Synthetic development of new 3-(4-arylmethylamino)butyl-5-arylidene-rhodanines under microwave irradiation and their effects on tumor cell lines and against protein kinases

Article abstract of DOI:10.3390/molecules200712412

A new route to 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-one 9 was developed in six steps from commercial 1,4-diaminobutane 1 as starting material. The key step of this multi-step synthesis involved a solution phase "one-pot two-s

Full text of DOI:10.3390/molecules200712412

Molecules 2015, 20, 12412-12435; doi:10.3390/molecules200712412
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthetic Development of New 3-(4-Arylmethylamino)butyl-5-
arylidene-rhodanines under Microwave Irradiation and Their
Effects on Tumor Cell Lines and against Protein Kinases
Camille Déliko Dago 1,2, Christelle N´ta Ambeu 1,2, Wacothon-Karime Coulibaly 3,
Yves-Alain Békro 2, Janat Mamyrbékova 2, Audrey Defontaine 4, Blandine Baratte 4,
Stéphane Bach 4, Sandrine Ruchaud 4, Rémy Le Guével 5, Myriam Ravache 5, Anne Corlu 5
and Jean-Pierre Bazureau 1,*
1
Université de Rennes 1, Institut des Sciences Chimiques de Rennes (ISCR), UMR CNRS 6226,
Groupe ICMV, Bât. 10A, Campus de Beaulieu, 263 Avenue du Général Leclerc, CS 74205,
35042 Rennes Cedex, France; E-Mails: deliko.dago@univ-rennes1.fr (C.D.D.);
christelle.ambeu@univ-rennes1.fr (C.N.A.)
2
Laboratoire de Chimie Bio-Organique et de Substances Naturelles (LCBOSN),
Université Nangui Abrogoua, Abidjan 02, BP 802, Cote d'Ivoire;
E-Mails: bekro2001@yahoo.fr (Y.-A.B.); kojanova1926@hotmail.fr (J.M.)
3
UFR des Sciences Biologiques, Université de Péléforo Gon Coulibaly, Korhogo, BP 1328,
Cote d'Ivoire; E-Mail: wacothon@yahoo.fr
4
Station Biologique de Roscoff, USR 3151, CNRS-UPMC, Kinase Inhibitory Specialized Screening
facility, KISSf, Place George Teissier, BP 74, 29682 Roscoff, France;
E-Mails: audrey.defontaine@sb-roscoff.fr (A.D.); blandine.baratte@sb-roscoff.fr (B.B.);
stephane.bach@sb-roscoff.fr (S.B.); sandrine.ruchaud@sb-roscoff.fr (S.R.)
5
Université de Rennes 1, ImPACcell, SFR Biosit, Bât. 8, 2 Avenue du Professeur Léon Bernard,
CS 34317, 35043 Rennes Cedex, France; E-Mails: remy.leguevel@univ-rennes1.fr (R.L.G.);
myriam.ravache@gmail.com (M.R.); anne.corlu@univ-rennes1.fr (A.C.)
* Author to whom correspondence should be addressed;
E-Mail: jean-pierre.bazureau@univ-rennes1.fr; Tel.: +33-223-236-603; Fax: +33-223-236-374.
Academic Editor: Marilena Radoiu
Received: 27 May 2015 / Accepted: 30 June 2015 / Published: 8 July 2015
Abstract: A new route to 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-
4-one 9 was developed in six steps from commercial 1,4-diaminobutane 1 as starting
material. The key step of this multi-step synthesis involved a solution phase “one-pot

Molecules 2015, 20
12413
two-steps” approach assisted by microwave dielectric from N-(arylmethyl)butane-1,4-diamine
hydrochloride 6a–f (as source of the first point diversity) and commercial bis-(carboxymethyl)-
trithiocarbonate reagent 7 for construction of the rhodanine platform. This platform was
immediately functionalized by Knoevenagel condensation under microwave irradiation
with a series of aromatic aldehydes 3 as second point of diversity. These new compounds
were prepared in moderate to good yields and the fourteen synthetic products 9a–n have
been obtained with a Z-geometry about their exocyclic double bond. These new 5-arylidene
rhodanines derivatives 9a–n were tested for their kinase inhibitory potencies against four
protein kinases: Human cyclin-dependent kinase 5-p25, HsCDK5-p25; porcine Glycogen
Synthase Kinase-3, GSK-3α/β; porcine Casein Kinase 1, SsCK1 and human HsHaspin.
They have also been evaluated for their in vitro inhibition of cell proliferation (HuH7 D12,
Caco 2, MDA-MB 231, HCT 116, PC3, NCI-H727, HaCat and fibroblasts). Among of all
these compounds, 9j presented selective micromolar inhibition activity on SsCK1 and 9i
exhibited antitumor activities in the HuH7 D12, MDA-MBD231 cell lines.
Keywords: one-pot two-steps; Knoevenagel condensation; 5-arylidene rhodanine; protein
kinase; inhibitor; SsCK1; HsCDK5-p25; cell lines; Alzheimer’s disease; cancer
1. Introduction
During the last decades, the five-membered heterocycle rings (FMHRs) are considered “privileged
scaffolds” in medicinal chemistry [1]. Among the five-membered heterocycle rings, the 5-arylidene-2-
thioxo-1,3-thiazolidine-4-ones or 5-arylidene rhodanine derivatives represented particularly privileged
moieties in drug discovery because they have an inherent tendency for biological activity [2], such as
DDX3 inhibitor for HIV replication [3], as potent and selective inhibitors of the “atypical” dual-specificity
phosphatase (DSP) family member-JNK-stimulating phosphatase-1 (JSP-1) [4], as pancreatic cholesterol
esterase (CEase) inhibitor [5] with IC50 values ranging from 1.44 to 85 μM. To discover chemical probes
to further understand the function of human DNA polymerase λ in cancer, an inhibitor high-throughput
screening (HTS) using SYBR® Green-base assay [6] revealed that three 5-arylidene-2-thioxo-
1,3-thiazolidine-4-ones were identified as strong inhibitors. A new series of D-glutamic acid-base
Escherichia coli MurD inhibitors incorporating the 5-arylidene rhodanine scaffold have been designed,
synthesized and evaluated [7]. Substituted 5-arylidene-2-thioxo-1,3-thiazolidine-4-ones were able to
inhibit HIV replication in MT-4 cells at low micromolar concentration with an appreciable selectivity
index and are good scaffolds for the development of novel HIV-1 integrase inhibitors [8]. Recently, a
family of 5-arylalkylidene rhodanine derivatives presented antiviral activity against chikungunya virus
(LR2006_OPY1) in Vero cell culture by cytopathic effect CPE reduction assay [9]. For Alzheimer’s
disease, the FMHRs derived from 5-arylidene-2-thioxo-1,3-thiazolidine-4-ones have been described
for amyloid polypeptide fibril formation [10], regulation of Cathepsin D immuno-reactivity in the senile
plaques [11] and inhibition of tau aggregation [12].
Protein kinases represent an important class of enzymes that play an important role in the regulation
of various processes. These enzymes catalyze protein-phosphorylation on serine, threonine and tyrosine

Molecules 2015, 20
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residues, which are frequently deregulated in human diseases. Only the 518 human kinases have been
investigated as potential therapeutic targets [13]. Consequently, the search of protein-kinase inhibitors
represented interesting targets in the pharmaceutical industry for new therapeutic agents. Over the past
decade, our research group have investigated the chemical development of five-membered heterocycle
rings derived from marine alkaloid as low-molecular weight-inhibitors of dual specificity, tyrosine
phosphorylation-regulated kinases (DYRKs) and CLKs (cdc2-like kinases) [14–16], two families of
kinases involved in various diseases including Alzheimer’s disease (AD) [17], and also cancer [18–20].
Continuing in the effort to identify new DYRK inhibitors, particularly DYRK1A, we continued
to explore successively the synthesis of N,Nʹ-bis-(5-arylidene-4-oxo-3,5-dihydro-4H-imidazol-2-yl)
diamines [21], N,Nʹ-bis-(5-arylidene-4-oxo-4,5-dihydrothiazolidine-2-yl)aminopropylpiperazines [22] and
finally unsymmetrical linked bis-5-arylidene-rhodanine derivatives linked in N-3 position [23] as potential
kinases inhibitors (Figure 1). Among the three series of symmetrical or unsymmetrical N,Nʹ-diamines
bearing various platforms (imidazolidine-4-one or 2-thioxo-1,3-thiazolidine-4-one moieties), only one
of these compounds has shown nanomolar inhibition potency (IC50 40 nM) towards DYRK1A. Owing to
the fact that none of the unsymmetrical linked bis-5-arylidene rhodanines presented a significant activity
against representative tumoral cell lines, we decided to change one of the two heterocyclic platforms by
various (arylmethyl)aminobutyl moieties grafted on N-3 position of 5-arylidene-rhodanine derivatives
in order to obtain significant potential biological activities on tumoral cell lines and effects on some
protein kinases (HsCDK5-p25, SsGSK3α/β, SsCK1 and HsHaspin). Herein, we present the building of
this 3-(4-arylmethylamino)butyl-5-arylidene-rhodanine library in which the 5-arylidene-2-thioxo-1,3-
thiazolidine-4-one moiety was mostly carried out under microwave irradiation [24] and the biological
activities of these compounds.
R2
R1
O
R3 , R2
R2, R3
S
S
S
S
H
N
H
H
N
O
N
N
N
N
N
N
S
N
n
n
O
O
N
N
N
S
R1
R1
O
O
N
O
Unsymmetrical linked bis-5-arylidene
rhodanine derivatives
N
H
Symmetrical linked bis-5-arylidene
imidazoline-4-one derivatives
N,N'-Bis(5-arylidene-4-oxo-4,5-dihydrothiazolin-2-yl)
aminopropyl piperazine
DYRK1A, IC50: 40 nM
O
N
n = 0, 2 or
N
N
n = 2 or
N
N
or
N
Figure 1. Symmetrical linked bis-5-arylidene imidazoline-4-ones, symmetrical linked
bis-5-arylidene-1,3-thiazoline-4-ones and unsymmetrical linked bis-5-arylidene rhodanines
derivatives (as inhibitors of the proteine kinase DYRK1A or against cell proliferation on
cell tumor lines) identified and developed in our group.
2. Results and Discussion
2.1. Chemistry
Access to the planned 3-(4-arylphenylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-ones 9 is
outlined in Scheme 1. For this study, we selected a diamino linker 1 with a butyl chain in order to obtain
a good molecular flexibility between the 4-arylmethyl moiety and the 2-thioxo-1,3-thiazolidine-4-one

Molecules 2015, 20
12415
platform. A molecule with the lowest number of carbons for the diamino linker is more conformationally
restrained [25]. The 1,4-diamino-butane linker 1 was treated with di-tert-butyldicarbonate (Boc2O) in
1,4-dioxane at room temperature to afford mainly the mono-N-Boc protected amine 2 in good yield
(84%) [26]. To obtain a sufficient number of compounds suitable for a preliminary biological screening,
we privileged the transformation of N-Boc-1,4-diamino-butane 2 into mono protected N-arylmethyl
diamine 5 by reductive amination in two steps. The (4-arylmethylamino)butyl chain appended on the
N-3 position of the 2-thioxo-1,3-thiazolidine-4-one platform represent the first point of diversity for the
desired target compounds 9. Preparation of 4 was easily realized by reaction between of appropriate
arylaldehyde 3a–f with N-Boc diamine 2 in the presence of molecular sieves 3Å and, the condensation
was conducted in a solution of diethyl ether Et2O at room temperature during 24 h. Then, transformation
of arylaldimines 4 into mono protected N-arylmethyl diamines 5 could be readily accomplished in
good yields (85% to 98%) using NaBH4 (5 equiv.) in MeOH at 50 °C during 24 h. For the cleavage of
N-Boc group, the use of trifluoroacetic acid in dichloromethane is often efficient, but in our case, we
observed the formation of impurities resulting from uncontrolled degradation of 5 in this strong acidic
media. We thus preferred to use a more classical and practical approach by using a solution of
6M HCl. The deprotection was conducted in 1,4-dioxane at room temperature after 4 h of reaction time.
As can be seen from the data presented in Table 1, the N-(arylmethyl)butane-1,4-diamine hydrochloride
6a–f were efficiently prepared with electron-rich and electron-poor aldehydes 3a–f in yields ranging
from 72% to 98%.
H
H
i
iii
ii
NH2
N
O
N
O
H2N
H2N
N
R
O
O
O
R
1
2
4a-f
3a-f
S
S
H
NH2
6a-f, HCl
v
iv
N
O
N
N
N
N
R
H
R
R
H
H
S
O
O
5a-f
8a-f
7
S
S
R
HO2C
CO2H
S
S
vi
N
N
H
O
R
R
O
9a-n
3a-h
Scheme 1. Route used for the preparation of 3-(4-arylphenylamino)butyl-5-arylidene-2-
thioxo-1,3-thiazolidine-4-ones 9 via the “one-pot two-steps” reaction under microwave.
Reagents and reaction conditions: (i) (t-BuO2C)2O, 1,4-dioxane, 25 °C, 24 h. (ii) 3 1
equiv., molecular sieve 3Å, Et2O, 25 °C, 16 h. (iii) NaBH4 5 equiv., MeOH, 50 °C, 24 h.
(iv) HCl 6 M, 1,4-dioxane, 25 °C, 4 h. (v) 6 1 equiv., 7 1 equiv., Et3N 2 or 3 equiv., DME,
MWI, 90 °C, 15 or 30 min. (vi) 3 1 equiv., MWI, 110 °C, 15 or 30 min.
With the desired salts 6 in hand, we wished to examine the construction of the 2-thioxo-1,3-
thiazolidine-4-one platform followed by Knoevenagel condensation using arylaldehyde 3 under
microwave irradiation for installation of the 5-arylidene moiety. The choice of arylaldehydes 3 in
Knoevenagel condensation represent, in fact, the second point of diversity in the final structure of the
targeted compounds 9. In a previous work issued from our laboratory [23], we have developed a
“one-pot two-steps” method under microwave irradiation, which was applied for the synthesis of
unsymmetrical linked bis-arylidene rhodanines from symmetric diamines.

Molecules 2015, 20
Table 1. Results for the preparation of compounds 2, 4, 5, 6 and 9.
12416
Reaction Conditions under MWI a
Starting
Product
Yield b
Compound
Starting
Number of
Reaction Time
Reaction Time for
(%)
Product 6 equiv. for Et3N
for 8 c (min)
Condensation d (min)
2
4a
4b
4c
4d
4e
4f
-
-
-
-
-
-
-
-
-
84
97
99
98
98
98
99
93
99
85
90
97
98
84
97
98
98
72
92
10
6
3a
3b
3c
3d
3e
3f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5a
5b
5c
5d
5e
5f
4a
4b
4c
4c
4e
4f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6a
6b
6c
6d
6e
6f
5a
5b
5c
5d
5e
5f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9a
9b
9c
9d
9e
9f
3e
3b
3f
6b
6b
6b
6b
6d
6d
6d
6e
6e
6e
6e
6f
6f
6f
3
3
3
3
3
3
3
2
2
2
3
3
3
3
30
30
30
30
15
15
30
15
15
15
30
15
15
30
30
30
30
30
15
15
30
15
15
15
30
15
15
5
3g
3e
3f
30
21
9
9g
9h
9i
3d
3e
3f
5
59
59
34
46
15
7
9j
3h
3g
3e
3f
9k
9l
9m
9n
O
3g
O
30
34
O
O
O
O
O
O
O
O
O
O
O
O
R
O
O
Cl
HO
HO
3
3b
3c
3e
3h
3a
3d
3f
3g
a
Notes: Reaction realized in a tube (sealed with a snap cap) under microwave irradiation (μω) with the
c
Monowave® 300 Anton-Paar reactor. b Isolated yield. Reaction temperature: 90 °C for the preparation of 8
(1st period of microwave irradiation). d Reaction temperature: 110 °C for condensation reaction after addition
of 3 (2nd period of microwave irradiation).
Application of this methodology, based on a “modified Holmberg method”, first involved, in our
case, the reaction of the commercial bis-(carboxymethyl)-trithiocarbonate reagent 7 with the synthesized

Molecules 2015, 20
12417
N-(arylmethyl)butane-1,4-diamine hydrochloride 6 to afford the intermediate 8 and secondly, Knoevenagel
condensation to produce the desired 5-arylidene rhodanines 9. This “one-pot two-steps” methodology
was realized under microwave dielectric heating because the use of commercial scientific laboratory
microwave apparatus favoured higher product yields and significant rate enhancements compared to
reactions which run with conventional heating (i.e., in oil bath) [27]. After several experiments,
optimal reaction conditions for this “one-pot two-steps” synthesis involved reaction of a stoichiometric
mixture of N-(arylmethyl)butane-1,4-diamine hydrochloride 6 and commercial reagent 7 solubilized in
dimethoxyethane with one or two equivalents of triethylamine. This starting reaction mixture was
placed in a commercial glass tube closed with a snap cap and was irradiated with appropriate reaction
time (15 or 30 min.) at 90 °C. After this first period of microwave irradiation followed by a cooling
down to room temperature, aromatic aldehyde 3 was directly added to the crude suspension and was
submitted again to microwave dielectric heating at 110 °C during 15 or 30 min for condensation.
The desired compound 9 was obtained as precipitate after addition of methanol in the solventless
crude reaction mixture (after elimination of the volatile compounds in vacuo) and triturated, followed by
successive washings with deionized water, ethanol, ether and finally was recrystallized from absolute
EtOH to increase the quality of the precipitated product 9. A set of 14 pure compounds 9a–n was
1
13
prepared in 5%–59% yield (Table 1) and all the products 9 were characterized by H-, C-NMR and
HRMS before entering the biological tests. For the exocyclic double bond (CH=C) in C-5 position of all
the 3-(4-arylmethylamino)butyl-2-thioxo-1,3-thiazolidine-4-one 9a–n, it’s possible in theory to observe
E- and/or Z-geometrical isomers. Examination of their 1H-NMR spectra in DMSO-d6 showed only one
signal for the methylene proton (CH=) in the range 7.54–7.74 ppm at lower field values than those
expected for the E-isomers, which indicates that all the compounds 9a–n have the Z-configuration due to
13
the high degree of thermodynamic stability of this isomer [28,29]. In C-NMR, the signal of C-5 (C=)
appears in the range 120.0–127.5 ppm, and we also observed only one signal for the exocyclic methylene
proton (134.6 < δCH= < 135.9 ppm) that confirmed the presence of only the Z-geometrical isomer for
the targeted compounds 9a–n.
2.2. Biology
As an initial effort to investigate their in vitro bioactivity, the new synthesized 3-(4-arylmethylamino)
butyl-2-thioxo-1,3-thiazolidine-4-one 9a–n and also their precursors 6a–f were evaluated for their
in vitro inhibition of cell proliferation. For this study, we used a panel of seven representative tumoral
cell lines, namely HuH7 D12 (differential hepato cellular carcinoma), Caco 2 (differentiating colorectal
adenocarcinoma), MDA-MBD231 (prostate carcinoma), HCT 116 (actively proliferating colorectal
adenocarcinoma), PC3 (prostate carcinoma), NCI-H727 (lung carcinoma), HaCat keratinocyte and,
diploid skin fibroblasts as normal cell lines for control. Roscovitine, Doxorubicine and Taxol were also
used as positive controls and their IC50 values are compared with those obtained for compounds 6 and 9.
Results of the in vitro antiproliferative data activity are reported in Table 2. None of the N-(arylmethyl)
butane-1,4-diamines hydrochloride 6a–f presented a significant activity against the seven representative
tumoral cell lines. For the other compounds 9, the most active compound was clearly 9i (Figure 2) and
exhibited antitumor activities in the HuH7 D12, MDA-MBD231 and HaCat cell lines with IC50 values
lower than 10 μM (HuH7 D12 and MDA-MBD231: IC50 9 μM) and did not inhibit the growth of normal

Molecules 2015, 20
12418
fibroblasts (IC50 > 25 μM). In addition, compounds 9d, 9h, 9j and 9n presented moderate antitumor
activities (IC50 10–17 μM) in all cell lines of the panel but without selectivity.
Table 2. Antiproliferative activity of compounds 6(a–e) and 9(a–n) on six representative
tumor cell lines.
% of Survival a and IC50 (μM) of Selected Compounds b
Compound
Huh7 D12
102
Caco 2
114
MDA-MB231
104
HCT 116
112
PC3
100
NCI-H727
97
HaCat
97
Fibroblasts
93
6a
6b
107
114
100
112
100
86
106
100
6c
82
93
102
98
96
100
80
84
6d
107
111
99
111
98
92
96
93
6e
91
106
102
102
96
98
96
93
6f
88
109
99
96
97
92
94
105
9a
85
109
100
109
92
104
103
104
9b
105
108
97
102
99
90
104
167
9c
88
93
92
97
97
95
101
102
9d
7 (10)
74
23 (15)
97
15 (11)
97
1 (10)
99
41 (17)
85
11 (10)
102
2 (10)
86
29 (>25)
122
9e
9f
102
110
98
113
98
96
100
134
9g
111
117
104
118
98
95
110
100
9h
7 (10)
5 (9)
22 (11)
72
42 (14)
17 (13)
41 (27)
98
49 (10)
17 (9)
42 (19)
92
65 (13)
8 (14)
40 (22)
98
51 (13)
39 (14)
38 (19)
83
43 (>25)
9 (11)
33 (19)
96
10 (10)
6 (8)
21 (13)
80
80 (>25)
54 (>25)
77 (>25)
84
9i
9j
9k
9l
90
105
98
106
93
96
95
128
9m
97
107
99
103
96
98
94
149
9n
41 (15)
21 (15)
63 (0.03)
36 (0.003)
100 (>25)
28 (16)
3 (15)
43 (0.03)
29 (0.04)
100 (>25)
55 (15)
21 (12
82 (0.01)
41 (0.04)
100 (>25)
52 (21)
10 (9)
22 (0.03)
9 (<0.001)
100 (>25)
61 (19)
24 (13)
34
63 (17)
30 (43)
65
48 (15)
6 (11)
88 (0.02)
11 (0.001)
100 (>25)
86 (>25)
53 (125)
74 (>0.25)
76 (>25)
100 (>25)
Roscovitine
Doxorubicine
Taxol
DMSO
35 (<0.001)
59
100 (>25)
100 (>25)
Notes: a Percentage of survival measured at 25 μM (after 48 h using a single dose, triplicate). b IC50 values in
brackets are expressed in μM and are the average of three assays, standard error ± 0.5 μM.
O
OH
O
O
S
S
S
O
S
O
S
S
N
N
N
O
O
O
O
N
H
N
H
N
3
3
3
H
O
O
O
9i
9h
O
9d
OH
OH
S
S
S
S
O
N
O
O
N
O
O
N
N
3
H
3
H
O
O
9j
9n
Figure 2. Structure of compounds 9d, 9(h–j) and 9n, which are active against protein
kinases and/or tumor cell lines.

Molecules 2015, 20
12419
Then, we evaluated the intermediates salts 6a–f and fourteen final compounds 9a–n on four protein
kinases: HsCDK5-p25 (cyclin-dependant kinase 5-p25), SsGSK-3α/β (glycogen synthase kinase-3α/β)
SsCK1δ/ε (Caseine kinase 1) and HsHaspin as an essential mitotic kinase [30]. All assays were run in the
presence of 15 μM of ATP and appropriate substrates. IC50 values were determined from dose-response
curves. Concerning the library of the fourteen (5Z) 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-
1,3-thiazolidine-4-ones 9a–n and also the six diamines salts 6a–f, the results are given in Table 3.
Among all the products, the main part of their inhibition activity was focused on two proteins kinases
(SsCK1 and HsCDK5-p25) for four products, which exhibited inhibitory activity in the micromolar
range (SsCK1: 1.4 μM < IC50 < 6.6 μM; HsCDK5-p25: IC50 1.2 μM). For the compound 9j and 9n, we
observed micromolar inhibition activity on SsCK1 (IC50 values for 9j: 1.4 μM and 9n: 2 μM) with
good selectivity. It is interesting to note that these two compounds are respectively substituted with
an hydroxyl function on para-position of the 5-arylidene moiety and also with a similar bulky group
(1,3-benzodioxol-5-yl for 9j and 2,3-dihydro-benzo[1,4]dioxin-6-yl for 9n) on the arylmethylamino
moiety grafted on the N-3 butyl chain. On the contrary, the presence of 1,3-benzodioxol-5-yl (for 9h)
or 2,3-dihydro-benzo[1,4]dioxin-6-yl moiety (for 9i) in the 5-arylidene group induced a moderate loss
of inhibition activity for SsCK1 (IC50 for 9h: 6.6 μM and 9i: 5.4 μM) associated with micromolar
affinity for HsCDK5-p25 (IC50 for 9h: 1.1 μM and 9i: 1.3 μM). Compound 9i presented also inhibition
proliferation activity on Huh7 D12, MDA-MBD231 cell lines and is inactive against normal fibroblasts.
Table 3. Effects of the compounds 6a–f and 9a–n on the catalytic activity of four purified
protein kinases a.
Compound
HsCDK5/p25 SsGSK-3α/β SsCK1δ/ε
HsHaspin
>10
6a–f
9a–g
9h
>10
>10
1.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.6
5.1
1.4
>10
2
>10
>10
9i
1.3
>10
9j
>10
>10
10
>10
9k–m
9n
>10
>10
Note: a Compounds were tested at various concentrations on each kinase as described in Experimental Section.
IC50 values, calculated from the dose-response curves, are reported in μM > 10, inhibitory but IC50 > 10 μM.
3. Experimental Section
3.1. Chemistry Section
3.1.1. General Section
Melting points were determined on a Kofler melting point apparatus and were uncorrected. Thin-layer
chromatography (TLC) was accomplished on 0.2-mm precoated plates of silica gel 60 F-254 (Merck,
Fontenay-sous-Bois, France). Visualization was made with ultraviolet light (254 and 365 nm) or with a
fluorescence indicator. 1H-NMR spectra were recorded on BRUKER AC 300 P (300 MHz) spectrometer,
13C-NMR spectra on a BRUKER AC 300 P (75 MHz) spectrometer. Chemical shifts are expressed in
parts per million downfield from tetramethylsilane as an internal standard. Data are given in the

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following order: δ value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad),
number of protons, coupling constants J is given in Hertz. The mass spectra (HRMS) were taken
respectively on a MS/MS ZABSpec Tof Micromass (EBE TOF geometry) at an ionizing potential of
8 eV and on a VARIAN MAT 311 at an ionizing potential of 70 eV in the Centre Régional de Mesures
Physiques de l’Ouest (CRMPO, Rennes, France). Reactions under microwave irradiations were realized
in the Anton Paar Monowave 300® microwave reactor (Anton-Paar, Courtaboeuf, France) using
borosilicate glass vials of 10 mL equipped with snap caps (at the end of the irradiation, cooling reaction
was realized by compressed air). The microwave instrument consists of a continuous focused microwave
power output from 0 to 800 W for this Monowave 300® apparatus. All the experiments in the microwave
reactor were performed using a stirring option. The target temperature was reached with a ramp of
5 min and the chosen microwave power stayed constant to hold the mixture at this temperature. The
reaction temperature is monitored using calibrated infrared sensor and the reaction time included the
ramp period. The microwave irradiation parameters (power and temperature) were monitored by the
Monowave software package of the Monowave 300® microwave reactor. Solvents were evaporated
with a BUCHI rotary evaporator. All reagents and solvents were purchased from Acros, Sigma-Aldrich
Chimie (Saint-Quentin Fallavier, France), TCI France and Fluka France and were used without
further purification.
tert-Butyl (4-aminobutyl)carbamate (2). In a 250 mL two-necked round-bottomed flask, provided
with magnetic stirrer and condenser, commercial 1,4-diaminobutane 1 (14.3 mL, 12.5 g, 0.14 mol) was
solubilized in 69 mL of 1,4-dioxane at room temperature. To this mixture was added dropwise a solution
of commercial di-tert-butyldicarbonate (6.5 g, 30 mmol) in 1,4-dioxane (85 mL) over a period of 3 h.
After vigorous stirring at 25 °C during a 12 h period, the volatile compounds of the reaction mixture
were eliminated in vacuo and into the crude reaction mixture was poured 150 mL of deionized water.
The mixture was extracted with methylene chloride (5 × 50 mL), organic phases were collected and dried
over magnesium sulfate. The filtrate was concentrated in a rotary evaporator under reduced pressure and
was dried under high vacuum (10−2 Torr) at 25 °C for 10 min. The desired carbamate 2 (1.58 g) was
1
obtained as a colourless mobile oil in 84% yield and was further used without purification. H-NMR
(DMSO-d6) δ: 1.34 (s, 9H, Me3CO); 1.40 (m, 4H, CH2); 2.61 (t, 2H, J = 6.7 Hz, CH2); 3.02 (m, 2H,
CH2NH); 4.93 (br s, 2H, NH2); 5.71 (br s, 1H, NH). 13C-NMR (DMSO-d6) δ: 27.2 (CH2); 28.4
(OC(CH3)3); 29.5 (CH2); 40.2 (CH2); 41.1 (CH2); 78.9 (OC(CH3)3); 156.1 (C=O). HRMS, m/z:
189.1600 found (calculated for C9H21N2O2 [M + H]+ requires 189.1603).
3.1.2. Standard Procedure for the Preparation of Aldimines 4a–f from tert-Butyl (4-aminobutyl)
Carbamate 2 and Aromatic Aldehyde 3a–f
In a 100 mL two-necked round-bottomed flask, provided with magnetic stirrer and condenser,
containing a solution of tert-butyl (4-aminobutyl)carbamate 2 (1.97 g, 10.5 mmol) in anhydrous ether
(50 mL) was added dropwise during 30 min, a suspension of commercial aldehyde (10 mmol) in anhydrous
ether (30 mL) and molecular sieves (2 g, 3 Å, 8–12 mesh). The crude reaction mixture is stirred vigorously
for 16 h at room temperature until the disappearance of aromatic aldehyde 3 controlled by thin-layer
chromatography on 0.2-mm precoated plates of silica gel 60 F-254 (Merck). The crude reaction mixture

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was filtered on filter paper and then concentrated in a rotary evaporator under reduced pressure. The
crude residue was dried under high vacuum (10−2 Torr) at 25 °C for 10 min. The desired aldimine 4 was
obtained as yellowish viscous oil and was further used without purification.
[4-(Benzylidene-amino)-butyl]-carbamic acid tert-butyl ester (4a). Compound 4a was prepared in 97%
yield (2.68 g) from benzaldehyde 3a (1.06 g, 10 mmol) according to the standard procedure as yellowish
1
viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.40–1.48 (m, 2H, CH2, H-2); 1.54–1.62 (m,
2H, CH2, H-3); 2.96 (q, 2H, J = 6.8 Hz, CH2NH, H-1); 3.55 (t, 2H, J = 6.8 Hz, CH2N=, H-4); 6.79–6.83
(br s, 1H, NH); 7.41–7.46 (m, 3H, H-3ʹ, H-4ʹ, H-5ʹ, Ar); 7.71–7.75 (m, 2H, H-2ʹ, H-6ʹ, Ar); 8.32 (s, 1H,
N=CH). 13C-NMR (DMSO-d6) δ: 27.3 (C-2); 27.8 (C-3); 28.2 (OC(CH3)3); 39.6 (C-1); 60.1 (C-4); 77.2
(OC(CH3)3); 127.7 (C-2ʹ, C-6ʹ, Ar); 128.5 (C-3ʹ, C-5ʹ, Ar); 130.4 (C-4ʹ, Ar); 136.1 (C-1ʹ, Ar); 155.5
(C=O); 160.5 (N=CH).
{4-[(4-Methoxy-benzylidene)-amino]-butyl}-carbamic acid tert-butyl ester (4b). Compound 4b was
prepared in 99% yield (3.03 g) from 4-methoxybenzaldehyde 3b (1.36 g, 10 mmol) according to the
1
standard procedure as yellowish viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.32–1.46
(m, 2H, CH2, H-2); 1.52–1.61 (m, 2H, CH2, H-3); 2.94 (q, 2H, J = 6.9 Hz, CH2NH, H-1); 3.50 (t, 2H,
J = 6.8 Hz, CH2N=, H-4); 3.80 (s, 3H, OCH3); 6.77–6.82 (br s, 1H, NH); 6.98 (d, 2H, J = 8.8 Hz, H-2ʹ,
H-6ʹ, Ar); 7.66 (d, 2H, J = 8.8 Hz, H-3ʹ, H-5ʹ, Ar); 8.24 (s, 1H, N=CH). 13C-NMR (DMSO-d6) δ: 27.4
(C-2); 28.0 (C-3); 28.2 (OC(CH3)3); 39.6 (C-1); 55.2 (OCH3); 60.1 (C-4); 77.3 (OC(CH3)3); 114.0 (C-2ʹ,
C-6ʹ, Ar); 129.0 (C-1ʹ, Ar); 129.3 (C-3ʹ, C-5ʹ, Ar); 155.5 (C=O); 159.7 (N=CH); 161.0 (C-4ʹ, Ar).
{4-[(2-Chloro-benzylidene)-amino]-butyl}-carbamic acid tert-butyl ester (4c). Compound 4c was
prepared in 98% yield (3.05 g) from 2-chlorobenzaldehyde 3c (1.41 g, 10 mmol) according to the
1
standard procedure as yellowish viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.34–1.48
(m, 2H, CH2, H-2); 1.56–1.63 (m, 2H, CH2, H-3); 2.96 (q, 2H, J = 6.7 Hz, CH2NH, H-1); 3.61 (t, 2H,
J = 6.8 Hz, CH2N=, H-4); 6.78–6.82 (br s, 1H, NH); 7.36–7.52 (m, 3H, H-4ʹ, H-5ʹ, H-6ʹ, Ar);
7.94–7.97 (m, 1H, H-3ʹ, Ar); 8.65 (s, 1H, N=CH). 13C-NMR (DMSO-d6) δ: 27.3 (C-2); 27.7 (C-3); 28.2
(OC(CH3)3); 39.6 (C-1); 60.4 (C-4); 77.2 (OC(CH3)3); 127.4 (C-5ʹ, Ar); 128.0 (C-4ʹ, Ar); 129.8 (C-6ʹ,
Ar); 132.0 (C-3ʹ, Ar); 132.7 (C-1ʹ, C-2ʹ, Ar); 133.8 (C-1ʹ, C-2ʹ, Ar); 155.5 (C=O); 156.6 (N=CH).
{4-[(3-Methoxy-benzylidene)-amino]-butyl}-carbamic acid tert-butyl ester (4d). Compound 4d was
prepared in 98% yield (3.03 g) from 3-methoxybenzaldehyde 3d (1.36 g, 10 mmol) according to the
1
standard procedure as yellowish viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.36–1.45
(m, 2H, CH2, H-2); 1.54–1.64 (m, 2H, CH2, H-3); 2.96 (q, 2H, J = 6.7 Hz, CH2NH, H-1); 3.54 (t, 2H,
J = 6.7 Hz, CH2N=, H-4); 3.78 (s, 3H, OCH3), 6.76–6.82 (br s, 1H, NH); 6.99–7.03 (m, 1H, H-5ʹ, Ar);
13
7.27–7.38 (m, 3H, H-2ʹ, H-4ʹ, H-6ʹ, Ar); 8.29 (s, 1H, N=CH). C-NMR (DMSO-d6) δ: 27.3 (C-2);
27.8 (C-3); 28.2 (OC(CH3)3); 39.6 (C-1); 55.0 (OCH3); 60.1 (C-4); 77.2 (OC(CH3)3); 111.9 (C-5ʹ, Ar);
116.6 (C-6ʹ, Ar); 120.6 (C-4ʹ, Ar); 129.6 (C-2ʹ, Ar); 137.6 (C-1ʹ, Ar); 155.5 (C=O); 159.4 (C-3ʹ, Ar);
160.4 (C=N).
{4-[(Benzo[1,3]dioxol-5-ylmethylene)-amino]-butyl}-carbamic acid tert-butyl ester (4e). Compound
4e was prepared in 98% yield (3.13 g) from 3,4-methylenedioxybenzaldehyde 3e (1.51 g, 10 mmol)

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1
according to the standard procedure as yellowish viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H,
Me3C); 1.30–1.46 (m, 2H, CH2, H-2); 1.51–1.61 (m, 2H, CH2, H-3); 2.94 (q, 2H, J = 6.9 Hz, CH2NH,
H-1); 3.49 (t, 2H, J = 6.7 Hz, CH2N=, H-4); 6.07 (s, 2H, H-2ʹ, Ar), 6.77–6.82 (br s, 1H, NH); 6.97 (d,
1H, J = 8 Hz, H-6ʹ, H-7ʹ, Ar); 7.16–7.20 (m, 1H, H-6ʹ, H-7ʹ, Ar); 7.27–7.28 (m, 1H, H-4ʹ, Ar), 8.20 (s,
1H, N=CH). 13C-NMR (DMSO-d6) δ: 27.3 (C-2); 27.9 (C-3); 28.2 (OC(CH3)3); 39.6 (C-1); 59.8 (C-4);
77.4 (OC(CH3)3); 101.4 (C-2ʹ, Ar); 105.8 (C-6ʹ, Ar); 108.1 (C-7ʹ, Ar); 124.0 (C-4ʹ, Ar); 130.9 (C-5ʹ,
Ar); 147.8 (C-3aʹ, C-7aʹ, Ar); 149.3 (C-3aʹ, C-7aʹ, Ar); 155.5 (C=O); 160.0 (C=N).
{4-[(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethylene)-amino]-butyl}-carbamic acid tert-butylester (4f).
Compound 4f was prepared in 99% yield (3.31 g) from 2,3-dihydro-benzo[1,4]dioxin-6-carbaldehyde
3f (1.50 g, 10 mmol) according to the standard procedure as yellowish viscous oil. 1H-NMR (DMSO-d6) δ:
1.37 (s, 9H, Me3C); 1.34–1.45 (m, 2H, CH2, H-2); 1.51–1.60 (m, 2H, CH2, H-3); 2.94 (q, 2H, J = 6.8 Hz,
CH2NH, H-1); 3.50 (t, 2H, J = 6.7 Hz, CH2N=, H-4); 4.23–4.30 (m, 4H, H-2ʹ, H-3ʹ, Ar), 6.77–6.81
(br s, 1H, NH); 6.88–6.91 (m, 1H, H-7ʹ, Ar); 7.18–7.21 (m, 2H, H-5ʹ, H-8ʹ, Ar); 8.17 (s, 1H, N=CH).
13C-NMR (DMSO-d6) δ: 27.3 (C-2); 27.9 (C-3); 28.2 (OC(CH3)3); 39.6 (C-1); 60.0 (C-4); 63.9 (C-2ʹ,
Ar), 64.2 (C-3ʹ, Ar), 77.2 (OC(CH3)3); 116.0 (C-7ʹ, Ar); 117.1 (C-8ʹ, Ar); 121.2 (C-5ʹ, Ar); 129.8 (C-6ʹ,
Ar); 143.4 (C-4aʹ, C-8aʹ, Ar); 145.4 (C-4aʹ, C-8aʹ, Ar); 155.5 (C=O); 159.6 (C=N).
3.1.3. Standard Procedure for Reduction of Aldimines 4a–f into N-Boc Monoprotected Diamines 5a–f
In a 50 mL two-necked round-bottomed flask, provided with a magnetic stirrer and reflux condenser,
compound 4 (5 mmol) was dissolved in methanol p.a. (30 mL) under vigorous stirring and cooled at
0 °C. To this solution was added by small portions commercial sodium borohydride NaBH4 (0.95 g,
25 mmol) over a period of 20 min. The resulting suspension was stirred at 50 °C for 24 h (monitored
by TLC on 0.2-mm precoated plates of silica gel 60 F-254, Merck). After cooling down to room
temperature, the volatile compounds were removed in a rotary evaporator under reduced pressure, then
deionized water (40 mL) was added in one portion to the crude residue. The mixture was transferred to a
separating funnel and was extracted with dichloromethane (3 × 50 mL). The combined organic phases
were dried over magnesium sulphate MgSO4, filtered on filter paper and the solvent was eliminated
in vacuo. The crude residue was dried under high vacuum (10−2 Torr) at 25 °C for 2 h. The desired
compound 5 was obtained as yellowish viscous oil and was further used without purification.
(4-Phenylmethylamino-butyl)-carbamic acid tert-butyl ester (5a). Compound 5a was prepared in 93%
yield (1.25 g) from [4-(benzylidene-amino)-butyl]-carbamic acid tert-butyl ester 4a (1.38 g, 5 mmol)
according to the standard procedure that gave 5a as yellowish viscous oil. 1H-NMR (DMSO-d6) δ: 1.37
(s, 9H, Me3C); 1.34–1.42 (m, 4H, CH2, H-2, H-3); 2.45 (t, 2H, J = 6.2 Hz, CH2NH, H-4); 2.89–2.91 (m,
2H, CH2NH, H-1); 3.66 (s, 2H, ArCH2NH); 6.78–6.82 (br s, 1H, NHCO); 7.18–7.13 (m, 1H, H-4ʹ, Ar);
13
7.26–7.33 (m, 4H, H-2ʹ, H-3ʹ, H-5ʹ, H-6ʹ, Ar). C-NMR (DMSO-d6) δ: 27.4 (C-3); 26.8 (C-2); 39.8
(C-1); 48.4 (C-4); 53.0 (ArCH2NH); 77.2 (OC(CH3)3); 126.3 (C-4ʹ, Ar); 127.8 (C-2ʹ, C-6ʹ, Ar); 128.0
(C-3ʹ, C-5ʹ, Ar); 141,0 (C-1ʹ, Ar); 155,5 (C=O).
[4-(4-Methoxy-phenylmethylamino)-butyl]-carbamic acid tert-butyl ester (5b). Compound 5b was
prepared in 99% yield (1.52 g) from {4-[(4-methoxy-benzylidene)-amino]-butyl}-carbamic acid

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tert-butyl ester 4b (1.53 g, 5 mmol) according to the standard procedure that gave 5b as yellowish
1
viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.33–1.41 (m, 4H, H-2, H-3); 2.45 (t, 2H,
J = 6.5 Hz, CH2NH, H-4); 2.88–2.90 (m, 2H, CH2NHCO, H-1); 3.60 (s, 2H, ArCH2NH); 3.72 (s, 3H,
OCH3); 6.76–6.80 (br s, 1H, NHCO); 6.85 (d, 2H, J = 8.5 Hz, H-2ʹ, H-6ʹ, Ar); 7.22 (d, 2H, J = 8.6 Hz,
13
H-3ʹ, H-5ʹ, Ar). C-NMR (DMSO-d6) δ: 26.6 (C-2); 27.4 (C-3); 28.2 (OC(CH3)3); 39.8 (C-4); 48.2
(C-1); 52.3 (ArCH2NH); 54.9 (OCH3); 77.2 (OC(CH3)3); 113.4 (C-2ʹ, C-6ʹ, Ar); 129.0 (C-3ʹ, C-5ʹ, Ar);
132.6 (C-1ʹ, Ar); 155.5 (C=O); 158.0 (C-4ʹ, Ar).
[4-(2-Chloro-phenylmethylamino)-butyl]-carbamic acid tert-butyl ester (5c). Compound 5c was
prepared in 85% yield (1.33 g) from {4-[(2-chloro-benzylidene)-amino]-butyl}-carbamic acid tert-butyl
ester 4c (1.55 g, 5 mmol) according to the standard procedure that gave 5c as yellowish viscous oil.
1H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.34–1.43 (m, 4H, CH2, H-2, H-3, 4H); 2.47–2.52 (m, 2H,
ArCH2NH, H-4); 2.88–2.92 (m, 2H, CH2NHCO, H-1); 3.75 (s, 2H, ArCH2NH); 6.77–6.80 (br s, 1H,
NHCO); 7.21–7.33 (m, 2H, H-4ʹ, H-5ʹ, Ar); 7.37–7.4 (m, 1H, H-6ʹ, Ar); 7.5–7.53 (m, 1H, H-3ʹ, Ar).
13C-NMR (DMSO-d6) δ: 26.7 (C-2); 27.4 (C-3); 28.2 (OC(CH3)3); 39.8 (C-4); 48.5 (C-1); 50.0
(ArCH2NH); 77.2 (OC(CH3)3); 126.9 (C-5ʹ, Ar); 128.0 (C-4ʹ, Ar); 128.9 (C-6ʹ, Ar); 129.6 (C-3ʹ, Ar);
132.5 (C-1ʹ, Ar); 138.2 (C-2ʹ, Ar); 155.5 (C=O).
[4-(3-Methoxy-phenylmethylamino)-butyl]-carbamic acid tert-butyl ester (5d). Compound 5d was
prepared in 90% yield (1.39 g) from {4-[(3-methoxy-benzylidene)-amino]-butyl}-carbamic acid
tert-butyl ester 4d (1.53 g, 5 mmol) according to the standard procedure that gave 5d as pale pink
1
viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.34–1.46 (m, 4H, H-2, H-3); 2.45 (t, 2H,
J = 6.4 Hz, CH2NH, H-4); 2.89–2.91 (m, 2H, CH2NHCO, H-1); 3.64 (s, 2H, ArCH2NH); 3.74 (s, 3H,
OCH3); 6.75–6.80 (br s, 1H, NHCO); 6.75–6.91 (m, 3H, H-4ʹ, H-5ʹ, H-6ʹ, Ar); 7.17–7.22 (m, 1H,
13
H-2ʹ, Ar). C-NMR (DMSO-d6) δ: 26.8 (C-2); 27.4 (C-3); 28,2 (OC(CH3)3); 39.8 (C-4); 48.4 (C-1);
52.9 (ArCH2NH); 54.8 (OCH3); 77.2 (OC(CH3)3); 111.8 (C-5ʹ, Ar); 113.2 (C-6ʹ, Ar); 120.0 (C-4ʹ, Ar);
128.9 (C-2ʹ, Ar); 142.8 (C-1ʹ, Ar); 155.5 (C=O); 159.2 (C-3ʹ, Ar).
{4-[(Benzo[1,3]dioxol-5-ylmethyl)-amino]-butyl}-carbamic acid tert-butyl ester (5e). Compound 5e was
prepared in 97% yield (1.56 g) from {4-[(benzo[1,3]dioxol-5-ylmethylene)-amino]-butyl}-carbamic acid
tert-butyl ester 4e (1.60 g, 5 mmol) according to the standard procedure that gave 5e as yellowish
1
viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.23–1.40 (m, 4H, H-2, H-3); 2.43 (t, 2H,
J = 6.5 Hz, CH2NH, H-4); 2.89–2.91 (m, 2H, CH2NHCO, H-1); 3.64 (s, 2H, ArCH2NH); 5.96 (s, 2H,
H-2ʹ, Ar); 6.73–6.82 (br s, 1H, NHCO); 6.73–6.90 (m, 3H, H-4ʹ, H-6ʹ, H-7ʹ, Ar). 13C-NMR (DMSO-d6)
δ: 26.8 (C-2); 27.4 (C-3); 28.2 (OC(CH3)3); 39.8 (C-4); 48.1 (C-1); 52.7 (ArCH2NH); 77.2 (OC(CH3)3);
100.6 (C-2ʹ, Ar); 107.7 (C-6ʹ, Ar); 108.2 (C-7ʹ, Ar); 120.8 (C-4ʹ, Ar); 135.0 (C-5ʹ, Ar); 145.7 (C-3aʹ,
C-7aʹ, Ar); 147.1 (C-3aʹ, C-7aʹ, Ar); 155.5 (C=O).
{4-[(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethyl)-amino]-butyl}-carbamic acid tert-butyl ester (5f).
Compound 5f was prepared in 88% yield (1.48 g) from {4-[(2,3-dihydro-benzo[1,4]dioxin-6-ylmethylene)-
amino]-butyl}-carbamic acid tert-butylester 4f (1.67 g, 5 mmol) according to the standard procedure
1
that gave 5f as yellowish viscous oil. H-NMR (DMSO-d6) δ: 1.37 (s, 9H, Me3C); 1.33–1.43 (m, 4H,
H-2, H-3); 2.42 (t, 2H, J = 6.4 Hz, CH2NH, H-4); 2.88–2.90 (m, 2H, CH2NHCO, H-1); 3.53 (s, 2H,

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ArCH2NH); 4.20 (s, 4H, H-2ʹ, H-3ʹ, Ar); 6.75–6.80 (br s, 1H, NHCO); 6.75–6.80 (m, 3H, H-5ʹ, H-7ʹ,
H-8ʹ, Ar). 13C-NMR (DMSO-d6) δ: 26.8 (C-2); 27.4 (C-3); 28.2 (OC(CH3)3); 39.8 (C-4); 48.2 (C-1); 52.3
(ArCH2NH); 63.9 (C-2ʹ, C-3ʹ, Ar); 64.0 (C-2ʹ, C-3ʹ, Ar); 77.2 (OC(CH3)3); 116.4 (C-7ʹ, Ar); 116.5 (C-8ʹ,
Ar); 120.6 (C-5ʹ, Ar); 134.0 (C-6ʹ, Ar); 141.9 (C-4aʹ, C-8aʹ, Ar); 143.0 (C-4aʹ, C-8aʹ, Ar); 155.5 (C=O).
3.1.4. Standard Procedure for the Preparation of Hydrochloride Salts 6a–f after Deprotection of N-Boc
Monoprotected Diamines 5a–f
In a 100 mL two-necked round-bottomed flask provided with a magnetic stirrer and condenser,
N-Boc monoprotected diamine 5 (12.1 mmol, 1 equiv.) was solubilized in 1,4-dioxane (80 mL) at room
temperature under vigorous stirring for 10 min. To this homogeneous solution was added dropwise for
1 h a solution of 6 M HCl (20 mL). The reaction mixture was stirred during 4 h at 25 °C and was
concentrated in a rotary evaporator under reduced pressure for elimination of volatile compounds.
To the crystallized crude reaction mixture was added 60 mL of anhydrous Et2O and after triturating,
the insoluble salt 6 was collected by filtration, on a Büchner funnel (porosity N°4) then washed with
6 × 10 mL of Et2O. The desired salt 6 was dried under high vacuum (10−2 Torr) at 25 °C for 1 h that
gave a yellowish or white powder and was further used without purification.
N-1-Phenylmethyl-butane-1,4-diamine hydrochloride (6a). Compound 6a was prepared in 84%
yield (2.18 g) from (4-phenylmethylamino-butyl)-carbamic acid tert-butyl ester 5a (3.37 g, 12.1 mmol)
1
according to the standard procedure as yellowish powder. Mp > 260 °C. H-NMR (DMSO-d6) δ:
1.57–1.67 (m, 2H, CH2, H-3); 1.70–1.81 (m, 2H, CH2, H-2); 2.79 (t, 2H, J = 7.3 Hz, CH2NH, H-4);
2.89 (t, 2H, J = 7.5 Hz, CH2NH2, H-1); 4.10 (s, 2H, CH2NH, H-4); 7.4–7.45 (m, 3H, H-3ʹ, H-4ʹ, H-5ʹ,
Ar); 7.58–7.62 (m, 2H, H-2ʹ, H-6ʹ, Ar); 8.19 (br s, 2H, NH2); 9.57 (br s, 1H, NH). 13C-NMR (DMSO-d6)
δ: 22.3 (C-3); 24.0 (C-2); 38.0 (C-4); 45.5 (C-1); 49.7 (ArCH2NH); 128.5 (C-3ʹ, C-5ʹ, Ar); 128.8 (C-4ʹ,
Ar); 130.1 (C-2ʹ, C-6ʹ, Ar); 132.0 (C-1ʹ, Ar).
N-1-(4-Methoxy-phenylmethyl)-butane1,4-diamine hydrochloride (6b). Compound 6b was prepared in
97% yield (2.87 g) from (4-methoxyphenylmethylamino-butyl)-carbamic acid tert-butyl ester 5b (3.73 g,
1
12.1 mmol) according to the standard procedure as pale brownish powder. Mp > 260 °C. H-NMR
(DMSO-d6) δ: 1.57–1.66 (m, 2H, H-3); 1.69–1.79 (m, 2H, H-2); 2.77–2.84 (m, 4H, H-1, H-4); 3.76 (s,
3H, OCH3); 4.03 (s, 2H, ArCH2NH); 6.97 (d, 2H, J = 8.7 Hz, H-2ʹ, H-6ʹ, Ar); 7.51 (d, 2H, J = 8.7 Hz,
13
H-3ʹ, H-5ʹ, Ar), 8.15 (br s, 2H, NH2); 9.41 (br s, 1H, NH). C-NMR (DMSO-d6) δ: 22.3 (C-3); 24.0
(C-2); 38.0 (C-4); 45.3 (C-1); 49.2 (ArCH2NH); 55.2 (OCH3); 113.9 (C-2ʹ, C-6ʹ, Ar); 123.7 (C-1ʹ, Ar);
131.6 (C-3ʹ, C-5ʹ, Ar); 159.6 (C-4ʹ, Ar).
N-1-(2-Chloro-phenylmethyl)-butane1,4-diamine hydrochloride (6c). Compound 6c was prepared in
98% yield (2.95 g) from (2-chlorophenylmethylamino-butyl)-carbamic acid tert-butyl ester 5c (3.79 g,
12.1 mmol) according to the standard procedure as pale brownish powder. Mp = 206–208 °C.
1H-NMR (DMSO-d6) δ: 1.60–1.69 (m, 2H, H-3); 1.74–1.84 (m, 2H, H-2); 2.73–2.84 (m, 2H, CH2NH,
H-4); 2.93–3.02 (m, 2H, CH2NH2, H-1); 4.23 (t, 2H, J = 5.9 Hz, ArCH2NH); 7.39–7.48 (m, 2H, H-4ʹ,
H-5ʹ, Ar); 7.52–7.56 (m, 1H, H-6ʹ, Ar); 7.81–7.85 (m, 1H, H-3ʹ, Ar); 8.18 (br s, 2H, NH2, 2H); 9.68

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(br s, 1H, NH). 13C-NMR (DMSO-d6) δ: 22.3 (C-3); 24.0 (C-2); 38.0 (C-4); 46.1 (C-1); 46.7 (ArCH2NH);
127.5 (C-5ʹ, Ar); 129.5 (C-4ʹ, Ar); 129.9 (C-1ʹ, Ar); 130.7 (C-6ʹ, Ar); 131.9 (C-3ʹ, Ar); 133.5 (C-2ʹ, Ar).
N-1-(3-Methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride (6d). Compound 6d was prepared in
98% yield (2.98 g) from [4-(3-methoxy-phenylmethylamino)-butyl]-carbamic acid tert-butyl ester 5d
(3.73 g, 12.1 mmol) according to the standard procedure as pale brownish powder. Mp = 190–192 °C.
1H-NMR (DMSO-d6) δ: 1.58–1.67 (m, 2H, H-3); 1.71–1.81 (m, 2H, H-2); 2.78 (t, 2H, J = 7.3 Hz,
CH2NH, H-4); 2.87 (t, 2H, J = 7.6 Hz, CH2NH2, H-1); 3.77 (s, 3H, OCH3); 4.08 (t, 2H, J = 5.6 Hz,
ArCH2NH); 6.94–6.97 (m, 1H, H-5ʹ, Ar); 7.12–7.14 (m, 1H, H-6ʹ, Ar); 7.29–7.35 (m, 2H, H-2ʹ, H-4ʹ,
13
Ar); 8.18 (br s, 2H, NH2); 9.58 (br s, 1H, NH). C-NMR (DMSO-d6) δ: 22.3 (C-3); 24.0 (C-2); 38.0
(C-4); 45.5 (C-1); 49.7 (ArCH2NH); 55.2 (OCH3); 114.5 (C-5ʹ, Ar); 115.5 (C-6ʹ, Ar); 122.0 (C-4ʹ, Ar);
129.6 (C-2ʹ, Ar); 133.4 (C-1ʹ, Ar); 159.2 (C-3ʹ, Ar).
N-1-Benzo[1,3]dioxol-5-ylmethyl-butane-1,4-diamine hydrochloride (6e). Compound 6e was prepared
in 72% yield (2.25 g) from {4-[(benzo[1,3]dioxol-5-ylmethyl)-amino]-butyl}-carbamic acid tert-butyl
ester 5e (3.90 g, 12.1 mmol) according to the standard procedure as pale brownish powder. Mp > 260 °C.
1H-NMR (DMSO-d6) δ: 1.58–1.67 (m, 2H, H-3); 1.71–1.81 (m, 2H, H-2); 2.78 (t, 2H, J = 7.3 Hz,
CH2NH, H-4); 2.87 (t, 2H, J = 7.6 Hz, CH2NH2, H-1); 3.77 (s, 3H, OCH3); 4.08 (t, 2H, J = 5.6 Hz,
ArCH2NH); 6.94–6.97 (m, 1H, H-5ʹ, Ar); 7.12–7.14 (m, 1H, H-6ʹ, Ar); 7.29–7.35 (m, 2H, H-2ʹ, H-4ʹ,
13
Ar); 8.18 (br s, 2H, NH2); 9.58 (br s, 1H, NH). C-NMR (DMSO-d6) δ: 22.3 (C-3); 24.0 (C-2); 38.0
(C-4); 45.2 (C-1); 49.4 (ArCH2NH); 101.2 (C-2ʹ, Ar); 108.2 (C-6ʹ, Ar); 110.4 (C-7ʹ, Ar); 124.1 (C-4ʹ,
Ar); 125.4 (C-5ʹ, Ar); 147.2 (C-3aʹ, C-7aʹ, Ar); 147.6 (C-3aʹ, C-7aʹ, Ar).
N-1-(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethyl)-butane-1,4-diamine hydrochloride (6f). Compound 6e
was prepared in 92% yield (3.04 g) from {4-[(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)-amino]-
butyl}-carbamic acid tert-butyl ester 5f (4.07 g, 12.1 mmol) according to the standard procedure as
pale brownish powder. Mp = 239–241 °C. 1H-NMR (DMSO-d6) δ: 1.57–1.66 (m, 2H, H-3); 1.68–1.78
(m, 2H, H-2); 2.73–2.82 (m, 4H, H-1, H-4); 3.97 (t, 2H, J = 5.4 Hz, ArCH2NH); 4.24 (s, 4H,
OCH2CH2O); 6.87 (d, 1H, J = 8.2 Hz, H-7ʹ, Ar); 7.01–7.04 (m, 1H, H-8ʹ, Ar); 7.15 (m, 1H, H-5ʹ, Ar);
8.18 (br s, 2H, NH2); 9.43 (br s, 1H, NH). 13C-NMR (DMSO-d6) δ: 22.3 (C-3); 24.0 (C-2); 38.0 (C-4);
45.2 (C-1); 49.1 (ArCH2NH); 64.0 (C-2ʹ, Ar); 64.1 (C-3ʹ, Ar); 117.0 (C-7ʹ, Ar); 119.0 (C-8ʹ, Ar); 123.2
(C-5ʹ, Ar); 124.7 (C-6ʹ, Ar); 143.1 (C-4aʹ, C-8aʹ, Ar); 143.8 (C-4aʹ, C-8aʹ, Ar).
3.1.5. Standard Procedure for the Preparation of N-Substituted 5-Arylidene Rhodanine Derivatives
9a–f under Microwave Irradiation Using “One-Pot Two-steps” Protocol
In a 10 mL glass tube were placed successively bis-(carboxymethyl)trithiocarbonate 7 (0.11 g,
0.50 mol, 1 equiv.), dimethoxyethane (2 mL), triethylamine (135 mL, 101 mg, 1 mmol, 2 equiv. or
202 mL, 152 mg, 1.5 mmol, 3 equiv.) and hydrochloride salt 6 (0.5 mmol, 1 equiv.). The glass tube was
sealed with a snap cap and placed in the Monowave® 300 Anton Paar microwave cavity (p = 800 Watt).
The reaction mixture was irradiated at 90 °C for 15–60 min. under vigorous magnetic stirring. After
microwave dielectric heating, the crude reaction mixture was allowed to cool down at room temperature,
aldehyde 3 (0.5 mol, 1 equiv.) was added to the cooled reaction mixture which was immediately

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submitted to microwave irradiation at 110 °C for 5–30 min. After cooling down to room temperature,
the volatile compounds of the reaction mixture were removed in a rotary evaporator under reduced
pressure. To the crude reaction mixture was added 4 mL of MeOH and after triturating, the insoluble
product 9 was collected by filtration on a Büchner funnel (porosity N°4), washed with deionized water
(5 mL), triturated and mixing with ethanol (3 × 5 mL) during 3 h, washed successively with ethanol
(5 mL) and anhydrous ether (5 × 2 mL), then dried under high vacuum (10−2 Torr) at 25 °C for 1 h. The
desired compound 9 was eventually purified by recrystallization in EtOH after control by 1H-NMR in
solution of CDCl3/TFA (98:2).
(5Z)3-[4-(4-Methoxyphenylmethylamino)butyl]-5-(1,3-benzodioxol-5-ylmethylene)-2-thioxo-1,3-
thiazolidin-4-one (9a). According to the standard procedure, compound 9a was prepared in 10% yield
(23 mg) from N-1-(4-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6b (122.3 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and piperonaldehyde 3e
(75.1 mg, 0.5 mmol, 1 equiv.) after 30 min. at 90 °C, followed by a second reaction time of 30 min. at
110 °C (for condensation step with 3e), which gave 9a as a yellowish powder. Mp = 170–229 °C
1
(decomposition). H-NMR (CDCl3/TFA 98:2) δ: 1.61–1.69 (m, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.05–3.11 (m,
2H, ArCH2NH); 3.73 (s, 3H, OCH3); 4.00–4.08 (m, 4H, CH2, H-1ʹʹ, H-4ʹʹ), 5.97 (s, 2H, CH2, H-2ʹ);
6.80–7.16 (m, 7H, H-2ʹʹʹ, H-3ʹʹʹ, H-5ʹʹʹ, H-6ʹʹʹ, H-4ʹ, H-6ʹ, H-7ʹ, Ar); 7.55 (br s, 1H, NH); 7.61 (s, 1H,
HC=C). 13C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ, 3ʹʹ); 23.8 (C-3ʹʹ, 2ʹʹ); 44.2 (C-4ʹʹ, C-1ʹʹ); 47.0
(ArCH2NH); 52.1 (C-1ʹʹ, C-4ʹʹ); 55.5 (OCH3); 102.2 (C-2ʹ); 109.4 (C-2ʹʹʹ, C-6ʹʹʹ); 109.5 (C-3ʹʹʹ, C-5ʹʹʹ);
115.0 (C-6ʹ); 120.0 (C-1ʹʹʹ); 127.5 (C=CH); 128.2 (C-7ʹ); 131.2 (C-4ʹ); 135.0 (C=CH); 148.8 (C-5ʹ);
148.9 (C-4ʹʹʹ); 150.6 (C-3ʹa); 150.9 (C-7ʹa); 169.4 (C=O, C-4); 192.9 (C=S, C-2). HRMS, m/z:
457.1256 found (calculated for C23H25N2O4S2 [M + H]+ requires 457.1253).
(5Z)3-[4-(4-Methoxyphenylmethylamino)butyl]-5-(4-methoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-
one (9b). According to the standard procedure, compound 9b was prepared in 6% yield (13.3 mg) from
N-1-(4-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6b (122.3 mg, 0.5 mmol, 1 equiv.),
triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 4-methoxybenzaldehyde 3b (68.1 mg, 0.5 mmol,
1 equiv.) after 30 min. at 90 °C, followed by a second reaction time of 30 min. at 110 °C (for
1
condensation step with 3b), which gave 9b as a yellowish powder. Mp = 253–255 °C. H-NMR
(CDCl3/TFA 98:2) δ: 1.67–1.73 (m, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.07–3.13 (m, 2H, ArCH2NH); 3.81 (s, 6H,
ArOCH3); 4.04–4.11 (s, 4H, CH2, H-1ʹʹ, H-4ʹʹ), 6.94 (dd, 4H, J = 8.8 Hz, H-2ʹ, H-6ʹ, H-2ʹʹʹ, H-6ʹʹʹ, Ar);
7.40 (d, 4H, J = 8.8 Hz, H-3ʹ, H-5ʹ, H-3ʹʹʹ, H-5ʹʹʹ, Ar); 7.60 (br s, 1H, NH); 7.67 (s, 1H, CH=).
13C-NMR (CDCl3/TFA 98:2) δ: 24.3 (C-2ʹʹ, C-3ʹʹ); 44.0 (C-1ʹʹ, C-4ʹʹ); 46.8 (ArCH2NH); 55.6 (C-4ʹ,
OCH3, C-4ʹʹʹ, OCH3); 115.1 (C-2ʹ, C-6ʹ, C-2ʹʹʹ, C-6ʹʹʹ); 119.6 (C-1ʹ, C-1ʹʹʹ); 126.0 (C=); 133.1 (C-3ʹ,
C-5ʹ, C-3ʹʹʹ, C-5ʹʹʹ); 130.5 (C-5ʹ, C-5ʹʹʹ); 134.6 (CH=); 162.0 (C-4ʹ, C-4ʹʹʹ); 169.0 (C=O, C-4); 193.2
(C=S, C-2). HRMS, m/z: 443.1463 found (calculated for C23H27N2O3S2 [M + H]+ requires 443.1460).
(5Z)3-[4-(4-Methoxyphenylmethylamino)butyl]-5-(2,3-dihydro-1,4-benzodioxin-6-ylmethylene)-2-
thioxo-1,3-thiazolidin-4-one (9c). According to the standard procedure, compound 9c was prepared in
5% yield (11.8 mg) from N-1-(4-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6b (122.3 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 2,3-dihydro-1,4-benzodioxin-

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6-carboxaldehyde 3f (82.1 mg, 0.5 mmol, 1 equiv.) after 30 min. at 90 °C, followed by a second reaction
time of 30 min. at 110 °C (for condensation step with 3f), which gave 9c as a yellowish powder.
1
Mp = 255–261 °C. H-NMR (CDCl3/TFA 98:2) δ: 1.68 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.03–3.08 (m, 2H,
ArCH2NH); 3.73 (s, 3H, OCH3); 4.07 (s, 4H, CH2, H-1ʹʹ, H-4ʹʹ), 4.20–4.25 (m, 2H, CH2, H-2ʹ, H-3ʹ);
6.84–6.97 (m, 7H, H-5ʹ, H-7ʹ, H-8ʹ, H-2ʹʹʹ, H-3ʹʹʹ, H-5ʹʹʹ, H-6ʹʹʹ, Ar); 7.58 (s, 1H, CH=); 7.60 (br s, 1H,
NH). 13C-NMR (CDCl3/TFA 98:2) δ: 24.2 (C-2ʹʹ, C-3ʹʹ); 44.1 (C-4ʹʹ, C-1ʹʹ); 47.1 (ArCH2NH); 55.0
(OCH3); 64.2 (C-2ʹ); 64.8 (C-3ʹ); 118.4 (C-5ʹ, C-7ʹ, C-8ʹ); 119.7 (C-2ʹʹʹ, C-6ʹʹʹ); 120.3 (C=); 125.7
(C-3ʹʹʹ, C-5ʹʹʹ); 126.8 (C-6ʹ); 135.0 (CH=); 143.9 (C-8ʹa); 146.7 (C-4ʹa); 169.5 (C=O, C-4); 193.2 (C=S,
C-2). HRMS, m/z: 471.1412 found (calculated for C24H27N2O4S2 [M + H]+ requires 471.1415).
(5Z)3-[4-(4-Methoxyphenylmethylamino)butyl]-5-(4-hydroxy-3-methoxybenzylidene)-2-thioxo-1,3-
thiazolidin-4-one (9d). According to the standard procedure, compound 9d was prepared in 30% yield
(68.8 mg) from N-1-(4-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6b (122.3 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 4-hydroxy-3-
methoxybenzaldehyde 3g (76.1 mg, 0.5 mmol, 1 equiv.) after 30 min. at 90 °C, followed by a second
reaction time of 30 min. at 110 °C (for condensation step with 3g), which gave 9d as a yellowish
powder. Mp = 191–193 °C. 1H-NMR (CDCl3/TFA 98:2) δ: 1.69 (m, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.06–3.07
(m, 2H, ArCH2NH); 3.72 (s, 3H, C-4ʹʹʹ, OCH3); 3.86 (s, 3H, C-3ʹ, OCH3); 4.04–4.08 (m, 4H, CH2,
H-1ʹʹ, H-4ʹʹ), 6.81–7.17 (m, 7H, H-2ʹ, H-5ʹ, H-6ʹ, H-2ʹʹʹ, H-3ʹʹʹ, H-5ʹʹʹ, H-6ʹʹʹ, Ar); 7.55 (br s, 1H, NH);
7.60 (s, 1H, CH=C). 13C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ, C-3ʹʹ); 23.9 (C-2ʹʹ, C-3ʹʹ); 43.1 (C-1ʹʹ,
C-4ʹʹ); 47.0 (ArCH2NH); 55.5 (ArOCH3); 56.1 (ArOCH3); 112.6 (C-6ʹ); 115.0 (C-2ʹʹʹ, C-6ʹʹʹ); 115.6
(C-2ʹ); 118.9 (C-1ʹʹʹ); 121.2 (C=); 125.9 (C-1ʹ); 126.8 (C-5ʹ); 131.2 (C-3ʹʹʹ, C-5ʹʹʹ); 135.9 (CH=); 147.2
(C-4ʹʹʹ); 148.9 (C-3ʹ); 160.8 (C-4ʹ); 169.4 (C=O, C-4); 193.1 (C=S, C-2). HRMS, m/z: 459.1412 found
(calculated for C23H27N2O4S2 [M + H]+ requires 459.1416).
(5Z)3-[4-(3-Methoxyphenylmethylamino)butyl]-5-(1,3-benzodioxol-5-ylmethylene)-2-thioxo-1,3-
thiazolidin-4-one (9e). According to the standard procedure, compound 9e was prepared in 21% yield
(48.0 mg) from N-1-(3-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6d (122.3 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and piperonaldehyde 3e (75.1 mg,
0.5 mmol, 1 equiv.) after 15 min. at 90 °C, followed by a second reaction time of 15 min. at 110 °C (for
condensation step with 3e), which gave 9e as a yellowish powder. Mp = 178–246 °C (decomposition).
1H-NMR (CDCl3/TFA 98:2) δ: 1.82 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.19–3.22 (m, 2H, ArCH2NH); 3.83 (s,
3H, OCH3); 4.13-4.23 (m, 4H, CH2, H-1ʹʹ, H-4ʹʹ), 6.10 (s, 2H, CH2, H-2ʹ); 6.92–7.28 (m, 6H, H-2ʹʹʹ,
H-4ʹʹʹ, H-6ʹʹʹ, H-4ʹ, H-6ʹ, H-7ʹ, Ar), 7.33–7.38 (m, 1H, H-5ʹʹʹ, Ar), 7.55 (br s, 1H, NH); 7.70 (s, 1H,
13
CH=). C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ, C-3ʹʹ); 23.9 (C-3ʹʹ, C-2ʹʹ); 42.9 (C-4ʹʹ, C-1ʹʹ); 46.8
(ArCH2NH); 52.1 (C-1ʹʹ, C-4ʹʹ); 55.3 (OCH3); 102.1 (C-2ʹ); 109.3 (C-6ʹʹʹ); 109.4 (C-5ʹʹʹ); 114.9 (C-4ʹʹʹ);
115.9 (C-2ʹʹʹ); 119.8 (C-1ʹʹʹ); 121.8 (C-6ʹ); 127.4 (C=); 127.6 (C-5ʹ); 127.9 (C-7ʹ); 130.7 (C-4ʹ); 134.5
(CH=); 148.8 (C-7ʹa); 150.5 (C-3ʹa); 160.3 (C-3ʹʹʹ); 168.5 (C=O, C-4); 193.1 (C=S, C-2). HRMS, m/z:
457.1259 found (calculated for C23H25N2O4S2 [M + H]+ requires 457.1257).
(5Z)3-[4-(3-Methoxyphenylmethylamino)butyl]-5-(2,3-dihydro-1,4-benzodioxin-6-ylmethylene)-2-
thioxo-1,3-thiazolidin-4-one (9f). According to the standard procedure, compound 9c was prepared in 9%

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yield (21.2 mg) from N-1-(3-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6d (122.3 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 2,3-dihydro-1,4-
benzodioxin-6-carboxaldehyde 3f (82.1 mg, 0.5 mmol, 1 equiv.) after 15 min. at 90 °C, followed by a
second reaction time of 15 min. at 110 °C (for condensation step with 3f), which gave 9f as
a yellowish powder. Mp = 247–251 °C. 1H-NMR (CDCl3/TFA 98:2) δ: 1.84 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ);
3.04–3.12 (m, 2H, ArCH2NH); 3.95 (s, 3H, OCH3); 4.23 (s, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 4.37–4.40 (m, 4H,
H-2ʹ, H-3ʹ); 6.99–7.28 (m, 6H, H-2ʹʹʹ, H-4ʹʹʹ, H-5ʹʹʹ, H-6ʹʹʹ, H-5ʹ, H-7ʹ, H-8ʹ, Ar); 7.60 (br s, 1H, NH);
7.74 (s, 1H, CH=). 13C-NMR (CDCl3/TFA 98:2) δ: 24.1 (C-2ʹʹ, C-3ʹʹ); 44.1 (C-1ʹʹ, C-4ʹʹ); 47.3
(ArCH2NH); 64.2 (C-2ʹ); 64.8 (C-3ʹ); 118.5 (C-5ʹ, C-7ʹ, C-8ʹ); 119.8 (C-4ʹʹʹ, C-6ʹʹʹ); 125.9 (C-2ʹʹʹ,
C-5ʹʹʹ); 126.8 (C=); 135.3 (CH=); 143.8 (C-8ʹa); 146.6 (C-4ʹa); 168.5 (C=O, C-4); 193.3 (C=S, C-2).
HRMS, m/z: 471.1412 found (calculated for C24H27N2O4S2 [M + H]+ requires 471.1415).
(5Z)3-[4-(3-Methoxyphenylmethylamino)butyl]-5-(3-methoxybenzylidene)-2-thioxo-1,3-thiazolidin-4-
one (9g). According to the standard procedure, compound 9g was prepared in 5% yield (11.1 mg) from
N-1-(3-methoxy-phenylmethyl)-butane-1,4-diamine hydrochloride 6d (122.3 mg, 0.5 mmol, 1 equiv.),
triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 3-methoxybenzaldehyde 3d (68.1 mg, 0.5 mmol,
1 equiv.) after 30 min. at 90 °C, followed by a second reaction time of 30 min at 110 °C (for condensation
1
step with 3d), which gave 9g as a yellowish powder. Mp = 225–227 °C. H-NMR (CDCl3/TFA 98:2)
δ: 1.73 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.06–3.10 (m, 2H, ArCH2NH); 3.80 (s, 6H, ArOCH3); 4.11 (s, 4H, CH2,
H-1ʹʹ, H-4ʹʹ), 6.93–6.96 (m, 4H, H-2ʹ, H-6ʹ, H-2ʹʹʹ, H-6ʹʹʹ, Ar); 7.04 (d, 2H, J = 7.7 Hz, H-4ʹ, H-4ʹʹʹ, Ar),
7.32 (m, 2H, H-5ʹ, H-5ʹʹʹ, Ar), 7.55 (br s, 1H, NH); 7.66 (s, 1H, CH=). 13C-NMR (CDCl3/TFA 98:2) δ:
24.3 (C-2ʹʹ, C-3ʹʹ); 44.0 (C-1ʹʹ, C-4ʹʹ); 47.1 (ArCH2NH); 55.5 (ArOCH3); 115.3 (C-6ʹ, C-6ʹʹʹ); 117.3
(C-2ʹ, C-2ʹʹʹ); 123.1 (C=); 123.6 (C-4ʹ, C-4ʹʹʹ); 130.5 (C-5ʹ, C-5ʹʹʹ); 134.0 (CH=); 134.5 (C-1ʹ, C-1ʹʹʹ);
160.0 (C-3ʹ, C-3ʹʹʹ); 168.5 (C=O, C-4); 193.3 (C=S, C-2). HRMS, m/z: 443.1463 found (calculated for
C23H27N2O3S2 [M + H]+ requires 443.1465).
(5Z)3-[4-(1,3-Benzodioxol-5-ylmethylamino)butyl]-5-(1,3-benzodioxol-5-ylmethylene)-2-thioxo-1,3-
thiazolidin-4-one (9h). According to the standard procedure, compound 9h was prepared in 59% yield
(139 mg) from N-1-benzo[1,3]dioxol-5-ylmethyl-butane-1,4-diamine hydrochloride 6e (129.4 mg,
0.5 mmol, 1 equiv.), triethylamine (135 μL, 101 mg., 1 mmol, 2 equiv.) and piperonaldehyde 3e (75.1 mg,
0.5 mmol, 1 equiv.) after 15 min. at 90 °C, followed by a second reaction time of 15 min. at 110 °C
1
(for condensation step with 3e), which gave 9h as a yellowish powder. Mp = 245–256 °C. H-NMR
(CDCl3/TFA 98:2) δ: 1.74 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.04–3.05 (m, 2H, ArCH2NH); 4.05–4.07 (m, 4H,
CH2, H-1ʹʹ, H-4ʹʹ); 5.91 (s, 2H, CH2, H-2ʹʹʹ); 6.00 (s, 2H, CH2, H-2ʹ); 6.72–7.01 (m, 6H, H-4ʹ, H-6ʹ,
13
H-7ʹ, H-4ʹʹʹ, H-6ʹʹʹ, H-7ʹʹʹ, Ar); 7.58 (s, 1H, CH=); 7.65 (br s, 1H, NH);. C-NMR (CDCl3/TFA 98:2)
δ: 23.1 (C-2ʹʹ, C-3ʹʹ); 24.0 (C-3ʹʹ); 43.1 (C-1ʹʹ, C-4ʹʹ); 46.5 (ArCH2NH); 51.9 (C-4ʹʹ, C-1ʹʹ); 101.8 (C-2ʹ,
C-2ʹʹʹ); 102.2 (C-2ʹʹʹ, C-2ʹ); 109.0 (C-6ʹʹʹ); 109.3 (C-6ʹ); 109.4 (C-7ʹʹʹ); 109.8 (C-7ʹ); 119.8 (C-5ʹʹʹ);
122.6 (C=); 124.3 (C-4ʹʹʹ); 127.4 (C-4ʹ); 128.1 (C-5ʹ); 134.9 (CH=); 148.6 (C-7ʹa); 148.9 (C-3ʹa); 149.2
(C-7ʹʹʹa); 150.6 (C-3ʹʹʹa); 168.9 (C=O, C-4); 193.0 (C=S, C-2). HRMS, m/z: 471.1048 found
(calculated for C23H23N2O5S2 [M + H]+ requires 471.1049).

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(5Z)3-[4-(1,3-Benzodioxol-5-ylmethylamino)butyl]-5-(2,3-dihydro-1,4-benzodioxin-6-ylmethylene)-2-
thioxo-1,3-thiazolidin-4-one (9i). According to the standard procedure, compound 9i was prepared in
59% yield (143 mg) from N-1-benzo[1,3]dioxol-5-ylmethyl-butane-1,4-diamine hydrochloride 6e
(129.4 mg, 0.5 mmol, 1 equiv.), triethylamine (135 μL, 101 mg., 1 mmol, 2 equiv.) and 2,3-dihydro-
1,4-benzodioxin-6-carboxaldehyde 3f (82.1 mg, 0.5 mmol, 1 equiv.) after 15 min. at 90 °C, followed
by a second reaction time of 15 min. at 110 °C (for condensation step with 3f), which gave 9i as
a yellowish powder. Mp = 253–257 °C. 1H-NMR (CDCl3/TFA 98:2) δ: 1.68 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ);
3.05–3.06 (m, 2H, ArCH2NH); 4.03–4.04 (m, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 4.20–4.25 (m, 4H, CH2, H-2ʹ,
H-3ʹ); 5.89 (s, 2H, CH2, H-2ʹʹʹ); 6.70 (s, 3H, H-4ʹʹʹ, H-6ʹʹʹ, H-7ʹʹʹ, Ar); 6.85–6.96 (m, 3H, H-5ʹ, H-7ʹ,
H-8ʹ, Ar); 7.50 (br s, 1H, NH); 7.55 (s, 1H, CH=). 13C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ, C-3ʹʹ);
23.8 (C-3ʹʹ, C-2ʹʹ); 43.1 (C-1ʹʹ, C-4ʹʹ); 47.0 (ArCH2NH); 52.4 (C4ʹʹ, C-1ʹʹ); 64.2 (C-2ʹ, C-3ʹ); 64.8 (C-3ʹ,
C-2ʹ); 101.8 (C-2ʹʹʹ); 109.1 (C-6ʹʹʹ); 109.5 (C-7ʹʹʹ); 118.5 (C-7ʹ); 119.7 (C-5ʹʹʹ); 119.8 (C-8ʹ); 122.3
(C=); 124.1 (C-4ʹʹʹ); 125.9 (C-5ʹ); 126.6 (C-6ʹ); 135.5 (CH=); 144.0 (C-7ʹʹʹa); 146.9 (C-3ʹʹʹa); 148.6
(C-8ʹa); 149.4 (C-4ʹa); 169.5 (C=O, C-4); 193.2 (C=S, C-2). HRMS, m/z: 485.1205 found (calculated
for C24H25N2O5S2 [M + H]+ requires 485.1203).
(5Z)3-[4-(1,3-Benzodioxol-5-ylmethylamino)butyl]-5-(4-hydroxy-benzylidene)-2-thioxo-1,3-thiazolidin-4-
one (9j). According to the standard procedure, compound 9j was prepared in 34% yield (75.2 mg) from
N-1-benzo[1,3]dioxol-5-ylmethyl-butane-1,4-diamine hydrochloride 6e (129.4 mg, 0.5 mmol, 1 equiv.),
triethylamine (135 μL, 101 mg, 1 mmol, 2 equiv.) and 4-hydroxybenzaldehyde 3h (61.1 mg, 0.5 mmol,
1 equiv.) after 15 min. at 90 °C, followed by a second reaction time of 15 min. at 110 °C (for condensation
1
step with 3h), which gave 9i as a orange powder. Mp = 93–124 °C (decomposition). H-NMR
(CDCl3/TFA 98:2) δ: 1.59–1.73 (m, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.01–3.13 (m, 2H, ArCH2NH); 4.00–4.05
(m, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 5.88 (s, 2H, CH2, H-2ʹʹʹ); 6.66–6.69 (m, H-4ʹʹʹ, H-6ʹʹʹ, H-7ʹʹʹ, Ar); 6.86 (dd,
2H, J = 8.7 Hz, H-2ʹ, H-6ʹ, Ar); 7.32 (dd, 2H, J = 8.7 Hz, H-3ʹ, H-5ʹ, Ar); 7.55 (br s, 1H, NH); 7.60
13
(s, 1H, CH=, 1H). C-NMR (CDCl3/TFA 98:2) δ: 22.5 (C-2ʹʹ, C-3ʹʹ); 23.2 (C-3ʹʹ, C-2ʹʹ); 42.5 (C-1ʹʹ,
C-4ʹʹ); 46.4 (ArCH2NH); 51.9 (C-4ʹʹ, C-1ʹʹ); 101.2 (C-2ʹʹʹ); 108.5 (C-6ʹʹʹ); 108.8 (C-7ʹʹʹ); 116.1 (C-2ʹ,
C-6ʹ); 118.6 (C-5ʹʹʹ); 121.7 (C=); 123.4 (C-4ʹʹʹ); 125.6 (C-1ʹ); 132.9 (C-3ʹ, C-5ʹ); 134.8 (CH=); 148.0
(C-7ʹʹʹa); 148.8 (C-3ʹʹʹa); 157.6 (C-4ʹ); 169.0 (C=O, C-4); 192.6 (C=S, C-2). HRMS, m/z: 443.1099
found (calculated for C22H23N2O4S2 [M + H]+ requires 443.1095).
(5Z) 3-[4-(1,3-Benzodioxol-5-ylmethylamino)butyl]-5-(4-hydroxy-3-methoxybenzylidene)-2-thioxo-1,3-
thiazolidin-4-one (9k). According to the standard procedure, compound 9k was prepared in 46% yield
(109 mg) from N-1-benzo[1,3]dioxol-5-ylmethyl-butane-1,4-diamine hydrochloride 6e (129.4 mg,
0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 4-hydroxy-3-
methoxybenzaldehyde 3g (76.1 mg, 0.5 mmol, 1 equiv.) after 30 min. at 90 °C, followed by a second
reaction time of 30 min. at 110 °C (for condensation step with 3g), which gave 9k as a red powder.
1
Mp = 200–203 °C. H-NMR (CDCl3/TFA 98:2) δ: 1.71 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.05–3.06 (m, 2H,
ArCH2NH); 3.88 (s, 3H, OCH3); 4.04 (s, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 5.91 (s, 2H, CH2, H-2ʹʹʹ); 6.74 (s, 3H,
H-4ʹʹʹ, H-6ʹʹʹ, H-7ʹʹʹ, Ar); 6.87–7.02 (m, 3H, H-2ʹ, H-5ʹ, H-6ʹ, Ar); 7.58 (s, 1H, CH=); 7.65 (br s, 1H,
NH). 13C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ, C-3ʹʹ); 23.9 (C-3ʹʹ, C-2ʹʹ); 42.9 (C-1ʹʹ, C-4ʹʹ);
46.6 (ArCH2NH); 52.1 (C-4ʹʹ, C-1ʹʹ); 56.1 (OCH3); 101.8 (C-2ʹʹʹ); 109.0 (C-6ʹʹʹ); 109.6 (C-7ʹʹʹ); 112.3

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(C-6ʹ); 115.5 (C-2ʹ); 119.1 (C-5ʹʹʹ); 122.7 (C=); 124.0 (C-5ʹ); 125.8 (C-1ʹ); 126.5 (C-4ʹʹʹ); 134.9 (CH=);
147.1 (C-3ʹ); 148.6 (C-7ʹʹʹa); 148.8 (C-4ʹ); 149.2 (C-3ʹʹʹa); 168.5 (C=O, C-4); 193.2 (C=S,
C-2). HRMS, m/z: 473.1205 found (calculated for C23H25N2O5S2 [M + H]+ requires 473.1206).
(5Z)3-[4-(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethylamino)butyl]-5-(1,3-benzodioxol-5-ylmethylene)-2-
thioxo-1,3-thiazolidin-4-one (9l). According to the standard procedure, compound 9l was prepared in
15% yield (36.3 mg) from N-1-(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)-butane-1,4-diamine
hydrochloride 6f (136.4 mg, 0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.)
and piperonaldehyde 3e (75.1 mg, 0.5 mmol, 1 equiv.) after 15 min. at 90 °C, followed by a second
reaction time of 15 min. at 110 °C (for condensation step with 3e), which gave 9l as a yellowish
1
powder. Mp = 172–233 °C (decomposition). H-NMR (CDCl3/TFA 98:2) δ: 1.66–1.69 (m, 4H, CH2,
H-2ʹʹ, H-3ʹʹ); 3.03–3.10 (m, 2H, ArCH2NH); 4.03–4.07 (m, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 4.16–4.19 (m, 4H,
CH2, H-2ʹʹʹ, H-3ʹʹʹ); 5.97 (s, 2H, CH2, H-2ʹ); 6.66–7.00 (m, 6H, H-4ʹ, H-6ʹ, H-7ʹ, H-5ʹʹʹ, H-7ʹʹʹ, H-8ʹʹʹ,
13
Ar); 7.55 (br s, 1H, NH); 7.59 (s, 1H, CH=). C-NMR (CDCl3/TFA 98:2) δ: 23.8 (C-2ʹʹ, C-3ʹʹ); 24.2
(C-3ʹʹ); 43.1 (C-1ʹʹ, C-4ʹʹ); 47.0 (ArCH2NH); 52.1 (C-4ʹʹ, C-1ʹʹ); 64.2 (C-2ʹʹʹ); 64.4 (C-3ʹʹʹ); 102.2
(C-2ʹ); 109.4 (C-7ʹʹʹ); 109.5 (C-8ʹʹʹ); 118.5 (C-5ʹʹʹ); 119.5 (C-6ʹʹʹ); 122.0 (C=); 122.9 (C-6ʹ); 127.3
(C-5ʹ); 128.2 (C-7ʹ); 135.0 (C-4ʹ); 135.5 (CH=); 143.9 (C-4ʹʹʹa); 145.1 (C-8ʹʹʹa); 150.6 (C-3ʹa); 150.8
(C-7ʹa); 168.5 (C=O, C-4); 193.0 (C=S, C-2). HRMS, m/z: 485.1205 found (calculated for
C24H25N2O5S2 [M + H]+ requires 485.1206).
(5Z)3-[4-(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethylamino)butyl]-5-(2,3-dihydro-benzo[1,4]dioxin-6-
ylmethylene)-2-thioxo-1,3-thiazolidin-4-one (9m). According to the standard procedure, compound 9m
was prepared in 7% yield (17.5 mg) from N-1-(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)-butane-1,4-
diamine hydrochloride 6f (136.4 mg, 0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3
equiv.) and 2,3-dihydro-1,4-benzodioxin-6-carboxaldehyde 3f (82.1 mg, 0.5 mmol, 1 equiv.) after 15
min. at 90 °C, followed by a second reaction time of 15 min. at 110 °C (for condensation step with 3f),
1
which gave 9m as a yellowish powder. Mp = 170–243 °C (decomposition). H-NMR (CDCl3/TFA
98:2) δ: 1.64–1.69 (m, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.04–3.10 (m, 2H, ArCH2NH); 4.03–4.08 (m, 4H, CH2,
H-1ʹʹ, H-4ʹʹ); 4.16–4.23 (m, 8H, CH2, H-2ʹ, H-3ʹ, H-2ʹʹʹ, H-3ʹʹʹ); 6.75–7.57 (m, 6H, H-5ʹ, H-7ʹ, H-8ʹ,
H-5ʹʹʹ, H-7ʹʹʹ, H-8ʹʹʹ, Ar); 7.60 (br s, 1H, NH); 8.77 (s, 1H, CH=). 13C-NMR (CDCl3/TFA 98:2) δ: 24.2
(C-2ʹʹ, C-3ʹʹ); 44.0 (C-1ʹʹ, C-4ʹʹ); 64.2 (C-2ʹ, C-2ʹʹʹ); 64.8 (C-3ʹ, C-3ʹʹʹ); 118.4 (C-7ʹ, C-7ʹʹʹ); 119.6 (C-8ʹ,
C-8ʹʹʹ); 120.3 (C=); 125.6 (C-5ʹ, C-5ʹʹʹ); 126.8 (C-6ʹ, C-6ʹʹʹ); 134.5 (CH=); 144.0 (C-8ʹa, C-8ʹʹʹa); 146.6
(C-4ʹa, C-4ʹʹʹa); 166.6 (C=O, C-4); 182.5 (C=S, C-2). HRMS, m/z: 499.1364 found (calculated for
C25H27N2O5S2 [M + H]+ requires 499.1366).
(5Z)3-[4-(2,3-Dihydro-benzo[1,4]dioxin-6-ylmethylamino)butyl]-5-(4-hydroxy-3-methoxybenzylidene)-2-
thioxo-1,3-thiazolidin-4-one (9n). According to the standard procedure, compound 9n was prepared in 34%
yield (82.7 mg) from N-1-(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)-butane-1,4-diamine hydrochloride 6f
(136.4 mg, 0.5 mmol, 1 equiv.), triethylamine (202 μL, 152 mg, 1.5 mmol, 3 equiv.) and 4-hydroxy-3-
methoxybenzaldehyde 3g (76.1 mg, 0.5 mmol, 1 equiv.) after 30 min. at 90 °C, followed by a second
reaction time of 30 min. at 110 °C (for condensation step with 3g), which gave 9n as a red powder.
Mp = 202–204 °C. 1H-NMR (CDCl3/TFA 98:2) δ: 1.69 (s, 4H, CH2, H-2ʹʹ, H-3ʹʹ); 3.06 (s, 2H, ArCH2NH);

Molecules 2015, 20
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3.86 (s, 3H, OCH3); 4.03 (s, 4H, CH2, H-1ʹʹ, H-4ʹʹ); 4.16 (s, 4H, CH2, H-2ʹʹʹ, H-3ʹʹʹ); 6.67–7.03 (m, 6H,
H-2ʹ, H-5ʹ, H-6ʹ, H-5ʹʹʹ, H-7ʹʹʹ, H-8ʹʹʹ, Ar); 7.60 (s, 1H, CH=). 13C-NMR (CDCl3/TFA 98:2) δ: 23.1 (C-2ʹʹ
or 3ʹʹ); 23.8 (C-3ʹʹ ou C-2ʹʹ); 43.1 (C-1ʹʹ ou 4ʹʹ); 47.0 (ArCH2NH); 52.1 (C-4ʹʹ, C-1ʹʹ); 56.1 (OCH3);
64.4 (C-2ʹʹʹ, C-3ʹʹʹ); 64.4 (C-3ʹʹʹ, C-2ʹʹʹ); 112.6 (C-6ʹ); 115.6 (C-2ʹ); 118.4 (C-7ʹʹʹ); 118.6 (C-8ʹʹʹ); 119.0
(C-6ʹʹʹ); 122.0 (C=); 122.9 (C-5ʹʹʹ); 125.9 (C-1ʹ); 126.8 (C-5ʹ); 135.9 (CH=); 143.9 (C-8ʹʹʹa); 145.1
(C-3ʹa); 147.2 (C-4ʹʹʹa); 148.9 (C-4ʹ);169.4 (C=O, C-4); 193.1 (C=S, C-2). HRMS, m/z: 487.1361 found
(calculated for C24H27N2O5S2 [M + H]+ requires 487.1359).
3.2. Biochemistry Section
3.2.1. Protein Kinase Assay Buffers
Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg heparin/mL.
Buffer B: 60 mM β-glycerophosphate, 15 mM p-nitrophenyl-phosphate, 25 mM Mops (pH 7.2),
5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1 mM phenylphosphate.
3.2.2. Kinase Preparations and Assays
Kinase activities for each enzyme were assayed in buffer A (25 mM Tris-HCl pH 7.5, 10 mM MgCl2,
1 mM EGTA, 1 mM DTT, 50 µg/mL Heparin, BSA 0.15 mg/mL) or B (60 mM β-glycerophosphate,
30 mM p-Nitrophenylphosphate, 25 mM MOPS, 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 0.1 mM Na
vanadate), with their corresponding substrates, in the presence of 15 µM [γ-33P] ATP (3000 Ci/mmol;
10 mCi/mL) in a final volume of 30 µL. After 30 min incubation at 30 °C, the reaction was stopped by
harvesting, using a FilterMate harvester (Packard, Meriden, CT, USA), onto P81 phosphocellulose
papers (GE Healthcare, Velizy-Villacoublay, France) which were washed in 1% phosphoric acid.
Scintillation fluid was added and the radioactivity measured in a Packard counter. Blank values were
subtracted and activities calculated as pmoles of phosphate incorporated during the 30 min incubation.
The activities were expressed in a percentage of the maximal activity, i.e., in the absence of inhibitors.
Controls were performed with appropriate dilutions of DMSO. CK1 and Haspin peptide substrates
were obtained from Proteogenix (Oberhausbergen, France).
CDK5/p25 (human, recombinant) was prepared as previously described [31]. Its kinase activity was
assayed in buffer B, with 1 mg histone H1/mL.
Casein kinase 1 (CK1δ/ε) (porcine brain, native) was assayed with 0.67 µg of CKS peptide
(RRKHAAIGpSAYSITA) [32].
GSK-3α/β (porcine brain, native) was assayed, as described for CDK5/p25 but in Buffer A and using a
GSK-3 specific substrate (GS-1: YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated
serine) [33]. GS-1 was synthesized by Millegen (Labege, France).
Haspin kinase domain (HsHaspin-kd aa 470 to 798) encoding cDNA, obtain by RT-PCR, was cloned
into pGex-6P-3. The fusion protein was expressed in Escherichia coli strain BL21-KRX (Promega,
Madison, WI, USA) and purified by affinity chromatography on glutathione-agarose beads (Sigma).
Haspin-kd activity was assayed with 3 µM Histone H3 (1-21) peptide, a specific Haspin substrate,
(ARTKQTARKSTGGKAPRKQLA), in Buffer H (MOPS 25 mM pH 7.5; 10 mM MgCl2).

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3.3. Cell Culture and Survival Assays
Skin diploid fibroblastic cells were provided by BIOPREDIC International Company (Rennes, France).
Caco2 (Ref ECACC: 86010202), Huh-7D12 (Ref ECACC: 01042712), MDA-MB-231 (Ref ECACC:
92020424), HCT-116 (Ref ECACC: 91091005), PC3 (Ref ECACC: 90112714), NCI-H727 (Ref ECACC:
94060303) cell lines were obtained from the ECACC collection and HaCaT (from Cell Lines Service,
Eppelheim, Germany). Cells were grown according to ECACC recommendations [34]. The toxicity
test of the compounds on these cells was as follows: 2 × 103 cells for HCT-116 cells or 4 × 103 for
the other cells were seeded in 96 multiwell plates in triplicate and left for 24 h for attachment, spreading
and growing. Then, cells were exposed for 48 h to increasing concentrations of the compounds, ranging
from 0.1 to 25 μM in a final volume of 120 μL of culture medium. Cells were fixed in cooled solution
of 90% ethanol/5% acetic acid, nuclei were stained with Hoechst 3342 (Sigma) and counted using
automated imaging analysis (Cellomics Arrayscan VTI/HCS Reader, Thermo/Scientific, Waltham, MA,
USA). The IC50 were graphically determined.
4. Conclusions
In summary, we have developed, in this preliminary project, a new route to (5Z) 3-(4-arylmethylamino)
butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-ones 9. Starting from commercial butane-1,4-diamine,
the process involved six steps and the key step is a solution phase “one-pot two-steps” approach for the
construction of the 2-thioxo-1,3-thiazolidine-4-one platform under microwave dielectric heating followed
by Knoevenagel condensation for installation of the 5-arylidene moiety as second point of diversity.
This methodology offered the possibility of preparing a library of fourteen new compounds in moderate
to good yields and, the targeted compounds 9a–n have been built with a Z-geometry. The in vitro
inhibition of cell proliferation was carried out on a panel of seven representative tumoral cell lines and
the compounds 9a–n were also evaluated against four protein kinases. Among all of these compounds,
the compound 9j turned out to be interesting because it presented selective micromolar inhibition activity
on SsCK1 (IC50 1.4 μM). Molecules 9h and 9i were also bioactive on SsCK1 and HsCDK5-p25. The
current results are the starting point of a new larger program within our group to investigate intensively
the biological properties of these new inhibitors with potential application in Alzheimer’s disease or
in cancer.
Acknowledgments
Two of us (C.D.D. and C.N.A.) wish to thank the “Benian International Fondation”, “ElecBTP” and
the “Ministère de l’Enseignement Supérieur et de la Recherche de la Côte d’Ivoire” for their respective
grants. Financial support of this program carried out under the French National Cancer Institute
“Cancéropôle Grand Ouest” by contract “Ion Channel-Network CGO 2012”, is gratefully acknowledged.
Author Contributions
J.-P.B. was the manager of the project. J.-P.B., Y.-A.B. and J.M. conceived and designed the
experiments. C.D.D., C.N.A. and W.-K.C. performed the chemical experiments. A.D. and B.B. performed
the kinase assays. S.B. and S.R. analyzed the data of kinase assays. M.R. and R.L.G. performed the

Molecules 2015, 20
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antiproliferative activity assays. R.L.G. and A.C. analyzed the data of antiproliferative assays and
J.-P.B. wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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