Novel isoquinoline derivatives as antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2013.03.042

The wide variety of potent biological activities of natural and synthetic isoquinoline alkaloids encouraged us to develop novel antimicrobial isoquinoline compounds. We synthesized a variety of differently functionalized 1-pentyl-6,7-dimethoxy-1,2,3,4-tet

Full text of DOI:10.1016/j.bmc.2013.03.042

Bioorganic & Medicinal Chemistry 21 (2013) 3221–3230
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel isoquinoline derivatives as antimicrobial agents
Abraham Galán a, Laura Moreno a, Javier Párraga a, Ángel Serrano a, Ma Jesús Sanz b,c, Diego Cortes a,
Nuria Cabedo d,
⇑
a Departamento de Farmacología, Laboratorio de Farmacoquímica, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46013 Valencia, Spain
c Institute of Health Research-INCLIVA, University Clinic Hospital of Valencia, Valencia, Spain
d Centro de Ecología Química Agrícola-Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, Edificio 6C, 46022 Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The wide variety of potent biological activities of natural and synthetic isoquinoline alkaloids encouraged
us to develop novel antimicrobial isoquinoline compounds. We synthesized a variety of differently func-
tionalized 1-pentyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines (THIQs), including dihydroisoquino-
linium salts (2 and 5), methyl pentanoate-THIQ (6), 1-pentanol-THIQ (7), ester derivatives (8–15) and
carbamate derivatives (16–23). We employed classic intramolecular Bischler–Napieralski cyclodehydra-
tion to generate the isoquinoline core. All the structures were characterized by nuclear magnetic reso-
nance and mass spectrometry. The bactericide and fungicide activities were evaluated for all the
synthesized compounds and structure-activity relationships were established. Many compounds exhib-
ited high and broad-range bactericidal activity. Fluorophenylpropanoate ester 13 and the halogenated
phenyl- (17, 18) and phenethyl carbamates (21, 22) exerted the most remarkable bactericidal activity.
However, few compounds displayed antifungal activity against most of the fungi tested. Among them,
chlorinated derivatives like chlorobenzoate and chlorophenylpropanoate esters (10 and 14, respectively)
and chlorophenethyl carbamate 22, exhibited the greatest antifungal activity.
Received 7 December 2012
Revised 1 March 2013
Accepted 11 March 2013
Available online 2 April 2013
Keywords:
Esters and carbamates 1-alkyl-isoquinolines
Bactericide
Fungicide
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
this background, we decided to synthesize sixteen new N-
methyl-6,7-dimethoxy-1,2,3,4-THIQs with a functionalized pentyl
Isoquinoline alkaloids are natural and synthetic products with a
wide variety of potent biological activities,1,2 including inhibition
of cell proliferation,3,4 b-adrenergic receptor antagonism,5 and
inhibition of enzymes related to the metabolism of catecholamines
and indoleamines, such as monoamino oxidase.6 In the last few
decades, many natural tetrahydroisoquinolines (THIQs) with po-
tent antitumor and antimicrobial activities have been isolated.7,8
Recently, some potent antifungal THIQs have been synthesized
chain at the 1-position, containing functionalized esters or carba-
mates. Among all the methods described for synthesizing the iso-
quinoline core, we decided to apply Bischler–Napieralski
cyclodehydration.2,17,18 Then, we performed an N-alkylation of
the unstable imine and a subsequent reduction to obtain N-
methyl-1-substituted-1,2,3,4-THIQs. Once synthesized, we next
investigated the potential antibacterial and antifungal activities
of these N-methyl-1-substituted-THIQs, including the new 1-pen-
tyl-THIQs (eight esters and eight carbamates), as well as some of
their precursors. Taken into account the results obtained in the
biological assays, we analyzed the relevance of carbamate, ester
or alcohol groups at the end of the alkyl chain at the 1-position
of the THIQ nucleus. Therefore, we have been able to establish a
structure–antimicrobial activity relationship among all the synthe-
sized THIQs.
based on the structure of lanosterol 14a-demethylase (CYP51), a
fungal key enzyme involved in sterol biosynthesis.9,10 In addition,
some potent antibacterial 1-aryl-6,7-dimethoxy-1,2,3,4-THIQs
have been synthesized in recent years.11
Our research group has long since focused on isolating and syn-
thesizing isoquinoline-containing compounds with dopaminer-
gic12–15 and antitumor4 activity. However, since the development
of resistance to classic antibiotics is a cause for concern for both
agricultural pest control and human health, we have recently de-
voted our research efforts to find new antimicrobial agents. There-
fore, we studied the bactericidal and fungicidal effects of new
synthetic antimicrobial pyrrolo[2,1-a]isoquinolin-3-ones.16 Given
2. Results and discussion
2.1. Chemistry
The approach used to synthesize 1-butyl-THIQ, and the
differently functionalized 1-pentyl-THIQs, was based on the
preparation of N-(3,4-dimethoxy-phenthyl)pentanamide 1 and
⇑
Corresponding author. Tel.: +34 963879058; fax: +34 963879059.
E-mail addresses: ncabedo@uv.es, ncabedo@ceqa.upv.es (N. Cabedo).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.042

3222
A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
N-(3,4-dimethoxyphenethylamino)-6-oxohexanoate 4, respec-
tively. Both amides were obtained under Schotten–Baumann con-
ditions with subsequent Bischler–Napieralski cyclodehydration to
generate the isoquinoline core (Schemes 1 and 2). Then, b-(3,4-
dimethoxyphenyl)ethylamine was condensed with valeryl chloride
or methyl adipoyl chloride to give the corresponding amide 1 or 4,
respectively. These amides were next treated with POCl3 to gener-
ate the appropriate dihydroisoquinoline 10 or 40. These unstable
imines 10 and 40 were N-alkylated with MeI to obtain the expected
3,4-dihydroisoquinolinium salt 2 and 5, respectively. Finally, these
imoniums were reduced with NaBH4 to give the corresponding
THIQs 3 and 6. Subsequently, the terminal methyl ester of THIQ
6 was reduced to a primary alcohol with LiAlH4 to generate 1-pent-
anol-THIQ 7.
2.2.1. Structure–bactericidal activity relationships
Initially, we examined 1-alkyl-isoquinolines with DHIQ and
THIQ nucleus, resulting both 1-butyl-3,4-dihydroisoquinolinium
salt 2 and 1-butyl-THIQ 3 active against all the bacterial strains
tested, except for E. coli 100. The introduction of a methyl ester
in the alkyl chain at 1-position gave the DHIQ 5 and its analogous
THIQ 6 that were active against few bacterial strains, however
THIQ 6 displayed bactericidal effects against S. aureus and E. coli
100. In view of these results, the presence of a methyl ester group
reduced the bactericidal spectrum. Interestingly, the common pre-
cursor 1-pentanol-THIQ 7 exhibited the highest activity against all
the strains tested showing remarkable effects against E. coli 100
(62% inhibition). Despite these findings, it lacked of any relevant
activity against B. cereus.
Owing to the chemical versatility of the terminal alcohol of THIQ
7, we decided to synthesize the 1-pentyl-THIQs functionalized with
esters (8–15) and carbamates (16–23) (Schemes 3 and 4). 1-Penta-
nol-THIQ 7 was reacted with acid chlorides in the presence of 4-
DMAP and triethylamine to obtain the new esters compounds (8–
15). Moreover, 1-pentanol-THIQ 7 reacted with the corresponding
isocyanates to yield the corresponding carbamates (16–23). All
the compounds’ structures were elucidated by NMR spectra and
ESMS spectrometry data. It should be emphasized that, to the best
of our knowledge, products 2–23 are reported for the first time. Fur-
thermore, we decided to explore the influence of a variety of func-
tionalized substituents at the 1-position in the THIQ skeleton,
including carbamate and ester groups on antimicrobial activity.
Next, to find more potent bactericidal THIQs, we examined the
introduction of ester and carbamate functions in the alkyl chain at
1-position as well as substituent effects at the para-position of the
phenyl group of these ester and carbamate THIQs. All the benzoate
esters of the 1-pentyl-THIQs bearing different substituents in the
phenyl group (8–15) exhibited relevant activity against all the bac-
teria tested. In general, they showed the following activity-grow-
ing trend: Ph (8) < PhOMe (11) < PhCl (10) < PhF (9), emphasizing
that THIQ 9 with a fluorinated benzoate exhibited the highest
activity against S. typhi (61%). Furthermore, phenylpropanoate
derivatives 12–15, in which two methylenes were placed between
the ester group and the substituted aromatic ring, displayed broad
antibacterial activity being effective against all the strains tested.
Therefore, the conformational flexibility provided by the two addi-
tional carbons did not significantly improve the bactericidal activ-
ity of these compounds. Similarly to its analogs, the fluorinated
compound 13 showed the strongest bactericidal activity against
most of the strains tested. Its activity against B. cereus and E. coli
100 was noteworthy (49% and 46%, respectively). Regarding the
carbamate series, all the 1-pentyl-THIQs 16–23, showed wide
broad-range of bactericidal activity (Table 3). In this context, THIQ
16 exhibited 59% of antibacterial activity against E. coli 100, while
the fluorinated analog 17 showed 64% activity against E. faecalis
and E. carotovora. Methoxylated THIQ 19 exhibited 51% activity
against S. aureus and 52% activity against E. coli 405, while the chlo-
rinated compound 18 displayed greater antibacterial activity
against S. typhi and E. coli 405 (67% and 68%, respectively). Com-
pared with analogs 16–19, the THIQs 20–23 possessing two carbon
2.2. Antimicrobial activity
All the THIQs (2, 3, 5–23) were tested in vitro for their antimi-
crobial activity against several human pathogenic bacteria and
economically relevant phytopathogenic fungi. The inhibition zones
of bacterial and fungal growth caused by the synthesized com-
pounds are outlined in Tables 1–5.
The bacterial strains used in the antibacterial assay were three
Gram-positive (Bacillus cereus, Staphylococcus aureus and Entero-
coccus faecalis) and four Gram-negative (Salmonella typhi, Esche-
richia coli 100, E. coli 405 and Erwinia carotovora). The following
phytopathogenic fungal strains were used to evaluate the anti-
fungal activity: Fusarium culmorum, Geotrichum candidum, Tricho-
derma viride, Phytophthora citrophthora and Aspergillus parasiticus.
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
(b)
(a)
3
6
1
A
B
NH2
NH
N
O
7
4
1
1'
β-(3,4-dimethoxyphenyl)
ethylamine
4'
1'
1 (98 %)
(c)
H3CO
H3CO
H3CO
H3CO
NCH3
NCH3
(d)
3 (61 %)
2 (97%)
Scheme 1. Synthesis of N-methyl-1-butyl-6,7-dimethoxy-1,2,3,4-THIQ (3). Reagents and conditions: (a) b-(3,4-dimethoxyphenyl)ethylamine, valeryl chloride, NaOH 5%,
CH2Cl2, rt, 2 h; (b) POCl3, CH3CN, reflux, 1 h; (c) MeI, acetone, reflux, 3 h; (d) NaBH4, MeOH, rt, 2 h.

A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
3223
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
(a)
(b)
A
B
NH2
NH
N
O
β-(3,4-dimethoxyphenyl)
ethylamine
O
O
4'
O
O
4
(91 %)
(c)
H3CO
H3CO
H3CO
H3CO
H3CO
NCH3
NCH3
NCH3
H3CO
(e)
(d)
O
O
O
O
HO
5
7 (87 %)
6 (58 %)
(96 %)
Scheme 2. Synthesis of N-methyl-1-pentanol-6,7-dimethoxy-1,2,3,4-THIQ (7). Reagents and conditions: (a) b-(3,4-dimethoxyphenyl)ethylamine, methyl adipoyl chloride,
NaOH 5%, CH2Cl2, rt, 2 h; (b) POCl3, CH3CN, reflux, 1 h; (c) MeI, acetone, reflux, 3 h; (d) NaBH4, MeOH, rt, 2 h; (e) LiAlH4, Et2O, THF, reflux, 2 h.
O
R= F
R= Cl
OH
Cl
R= OCH3
R
(b)
Cl
O
O
R= H
R= F
R= Cl
R
R
R= OCH3
H3CO
H3CO
H3CO
6
7
6
NCH3
NCH3
1'
H3CO
7
1
1
(a)
(a)
1'
7
1-pentanol-THIQ
5'
5'
O
O
8'
1''
O
O
1''
8:
9'
R
4''
R= H (67 %)
9: R= F (80 %)
R= Cl (66 %)
11: R= OCH3 (32 %)
R
4''
12: R= H
(65 %)
R= F (51 %)
14: R= Cl (10 %)
15:
10:
13:
R= OCH3 (14 %)
Scheme 3. Synthesis of esters N-methyl-1-pentyl-6,7-dimethoxy-1,2,3,4-THIQs (8–15). Reagents and conditions: (a) 4-DMAP, Et3N, CH2Cl2, rt, 5 h; (b) SOCl2, CH2Cl2, reflux,
3 h.
atom units linking the carbamate group and the corresponding
phenyl substituent, displayed similar bactericidal activity trends.
Of note, the halogenated phenyl derivatives including the fluori-
nated 21, and the mainly chlorinated 22, displayed the largest inhi-
bition zones against most of the strains tested with remarkable
results. In regard to this, the fluorinated phenyl derivative 21
was active by 61% and 62% against S. typhi and E. coli 405,
respectively, and its chlorinated analog 22 exhibited a higher bac-
tericidal activity against the same bacteria (75%).
2.2.2. Structure–fungicidal activity relationships
SAR studies were carried out against several phytopathogenic
fungi. In this case, we observed that 1-butyl-tetrahydroisoquino-
line
3 was only slightly active against F. culmorum and 3,

3224
A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
1-pentanol-THIQ
7
O
C
N
O
C
N
R
(a)
R= H
R= F
R= Cl
R= H
R= F
R= Cl
R
R= OCH3
R= OCH3
H3CO
H3CO
H3CO
H3CO
6
6
NCH3
NCH3
7
7
1
1
1'
1'
5'
5'
O
O
HN
O
HN
O
9'
1''
10'
1''
16: R= H (75 %)
17:
20:
R= H (66 %)
21: R= F (67 %)
22:
R= F (22 %)
18: R= Cl (64 %)
19:
4''
R= Cl (67 %)
23: R= OCH3 (48 %)
R
4''
R= OCH3 (73 %)
R
Scheme 4. Synthesis of carbamates N-methyl-1-pentyl-6,7-dimethoxy-1,2,3,4-THIQs (16–23). Reagents and condition: (a) CH2Cl2, reflux, 24 h.
Table 1
Strains
Bactericidal activity
Inhibition zone (mm) 24 h (means SE)a
2b
3b
5b
6b
7b
Tetracyclineb
B. cereus
S. aureus
7.0 0A
7.0 0A
6.0 0.5A
6.0 0AB
7.0 0A
6.33 0.41A
6.33 0.41A
0
0
23.33 0.41B
27.0 0.71D
31.17 0.89C
24.33 0.41C
25.67 0.41C
34.67 1.45B
25.67 0.41C
0
0
0
0
0
0
0
0
5.50 0B
10.67 1.08C
10.33 1.08B
12.33 0.82B
11.33 1.08B
8.0 0.71A
E. faecalis
S. typhi
E. coli 405
E. carotovora
E. coli 100
7.0 0A
0
0
0
0
0
0
0
0
6.75 0.25A
7.0 0A
6.25 0.25A
6.0 0A
7.0 0A
6.0 0A
5.50 0A
0
0
0
0
0
0
5.83 0.20A
16.0 0B
a
Each value represents the average and the standard error of three independent experiments. Within each line, the mean values labelled with a different superscript (A–D)
show statistically significant differences (P <0.05).
b
Dose: 0.2 mg/disk.
4-dihydroisoquinolinium salts 2 and 5 didn’t show any antifungal
activity, except the fluorinated THIQ 17, which was inactive for all
the fungal strains tested. Finally, the presence of two-carbons be-
tween the carbamate and aromatic ring seemed to be detrimental
for antifungal activity with the exception of the chlorinated THIQ
22, which showed a wider fungicidal spectrum.
activity neither at 10 l l
g/mm2 nor at 20 g/mm2. Similarly, THIQs 6
and 7, with methyl ester and alcohol groups, respectively, did not
display antifungal activity at the doses tested. The introduction
of an aromatic lipophilic group in the alkyl chain at 1-position such
as benzoate and phenylpropanoate esters and phenyl and phen-
ethyl-carbamates was essential to develop active THIQs. Results
showed that all the 1-pentyl-THIQs bearing phenyl esters (8–15)
showed antifungal activity against some of the strains. The unhalo-
genated THIQ 8, the fluorinated THIQ 9 and the methoxylated THIQ
11 were all active against F. culmorum, T. viride and G. candidum.
Interestingly, the chlorinated THIQ 10 displayed the highest anti-
fungal activity against all the fungi tested, except for A. parasiticus.
Additionally, phenylpropanoate THIQs 12–15 were more active
than their corresponding benzoate analogs (8–11). These results
suggest that conformational flexibility with a lengthened structure
was, in general, beneficial for fungicidal activity. Regarding the car-
bamate series, all the 1-pentyl-THIQs (16–23) exhibited antifungal
In conclusion, most of these 1-substituted-THIQs displayed
both fungicidal and (mostly) bactericidal activity. Structure–bacte-
ricidal activity relationships revealed that 1-butyl-isoquinolines
with DHIQ and THIQ nucleus (2 and 3, respectively), possessed
moderate bactericidal activity. The introduction of a methyl ester
group in the alkyl chain at 1-position such as DHIQ 5 and THIQ
6, seemed to reduce the bactericidal spectrum. However, different
substituents affording an alcohol function such as 1-pentanol-THIQ
7 and also several lipophilic 1-pentyl-THIQs with the phenyl ester
(8–15) and phenyl carbamate (16–23) moieties, displayed signifi-
cant bioactivity against all the bacteria tested. Structure–fungicidal
activity relationships showed that the introduction in the alkyl
chain at 1-position of ester and carbamate functions attached to

A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
3225
Table 2
Strains
Bactericidal activity
Inhibition zone (mm) 24 h (means SE)a
8b
9b
10b
11b
12b
13b
14b
15b
Tetracyclineb
B. cereus
S. aureus
10.83 1.59A
10.67 0.82AB
9.83 0.73A
9.67 0.41A
9.83 0.54A
12.17 1.14A
11.0 0.71AB
13.0 0.71B
13.67 1.08C
13.67 0.41B
14.83 0.20B
13.67 0.41B
13.50 0.35AB
14.0 0.71C
13.0 0B
10.33 0.41A
11.33 0.41BC
11.33 0.41C
11.33 0.41C
11.33 0.41C
10.33 1.08A
10.83 0.20A
10.33 0.41A
9.67 0.41AE
8.67 0.41DE
8.67 0.41DE
8.67 0.41D
10.67 0.41A
11.33 0.41AB
11.33 0.41AB
10.33 0.41AB
9.33 0.41AD
9.17 0.20AD
8.83 0.54D
11.0 0A
8.50 0.71C
7.83 0.20F
7.0 0F
23.33 0.41D
27.0 0.71G
31.17 0.89G
24.33 0.41F
25.67 0.41F
34.67 1.45B
25.67 0.41F
12.67 0.41CD
11.67 0.41C
11.50 0.35C
11.50 0.35C
12.67 0.41A
13.67 0.20C
8.67 0.41EF
8.33 0.20E
8.17 0.41E
9.17 0.54AD
11.0 0A
E. faecalis
S. typhi
E. coli 405
E. carotovora
E. coli 100
8.0 0.35E
7.33 0.20E
11.33 0.82A
7.0 0E
11.67 0.41A
11.83 0.20B
8.67 0.20D
a
Each value represents the average and the standard error of three independent experiments. Within each line, the mean values labelled with a different superscript (A–G)
show statistically significant differences (P <0.05).
b
Dose: 0.2 mg/disk.
Table 3
Strains
Bactericidal activity
Inhibition zone (mm) 24 h (means SE)a
16b
17b
18b
19b
10.33 0.41CD 10.17 0.20C
13.67 0.41A
20b
21b
22b
23b
Tetracyclineb
B. cereus
S. aureus
E. faecalis
S. typhi
12.67 1.08A
15.0 0.93ABC
14.17 0.89A
12.0 1.87AB
12.33 1.74AB
14.33 0.41B
14.33 0.41B
11.33 0.41DE
14.67 0.41B
12.0 0AE
23.33 0.41F
27.0 0.71E
31.17 0.89F
14.0 1.41AB 17.33 0.41C
14.0 0.71AB 15.33 0.41ABC 16.33 0.41BC
9.0 1.41D
11.0 0.71E
20.0 1.87B
18.0 0.71BC 14.67 0.41A
13.67 0.82A
15.0 0.71AD
17.0 0.71CD
18.33 0.41E
17.67 0.41C
18.33 0.82B
14.67 0.82CD 16.33 0.41CE 11.33 0.41A
13.50 0.35BC 14.83 0.20CD
13.33 0.82BC 24.33 0.41F
E. coli 405
12.0 0.71A
17.33 0.41C
12.50 0.35AC
13.33 0.41AB
9.67 0.41A
14.0 0.71BD
11.0 0A
16.0 0.71CD
11.67 0.41A
11.33 0.41DE
13.33 0.41AB
17.0 1.22BC 34.67 1.45B
12.33 0.41BC 10.67 0.41E 25.67 0.41F
25.67 0.41E
E. carotovora 10.67 0.41A
22.33 0.82B
E. coli 100
15.17 0.20A
12.33 0.41BC 13.17 0.54B
11.67 0.41CD 10.67 0.41E
a
Each value represents the average and the standard error of three independent experiments. Within each line, the mean values labelled with a different superscript (A–F)
show statistically significant differences (P <0.05).
b
Dose: 0.2 mg/disk.
Table 4
Strains
Antifungal activity
Inhibition zone (mm) 72 h (means SE)a
3b
8b
9b
10b
11b
12b
13b
14b
15b
Benomyl
F. culmorum
G. candidum
T. viride
P. citrophthora
Parasiticus
5.50 0A
7.0 0BC
8.0 0D
7.0 0A
7.0 0A
6.67 0.41B
12.33 0B
9.0 0.50C
10.33 0A
5.50 0.25A
7.33 0.25AC
5.50 0.61D
6.33 0.41B
7.83 0.20CD
8.0 0.71ABC
6.67 0.41B
7.67 0.41CD
8.33 0.82D
8.33 0.41BC
6.0 0B
9.5 0.5E
8.5 0.5D
9.0 0C
6.5 0.5B
5.75 0.25E
6.0 0D
18.0 0F,c
0
0
0
0
0
0
0
0
6.75 0.25A
7.25 0.25AB
0
0
22.0 1.0E,d
24.5 1.5D,e
18.0 1.0d
0
0
0
0
0
0
0
0
0
0
0
0
8.5 0.5C
0
0
0
0
0
0
0
0
0
0
0
0
Compounds 2 and 5–7 did not exert antifungal activity.
a
Each value represents the average and the standard error of three independent experiments. Within each line, the mean values labeled with a different superscript (A–F)
show statistically significant differences (P <0.05).
b
Dose: 0.2 mg/disk.
c
Dose: 10
l
g/disk.
g/disk.
g/disk.
d
e
Dose: 1
l
Dose: 1.5
l
different phenyl moieties was essential to develop new antifungal
THIQs. In addition and on a regular basis, 1-pentyl-THIQs bearing
esters (8–15) displayed generally more antifungal activity than
the corresponding THIQs with carbamates (16–23).
chromatography. Quoted yields were of purified compounds. Elec-
trospray mass spectrometry (ESMS) was recorded in a Esquire
3000 Plus spectrometer instrument (Bruker). Nuclear magnetic
resonance (1H and 13C NMR) spectra were recorded with CDCl3
as solvent in a Bruker DPX-500 spectrometer. Multiplicities of 13C
NMR resonances were assigned by DEPT experiments. 1H and 13C
assignments were realized by mono and bidimensional techniques
(COSY 45, HMQC, HSQC and HMBC).
3. Materials and methods
3.1. Synthetic procedures
3.1.2. General procedure for the synthesis of amides (1 and 4)
Corresponding alkyl chloride (16.6 mmol) was added dropwise
at 0 °C to a solution of b-(3,4-dimethoxyphenyl)ethylamine (3.0 g,
16.6 mmol) in CH2Cl2 (30 mL) and 5% aqueous NaOH (15 mL). The
reaction was stirred at room temperature for 2 h. After extraction
with CH2Cl2, the combined organic phases were washed with brine
3.1.1. General
Reagents were purchased from Sigma–Aldrich Chemical Com-
pany (St. Louis, MO, USA) and solvents from Scharlab (Barcelona,
Spain). All the reactions were monitored by analytical thin layer
chromatography with silica gel 60 F254 (Merck 5554). Residues
were purified by silica gel 60 (40–63 lm, Merck 9385) column

3226
A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
Table 5
Strains
Antifungal activity
Inhibition zone (mm) 72 h (means SE)a
16b
18b
19b
20b
21b
22b
23b
Benomyl
F. culmorum
G. candidum
T. viride
P. citrophthora
Parasiticus
7.0 0A
7.33 0.41A
10.33 0.41B
8.67 0.82B
0
0
0
0
0
0
15.0 3.0B
10.0 0.0B
7.0 0AB
0
0
18.0 0B,c
6.25 0.25A
6.67 0.41A
7.67 0.41C
7.67 0.41C
7.0 0AC
0
0
5.75 0.25A
6.17 0.54A
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
22.0 1.0C,d
24.5 1.5B,e
18.0 1.0d
0
0
0
0
0
0
0
0
0
0
6.25 0.25A
0
0
Compound 17 did not display antifungal activity.
a
Each value represents the average and the standard error of three independent experiments. Within each line, the mean values labeled with a different superscript (A–D)
show statistically significant differences (P <0.05).
b
Dose: 0.2 mg/disk.
c
Dose: 10
l
g/disk.
g/disk.
g/disk.
d
e
Dose: 1
l
Dose: 1.5
l
and H2O, dried over anhydrous Na2SO4, filtered and evaporated to
dryness. The residue was purified (CH2Cl2/MeOH 95:5) to obtain
the corresponding amide.
3.1.3.1. N-Methyl-1-butyl-6,7-dimethoxy-3,4-dihydroisoquino-
linium (2). Under the same conditions described above and
using amide 1 (2.0 g, 7.5 mmol) as starting material, 1.9 g of a yel-
low powder 2 (97%) were obtained. 1H NMR (500 MHz, CDCl3): d
7.18 (s, 1H, H-8), 6.91 (s, 1H, H-5), 4.14 (t, J = 7.8 Hz, 2H, H-3),
3.99 and 3.91 (2s, 6H, OCH3-6 and OCH3-7), 3.91 (s, 3H, NCH3),
3.27 (t, J = 7.8 Hz, 2H, H-4), 3.21–3.16 (m, 2H, H-10), 1.72–1.63
(m, 2H, H-20), 1.56–1.47 (m, 2H, H-30), 1.00–0.95 (m, 3H, H-40);
13C NMR (125 MHz, CDCl3): d 177.1 (C-1), 156.4 (C-6), 148.7 (C-
7), 133.4 (C-4a), 118.6 (C-8a), 112.0 (CH-8), 111.1 (CH-5), 56.9
(OCH3-6), 56.6 (OCH3-7), 53.3 (CH2-3), 45.9 (NCH3), 31.7 (CH2-10),
29.7 (CH2-20), 26.0 (CH2-4), 22.8 (CH2-30), 13.6 (CH2-40); ESMS m/
z (%) 263 [M+H]+ (31).
3.1.2.1. N-(3,4-Dimethoxyphenethyl)pentanamide (1).
Under
the conditions depicted above and using valeryl chloride (2.0 mL,
16.6 mmol), 4.3 g of amide 1 as a white powder (98%) were obtained.
1H NMR (500 MHz, CDCl3): d 6.80–6.77 (m, 1H, H-5), 6.72–6.68 (m,
2H, H-2, H-6), 5.55 (brs, 1H, CONH), 3.84 (s, 6H, OCH3-3, OCH3-4),
3.47 (dd, J = 6.9, 12.9 Hz, 2H, H-a), 2.74 (t, J = 6.9 Hz, 2H, H-b), 2.10
(t, J = 6.9 Hz, 2H, H-10), 1.60–1.51 (m, 2H, H-20), 1.34–1.24 (m, 2H,
H-30), 0.87 (t, J = 7.5 Hz, 3H, H-40); 13C NMR (125 MHz, CDCl3): d
173.1 (CONH), 149.0 (C-3), 147.6 (C-4), 131.4 (C-1), 120.6 (CH-6),
111.9 (CH-2), 111.3 (CH-5), 55.9 (OCH3-3), 55.8 (OCH3-4), 40.5
(CH2-a
), 36.5 (CH2-10), 35.2 (CH2-b), 27.8 (CH2-20), 22.3 (CH2-30),
3.1.3.2.
dihydroisoquinolinium (5).
N-Methyl-1-(methylpentanoate)-6,7-dimethoxy-3,4-
Under the same conditions de-
13.7 (CH2-40); ESMS m/z (%) 266 [M+H]+ (5).
scribed in the general procedure and using 4 (2.0 g, 6.2 mmol) as
starting material, 1.9 g of a yellow oil 5 (96%) were obtained. 1H
NMR (500 MHz, CDCl3): d 7.22 (s, 1H, H-8), 6.90 (s, 1H, H-5), 4.11
(t, J = 7.3 Hz, 2H, H-3), 3.96 and 3.93 (2s, 6H, OCH3-6 and OCH3-
7), 3.91 (s, 3H, NCH3), 3.60 (s, 3H, COOCH3), 3.30–3.20 (m, 4H, H-
4, H-10), 2.38 (t, J = 6.3 Hz, 2H, H-40), 1.84–1.72 (m, 4H, H-20, H-
30); 13C NMR (125 MHz, CDCl3): d 176.6 (C-1), 173.2 (CO), 156.3
(C-6), 148.7 (C-7), 133.3 (C-4a), 118.5 (C-8a), 111.9 (CH-8), 111.0
(CH-5), 56.9 (OCH3-7), 56.7 (OCH3-6), 53.2 (CH2-3), 51.6 (COOCH3),
46.0 (NCH3), 32.7 (CH2-40), 31.7 (CH2-10), 26.8 (CH2-20), 25.9 (CH2-
4), 24.6 (CH2-30); ESMS m/z (%) 320 [M]+ (11).
3.1.2.2. Methyl N-(3,4-dimethoxyphenethylamino)-6-oxohex-
anoate (4).
Under the same conditions described in the gen-
eral procedure and using methyl adipoyl chloride (2.6 mL,
16.6 mmol), 4.9 g of amide 4 as a yellow powder (91%) were ob-
tained. 1H NMR (500 MHz, CDCl3): d 6.77 (d, J = 8.0 Hz, 1H, H-5),
6.72–6.67 (m, 2H, H-2, H-6), 5.71 (s, 1H, CONH), 3.82 and 3.80
(2s, 6H, OCH3-3 and OCH3-4), 3.62 (s, 3H, COOCH3), 3.46 (q,
J = 7.1 Hz, 2H, H-a), 2.72 (t, J = 7.1 Hz, 2H, H-b), 2.28 (t, J = 6.9 Hz,
2H, H-40), 2.11 (t, J = 6.0 Hz, 2H, H-10), 1.65–1.53 (m, 4H, H-20, H-
30); 13C NMR (125 MHz, CDCl3): d 173.8 (CO), 172.4 (CONH),
148.9 (C-3), 147.6 (C-4), 131.3 (C-1), 120.5 (CH-6), 111.8 (CH-5),
111.3 (CH-2), 55.8 (OCH3-3, OCH3-4), 51.4 (COOCH3), 40.6 (CH2-
3.1.4. General procedure for the synthesis of N-methyl-1-
substituted-6,7-dimethoxy-1,2,3,4-THIQs (3 and 6)
a
), 36.1 (CH2-10), 35.2 (CH2-b), 33.5 (CH2-40), 25.0 (CH2-20), 24.3
(CH2-30); ESMS m/z (%) 324 [M+H]+ (5).
A
solution of the corresponding dihydroisoquinolinium
(1.9 mmol) in MeOH (30 mL) at 0 °C was treated with NaBH4
(216 mg, 5.7 mmol). The reaction mixture was stirred at room tem-
perature for 2 h. Then, H2O (15 mL) was added dropwise and the
organic solvent was removed under reduced pressure. The aqueous
mixture was extracted with CH2Cl2 and washed with brine and
H2O. A combination of the organic phases was dried over anhy-
drous Na2SO4, filtered and evaporated to dryness. The residue
was purified (CH2Cl2/MeOH 93:7) to obtain the corresponding
THIQ.
3.1.3. General procedure for the synthesis of N-methyl-1-
substituted-6,7-dimethoxy-3,4-dihydroisoquinolinium (2 and
5)
A solution of the corresponding amide (7.5 mmol) in dry aceto-
nitrile (30 mL) was treated with POCl3 (3.5 mL, 37.7 mmol) and re-
fluxed for 1 h under a N2 atmosphere. Then, the reaction mixture
was evaporated to dryness. The residue was dissolved in H2O
(10 mL), basified to pH ꢀ 9 and extracted with CH2Cl2. The organic
phase was washed with brine and H2O, dried over anhydrous
NaSO4, filtered and evaporated to dryness. A reddish oil, corre-
sponding to an imine, was obtained and directly used in the next
reaction. The imine was dissolved in dry acetone (30 mL) and trea-
ted with MeI (1.7 mL, 26.6 mmol). The reaction mixture was stirred
and refluxed for 3 h under a N2 atmosphere. Then, the solvent was
evaporated to dryness and the residue was purified (CH2Cl2/MeOH
9:1) to obtain the corresponding dihydroisoquinolinium salt.
3.1.4.1. N-Methyl-1-butyl-6,7-dimethoxy-1,2,3,4-THIQ (3).
Under
the same conditions described above and using 2(0.5 g, 1.9 mmol) as start-
ing material, 303 mg of a translucent oil 3 (61%) were obtained. 1H NMR
(500 MHz, CDCl3): d 6.52 (s, 1H, H-8), 6.49 (s, 1H, H-5), 3.77 and 3.76 (2s,
6H, OCH3-6 and OCH3-7), 3.30 (t, J = 5.4 Hz, 1H, H-1), 3.08–3.00 (m, 1H,
Ha-3), 2.71–2.54 (m, 3H, Hb-3, H-4), 2.37 (s, 3H, NCH3), 1.71–1.64 (m,
2H, H-10), 1.38–1.15 (m, 4H, H-20, H-30), 0.82 (t, J = 7.1 Hz, 3H, H-40); 13C

A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
3227
NMR (125 MHz, CDCl3): d146.9 (C-6, C-7), 130.1 (C-8a), 126.3 (C-4a), 111.0
(CH-5), 110.0 (CH-8), 63.11 (CH-1), 55.6 (OCH3-6), 55.4 (OCH3-7), 48.0
(CH2-3), 42.5 (NCH3), 34.4 (CH2-10), 27.5 (CH2-20), 25.3 (CH2-4), 22.7
(CH2-30), 13.8 (CH2-40); ESMS m/z (%) 264 [M+H]+ (39).
166.6 (CO), 147.7 (C-6), 147.4 (C-7), 132.8 (C-400), 130.5 (C-100),
129.5 (CH-200, CH-600), 128.4 (C-8a), 128.3 (CH-300, CH-500), 125.3
(C-4a), 111.4 (CH-8), 110.3 (CH-5), 65.0 (CH2-50), 63.2 (CH-1),
56.0 (OCH3-6), 55.8 (OCH3-7), 46.9 (CH2-3), 41.7 (NCH3), 34.8
(CH2-10), 28.7 (CH2-40), 26.3 (CH2-30), 25.6 (CH2-20), 24.5 (CH2-4);
ESMS m/z (%) 398 [M+H]+ (29).
3.1.4.2. N-Methyl-1-(methylpentanoate)-6,7-dimethoxy-1,2,3,4-
THIQ (6).
Under the same conditions described in the general
procedure and using 5 (1.0 g, 3.1 mmol) as starting material,
576 mg of a translucent oil 6 (58%) were obtained. 1H NMR
(500 MHz, CDCl3): d 6.55 (s, 1H, H-5), 6.54 (s, 1H, H-8), 3.83 (s,
6H, OCH3-6, OCH3-7), 3.63 (s, 3H, COOCH3), 3.35 (t, J = 5.5 Hz, 1H,
H-1), 3.11–3.04 (m, 1H, Ha-3), 2.76–2.69 (m, 1H, Ha-4), 2.68–
2.60 (m, 2H, Hb-3, Hb-4), 2.46 (s, 3H, NCH3), 2.28 (t, J = 7.6 Hz,
2H, H-40), 1.76–1.71 (m, 2H, H-10), 1.66–1.56 (m, 2H, H-30), 1.46–
1.39 (m, 1H, Ha-20), 1.31–1.24 (m, 1H, Hb-20); 13C NMR
(125 MHz, CDCl3): d 174.2 (CO), 147.2 (C- 6), 147.1 (C-7), 130.0
(C-8a), 126.6 (C-4a), 111.2 (CH-5), 110.1 (CH-8), 63.1 CH-1), 55.9
OCH3-7), 55.7 (OCH3-6), 51.4 (COOCH3), 48.2 (CH2-3), 42.7
(NCH3), 34.6 (CH2-10), 34.0 (CH2-40), 25.5 (CH2-4), 25.2 (CH2-30),
25.0 (CH2-20); ESMS m/z (%) 322 [M+H]+ (2).
3.1.6.2. N-Methyl-1-[(400-fluorobenzoate)pentyl]-6,7-dimethoxy-
1,2,3,4-THIQ (9).
general procedure,
Under the same conditions described in the
and 4-fluorobenzoyl chloride (60 L,
7
l
0.5 mmol) reacted to obtain 166 mg of the yellow oil 9 (80%). 1H
NMR (500 MHz, CDCl3): d 7.98–7.90 (m, 2H, H-200, H-600), 7.05–
7.00 (m, 2H, H-300, H-500), 6.51 (s, 1H, H-5), 6.49 (s, 1H, H-8), 4.18
(t, J = 6.6 Hz, 2H, H-50), 3.55 and 3.54 (2s, 6H, OCH3-6 and OCH3-
7), 3.49 (t, J = 5.7 Hz, 1H, H-1), 3.20–3.11 (m, 1H, Ha-3), 2.81–
2.70 (m, 1H, Hb-3), 2.70–2.62 (m, 2H, H-4), 2.44 (s, 3H, NCH3),
1.86–1.77 (m, 1H, Ha-10), 1.72–1.62 (m, 3H, Hb-10, H-40), 1.45–
1.30 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d 165.2 (C-
400, d, J = 251 Hz), 165.2 (CO), 147.3 (C-6), 147.0 (C-7), 131.5 (CH-
200, CH-600, d, J = 9 Hz), 128.1 (C-8a), 126.4 (C-4a), 125.1 (C-100),
114.6 (CH-300, CH-500, d, J = 21 Hz), 111.1 (CH-8), 110.1 (CH-5),
64.8 (CH2-50), 62.8 (CH-1), 55.7 (OCH3-6), 55.5 (OCH3-7), 46.7
(CH2-3), 41.4 (NCH3), 34.3 (CH2-10), 28.3 (CH2-40), 25.9 (CH2-20),
25.2 (CH2-30), 24.2 (CH2-4); ESMS m/z (%) 416 [M+H]+ (17).
3.1.5. Synthesis of N-methyl-1-pentanol-6,7-dimethoxy-1,2,3,4-
THIQ (7)
A solution of N-methyl-1-(methylpentanoate)-6,7-dimethoxy-
1,2,3,4-THIQ 6 (545 mg, 1.7 mmol) in dry THF (30 mL) was added
to a stirred suspension of LiAlH4 (69 mg; 1.8 mmol) in anhydrous
Et2O (6 mL) under a N2 atmosphere and the mixture was refluxed
for 2 h. Thereafter, the reaction mixture was cooled and H2O
(15 mL) was added dropwise to destroy the excess of LiAlH4. The
solvent was evaporated to obtain 431 mg of a pink oil 7 (87%).
1H NMR (500 MHz, CDCl3): d 6.55 (s, 1H, H-5), 6.54 (s, 1H, H-8),
3.83 (s, 6H, OCH3-6, OCH3-7), 3.59 (t, J = 6.6 Hz, 2H, H-50), 3.37 (t,
J = 5.5 Hz, 1H, H-1), 3.13–3.05 (m, 1H, Ha-3), 2.77–2.69 (m, 1H,
Ha-4), 2.68–2.61 (m, 2H, Hb-3, Hb-4), 2.42 (s, 3H, NCH3), 1.77-
1.70 (m, 2H, H-10), 1.58–1.50 (m, 2H, H-40), 1.47–1.26 (m, 4H, H-
20, H-30); 13C NMR (125 MHz, CDCl3): d 147.2 (C-6), 147.1 (C-7),
130.1 (C-8a), 126.5 (C-4a), 111.2 (CH-5), 110.2 (CH-8), 63.25 (CH-
1), 62.7 (CH2-50), 56.0 (OCH3-7), 55.8 (OCH3-6), 48.0 (CH2-3), 42.7
(NCH3), 34.9 (CH2-10), 32.5 (CH2-40), 25.9 (CH2-4), 25.3 (CH2-30),
25.2 (CH2-20); ESMS m/z (%) 294 [M+H]+ (14).
3.1.6.3.
thoxy-1,2,3,4-THIQ (10).
N-Methyl-1-[(400-chlorobenzoate)pentyl]-6,7-dime-
Under the same conditions de-
scribed above, 7 and 4-chlorobenzoyl chloride (0.1 mL, 0.8 mmol)
reacted to obtain 143.3 mg of yellow oil 10 (66%). 1H NMR
(500 MHz, CDCl3): d 7.97–7.91 (m, 2H, H-200, H-600), 7.42–7.35 (m,
2H, H-300, H-500), 6.56 (s, 1H, H-8), 6.55 (s, 1H, H-5), 4.28 (t,
J = 6.7 Hz, 2H, H-50), 3.83 and 3.82 (2s, 6H, OCH3-6 and OCH3-7),
3.38 (t, J = 5.4 Hz, 1H, H-1), 3.15–3.04 (m, 1H, Ha-3), 2.77–2.61
(m, 3H, Hb-3, H-4), 2.43 (s, 3H, NCH3), 1.79–1.69 (m, 4H, H-10, H-
40), 1.51–1.30 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d
165.7 (CO), 147.2 (C-6, C-7), 139.2 (C-100), 130.9 (CH-200, CH-600),
129.9 (C-8a), 128.9 (C-400), 128.6 (CH-300, CH-500), 126.5 (C-4a),
111.2 (CH-5), 110.1 (CH-8), 65.3 (CH2-50), 63.3 (CH-1), 56.0
(OCH3-6), 55.7 (OCH3-7), 48.2 (CH2-3), 42.7 (NCH3), 34.9 (CH2-10),
28.7 (CH2-40), 26.3 (CH2-30), 25.5 (CH2-4), 25.2 (CH2-20); ESMS m/
z (%) 432 [M+H]+ (12).
3.1.6. General procedure for the synthesis of esters N-methyl-1-
pentyl-6,7-dimethoxy-1,2,3,4-THIQ (8–15)
A solution of the corresponding benzoyl chloride (0.3 mmol) in
dry CH2Cl2 (5 mL) was added dropwise at 0 °C to a stirred solution
3.1.6.4.
thoxy-1,2,3,4-THIQ (11).
scribed in the general procedure,
N-Methyl-1-[(400-methoxybenzoate)pentyl]-6,7-dime-
Under the same conditions de-
and 4-methoxybenzoyl
7
of N-methyl-1-pentanol-6,7-dimethoxy-1,2,3,4-THIQ
0.3 mmol), 4-(dimethylamino)pyridine (12 mg, 0.1 mmol) and tri-
ethylamine (42 L, 0.3 mmol) in dry CH2Cl2 (20 mL). The reaction
mixture was stirred at room temperature under a N2 atmosphere
for 5 h. Next, the reaction mixture was extracted with CH2Cl2 and
washed with H2O. The organic layer was dried over anhydrous
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied (toluene/AcOEt/MeOH 6:3:1) to obtain the corresponding
THIQ ester.
7
(88 mg,
chloride (0.1 mL, 0.8 mmol) reacted to obtain 68 mg of the yellow
1
oil 11 (32%). H NMR (500 MHz, CDCl3): d 7.98–7.92 (m, 2H, H-300,
l
H-500), 6.91–6.86 (m, 2H, H-200, H-600), 6.56 (s, 1H, H-5), 6.55 (s, 1H,
H-8), 4.24 (t, J = 6.6 Hz, 2H, H-50), 3.83 (s, 9H, OCH3-6, OCH3-7,
OCH3-400), 3.51 (t, J = 5.7 Hz, 1H, H-1), 3.24–3.14 (m, 1H, Ha-3),
2.84–2.64 (m, 3H, Hb-3, H-4), 2.49 (s, 3H, NCH3), 1.91–1.82 (m,
1H, Ha-10), 1.79–1.68 (m, 3H, Hb-10, H-40), 1.53–1.37 (m, 4H, H-
13
20, H-30); C NMR (125 MHz, CDCl3): d 166.3 (CO), 163.2 (C-400),
147.5 (C-6), 147.3 (C-7), 131.4 (CH-300, CH-500), 128.9 (C-8a), 125.6
(C-4a), 122.9 (C-100), 113.2 (CH-200, CH-600), 111.3 (CH-5), 110.3
(CH-8), 64.6 (CH2-50), 63.1 (CH-1), 56.0 (OCH3-6), 55.8 (OCH3-7),
47.1 (CH2-3), 41.9 (NCH3), 34.8 (CH2-10), 28.7 (CH2-40), 26.3 (CH2-
30), 25.5 (CH2-20), 24.6 (CH2-4); ESMS m/z (%) 428 [M+H]+ (14).
3.1.6.1.
THIQ (8).
N-Methyl-1-(pentylbenzoate)-6,7-dimethoxy-1,2,3,4-
Under the same conditions described above, 7 and
benzoyl chloride (35.0 lL, 0.3 mmol) reacted to obtain 80 mg of a
yellow oil 8 (67%). 1H NMR (500 MHz, CDCl3): d 8.04–8.00 (m,
2H, H-200, H-600), 7.57–7.50 m, 1H, H-400), 7.46–7.39 (m, 2H, H-300,
H-500), 6.58 (s, 1H, H-5), 6.56 (s, 1H, H-8), 4.28 (t, J = 6.6 Hz, 2H,
H-50), 3.85 and 3.84 (2s, 6H, OCH3-6 and OCH3-7), 3.58 (t,
J = 5.7 Hz, 2H, H-1), 3.30–3.20 (m, 1H, Ha-3), 2.91–2.85 (m, 1H,
Hb-3), 2.85–2.78 (m, 1H, Ha-4), 2.78–2.70 (m, 1H, Hb-4), 2.54 (s,
3H, NCH3), 1.97–1.85 (m, 1H, Ha-10), 1.80–1.71 (m, 3H, Hb-10, H-
40), 1.54–1.39 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d
3.1.6.5.
thoxy-1,2,3,4-THIQ (12).
N-Methyl-1-[(phenylpropanoate)pentyl]-6,7-dime-
Under the same conditions de-
scribed above, 7 and hydrocinnamoyl chloride (0.1 mL, 0.8 mmol)
reacted to obtain 139 mg of yellow oil 12 (65%). 1H NMR
(500 MHz, CDCl3): d 7.30–7.23 (m, 2H, H-200, H-600), 7.21–7.15 (m,
3H, H-300, H-400, H-500), 6.56 (s, 1H, H-5), 6.55 (s, 1H, H-8), 4.03 (t,
J = 6.7 Hz, 2H, H-50), 3.83 (s, 6H, OCH3-6, OCH3-7), 3.37 (t,

3228
A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
J = 5.4 Hz, 1H, H-1), 3.14–3.05 (m, 1H, Ha-3), 2.93 (t, J = 7.8 Hz, 2H,
H-90), 2.78–2.63 (m, 3H, Hb-3, H-4), 2.60 (t, J = 7.8 Hz, 2H, H-80),
2.44 (s, 3H, NCH3), 1.77–1.69 (m, 2H, H-10), 1.63–1.53 (m, 2H, H-
40), 1.47–1.24 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d
172.8 (CO), 147.1 (C-6, C-7), 140.4 (C-100), 129.9 (C-8a), 128.3
(CH-200, CH-600), 128.1 (CH-300, CH-500), 126.5 (C-4a), 126.2 (CH-400),
111.2 (CH-5), 110.1 (CH-8), 64.5 (CH2-50), 63.2 (CH-1), 55.9
(OCH3-6), 55.7 (OCH3-7), 48.1 (CH2-3), 42.6 (NCH3), 35.8 (CH2-80),
34.8 (CH2-10), 30.8 (CH2-90), 28.5 (CH2-40), 26.1 (CH2-30), 25.4
(CH2-4), 25.1 (CH2-20); ESMS m/z (%) 426 [M+H]+ (14).
(m, 2H, H-200, H-600), 6.84–6.78 (m, 2H, H-300, H-500), 6.56 (s, 1H, H-
5), 6.55 (s, 1H, H-8), 4.03 (t, J = 6.7 Hz, 2H, H-50), 3.84 (s, 6H,
OCH3-6, OCH3-7), 3.77 (s, 3H, OCH3-400), 3.38 (t, J = 5.4 Hz, 1H, H-
1), 3.15–3.06 (m, 1H, Ha-3), 2.87 (t, J = 7.8 Hz, 2H, H-90), 2.79–
2.71 (m, 1H, Ha-4), 2.71–2.63 (m, 2H, Hb-3, Hb-4), 2.57 (t,
J = 7.8 Hz, 2H, H-80), 2.44 (s, 3H, NCH3), 1.78–1.69 (m, 2H, H-10),
1.62–1.53 (m, 2H, H-40), 1.47–1.23 (m, 4H, H-20, H-30); 13C NMR
(125 MHz, CDCl3): d 173.0 (CO), 158.0 (C-400), 147.2 (C-6, C-7),
132.6 (C-100), 129.9 (C-8a), 129.2 (CH-200, CH-600), 126.4 (C-4a,)
113.8 (CH-300, CH-500), 111.3 (CH-5), 110.2 (CH-8), 64.5 (CH2-50),
63.3 (CH-1), 56.0 (OCH3-6), 55.8 (OCH3-7), 55.2 (OCH3-400), 48.1
(CH2-3), 42.7 (NCH3), 36.1 (CH2-80), 34.9 (CH2-10), 30.1 (CH2-90),
28.6 (CH2-40), 26.2 (CH2-20), 25.4 (CH2-30), 25.2 (CH2-4); ESMS m/
z (%) 456 [M+H]+ (15).
3.1.6.6. N-Methyl-1-[(400-fluorophenylpropanoate)pentyl]-6,7-
dimethoxy-1,2,3,4-THIQ (13).
A solution of 3-(4-fluoro-
phenyl)propionic acid (300 mg, 1.8 mmol) in dry CH2Cl2 (20 mL)
was treated with thionyl chloride (2.2 mL, 30.2 mmol). The reac-
tion mixture was refluxed for 3 h and the solvent was then re-
moved until dryness. A translucent oil, corresponding to 3-(4-
fluorophenyl)propanoyl chloride, was obtained and used directly
in the next reaction under the same conditions described in the
general procedure. Therefore, 7 (314.5 mg, 1.1 mmol) and 3-(4-
fluorophenyl)propanoyl chloride (300 mg, 1.6 mmol) reacted to
obtain 251 mg of a yellow oil 13 (51%). 1H NMR (500 MHz, CDCl3):
d 7.15–7.09 (m, 2H, H-200, H-600), 6.95–6.89 (m, 2H, H-300, H-500), 6.55
(s, 1H, H-5), 6.53 (s, 1H, H-8), 4.01 (t, J = 6.7 Hz, 2H, H-50), 3.82 and
3.81 (2s, 6H, OCH3-6 and OCH3-7), 3.35 (t, J = 5.4 Hz, 1H, H-1),
3.12–3.03 (m, 1H, Ha-3), 2.88 (t, J = 7.7 Hz, 2H, H-90), 2.74–2.60
(m, 3H, Hb-3, H-4), 2.56 (t, J = 7.7 Hz, 2H, H-80), 2.41 (s, 3H,
NCH3), 1.75–1.67 (m, 2H, H-10), 1.59–1.52 (m, 2H, H-40), 1.44–
1.21 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d 172.6
(CO), 161.3 (C-400, d, J = 242 Hz), 147.1 (C-6, C-7), 136.1 (C-100),
130 (CH-200, CH-600, d, J = 8 Hz), 126.5 (C-8a, C-4a), 115.0 (CH-300,
CH-500, d, J = 21 Hz), 111.2 (CH-8), 110.0 (CH-5), 64.5 (CH2-50),
63.2 (CH-1), 55.9 (OCH3-6), 55.6 (OCH3-7), 48.1 (CH2-3), 42.6
(NCH3), 35.8 (CH2-80), 34.7 (CH2-10), 30.0 (CH2-90), 28.5 (CH2-40),
26.1 (CH2-4), 25.4 (CH2-20), 25.0 (CH2-30); ESMS m/z (%) 444
[M+H]+ (19).
3.1.7. General procedure for the synthesis of carbamates N-
methyl-1-[(substituted carbamate)pentyl]-6,7-dimethoxy-
1,2,3,4-THIQs (16–23)
A mixture of N-methyl-1-pentanol-6,7-dimethoxy-1,2,3,4-THIQ
(7, 0.4 mmol) and the corresponding isocyanate (1.2 mmol) in
CH2Cl2 (20 mL) was refluxed under a N2 atmosphere for 24 h. The
reaction mixture was extracted with CH2Cl2 and washed with brine
and H2O. The organic phase was dried over anhydrous Na2SO4, fil-
tered and evaporated to dryness. The residue was purified (CH2Cl2/
MeOH 97:3) to obtain the corresponding THIQ carbamate.
3.1.7.1.
thoxy-1,2,3,4-THIQ (16).
N-Methyl-1-[(phenylcarbamate)-pentyl]-6,7-dime-
A mixture of 7 (118 mg, 0.4 mmol)
and phenyl isocyanate (0.13 mL, 1.2 mmol) was subjected to simi-
lar conditions to those above described to obtain 124 mg of a dark
yellow oil 16 (75%). 1H NMR (500 MHz, CDCl3): d 7.41–7.35 (m, 2H,
H-300, H-500), 7.30–7.25 (m, 2H, H-200, H-600), 7.05–7.00 (m, 1H, H-400),
6.86 (s, 1H, NH), 6.56 (s, 1H, H-5), 6.55 (s, 1H, H-8), 4.12 (t,
J = 6.6 Hz, 2H, H-50), 3.83 (s, 6H, OCH3-6, OCH3-7), 3.41 (t,
J = 5.4 Hz, 1H, H-1), 3.17–3.08 (m, 1H, Ha-3), 2.79–2.63 (m, 3H,
Hb-3, H-4), 2.45 (s, 3H, NCH3), 1.83–1.70 (m, 2H, H-10), 1.65 (qt,
J = 6.9 Hz, 2H, H-40), 1.49–1.30 (m, 4H, H-20, H-30); 13C NMR
(125 MHz, CDCl3): d 153.7 (CO), 147.3 (C-6), 147.2 (C-7), 138.0
(C-100), 129.6 (C-8a), 128.9 (CH-200, CH-600), 126.3 (C-4a), 123.2
(CH-400), 118.6 (CH-300, CH-500), 111.3 (CH-8), 110.2 (CH-5), 65.3
(CH2-50), 63.3 (CH-1), 56.0 (OCH3-6), 55.8 (OCH3-7), 48.0 (CH2-3),
42.5 (NCH3), 34.8 (CH2-10), 28.8 (CH2-40), 26.2 (CH2-30), 25.3
(CH2-4), 25.2 (CH2-20); ESMS m/z (%) 413 [M+H]+ (36).
3.1.6.7. N-Methyl-1-[(400-chlorophenylpropanoate)pentyl]-6,7-
dimethoxy-1,2,3,4-THIQ (14).
A solution of 3-(4-chloro-
phenyl)propionic acid (111 mg, 0.6 mmol) was treated with thio-
nyl chloride (0.7 mL, 9.6 mmol), as described for 13. Then, a
solution of 3-(4-chlorophenyl)propanoyl chloride (102 mg,
0.5 mmol) and 7 (118 mg, 0.4 mmol) reacted under the same con-
ditions described in the general procedure to obtain 19 mg of a yel-
low oil 14 (10%). 1H NMR (500 MHz, CDCl3): d 7.23 (d, J = 8.3 Hz,
2H, H-300, H-500), 7.12 (d, J = 8.3 Hz, 2H, H-200, H-600), 6.56 (s, 1H, H-
5), 6.55 (s, 1H, H-8), 4.02 (t, J = 6.7 Hz, 2H, H-50), 3.84 (s, 6H,
OCH3-6, OCH3-7), 3.40 (t, J = 5.1 Hz, 1H, H-1), 3.17–3.08 (m, 1H,
Ha-3), 2.90 (t, J = 7.7 Hz, 2H, H-90), 2.80–2.64 (m, 3H, Hb-3, H-4),
2.58 (t, J = 7.7 Hz, 2H, H-80), 2.45 (s, 3H, NCH3), 1.80–1.68 (m, 2H,
H-10), 1.62–1.53 (m, 2H, H-40), 1.36–1.22 (m, 4H, H-20, H-30); 13C
NMR (125 MHz, CDCl3): d 172.7 (CO), 147.3 (C-6), 147.2 (C-7),
139.0 (C-100), 132.0 (C-8a, C-400), 129.7 (CH-200, CH-600), 128.6 (CH-
300, CH-500), 126.6 (C-4a), 111.3 (CH-5), 110.2 (CH-8), 64.7 (CH2-
50), 63.3 (CH-1), 56.0 (OCH3-6), 55.8 (OCH3-7), 48.0 (CH2-3), 42.6
(NCH3), 35.7 (CH2-80), 34.9 (CH2-10), 30.3 (CH2-90), 28.6 (CH2-40),
26.2 (CH2-20, CH2-30), 25.3 (CH2-4); ESMS m/z (%) 460 [M+H]+ (32).
3.1.7.2.
dimethoxy-1,2,3,4-THIQ (17).
N-Methyl-1-[(400-fluorophenylcarbamate)pentyl]-6,7-
mixture of (84 mg,
A
7
0.3 mmol) and 4-fluorophenyl isocyanate (0.1 mL, 0.9 mmol) was
subjected to similar conditions to those described in the general
procedure to obtain 28 mg of a dark yellow oil 17 (22%). 1H NMR
(500 MHz, CDCl3): d 7.34–7.24 (m, 2H, H-200, H-600), 6.94–6.88 (m,
2H, H-300, H-500), 6.50 (s, 1H, H-5), 6.49 (s, 1H, H-8), 4.05 (t,
J = 6.5 Hz, 2H, H-50), 3.77 (s, 6H, OCH3-6, OCH3-7), 3.50–3.43 (m,
1H, H-1), 3.18–3.10 (m, 1H, Ha-3), 2.77–2.66 (m, 3H, Hb-3, H-4),
2.45 (s, 3H, NCH3), 1.83–1.74 (m, 1H, Ha-10), 1.73–1.65 (m, 1H,
Hb-10), 1.62–1.55 (m, 2H, H-40), 1.46–1.27 (m, 4H, H-20, H-30); 13C
NMR (125 MHz, CDCl3): d 163.0 (C-400, d, J = 243 Hz), 153.9 (CO),
147.6 (C-6), 147.4 (C-7), 134.1 (C-100), 129.4 (C-8a), 125.5 (C-4a),
120.3 (CH-200, CH-600, d, J = 8 Hz), 115.5 (CH-300, CH-500, d,
J = 23 Hz), 111.3 (CH-5), 110.2 (CH-8), 65.4 (CH2-50), 63.5 (CH-1),
56.0 (OCH3-7), 55.8 (OCH3-6), 47.6 (CH2-3), 42.1 (NCH3), 34.8
(CH2-10), 28.7 (CH2-40), 26.3 (CH2-30), 25.3 (CH2-20), 24.8 (CH2-4);
ESMS m/z (%) 431 [M+H]+ (45).
3.1.6.8. N-Methyl-1-[(400-methoxyphenylpropanoate)-pentyl]-
6,7-dimethoxy-1,2,3,4-THIQ (15).
A
solution of 3-(4-
methoxyphenyl)propionic acid (198 mg, 1.1 mmol) in dry CH2Cl2
(20 mL) was treated with thionyl chloride (1.4 mL, 19.2 mmol), as
described for 13. Then, a solution of 3-(4-methoxyphenyl)propa-
noyl chloride (198.7 mg, 1.0 mmol) and 7 reacted under the same
conditions described in the general procedure to obtain 46 mg of
the yellow oil 15 (14%). 1H NMR (500 MHz, CDCl3): d 7.13–7.07
3.1.7.3. N-Methyl-1-[(400-chlorophenylcarbamate)-pentyl]-6,7-
dimethoxy-1,2,3,4-THIQ (18).
A mixture of 7 (150 mg,
0.5 mmol) and 4-chlorophenyl isocyanate (0.2 mL, 1.5 mmol) was

A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
3229
subjected to similar conditions to those above described to obtain
144 mg of a yellow oil 18 (64%). 1H NMR (500 MHz, CDCl3): d 7.32
(d, J = 8.4 Hz, 2H, H-300, H-500), 7.23 (d, J = 8.4 Hz, 2H, H-200, H-600),
6.92 (s, 1H, NH), 6.56 (s, 1H, H-5), 6.55 (s, 1H, H-8), 4.11 (t,
J = 6.6 Hz, 2H, H-50), 3.83 and 3.82 (2s, 6H, OCH3-6 and OCH3-7),
3.37 (t, J = 5.3 Hz, 1H, H-1), 3.13–3.06 (m, 1H, Ha-3), 2.77–2.62
(m, 3H, Hb-3, H-4), 2.43 (s, 3H, NCH3), 1.78–1.71 (m, 2H, H-10),
1.67–1.59 (m, 2H, H-40), 1.47–1.27 (m, 4H, H-20, H-30); 13C NMR
(125 MHz, CDCl3): d 153.6 (CO), 147.2 (C-6), 147.1 (C-7), 136.7
(C-100), 129.9 (C-8a), 128.9 (CH-200, CH-600), 128.1 (C-400), 126.6 (C-
4a), 119.8 (CH-300, CH-500), 111.3 (CH-5), 110.2 (CH-8), 65.5 (CH2-
50), 63.3 (CH-1), 56.0 (OCH3-6), 55.7 (OCH3-7), 48.2 (CH2-3), 42.7
(NCH3), 34.8 (CH2-10), 28.8 (CH2-40), 26.2 (CH2-4), 25.3 (CH2-20,
CH2-30); ESMS m/z (%) 447 [M+H]+ (100).
8), 110.2 (CH-5), 64.9 (CH2-50), 63.3 (CH-1), 56.0 (OCH3-7), 55.7
(OCH3-6), 48.1 (CH2-3), 42.7 (NCH3), 42.1 (CH2-90), 35.3 (CH2-
100), 34.9 (CH2-10), 29.0 (CH2-40), 26.2 (CH2-4), 25.3 (CH2-20, CH2-
30); ESMS m/z (%) 459 [M+H]+ (100).
3.1.7.7.
6,7-dimethoxy-1,2,3,4-THIQ (22).
N-Methyl-1-[(400-chlorophenethylcarbamate)pentyl]-
Finally, 244 mg of
7
(0.8 mmol) was subjected to similar conditions to those above de-
scribed, using 4-chlorophenethyl isocyanate (0.4 mL, 2.5 mmol), to
obtain 253 mg of the yellow oil 22 (67%). 1H NMR (500 MHz,
CDCl3): d 7.27 (d, J = 8.3 Hz, 2H, H-300, H-500), 7.12 (d, J = 8.3 Hz,
2H, H-200, H-600), 6.56 (s, 2H, H-5, H-8), 4.70 (s, 1H, NH), 4.02 (t,
J = 6.5 Hz, 2H, H-50), 3.85 and 3.84 (2s, 6H, OCH3-6 and OCH3-7),
3.43–3.34 (m, 3H, H-1, H-90), 3.15–3.07 (m, 1H, Ha-3), 2.80–2.75
(m, 2H, H-100), 2.75–2.64 (m, 3H, Hb-3, H-4), 2.45 (s, 3H, NCH3),
1.80–1.69 (m, 2H, H-10), 1.62–1.53 (m, 2H, H-40), 1.49–1.38 (m,
1H, Ha-20), 1.38–1.23 (m, 3H, Hb-20, H-30); 13C NMR (125 MHz,
CDCl3): d 156.6 (CO), 147.2 (C-6, C-7), 137.3 (C-100), 132.2 (C-400),
130.1 (CH-200, CH-600), 129.9 (C-8a), 128.7 (CH-300, CH-500), 126.5
(C-4a), 111.3 (CH-5), 110.2 (CH-8), 65.0 (CH2-50), 63.3 (CH-1),
56.0 (OCH3-7), 55.8 (OCH3-6), 48.1 (CH2-3), 42.7 (NCH3), 41.9
(CH2-90), 35.5 (CH2-100), 34.9 (CH2-10), 29.0 (CH2-40), 26.2 (CH2-
30), 25.4 (CH2-20), 25.2 (CH2-4); ESMS m/z (%) 475 [M+H]+ (7).
3.1.7.4. N-Methyl-1-[(400-methoxyphenylcarbamate)pentyl]-6,7-
dimethoxy-1,2,3,4-THIQ (19).
A mixture of 7 (150 mg,
0.5 mmol) and 4-methoxyphenyl isocyanate (0.2 mL, 1.5 mmol)
was subjected to similar conditions to those above described to ob-
tain 160 mg of a yellow oil 19 (73%). 1H NMR (500 MHz, CDCl3): d
7.30–7.23 (m, 2H, H-200, H-600), 6.83–6.79 (m, 2H, H-300, H-500), 6.55
(s, 1H, H-5), 6.54 (s, 1H, H-8), 4.09 (t, J = 6.6 Hz, 2H, H-50), 3.82 (s,
6H, OCH3-6, OCH3-7), 3.75 (s, 3H, OCH3-400), 3.37 (t, J = 5.4 Hz, 1H,
H-1), 3.13–3.05 (m, 1H, Ha-3), 2.77–2.61 (m, 3H, Hb-3, H-4), 2.42
(s, 3H, NCH3), 1.77–1.70 (m, 2H, H-10), 1.62 (p, J = 6.63 Hz, 2H, H-
40), 1.48–1.27 (m, 4H, H-20, H-30); 13C NMR (125 MHz, CDCl3): d
155.7 (C-400), 154.1 (CO), 147.1 (C-6, C-7), 131.1 (C-100), 129.9 (C-
8a), 128.5 (C-4a), 120.5 (CH-200, CH-600), 114.1 (CH-300, CH-500),
111.2 (CH-5), 110.1 (CH-8), 65.1 (CH2-50), 63.2 (CH-1), 56.0
(OCH3-6), 55.7 (OCH3-7), 48.1 (CH2-3), 42.7 (NCH3), 34.7 (CH2-10),
28.8 (CH2-40), 26.2 (CH2-4), 25.2 (CH2-20, CH2-30); ESMS m/z (%)
443 [M+H]+ (100).
3.1.7.8. N-Methyl-1-[(400-methoxyphenethylcarbamate)pentyl]-
6,7-dimethoxy-1,2,3,4-THIQ (23).
Finally, 162 mg of
7
(0.6 mmol) was subjected to similar conditions to those described
in the general procedure, using 4-methoxyphenethyl isocyanate
(0.3 mL, 1.7 mmol), to obtain 136 mg of the yellow oil 23 (48%).
1H NMR (500 MHz, CDCl3): d 7.09 (d, J = 8.5 Hz, 2H, H-200, H-600),
6.84 (d, J = 8.5 Hz, 2H, H-300, H-500), 6.55 (s, 2H, H-5, H-8), 4.68 (s,
1H, NH), 4.01 (t, J = 6.2 Hz, 2H, H-50), 3.84 and 3.83 (2s, 6H,
OCH3-6 and OCH3-7), 3.77 (s, 3H, OCH3-400), 3.43–3.33 (m, 3H, H-
1, H-90), 3.15–3.08 (m, 1H, Ha-3), 2.78–2.63 (m, 5H, Hb-3, H-4,
H-100), 2.45 (s, 3H, NCH3), 1.81–1.68 (m, 2H, H-10), 1.64–1.53 (m,
2H, H-40), 1.47–1.37 (m, 1H, Ha-20), 1.37- 1.23 (m, 3H, Hb-20, H-
3.1.7.5.
thoxy-1,2,3,4-THIQ (20).
N-Methyl-1-[(phenethylcarbamate)pentyl]-6,7-dime-
A mixture of 7 (150 mg, 0.5 mmol)
and phenethyl isocyanate (0.2 mL, 1.5 mmol) was subjected to
similar conditions to those described in the general procedure to
obtain 146 mg of a yellow oil 20 (66%). 1H NMR (500 MHz, CDCl3):
d 7.31–7.25 (m, 2H, H-200, H-600), 7.24–7.14 (m, 3H, H-300, H-400, H-
500), 6.56 (s, 1H, H-5), 6.55 (s, 1H, H-8), 4.71 (s, 1H, NH), 4.01 (t,
J = 6.3 Hz, 2H, H-50), 3.84 and 3.83 (2s, 6H, OCH3-6 and OCH3-7),
3.45–3.39 (m, 2H, H-90), 3.37 (t, J = 5.4 Hz, 1H, H-1), 3.13–3.05
(m, 1H, Ha-3), 2.83–2.76 (m, 2H, H-100), 2.77–2.62 (m, 3H, Hb-3,
H-4), 2.43 (s, 3H, NCH3), 1.78–1.69 (m, 2H, H-10), 1.64–1.52 (m,
2H, H-40), 1.46–1.25 (m, 4H, H-20, H-30); 13C NMR (125 MHz,
CDCl3): d 156.6 (CO), 147.2 (C-6, C-7), 138.8 (C-100), 130.1 (C-8a),
128.8 (CH-200, CH-600), 128.5 (CH-300, CH-500), 126.5 (CH-400), 126.3
(C-4a), 111.2 (CH-5), 110.2 (CH-8), 64.9 (CH2-50), 63.3 (CH-1),
56.0 (OCH3-6), 55.7 (OCH3-7), 48.1 (CH2-3), 42.7 (NCH3), 42.1
(CH2-90), 36.1 (CH2-100), 34.9 (CH2-10), 29.0 (CH2-40), 26.2 (CH2-
4), 25.3 (CH2-20, CH2-30); ESMS m/z (%) 441 [M+H]+ (100).
13
30); C NMR (125 MHz, CDCl3): d 158.2 (C-400), 156.6 (CO), 147.3
(C-6), 147.2 (C-7), 130.8 (C-100), 129.7 (C-8a, CH-300, CH-500), 126.3
(C-4a,), 114.0 (CH-200, CH-600), 111.3 (CH-5), 110.2 (CH-8), 64.9
(CH2-50), 63.4 (CH-1), 56.0 (OCH3-6), 55.8 (OCH3-7), 55.2 (OCH3-
400), 48.0 (CH2-3), 42.6 (NCH3), 42.3 (CH2-90), 35.2 (CH2-100), 34.9
(CH2-10), 29.0 (CH2-40), 26.2 (CH2-20), 25.3 (CH2-4, CH2-30); ESMS
m/z (%) 471 [M+H]+ (8).
3.2. Antimicrobial activities
3.2.1. Target microorganisms
Antifungal activity was measured against five phytopathogenic
fungi: Aspergillus parasiticus (CECT 2681), Trichoderma viride (CECT
2423), Fusarium culmorum (CCM 172), Phytophthora citrophthora
(CECT 2353) and Geotrichum candidum (CCM 245). Antibacterial
activity was determined against seven human pathogenic bacteria:
Bacillus cereus (CECT 148), Staphylococcus aureus (CECT 86), Entero-
coccus faecalis (CECT 481), Salmonella typhi (CECT 409), Escherichia
coli (CECT 405), Escherichia coli (CECT 100) and Erwinia carotovora
(CECT 225). The species were provided by the Spanish Type Culture
Collection (CECT) or by the Cathedra Collection of Microbiology
(CCM) of the Biotechnology Department (Polytechnic University
of Valencia).
3.1.7.6.
6,7-dimethoxy-1,2,3,4-THIQ (21).
N-Methyl-1-[(400-fluorophenethylcarbamate)-pentyl]-
A mixture of 7 (150 mg,
0.5 mmol) and 4-fluorophenethyl isocyanate (0.2 mL, 1.5 mmol)
was subjected to similar conditions to those above described to ob-
tain 153 mg of a yellow oil 21 (67%). 1H NMR (500 MHz, CDCl3): d
7.17–7.08 (m, 2H, H-200, H-600), 7.00–6.94 (m, 2H, H-300, H-500), 6.55
(s, 1H, H-5), 6.54 (s, 1H, H-8), 4.72 (s, 1H, NH), 4.00 (t, J = 6.4 Hz,
2H, H-50), 3.83 and 3.81 (2s, 6H, OCH3-6 and OCH3-7), 3.41–3.31
(m, 3H, H-1, H-90), 3.13–3.04 (m, 1H, Ha-3), 2.81–2.60 (m, 5H,
Hb-3, H-4, H-100), 2.42 (s, 3H, NCH3), 1.76–1.68 (m, 2H, H-10),
1.62–1.52 (m, 2H, H-40), 1.45–1.23 (m, 4H, H-20, H-30); 13C NMR
(125 MHz, CDCl3): d 161.6 (C-400, d, J = 243 Hz), 156.6 (CO), 147.2
(C-6, C-7), 134.4 (C-100), 130.1 (CH-200, CH-600, d, J = 8 Hz), 130.0
(C-8a), 126.5 (C-4a), 115.3 (CH-300, CH-500, d, J = 21 Hz), 111.2 (CH-
3.2.2. Antimicrobial assays
The assays were carried out in triplicate. Each compound was
assayed against each microorganism using the paper disk-agar dif-
fusion method as previously described.19 The doses used in the as-
says for all compounds with the isoquinoline core (2, 3, 5–23) were
10 and 20 l
g/mm2 (0.2 and 0.4 mg/disk, respectively). Fungal

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A. Galán et al. / Bioorg. Med. Chem. 21 (2013) 3221–3230
species were seeded in sterile Petri dishes containing Potato Dex-
trose Agar culture medium (Difco) and were incubated for 7 days
at 28 °C. To obtain suspensions of ꢁ106 conidia/mL, tween 80
(0.05%) in sterile distilled water solution was used. Then, 1 mL of
conidia suspensions were added to 15 mL of PDA in sterile Petri
dishes. Whatmann disks (No. 113, 0.5 cm diameter) were impreg-
nated with the products to test at appropriate concentrations. After
medium’s solidification, four impregnated Whatmann disks were
placed in the Petri dishes. Disks impregnated just with the vehicle
were used as negative controls and those with benomyl (methyl-1-
[butylcarbamoyl]-2-benzimidazolecarbamate) (Sigma) at different
concentrations based on the fungal species assayed, were used as
positive controls. Antifungal activity was determined by measuring
the inhibition zone developed around the paper disk. For evalua-
tion of the bactericidal activity, 24-h cultures of each bacterium
(maintained in inclined tubes on solid culture medium) were reac-
tivated with Nutrient Broth (Difco) and then incubated for a further
24 h at 28 or 37 °C, depending on the bacteria investigated. Next,
1 mL of each bacterial suspension was placed in sterile Petri plates
and 15 mL of Plate Count Agar (Difco) culture medium added. After
solidification of the medium, four Whatmann paper disks (No. 113,
0.5 cm diameter), previously impregnated with the products to test
were placed in the dishes. The plates were incubated for 24 h in the
dark at 28 or 37 °C, based on the bacteria investigated. Disks
impregnated just with the vehicle were used as negative controls.
Acknowledgments
A.G. was the recipient of a fellowship from FPU program of
Spanish Ministry of Education, Culture and Sport. This study was
supported by grants SAF2011-23777, Spanish Ministry of Economy
and Competitiveness, RIER RD08/0075/0016, Carlos III Health Insti-
tute, Spanish Ministry of Health and the European Regional Devel-
opment Fund (FEDER).
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Variance analysis (one-way ANOVA) was performed for the
antifungal and antibacterial results (Tables 1–5). Least significant
difference (LSD) test was used to compare means and F test was
performed to determine statistically significant differences
(P <0.05) by Statgraphics Centurion XVI version.