Synthesis of zanthoxylamide protoalkaloids and their in silico ADME-Tox screening and in vivo toxicity assessment in zebrafish embryos

Article abstract of DOI:10.1016/j.ejps.2018.10.028

Inspired by the simple and attractive structure of zanthoxylamide protoalkaloids: armatamide, rubecenamide, lemairamin, rubemamine and zanthosine; isolated from plants of the genus Zanthoxylum. We report the synthesis of a series of 29 substituted N-pheny

Full text of DOI:10.1016/j.ejps.2018.10.028

Accepted Manuscript
Synthesis of zanthoxylamide protoalkaloids and their in silico
ADME-Tox screening and in vivo toxicity assessment in zebrafish
embryos
Carlos E. Puerto Galvis, Vladimir V. Kouznetsov
PII:
DOI:
Reference:
S0928-0987(18)30481-0
https://doi.org/10.1016/j.ejps.2018.10.028
PHASCI 4732
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
4 September 2018
25 October 2018
30 October 2018
Please cite this article as: Carlos E. Puerto Galvis, Vladimir V. Kouznetsov , Synthesis
of zanthoxylamide protoalkaloids and their in silico ADME-Tox screening and in vivo
toxicity assessment in zebrafish embryos. Phasci (2018), https://doi.org/10.1016/
j.ejps.2018.10.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Research Paper
Synthesis of zanthoxylamide protoalkaloids and their In silico
ADME-Tox screening and In vivo toxicity assessment in
Zebrafish Embryos
Carlos E. Puerto Galvisa,b and Vladimir V. Kouznetsova*
a Laboratorio de Química Orgánica y Biomolecular, CMN, Universidad Industrial de Santander.
Parque Tecnológico Guatiguará, Km 2 Vía Refugio, Piedecuesta 681011, Colombia. Tel: +57 7
634 4000 Ext. 3593.
b Laboratorio de Química Orgánica Aplicada, Universidad Manuela Beltrán, Cl. 33 # 26-34,
Bucaramanga, Colombia, PBox, 680002. Fax/Tel: +57 7 6525202.
Keywords: Zanthoxylamide protoalkaloids, Umami amides, ADME-Tox properties,
Zebrafish embryo model, Toxicology profile
Abstract
Inspired by the simple and attractive structure of zanthoxylamide protoalkaloids:
armatamide, rubecenamide, lemairamin, rubemamine and zanthosine; isolated from plants of
the genus Zanthoxylum. We report the synthesis of a series of 29 substituted N-phenylethyl
cinnamamides through the direct amidation of a variety of cinnamic acids with a broad range
of phenylethylamines promoted by tris-(2,2,2-trifluoroethyl) borate (B(OCH2CF3)3) in
excellent yields and under mild reaction conditions. Then, the toxicological profile of the
prepared compounds was studied through in silico computational methods, analyzing eight
toxicity risks (hepatotoxicity, mutagenic, carcinogenicity, tumorigenic, immunotoxicity,
cytotoxicity, irritant and reproductive effects) and two toxicity targets (AOFA and PGH1),
while the acute toxicity toward zebrafish embryos (96 hpf-LC50, 50% lethal concentration)
was also determined in the present study. From the results of the toxicity tests, we concluded
that zanthoxylamide protoalkaloids can be classified as slightly toxic compounds, with a
LC50 values around 217 µM that gave an understanding of their toxicity on living organisms
and their possible environmental impact.

ACCEPTED MANUSCRIPT
1. Introduction
With over 546 members, plants of the genus Zanthoxylum (Rutaceae family) are one of the
most distributed plants around the world [1]. Almost half of these species, can be found in
tropical and temperate regions of North America, South America, Africa, Asia, and Australia
[2]. Traditionally, the bark, stems, fruits, roots and seeds of these plants have been used in
cooking, as a culinary spice, and in folk medicine, to treat carminative, anthelmintic, fever,
dyspepsia and stomachic disorders [3]. These properties have motivated several
phytochemical studies that have allowed the isolation and identification of relevant
secondary metabolites like alkaloids [4], coumarins [5], flavonoids [6], limonoids [7], and
volatile oils [8,9] with interesting phytochemical and biological activities, such as
antimicrobial, antidiarrheal, antileishmanial, antiprotozoal, anticholinesterase, larvicidal and
antioxidant activities [2, 10-12].
Within the mentioned natural products, a vast variety of amides can be found in the genus
Zanthoxylum, and among them, those metabolites derived from cinnamic acids and L-
phenylalanine are known as zanthoxylamide protoalkaloids. Representative members of this
family are: armatamide 1 [13], rubecenamide 2 [14], lemairamin 3 [15], rubemamine 4 [16]
and zanthosine 5 [17], have been recognized for their biological properties (Fig. 1) [18-19].
Fig. 1. Representative zanthoxylamide protoalkaloids.
Recently, Frerot and co-workers described a structure-taste relationship of cinnamamides
against the umami taste, which is mediated by the TAS1R1-TAS1R3 receptor belonging to
the family of glutamates receptors [14]. While Ley and his group carried out in vitro and in
vivo studies with zanthoxylamide protoalkaloids 1-5 that demonstrated the importance of the
3,4-dimethoxycinnamic moiety to function as umami taste modulators through their affinity
to the TAS1R1-TAS1R3 receptors [20]. Other in vivo studies have been reported for extracts
and essential oils from the zanthoxylum genus were zanthoxylamide protoalkaloids 1-5 are

ACCEPTED MANUSCRIPT
present [21-23], however, in silico and in vivo toxicological profiles of these protoalkaloids
are practically unknown to best of our knowledge.
The promising umami properties of protoalkaloids 1-5 suggest that in a near future these
compounds have potential use in human food preparations, putting in evidence the need of
development a large-scale protocol for their preparation. However, the approaches employed
so far suffered the same drawbacks that have accompanied the amide bond formation during
the last decades: the previous activation of carboxylic acids with toxic coupling reagents,
high temperatures and long reaction times, multiple steps, poor reagents solubility in
common solvents and low to moderate yields in protocols that generates a great quantity of
waste [24-28].
Thereby, and taking into account the very low concentration and the unknown toxicological
properties of zanthoxylamides 1-5, and their analogues in Zanthoxylum genus, we wish to
report an efficient and green procedure for the direct amidation of cinnamic acids mediated
by a simple borate ester for the synthesis of diverse zanthoxylamide protoalkaloids to perform
in silico and in vivo toxicological studies using the zebrafish embryo model, which accurately
predict the relative oral toxicity of zanthoxylamide protoalkaloids after chemical exposure.
2. Results and discussion
2.1 Synthesis
Selecting phenylethylamine 6a and cinnamic acid 7a as model substrates, the first attempt
to obtain the corresponding N-phenylethyl cinnamamide 8a was conducted based on our
previous reports, where boric acid (H3BO3) resulted to be an efficient catalyst for this
amidation reaction [29-30]. By using this catalyst (15 mol %), compound 8a was obtained in
86 % yield after 24 hours (Table 1, entry 1), and seeking for a greener protocol, silica gel
was then used as a heterogeneous catalyst guided by Gamba-Sanchez’s work, but the desired
product was isolated in moderate yield, 68 % (Table 1, entry 2) [31]. Although, the good
results observed with H3BO3, we noticed that substituted cinnamic acids were insoluble in
toluene, even at high temperatures, which would be the major drawbacks in our objective to
expand and increase the structural diversity of the zanthoxylamide protoalkaloids. Thus, we
turned our attention to explore other amidation reactions performed in more polar solvents.
Hall and co-workers reported the high catalytic activity of a synthetic arylboronic acid,
known as a MIBA (5-methoxy-2-iodophenylboronic acid), which efficiently catalysed the
coupling of carboxylic acid and amines at room temperature in CH2Cl2 [32]. Nevertheless,
cinnamamide 8a was obtained in poor yield (31 %) after using 10 mol % of MIBA for 48
hours (Table 1, entry 3). Motivated by the attractive catalytic activity of boron compounds,
Sheppard and co-workers investigated the direct amidation reaction promoted by a
stoichiometric excess of the tris-(2,2,2-trifluoroethyl) borate (B(OCH2CF3)3) (2 equiv. of
boron catalyst peer 1 equiv. of reactants) in acetonitrile as a solvent [33]. In our hands, when
we applied this methodology, the desired product 8a was isolated in good yield, 88% (Table

ACCEPTED MANUSCRIPT
1, entry 4), nevertheless, employing an excess of this borate would not be profitable at large
scale, and for that reason the borate loading was reduced to 1.5 equiv., observing a slightly
decrease in the yield of 8a (82 %) (Table 1, entry 5). With the aim of continuing reducing the
catalyst/substrate ratio, an experiment with equivalent amount of B(OCH2CF3)3 was carried
out increasing the reaction temperature to 100 °C, which furnished the cinnamamide 8a with
a 90% yield (Table 1, entry 6). This result encouraged us to lowering the amount of
B(OCH2CF3)3 to 0.5 equiv. and for our delight, the desired N-phenylethyl cinnamamide 8a
was isolated in 98 % yield after 24 hours at 100 °C (Table 1, entry 7). Finally, we found that
decreasing the catalyst loading to 0.1 equiv. dramatically reduce the yield to 78 % (Table 1,
entry 8).
Table 1 Optimization of reaction conditions for the synthesis of cinnamamide 8a.a
Entry
Cat. (equiv.)
Solvent, [M]
Temp.
(°C)
Yield,
b
%
1
2
3
4
5
6
7
8
H3BO3 (0.07)
SiO2 (20 % p/p)
MIBA (0.05)
B(OCH2CF3)3 (2)
B(OCH2CF3)3 (1.5)
B(OCH2CF3)3 (1)
B(OCH2CF3)3 (0.5)
B(OCH2CF3)3 (0.1)
Toluene (0.05)
Toluene (0.05)
CH2Cl2 (0.02)
CH3CN (0.5)
CH3CN (0.5)
CH3CN (0.5)
CH3CN (0.5)
CH3CN (0.5)
110
110
25
80
80
100
100
100
86
68
31c
88
82
90
98
78
a Reactions performed in a 0.5 mmol scale: phenylethylamine 6a (0.5 mmol), cinnamic acid 7a (0.5 mmol), catalyst, solvent
and temperature for 24 h. b Isolated yield after column chromatography (SiO2). c Reaction carried out with pre-activated 4Å
molecular sieves for 48 h.
B(OCH2CF3)3 acts as a Lewis acid to activate the cinnamic acid and form a boronate species
susceptible to the nucleophilic attack from the amine [34]. But probably, an excess of this
catalyst in the reaction media is affecting the nucleophilic nature of the amine by favouring
an acid-base interaction, equilibrium that could be controlled by the temperature in which the
reaction is performed. Thus, we found an optimal catalyst loading to specifically mediate the
coupling between commercially available cinnamic acids and phenylethylamines.
Having the optimized reaction conditions for the direct amidation reaction of cinnamic
acids in hand, we began to study the substrate scope (Table 2). First, the reactivity of various
phenylethylamines bearing diverse substituents on the aromatic ring was examined. We
found that in general, the electronic or steric nature of these substituents did not influence the
reactivity of the amines 6a-6h, even the heteroaromatic derivative 6h, gave cinnamamide
analogues 8a-8h in good to excellent yields (64-98 %). The same pattern was observed for
the amides 8i-8o, which were obtained in good yields (61-93 %) from the corresponding 3,4-
(methylenedioxy) cinnamic acid 7b. It is worth noting that in cases of cinnamamides 8c and
8k the poor yields, 64 and 61 % respectively, are due to the low boiling point of the amine

ACCEPTED MANUSCRIPT
6c (50-52 °C, Aldrich reagent) instead of the nature of the reaction (Table 2). Methoxy-
substituted cinnamic acids 7c-7d furnished the respective cinnamamides 8p-8t in excellent
yields (81-92 %), while O-acetyl cinnamic derivatives 7e-7h, where the hydroxyl group was
acetylated in order to prevent the ester formation, gave good results as well and the desired
amides 8u-8z were isolated in 88-92 % yield. (Table 2). Finally, tryptamine cinnamamides
8aa (83 %) and 8ab (86 %), analogues to moschamine alkaloid isolated from Carthamus
tinctorius L. [35], were also easily prepared, putting in evidence the robustness of the
developed protocol (Table 2).
Table 2 Substrate scope for the direct amidation reaction between phenylethylamines 6a-i and
cinnamic acids 7a-h mediated by B(OCH2CF3)3.a
a
Reaction conditions: phenylethylamine 6a-6i (2 mmol), cinnamic acid 7a-7h (2 mmol), B(OCH2CF3)3 (0.5
equiv), CH3CN (0.5 M), 100 °C, 24 h. b Isolated yield after column chromatography (SiO2).

ACCEPTED MANUSCRIPT
2.2 In silico Toxicology Profile
The in silico study of the synthetized compounds 8a-8ab were first estimated with Osiris,
using the stand-alone DataWarrior program [36], determining that the tested cinnamamides
fulfil the Lipinski “rule of five”, showing i) a octanol/water partition coefficient (LogP) ≤ 5;
ii) a molecular weight ≤ 500 Daltons; iii) a number of hydrogen bond acceptors (nNO) ≤ 10;
iv) a number of hydrogen bond donors (nNHOH) ≤ 5; v) a number of rotable bonds (nRB) ≤
10; and vi) topological polar surface area (TPSA) ≤ 140 (Table 3) [37].
Table 3. Molecular descriptors calculated for molecules 8a-8ab according to the Lipinski’s Rule
using the DataWarrior program.
Comp.
8a
8b
8c
8d
8e
MWa
cLogPb cLogSc HBAd HBDe TPSAf
251.328 3.2758 -3.426
285.773 3.8818 -4.162
269.318 3.3766 -3.74
281.354 3.2058 -3.444
281.354 3.2058 -3.444
281.354 3.2058 -3.444
311.380 3.1358 -3.462
252.316 2.3289 -2.655
295.337 3.3872 -4.137
329.782 3.9932 -4.873
313.327 3.488 -4.451
325.363 3.3172 -4.155
325.363 3.3172 -4.155
325.363 3.3172 -4.155
355.389 3.2472 -4.173
296.325 2.4403 -3.366
341.406 3.0658 -3.48
341.406 3.0658 -3.48
371.432 2.9958 -3.498
371.432 2.9958 -3.498
401.457 2.9258 -3.516
339.390 3.1925 -3.744
369.416 3.1225 -3.762
369.416 3.1225 -3.762
399.442 3.0525 -3.78
399.442 3.0525 -3.78
429.467 2.9825 -3.798
290.365 3.3152 -3.951
334.374 3.4266 -4.662
2
2
2
3
3
3
4
3
4
4
4
5
5
5
6
5
5
5
6
6
7
5
6
6
7
7
8
3
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
29.1
29.1
29.1
38.33
38.33
38.33
47.56
41.99
47.56
47.56
47.56
56.79
56.79
56.79
66.02
60.45
56.79
56.79
66.02
66.02
75.25
64.63
73.86
73.86
83.09
83.09
92.32
44.89
63.35
8f
8g
8h
8i
8j
8k
8l
8m
8n
8ñ
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
8z
8aa
8bb
a Molecular Weight (g/mol). b Logarithm of the partition coefficient between n-octanol and water. c Logarithm
of the solubility measured in mol/L. d Number of hydrogen-bond acceptors. e Number of hydrogen-bond donors.
f Polar Surface Area (Å2).
The toxicity risks assessed with the DataWarrior program indicated that the evaluated
cinnamamides 8a-8ab show a very low potential of being classified as mutagenic,
tumorgenic or irritant compounds, except for molecules 8w and 8x, derived from feluric acid,

ACCEPTED MANUSCRIPT
which exhibit a high predisposition of being irritants (Table 4). The main toxicity risk
manifested by the cinnamamides 8a-8ab is their possible reproductive effects. In almost all
cases, the α,β-unsaturated amide core showed a moderate risk (Table 4). However, the risk
reaches high levels when the 3,4-methylenedioxy fragment is present in the aromatic ring of
the cinnamic moiety, compounds 8i-8o, or when this ring is substituted by the acetyl function,
derivatives 8u and 8v. But in the last cases, this effect is restraint by the presence of methoxy
groups (Table 4).
Finally, the drug-likeness score, which refers to the bioactive substructure fragments
associated with approved drugs was also calculated with the DataWarrior program. Although
that the best drug-likeness score was showed by compound 8k (1.1130), values ≥ 1.5 are
considered good, almost all cinnamamides 8a-8ab exhibited positive values, which state that
these molecules contain common fragments present in commercial drugs (Table 4).
Table 4. Predictive toxicity risks estimated with the DataWarrior program.a
Comp. Muta. Tumor. Irrit. Reprod. Druglikeness
effects
0.2365
0.3116
-1.1035
0.3239
0.3239
0.3239
0.3239
0.2365
0.2270
0.3075
1.1130
0.3297
0.3297
0.3297
0.3297
0.2270
0.3233
0.3233
0.3233
0.3233
0.3233
0.3033
0.3033
0.3033
0.3033
0.3033
0.3033
0.8109
0.7993
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8ñ
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
8z
8aa
8bb
a Muta: mutagenic; Tumor: tumorgenic; Irrit: irritant; Reprod. effects: reproductive effects.
: None risk.
: Moderate risk.
: High risk.

ACCEPTED MANUSCRIPT
The in silico toxicity prediction of compounds 8a-8ab was complemented with the ProTox
II program [38]. This web server allowed the estimation of hepatotoxicity [39],
carcinogenicity [40], immunotoxicity [41], mutagenicity [42] and cytotoxicity [43] risks of
the synthetized cinnamamides (Table 5).
In general, none potential risk of hepatotoxicity and cytotoxicity was predicted for the
evaluated compounds 8a-8ab (Table 5). A slightly risk of carcinogenicity was estimated for
fluorine cinnamamide 8d and derivatives 8i-8n possessing the 3,4-methylenedioxy fragment,
while amides 8m-ñ, which also have this fragment and were mono- or di-methoxy
substituted, displayed a slightly risk of being mutagenic, in contrast to the predicted risk
suggested by the DataWarrior program (Tables 4 and 5). On the other hand, the α,β-
unsaturated amide core can be considered as an toxicophore subunit that affects the immune
system, since almost all cinnamamides 8a-8ab exhibit a high probability of being
immunotoxic compounds (Table 5).
Additionally, the binding affinity of compounds 8a-8ab were assessed against sixteen
different biological targets: adenosine A2a receptor, adrenergic beta 2 receptor, androgen
receptor, amine oxidase A, corticotropin-releasing hormone receptor 1, dopamine D3
receptor, estrogen receptor 1 and 2, glucocorticoid receptor, histamine H1 receptor, nuclear
receptor subfamily 1 group I member 2, opiod receptor kappa 1, progesterone receptor,
phosphodiesterase 4D and prostaglandin G/H synthase 1. Among them, cinnamamides 8a-
8ab displayed a good affinity to amine oxidase A (AOFA) and prostaglandin G/H synthase
1 (PGH1) (Table 5).
Table 5. Toxicity risks and targets predicted with the ProTox II program.a
AOFA PGH1
Comp. Hepa. Carci. Immu. Muta. Cyto.
(%)
(%)
-
-
-
-
-
8a
8b
8c
8d
8e
8f
8g
8h
8i
42.1
-
-
45.9
45.9
48.9
46.6
83.1b
76.7b
78.6b
78.0b
81.6b
82.4b
80.1b
46.6
72.1
48.9
72.1
72.1
25.5
25.5
33.7
32.7
34.0
34.0
34.0
34.0
34.0
34.0
34.0
32.7
56.6
33.7
52.6
52.6
8j
8k
8l
8m
8n
8ñ
8o
8p
8q
8r
8s

ACCEPTED MANUSCRIPT
72.1
45.9
48.9
69.3
69.3
69.3
69.3
-
52.6
25.5
33.7
50.7
50.7
50.7
50.7
82.7b
79.4b
8t
8u
8v
8w
8x
8y
8z
8aa
8bb
-
a Hepa: hepatotoxicity; Carci: carcinogenicity; Immu: immunotoxicity; Muta: mutagenicity; Cyto: cytotoxicity.
: Inactive.
: Slightly inactive. : Slightly active.
: Active. b Similarity with known ligands for this
toxicity target.
The percentage of affinity of these derivatives, as a pharmacophoric unit, to the active site
of these toxicity targets resulted above 42 % for AOFA and 25 % for PGH1 (Table 5).
However, compounds that showed high affinity to the AOFA target were cinnamamides 8m-
ñ, and in Table 4 the percentage of similarity with the known ligands to this target is
described, being the amide 8i the most promising compound, while the affinity for PGH1
target is increased by tryptamine derivatives 8aa and 8ab, which (especially, amide 8aa)
would be a good candidate in further and specialized biological studies (Table 5).
2.3 In Vivo Toxicity Assessment in Zebrafish Embryos
The Zebrafish (Danio rerio) has gain global acceptance as a modern experimental
vertebrate model for assessing the toxicity of diverse synthetic and natural small molecules
(SMs) [44-47]. After the Organization for Economic Cooperation and Development (OECD)
formalized the guideline for testing chemicals on fish embryos, under the Test Guideline 236:
Fish Embryo Acute Toxicity test (FET) [48], Ali and co-workers described and standardized
the toxicological assessment of SMs using zebrafish embryos in what is nowadays known as
the ZFET test [49]. Thus, these previously reported methodologies were adapted in our
laboratory with some modifications for the toxicological assessment of the zanthoxylamide
protoalkaloids 8a-8ab and seven active principles from recognized and commercial drugs
(Table 6) [50].
Table 6. Zebrafish embryo LC50 values found for the zanthoxylamide protoalkaloids 8a-8ab and the
selected positive controls.
a
Zebrafish LC50
Aquatic animal
Comp.
acute toxicityb
μmol/L ± SEM mg/L ± SEM
184.18 ± 6.94
179.21 ± 5.95
321.20 ± 7.53
213.97 ± 4.22
254.34 ± 2.97
296.81 ± 3.92
263.67 ± 5.86
43.9 ± 1.7
51.2 ± 1.7
86.5 ± 2.0
60.2 ± 1.2
71.6 ± 0.8
83.5 ± 1.1
82.1 ± 1.8
ST
ST
ST
ST
ST
ST
ST
8a
8b
8c
8d
8e
8f
8g

ACCEPTED MANUSCRIPT
285.84 ± 1.19
150.42 ± 8,63
153.22 ± 5.56
242.81 ± 9.15
191.46 ± 8.90
213.91 ± 4.08
245.29 ± 2.51
226.94 ± 0.41
254.26 ± 2.07
203.48 ± 6.17
214.30 ± 1.60
157.27 ± 5.96
248.16 ± 6.94
272.51 ± 5.06
184.64 ± 4.52
192.55 ± 5.61
164.24 ± 8.34
177.44 ± 1.30
211.06 ± 7.56
162.05 ± 7.08
23.54 ± 1.97
18.60 ± 6.94
3.84 ± 0.21c
72.1 ± 0.3
44.4 ± 2.5
50.5 ± 1.8
76.1 ± 2.9
62.3 ± 2.9
69.6 ± 1.3
79.8 ± 0.8
80.7 ± 0.1
75.3 ± 0.6
69.5 ± 2.1
73.2 ± 0.5
58.4 ± 2.2
92.2 ± 2.6
109.4 ± 2.0
62.7 ± 1.5
71.1 ± 2.1
60.7 ± 3.1
70,9 ± 0.5
84.3 ± 3.0
69.6 ± 3.0
6.8 ± 0.6
ST
ST
ST
ST
ST
ST
ST
ST
ST
ST
ST
ST
ST
PN
ST
ST
ST
ST
ST
ST
MT
MT
T
8h
8i
8j
8k
8l
8m
8n
8ñ
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
8z
8aa
6.2 ± 2.3
8bb
Camptothecin 9
Imiquimod 10
Orlistat 11
Ribavirin 12
Propranolol 13
BHA 14
Eugenol 15
0.001 ± 0.1
84.3 ± 1.2
43.1 ± 1.2
84.1 ± 1.0
4,6 ± 0.2
350.85 ± 4.99
86.99 ± 2.49
344.58 ± 4.23
17.92 ± 0.79
90.25 ± 1.15
113.88 ± 0.54
ST
ST
ST
MT
ST
ST
16,3 ± 0.2
18.7 ± 0.1
a LC50 values are the mean ± SEM of three different experiments in triplicate. b Toxicity scale (mg/L) = super
toxic 0-0.1 (T), highly toxic 0.1-1 (HT), moderately toxic 1-10 (MT), slightly toxic 10-100 (ST), practically
nontoxic 100-1000 (PN) and relatively harmless >1000 (RH). c LC50 values in nmol/L.
Camptothecin 9, an anti-cancer drug against a wide range of tumours by inhibiting both
DNA and RNA synthesis [51]; imiquimod 10, used in the treatment of common warts and
molluscum contagiosumin [52]; orlistat 11, a phospholipase A2 inhibitor used in obesity
treatment [53]; ribavirin 12, an antiviral drug used to treat RSV infection and hepatitis C
[54]; propranolol 13, a beta blocker agent used to treat high blood pressure and irregular heart
rhythms [55]; butylated hydroxyanisole (BHA) 14, an antioxidant compound [56]; and
eugenol 15, an fish anaesthetic [57]; were selected as a positive controls (Table 6).
Within cinnamamides 8a-8h, obtained from cinnamic acid 7a, the unsubstituted 8a and the
chlorine 8b derivatives resulted to be the most toxic compounds with a LC50 values of 184.1
and 179.2 µM, respectively, while the fluorine compound 8c was the less toxic amide (LC50
= 321.2 µM) (Table 6).
Comparing these results with the obtained LC50 values for the series of cinnamamides 8i-
8o, derived from the 3,4-methylenedioxy cinnamic acid 7b, the toxicity displayed by these
derivatives slightly increased from the unsubstituted derivatives 8a-8h, independently of the

ACCEPTED MANUSCRIPT
substituent from the respective amine 6a-6h. Thus, the 3,4-methylenedioxy group could be
considered as the main toxicophoric subunit for this kind of compounds (Table 6).
Particularly, cinnamamide 8r resulted to be one of the most toxic compound among the
series 8a-8z with a LC50 = 157.2 µM. This derivative has four methoxy groups distributed
equally in both aromatic rings of the cinnamamide core. Analysing the effect of methoxy
groups on the exhibited toxicity by derivatives 8p-8t, 3,4-dimethoxy compounds 8p-8r
resulted more toxic than 3,4,5-trimetoxy derivatives 8s and 8t (Table 6).
In general, O-acetyl cinnamamides 8u-8z resulted toxic, even more when this group was in
para- position of the corresponding cinnamic acid 7e, 7f and 7h, while in the case of
compound 8y, with the O-acetyl group in meta- position from cinnamic acid 7g, the toxicity
slightly decreased in comparison with its analogues with a LC50 value of 211.0 µM (Table
6).
Finally, tryptamine derivatives 8aa and 8ab resulted to be the most lethal compounds,
where the toxicity of the 3,4-methylenedioxy subunit is confirmed since amide 8ab (LC50 =
18.6 µM) displayed a slightly mortality in contrast to its unsubstituted analogue 8aa (LC50 =
23.5 µM) (Table 6). Without any doubt, the toxicity exhibited by these compounds is closely
related with the presence of the indole fragment, perhaps due to its affinity to several
biological targets, especially those associated with cell death and cytotoxic properties [58].
In concordance with the toxicity rating scale established by Fish and Wildlife service
(FSW), the LC50 values of cinnamamides 8a-8ab were also expressed in mg/L in order to
classify them according to their aquatic acute toxicity (Table 5). In almost all the cases,
zanthoxylamide protoalkaloids 8a-8z were classified as slightly toxic agents possessing a
LC50 between 43 and 109 mg/L, while both cinnamamides 8aa and 8ab, derived from
tryptamine 6i, were ranked as moderate toxic compounds for aquatic animals according to
the FSW scale (Table 6) [59].
During their study, Ley and co-workers investigated the ability of armatamide 8n,
zanthosine 8ñ and rubemamine 8r to activate the heterodimeric umami receptor in a
heterologous cell system via calcium imaging. Nevertheless, only rubemamine 8r activated
the umami receptor hTAS1R1-rTas1r3 with an effective concentration (EC50) of 44 µM [20].
Since the main application of this amide will be in culinary preparations for human
consumption, the toxicity and adverse effects induced by 8r needs to be well described.
Fortunately, we found that its LC50 is 3.5-fold above its EC50, 157 µM vs. 44 µM (Table 6).
From the selected positive controls 9-12, camptothecin 9 resulted to be the most lethal
compound with a LC50 of 3.8 nM, while imiquimod 10 was the less toxic agent among this
series of molecules with a LC50 of 350.8 µM (Table 6). On average, the LC50 showed by
zanthoxylamide protoalkaloids 8a-8z was 217 µM, and with the toxicity data of the selected
reference compounds, a toxicity scale was established in which the average LC50 for 8a-8z
fit in the safe region (Fig. 2). In contrast, tryptamine cinnamamides 8aa and 8ab were
separated from this group since they exhibited a higher toxicity, locating them in the slightly
toxic region with an average LC50 value of 21 µM (Fig. 2).

ACCEPTED MANUSCRIPT
Fig. 2. Toxicity scale established with the selected drugs 9-15 and the average LC50 for
zanthoxylamide protoalkaloids 8a-8z and cinnamamides 8aa-8ab.
3. Conclusions
We have developed an efficient and versatile methodology for the synthesis of N-
phenylethyl cinnamamides promoted by the B(OCH2CF3)3 in good to excellent yields. Our
protocol showed excellent functional group tolerance for a wide range of phenylethylamines
and cinnamic acids, demonstrating a high robustness, employing a polar solvent, without
desiccant agents, with a lower catalyst loading in comparison to previous reports, and thus it
was performed under mild reaction conditions. Within this study, five recognized
zanthoxylamide protoalkaloids were also prepared in excellent yields in contrast to the recent
literature reports: lemairamin 8g, armatamide 8n, zanthosine 8ñ, rubecenamide 8q and
rubemamine 8r, which will allow its large-scale production for industry and biological
applications.
Due to the promising use of these zanthoxylamide protoalkaloids, especially rubemamine
8r, as a culinary spice for human consumption, we described the toxicity of these
cinnamamides, which proven to be mildly toxic in the zebrafish embryo model and not
represent a potential risk as hepatotoxicity, mutagenic, carcinogenicity, tumorigenic,
cytotoxicity, irritant and reproductive effects, accordingly to the in silico computational
methods: DataWarrior and ProTox II programs. Although the majority of these
cinnamamides displayed a high immunotoxic profile, a deep and advance study in this field
is required. However, our work will be a good start point for further investigations related
with the chemistry and biology of these interesting protoalkaloids.

ACCEPTED MANUSCRIPT
4. Experimental section
4.1 Chemistry
Unless otherwise noted, all reactions have been carried out with distilled and dried solvents.
All work-up and purification procedures were carried out with reagent grade solvents
(purchased from Aldrich and Merck) in air. Thin-layer chromatography (TLC) was
performed using Merck silica gel 60 F254 precoated plates (0.25 mm). Column
chromatography was performed on Biotage® Automated Liquid Chromatography System
Isolera One® using Biotage® SNAP Ultra 25 um HP-Sphere 10 g silica gel cartridges.
Infrared (FT-IR) spectra were recorded on a Lumex Infralum FT-02 spectrometer, and
the wave numbers of the absorption peaks are listed in cm-1. Peaks/Bands are characterized
according to the functional group. 1H NMR spectra were recorded on a Bruker Avance-400
(400 MHz) spectrometer. Chemical shifts are reported in ppm with the solvent resonance as
the internal standard (CDCl3: δ 7.26 ppm; DMSO-d6: δ 2.50 ppm). Data were reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of
doublets, br = broad, m = multiplet), coupling constants (Hz) and integration. 13C NMR
spectra were recorded on a Bruker Avance-400 (400 MHz) spectrometer with complete
proton decoupling. Chemical shifts are reported in ppm from solvent resonance as the internal
standard (CDCl3: δ 77.00 ppm; DMSO-d6: δ 40.45 ppm). On DEPT-135 spectra, the signals
of CH3 and CH carbons are shown with positive phase (+), while CH2 carbons are shown
with negative phase (-). Quaternary carbons are not shown.
High Resolution Mass Spectra (HRMS) were measured on a Thermo Scientific LTQ
Orbitrap XL apparatus. Melting points were measured on a Fisher Johns melting point
apparatus and are uncorrected.
4.2.1 General procedure for the synthesis of zanthoxylamide protoalkaloids 8a-8ab
In a 50 mL vial equipped with a magnetic stir bar was added the respective cinnamic acid
7a-7h (2 mmol), the vial was sealed and the air was evacuated establishing an Argon
atmosphere and anhydrous MeCN (8 mL, 0.5 M) was added. Then, B(OCH2CF3)3 (1 mmol,
0.5 equiv.) was added dropwise into the stirred solution at room temperature. After 10
minutes, the corresponding phenylethylamine 6a-6i was added in one portion and the reaction
mixture was stirred at 100 °C for 24 hours. After cooling to room temperature, the mixture
was extracted with AcOEt (3 x 20 mL). The organic layers were combined, dried over with
Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified
by silica gel column chromatography (20-30% AcOEt in petroleum ether) to furnished the
desired zanthoxylamide protoalkaloids 8a-8ab.
4.2 In silico and bioinformatic studies
In silico analysis for the calculation of the molecular descriptors, toxicity prediction as well
as the calculation of the druglikeness were performed using the stand-alone DataWarrior

ACCEPTED MANUSCRIPT
program. The information about the methods used to evaluate each property is available on
the program web site http://www.openmolecules.org.
The toxicity risks and the affinity of the selected toxic targets were predicted with the
ProTox II program, free available online at [http://tox.charite.de/protox_II/]
4.3 In vivo toxicity assessment in zebrafish embryos
Wild-type adult zebrafish of both sexes were separated in two tanks (30 L each), according
to their gender, at 26 ± 2 °C under natural light-dark photoperiods. Fishes were feed twice
daily and the water quality was recorded weekly, in order to acclimate the fishes for at least
two weeks before experiments begin. For the reproduction of the adult fishes, small breeding
tanks were set up in the evening previous to experiment, each containing three males and one
female specimen. The tanks were isolated until next morning when the lights switch on and
the natural mating occurs, without any perturbation.
The adult fishes were returned to their corresponding tank and the embryos were collected,
pooled and washed with E3 medium and transferred into a 92 mm glass Petri dish. Further,
dead, delayed, malformed and unfertilized embryos were identified under a dissecting
microscope and removed by select aspiration with a pipette. This last procedure was repeated
at 12 and 20 hpf in order to remove the unfit embryos. Throughout this period of time, the
embryos were kept at 28 ± 2 °C in an incubator under natural light-dark photoperiods.
The selected embryos of 24 hpf from the Petri dish were gently distributed into 96-well
plates, placing a single embryo and 200 μL of E3 medium per well.
Adult zebrafish were care and used according to the Guide of the National Institute of
Health for Care and Use of Laboratory Animals, keep them healthy and free of any signs of
disease. The Ethics and Research Committee of the Heart Institute of Bucaramanga approved
the protocol under the Acta Number 050 of May 26 of 2015.
4.3.1 Determination of zebrafish embryo LC50
For this experiment, in total 72 embryos were required per sample in order to run three
independent experiments in three different plates, and each compound was evaluated three
times in the same plate, allowing the evaluation of four samples peer plate. In the range of
concentrations established by a geometric series, starting from 12.5 and finishing in 1250
μM, the determination of the LC50 (expressed in μmol of compound/L of solution) was based
on the cumulative mortality after 72 hours of chemical exposure (96 hpf). Each embryo was
examined under a dissecting microscope and the statistical analysis was made using
Regression Probit analysis with SPSS for windows version 19.0. Data are expressed as the
standard error of the mean (SEM) of three different experiments in triplicate.
Conflicts of interest
There are no conflicts to declare.

ACCEPTED MANUSCRIPT
Acknowledgements
We thank the Colombian Institute for Science and Research (COLCIENCIAS) under the project
No. RC-0346-2013 for the financial support. CEPG acknowledges the fellowship given by the
doctoral program COLCIENCIAS-Conv. 617.
Appendix A. Supplementary data
1
13
Detailed experimental procedures, characterization data and copies of the H NMR, C NMR and
DEPT-135 charts for the synthetized compounds can be found at http://XXXX
References
[1] M. Zhang, J. Wang, L. Zhu, T. Li, W. Jiang, J. Zhou, W. Peng, C. Wu, Zanthoxylum
bungeanum Maxim. (Rutaceae): A Systematic Review of Its Traditional Uses, Botany,
Phytochemistry, Pharmacology, Pharmacokinetics, and Toxicology, Int. J. Mol. Sci. 18 (2017)
2172.
[2] R. Supabphol, J. Tangjitjareonkun, Chemical Constituents and Biological Activities of
Zanthoxylum limonella (Rutaceae): A Review, Trop. J. Pharm. Res. 13 (2014) 2119-2130.
[3] J.J. Chruma, D.J. Cullen, L. Bowman, P.H. Toy, Polyunsaturated fatty acid amides from the
Zanthoxylum genus - from culinary curiosities to probes for chemical biology, Nat. Prod. Rep.
35 (2018) 54-74.
[4] Q.B. Xiong, D. Shi, H. Yamamoto, M. Mizuo, Alkylamides from pericarps of Zanthoxylum
bungeanum, Phytochemistry, 46 (1997) 1123-1126.
[5] X. Yang, Aroma Constituents and Alkylamides of Red and Green Huajiao (Zanthoxylum
bungeanum and Zanthoxylum schinifolium), J. Agric. Food Chem. 56 (2008) 1689-1696.
[6] L.C. Yang, R. Li, J. Tan, Z.T. Jiang, Polyphenolics Composition of the Leaves of Zanthoxylum
bungeanumMaxim. Grown in Hebei, China, and Their Radical Scavenging Activities, J. Agric.
Food Chem. 61 (2013) 1772-1778.
[7] L. Xia, J.M. You, G.L. Li, Z.W. Sun, Y.R. Suo, Compositional and Antioxidant Activity
Analysis of Zanthoxylum bungeanum Seed Oil Obtained by Supercritical CO2 Fluid Extraction,
J. Am. Oil Chem. Soc. 88 (2011) 23-32.
[8] T.P Singh, O.M. Singh, Phytochemical and pharmacological profile of Zanthoxylum armatum
DC. - An overview, Indian J. Nat. Prod. Resour. 2 (2011) 275-285.
[9] Barkatullah, M. Ibrar, N. Muhammad, A. Khan, S.A. Khan, S. Zafar, S. Jan, N. Riaz, Z. Ullah,
U. Farooq, J. Hussain, Pharmacognostic and phytochemical studies of Zanthoxylum armatum
DC, Pak. J. Pharm. Sci. (30) 2017 429-438.
[10] J.S. Negi, V.K. Bisht, A.K. Bhandari, P. Singh, R.C. Sundriyal, Chemical constituents and
biological activities of the genus Zanthoxylum: A review, Afr. J. Pure Appl. Chem. 5 (2011)
412-416.
[11] F. Alam, Q. N. us Saqib, A. Waheed, Cytotoxic activity of extracts and crude saponins
from Zanthoxylum armatum DC. against human breast (MCF-7, MDA-MB-468) and colorectal
(Caco-2) cancer cell lines, BMC Complement. Altern. Med. 17 (2017) 368.

ACCEPTED MANUSCRIPT
[12] C.H. Wijaya, F.I. Napitupulu, V. Karnady, S. Indariani, A review of the bioactivity and flavor
properties of the exotic spice “andaliman” (Zanthoxylum acanthopodium DC.), Food Rev. Int.
(2018) DOI: 10.1080/87559129.2018.1438470.
[13] N.K. Kalia, B. Singh, R.P Sood, A New Amide from Zanthoxylum armatum, J. Nat. Prod. 62
(1999) 311-312.
[14] E. Frerot, N. Neirynck, I. Cayeux, Y. Yuan, Y.M. Yuan, New Umami Amides: Structure–Taste
Relationship Studies of Cinnamic Acid Derived Amides and the Natural Occurrence of an
Intense Umami Amide in Zanthoxylum piperitum, J. Agric. Food Chem. 63 (2015) 7161-7168.
[15] S.K. Adesina, T.A. Olugbade, D.D. Akinwusi, D.J. von Willert, New amides from Zanthoxylum
lemairie pericarps, Planta Med. 63 (1997) 286-287.
[16] F. Cutillo, B. D’Abrosca, M. DellaGreca, C. Di Marino, A. Golino, L. Previtera, A. Zarrelli,
Cinnamic acid amides from Chenopodium album: effects on seeds germination and plant growth,
Phytochemistry 64 (2003) 1381-1387.
[17] S.K. Adesina, O.A. Olatunji, D. Bergenthal, J. Reisch, Natural product chemistry. Part 122.
Transcinnamoylamides from Zanthoxylum rubescens pericarbs. Pharmazie 43 (1988) 517-518.
[18] A. Gunia-Krzyżak, K. Pańczyk, A.M. Waszkielewicz, H. Marona, Cinnamamide Derivatives for
Central and Peripheral Nervous System Disorders—A Review of Structure–Activity
Relationships, ChemMedChem. 10 (2015) 1302-1325.
[19] A. Balsamo, P. Crotti, A. Lapucci, B. Macchia, F. Macchia, A. Cuttica, N. Passerini, Structure-
activity relationship in cinnamamides. 3. Synthesis and anticonvulsant activity evaluation of
some derivatives of (E)- and (Z)-m-(trifluoromethyl)cinnamamide, J. Med. Chem. 24 (1981)
525-532.
[20] M. Backes, K. Obst, J. Bojahr, A. Thorhauer, N. Roudnitzky, S. Paetz, K.V. Reichelt, G.E.
Krammer, W. Meyerhof, J.P. Ley, Rubemamine and Rubescenamine, Two Naturally
Occurring N-Cinnamoyl Phenethylamines with Umami-Taste-Modulating Properties, J. Agric.
Food Chem. 63 (2015) 8694-8704.
[21] Barkatullah, M. Ibrar, N. Muhammad, Evaluation of Zanthoxylum armatum DC for in-vitro and
in-vivo pharmacological screening, Afr. J. Pharm. Pharmacol. 5 (2011) 1718-1723.
[22] L. Márquez, J. Agüero, I. Hernández, G. Garrido, I. Martínez, R. Diéguez, S. Prieto, Y. Rivas,
J. Molina-Torres, M. Curini, R. Delgado, Anti-inflammatory Evaluation and Phytochemical
Characterization of some Plants of the Zanthoxylum genus, Acta Farm. Bonaerense, 24 (2005)
325-330.
[23] S. Sharma, V.P. Rasal, R.K. Joshi, P.A. Patil, In vivo Evaluation of Antiasthmatic Activity of
the Essential Oil of Zanthoxylum armatum, Indian J. Pharm. Sci. 80 (2018) 383-390.
[24] J.S. Carey, D. Laffan, C. Thomson, M.T. Williams, Analysis of the reactions used for the
preparation of drug candidate molecules, Org. Biomol. Chem. 4 (2006) 2337-2347.
[25] V.R. Pattabiraman, J.M. Bode, Rethinking amide bond synthesis, Nature 480 (2011) 471-479.
[26] A. Ojeda-Porras, D. Gamba-Sánchez, Recent Developments in Amide Synthesis Using
Nonactivated Starting Materials, J. Org. Chem. 81 (2016) 11548-11555.
[27] R. Marcia de Figueiredo, J.-S. Suppo, J.-M. Campagne, Nonclassical Routes for Amide Bond
Formation, Chem. Rev. 116 (2016) 12029-12122.
[28] J.R. Dunetz, J. Magano, G.A. Weisenburger, Large-Scale Applications of Amide Coupling
Reagents for the Synthesis of Pharmaceuticals, Org. Process Res. Dev. 20 (2016) 140-177.

ACCEPTED MANUSCRIPT
[29] J.G. Hernández Barajas, L.Y. Vargas Méndez, V.V. Kouznetsov, E.E. Stashenko, Efficient
Synthesis of New N-Benzyl- or N-(2-Furylmethyl)cinnamamides Promoted by the ‘Green’
Catalyst Boric Acid, and Their Spectral Analysis, Synthesis 3 (2008) 377-382.
[30] C.E. Puerto Galvis, J.G. Hernandez Barajas, V.V. Kouznetsov, Yb(OTf)3-Catalyzed
Bromination Reactions of Natural Product-like N-Benzyl Cinnamamides: A Facile Route to
Diverse N-Substituted Amides of Pharmacological Interest, Curr. Org. Chem. 17 (2013) 1545-
1554.
[31] A. Ojeda-Porras, A. Hernández-Santana, D. Gamba-Sánchez, Direct amidation of carboxylic
acids with amines under microwave irradiation using silica gel as a solid support, Green Chem.
17 (2015) 3157-3163.
[32] N. Gernigon, R.M. Al-Zoubi, D.G. Hall, Direct Amidation of Carboxylic Acids Catalyzed
by ortho-Iodo Arylboronic Acids: Catalyst Optimization, Scope, and Preliminary Mechanistic
Study Supporting a Peculiar Halogen Acceleration Effect, J. Org. Chem. 77 (2012) 8386-8400.
[33] R.M. Lanigan, P. Starkov, T.D. Sheppard, Direct Synthesis of Amides from Carboxylic Acids
and Amines Using B(OCH2CF3)3, J. Org. Chem. 78 (2013) 4512-4523.
[34] T. Marcelli, Mechanistic insights into direct amide bond formation catalyzed by boronic acids:
halogens as Lewis bases, Angew. Chem. Int. Ed. Engl. 49 (2010) 6840-6843.
[35] G.-H. Kang, E.-J. Chang, S.-W. Choi, Antioxidative Activity of Phenolic Compounds in Roasted
Safflower (Carthamus tinctorius L.) Seeds, J. Food Sci. Nutr. 4 (1999) 221-225.
[36] T. Sander, J. Freyss, M. von Korff, C. Rufener, DataWarrior: An Open-Source Program For
Chemistry Aware Data Visualization And Analysis, J. Chem. Inf. Model. 55 (2015) 460-473.
[37] C.A. Lipinski. Lead- and drug-like compounds: the rule-of-five revolution, Drug Discov Today
Technol. 1 (2004) 337-341.
[38] M.N. Drwal, P. Banerjee, M. Dunkel, M.R. Wettig, R. Preissner, ProTox: a web server for the
in silico prediction of rodent oral toxicity, Nucleic Acids Res. 42 (2014) W53-W58.
[39] J. Liu, K. Mansouri, R.S. Judson, M.T. Martin, H. Hong, M. Chen, X. Xu, R.S. Thomas, I. Shah,
Predicting hepatotoxicity using ToxCast in vitro bioactivity and chemical structure, Chem. Res.
Toxicol. 28 (2015) 738-751.
[40] L.S. Gold, T.H. Slone, N.B. Manley, G.B. Garfinkel, E.S. Hudes, L. Rohrbach, B.N. Ames, The
Carcinogenic Potency Database: analyses of 4000 chronic animal cancer experiments published
in the general literature and by the U.S. National Cancer Institute/National Toxicology Program,
Environ. Health Perspect. 96 (1991) 11-15.
[41] A.K. Schrey, J. Nickel-Seeber, M.N. Drwal, P. Zwicker, N. Schultze, B. Haertel, R. Preissner,
Computational prediction of immune cell cytotoxicity, Food Chem. Toxicol. 107 (2017) 150-
166.
[42] K. Hansen, S. Mika, T. Schroeter, A. Sutter, A. ter Laak, T. Steger-Hartmann, N. Heinrich, K.R.
Müller, Benchmark Data Set for in Silico Prediction of Ames Mutagenicity, J. Chem. Inf. Model.
49 (2009) 2077-2081.
[43] A.P. Bento, A. Gaulton, A. Hersey, L.J. Bellis, J. Chambers, M. Davies, F.A. Krüger, Y. Light,
L. Mak, S. McGlinchey, M. Nowotka, G. Papadatos, R. Santos, J.P. Overington, The ChEMBL
bioactivity database: an update, Nucleic Acids Res. 42 (2014) D1083- D1090.
[44] A.J. Hill, H. Teraoka, W. Heideman, R.E. Peterson, Zebrafish as a model vertebrate for
investigating chemical toxicity, Toxicol Sci. 86 (2005) 6-19.

ACCEPTED MANUSCRIPT
[45] Y.-J. Dai, Y.-F. Jia, N. Chen, W.-P. Bian, Q.-K. Li, Y.-B. Ma, Y.-L. Chen, D.-S. Pei, Zebrafish
as a model system to study toxicology, Environ. Toxicol. Chem., 33 (2014) 11-17.
[46] Y. Nishimura, A. Inoue, S. Sasagawa, J. Koiwa, K. Kawaguchi, R. Kawase, T. Maruyama, S.
Kim, T. Tanaka, Using zebrafish in systems toxicology for developmental toxicity testing,
Congenit. Anom., 56 (2016) 18-27.
[47] K.Y. Lee, G.H. Jang, C.H. Byun, M. Jeun, P.C. Searson, K.H. Lee, Zebrafish models for
functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical
applications, Biosci. Rep. 37 (2017) BSR20170199.
[48] OECD Guidelines for the testing of chemicals. Section 2: effects on biotic systems test no. 236:
Fish embryo acute toxicity (FET) test, Paris, France: Organization for Economic Cooperation
and Development (2013).
[49] S. Ali, H.G.J. van Mil, M.K. Richardson, Large-Scale Assessment of the Zebrafish Embryo as a
Possible Predictive Model in Toxicity Testing, PLoS One, 6 (2011) e21076.
[50] C.A. Martínez Bonilla, C.E. Puerto Galvis, L.Y. Vargas Méndez, V.V. Kouznetsov, Ce(SO4)2-
catalysed the highly diastereoselective synthesis of tetrahydroquinolines via an imino Diels
Alder ABB′ type reaction and their in vivo toxicity and imaging in zebrafish embryos, RSC Adv.
6 (2016) 37478-37486.
[51] C. Song, S. Song, H. Liang, X. Liu, Effect of Camptothecin on the Embryonic Development and
Angiogenesis of Zebrafish Embryos, Adv. Mat. Res. 750 (2013) 1472-1475.
[52] C.H. Kim, J.H. Ahn, S.U. Kang, H.S. Hwang, M.H. Lee, J.H. Pyun, H.Y. Kang, Imiquimod
induces apoptosis of human melanocytes, Arch. Dermatol. Res. 302 (2010) 301-306.
[53] J.D. Clifton, E. Lucumi, M.C. Myers, A. Napper, K. Hama, S.A. Farber, A.B. Smith III, D.M.
Huryn, S.L. Diamond, M. Pack, Identification of Novel Inhibitors of Dietary Lipid Absorption
Using Zebrafish, PLoS ONE, 5 (2010) e12386.
[54] D. Morick, A. Saragovi, Inhibition of nervous necrosis virus by ribavirin in a zebrafish larvae
model, Fish Shellfish Immunol. 60 (2017) 537-544.
[55] S.R. Raj, B.K. Black, I. Biaggioni, S.Y. Paranjape, M. Ramirez, W.D. Dupont, D. Robertson,
Propranolol decreases tachycardia and improves symptoms in the postural tachycardia
syndrome: less is more, Circulation 120 (2009) 725-734.
[56] G.M. Williams, M.J. Iatropoulos, J. Whysner, Safety assessment of butylated hydroxyanisole
and butylated hydroxytoluene as antioxidant food additives, Food Chem. Toxicol. 37 (1999)
1027-1038.
[57] J. Grush, D.L. Noakes, R.D. Moccia, The efficacy of clove oil as an anesthetic for the zebrafish,
Danio rerio (Hamilton), Zebrafish 1 (2004) 46-53.
[58] V. Sharma, P. Kumar, D. Pathak, Biological importance of the indole nucleus in recent years: A
comprehensive review, J. Heterocyclic Chem. 47 (2010) 491-502.
[59] I. Juneidi, M. Hayyan, M.A. Hashim, Evaluation of toxicity and biodegradability for cholinium-
based deep eutectic solvents, RSC Adv. 5 (2015) 83636-83647.

ACCEPTED MANUSCRIPT
GA
Synthesis of zanthoxylamide protoalkaloids and their In silico ADME-Tox
screening and In vivo toxicity assessment in Zebrafish Embryos
Carlos E. Puerto Galvis and Vladimir V. Kouznetsov*