Searching for new agents active against Candida albicans biofilm: A series of indole derivatives, design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.01.012

Candida albicans biofilm represents a major clinical problem due to its intrinsic tolerance to anti-fungal compounds and it has been highly related to infections in catheterized patients. Few compounds are described as able to inhibit biofilm formation or to interfere with preformed biofilm of C. albicans. Here we report the in vitro evaluation of anti-biofilm activity on C. albicans ATCC 10231 of a series of new and already known amine and amide indole derivatives. Among the studied compounds, fifteen resulted active on C. albicans ATCC 10231 biofilm, with BMIC₅₀ ≤ 16 μg/mL. Three of them (7, 23 and 33) showed a selectivity towards mature biofilm and the most active of them was the compound 23 (BMIC₅₀ = 4 μg/mL). On the other hands, two different compounds (21 and 22) were selective towards biofilm formation with BMIC₅₀ values of 8 μg/mL. Otherwise, compounds 16 and 17 resulted active on biofilm formation, with BMIC₅₀ of 8 μg/mL and 2 μg/mL respectively, and on mature biofilm with BMIC₅₀ of 2 μg/mL. These two last compounds also showed an interesting activity towards the planktonic cells of C. albicans. A selection of the more active compounds was also evaluated on different C. albicans strains (PMC1042, PMC1083 and ATCC 10261), showing a comparable or higher anti-biofilm activity, especially on mature biofilm. In vivo toxicity studies using the Galleria mellonella larvae, were finally carried out on more active indole derivatives, showing that they are poorly toxic even at the highest concentrations tested (500–1000 μg/mL).

Full text of DOI:10.1016/j.ejmech.2019.01.012

Accepted Manuscript
Searching for new agents active against Candida albicans biofilm: A series of indole
derivatives, design, synthesis and biological evaluation
Fabiana Pandolfi, Federica D'Acierno, Martina Bortolami, Daniela De Vita, Fabio
Gallo, Alessandra De Meo, Roberto Di Santo, Roberta Costi, Giovanna Simonetti,
Luigi Scipione
PII:
S0223-5234(19)30018-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.012
EJMECH 11019
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2018
Revised Date: 14 September 2018
Accepted Date: 6 January 2019
Please cite this article as: F. Pandolfi, F. D'Acierno, M. Bortolami, D. De Vita, F. Gallo, A. De Meo, R.
Di Santo, R. Costi, G. Simonetti, L. Scipione, Searching for new agents active against Candida albicans
biofilm: A series of indole derivatives, design, synthesis and biological evaluation, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.012.
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Searching for new agents active against Candida albicans biofilm: A series of indole
ACCEPTED MANUSCRIPT
derivatives, design, synthesis and biological evaluation
a
a
Fabiana Pandolfi , Federica D’Acierno b, Martina Bortolami , Daniela De Vita a, Fabio Gallo a, Alessandra
De Meo a, Roberto Di Santo c, Roberta Costi c, Giovanna Simonetti b*#, Luigi Scipione a*#
a Department of “Chimica e Tecnologie del Farmaco”, Sapienza University of Rome, Piazzale Aldo Moro, 5,
00185 Rome, Italy
b Department of “Sanità Pubblica e Malattie Infettive”, Sapienza University of Rome, Piazzale Aldo Moro, 5,
00185 Rome, Italy
c
“Istituto Pasteur-Fondazione Cenci Bolognetti”, Department of “Chimica e Tecnologie del Farmaco”,
Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
* Corresponding authors: Giovanna Simonetti (giovanna.simonetti@uniroma1.it); Luigi Scipione
(luigi.scipione@uniroma1.it).
# The authors Giovanna Simonetti and Luigi Scipione equally contributed to this work.
Abstract
Candida albicans biofilm represents a major clinical problem due to its intrinsic tolerance to anti-fungal
compounds and it has been highly related to infections in catheterized patients. Few compounds are described
as able to inhibit biofilm formation or to interfere with preformed biofilm of C. albicans. Here we report the
in vitro evaluation of anti-biofilm activity on C. albicans ATCC 10231 of a series of new and already known
amine and amide indole derivatives. Among the studied compounds, fifteen resulted active on C. albicans
ATCC 10231 biofilm, with BMIC50 ≤ 16 µg/mL. Three of them (7, 23 and 33) showed a selectivity towards
mature biofilm and the most active of them was the compound 23 (BMIC50 = 4 µg/mL). On the other hands,
two different compounds (21 and 22) were selective towards biofilm formation with BMIC50 values of 8
µg/mL. Otherwise, compounds 16 and 17 resulted active on biofilm formation BMIC50 of 8 µg/mL and 2
µg/mL respectively and on mature biofilm with BMIC50 of 2 µg/mL. These two last compounds also showed
an interesting activity towards the planktonic cells of C. albicans. A selection of the more active compounds
was also evaluated on different C. albicans strains (PMC1042, PMC1083 and ATCC 10261), showing a
comparable or higher anti-biofilm activity, especially on mature biofilm. In vivo toxicity studies using the

Galleria mellonella larvae, were finally carried out on more active indole derivatives, showing that they are
ACCEPTED MANUSCRIPT
poorly toxic even at the highest concentrations tested (500-1000 µg/mL).
Keywords
Indole derivatives; Candida albicans biofilm; Galleria mellonella.
1. Introduction
Microorganisms, in their natural habitats, are found attached to surfaces and not only as free-floating
(planktonic) organisms. Candida albicans is a part of the normal microbiota of humans, which allows the
contact with most medical devices and host surfaces, permitting formation of robust biofilms [1]. Candida
biofilms lead to recurring infections and in some cases death [2] and it has been estimated that it may be
responsible for up to 65% of infections in catheterized patients [3]. C. albicans biofilm presents a significant
clinical problem, with current treatment options severely limited by the intrinsic tolerance of fungal biofilms
for anti-fungal [4]; as a matter of fact, biofilms of C. albicans are less susceptible to many antifungal drugs,
including fluconazole [5]. Few molecules were reported in literature to inhibit or to prevent the C. albicans
biofilm formation; therefore, there is a strong need for the development of novel antifungal biofilm strategies.
In previous works, we focused our attention on antifungal compounds, particularly, we studied compounds
active against C. albicans planktonic cells [6, 7] and biofilm [8]; studying a set of cinnamic acid derivatives,
we identified a new indole compound which was active against biofilm formation of C. albicans with a MIC
of 8 µg/mL [9].
The analysis of literature on the anti-biofilm activity of indolic compounds evidenced few data, such as the
paper of Oh and co-workers [10] that reports the ability of indole and indole-3-acetonitrile to repress the
biofilm formation in C. albicans interfering with quorum sensing and to inhibit the yeast filamentation.
Recently Vila and Lopez-Ribot have identified the indole derivative MMV688768, as the most potent
compound belonging to a synthetic library, which was able to reduce the metabolic cell activity within the C.
albicans preformed biofilm [11].
2

On these bases we decided to explore the anti-biofilm activity of the indole moiety and, therefore, we have
ACCEPTED MANUSCRIPT
selected a set of indole amine and amide derivatives with the aim to identify new compounds able to interfere
with the C. albicans biofilm. We have selected small indole molecules already known in the literature or
commercially available and furthermore, we have designed and synthesized some new original compounds.
In particular, tryptamine has been chosen as starting compound and its structure was modified as outlined in
Chart 1. A set of amine derivatives was obtained by binding the amine function of the tryptamine to an
aromatic group, such as naphthyl or variously substituted benzylic group or to a heterocycle such as pyridine,
nitrofuran and indole. A further series of amines was obtained by molecular duplication starting from
tryptamine, coupled with terephthalaldehyde or from 1H-indole-3-carbaldehyde coupled with linear alkyl-
diamines.
Moreover, we synthesized a series of amide derivatives, containing a set of compounds analogous to some of
the amine derivatives and a set of amides obtained by conjugation with some representative nonsteroidal
antiinflammatory drugs (NSAIDs), as some authors reported the inhibitory activities of NSAIDs against C.
albicans biofilm [12, 13].
3

H
N
H
N
R
R
Amide derivatives
Amine derivatives
ACCEPTED MANUSCRIPT
O
N
H
N
H
NSAIDs coniugates
R
5
H
N
R
4
27: R =
28: R =
29: R =
30: R =
31 : R =
36: R =
14: R =
R =
R
R
1
3
R
2
15: R =
16: R =
17: R =
18 : R =
19 : R =
20 : R =
21 : R =
22: R =
N
1: R1=R2=R3=R4=R5=H
NO
2
O
2: R1=R2=R4=R5=H; R3=N(CH3)2
3: R1=R2=R4=R5=H; R3=Br
37: R =
N
N
4: R1=R3=R4=R5=H; R2=Br
O
5: R2=R3=R4=R5=H; R1=Br
6: R1=R2=R4=R5=H; R3=NO2
7: R1=NO2; R2=R3=R4=R5=H
8: R1=R2=R4=R5=H; R3=Cl
O
38: R =
HN
9: R1=R3=R4=R5=H; R2=OH
10: R1=R4=R5=H; R2=OCH3; R3=OH
11: R2=R3=R4=H; R1=NO2; R5=Cl
12: R1=R4=R5=H; R2=NO2; R3=OCH3
13: R1=R5=H; R2=R3=R4=OCH3
N
N
N
N
N
N
N
32 : R =
33 : R =
34 : R =
35: R =
39: R =
HN
CF
3
Molecular duplication
H
N
NO
2
O
O
H
N
N
H
40 : R =
N
H
N
H
24
N
O
NO
N
Cl
2
N
H
N
H
O
n
N
H
N
H
25: n = 2
26: n = 3
H
N
H
N
NO2
O
O
N
N
N
H
N
H
N
23
N
H
41
Chart 1: designed indole derivatives
All synthesized compounds were evaluated for the activity against planktonically grown cells of C.
albicans as well as on preformed and forming C. albicans biofilm. Finally, the most active compounds
were selected in order to evaluate the in vivo toxicity using the larvae of the greater wax moth, Galleria
mellonella.
2. Results and discussions
2.1 Chemistry
In the group of studied compounds, some are known in the literature, others are commercially available but
synthesized on request and not easily purchased through our usual suppliers, others are not known or
described in literature. All the compounds have been re-synthesized, in some cases according to the
procedures described in the literature, as for the amine derivatives 1 [14], 3 [15], 10 [16], 17 [16], 18 [17], 21
4

[18], 22 [18], and 23 [19]; in the other cases the target compounds were obtained by alternative methods or
ACCEPTED MANUSCRIPT
by modification of literature procedure, as specified for each compound. The amine derivatives were
prepared following two synthetic methods, illustrated in the Schemes 1 and 2. The compounds 2, 6, 7, 9, and
11-16, were synthesized by direct reductive amination modifying the procedures reported in literature [20].
The reaction has been carried out in MeOH using a small amount of acetic acid as catalyst and α-picoline-
borane as reductive agent. Equimolar amounts of amine, aldehyde, and α-picoline-borane were generally
used and the reaction was monitored by IR spectroscopy to evaluate the disappearance of the carbonyl
stretching band of the aldehyde function around at 1690 cm-1 and by TLC. At this point 10% aqueous HCl
was added and the obtained aqueous acid solution was neutralized to pH = 7 by the addition of saturated
aqueous Na2CO3. The obtained residues were purified by chromatography on a silica gel column and/or by
crystallization. The structure of synthesized compounds was confirmed by spectroscopic analysis, as the
appearance of the benzyl methylene singlet between 4.05 and 3.64 ppm in the 1H-NMR spectra.
R
5
R
4
R =
O
R
R
1
H
N
R
3
H
N
R
H
2
R
NH
2
14: R =
15: R =
16: R =
2
6
7
: R1=R2=R4=R5=H; R3=N(CH3)2
: R1=R2=R4=R5=H; R3=NO2
: R1=NO2; R2=R3=R4=R5=H
a
N
H
N
H
2, 6, 7, 9, 11-16
9: R1=R3=R4=R5=H; R2=OH
NO
2
O
11: R2=R3=R4=H; R1=NO2; R5=Cl
12: R1=R4=R5=H; R2=NO2; R3=OCH3
13: R1=R5=H; R2=R3=R4=OCH3
Scheme 1. Synthetic procedure for amines 2, 6, 7, 9, 11-16 (Method A). Reagents and conditions: a) pic-BH3, AcOH, MeOH, 2h,
rt.
The amine derivatives 4, 5, 8, 19, 20, 24-26 were synthesized following the method B procedure. Amine
reagent and appropriate aldehyde were dissolved in the required amount of dichloromethane and molecular
sieves were added as desiccant agents. The reaction was monitored by IR spectroscopy to confirm the imine
formation, with stretching absorption band around 1640 cm-1, and the disappearance of the aldehyde band at
about 1700 cm-1. The obtained imine intermediate, after removal of molecular sieves and solvent, was
5

reduced without further purification, by treatment with NaBH4 in methanol. The obtained crude residues
ACCEPTED MANUSCRIPT
were purified by chromatography on a silica gel column and/or by crystallization. The structure of the
synthesized compounds was confirmed by spectroscopic analysis, as the appearance of the benzyl methylene
singlet between 3.80 and 3.74 ppm in the 1H-NMR spectra.
4: R=
5: R=
8: R=
O
Br
N
R
19: R=
20: R=
H
N
NH
H
2
R
Br
N
H
a
,
b
N
H
N
4, 5, 8, 19, 20
Cl
H
N
O
a b
,
NH
H
O
2
+
N
H
H
N
H
N
H
N
H
O
24
H
a b
,
H
N
H N
2
NH
+
2
N
n
H
n
N
H
N
H
N
H
25: n = 2
26: n = 3
Scheme 2. Synthetic procedure for amines 4, 5, 8, 19, 20, 24-26 (Method B). Reagents and conditions: a) molecular sieves (4Å),
CH2Cl2, 12h, rt; b) NaBH4, MeOH, 2h, rt.
The amide derivative 27 was prepared as described by Honegr and coll. [21] and amides 29, 30 and 34,
already described in the literature [22, 23] were prepared in higher or equivalent yields than those described
but using milder reagents and conditions. The amides 28-41 were prepared following the procedure
illustrated in the Schemes 3 and 4 The synthesis was carried out using carbonyldiimidazole (CDI) as
.
activating agent of the carboxylic acid function. Although the reactivity of the CDI-carboxylic acid
intermediate is lower than acid chlorides, CDI is more controllable and allows to avoid the use of more
reactive activating agents, such as thionyl chloride, which may cause cross reactions. Therefore, CDI was
6

suspended in ethyl acetate and the opportune carboxylic acid was added, the reaction was heated to reflux to
ACCEPTED MANUSCRIPT
improve solubility of the reagents and it was monitored by IR spectroscopy evaluating the appearance of two
carbonyl stretching bands around 1735 and 1700 cm-1, concerning the CDI-carboxylic acid intermediate.
At this point tryptamine was added and the reaction was refluxed until the appearance of the amide carbonyl
stretching band in the IR spectra between 1634 and 1650 cm-1. The compounds were purified by
chromatography on a silica gel or alumina column and/or by crystallization. Also in this case, the structure of
1
final compounds was confirmed by spectroscopic analysis; as an example in the H-NMR spectra of amide
derivatives was observed the shift of the signal due to the -CH2-NH- group from 2.67 ppm (tryptamine) to
3.62-3.55 ppm (amide derivatives).
N
N
N
N
N
31
29
30
28
N
NO
2
O
O
N
32
N
H
33
34
35
H
N
O
a, b
R
OH
R
R =
O
O
N
H
HN
28-40
36
38
37
O
N
N
HN
CF
3
O
Cl
39
40
Scheme 3. Synthesis of amide compounds (28-40). Reagents and conditions: a) CDI, AcOEt, 5h, reflux; b) Tryptamine, 12h,
reflux.
H
N
H
N
O
O
O
O
a, b
HO
OH
N
H
N
H
N
N
41
Scheme 4. Synthesis of amide 41. Reagents and conditions: a) CDI, AcOEt, 5h, reflux; b) Tryptamine, 12h, reflux.
7

ACCEPTED MANUSCRIPT
The detailed synthetic procedures, the analytical and spectroscopic data of the new and the re-synthesized
compounds are reported in the experimental section and are in agreement with the proposed structures.
2.2 Microbiological assays
The synthesized compounds 1–41 have been screened against C. albicans planktonic cells and against C.
albicans biofilm, in formation and mature (24 h), according to the literature procedures [24, 25], using C.
albicans ATCC 10231, a strain sensitive to fluconazole on planktonic cells (0.5 µg/mL) and resistant in the
different phases of biofilm formation (BMIC50 128 µg/mL on biofilm formation and >128 µg/mL on mature
biofilm). All the compounds showing anti-biofilm and anti-fungal activity with BMIC50 and MIC50 values
lower than 64 µg/mL were reported in the Table 1.
Table 1. Antifungal activity of the amine and amide derivatives against C. albicans ATCC 10231 biofilms and planktonic cells.
R4
R5
R3
R2
H
N
BMIC50
(µg/mL)
MIC50
(µg/mL)
R1
N
H
Mature
biofilm
Biofilm
formation
Planktonic
cells
Compound
R1
R2
R3
R4
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
32
16
8
32
>128
>128
64
1
2
N(CH3)2
32
8
H
H
Br
H
3
H
Br
H
32
16
32
8
4
64
4
Br
H
H
16
32
64
16
16
128
128
5
H
NO2
H
>128
>128
32
6
NO2
H
H
7
H
Cl
32
8
8
NO2
H
H
H
>128
>128
11
12
NO2
OCH3
32
H
N
R
N
H
R
8

16
16
>128
16
15
16
17
21
22
NO2
ACCEPTED MANUSCRIPT
O
2
2
8
2
8
8
O
4
64
64
NO
2
>128
>128
N
N
NO2
4
8
>128
16
>128
>128
23
24
N
H
H
N
H
N
N
H
N
H
H
N
R
O
N
H
R
32
32
>128
27
32
64
16
8
32
>128
128
16
>128
>128
>128
>128
28
29
33
36
N
N
NO2
O
>128
>128
128
128
>128
0.5
Tryptamine
Fluconazole
The inhibition of biofilm formation and destruction of pre-formed biofilm were evaluated by measuring the metabolic activity of
cells within the biofilm (XTT assay). The BMIC end point for biofilm is based on the lowest drug concentration producing a
decrease of 50% metabolic activity relative to the untreated growth control. MIC end point for planktonic cells is based on lowest
drug concentration that prevented 50% of growth with respect to the untreated control. At least two experiments were performed on
two separate dates for each compound tested in triplicate. The results were expressed as median. Compounds showing BMIC50 and
MIC50 values higher than 64 µg/mL are not reported in the table.
In the first set of amine derivatives of the tryptamine compounds (1-13) we identified ten active compounds
(1-8, 11 and 12) which differ for the presence of various substituent on the phenyl ring. The compound 1, that
does not present substituents on the benzyl group, has a good activity both on biofilm formation and on
9

mature biofilm (BMIC50 = 32 µg/mL); taking it as reference, we try to outline structure-activity relationships
ACCEPTED MANUSCRIPT
(SAR). The presence of a halogen, as bromine or chlorine, on the phenyl ring produces an increase of the
anti-biofilm activity, in particular on forming biofilm, as can be observed for compounds 3, 4, 5, and 8.
Furthermore, bromine was slightly more effective than chlorine in increasing the anti-biofilm activity, as
shown by the comparison between 3 and 8. The introduction of a nitro group in para position on phenyl
(compound 6) retains the same activity of compound 1, but, if the nitro group is placed in ortho position, the
activity on mature biofilm enhances as the BMIC50 value decreases from 32 to 8 µg/mL (compound 7).
Combining the presence of the nitro group and chlorine on the phenyl ring (11) also the anti-biofilm activity
was combined; indeed, 11 possess the same activity of 7 on mature biofilm (BMIC50 = 8 µg/mL) and the
same activity of 8 on biofilm formation (BMIC50 = 16 µg/mL).
On the other hand, the introduction of a hydroxy and/or methoxy groups on the phenyl ring (9, 10 and 13)
dramatically reduced the anti-biofilm activity (BMIC50 > 128 µg/mL), compared to the compound 1; in the
same way, the simultaneous presence of the nitro group and a methoxy group (12) decreases the activity on
biofilm formation (BMIC50 = 128 µg/mL). Finally, compound 2, containing a dimethylamino group on the
phenyl ring, shows an interesting activity on mature biofilm, with a BMIC50 value of 16 µg/mL. In general,
these amine derivatives do not show activity on planktonic cells, except for 3, 4 (MIC50 = 64 µg/mL both)
and 8 (MIC50 = 32 µg/mL), consequently it is reasonable to imagine that these compounds could interfere
with some specific biofilm mechanisms.
Tryptamine, tested as reference molecule, does not show any activity, either on planktonically grown cells or
in formation and mature biofilms, as reported in Table 1. This evidence suggests that the structural
modification of the primary amine function of tryptamine is a key requirement to obtain the anti-biofilm
activity.
In a second set of amine derivatives (14-23) the phenyl group was replaced with various aromatic and
heteroaromatic rings (14-20), with cinnamic or 4-nitrocinnamic groups (21, 22) or modified by introducing a
piperazine spacer (23). The activity data showed in the Table 1 indicate that the nitrofurane derivative (15)
has a good activity on mature biofilm and on biofilm formation with BMIC50 of 16 µg/mL. Moreover, the
naphthyl (16) and the 4-phenoxybenzyl derivatives (17) are very active against biofilm in formation (BMIC50
10

= 8 and 2 µg/mL, respectively) as well as on mature biofilm (BMIC50 = 2 µg/mL, both) and they are also
ACCEPTED MANUSCRIPT
active on planktonic cells (16 and 4 µg/mL, respectively), though less than fluconazole (0.5 µg/mL). These
data also suggest that the anti-biofilm activity of compounds 16 and 17 could be due to a combined
mechanism of action directed on planktonic cells and on the biofilm. The replacement of benzyl group of the
compound 1 with cinnamic moiety, to obtain compounds 21 and 22, has produced an enhancement of the
anti-biofilm activity, selectively against biofilm in formation, with the reduction of BMIC50 value from 32
µg/mL to 8 µg/mL.
The activities of compounds 14, 18-20 on biofilm mature and in formation, as well on planktonic cells,
ranging from 64 to >128 µg/mL, indicates that the replacement of the phenyl group of the reference
compound 1 with other heterocyclic groups, as indole (14) and pyridines (18-20), resulted in a significant
reduction of the anti-biofilm activity. The introduction of a piperazine group between indole and nitrophenyl
functions to obtain compound 23 has produced an enhancement of the anti-biofilm activity, selectively
against mature biofilm, with BMIC50 value of 4 µg/mL.
In the set of amide derivatives (27-35) only four compounds showed MIC50 and BMIC50 values lower than
64 µg/mL (Table 1). It can be observed that the amide derivative 27 corresponding to amine compounds 1
retains the activity both on biofilm formation and on mature biofilm with a BMIC50 value of 32 µg/mL.
Among the pyridine derivatives (28-30) only compound 28 has the same activity of 27, also significantly
better than to the analogous amine derivative (18); the others pyridines present BMIC50 value range from 64
to >128 µg/mL on mature biofilm, on biofilm formation, as well on planktonic cells. The replacement of
phenyl group with nitrofurane moiety to obtain compound 34 have produced an enhancement of the anti-
biofilm activity, selectively against biofilm in formation, with the reduction of BMIC50 value from 32 µg/mL
to 16 µg/mL.
Moreover, the substitution of phenyl with pyrazine (32), indole (34), or isoxazole (35) rings resulted in a
significant reduction of the anti-biofilm activity.
The amide bond was useful to conjugate the tryptamine moiety to NSAIDs, as ibuprofen, ketoprofen,
mefenamic acid, niflumic acid and indomethacin to obtain the amide derivatives 36-40. In literature, as
described above, it is reported the bacterial and fungal anti-biofilm activity of some NSAIDs, but only the
11

hybrid between tryptamine and ibuprofen (36) shows a good activity on mature and in formation biofilm,
ACCEPTED MANUSCRIPT
with BMIC50 value of 8 and 16 µg/mL, respectively. Finally, all the amide compounds do not show activity
against C. albicans planktonic cells.
The duplication of the indole moiety to obtain diamine derivatives (24-26), induced different effects on anti-
biofilm activity that depend on the nature of the spacer. The presence of an aromatic linker increased the
activity, as can be noticed for compound 24, that possess BMIC50 values of 8 and 16 µg/mL on mature and
formation biofilm respectively; otherwise, an aliphatic spacer decreases significantly the activity as in the
cases of compounds 25 and 26 (BMIC50 > 128 µg/mL).
Lastly, the duplication of indole moiety with insertion of a pyridine to obtain diamide derivative 41, on the
contrary, does not produce activity on mature and formation biofilm and on planktonic cells (BMIC50 > 128
µg/mL).
The most active compounds were also tested on different strains of C. albicans, coming from the American
Type Culture Collection (C. albicans ATCC 10261) and from the Pharmaceutical Microbiology Culture
Collection (C. albicans PMC 1042 and PMC 1082) (Table 2).
Table 2. Antifungal activity of selected amine and amide derivatives against biofilm formation, mature biofilm and planktonic cells
of C. albicans PMC1042, PMC1083 and ATCC 10261.
C. albicans PMC1042
C. albicans PMC1082
C. albicans ATCC10261
BMIC50
(µg/mL)
MIC50
(µg/mL)
BMIC50
(µg/mL)
MIC50
BMIC50
(µg/mL)
BMIC50
(µg/mL)
(µg/mL)
Mature
Biofilm
Planktonic Mature
Biofilm
Planktonic Mature
Biofilm
Planktonic
Compound
biofilm formation
cells
16
biofilm formation
cells
32
biofilm
formation
cells
32
2
2
8
8
4
2
8
8
8
8
8
8
3
128
64
32
32
16
2
8
2
8
16
8
8
16
17
2
32
8
>256
>256
>256
8
2
32
8
>256
>256
>256
0.125
8
32
4
>256
256
>256
2
23
2
4
4
24
36
2
8
4
8
8
8
>64
>64
>64
>64
>64
>64
Fluconazole
The inhibition of biofilm formation and destruction of pre-formed biofilm were evaluated by measuring the metabolic activity of
cells within the biofilm (XTT assay). The BMIC end point for biofilm is based on the lowest drug concentration producing a
decrease of 50% metabolic activity relative to the untreated growth control. MIC end point for planktonic cells is based on lowest
drug concentration that prevented 50% of growth with respect to the untreated control. At least two experiments were performed on
two separate dates for each compound tested in triplicate. The results were expressed as median.
12

The compounds 3, 16, 17, 23, 24 and 36 were active also towards biofilm from C. albicans ATCC 10261,
ACCEPTED MANUSCRIPT
PMC 1042 and PMC 1082 strains, and showed, in most cases, BMIC50 values lower than those observed on
ATCC 10231 strain. The obtained results indicate that these indole derivatives are more effective on mature
biofilm than on biofilm formation, suggesting that they could act more selectively in the advanced stages of
biofilm formation, rather than in the early stages.
The evaluation of selective toxicity is an extremely important and preliminary phase in the development of
new compounds of pharmaceutical interest; for this reason, we decided to study in vivo toxicity, as described
by Dolan et al. [26], using the larvae of the Greater wax moth, Galleria mellonella. McCann et al. have
demonstrated that the toxicity exhibited in G. mellonella was similar to that observed in Swiss mice [27]. A
selection of the synthesized derivatives that exhibited good anti-biofilm activity (3, 7, 16, 17, 23, 24 and 36)
was chosen for the evaluation of their toxicity. Almost all compounds tested displayed a 100% survival rate
of G. mellonella larvae. Compounds 3 and 23 at the concentration range of 5-500 µg/mL and compound 36 at
the concentration range of 10-1000 µg/mL, displayed a 100% survival rate after 72 h. Compounds 16 and 24
at the concentration of 50 µg/mL and compound 7 at the concentration of 50 and 100 µg/mL displayed a 90%
survival rate after 72 h and 100% survival at the other concentrations after 72 h (statistically not significant,
Student’s t test, P> 0.1). Compound 17 at the concentration of 500 µg/mL displayed a 90% survival rate after
24 h, a 70% survival rate after 72 h (Student’s t test, P<0.05) and 100% survival for the other concentrations
after 72 h. (Table 3).
Table 3. Survival rate of Galleria mellonella larvae (expressed as %) at 24, 48 and 72 h after the injection of the indole derivatives
3, 7, 16, 17, 23, 24 and 36.
Dosage
G. mellonella survival (%)b
Dosage
G. mellonella survival (%)b
Cmpd
3
concentration
Cmpd
23
concentration
24h
48h
72h
24h
48h
72h
(µg/mL)a
500
100
50
(µg/mL)a
500
100
50
100
100
100
100
100
100
90 *
90 *
100
100
100
100
100
100
90 *
90 *
100
100
100
100
100
100
90 *
90 *
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90 *
100
100
100
100
100
100
100
90 *
10
10
5
5
500
100
50
500
100
50
7
24
13

10
5
100
100
100
100
90 *
100
100
90 **
100
100
100
100
100
100
10
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
ACCEPTED MANUSCRIPT
100
100
100
90 *
100
100
70 **
100
100
100
100
100
100
100
90 *
100
100
70 **
100
100
100
100
5
500
100
50
10
5
1000
500
100
50
16
17
36
10
500
100
50
10
5
a Compounds were dissolved in sterile water (containing less than 1% v/v of DMSO) and the volume injected into the last, left pro-
b
leg, of the G. mellonella larvae was 20 µL; The results represent the mean percentage of survival, of two independent
experiments, of G. mellonella larvae, as a function of the administered dosage; * Student’s t test, P > 0.1; ** Student’s t test, P <
0.05.
3. Conclusions
The synthesis of indole derivatives has allowed to identify new compounds endowed of anti-biofilm activity.
In general, it was observed that the halogen derivatives have shown higher inhibition activity both of mature
and of in formation biofilms, in particular the bromine derivatives are more effective than chlorine ones. For
almost all nitro derivatives, except for 22, a higher activity against mature biofilm was observed; this could
be related to the ability of these compounds to generate ROS. In fact, it is known from the literature that
compounds able to induce the production of ROS possess a potential anti-biofilm activity [28, 29].
Many of the synthesized compounds showed higher activity towards biofilm (both mature and in formation)
if compared with planktonic cells. This fact could be related to a different susceptibility due to the phenotypic
and physiologic differences between biofilm (sessile cells) and non-adhered (planktonic cells). Moreover,
this selectivity could be explained considering that the molecules could act by preventing the adhesion of the
cells to form biofilm and/or favoring the release of the cells in planktonic form which would result in a lesser
amount of experimentally determined biofilm.
Finally other interesting compounds are the naphthyl (16) and the 4-phenoxybenzyl (17) derivatives, that
showed high activity against C. albicans ATCC 10231 mature biofilm (BMIC50 2 µg/mL) and biofilm in
formation (BMIC50 8 µg/mL and 2 µg/mL respectively) as well as on planktonic cells (MIC50 16 and 4
µg/mL respectively) though less than fluconazole (MIC50 0.5 µg/mL). These data also suggest that the anti-
14

biofilm activity of compounds 16 and 17 could be due to a combined action on planktonic cells and on the
ACCEPTED MANUSCRIPT
biofilm; indeed, they could be considered good lead compounds to develop new antifungal drugs.
Promising anti-biofilm compounds have been identified and the combination of these compounds with
established antifungal agents could have additive or synergistic action that should be studied.
Further investigations into the development of efficient anti-biofilm agents against other microorganisms
based on these results are still in progress.
4. Experimental section
4.1 Chemistry
4.1.1 Materials and methods
Reagents and solvents were of analytical grade and were purchased from Sigma-Aldrich (Milano, Italy).
Melting points were determined on Tottoli apparatus (Buchi) and are uncorrected. Infrared spectra were
recorded on a Perkin Elmer Spectrum One FT-IR spectrometer equipped with an ATR system. 1H-NMR and
13C-NMR spectra were acquired on a Bruker AVANCE-400 spectrometer at 9.4 Tesla, in DMSO-d6, CD3OD,
CDCl3, CD2Cl2 and Acetone- d6 at 27 °C; chemical shift values are given in δ (ppm) with respect to the
solvent residual peak (DMSO = 2.50 ppm, CD3OD = 3.31 ppm, CDCl3 = 7.26, CD2Cl2 = 5.32 ppm, Acetone
= 2.05 ppm). Coupling constants are given in Hz. The following abbreviation were used: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, s br = singlet broad, dd = doublet of doublets, dt = doublet of
triplets. Mass spectra were recorded on a ThermoFinnigan LCQ Classic LC/MS/MS ion trap equipped with
an ESI source and a syringe pump. Samples (10-4-10-5 M in MeOH/H2O 90:10) were infused in the
electrospray system at a flow rate of 5-10 µl min-1. When necessary, 50 µL of 10-2 M aqueous HCOOH or 10-
2
M aqueous NH3 were added to the sample solutions, in order to promote the analyte ionization. Elemental
analyses were obtained by a PE 2400 (Perkin-Elmer) analyser; analyses indicated by the symbols of the
elements or functions were within ± 0.4 % of the theoretical values.
4.1.2 General procedure for the synthesis of amine compounds
15

Method A: Tryptamine and the opportune aldehyde (1:1 molar ratio) were dissolved in 10 mL of MeOH–
ACCEPTED MANUSCRIPT
AcOH (10:1), pic-BH3 was added, and the reaction mixture was stirred for 2 h at room temperature. Then
MeOH was evaporated under reduce pressure and HCl (10 %, 10 mL) was added to the residue. The aqueous
solution was stirred for 0.5 h at room temperature, then cooled in an ice bath and Na2CO3 (≈ 2.5 g) and H2O
(10 mL) were added until pH = 7. The aqueous solution was extracted with AcOEt (3 x 30 mL) and the
combined organic layers were washed with brine (15 mL), dried over Na2SO4 and concentrated under
reduced pressure.
Method B: Tryptamine, the opportune aldehyde (1:1 or 2:1 molar ratio) and molecular sieves (4 Å) in
CH2Cl2 (20 mL) were stirred at rt for 12 h. The mixture was then filtered on a celite pad, concentrated and
treated with NaBH4 in MeOH (30 mL) at rt for 2 h. After reaction MeOH was evaporated under reduced
pressure, H2O (15 mL) was added and the mixture was extracted with CH2Cl2 (3 x 20 mL). The organic
layers were dried over Na2SO4 and the solvent was removed under vacuum.
4.1.2.1 4-(((2-(1H-indol-3-yl)ethyl)amino)methyl)-N,N-dimethylaniline (2)
Compound 2 was prepared using tryptamine (0.208 g, 1.30 mmol), 4-(dimethylamino)benzaldehyde (0.194 g,
1.30 mmol) and pic-BH3 (0.139 g, 1.30 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using AcOEt/MeOH (1:1) to give a yellow-orange solid, mp
1
98-99°C (Rf = 0.24, 80.5 mg, 21 % yield). IR (cm-1): νN-H 3281, finger print 1215, 1170, 1097. H-NMR
(DMSO-d6) δ (ppm): 10.75 (s br, 1H, -NH- indole); 7.52 (d, J = 8.0 Hz, 1H, indole proton), 7.31 (d, J = 8.0
Hz, 1H, indole proton); 7.12-7.10 (m, 3H, aromatic protons); 7.04 (t, J = 7.2 Hz, 1H, indole proton); 6.94 (t, J
= 7.1 Hz, 1H, indole proton); 6.65 (d, J = 8.4 Hz, 2H, aromatic protons); 4.10 (bs, 1H, NH amine); 3.60 (s,
13
2H, Ph-CH2-NH-); 2.84 (s, 6H, -N(CH3)2); 2.84-2.74 (m, 4H, -CH2- CH2-). C-NMR (MeOD) δ (ppm):
151.6, 138.2, 130.4, 128.6, 128.5, 123.4, 122.4, 119.6, 119.3, 114.2, 113.4, 112.3, 53.7, 50.0, 41.1, 26.0. ESI-
MS (m/z): (M+H)+ = 293.47. Anal. C19H23N3 (C, H, N).
4.1.2.2 N-(3-bromobenzyl)-2-(1H-indol-3-yl)ethanamine (4)
Compound 4 was prepared using tryptamine (0.160 g, 1.00 mmol), 3-bromobenzaldehyde (117 µL, 1.00
mmol) and NaBH4 (0.076 g, 2.00 mmol), following the method B procedure. The obtained residue was
16

subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give a pink solid, mp 64-66°C
ACCEPTED MANUSCRIPT
1
(Rf = 0.37, 198 mg, 60 % yield). IR (cm-1): νN-H 3289, finger print 1232, 1110, 1076, 1068. H-NMR
(DMSO-d6) δ (ppm): 10.77 (s, 1H, -NH- indole); 7.55 (s, 1H, aromatic proton); 7.49 (d, J = 7.8 Hz, 1H,
indole proton); 7.41 (dd, J = 7.8 Hz, 1H, aromatic proton); 7.32 (d, J = 7.9 Hz, 2H, aromatic protons); 7.25 (t,
J = 7.7 Hz, 1H, aromatic proton); 7.12 (d, J = 2.1 Hz, 1H, indole proton); 7.05 (t, J = 7.2 Hz, 1H, indole
proton); 6.96 (t, J = 7.3 Hz, 1H, indole proton); 3.74 (s, 2H, Ph-CH2-NH-); 2.84 (t, J = 6.8 Hz, -CH2-CH2-);
13
2.77 (t, J = 6.4 Hz, 2H, -CH2- CH2). C-NMR (CDCl3) δ (ppm): 142.8, 136.4, 131.1, 130.0, 129.9, 127.4,
126.7, 122.5, 122.1, 122.0, 119.4, 118.9, 113.8, 111.2, 53.3, 49.3, 25.8. ESI-MS (m/z): (M+H)+ = 328.67
(85); 330.80 (100). Anal. C17H17BrN2 (C, H, N).
4.1.2.3 N-(2-bromobenzyl)-2-(1H-indol-3-yl)ethanamine (5)
Compound 5 was prepared using tryptamine (0.160 g, 1.00 mmol), 2-bromobenzaldehyde (116 µL, 1.00
mmol) and NaBH4 (0.076 g, 2.00 mmol), following the method B procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give a pink solid, mp 95-96°C
(Rf = 0.43, 124 mg, 38 % yield). IR (cm-1): νN-H 3265, finger print 1321, 1218, 1087, 926. 1H-NMR (DMSO-
d6) δ (ppm): 10.78 (s, 1H, -NH- indole); 7.56 (d, J = 7.9 Hz, 1H, indole proton); 7.52 – 7.49 (m, 2H, aromatic
protons); 7.36-7.32 (m, 2H, aromatic protons); 7.18 (td, J1 = 7.7 Hz, J2 = 1.6 Hz, 1H, aromatic proton); 7.14
(d, J = 2.1 Hz, 1H, indole proton); 7.05 (t, J = 7.2 Hz, 1H, indole proton); 6.96 (t, J = 7.4 Hz, 1H, indole
proton); 3.80 (s, 2H, Ph-CH2-NH-); 2.89-2.82 (m, 4H, -CH2- CH2- NH-). 13C-NMR (MeOD) δ (ppm): 138.1,
136.9, 132.5, 130.3, 128.7, 127.3, 127.2, 123.5, 122.2, 121.0, 118.2, 117.9, 111.8, 110.9, 52.7, 48.5, 24.8.
ESI-MS (m/z): (M+H)+ = 328.80 (100); 330.87 (80). Anal. C17H17BrN2 (C, H, N).
4.1.2.4 2-(1H-indol-3-yl)-N-(4-nitrobenzyl)ethanamine (6)
Compound 6 was prepared using tryptamine (0.301 g, 1.88 mmol), 4-nitrobenzaldehyde (0.284 g, 1.88
mmol) and pic-BH3 (0.201 g, 1.88 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using initially CHCl3 and subsequently CHCl3/MeOH (9:1) to
give a yellow solid, mp 55-57°C (Rf = 0.27, 453 mg, 82 % yield). IR (cm-1): νN-H 3416, νNO2 1513 and 1345,
17

finger print 1231, 1106, 1073. 1H-NMR (DMSO-d6) δ (ppm): 10.77 (s br, 1H, -NH- indole); 8.16 (d, J = 8.7
ACCEPTED MANUSCRIPT
Hz, 2H, aromatic protons); 7.61 (d, J = 8.6 Hz, 2H, aromatic protons); 7.49 (d, J = 7.8 Hz, 1H, indole
proton); 7.32 (d, J = 8.1 Hz, 1H, indole proton); 7.13 (d, J = 1.7 Hz, 1H, indole proton); 7.05 (t, J = 7.0 Hz,
1H, indole proton); 6.95 (t, J = 7.7 Hz, 1H, indole proton); 3.88 (s, 2H, Ph-CH2-NH-); 2.8 (t, J = 6.8 Hz, 2H,
13
-CH2- CH2- NH-); 2.79 (t, J = 6.8 Hz, 2H, -CH2- CH2- NH-). C-NMR (DMSO-d6) δ (ppm): 150.1, 146.7,
136.7, 129.2, 127.7, 123.7, 123.0, 121.3, 118.7, 118.5, 112.9, 111.8, 52.6, 50.0, 26.0. ESI-MS (m/z): (M+H)+
= 295.87. Anal. C17H17N3O2 (C, H, N).
4.1.2.5 2-(1H-indol-3-yl)-N-(2-nitrobenzyl)ethanamine (7)
Compound 7 was prepared using tryptamine (0.160 g, 1.00 mmol), 2-nitrobenzaldehyde (0.151 g, 1.00
mmol) and pic-BH3 (0.107 g, 1.00 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using AcOEt/MeOH (9:1) to give a brownish solid, mp 70-
73°C (Rf = 0.58, 53 mg, 18 % yield). IR (cm-1): νN-H 3288, νNO2 1515 and 1340, finger print 1230, 1105,
1073. 1H-NMR (MeOD) δ (ppm): 7.94 (d, J = 8.1 Hz, 1H, aromatic proton); 7.60 (t, J = 7.2 Hz, 1H, aromatic
proton); 7.53-7.44 (m, 3H, aromatic protons); 7.33 (d, J = 8.1 Hz, 1H, indole proton); 7.09-7.04 (m, 2H,
indole protons); 6.94 (t, J = 7.4 Hz, 1H, indole proton); 4.01 (s, 2H, Ph-CH2-NH-); 3.00-2.92 (m, 4H, -CH2-
13
CH2-). C-NMR (MeOD) δ (ppm): 149.0, 136.9, 133.9, 133.0, 131.6, 128.2, 127.2, 124.5, 122.1, 121.0,
118.2, 117.8, 111.8, 110.9, 49.7, 48.7, 24.8. ESI-MS (m/z): (M+H)+ = 295.80. Anal. C17H17N3O2 (C, H,
N).
4.1.2.6 N-(4-chlorobenzyl)-2-(1H-indol-3-yl)ethanamine (8)
Compound 8 was prepared using tryptamine (0.160 g, 1.00 mmol), 4-chlorobenzaldehyde (0.141 g, 1.00
mmol) and NaBH4 (0.076 g, 2.00 mmol), following the method B procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2/MeOH (8:2) to give a white solid, mp 83-85°C
(Rf = 0.53, 219 mg, 77 % yield). IR (cm-1): νN-H 3416, finger print 1218, 1092, 1013. 1H NMR (DMSO-d6) δ
(ppm): 10.77 (s, 1H, -NH- indole); 7.48 (d, J = 7.8 Hz, 1H, indole proton); 7.35 – 7.31 (m, 5H, aromatic
protons); 7.12 (d, J = 2.1 Hz, 1H, indole proton); 7.05 (t, J = 7.3 Hz, 1H, indole proton); 6.96 (t, J = 7.3 Hz,
13
1H, indole proton); 3.74 (s, 2H, Ph-CH2-NH-); 2.87-2.76 (m, 4H, -CH2- CH2- NH-). C NMR (MeOD) δ
18

(ppm): 137.6, 136.9, 132.6, 129.7, 128.1, 127.2, 122.1, 121.1, 118.3, 117.9, 111.9, 111.0, 51.9, 48.7, 24.6.
ACCEPTED MANUSCRIPT
ESI-MS (m/z): (M+H)+ = 284.93 (100); 286.87 (30). Anal. C17H17ClN2 (C, H, N).
4.1.2.7 3-(((2-(1H-indol-3-yl)ethyl)amino)methyl)phenol (9)
Compound 9 was prepared using tryptamine (0.528 g, 3.30 mmol), 3-hydroxybenzaldehyde (0.402 g, 3.30
mmol) and pic-BH3 (0.353 g, 3.30 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using AcOEt/MeOH (1:1) to give a yellowish solid, mp 132-
1
134°C (Rf = 0.36, 100 mg, 11 % yield). IR (cm-1): νN-H 3445, νOH 3326, finger print 1275, 1160, 1084. H-
NMR (DMSO-d6) δ (ppm): 10.82 (s br,1H, -NH- indole); 7.53 (d, J = 7.8 Hz, 1H, indole proton); 7.36 (d, J =
7.9 Hz, 1H, indole proton); 7.16-7.07 (m, 3H, aromatic protons); 6.99 (t, J = 7.2 Hz, 1H, indole proton);
6.80-6.76 (m, 2H, aromatic protons); 6.65 (d, J = 7.5 Hz, 1H, aromatic proton); 3.71 (s, 2H, Ph-CH2-NH-);
13
2.89-2.93 (m, 4H, -CH2- CH2-). C-NMR (DMSO-d6) δ (ppm): 157.7, 142.5, 136.7, 129.4, 127.7, 123.0,
121.3, 119.0, 118.8, 118.6, 115.3, 114.0, 112.9, 111.8, 53.2, 49.9, 25.7. ESI-MS (m/z): (M+H)+ = 265.13.
Anal. C17H18N2O (C, H, N).
4.1.2.8 N-(2-chloro-6-nitrobenzyl)-2-(1H-indol-3-yl)ethanamine (11)
Compound 11 was prepared using tryptamine (0.160 g, 1.00 mmol), 2-chloro-6-nitrobenzaldehyde (0.185 g,
1.00 mmol) and pic-BH3 (0.107 g, 1.00 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2/MeOH (9.5:0.5) and then crystallized from
methanol to give a yellow solid, mp 127-129°C (Rf = 0.40, 66 mg, 20 % yield). IR (cm-1): νN-H 3283, νNO2
1
1525 and 1349, finger print 1109, 1080, 1006. H-NMR (DMSO-d6) δ (ppm): 10.78 (s, 1H, -NH- indole);
7.81-7.74 (m, 2H, aromatic proton), 7.49 (t, J = 8.0 Hz, 1H, aromatic proton); 7.43 (d, J = 7.6 Hz, 1H, indole
proton); 7.30 (d, J = 8.0 Hz, 1H, indole proton); 7.08 (s, 1H, indole proton); 7.03 (t, 1H, J = 7.2 Hz, indole
13
proton); 6.93 (t, J = 7.2 Hz, 1H, indole proton); 4.03 (s, 2H, Ph-CH2-NH-); 2.75 (m, 4H, -CH2- CH2-). C-
NMR (DMSO-d6) δ (ppm): 152.1, 136.7, 135.5, 133.8, 132.7, 129.8, 127.6, 123.6, 123.0, 121.3, 118.6,
118.5, 112.6, 111.8, 50.3, 46.4, 25.9. ESI-MS (m/z): (M+H)+ = 329.73. Anal. C17H16ClN3O2 (C, H, N).
4.1.2.9 2-(1H-indol-3-yl)-N-(4-methoxy-3-nitrobenzyl)ethanamine (12)
19

Compound 12 was prepared using tryptamine (0.160 g, 1.00 mmol), 4-methoxy-3-nitrobenzaldehyde (0.181
ACCEPTED MANUSCRIPT
g, 1.00 mmol) and pic-BH3 (0.107 g, 1.00 mmol), following the method A procedure. The obtained residue
was subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give an off-white solid, mp
71-73°C (Rf = 0.30, 193 mg, 59 % yield). IR (cm-1): νN-H 3413, νNO2 1526 and 1344, finger print 1280, 1259,
1
1018. H-NMR (DMSO-d6) δ (ppm): 10.78 (s, 1H, -NH- indole); 7.83 (d, J = 1.6 Hz, 1H, indole proton);
7.60 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1H, aromatic proton); 7.48 (d, J = 7.6 Hz, 1H, indole proton); 7.33-7.29
(m, 2H, aromatic protons); 7.12 (s, 1H, indole proton); 7.05 (t, J = 7.6 Hz, 1H, indole proton); 6.95 (t, J = 8.0
Hz, 1H, indole proton); 5.76 (s, 1H, -NH- amine); 3.90 (s, 3H, –OCH3); 3.74 (s, 2H, Ph-CH2-NH-); 2.84 (t, J
= 6.8 Hz, 2H, CH2- CH2- NH-); 2.76 (t, J = 6.4 Hz, 2H, CH2- CH2- NH-). 13C-NMR (MeOD) δ (ppm): 151.7,
139.6, 136.8, 134.0, 131.1, 127.2, 124.8, 122.2, 121.1, 118.3, 117.9, 113.5, 111.7, 110.9, 55.7, 51.2, 48.6,
24.5. ESI-MS (m/z): (M+H)+ = 325.80. Anal. C18H19N3O3 (C, H, N).
4.1.2.10 2-(1H-indol-3-yl)-N-(3,4,5-trimethoxybenzyl)ethanamine (13)
Compound 13 was prepared using tryptamine (0.327 g, 2.04 mmol), 3,4,5-trimethoxybenzaldehyde (0.400 g,
2.04 mmol) and pic-BH3 (0.218 g, 2.04 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using AcOEt/MeOH (1:1) to give a white solid, mp 69-71°C
(Rf = 0.35, 383 mg, 55 % yield). IR (cm-1): νN-H 3386, finger print 1237, 1128, 1000. 1H-NMR (DMSO-d6) δ
(ppm): 10.77 (s br, 1H, -NH- indole); 7.49 (d, J = 7.8 Hz, 1H, indole proton); 7.31 (d, J = 8.1 Hz, 1H, indole
proton); 7.13 (s, 1H, indole proton); 7.04 (t, J = 7.2 Hz, 1H, indole proton); 6.95 (t, J = 7.7, 1H, indole
proton); 6.61 (s, 2H, phenyl protons); 3.71 (s, 6H, m-OCH3); 3.68 (s, 2H, Ph-CH2-NH-); 3.61 (s, 3H, p-
13
OCH3); 2.87-2.77 (m, 4H, CH2- CH2-). C-NMR (DMSO-d6) δ (ppm): 153.1, 137.2, 136.7, 136.4, 127.8,
123.0, 121.3, 118.8, 118.5, 113.1, 111.8, 105.3, 60.4, 56.1, 53.4, 50.0, 25.9. ESI-MS (m/z): (M+H)+ =
340.70. Anal. C20H24N2O3 (C, H, N).
4.1.2.11 2-(1H-indol-3-yl)-N-(1H-indol-3-ylmethyl)ethanamine (14)
Compound 14 was prepared using tryptamine (0.301 g, 1.88 mmol), 1H-indole-3-carbaldehyde (0.273 g, 1.88
mmol) and pic-BH3 (0.201 g, 1.88 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using AcOEt/MeOH (1:1) to give a yellow solid, mp 78-80°C
20

(Rf = 0.35, 148 mg, 27 % yield). IR (cm-1): νN-H 3395, finger print 1225, 1092, 1007. 1H-NMR (DMSO-d6) δ
ACCEPTED MANUSCRIPT
(ppm): 10.81 (s br, 1H, -NH- indole); 10.75 (s br, 1H, -NH- indole); 7.56 (d, J = 7.8 Hz, 1H, indole proton);
7.50 (d, J = 7.8 Hz, 1H, indole proton); 7.34-7.31 (m, 2H, indole protons); 7.20 (d, J = 2.4 Hz 1H, indole
proton); 7.11 (d, J = 2.0 Hz 1H, indole proton); 7.05 (t, J = 7.2 Hz 2H, indole protons); 6.95 (t, J = 7.8 Hz,
2H, indole protons); 4.11 (bs, 1H, NH amine); 3.89 (s, 2H, Ph-CH2-NH-); 2.87 (s, 4H, CH2- CH2-). 13C-NMR
(MeOD) δ (ppm): 147.7, 145.6, 136.9, 130.9, 129.8, 127.2, 122.1, 121.0, 118.3, 117.9, 114.7, 114.3, 112.2,
111.8, 111.7, 110.9, 54.9, 52.5, 24.4. ESI-MS (m/z): (M+H)+ = 289.73. Anal. C19H19N3 (C, H, N).
4.1.2.12 2-(1H-indol-3-yl)-N-((5-nitrofuran-2-yl)methyl)ethanamine (15)
Compound 15 was prepared using tryptamine (0.248 g, 1.55 mmol), 5-nitrofuran-2-carbaldehyde (0.220 g,
1.55 mmol) and pic-BH3 (0.166 g, 1.55 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2 to give a yellow solid, mp 98-100°C (Rf = 0.27,
69 mg, 16 % yield). IR (cm-1): νN-H 3432, νNO2 1494 and 1354, finger print 1241, 1019. 1H-NMR (DMSO-d6)
δ (ppm): 10.76 (s, 1H, -NH- indole); 7.65 (m, 1H, indole proton); 7.32 (d, J = 7.7 Hz, 1H, indole proton);
7.14 (s, 1H, indole proton); 7.04 (t, J = 7.8 Hz, 1H, indole proton); 6.95 (t, J = 7.8 Hz, 1H, indole proton);
6.76 (m, 2H, furane protons); 3.74 (s, 2H, Ph-CH2-NH-); 2.82 (m, 4H, CH2- CH2-). 13C-NMR (DMSO-d6) δ
(ppm): 157.8, 151.8, 136.6, 127.6, 123.2, 121.3, 118.7, 118.6, 114.4, 113.3, 112.4, 111.8, 54.6, 50.2, 23.3.
ESI-MS (m/z): (M+H)+ = 285.93. Anal. C15H15N3O3 (C, H, N).
4.1.2.13 2-(1H-indol-3-yl)-N-(naphthalen-2-ylmethyl)ethanamine (16)
Compound 16 was prepared using tryptamine (0.160 g, 1.00 mmol), naphthalene-2-carbaldehyde (0.156 g,
1.00 mmol) and pic-BH3 (0.107 g, 1.00 mmol), following the method A procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2/MeOH (9.5:0.5) to give a white solid, mp 97-
99°C (Rf = 0.81, 140 mg, 46 % yield). IR (cm-1): νN-H 3417, finger print 1230, 1104, 1076. 1H-NMR (MeOD)
δ (ppm): 7.77-7.69 (m, 3H, aromatic protons); 7.60 (s, 1H, aromatic protons); 7.50 (d, J = 8.0 Hz, 1H, indole
proton); 7.42-7.39 (m, 2H, aromatic protons); 7.35 (d, J = 8.4 Hz, 1H, indole proton); 7.31 (dd, J1 = 1.6 Hz,
J2 = 8.4 Hz, 1H, aromatic proton); 7.19-7.07 (m, 1H, indole proton); 7.03 (s, 1H, indole proton); 6.99-6.95
(m, 1H, indole proton); 3.84 (s, 2H, Ph-CH2-NH-); 2.97 (t; J = 6.4 Hz, 2H, CH2- CH2- NH-); 2.91 (t, J = 6.8
21

Hz, 2H, CH2- CH2- NH-). 13C-NMR (MeOD) δ (ppm): 136.9, 136.2, 133.5, 132.8, 127.8, 127.4, 127.3, 127.2,
ACCEPTED MANUSCRIPT
126.6, 126.2, 125.7, 125.4, 122.1, 121.1, 118.3, 118.0, 111.9, 110.9, 52.7, 48.7, 24.6. ESI-MS (m/z): (M+H)+
= 300.88. Anal. C21H20N2 (C, H, N).
4.1.2.14 2-(1H-indol-3-yl)-N-(pyridin-3-ylmethyl)ethanamine (19)
Compound 19 was prepared using tryptamine (0.160 g, 1.00 mmol), pyridine-3-carbaldehyde (94 µL, 1.00
mmol) and NaBH4 (0.076 g, 2.00 mmol), following the method B procedure. The obtained product was an
1
orange oil (247 mg, 98 % yield). IR (cm-1): νN-H 3406, finger print 1231, 1100, 1029, 1010. H-NMR
(DMSO-d6) δ (ppm): 10.78 (s, 1H, -NH- indole); 8.52 (d, J = 1.7 Hz, 1H, pyridine proton); 8.43 (dd, J1 =
71.6 Hz, J2 = 4.8 Hz, 1H pyridine proton); 7.73 (d, J = 7.8, 1H, pyridine proton); 7.49 (d, J = 7.8 Hz, 1H,
indole proton); 7.32 (dd, J1 = 2.4 Hz, J2 = 5.6 Hz, 2H, pyridine proton); 7.13 (d, J = 2.1 Hz, 1H, indole
proton); 7.05 (t, J = 7.3 Hz, 1H, indole proton); 6.95 (t, J = 7.4 Hz, 1H, indole proton); 3.76 (s, 2H, Py-CH2-
13
NH-); 2.86-2.76 (m, 4H, -CH2- CH2- NH-). C-NMR (MeOD) δ (ppm): 148.8, 147.4, 137.0, 136.8, 135.5,
127.2, 123.8, 122.1, 121.0, 118.2, 117.8, 111.9, 110.9, 49.9, 48.9, 24.7. ESI-MS (m/z): (M+H)+ = 252.00.
Anal. C16H17N3 (C, H, N).
4.1.2.15 2-(1H-indol-3-yl)-N-(pyridin-4-ylmethyl)ethanamine (20)
Compound 20 was prepared using tryptamine (0.160 g, 1.00 mmol), pyridine-4-carbaldehyde (95 µL, 1.00
mmol) and NaBH4 (0.076 g, 2.00 mmol), following the method B procedure. The obtained product was an
ocra yellow solid (128 mg, 51 % yield). IR (cm-1): νN-H 3301, finger print 1220, 1119, 1062, 1002. 1H-NMR
(DMSO-d6) δ (ppm): 10.79 (s, 1H, -NH- indole); 8.47 (d, J = 5.7 Hz, 2H, pyridine protons); 7.49 (d, J = 7.8
Hz, 1H, indole proton); 7.34-7.31 (m, 3H, aromatic protons); 7.13 (d, J = 1.9 Hz, 1H, indole proton); 7.05 (t,
J = 7.5 Hz, 1H, indole proton); 6.96 (t, J = 7.4 Hz, 1H, indole proton); 3.77 (s, 2H, Py-CH2-NH-); 2.87-2.76
13
(m, 4H, -CH2- CH2- NH-). C-NMR (MeOD) δ (ppm): 150.0, 148.5, 136.9, 127.3, 123.4, 122.2, 121.1,
118.3, 117.9, 112.0, 111.0, 51.4, 49.0, 24.8. ESI-MS (m/z): (M+H)+ = 252.07. Anal. C16H17N3 (C, H, N).
4.1.2.16 N,N'-(1,4-phenylenebis(methylene))bis(2-(1H-indol-3-yl)ethanamine) (24)
22

Compound 24 was prepared using tryptamine (0.320 g, 2.00 mmol), benzene-1,4-dicarbaldehyde (0.134 g,
ACCEPTED MANUSCRIPT
1.00 mmol) and NaBH4 (0.114 g, 3.00 mmol), following the method B procedure. The obtained residue was
subjected to silica gel column chromatography using CH2Cl2/MeOH (8:2) to give an off-white solid, mp 144-
145°C (Rf = 0.32, 114 mg, 26 % yield). IR (cm-1): νN-H 3314, finger print 1241, 1019. 1H-NMR (DMSO-d6) δ
(ppm): 10.76 (s, 2H, -NH- indole); 7.48 (d, J = 7.5 Hz, 2H, aromatic protons); 7.32 (d, J = 8.2 Hz, 2H,
aromatic protons); 7.23 (s, 4H, indole protons); 7.11 (s, 2H, indole protons); 7.04 (t, J = 6.2 Hz, 2H, indole
protons); 6.94 (t, J = 6.6 Hz, 2H, indole protons); 3.70 (s, 4H, Ph-CH2-NH-); 2.84-2.78 (m, 8H, -CH2- CH2-).
13C-NMR (DMSO-d6) δ (ppm): 139.6, 136.7, 128.1, 127.7, 123.0, 121.3, 118.8, 118.6, 113.1, 111.8, 53.2,
50.0, 25.9. ESI-MS (m/z): (M+H)+ = 422.93. Anal. C28H30N4 (C, H, N).
4.1.2.17 N1,N4-bis((1H-indol-3-yl)methyl)butane-1,4-diamine (25)
1H-indole-3-carbaldehyde (0.290 g, 2 mmol), butane-1,4-diamine (0.088 g, 1 mmol), molecular sieves (4 Å)
in CH2Cl2 (15 mL) and MeOH (5 mL) were stirred at rt for 12 h. The mixture was then filtered on a celite
pad, concentrated and treated with NaBH4 (0.302 g, 8 mmol) in MeOH (60 mL) at rt for 2 h. After reaction
MeOH was evaporated under reduced pressure, H2O (15 mL) was added and the mixture was extracted with
CH2Cl2 (3 x 20 mL). The organic layers were dried over Na2SO4 and the solvent was removed under vacuum.
The yellow solids obtained were washed with diethyl ether (5x1 mL) to give the pure 25 as a white solid, mp
1
125-126°C (94 mg, 25 % yield). IR (cm-1): νN-H 3170, finger print: 1235, 1124, 1109, 1009. H-NMR
(DMSO-d6) δ (ppm): 10.83 (s, 2H, -NH- indole); 7.59 (d, J = 7.8 Hz, 2H, indole protons); 7.34 (d, J = 8.0 Hz,
2H, indole protons); 7.20 (d, J = 1.9 Hz, 2H, indole protons); 7.06 (t, J = 7.1 Hz, 2H, indole protons); 6.96 (t,
J = 7.4 Hz, 2H, indole protons); 3.82 (s, 4H, Ar-CH2-NH-); 2.57-2.54 (m, 4H, -NH-CH2-CH2-CH2-CH2-NH-
); 1.49-1.47 (m, 4H, -NH-CH2-CH2-CH2-CH2-NH-). 13C-NMR (MeOD) δ (ppm): 136.7, 127.0, 123.7, 121.3,
118.7, 117.7, 111.4, 111.1, 48.0, 43.0, 26.7. ESI-MS (m/z): (M+H)+ = 346.47. Anal. C22H26N4 (C, H, N).
4.1.2.18 N1,N5-bis((1H-indol-3-yl)methyl)pentane-1,5-diamine (26)
1H-indole-3-carbaldehyde (0.290 g, 2 mmol), pentane-1,5-diamine (117 µL, 1 mmol), molecular sieves (4 Å)
in CH2Cl2 (15 mL) and MeOH (5 mL) were stirred at rt for 12 h. The mixture was then filtered on a celite
pad, concentrated and treated with NaBH4 (0.302 g, 8 mmol) in MeOH (60 mL) at rt for 2 h. After reaction
23

MeOH was evaporated under reduced pressure, H2O (15 mL) was added and the mixture was extracted with
ACCEPTED MANUSCRIPT
CH2Cl2 (3 x 20 mL). The organic layers were dried over Na2SO4 and the solvent was removed under vacuum.
The yellow solids obtained were washed with diethyl ether (5x1 mL) to give the pure 26 as a yellowish solid,
104-105 °C (93 mg, 26 % yield). IR (cm-1): νN-H 3404, finger print: 1225, 1105, 1010. 1H-NMR (DMSO-d6) δ
(ppm): 10.82 (s, 2H, -NH- indole); 7.59 (d, J = 7.8 Hz, 2H, indole protons); 7.34 (d, J = 8.0 Hz, 2H, indole
protons); 7.20 (s, 2H, indole protons); 7.06 (t, J = 7.4 Hz, 2H, indole protons); 6.96 (t, J = 7.4 Hz, 2H, indole
protons); 3.82 (s, 4H, Ar-CH2-NH-); 2.56-2.54 (m, 4H, –NH-CH2-CH2-CH2-CH2-CH2-NH- partially covered
by DMSO signal); 1.47-1.40 (m, 4H, –NH-CH2-CH2-CH2-CH2-CH2-NH-); 1.35-1.30 (m, 2H, –NH-CH2-
CH2-CH2-CH2-CH2-NH-). 13C-NMR (MeOD) δ (ppm): 136.7, 127.0, 123.4, 121.1, 118.6, 117.8, 112.0,
111.0, 48.2, 43.4, 28.6, 24.7. ESI-MS (m/z): (M+H)+ = 360.87. Anal. C23H28N4 (C, H, N).
4.1.2.19 N-(2-(1H-indol-3-yl)ethyl)pyridine-2-carboxamide (28)
Compound 28 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), pyridine-2-carboxylic acid
(0.123 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
obtained residue was subjected to silica gel column chromatography using CH2Cl2/MeOH (9.8:0.2) to give a
yellowish solid, mp 132-134 °C (Rf = 0.54, 224 mg, 84 % yield). IR (cm-1): νN-H 3373 and 3247, νc=o 1662,
finger print 1431, 1339, 1228. 1H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH- indole); 8.90 (t, J = 5.9 Hz,
1H, -NH- amide); 8.63 (d, J = 4.5 Hz, 1H, pyridine proton); 8.06 (d, J = 7.7 Hz, 1H, pyridine proton); 7.99
(td, J1 = 7.6 Hz, J2 = 1.6 Hz, 1H, pyridine proton); 7.63-7.58 (m, 2H, aromatic protons); 7.34 (d, J = 8.1 Hz,
1H, indole proton); 7.20 (d, J = 8.1 Hz, 1H, indole proton); 7.07 (t, J = 7.30 Hz, 1H, indole proton); 6.98 (t, J
= 7.3 Hz, 1H, indole proton); 3.61 (m, 2H, -CH2-CH2-NH-CO-); 2.97 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO-
). 13C-NMR (CDCl3) δ (ppm): 164.4, 149.9, 148.0, 137.5, 136.5, 127.4, 126.2, 122.25, 122.20, 122.0, 119.3,
118.8, 112.9, 111.3, 39.8, 25.6. ESI-MS (m/z): (M+H)+ = 265.87. Anal. C16H15N3O (C, H, N).
4.1.2.20 N-(2-(1H-indol-3-yl)ethyl)pyridine-3-carboxamide (29)
Compound 29 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), pyridine-3-carboxylic acid
(0.123 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
obtained residue was subjected to aluminum oxide column chromatography using CH2Cl2/MeOH (9:1) to
24

give a pinkish solid, mp 148-150 °C (Rf = 0.62, 216 mg, 82 % yield). IR (cm-1): νN-H 3278, νc=o 1652, finger
ACCEPTED MANUSCRIPT
1
print 1420, 1310, 1109. H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH- indole); 9.00 (d, J = 2.0 Hz, 1H,
pyridine proton); 8.82 (t, J = 5.4 Hz, 1H, -NH- amide); 8.70 (dd, J1 = 4.80 Hz, J2 = 1.40 Hz, 1H, pyridine
proton); 8.18 (td, J1 = 7.90 Hz, J2 = 1.70 Hz, 1H, pyridine proton); 7.58 (d, J = 7.80 Hz, 1H, indole proton);
7.51 (dd, J1 = 7.9 Hz, J2 = 4.8 Hz, 1H, pyridine proton); 7.35 (d, J = 8.1 Hz, 1H, indole proton); 7.20 (d, J =
1.8 Hz, 1H, indole proton); 7.07 (t, J = 7.4 Hz, 1H, indole proton); 6.98 (t, J = 7.4 Hz, 1H, indole proton);
13
3.59-3.54 (m, 2H, -CH2-CH2-NH-CO-); 2.97 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO-). C-NMR (MeOD) δ
(ppm): 166.4, 151.1, 147.6, 136.8, 135.6, 130.9, 127.5, 123.7, 122.1, 121.0, 118.2, 117.9, 111.9, 110.9, 40.9,
24.7. ESI-MS (m/z): (M+H)+ = 265.80. Anal. C16H15N3O (C, H, N).
4.1.2.21 N-(2-(1H-indol-3-yl)ethyl)pyridine-4-carboxamide (30)
Compound 30 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), pyridine-4-carboxylic acid
(0.123 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
obtained residue was subjected to aluminum oxide column chromatography using CH2Cl2/MeOH (9:1) to
give a yellow brilliant solid, mp 166-168 °C (Rf = 0.67, 230 mg, 87 % yield). IR (cm-1): νN-H 3163, νc=o 1654,
finger print 1402, 1304, 1213. 1H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH- indole); 8.92 (t, J = 5.4 Hz,
1H, -NH- amide); 8.72 (dd, J1 = 4.5 Hz, J2 = 1.5 Hz, 2H, pyridine protons); 7.75 (dd, J1 = 4.5 Hz, J2 = 1.5
Hz, 2H, pyridine protons); 7.58 (d, J = 7.80 Hz, 1H, indole proton); 7.35 (d, J = 8.1 Hz, 1H, indole proton);
7.19 (d, J = 2.0 Hz, 1H, indole proton); 7.07 (t, J = 7.4 Hz, 1H, indole proton); 6.98 (t, J = 7.3 Hz, 1H, indole
13
proton); 3.59-3.53 (m, 2H, -CH2-CH2-NH-CO-); 2.97 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO). C-NMR
(MeOD) δ (ppm): 166.3, 149.3, 142.9, 136.8, 127.5, 122.1, 121.6, 121.0, 118.2, 117.9, 111.8, 110.9, 40.9,
24.7. ESI-MS (m/z): (M+H)+ = 265.80. Anal. C14H11NO7 (C, H, N).
4.1.2.22 N-(2-(1H-indol-3-yl)ethyl)-6-methylpyridine-3-carboxamide (31)
Compound 31 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), 6-methylpyridine-3-
carboxylic acid (0.137 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described
for 27. The obtained residue was subjected to aluminum oxide column chromatography using CH2Cl2/MeOH
25

(9:1) to give a pinkish solid, mp 127-130 °C (Rf = 0.75, 275 mg, 98 % yield). IR (cm-1): νN-H 3163, νc=o 1654,
ACCEPTED MANUSCRIPT
finger print 1213, 1063, 1002. 1H-NMR (DMSO-d6) δ (ppm): 10.83 (s, 1H, -NH- indole); 8.88 (d, J = 2.1 Hz,
1H, pyridine proton); 8.73 (t, J = 5.5 Hz, 1H, -NH- amide); 8.08 (dd, J1 = 8.1 Hz, J2 = 2.3 Hz, 1H, pyridine
protons); 7.58 (d, J = 7.8 Hz, 1H, indole proton); 7.36 (d, J = 3.3 Hz, 1H, indole proton); 7.34 (d, J = 3.3 Hz,
1H, pyridine proton); 7.19 (d, J = 2.0 Hz, 1H, indole proton); 7.07 (t, J = 7.2 Hz, 1H, indole proton); 6.98 (t,
J = 7.3 Hz, 1H, indole proton); 3.57-3.52 (m, 2H, -CH2-CH2-NH-CO-); 2.96 (t, J = 7.5 Hz, 2H, -CH2-CH2-
13
NH-CO-); 2.52 (s, partially covered by DMSO signal -CH3). C-NMR (MeOD) δ (ppm): 166.4, 161.0,
147.1, 136.8, 135.9, 128.0, 127.5, 123.2, 122.1, 121.0, 118.3, 118.0, 111.9, 110.9, 40.8, 24.8, 22.5. ESI-MS
(m/z): (M+H)+ = 279.93. Anal. C17H17N3O (C, H, N).
4.1.2.23 N-(2-(1H-indol-3-yl)ethyl)pyrazine-2-carboxamide (32)
Compound 32 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), pyrazine-2-carboxylic acid
(0.124 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
obtained residue was subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give a
light orange solid, mp 184-186 °C (Rf = 0.62, 66 mg, 25 % yield). IR (cm-1): νN-H 3381 and 3253, νc=o 1655,
finger print 1225, 1094, 1020. 1H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH- indole); 9.21 (d, J = 1.1 Hz,
1H, pyrazine proton); 9.05 (t, J = 5.8 Hz, 1H, -NH- amide); 8.87 (d, J = 2.4 Hz, 1H, pyrazine proton), 8.73
(m, 1H, pyrazine proton), 7.61 (d, J = 7.8 Hz, 1H, indole proton); 7.34 (d, J = 8.1 Hz, 1H, indole proton);
7.20 (d, J = 1.9 Hz, 1H, indole proton); 7.07 (t, J = 7.3 Hz, 1H, indole proton); 6.98 (t, J = 7.4 Hz, 1H, indole
13
proton); 3.64-3.59 (m, 2H, -CH2-CH2-NH-CO-); 2.98 (t, J = 7.50 Hz, 2H, -CH2-CH2-NH-CO-). C-NMR
(DMSO-d6) δ (ppm): 163.2, 147.9, 145.3, 143.9, 143.8, 136.7, 127.7, 123.1, 121.4, 118.8, 118.7, 112.1,
111.8, 40.2*, 25.6. *partially covered by DMSO signal. ESI-MS (m/z): (M+H)+ = 266.73. Anal.
C15H14N4O (C, H, N).
4.1.2.24 N-(2-(1H-indol-3-yl)ethyl)-5-nitrofuran-2-carboxamide (33)
Compound 33 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), 5-nitrofuran-2-carboxylic acid
(0.157 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
26

obtained residue was subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give an
ACCEPTED MANUSCRIPT
orange solid, mp 157-159 °C (Rf = 0.77, 116 mg, 39 % yield). IR (cm-1): νN-H 3420 and 3335, νc=o 1657, νNO2
1353, finger print 1270, 1017. 1H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH- indole); 9.03 (t, J = 5.7 Hz,
1H, -NH- amide); 7.76 (d, J = 3.9 Hz, 1H, furane proton); 7.58 (d, J = 7.8 Hz, 1H, indole proton); 7.39 (d, J =
3.9 Hz, 1H, furane proton); 7.34 (d, J = 8.1 Hz, 1H, indole proton); 7.19 (d, J = 2.1 Hz, 1H, indole proton);
7.07 (t, J = 7.1 Hz, 1H, indole proton); 6.99 (t, J = 7.1 Hz, 1H, indole proton); 3.57-3.52 (m, 2H, -CH2-CH2-
NH-CO-); 2.95 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO-). 13C-NMR (DMSO-d6) δ (ppm): 156.5, 151.8, 149.0,
136.7, 127.7, 123.2, 121.4, 118.7, 118.7, 115.9, 114.0, 111.9, 111.9, 40.3* 25.4, *partially covered by DMSO
signal. ESI-MS (m/z): (M-H)- = 297.93. Anal. (C15H13N3O4 (C, H, N).
4.1.2.25 N-(2-(1H-indol-3-yl)ethyl)-1H-indole-2-carboxamide (34)
Compound 34 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1 mmol), 1H-indole-2-carboxylic acid
(0.161 g, 1 mmol) and tryptamine (0.160 g, 1 mmol), following the same procedure described for 27. The
obtained residue was subjected to silica gel column chromatography using CH2Cl2/MeOH (9:1) to give a
white solid, mp 202-204 °C (Rf = 0.68, 152 mg, 50 % yield). IR (cm-1): νN-H 3412 and 3247, νc=o 1637, finger
print 1257, 1100. 1H-NMR (DMSO-d6) δ (ppm): 11.61 (s, 1H, -NH- indole); 10.83 (s, 1H, -NH- indole); 8.64
(t, J = 5.6 Hz, 1H, -NH- amide); 7.61 (d, J = 7.9 Hz, 2H, indole protons); 7.43 (d, J = 8.2 Hz, 1H, indole
proton); 7.35 (d, J = 8.0 Hz, 1H, indole proton); 7.20-7.15 (m, 2H, indole protons); 7.12-6.94 (m, 4H, indole
13
protons); 3.61-3.56 (m, 2H, -CH2-CH2-NH-CO-); 2.98 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO-). C-NMR
(DMSO-d6) δ (ppm): 161.6, 136.9, 136.7, 132.5, 127.7, 127.6, 123.6, 123.1, 121.9, 121.4, 120.1, 118.8,
118.7, 112.8, 112.3, 111.9, 102.7, 40.3*, 25.8. *partially covered by DMSO signal. ESI-MS (m/z): (M+H)+ =
303.93. Anal. C19H17N3O (C, H, N).
4.1.2.26 N-(2-(1H-indol-3-yl)ethyl)-1,2-oxazole-5-carboxamide (35)
Compound 35 was prepared using 1,1-carbonyldiimidazole (0.107 g, 0.66 mmol), 1,2-oxazole-5-carboxylic
acid (0.075 g, 0.66 mmol) and tryptamine (0.106 g, 0.66 mmol), following the same procedure described for
27. The obtained residue was subjected to aluminum oxide column chromatography using AcOEt/MeOH
27

(9:1) and then crystallized from methanol to give a light orange solid, mp 127-129 °C (Rf = 0.83, 61 mg, 24
ACCEPTED MANUSCRIPT
1
% yield). IR (cm-1): νN-H 3383 and 3255, νc=o 1656, finger print 1296, 1205. H-NMR (DMSO-d6) δ (ppm):
10.84 (s, 1H, -NH- indole); 9.09 (t, J = 5.5 Hz, 1H, -NH- amide); 8.74 (d, J = 1.8 Hz, 1H, oxazole proton);
7.57 (d, J = 7.9 Hz, 1H, indole proton); 7.34 (d, J = 8.0 Hz, 1H, indole proton); 7.19 (d, J = 2.0 Hz, 1H,
indole proton); 7.08-7.04 (m, 2H, oxazole + aromatic protons); 6.98 (t, J = 7.4 Hz, 1H, indole proton); 3.57-
13
3.52 (m, 2H, -CH2-CH2-NH-CO-); 2.96 (t, J = 7.5 Hz, 2H, -CH2-CH2-NH-CO-). C-NMR (DMSO-d6) δ
(ppm): 163.4, 156.0, 152.1, 136.7, 127.7, 123.2, 121.4, 118.74, 118.69, 111.94, 111.87, 106.2, 40.2*, 25.3.
*partially covered by DMSO signal. ESI-MS (m/z): (M+H)+ = 255.87. Anal. C14H13N3O2 (C, H, N).
4.1.2.27 N-(2-(1H-indol-3-yl)ethyl)-2-(4-isobutylphenyl)propanamide (36)
Compound 36 was prepared using 1,1-carbonyldiimidazole (0.081 g, 0.50 mmol), 2-(4-(2-
methylpropyl)phenyl)propanoic acid (0.103 g, 0.50 mmol) and tryptamine (0.080 g, 0.50 mmol), following
the same procedure described for 27. The obtained residue was subjected to aluminum oxide column
chromatography using CH2Cl2/MeOH (9.5:0.5) to give a white solid, mp 102-105 °C (Rf = 0.90, 116 mg, 67
% yield). IR (cm-1): νc=o 1648, finger print 1234, 1213. 1H-NMR (DMSO-d6) δ (ppm): 10.76 (s br, 1H, -NH-
indole); 8.02 (bs, 1H, NH amide); 7.49 (d, J = 7.6 Hz, 1H, indole proton); 7.31 (d, J = 7.8 Hz, 1H, indole
proton); 7.20 (d, J = 8.0 Hz, 2H, aromatic protons); 7.08-7.03 (m, 4H, aromatic + indole protons); 6.95 (t, J =
7.6 Hz, 1H, indole proton); 3.53 (q, J = 7.1 Hz, 1H, -CH-CH3); 3.31-3.27 (m, partially obscured by HDO
signal -CH2-CH2-NH-); 2.77 (t, J = 7.6 Hz, 2H, -CH2-CH2-NH-); 2.40 (d, J = 7.2 Hz, 2H, -CH2-CH-(CH3)2);
1.83-1.76 (m, 1H, -CH2-CH-(CH3)2); 1.30 (d, J = 7.0 Hz, 3H, -CH-CH3); 0.85 (d, J = 6.6 Hz, 6H, -CH2-CH-
(CH3)2). 13C-NMR (DMSO-d6) δ (ppm): 173.7, 140.1, 139.6, 136.7, 129.2, 127.7, 127.4, 123.1, 121.3, 118.7,
118.6, 112.2, 111.8, 45.2, 44.7, 44.2 *, 30.0, 25.6, 22.6, 19.1, *partially covered by DMSO signal. ESI-MS
(m/z): (M+H)+ = 348.93. Anal. C23H28N2O (C, H, N).
4.1.2.28 N-(2-(1H-indol-3-yl)ethyl)-2-(4-benzoylphenyl)propanamide (37)
Compound 37 was prepared using 1,1-carbonyldiimidazole (0.107 g, 0.66 mmol), 2-(3-
benzoylphenyl)propanoic acid (0.150 g, 0.59 mmol) and tryptamine (0.095 g, 0.59 mmol), following the
28

same procedure described for 27. The obtained residue was subjected to aluminum oxide column
ACCEPTED MANUSCRIPT
chromatography using CHCl3/MeOH (9.5:0.5) and then it was washed with diethyl ether (3x1 mL) to give a
yellowish oil (Rf = 0.92, 80 mg, 27 % yield). IR (cm-1): νc=o 1649, finger print 1285, 1064. 1H-NMR (MeOD)
δ (ppm): 7.75-7.73 (m, 3H, aromatic + indole protons); 7.66 - 7.44 (m, 7H, aromatic protons); 7.31 (d, J = 8.2
Hz, 1H, indole proton); 7.07 (t, J = 7.2 Hz, 1H, indole proton); 6.97 (t, J = 7.2 Hz, 1H, indole proton); 6.90
(s, 1H, aromatic proton); 3.68 (q, J = 7.1 Hz, 1H, -CH-CH3); 3.55-3.39 (m, 2H, -CH2-CH2-NH-); 2.90 (t, J =
13
7.2, 2H, -CH2-CH2-NH-); 1.45 (d, J = 7.1, 3H, -CH3). C-NMR (MeOD) δ (ppm): 194.3, 171.1, 140.2,
138.4, 136.5, 136.1, 133.3, 132.4, 131.1, 130.3, 129.2, 128.9, 128.4, 127.4, 123.0, 121.7, 119.8, 118.8, 113.0,
111.2, 42.9, 42.1, 25.6, 15.4. ESI-MS (m/z): (M+H)+ = 397.16. Anal. C26H24N2O2 (C, H, N).
4.1.2.29 N-(2-(1H-indol-3-yl)ethyl)-2-((2,3-dimethylphenyl)amino)benzamide (38)
Compound 38 was prepared using 1,1-carbonyldiimidazole (0.162 g, 1.00 mmol), 2-(2,3-
dimethylanilino)benzoic acid (0.241 g, 1.00 mmol) and tryptamine (0.160 g, 1.00 mmol), following the same
procedure described for 27. The obtained residue was subjected to silica gel column chromatography using
CH2Cl2/MeOH (9.5:0.5) to give a pinkish solid, mp 162-164 °C (Rf = 0.87, 320 mg, 84 % yield). IR (cm-1):
νN-H 3410, 3379, 3302, νc=o 1625, finger print 1222, 1063. 1H-NMR (DMSO-d6) δ (ppm): 10.84 (s, 1H, -NH-
indole); 9.62 (s, 1H, -NH- aniline) 8.67 (t, J = 5.6 Hz, 1H, -NH- amide); 7.63-7.57 (m, 2H, aromatic protons);
7.34 (d, J = 8.0 Hz, 1H, indole proton); 7.24 (t, J = 7.3Hz, 1H, aromatic proton); 7.20 (d, J = 2.0 Hz, 1H,
indole proton); 7.14-7.03 (m, 3H, indole + aromatic protons); 7.02-6.90 (m, 2H, aromatic protons); 6.84 (d, J
= 8.3 Hz, 1H, aromatic proton); 6.73 (d, J = 7.4 Hz, 1H, indole proton); 3.58-3.53 (m, 2H, -CH2-CH2-NH-
13
CO-); 2.97 (t, J = 7.4 Hz, 2H, -CH2-CH2-NH-CO-); 2.28 (s, 3H, o-CH3); 2.12 (s, 3H, m-CH3). C-NMR
(CDCl3) δ (ppm): 169.8, 147.0, 139.6, 138.1, 136.5, 132.2, 130.8, 127.4, 127.3, 125.8, 125.6, 122.3, 122.2,
120.8, 119.6, 118.8, 117.3, 116.9, 115.0, 112.9, 111.4, 40.0, 25.3, 20.7, 13.9. ESI-MS (m/z): (M+H)+ =
383.79. Anal. C25H25N3O (C, H, N).
4.1.2.30 N-(2-(1H-indol-3-yl)ethyl)-2-((3-(trifluoromethyl)phenyl)amino)nicotinamide (39)
29