improper orientation.
Results obtained for complexes I–IV (Fig. 4 and Ta-
ble 3) indicate that the most favored interaction oc-
curs when the N atom of pyridine ring is acting as
an acceptor, while the CH3–OH group is the donor
counterpart (compare the energies obtained for com-
plexes I–IV, Table 3). These results are in agreement
with our experimental data, showing that compounds
possessing N and O groups at ring B are more active
in comparison to those molecules possessing a S
group.
Previous theoretical and crystallographic studies com-
paring oxygen and sulfur as H-bond acceptors showed
that oxygen donates its nr lone pair, whereas sulfur do-
nates its np lone pair.27,28 Our results are in agreement
with these results.

1856
F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
Ring
A
Ring
B
Connecting Chain
Scheme 2.
2.2.2. Role of the connecting chain. The lengthening of
the flexible connecting chain caused a dramatic decrease
in the antifungal activity. Compound 2, which is the
higher homologue of 3, was inactive. In addition, com-
pounds 32 and 33, possessing an extra methylene group
in the other side of the connecting chain, were also inac-
tive compounds. It should be noted that 32 is a higher
homologue of compound 15. These results suggest that
the distance between both aromatic rings A and B plays
a critical role for the antifungal activity.
In order to obtain more information about the role of
the allyl radical, we change the structure of the connect-
ing-chain moiety. Elimination of the allyl moiety result-
ed in a slight increase of antifungal activity (compare
compounds 7 and 14). A comparative conformational
study of these compounds was carried out from ab initio
RH/3-21G calculations. The potential energy surfaces
(PES) varying the torsional angles TA1 versus TA2 each
30ꢁ are shown in Figure 5. From this figure it is clear
that the molecular flexibility of compound 14 is signifi-
cantly higher than that obtained for compound 7 (note
the different sizes of the dark zones in the contour dia-
grams and the different deeps and slopes of the valleys
in the landscape diagrams). It appears that a more
flexible connecting chain could facilitate the binding
process.
Figure 2. Electrostatic potential-encoded electron density surfaces of
the core structures of compounds 7 and 15. The surfaces were
generated with Gaussian 03 after ab initio minimization with a 6-
31G(d,p) basis set. The coloring represents electrostatic potential with
red indicating the strongest attraction to a positive point charge and
blue indicating the strongest repulsion. The electrostatic potential is
the energy of interaction of the positive point charge with the nuclei
and electrons of a molecule. It provides a representative measure of
overall molecular charge distribution.
analysis, it becomes clear that replacing hydrogen with a
bromide (or another halogen group) leads to a consider-
able perturbation of the electron distribution of ring A
in these compounds. This electrostatic effect could offer
a complementary surface in the binding pocket. The
core structure of compound 21 could therefore represent
the necessary electronic configuration to achieve signifi-
cant antifungal activity.
2.2.3. Role of ring A. It appears that the presence of a
non-substituted phenyl ring A leads to antifungal com-
pounds (compounds 3, 7, 15, and 25). However, it
should be noted that the presence of different substitu-
ents like CH3 or halogen as R1 enhances the antifungal
activity (compare 17, 19, 20, and 21 with 15, and 27, 29,
30, and 31 with 25).
At this stage of our SAR study, some general trends
might be established. Compounds possessing both a hal-
ogen substituent (F, Cl or Br) at ring A and a heterocy-
cle as ring B (pyridine, thiophene or furan) were the
most active compounds in this series. Particularly note-
worthy was the lack of activity obtained for compounds
9–11, even though they possess halogen substituents at
ring A. However, it should be noted that these homoal-
lylamines have a non-substituted phenyl group as ring
B. Thus, the lack of activity of these compounds high-
lights the importance of apparently two active centers
for these compounds. Consequently, the antifungal
activity of homoallylamines and their analogues report-
ed here appears to be dependent on the combination of
at least, two binding sites, one of them (ring B) possess-
ing electronic characteristics to produce hydrogen bonds
or polar interactions.
According to these results, ring A seems to play a central
role in the recognition process. It was worthwhile to
investigate how the change in distribution of charge
due to the presence of CH3 or halogen substituents as
R1 influences its nature. The knowledge of the inherent
electronic properties of the ring A moiety of homoallyl-
amines is important for rationalizing their complemen-
tarity with the binding site. To explore the degree of
electronic changes in ring A due to the presence of
halogen substituents at the R1 positions, MEPs of com-
pounds 15, 17, and 21 were examined.
Electron density surfaces encoded with the electrostatic
potential obtained by ab initio computations demon-
strate that 17 and 21 have a closely related polarized
electron distribution at ring A (Figs. 6b and c). In com-
parison, the analogous graph for compound 15 shows a
very different and much less polarized electrostatic po-
tential surface in this ring (Fig. 6a). On the basis of this
Finally we wish to discuss some details about a putative
overall recognition process between the homoallylamine
molecule and its potential biological receptor. On the

F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
1857
Figure 3. Stereo-view showing the superimposition of compounds 6 and 7 using the pharmacophoric sites (CH3O group).
Table 3. Energies obtained at different levels of theory for the complexes and their components
Complex energy
(in hartrees)
Energy interaction of complexes
(in kcal molꢀ1
)
RHF/6-31G
RB3LYP/6-31++G**
RHF/6-31G
RB3LYP/6-
31++G**
In vacuo
IPCM
In vacuo
IPCM
I—(methanol/3-methyl-pyridine)
ꢀ400,615862
ꢀ498,470371
ꢀ382,550885
ꢀ705,208540
ꢀ403,364558
ꢀ501,855868
ꢀ385,101265
ꢀ708,072265
ꢀ403,377316
ꢀ501,863450
ꢀ385,109571
ꢀ708,080023
ꢀ7.43
ꢀ6.84
ꢀ6.54
ꢀ3.18
ꢀ4.61
ꢀ4.27
ꢀ3.96
ꢀ1.53
ꢀ3.01
ꢀ1.13
ꢀ1.45
0.90
II—(methanol/1-methoxy-4-methyl-benzene)
III—(methanol/2-methyl-furan)
IV—(methanol/2-methyl-thiophene)
Energy of interacting components (in hartrees)
3-Methyl-pyridine
1-Methoxy-4-methyl-benzene
2-Methyl-furan
ꢀ285,615852
ꢀ383,471302
ꢀ267,552296
ꢀ590,215311
ꢀ114,988166
ꢀ287,623346
ꢀ386,115199
ꢀ269,361090
ꢀ592,335970
ꢀ115,733858
ꢀ287,630355
ꢀ386,119499
ꢀ269,365107
ꢀ592,339296
ꢀ115,742157
2-Methyl-thiophene
Methanol
Interaction energies obtained for the four complexes are also shown in this table.
basis of our results and using the simple notion of recep-
tor-site occupancy, one may seek common chemical fea-
tures between homoallylamines and their structurally
related compounds to suggest chemical binding sites.
Thus, we propose that the OCH3 group or the lone pairs
of heteroatoms N, O or S atoms of ring B engage the
receptor at a specific site (site I, Fig. 7). If homoallyl-
amine engages the receptor at the site I, then it is possi-
ble to envisage that the rest of the homoallylamine
molecules contribute to the additional binding by inter-
acting with at least one accessory region. The molecular
structure of each of these compounds appears to be crit-
ical for antifungal activity. The fact that the activity is
markedly affected by altering the length of the connect-
ing chain suggests a co-operative effect between active
groups, and one may consider that the second aromatic
ring A makes a specific contribution to the binding via
an aromatic ring orientation (site II, Fig. 7). In fact,

1858
F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
Among them compounds 38 and 39 reported here and
some of their congeners previously reported exhibited
remarkable antifungal activity against dermatophyte
fungi. A detailed SAR study supported by theoretical
calculations helped us to identify and understand the
minimal structural requirements for the antifungal
action of these compounds.
On the basis of computational studies, it is possible to
depict the salient structural requirements of homoallyl-
amines and tetrahydroquinolines for displaying anti-
fungal activity:
• Presence of two aromatic rings (rings A and B).
• Presence of a heteroatom (with electronic lone pairs)
or CH3O groups on ring B.
• A particular length of the connecting chain.
• Presence of a halogen atom (particularly Br or Cl) as
R1 on ring A.
Figure 4. Spatial view of complexes I–IV obtained from RB3LYP/6-
31++G(d,p) calculations. The interactions are shown as involving
surface mesh.
there are various ways in which this moiety of the mol-
ecules may be involved. Thus, we may assume that a flat
portion of the receptor could allow binding with the aro-
matic ring A of the antifungal compound through dis-
persion forces (van der Waals). An aromatic residue of
the receptor could provide such a surface for example,
but this is speculative. For the conformationally restrict-
ed compounds reported here, the proper ‘‘anchoring’’ of
the benzene ring could be very important in a molecule
to facilitate the binding mechanism. Thus, a kind of
stepwise binding involving first site I followed by the rest
of the molecule (site II) seems a reasonable possibility.
We believe our results may be helpful in the structural
identification and understanding of the minimum struc-
tural requirements for these molecules and can provide a
guide in the design of compounds with these inhibitory
properties.
4. Experimental section
4.1. Chemistry
Melting points were determined with a Fisher–Johns
melting point apparatus and are not corrected. Infrared
spectra were recorded using KBr pellets on a Nicolet
Avatar Model 360 FTIR spectrophotometer. Nuclear
magnetic resonance spectra were measured on a Bruker
Since the molecules reported here are racemic, it is not
possible to establish the potential role of the sp3 chiral
carbon in the antifungal activity. It is clear that the
resolution of the more active compounds into the two
enantiomers could aid in establishing the difference
between the activity of each one. A next step in our
research will be the enantioselective syntheses of the
most interesting molecules of the series.
1
AM 400 instrument (400 MHz H NMR and 100 MHz
13C NMR), using chloroform-d as solvent and tetra-
methylsilane as internal standard. Chemical shifts (d)
and coupling constants (J) are reported in ppm and
Hz, respectively. A Hewlett–Packard (HP) 5890 A series
II Gas Chromatograph interfaced to a HP 5972 Mass
Selective Detector with a HP MS ChemStation Data
system was used for MS identification. Elemental analy-
ses were performed on a Perkin Elmer 2400 Series II
analyzer and were within 0.4 of theoretical values.
3. Conclusions
We report here a new group of homoallylamine and tet-
rahydroquinoline derivatives acting as antifungal agents
that adds information to previously reported studies.
Figure 5. Relaxed conformational potential energy surfaces (PES), E = E(TA1, TA2) of compounds 7 and 14. Top: energy landscape, bottom: energy
contour representations.

F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
1859
4.1.2. N-Benzyl-N-(4-ethylphenyl)amine (13). Yellow oil.
Yield 83%. IR (film): m 3417, 1615 cmꢀ1 (s, NH). MS m/z
(EI) 211 (M+, 63), 196 (70), 134 (11), 105 (4), 91 (100),
77 (10), 65 (14), 51 (4). Found: C, 85.10; H, 7.98; N,
6.91; calcd for C15H17N: C, 85.24; H, 7.66; N, 7.10.
4.1.3. N-(4-Methoxybenzyl)-N-phenylamine (14). Color-
less crystals. Mp 61–62 ꢁC. Yield 85%. IR (KBr): m
3418, 1604 cmꢀ1 (s, NH). 1H NMR: d 7.31 (2H, d,
J = 8.4 Hz, 3-HBn and 5-HBn), 7.19 (2H, t, J = 7.6 Hz,
3-HPh and 5-HPh), 6.89 (2H, d, J = 8.7 Hz, 2-HBn and
6-HBn), 6.75 (1H, t, J = 7.5 Hz, 4-HPh), 6.67 (2H, d,
J = 7.6 Hz, 2-HPh and 6-HPh), 4.27 (2H, s, –CH2–N),
3.82 (3H, s, CH3O); 13C NMR (CDCl3): d 158.9,
147.8, 131.1, 129.3, 129.2, 128.9, 128.8, 117.8, 114.0,
113.9, 113.2, 113.1, 55.3, 48.0. MS m/z (EI) 213 (M+,
29), 121 (100), 91 (4), 65 (3), 51 (3). Found: C, 78.55;
H, 7.20; N, 6.20; calcd for C14H15NO: C, 78.84; H,
7.09; N, 6.57.
4.1.4. N-(2-Furylmethyl)-N-phenylamine (34). Yellow li-
quid. Yield 99%. IR (film): m 3411, 1504 cmꢀ1 (s, NH).
1H NMR: d 7.40 (1H, dd, J = 2.0, 1.0 Hz, 5-HFu), 7.22
(2H, m, 3-HPh and 5-HPh), 6.78 (1H, tt, J = 7.5,
1.0 Hz, 4-HPh), 6.71 (2H, m, 2-HPh and 6-HPh), 6.35
(1H , dd, J = 3.0, 2.0 Hz, 4-HFu), 6.27 (1H, dd,
J = 3.0, 1.0 Hz, 3-HFu), 4.34 (2H, s, –CH2–N), 4.04
(1H, br s, NH); 13C NMR: d 152.7, 147.6, 141.9,
129.2, 118.0, 113.1, 110.3, 106.9, 41.4. MS m/z (EI)
173 (M+, 39), 172 (25), 144 (6), 81 (100), 65 (10), 53
(33). Found: C, 76.56; H, 6.33; N, 8.20; calcd for
C11H11NO: C, 76.28; H, 6.40; N, 8.09.
Figure 6. Electrostatic potential-encoded electron density surfaces of
compounds 15 (a), 17 (b), and 21 (c) The surfaces were generated with
Gaussian 03 after ab initio minimizations with a 6-31G(d,p) basis set.
The color-coded is shown at the left.
Site II
4.1.5. N-[(5-Methyl-2-furyl)methyl]-N-phenylamine (35).
Yellow liquid. Yield 90%. IR (film): m 3411, 1567 cmꢀ1
(s, NH); H NMR: d 7.22 (2H, m, 3-HPh and 5-HPh),
Hidrofobic
binding
R
A
1
N
H
6.77 (1H, tt, J = 7.0, 1.0 Hz, 4-HPh), 6.71 (2H, m, 2-
HPh and 6-HPh), 6.14 (1H, d, J = 3.0 Hz, 4-HFu), 5.93
(1H, dd, J = 3.0, 1.0 Hz, 3-HFu), 4.28 (2H, s, –CH2–
N), 3.95 (1H, br s, NH), 2.31 (3H, s, 5-CH3); 13C
NMR: d 151.5, 150.7, 147.7, 129.1, 117.9, 107.8,
106.1, 41.5, 13.5. MS m/z (EI) 187 (M+, 19), 95
(100), 77 (10), 65 (9), 51 (10), 43 (16). Found: C,
76.55; H, 7.15; N, 7.20; calcd for C12H13NO: C,
76.98; H, 7.00; N, 7.48.
B
OCH3
N
Site I
Polar
Interaction
Figure 7. Schematic representation of a reduced model for the active
sites of homoallylamines, showing the two putative binding sites (sites
I and II).
The reaction progress was monitored using thin layer
chromatography on a silufol UV254 TLC aluminum
sheet. Column chromatography (2 · 60 cm) was carried
out using Merck silica gel 60 (230–400 mesh). All re-
agents and solvents were purchased from Merck, Sigma,
and Aldrich Chemical Co., and were used without
further purification.
4.1.6. N-(2-Furylmethyl)-N-(4-methoxyphenyl)amine (36).
Yellow solid. Mp 41 ꢁC. Yield 95 %. IR (KBr): m
1
3407 cmꢀ1 (s, NH). H NMR: d 7.35 (1H, dd, J = 2.0,
1.0 Hz, 5-HFu), 6.80 (2H, m, 3-HPh and 5-HPh), 6.66
(2H, m, 2-HPh and 6-HPh), 6.33 (1H, dd, J = 3.0,
2.0 Hz, 4-HFu), 6.23 (1H, dd, J = 3.5, 1.0 Hz, 3-HFu),
4.28 (2H, s, –CH2–N), 3.63 (1H, br s, NH); 13C NMR:
d 153.0, 152.5, 141.8, 114.8, 114.6, 110.3, 106.9, 55.7,
42.4. MS m/z (EI) 203 (M+, 64), 202 (34), 122 (86), 108
(6), 95 (15), 81 (100), 65 (8), 53 (41). Found: C, 71.05;
H, 7.81; N, 6.33; calcd for C12H13NO2: C, 70.92; H,
6.45; N, 6.89.
Preparation of N-substituted anilines 12–14, 32–38, and
41–43 was realized according to reported methodolo-
gies.21,22 Compounds 12 and 13 are known.29
4.1.1. N-Benzyl-N-phenylamine (12). Yellow oil. Yield
85%. IR (film): m 3419, 1602 cmꢀ1 (s, NH). MS m/z (EI)
183 (M+, 61), 106 (19), 91 (100), 77 (23), 65 (25), 51 (16).
Found: C, 85.45; H, 7.01; N, 7.44; calcd for C13H13N:
C, 85.21; H, 7.15; N, 7.64.
4.1.7. N-(4-Methoxyphenyl)-N-[(5-methyl-2-furyl)methyl]-
amine (37). Yellow liquid. Yield 98%. IR (film): m
1
3411 cmꢀ1 (NH). H NMR: d 6.80 (2H, m, 3-HPh and
5-HPh), 6.67 (2H, m, 2-HPh and 6-HPh), 6.11 (1H, d,

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F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
J = 3.0 Hz, 4-HFu), 5.91 (1H, dd, J = 3.0, 1.0 Hz, 3-HFu),
4.21 (2H, s, –CH2–N), 3.76 (3H, s, 4-CH3O), 3.56 (1H, br
s, NH); 13C NMR: d 152.4, 151.4, 150.9, 141.9, 114.7,
107.7, 106.0, 55.6, 42.4, 13.45. MS m/z (EI) 217 (M+,
17), 123 (24), 108 (10), 95 (100), 77 (3), 65 (4), 51 (4),
43 (11). Found: C, 71.55; H, 7.05; N, 6.20; calcd for
C13H15NO2: C, 71.87; H, 6.96; N, 6.45.
124.9, 124.3, 117.1, 113.9 (2C), 49.5, 20.1. MS m/z (EI) 203
(M+, 31), 97 (100), 91 (7), 77 (7), 65 (6), 53 (8), 45 (6). Found:
C, 71.02; H, 6.67; N, 6.54; calcd for C12H13NS: C, 70.89; H,
6.45; N, 6.89.
4.1.13. N-(3-Methylphenyl)-N-(thien-2-ylmethyl)amine (43).
Yellow oil. Yield 97%. IR (film): m 3408 cmꢀ1 (s, NH). 1H
NMR: d 7.21 (1H, dd, J = 5.0, 1.0, 5-HThie), 6.98 (1H, dd,
J = 4.0, 3.0, 4-HThie), 6.97 (1H, t, J = 7.5, 5-HPh), 6.96 (1H,
dd, J = 4.0, 2.0, 3-HThie), 6.67 (1H, s, 2-HPh), 6.66 (1H, d,
J = 7.0, 6-HPh), 6.60 (1H, d, J = 7.0, 4-HPh), 4.69 (2H, s,
N–CH2–), 2.34 (3H, s, 3-CH3); 13C NMR: d 148.6,
142.9, 138.8, 129.0, 126.7, 124.6, 124.2, 118.1, 113.8,
110.3, 49.4, 21.9. MS m/z (EI) 203 (M+, 42), 97 (100), 91
(9), 77 (7), 65 (7), 53 (8), 45 (8). Found: C, 70.77; H,
6.76; N, 6.73; calcd for C12H13NS: C, 70.89; H, 6.45; N,
6.89.
4.1.8. N-(4-Bromophenyl)-N-(2-furylmethyl)amine (38).
Yellow solid. Mp 38 ꢁC. Yield 97%. IR (KBr): m
1
3415 cmꢀ1 (s, NH). H NMR: d 7.40 (1H, dd, J = 2.0,
1.0 Hz, 5-HFu), 7.29 (2H, m, 3-HPh and 5-HPh), 6.57
(2 H, m, 2-HPh and 6-HPh), 6.36 (1H, dd, J = 3.0,
2.0 Hz, 4-HFu), 6.26 (1H, dd, J = 3.0, 1.0 Hz, 3-HFu),
4.30 (2H, s, –CH2–N), 4.10 (1H, br s, NH); 13C NMR:
d 152.1, 146.5, 142.0, 131.8, 114.6, 110.3, 109.5, 107.1,
41.2. MS m/z (EI) 252 (M+, Br, 15), 251 (18), 91 (7),
81 (100), 63 (8), 53 (27). Found: C, 52.84; H, 4.20; N,
5.71; calcd for C11H10BrNO: C, 52.41; H, 4.00; N, 5.56.
Preparation of N-aryl-N-[1-(thien-2-yl)but-3-enyl]amines
44, 45 was realized according to reported
methodologies.16,17
4.1.9. N-(4-Chlorophenyl)-N-(2-furylmethyl)amine (39).
Yellow liquid. Yield 99%. IR (film): m 3415 cmꢀ1 (s,
NH). 1H NMR: d 7.38 (1H, dd, J = 2.0, 1.0 Hz, 5-
HFu), 7.14 (2H, m, 3-HPh and 5-HPh), 6.60 (2H, m, 2-
HPh and 6-HPh), 6.34 (1H dd, J = 3.5, 1.5 Hz, 4-HFu),
6.24 (1H, dd, J = 3.0, 1.0 Hz, 3-HFu), 4.29 (2H, s, –
CH2–N), 4.06 (1H, br s, NH); 13C NMR: d 152.2,
146.1, 142.0, 129.0, 122.5, 114.2, 110.3, 107.2, 41.4.
MS m/z (EI) 207 (M+, 35Cl, 23), 206 (14), 81 (100), 75
(5), 63 (3), 53 (17). Found: C, 63.87; H, 4.63; N, 7.03;
calcd for C11H10ClNO: C, 63.62; H, 4.85; N, 6.75.
4.1.14. N-(1-thien-2-yl)but-3-enyl]aniline (44). Yellow
viscous oil. Bp 124–126 ꢁC/1 mm Hg. Yield 70%. IR
(film): m 3405 (s, NH), 1639 (s, C@C), 920 cmꢀ1 (s,
1
@CH2). H NMR: d 7.18 (1H, dd, J = 4.0, 1.0 Hz, 5-
HThie), 7.14 (2H, t, J = 8.0 Hz, 3-HPh and 5-HPh), 6.99
(1H, d, J = 4.0 Hz, 4-HThie), 6.95 (1H, dd, J = 4.0,
1.0 Hz, 3-HThie), 6.71 (1H, t, J = 8.0 Hz, 4-HPh), 6.61
(2H, d, J = 8.0 Hz, 2-HPh and 6-HPh), 5.82 (1H, m, –
CH@), 5.19 (2H, m, @CH2), 4.72 (1H, t, J = 8.0 Hz, 1-
H ), 2.67 (2H, m, –CH2–); 13C NMR: d 148.6, 147.0,
134.0, 129.1, 126.8, 123.7, 123.4, 118.0, 113.6, 53.4,
43.1. MS m/z (EI) 229 (M+, 4), 188 (100), 104 (21), 97
(5), 91 (4), 83 (1), 77 (25), 63 (2). Found: C, 73.55; H,
6.34; N, 6.20; calcd for C14H15NS: C, 73.32; H, 6.59;
N, 6.11.
4.1.10. N-(4-Fluorophenyl)-N-(2-furylmethyl)amine (40).
Yellow liquid. Yield 99%. IR (film): m 3413 cmꢀ1 (s,
NH). 1H NMR: d 7.39 (1H, dd, J = 2.0, 1.0 Hz, 5-
HFu), 6.91 (2H, m), 6.63 (2H, m, 3-HPh and 5-HPh),
6.34 (1H, dd, J = 3.0, 2.0 Hz, 2-HPh and 6-HPh), 6.24
(1H, dd, J = 3.5, 1.0 Hz, 4-HFu), 4.29 (2H, s, –CH2–
N), 3.92 (1H, br s, NH); 13C NMR: d 157.3, 154.9,
152.5, 143.9, 141.9, 115.7, 115.5, 114.1, 110.3, 107.0,
42.0. MS m/z (EI) 191 (M+, 30), 190 (19), 95 (8), 81
(100), 75 (5), 53 (20). Found: C, 69.35; H, 5.43; N,
7.54; calcd for C11H10FNO: C, 69.10; H, 5.27; N, 7.33.
4.1.15.
N-4-Methylphenyl-N-(1-thien-2-yl)but-3-enyl]-
amine (45). Yellow viscous oil. Bp 120–122 ꢁC/1 mm Hg.
Yield 77%. IR (film): m 3403 (s, NH), 1639 (s, C@C),
1
919 cmꢀ1 (s, @CH2). H NMR: d 7.17 (1H, dd, J = 4.0,
1.0 Hz, 5-HThie), 6.99 (1H, dd, J = 4.0, 1.0 Hz, 4-HThie),
6.97 (1H, t, J = 4.0 Hz, 3-HThie), 6.96 (2H, d,
J = 8.0 Hz, 3-HPh and 5-HPh), 6.55 (2H, d, J = 8.0 Hz,
2-HPh and 6-HPh), 5.81 (1H, m, –CH@), 5.19 (2H, m,
@CH2), 4.69 (1H, t, J = 8.0 Hz, 1-H), 2.67 (2H, m,
–CH2–), 2.23 (3H, s, 4-CH3); 13C NMR: d 148.9, 144.7,
129.6, 134.2, 127.2, 126.7, 123.7, 123.4, 118.51, 113.8,
53.8, 43.1, 20.4. MS m/z (EI) 243 (M+, 5), 202 (100), 186
(2), 118 (13), 111 (1), 105 (2), 97 (5), 91 (20), 77 (5). Found:
C, 73.87; H, 7.22; N, 5.63; calcd for C15H17NS: C, 74.03;
H, 7.04; N, 5.76.
4.1.11. N-(Thien-2-ylmethyl)aniline (41). Yellow oil. Yield
1
95%. IR (film): m 3411 cmꢀ1 (s, NH). H NMR: d 7.23
(2H, t, J = 8.0, 3-HPh and 5-HPh), 7.19 (1H, dd, J = 4.0,
1.0, 5-HThie), 6.96 (1H, dd, J = 4.0, 1.0, 3-HThie), 6.95
(1H, t, J = 4.0, 4-HThie), 6.82 (2H, d, J = 8.0, 2-HPh and
6-HPh), 6.75 (1H, d, J = 8.0, 4-HPh), 4.68 (2H, s, –CH2–
N); 13C NMR: d 148.4, 142.7, 126.7, 124.6, 124.2, 116.5,
129.1 (2C), 113.1 (2C), 49.5. MS m/z (EI) 189 (M+, 33),
97 (100), 91 (3), 77 (15), 65 (9), 53 (10), 45 (15). Found:
C, 69.73; H, 5.45; N, 7.52; calcd for C11H11NS: C, 69.80;
H, 5.86; N, 7.40.
4.1.16. Microorganisms and media. For the antifungal
evaluation, strains from the American Type Culture
Collection (ATCC), Rockville, MD, USA, and CERE-
4.1.12. N-(4-Methylphenyl)-N-(thien-2-ylmethyl)amine (42).
Yellow oil. Yield 96%. IR (film): m 3408 cmꢀ1 (s, NH). 1H
NMR: d 7.20 (1H, dd, J = 4.0, 1.0, 5-HThie), 7.06 (2H, d,
J = 8.0, 3-HPh and 5-HPh), 6.97 (1H, t, J = 4.0, 3-HThie),
6.94 (1H, dd, J = 4.0, 1.0, 4-HThie), 6.76 (2H, d, J = 8.0, 2-
HPh and 6-HPh), 4.66 (2H, s, –CH2–N), 2.28 (3H, s, 4-
CH3); 13C NMR: d 148.43 (1C), 142.7, 128.1 (2C), 126.8,
´
MIC (C), Centro de Referencia Micologica, Facultad
´
´
de Ciencias Bioquımicas y Farmaceuticas, Suipacha
531-(2000)-Rosario, Argentina, were used: Microsporum
canis C 112, Microsporum gypseum C 115, Trichophyton
rubrum C 110, Trichophyton mentagrophytes ATCC
9972, and Epidermophyton floccosum C 114. Strains were

F. D. Suvire et al. / Bioorg. Med. Chem. 14 (2006) 1851–1862
1861
grown on Sabouraud-chloramphenicol agar slants for
48 h at 30 ꢁC, maintained on slopes of Sabouraud-dex-
trose agar (SDA, Oxoid), and subcultured every 15 days
to prevent pleomorphic transformations. Inocula of
spore suspensions were obtained according to reported
procedures30 and adjusted to 1–5 · 103 spores with colo-
ny-forming units (CFU)/ml.
both on the interaction sites of the molecules with point
charges and on the comparative reactivities of these
sites.38–41 These MEPs were calculated from RHF/6-
31G(d,p) wave functions using the SPARTAN pro-
gram.42 All the calculations were performed on a cluster
of PC Pentium 4. Each MI optimizations takes about
48 h. Previous reports25,26 indicate that it is necessary
to include correlation energy corrections and solvent
effects in these calculations to obtain reliable results.
4.1.17. Antifungal susceptibility testing. The minimal
inhibitory concentration (MIC) of each extract was
determined by using broth microdilution techniques fol-
lowing the guidelines of the National Committee for
Clinical Laboratory Standards for filamentous fungi
(M-38A) (NCCLS, 2002).31 MIC values were deter-
mined in RPMI-1640 (Sigma, St. Louis, MO, USA) buf-
fered to a pH 7.0 with MOPS. Microtiter trays were
incubated at 35 ꢁC for yeasts and hialophyphomycetes,
and at 28–30 ꢁC for dermatophyte strains in a moist,
dark chamber, and MICs were recorded at 48 h for
yeasts, and at a time according to the control fungus
growth, for the rest of fungi. The susceptibilities of the
standard drugs Ketoconazole, Terbinafine, and Ampho-
tericin B were defined as the lowest concentration of
drug which resulted in total inhibition of fungal growth.
Acknowledgments
This work was supported by grants from Universidad
´
´
Nacional de San Luis and Agencia de Promocion Cientı-
fica y Tecnologica de la Argentina (PICTR #00260).
´
This work is part of the Iberoamerican Project PIBEA-
FUN X.7 (Search and development of new antifungal
agents) of CYTED (Iberoamerican Program of Science
and Technology for the Development) Subprogram X.
R. D. Enriz is a member of the CONICET (Argentina)
staff. V.K. thanks Instituto Colombiano para el Desarr-
´
´
ollo de la Ciencia y la Tecnologıa ÔFrancisco Jose de
CaldasÕ (COLCIENCIAS, Proyecto: 1102-05-11429)
for financial support and Dr. Arilio R. Palma for pro-
viding some thiophene dervatives.
For the assay, extract stock solutions were twofold
diluted with RPMI-1640 from 250 to 0.98 lg/ml (final
volume = 100 ll) and a final DMSO concentration
61%. A volume of 100 ll of inoculum suspension was
added to each well with the exception of the sterility
control where sterile water was added to the well in-
stead. The MIC was defined as the minimum inhibitory
concentration of the extract which resulted in total inhi-
bition of the fungal growth.
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