Synthesis and biological evaluation of a new class of triazin-triazoles as potential inhibitors of human farnesyltransferase

Article abstract of DOI:10.1007/s11164-015-2131-1

A new synthesis of ethynyldimethoxytriazine 1, an important platform-compound for developing new chemical entities for anticancer research and for other biological applications, is described. Compound 1 was further reacted with azides 5a-i to provide triazin-triazoles 2a-i, which were tested on human farnesyltransferase and on the NCI-60 human tumor cell lines. Synthesis of other dimethoxytriazine derivatives 15 and 16, linked to a sp ² or a sp ³ carbon atom were also studied. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-015-2131-1

Res Chem Intermed
DOI 10.1007/s11164-015-2131-1
Synthesis and biological evaluation of a new class
of triazin–triazoles as potential inhibitors of human
farnesyltransferase
1
Liliana Lucescu1 Elena Bıcu Dalila Belei1
•
•
•
ˆ
3,4
3,4
•
Sergiu Shova2 Benoıt Rigo
Philippe Gautret
•
•
ˆ
Joelle Dubois Alina Ghinet1,3,4
5
•
¨
Received: 26 March 2015 / Accepted: 4 June 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A new synthesis of ethynyldimethoxytriazine 1, an important platform-
compound for developing new chemical entities for anticancer research and for other
biological applications, is described. Compound 1 was further reacted with azides
5a–i to provide triazin–triazoles 2a–i, which were tested on human farnesyltransferase
and on the NCI-60 human tumor cell lines. Synthesis of other dimethoxytriazine
derivatives 15 and 16, linked to a sp2 or a sp3 carbon atom were also studied.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2131-1)
contains supplementary material, which is available to authorized users.
&
Alina Ghinet
alina.ghinet@hei.fr
1
2
3
4
5
Department of Organic Chemistry, Faculty of Chemistry, ‘Al. I. Cuza’ University of Iasi, B-dul
Carol I, Nr. 11, Corp A, 700506 Iasi, Romania
‘Petru Poni’ Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley,
700487 Iasi, Romania
´ ˆ ´
Inserm, U995-LIRIC, Faculte de Medecine - Pole Recherche, Universite Lille 2, CHRU de
Lille, Place Verdun, 59045 Lille Cedex, France
´
´
UCLille, Laboratoire de Pharmacochimie, Hautes Etudes d’Ingenieur (HEI), 13 rue de Toul,
59046 Lille, France
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, UPR2301 CNRS,
Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
123

L. Lucescu et al.
Graphical Abstract
MeO
N
OMe
N
N
N
N
O
N
N
N
CO2H
MeO
N
1
OMe
Ar/Het
2a-i
target compounds
2c: IC50 (human FTase) = 38.6 µM
Keywords Dimethoxytriazine Á Triazole Á Triazin–triazole Á Farnesyltransferase
inhibitor Á Ethynyldimethoxytriazine
Introduction
Many s-triazine derivatives have been synthesized, particularly for their consider-
able therapeutic potential [1–13] and their antifungal [14–20] or herbicidal [21–23]
properties. In this context, very few dimethoxytriazines have been described
[24–34], and some of their 2-(alk-10-ynyl) derivatives such as I–III [35] present
interesting cytotoxic activities toward different tumor cell lines (Fig. 1). The
method utilized for the synthesis of these acetylenic compounds is the palladium
catalyzed cross-coupling between alk-1-ynes and 2-chloro-4,6-dimethoxy-1,3,5-
triazine [36–38]. As for their 2-alkyl analogs, they were often obtained by reacting
zinc bis(imino-bismethyl carbamate) 3 with acid chloride or anhydride [25, 26, 33,
˚
34], and we have reported that 4 A molecular sieves and pyridine can be
advantageously utilized in this reaction [39, 40]. The molecular structure of zinc salt
was initially described as 3a [24], but we recently proved from an RX study (see
‘Supporting information’ section) that the real structure was 3b,1 as suggested in a
patent [25, 26], but not confirmed (Fig. 2).
The farnesylated proteins play an important role in cell cycle progression [41].
Ras and Rho proteins are involved in the regulation of cyclin and CDK (cyclin-
dependent kinase) inhibitors. In addition, studies have shown that Rheb was a
protein involved in the cell cycle that also affected the Ras signaling pathway.
Therefore, it is necessary to design new farnesyltransferase inhibitors (FTIs) that
specifically inhibit the farnesylation of target proteins involved in cell proliferation.
In this field, our team already developed new families of human farnesyltransferase
(FTase) inhibitors [42–45]. FTase is a heterodimeric metalloenzyme with two
subunits: a (48 kDa) and b (45 kDa) [46]. To be activated, farnesyltransferase needs
1
CCDC-1045377 (for zinc derivative 3b) and CCDC-1045376 (for phenothiazine derivative 16) contain
the crystallographic data for this contribution. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
123

Synthesis and biological evaluation of a new class of…
MeO
N
OMe
OH
N
CN
N
N
N
N
N
N
N
N
N
N
N
O
N
MeO
N
OMe
MeO
N
OMe
MeO
N
OMe
MeO
N
1
OMe
Ar/Het
I
II
III
2a-i
IC50 (LOX IMVI) = 1.86 µM
melanoma cell line
IC50 (HCT-116) = 0.40 µM IC50 (OVCAR-4) = 1.86 µM
ovarian cancer cell line
target compounds
colon cancer cell line
Fig. 1 Structures of some biologically active dimethoxytriazines I–III [35] and target compounds 1 and
2a–i
O
O
O
N
O
O
N
O
HN
HN
HN
NH
NH
NH
NH
Zn2+
-
-
N Zn N
HN
O
O
3a
3b
Fig. 2 Structure of zinc salt 3: initially described structure 3a and X-ray proved structure 3b
a zinc and a magnesium atom. In order to block the activity of farnesyltransferase,
we became interested in the design and the synthesis of FTIs bearing chelating units.
Thus, after the successful investigation of compounds bearing complexing groups
(CN, CO2R) that bind the zinc atom of protein farnesyltransferase [42], we describe
here the study of compounds with a triazin–triazole unit as a potential zinc chelating
moiety.
Experimental
Materials and methods
Starting materials are commercially available. Melting points were measured on an
MPA 100 OptiMeltÒ apparatus and are uncorrected. Nuclear magnetic resonance
(NMR) spectra were acquired at 400 MHz for 1H NMR and 100 MHz for 13C NMR
on a Varian 400-MR spectrometer. Chemical shifts (d) are given in parts per million
123

L. Lucescu et al.
relative to CDCl3 (7.26; 77.1 ppm), DMSO-d6 (2.47; 40.0 ppm) or TMS. Splitting
patterns are designed: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m,
multiplet, and sym m, symmetric multiplet. Coupling constants J are reported in
Hertz (Hz). Thin layer chromatographies (TLC) were realized on Macherey–Nagel
silica gel plates with fluorescent indicator and were visualized under a UV-lamp at
254 and 366 nm. Column chromatographies were performed with a CombiFlash Rf
Companion (Teledyne-Isco System) using RediSep packed columns. IR spectra
were recorded on a Varian 640-IR FT-IR Spectrometer. Elemental analyses (C, H,
ˆ
N) of new compounds were determined on a Thermo Electron apparatus by ‘Pole
´
´ ´
Chimie Moleculaire’, Faculte de Sciences Mirande, Universite de Bourgogne,
Dijon, France.
Chemistry
We were recently involved in the synthesis of protein FTIs [43–45], and we decided
to synthesize products 2a–i (Fig. 1), in order to investigate the possibility for their
dimethoxytriazine linked to the triazole heterocycle to bind to the zinc atom of this
protein. However, we did not succeed in the activation of acids 7a–d [47, 48],2
whatever the conditions tested (SOCl2/DMF [49], oxalyl chloride/CH2Cl2/DMF
[50], TFAA/TFA [51]) (Scheme 1; Table 1). Thus, we considered obtaining the
target compounds 2a–i starting from acetylenic derivative 1 (Scheme 2). The
synthetic pathway started from propiolic acid 10, which after activation as acid
chloride, then treatment with salt 3b, furnished the target acetylenic derivative. The
instability and hazardous potential of the propioloyl chloride is described in the
literature [52]. Moreover, spontaneous inflammation sometimes occurring during
the isolation of propiolic acid chloride, was attributed to trace amounts of
monochloroacetylene formed during distillation [52, 53]. For this reason, we did not
try to isolate the pure propioloyl chloride 11 and we used it further without any
purification.
The reaction of 90 % pure propioloyl chloride 11 containing 10 % of addition
by-products 12 and 13, with salt 3b in our general conditions [39, 40] was
performed, leading to 55 % of acetylenic product 1 and 30 % of 2-[(E)-2-
chlorovinyl]-4,6-dimethoxy-1,3,5-triazine 14 (Scheme 2). Compound 1 was reacted
with azides 5a–i to provide the target compounds 2a–i in good yields (Scheme 2;
Table 2).
Because of the formation of secondary products (12–14) occurring during the
synthesis of propioloyl chloride 11 and the reaction with salt 3b in our general
conditions [39, 40] (Scheme 2), condensation of hydroxysuccinyl esters 20 and 22
with precursor 3b was studied. While starting from acids 10, 19 and 21, pyridinyl or
phenothiazinyl derivatives 15, and 16 were easily formed in medium to good yields,
and activation of propiolic acid 10 (CDI and N-hydroxysuccinimide) led only to the
addition of imidazole to the triple bound, providing compound 17 (Scheme 3); this
reaction is reminiscent of the formation of (Z) and (E)-3-chloroacryloyl chlorides 12
2
Acids 7a–d were obtained by saponification of the corresponding known esters.
123

Synthesis and biological evaluation of a new class of…
MeO
N
OMe
O
O
O
N
N
OH
R
O
N
N
N
N
N
N
N
N
N3
N
N
N
N
Br
O
O
O
O
O
O
(iii)
(i)
(ii)
(iv)
z
z
z
z
z
Z
5a-d
4a-d
a: Z=F
b: Z=Cl
c: Z=Br
d: Z=Me
6a-d
7a-d
8a-d (R=Cl)
9a-d (R=OCOCF3)
2a-d
Scheme 1 Initial envisaged chemical strategy for the isolation of triazin–triazoles 2a–d. Reagents and
conditions: i NaN3 (1.1–1.2 equiv), TBAB (0.11–0.15 equiv), CHCl3, H2O, room temperature (RT), 24 h,
58–90 % yield; ii ethyl propiolate (1 equiv), CuSO4Á5H2O (0.1 equiv), sodium ascorbate (0.3 equiv), t-
BuOH, MeCN, H2O, RT, 24 h, 33–71 % yield; iii 30 % KOH aq sln, EtOH, reflux, 15 min, 84–97 %
yield; iv conditions described below in Table 1
Table 1 Tested conditions for the activation of carboxylic acids 7a–d
Reagent
(no. equiv.)
Yielda
(%)
Entry
Solvent
Time (h) Temperature
SOCl2
(2)
4
1
DMF
reflux
0
O
O
2
reflux
2
CH2Cl2/DMF
0
Cl
Cl
(2)
SOCl2
(10)
3
4
-
2
2
reflux
reflux
0
0
SOCl2
(36)
DMF
O
O
5
F3C-COOH
26
reflux
0
F3C
O
CF3
(4)
Entries 1 and 5: Conditions described in reference [39] for the activation of a carboxylic acid group. Entry
2: Conditions described in reference [50]. Entry 3: Conditions described in reference [49]. Entry 4:
Conditions described in reference [51]
a
Tested conditions resulted in formation of insoluble material
and 13 from the addition of chloride ion to propioloyl chloride 11 (Scheme 2), and
the addition of the same ion to acetylenic compound 1 as described in Scheme 2.
In order to attest the withdrawing effect of the dimethoxytriazine unit, acetylenic
derivative 1 was reacted with diethylamine in dichloromethane at RT and provided
enamine 18 in a good 70 % yield (Scheme 3).
123

L. Lucescu et al.
OMe
N
OMe
3b
O
N
N
N
N
Cl
+
O
Cl
(i)
(ii)
CO2H
+
+
Cl
Cl
Cl
Cl
O
N
10
OMe
OMe
13 3%a
1 55%
14 30%
11 90%a
12 7%a
Mechanism of the dimethoxytriazine ring closure:
H
N
O
H
OMe
OMe
OMe
N
..
Cl
Cl
NH
N
-
..
-
:O:
..
N
- Cl
N
..
..
:
:
O
NH
NH
NH
OMe
OMe
11
OMe
OMe
N
OMe
N
OH
N
N
N
N
- H2O
OMe
H
OMe
1
OMe
N
N
OMe
OMe
N
N
N
N
N
N
X
N3
1
N
OMe
O
O
O
(iv)
(iii)
W
W
W
5a-i
4a-i
X=Cl or Br
2a-i
Mechanism of the triazole formation:
MeO
N
N
N
OMe
O
-
..
..
+
N
..
(iv)
N
N
W
N
OMe
N
5a-i
N
N
N
N
O
1
OMe
W
2a-i
Scheme 2 Synthesis and formation mechanism of 2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1 and of target
triazin–triazoles 2a–i. a 1H NMR yields. Reagents and conditions: i PCl5 (1 equiv), CH2Cl2, RT; ii zinc
˚
dimethylimidodicarbonimidate 3b (0.7 equiv), powdered 4 A molecular sieves, RT, 24 h; iii NaN3
(1.1–1.2 equiv), TBAB (0.11–0.15 equiv), CHCl3, H2O, RT, 24 h, 58–90 % yield; iv CuSO.45H2O (0.1
equiv), sodium ascorbate (0.2–0.3 equiv), t-BuOH, MeCN, H2O, RT, 24 h, 26–83 % yield
The crystal structure of triazine 16 is shown in Fig. 3. It comprises two
crystallographic independent but chemically identical molecules, denoted as A and
B (See ‘Supporting information’ section for full crystallographic data).
In both A and B asymmetric molecules, the phenothiazine units feature a
butterfly structure with a boat conformation for the central six-membered ring and
syn–anti conformation for the two dimethoxy-1,3,5-triazine fragments. The folding
dihedral angle between the benzene rings of the phenothiazine system is 140.65(6)°
123

Synthesis and biological evaluation of a new class of…
Table 2 Structure and isolated yields of triazin–triazoles 2a–i
Compound
2a
W
Yield Compound
W
Yield
MeO
MeO
52%
65%
58%
2f
79%
F
OMe
2b
2c
2g
2h
2i
61%
83%
N
S
Cl
Br
N
S
Cl
2d
2e
26%
38%
43%
Me
NC
N
N
and 140.83(5)° for the two crystallographic independent molecules A and B,
respectively. In the crystal packing, two asymmetric molecules are self-assembled
into one-dimensional supramolecular double-chains, which are mediated by
C–HÁÁÁO, C–HÁÁÁN hydrogen bonding and p–p stacking interactions. The perspective
view of the supramolecular architecture, along with the geometric parameters of the
H-bonds, is depicted in Figure S4. Two triazine rings of adjacent A and B
asymmetric units are stacked with Cg1ÁÁÁCg2 centroid-to-centroid distance of
˚
˚
3.3646(1) A and a perpendicular distance of 3.2756(9) A. Another significant p–p
stacking interaction are observed between Cg3 (the centroid of C1B–C6B ring) and
˚
Cg4 (the centroid of C1A–C6A ring) with Cg3ÁÁÁCg4 distance of 3.9108(1) A and a
˚
perpendicular distance of 3.2836(2) A. The main crystal structure motif can be
characterized as the parallel packing of the above-mentioned supramolecular
aggregates with no more intermolecular contacts significantly below the sum of the
van der Waals radii for the corresponding atoms.
2-Ethynyl-4,6-dimethoxy-1,3,5-triazine (1)
Under inert atmosphere, a solution of propioloyl chloride 11 (6.28 g, 70.9 mmol) in
anhydrous CH2Cl2 (35 mL) was added dropwise over 30 min to a stirred mixture of
˚
zinc dimethyl imidodicarbonimidate (16.47 g, 49.6 mmol) and powdered 4 A
molecular sieves in anhydrous CH2Cl2 (120 mL). After the complete addition, the
mixture was stirred at room temperature for 24 h. The mixture was then filtered, the
solid washed with CH2Cl2 and the filtrate washed with H2O and NaHCO3 saturated
123

L. Lucescu et al.
3b
(ii)
O
(i)
O
OMe
N
N
N
19
CO2H
N
N
N
O
N
O
20 17%
15 46%
OMe
3b
(ii)
S
S
N
S
(i)
N
N
N
CO2H
21
O
N
O
O
N
O
OMe
N
OMe
22 90%
16 72%
O
O
(i)
N
CO2H
N
O
O
N
10
17 39%
OMe
OMe
N
N
N
(iii)
N
N
+
NHEt2
Et2N
N
OMe
1
OMe
18 70%
Scheme 3 Synthesis of dimethoxytriazines 15 and 16, of unexpected addition product 17 and of addition
product 18. Reagents and conditions: i N-hydroxysuccinimide (1.2 equiv), carbonyldiimidazole (CDI) for
15 and 17 or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.2 equiv) for 16,
˚
CH2Cl2, RT, 24 h; ii zinc dimethylimidodicarbonimidate 3b (0.7 equiv), powdered 4 A molecular sieves,
CH2Cl2, RT, 24 h; iii CH2Cl2, RT, 15 h
solution, until complete neutralization of resulting HCl. Following drying (Na2SO4)
then evaporation in vacuo, the obtained solid was recrystallized from absolute
ethanol to give pure 2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1 as a beige solid in
55 % yield (6.43 g); mp (EtOH) 160–162 °C; Rf = 0.55 (EtOAc:n-heptane, 1:1).
1
IR m cm-1: 3163, 2116, 1537, 1464, 1403, 1346, 1231, 1128. H NMR (CDCl3,
400 MHz): d (ppm) 3.22 (s, 1H, CH), 4.06 (s, 6H, 2OCH3). 13C NMR (CDCl3,
100 MHz): d (ppm) 55.7 (2CH3), 78.8 (CH), 80.2 (C), 161.8 (C), 172.4 (2C).
Elemental analysis calcd (%) for C7H7N3O2: C 50.91, H 4.27, N 25.44; found: C
51.26, H 3.97, N 25.71.
2-((E)-2-Chlorovinyl)-4,6-dimethoxy-1,3,5-triazine (14) By-product from the syn-
thesis of triazine 1 isolated from the ethanol residue of the recrystallization of
compound 1. Beige solid; 30 % yield (4.28 g); mp (EtOH) 88–90 °C; Rf = 0.7
(EtOAc:n-heptane, 1:1). IR m cm-1: 3085, 2941, 1544, 1496, 1465, 1352, 1236,
123

Synthesis and biological evaluation of a new class of…
Fig. 3 View of the asymmetric unit in the crystal structure 16. Thermal ellipsoids are drawn at 50 %
probability level
1
1196. H NMR (DMSO-d6, 400 MHz): d (ppm) 4.05 (s, 6H, 2OCH3), 6.73 (d,
J = 13.4 Hz, 1H, CH), 7.78 (d, J = 13.4 Hz, 1H, CH). 13C NMR (DMSO-d6,
100 MHz): d (ppm) 55.3 (2CH3), 131.1 (CH), 135.4 (CH), 172.5 (2C), 172.6 (C).
Elemental analysis calcd (%) for C7H8ClN3O2: C 41.70, H 4.00, N 20.84; found: C
41.54, H 4.31, N 21.13.
General procedure for the synthesis of triazinyl–triazoles (2a–i)
A mixture of 2-ethynyl-4,6-dimethoxy-1,3,5-triazine (1) (1 equiv) and appropriate
azide 5 was dissolved (1 equiv) in t-BuOH:MeCN, 10:4. Sodium ascorbate (0.3
equiv), and water and hydrated copper (II) sulfate (0.1 equiv) were then added to the
reaction mixture. The resulting medium was stirred at room temperature for 24 h.
The obtained solid was filtered and washed with water and ammonium hydroxide,
and then with ethanol to provide pure triazinyl–triazoles (2a–i).
1-(4-Fluorophenyl)-2-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)etha-
none (2a) The general procedure was followed using 2-ethynyl-4,6-dimethoxy-
1,3,5-triazine 1 (0.19 g, 1.12 mmol), 2-azido-1-(4-fluorophenyl)ethanone 5a (0.2 g,
1.12 mmol), CuSO4Á5H2O (0.03 g, 0.1 mmol), sodium ascorbate (0.07 g,
0.3 mmol), t-BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to provide triazole
2a as a white solid in 52 % yield (0.2 g); mp (EtOH) 200–203 °C; Rf = 0.05
(EtOAc:n-hexane, 1:1). IR m cm-1: 1702, 1536, 1500, 1391, 1347, 1227, 1202,
1
1162. H NMR (CDCl3, 400 MHz): d (ppm) 4.04 (s, 6H, 2OCH3), 6.32 (s, 2H,
CH2), 7.47 (t, J = 8.4 Hz, 2H, ArH), 8.19 (m, 2H, ArH), 8.88 (s, 1H, ArH). 13C
NMR (CDCl3, 100 MHz): d (ppm) 55.0 (2CH3), 56.1 (CH2), 116.1 (d, J = 88.0 Hz,
2CH), 116.2 (CH), 130.3 (CH), 130.7 (d, J = 12.0 Hz, C), 131.3 (d, J = 36.0 Hz,
123

L. Lucescu et al.
2CH), 131.3 (CH), 164.3 (C), 166.8 (C), 168.4 (C), 172.4 (2C), 174.4 (C), 190.4 (C).
Elemental analysis calcd (%) for C15H13FN6O3: C 52.33, H 3.81, N 24.41; found: C
52.60, H 3.62, N 24.67.
1-(4-Chlorophenyl)-2-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)etha-
none (2b) The general procedure was followed using 2-ethynyl-4,6-dimethoxy-
1,3,5-triazine 1 (0.16 g, 1 mmol), 2-azido-1-(4-clorophenyl)ethanone 5b (0.2 g,
1 mmol), CuSO4Á5H2O (0.02 g, 0.1 mmol), sodium ascorbate (0.06 g, 0.3 mmol), t-
BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to provide triazole 2b as a beige
solid in 65 % yield (0.21 g); mp (EtOH) 189–192 °C; Rf = 0.06 (EtOAc:n-hexane,
1:1). IR m cm-1: 1705, 1536, 1500, 1387, 1360, 1225, 1199, 1086. 1H NMR
(DMSO-d6, 400 MHz): d (ppm) 4.04 (s, 6H, 2OCH3), 6.32 (s, 2H, CH2), 7.71 (d,
J = 9.2 Hz, 2H, ArH), 8.11 (d, J = 9.2 Hz, 2H, ArH), 8.88 (s, 1H, ArH). 13C NMR
(DMSO-d6, 100 MHz): d (ppm) 55.0 (2CH3), 56.2 (CH2), 129.1 (2CH), 130.1
(2CH), 130.3 (CH), 132.7 (C), 139.2 (C), 144.3 (C), 168.4 (C), 172.4 (2C), 190.9
(C). Elemental analysis calcd (%) for C15H13ClN6O3: C 49.94, H 3.63, N 23.30;
found: C 50.32, H 3.90, N 23.53.
1-(4-Bromophenyl)-2-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)etha-
none (2c) The general procedure was followed using 2-ethynyl-4,6-dimethoxy-
1,3,5-triazine 1 (0.16 g, 1 mmol), 2-azido-1-(4-brorophenyl)ethanone 5c (0.24 g,
1 mmol), CuSO4Á5H2O (0.02 g, 0.1 mmol), sodium ascorbate (0.06 g, 0.3 mmol), t-
BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to provide triazole 2c as a beige
solid in 58 % yield (0.22 g); mp (EtOH) 176–180 °C; Rf = 0.06 (EtOAc:n-hexane,
1
1:1). IR m cm-1: 1705, 1534, 1498, 1387, 1358, 1284, 1249, 1222, 1198. H NMR
(DMSO-d6, 400 MHz): d (ppm) 4.03 (s, 6H, 2OCH3), 6.31 (s, 2H, CH2), 7.85 (d,
J = 9.2 Hz, 2H, ArH), 8.30 (d, J = 9.2 Hz, 2H, ArH), 8.88 (s, 1H, ArH). 13C NMR
(DMSO-d6, 100 MHz): d (ppm) 55.8 (2CH3), 56.8 (CH2), 129.1 (C), 130.8 (2CH),
130.9 (CH), 132.7 (2CH), 133.7 (C), 144.9 (C), 169.1 (C), 173.0 (2C), 191.8 (C).
Elemental analysis calcd (%) for C15H13BrN6O3: C 44.46, H 3.23, N 20.74; found:
C 44.72, H 3.45, N 20.91.
2-(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)-1-p-tolylethanone (2d)
The general procedure was followed using 2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1
(0.24 g, 1.42 mmol), 2-azido-1-p-tolylethanone 5d (0.25 g, 1.42 mmol), CuSO4-
5H2O (0.035 g, 0.14 mmol), sodium ascorbate (0.08 g, 0.43 mmol), t-BuOH
(10 mL), MeCN (4 mL) and H2O (4 mL) to provide triazole 2d as a beige solid
in 38 % yield (0.18 g); mp (EtOH) 148–151 °C; Rf = 0.07 (EtOAc:n-hexane, 1:1).
IR m cm-1: 1696, 1580, 1542, 1496, 1461, 1391, 1344, 1287, 1253, 1225, 1182,
1
1105. H NMR (DMSO-d6, 400 MHz): d (ppm) 2.43 (s, 3H, CH3), 4.03 (s, 6H,
2OCH3), 6.36 (s, 2H, CH2), 8.12 (d, J = 8.4 Hz, 2H, ArH), 8.24 (d, J = 8.4 Hz,
2H, ArH), 8.87 (s, 1H, ArH). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 21.3 (CH3),
55.1 (2CH3), 56.1 (CH2), 128.3 (2CH), 129.5 (2CH), 130.3 (CH), 131.5 (C), 144.2
(C), 144.9 (C), 168.5 (C), 172.4 (2C), 191.2 (C). Elemental analysis calcd (%) for
C16H16N6O3: C 56.47, H 4.74, N 24.69; found: C 56.86, H 4.98, N 24.83.
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Synthesis and biological evaluation of a new class of…
4-(2-(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)acetyl)benzonitrile
(2e) The general procedure was followed using 2-ethynyl-4,6-dimethoxy-1,3,5-
triazine 1 (0.16 g, 1 mmol), 4-(2-azidoacetyl)benzonitrile 5e (0.2 g, 1 mmol),
CuSO4Á5H2O (0.025 g, 0.10 mmol), sodium ascorbate (0.06 g, 0.32 mmol), t-
BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to provide triazole 2e as a beige
solid in 43 % yield (0.16 g); mp (EtOH) 230–231 °C; Rf = 0.03 (EtOAc:n-hexane,
1
1:1). IR m cm-1: 2225, 1706, 1552, 1498, 1382, 1355, 1249, 1227, 1199, 1156. H
NMR (DMSO-d6, 400 MHz): d (ppm) 4.03 (s, 6H, 2OCH3), 6.36 (s, 2H, CH2), 8.12
(d, J = 8.4 Hz, 2H, ArH), 8.24 (d, J = 8.4 Hz, 2H, ArH), 8.87 (s, 1H, ArH). 13C
NMR (DMSO-d6, 100 MHz): d (ppm) 55.6 (2CH3), 57.0 (CH2), 116.5 (C), 118.5
(C), 129.3 (2CH), 130.7 (CH), 133.4 (2CH), 137.7 (C), 144.8 (C), 168.9 (C), 172.9
(2C), 191.9 (C). Elemental analysis calcd (%) for C16H13N7O3: C 54.70, H 3.73, N
27.91; found: C 54.51, H 3.59, N 27.77.
2-(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)-1-(3,4,5-trimethoxy-
phenyl)ethanone (2f) The general procedure was followed using 2-ethynyl-4,6-
dimethoxy-1,3,5-triazine 1 (0.29 g, 1.73 mmol), 2-azido-1-(3,4,5-trimethoxyphe-
nyl)ethanone 5f (0.43 g, 1.73 mmol), CuSO4Á5H2O (0.04 g, 0.17 mmol), sodium
ascorbate (0.10 g, 0.52 mmol), t-BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to
provide triazole 2f as a white solid in 79 % yield (0.56 g); mp (EtOH) 241–243 °C.
1
IR m cm-1: 2948, 1676, 1545, 1496, 1449, 1411, 1392, 1363, 1240, 1196, 1127. H
NMR (CDCl3, 400 MHz): d (ppm) 3.94 (s, 6H, 2OCH3), 3.96 (s, 3H, OCH3), 4.14
(s, 6H, 2OCH3), 5.92 (s, 2H, CH2), 7.25 (s, 2H, ArH), 8.87 (s, 1H, ArH). 13C NMR
(CDCl3, 100 MHz): d (ppm) 29.7 (CH2), 55.5 (2CH3), 56.1 (2CH3), 61.1 (CH3),
105.8 (2CH), 128.7 (2C), 130.4 (CH), 144.1 (C), 153.5 (2C), 172.9 (2C), 174.5 (C),
188.4 (C). Elemental analysis calcd (%) for C18H20N6O6: C 51.92, H 4.84, N 20.18;
found: C 52.16, H 5.01, N 20.15.
2-(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)-1-(10H-phenothiazin-
10-yl)ethanone (2g) The general procedure was followed using 2-ethynyl-4,6-
dimethoxy-1,3,5-triazine 1 (0.2 g, 1.21 mmol), 2-azido-1-(10H-phenothiazin-10-
yl)ethanone 5 g (0.34 g, 1.21 mmol), CuSO4Á5H2O (0.03 g, 0.12 mmol), sodium
ascorbate (0.07 g, 0.36 mmol), t-BuOH (10 mL), MeCN (4 mL) and H2O (4 mL) to
provide triazole 2 g as a white solid in 61 % yield (0.31 g); mp (H2O) 136–140 °C;
Rf = 0.07 (EtOAc:n-hexane, 1:1). IR m cm-1: 1687, 1545, 1498, 1460, 1353, 1282,
1
1256, 1180. H NMR (CDCl3, 400 MHz): d (ppm) 4.10 (s, 6H, 2OCH3), 5.42 (s,
2H, CH2), 7.31(t, J = 7.6 Hz, 2H, ArH), 7.39 (t, J = 7.6 Hz, 2H, ArH), 7.51 (d,
J = 7.6 Hz, 2H, ArH), 7.60 (d, J = 7.6 Hz, 2H, ArH), 8.57 (s, 1H, ArH). 13C NMR
(CDCl3, 100 MHz): d (ppm) 52.2 (CH2), 55.7 (2CH3), 126.7 (2CH), 127.6 (2CH),
128.2 (2CH), 129.7 (2CH), 130.2 (CH), 130.9 (2C), 137.8 (C), 144.6 (2C), 164.2
(C), 169.0 (C), 173.0 (2C). Elemental analysis calcd (%) for C21H17N7O3S: C 56.37,
H 3.83, N 21.91; found: C 56.67, H 3.94, N 22.07.
1-(2-Chloro-10H-phenothiazin-10-yl)-2-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1H-
1,2,3-triazol-1-yl)ethanone (2h) The general procedure was followed using
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2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1 (0.1 g, 0.61 mmol), 2-azido-1-(2-chloro-
10H-phenothiazin-10-yl)ethanone 5 h (0.2 g, 0.61 mmol), CuSO4Á5H2O (0.015 g,
0.06 mmol), sodium ascorbate (0.035 g, 0.18 mmol), t-BuOH (10 mL), MeCN
(4 mL) and H2O (4 mL) to provide triazole 2 h as a white solid in 83 % yield
(0.23 g); white solid; mp (H2O) 155–158 °C; Rf = 0.1 (EtOAc:n-hexane, 1:1). IR m
cm-1: 1689, 1543, 1501, 1458, 1353, 1286, 1249, 1179, 1103. 1H NMR (DMSO-d6,
400 MHz): d (ppm) 4.02 (s, 6H, 2OCH3), 5.77 (s, 2H, CH2), 7.39-7.51 (m, 3H,
ArH), 7.65 (d, J = 7.2 Hz, 2H, ArH), 7.80-7.88 (m, 2H, ArH), 8.82 (s, 1H, ArH).
13C NMR (DMSO-d6, 100 MHz): d (ppm) 52.7 (CH2), 56.1 (2CH3), 127.9 (CH),
128.6 (CH), 128.9 (2CH), 129.3 (CH), 130.3 (CH), 131.2 (2CH), 133.7 (2C), 139.2
(C), 140.1 (C), 145.1 (2C), 165.7 (C), 169.4 (C), 173.4 (2C). Elemental analysis
calcd (%) for C21H16ClN7O3S: C 52.34, H 3.35, N 20.35; found: C 52.48, H 3.40, N
20.58.
1-(1H-Indazol-1-yl)-2-(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-1-yl)etha-
none (2i) The general procedure was followed using 2-ethynyl-4,6-dimethoxy-
1,3,5-triazine 1 (0.1 g, 0.61 mmol), 2-azido-1-(2-chloro-10H-phenothiazin-10-
yl)ethanone 5i (0.2 g, 0.61 mmol), CuSO4Á5H2O (0.015 g, 0.06 mmol), sodium
ascorbate (0.035 g, 0.18 mmol), t-BuOH (10 mL), MeCN (4 mL) and H2O (4 mL)
to provide triazole 2i as a white solid in 26 % yield (0.14 g); white solid; mp
(EtOH) 195–199 °C; Rf = 0.08 (EtOAc:n-hexane, 1:1). IR m cm-1: 1708, 1582,
1
1557, 1468, 1434, 1385, 1344, 1331, 1234, 1173, 1149, 1121. H NMR (CDCl3,
400 MHz): d (ppm) 4.13 (s, 6H, 2OCH3), 6.19 (s, 2H, CH2), 7.43 (t, J = 7.6 Hz,
1H, ArH), 7.60 (t, J = 7.6 Hz, 1H, ArH), 7.90 (d, J = 7.6 Hz, 1H, ArH), 8.25 (s,
1H, ArH), 8.32 (d, J = 7.6 Hz, 1H, ArH), 8.64 (s, 1H, ArH). 13C NMR (CDCl3,
100 MHz): d (ppm) 52.4 (CH2), 55.4 (2CH3), 115.0 (CH), 121.4 (CH), 125.5 (CH),
129.1 (CH), 130.3 (CH), 141.7 (CH), 145.7 (2C), 164.5 (2C), 168.8 (2C), 172.8 (C).
Elemental analysis calcd (%) for C16H14N8O3: C 52.46, H 3.85, N 30.59; found: C
52.80, H 4.11, N 30.82.
Propioloyl chloride (11)
Propiolic acid 10 (5.0 g, 70.9 mmol) was added to a suspension of PCl5 (14.77 g,
70.9 mmol) in anhydrous CH2Cl2 (30 mL), under nitrogen. The obtained solution
was stirred at room temperature for 1 h, then concentrated in vacuo and used
without further purification to provide the propioloyl chloride 11 in 90 % yield
1
among with compounds 12 and 13. H NMR (CDCl3, 400 MHz): d (ppm) 3.76 (d,
J = 13.3 Hz, 1H, CH).
(E)-3-Chloroacryloyl chloride (12) By-product from the synthesis of propioloyl
chloride 11.
1
7 % NMR yield. H NMR (CDCl3, 400 MHz): d (ppm) 6.52 (d, J = 13.3 Hz,
1H, CH), 7.68 (d, J = 13.3 Hz, 1H, CH).
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Synthesis and biological evaluation of a new class of…
(Z)-3-Chloroacryloyl chloride (13) By-product from the synthesis of propioloyl
chloride 11.
3 % NMR yield. 1H NMR (CDCl3, 400 MHz): d (ppm) 6.60 (dd, J = 7.8,
1.2 Hz, 1H, CH), 6.87 (dd, J = 7.8, 1.2 Hz, 1H, CH).
x-Bromo or chloroacetophenones 4a–e were commercially available.
2-Bromo-1-(3,4,5-trimethoxyphenyl)ethanone (4f) and x-chloroacetophenones
4g and 4h were prepared as described previously by our team [43, 45].
1-(Chloroacetyl)-1H-indazole (4i)
To a solution of 1H-indazole (1.00 g, 8.47 mmol), and triethylamine (1.41 mL,
10.17 mmol) in toluene (20 mL), chloroacetyl chloride (1.01 mL, 12.71 mmol) was
added dropwise. The obtaining mixture was stirred at room temperature for 4 h. The
solvent was removed in vacuo and the resulting solid was recrystallized from
ethanol to give pure acylated indazole 4i as a cream solid in 78 % yield (1.28 g)
with the same physico-chemical properties as described in the literature [54, 55];
cream solid; mp (EtOH) 75–78 °C; Rf = 0.61 (EtOAc:n-hexane, 1:1). IR m cm-1
:
1
1742, 1497, 1386, 1356, 1336, 1270, 1203, 1131, 1063, 953, 897, 777. H NMR
(CDCl3, 400 MHz): d (ppm) 5.01 (s, 2H, CH2), 7.41 (t, J = 8.0 Hz, 1H, ArH), 7.61
(t, J = 8.0 Hz, 1H, ArH), 7.76 (d, J = 8.0 Hz, 1H, ArH), 8.17 (s, 1H, ArH), 8.44 (d,
J = 8.0 Hz, 1H, ArH). 13C NMR (CDCl3, 100 MHz): d 43.1 (CH2), 115.5 (CH),
121.3 (CH), 125.3 (CH), 126.4 (C), 130.2 (CH), 139.3 (C), 141.1 (CH), 166.0 (C).
Elemental analysis calcd (%) for C9H7ClN2O: C 55.54, H 3.63, N 14.39; found: C
55.86, H 3.84, N 14.61.
Azides 5a–e, 5g and 5h were prepared as described previously by our team [45,
56–58].
General procedure for the synthesis of azides 5f and 5i
To a solution of x-bromoacetophenone 4f or of x-chloroacetophenone 4i (1 equiv)
in chloroform, sodium azide (NaN3) (1.1–1.2 equiv), water, and tetrabutylammo-
nium bromide (TBAB) (0.11–0.15 equiv) were added. The reaction mixture was
stirred vigorously at room temperature for 24 h and then separated. The organic
layer was washed with water, dried (Na2SO4), and concentrated in vacuo. The
residue was titrated with ethanol and the resulting solid recrystallized from absolute
ethanol to give pure azide 5f or 5i.
2-Azido-1-(3,4,5-trimethoxyphenyl)ethanone (5f) The general procedure was used
with 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone 4f (0.5 g, 1.73 mmol), NaN3
(0.2 g, 3.07 mmol) and TBAB (0.12 g, 0.37 mmol) in chloroform (10 mL) and
water (10 mL) to provide pure azide 5f as a beige solid in 82 % yield (0.36 g); mp
(EtOH): 92–94 °C. Rf = 0.78 (EtOAc:n-C6H12 = 1:1). IR m cm-1: 2942, 2100,
1694, 1584, 1502, 1469, 1413, 1348, 1319, 1283, 1229, 1158, 1125, 1062. 1H NMR
(CDCl3, 400 MHz) d (ppm) 4.03 (s, 6H, 2OCH3), 4.04 (s, 3H, OCH3), 4.64 (s, 2H,
CH2), 7.25 (s, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d 54.7 (CH2), 56.4 (2CH3),
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L. Lucescu et al.
61.0 (CH3), 105.6 (2CH), 129.5 (C), 143.6 (C), 153.3 (2C), 192.1 (C). Elemental
analysis calcd (%) for C11H15N3O4: C 52.17, H 5.97, N 16.59; found: C 52.42, H
6.09, N 16.81.
1-(Azidoacetyl)-1H-indazole (5i) The general procedure was used with 1-(chlor-
oacetyl)-1H-indazole 4i (1.5 g, 7.71 mmol) in chloroform (15 mL), NaN3 (0.6 g,
9.23 mmol), water (15 mL), and TBAB (0.37 g, 1.15 mmol) to give pure
1-(azidoacetyl)-1H-indazole 5i as a beige solid in 69 % yield (1.1 g); mp (EtOH)
81–83 °C; Rf = 0.53 (EtOAc:n-hexane,1:1). IR m cm-1: 2121, 1702, 1504, 1469,
1430, 1346, 1265, 1215, 1168, 1146. 1H NMR (CDCl3, 400 MHz) d (ppm) 4.77 (s,
2H, CH2), 7.39 (t, J = 7.2 Hz, 1H, ArH), 7.59 (t, J = 7.2 Hz, 1H, ArH), 7.75 (d,
J = 8.0 Hz, 1H, ArH), 8.14 (s, 1H, ArH), 8.41 (dd, J = 8.4, 0.4 Hz, 1H, ArH). 13
C
NMR (CDCl3, 100 MHz): 51.7 (CH2), 115.1 (CH), 121.2 (CH), 125.1 (CH), 126.1
(C), 129.9 (CH), 138.0 (C), 141.0 (CH), 167.4 (C). Elemental analysis calcd (%) for
C9H9N5O: C 53.20, H 4.46, N 34.47; found: C 53.47, H 4.68, N 34.55.
General procedure for the synthesis of ethyl 1,2,3-triazole-4-carboxylates (6a–d)
A mixture of ethyl propiolate (1 equiv) and appropriate azide 5a–d was dissolved in
t-BuOH:MeCN = 10:4. Sodium ascorbate (0.2 equiv), water and copper sulfate (0.1
equiv) were then added. The reaction was stirred at room temperature for 24 h. The
obtained solid was filtered and washed with water and ammonium hydroxide, and
then with ethanol to provide pure triazoles (6a–d).
Ethyl 1-[2-(4-fluorophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (6a) The
general procedure was followed using ethyl propiolate (0.97 mL, 8.45 mmol),
2-azido-1-(4-fluorophenyl)ethanone 5a (1.51 g, 8.45 mmol), CuSO4Á5H2O (0.21 g,
0.84 mmol), sodium ascorbate (0.34 g, 1.62 mmol), t-BuOH (10 mL), MeCN
(4 mL) and H2O (4 mL) to provide triazole 6a as a white solid in 71 % yield
(2.02 g) with the same properties as described in the literature [47]; Rf = 0.38
(EtOAc:n-hexane,1:1). 1H NMR (DMSO-d6, 400 MHz): d (ppm) 1.32 (t,
J = 6.8 Hz, 3H, CO2CH2CH3), 4.34 (q, J = 6.8 Hz, 2H, CO2CH2CH3), 6.27 (s,
2H, CH2), 7.46 (t, J = 8.8 Hz, 2H, ArH), 8.18 (q, J = 6.4 Hz, 2H, ArH), 8.71 (s,
1H, ArH). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 14.1 (CH3), 56.1 (CH2), 60.5
(CH2), 116.1 (d, J = 86.8 Hz, 2CH), 130.6 (d, J = 74.3 Hz, C), 131.3 (d,
J = 40.0 Hz, 2CH), 138.8 (2C), 160.2 (CH), 164.3 (C), 166.8 (C), 190.2 (C).
Ethyl
1-[2-(4-chlorophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate
(6b)
The general procedure was followed using ethyl propiolate (0.83 mL, 8.2 mmol),
2-azido-1-(4-chlorophenyl)ethanone 5b (1.61 g, 8.2 mmol), CuSO4Á5H2O (0.21 g,
0.82 mmol), sodium ascorbate (0.33 g, 1.6 mmol), t-BuOH (15 mL), MeCN (6 mL)
and H2O (6 mL) to provide triazole 6b as a beige solid in 33 % yield (0.79 g) with
the same physico-chemical properties as described in the literature [47, 48]; mp
(EtOH) 181–184 °C; Rf = 0.33 (EtOAc:n-hexane,1:1). 1H NMR (DMSO-d6,
400 MHz): d (ppm) 1.32 (t, J = 7.2 Hz, 3H, CO2CH2CH3), 4.33 (q, J = 7.2 Hz,
123

Synthesis and biological evaluation of a new class of…
2H, CO2CH2CH3), 6.27 (s, 2H, CH2), 7.71 (d, J = 8.4 Hz, 2H, ArH), 8.09 (d,
J = 8.8 Hz, 2H, ArH), 8.70 (s, 1H, ArH). 13C NMR (DMSO-d6, 100 MHz): d
(ppm) 14.1 (CH3), 56.1 (CH2), 60.5 (CH2), 129.1 (2CH), 130.0 (2CH), 130.6 (CH),
132.6 (C), 138.8 (C), 139.2 (C), 160.2 (C), 190.7 (C).
Ethyl 1-[2-(4-bromophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (6c) The
general procedure was followed using ethyl propiolate (1.16 mL, 11.43 mol),
2-azido-1-(4-bromophenyl)ethanone 5c (2.75 g, 11.4 mol), CuSO4Á5H2O (0.29 g,
1.14 mol), sodium ascorbate (0.45 g, 2.28 mol), t-BuOH (20 mL), MeCN (8 mL)
and H2O (8 mL) to provide triazole 6c as a beige solid in 64 % yield (2.47 g) with
the same physico-chemical properties as described in the literature [47]; mp (EtOH)
180–184 °C; Rf = 0.3 (EtOAc:n-hexane,1:1).
Ethyl 1-[2-(4-methylphenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (6d)
The general procedure was followed using ethyl propiolate (1.68 mL, 16.66 mol),
2-azido-1-p-tolylethanone 5d (2.91 g, 16.66 mol), CuSO4Á5H2O (0.42 g, 1.66 mol),
sodium ascorbate (0.66 g, 3.33 mol), t-BuOH (20 mL), MeCN (8 mL) and H2O
(8 mL) to provide triazole 6d as a yellow solid in 54 % yield (2.99 g) with the same
physico-chemical properties as described in the literature [48]; mp (EtOH)
1
157–160 °C; Rf = 0.23 (EtOAc:n-hexane, 1:1). H NMR (DMSO-d6, 400 MHz):
d (ppm) 1.32 (t, J = 6.8 Hz, 3H, CO2CH2CH3), 2.43 (s, 3H, ArCH3), 4.33 (q,
J = 6.8 Hz, 2H, CO2CH2CH3), 6.23 (s, 2H, CH2), 7.42 (d, J = 8.0 Hz, 2H, ArH),
7.98 (d, J = 8.0 Hz, 2H, ArH), 8.72 (s, 1H, ArH). 13C NMR (DMSO-d6, 100 MHz):
d (ppm) 14.1 (CH3), 21.2 (CH3), 56.0 (CH2), 60.5 (CH2), 128.2 (2CH), 129.5 (2CH),
130.7 (CH), 131.3 (C), 138.7 (C), 144.9 (C), 160.2 (C), 191.0 (C).
General procedure for the synthesis of 1,2,3-triazole-4-carboxylic acids (7a–d)
KOH aq solution (30 %) was added to a suspension of ethyl 1,2,3-triazole-4-
carboxylate 6a–d (1 equiv) in EtOH. The resulting solution was heated at reflux for
15 min. After cooling to RT, the pH was adjusted to 4 with citric acid. The resulting
white solid was filtered and washed with ethanol to provide pure carboxylic acids
7a–d.
1-[2-(4-Fluorophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (7a) The
general procedure was followed using ethyl 1,2,3-triazole-4-carboxylate 6a (1.25 g,
4.2 mol), KOH (2.28 g, 40 mol), H2O (10 mL) and EtOH (20 mL) to provide
carboxylic acid 7a as a white solid in 97 % yield (1.12 g); mp (EtOH) 223–224 °C.
1H NMR (DMSO-d6, 400 MHz): d (ppm) 6.26 (s, 2H, CH2), 7.47 (t, J = 8.6 Hz,
2H, ArH), 8.62 (q, J = 8.6 Hz, 2H, ArH), 8.62 (s, 1H, ArH), 13.0 (s, 1H, COOH).
13C NMR (DMSO-d6, 100 MHz): d (ppm) 56.5 (CH2), 116.6 (d, J = 21.8 Hz,
2CH), 131.1 (d, J = 20.3 Hz, CH), 131.8 (d, J = 20.3 Hz, CH), 140.2 (C), 162.1
(C), 164.8 (CH), 167.3 (C), 190.8 (C). Elemental analysis calcd (%) for
C11H8FN3O3: C 53.02, H 3.24, N 16.86; found: C 53.39, H 3.41, N 17.02.
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1-[2-(4-Chlorophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (7b) The
general procedure was followed using ethyl 1,2,3-triazole-4-carboxylate 6b (0.8 g,
2.6 mol), KOH (1.35 g, 24.2 mol), H2O (5 mL) and EtOH (10 mL) to provide
carboxylic acid 7b as a white solid in 84 % yield (0.6 g); mp (EtOH) 231–234 °C.
1
IR m cm-1: 3121, 2998, 2954, 1682, 1589, 1542, 1438, 1232, 1092, 990. H NMR
(DMSO-d6, 400 MHz): d (ppm) 6.26 (s, 2H, CH2), 7.71 (d, J = 8.6 Hz, 2H, ArH),
8.10 (d, J = 8.6 Hz, 2H, ArH), 8.61 (s, 1H, ArH), 13.25 (s, 1H, COOH). 13C NMR
(DMSO-d6, 100 MHz): d (ppm) 56.6 (CH2), 129.6 (2CH), 130.6 (2CH), 131.0 (C),
133.1 (C), 139.7 (CH), 140.4 (C), 162.2 (C), 191.3 (C). Elemental analysis calcd
(%) for C11H8ClN3O3: C 49.73, H 3.04, N 15.82; found: C 49.98, H 3.32, N 15.97.
1-[2-(4-Bromophenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (7c) The
general procedure was followed using ethyl 1,2,3-triazole-4-carboxylate 6c (2.32 g,
6.85 mol), KOH (3.45 g, 61.6 mol), H2O (10 mL) and EtOH (20 mL) to provide
carboxylic acid 7c as a white solid in 91 % yield (1.93 g); mp (EtOH) 231–234 °C.
1
IR m cm-1: 3121, 2999, 2953, 1682, 1586, 1542, 1438, 1233, 1071. H NMR
(DMSO-d6, 400 MHz): d (ppm) 6.25 (s, 2H, CH2), 7.85 (d, J = 8.4 Hz, 2H, ArH),
8.01 (d, J = 8.8 Hz, 2H, ArH), 8.61 (s, 1H, ArH), 13.15 (s, 1H, COOH). 13C NMR
(DMSO-d6, 100 MHz): d (ppm) 56.5 (CH2), 128.9 (CH), 130.6 (2CH), 131.0 (C),
132.6 (2CH), 133.4 (C), 140.3 (C), 162.1 (C), 191.5 (C). Elemental analysis calcd
(%) for C11H8BrN3O3: C 42.60, H 2.60, N 13.55; found: C 42.81, H 2.51, N 13.74.
1-[2-(4-Methylphenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (7d) The
general procedure was followed using ethyl 1,2,3-triazole-4-carboxylate 6d (2.79 g,
0.01 mol), KOH (5.15 g, 0.09 mol), H2O (10 mL) and EtOH (20 mL) to provide
carboxylic acid 7d as a yellow solid in 87 % yield (2.5 g); mp (EtOH) 233–235 °C.
1
IR m cm-1: 3120, 1696, 1676, 1604, 1540, 1439, 1343, 1226, 1182, 1055, 949. H
NMR (DMSO-d6, 400 MHz): d (ppm) 2.42 (s, 3H, CH3), 6.22 (s, 2H, CH2), 7.43 (d,
J = 8.2 Hz, 2H, ArH), 7.98 (d, J = 8.2 Hz, 2H, ArH), 8.62 (s, 1H, ArH), 13.16 (s,
1H, COOH). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 21.7 (CH3), 56.4 (CH2),
128.8 (2CH), 130.0 (2CH), 131.1 (C), 131.9 (C), 140.1 (C), 145.4 (CH), 162.2 (C),
191.6 (C). Elemental analysis calcd (%) for C12H11N3O3: C 58.77, H 4.52, N 17.13;
found: C 58.63, H 4.38, N 17.40.
General procedure for the synthesis of triazines 15 and 16
Under an inert atm, triethylamine (1 equiv) was added dropwise to a homogeneous
mixture of activated ester 20 or 22 (1 equiv) and zinc dimethylimidodicarbonim-
idate 3b (0.6 equiv) in dichloromethane. After the complete addition, the mixture
was stirred at room temperature for 18 or 24 h. Then, the reaction mixture was
washed with water. The combined organic layers were dried (Na2SO4), filtered, and
the solvent evaporated. After evaporation, n-hexane was added to the crude residue.
The resulting solid was collected by filtration and recrystallized in n-hexane or
purified by flash chromatography to provide pure triazine 15 or 16.
123

Synthesis and biological evaluation of a new class of…
2,4-Dimethoxy-6-(pyridin-2-yl)-1,3,5-triazine (15) The general procedure was
used with triethylamine (0.25 mL, 1.82 mmol), activated ester 20 (0.4 g,
1.82 mmol) and zinc dimethylimidodicarbonimidate 3b (0.38 g, 1.09 mmol) in
dichloromethane (40 mL). After the complete addition, the mixture was stirred at
room temperature for 18 h. After evaporation of dichloromethane, n-hexane was
added to the crude residue. The resulting solid was collected by filtration to provide
triazine 15 as a white solid in 46 % yield (0.18 g); mp (n-C6H14) 93–96 °C;
Rf = 0.15 (EtOAc:n-hexane, 2:1). IR m cm-1: 1540, 1505, 1468, 1343, 1295, 1232,
1193, 1108, 1093. 1H NMR (CDCl3, 400 MHz): d (ppm) 4.15 (s, 6H, 2OCH3), 7.49
(t, J = 7.2 Hz, 1H, ArH), 7.88 (t, J = 7.2 Hz, 1H, ArH), 8.53 (d, J = 7.2 Hz, 1H,
ArH), 8.85 (d, J = 7.2 Hz, 1H, ArH). 13C NMR (CDCl3, 100 MHz): d (ppm) 55.5
(2CH3), 124.6 (2CH), 126.6 (CH), 137.2 (C), 150.2 (CH), 152.3 (C), 173.2 (2C).
Elemental analysis calcd (%) for C10H10N4O2: C 55.04, H 4.62, N 25.68; found: C
55.21, H 4.91, N 25.96.
10-(2-(4,6-Dimethoxy-1,3,5-triazin-2-yl)ethyl)-10H-phenothiazine (16) The gen-
eral procedure was used with triethylamine (0.11 mL, 0.81 mmol), activated ester
22 (0.3 g, 0.81 mmol) and zinc dimethyl imidodicarbonimidate 3b (0.16 g,
0.49 mmol) in dichloromethane. The mixture was stirred at room temperature for
24 h. The residue obtained upon evaporation was purified by chromatography
(EtOAc:n-hexane = 1:1) to provide pure 10-(2-(4,6-dimethoxy-1,3,5-triazin-2-
yl)ethyl)-10H-phenothiazine 16 as colorless crystals in 72 % yield (0.21 g); mp
(EtOAc:n-hexane) 89–92 °C; Rf = 0.68 (EtOAc:n-hexane, 1:1). IR m cm-1: 1559,
1
1547, 1496, 1459, 1443, 1382, 1353, 1251, 1232, 1194, 1114. H NMR (CDCl3,
400 MHz): d (ppm) 3.27 (t, J = 7.2 Hz, 2H, CH2), 3.98 (s, 6H, 2OCH3), 4.38 (bs,
2H, CH2), 6.82–7.05 (m, 4H, ArH), 7.05-7.22 (m, 4H, ArH). 13C NMR (CDCl3,
100 MHz): d (ppm) 36.1 (CH2), 44.7 (CH2), 55.5 (2CH3), 115.4 (2CH), 122.6
(2CH), 124.9 (2C), 127.3 (2CH), 127.4 (2CH), 144.7 (2C), 172.4 (2C), 180.6 (C).
Elemental analysis calcd (%) for C19H18N4O2S: C 62.28, H 4.95, N 15.29; found: C
62.32, H 5.04, N 15.48.
General procedure for the synthesis of activated esters (17, 20 and 22)
N-hydroxysuccinimide (1.2 equiv) and EDCI (1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride) or CDI (carbonyldiimidazole) (1.2 equiv) were added
to a suspension of carboxylic acid 10, 19 or 21 (1 equiv) in dichloromethane. The
reaction mixture was stirred at room temperature for 24 h. After concentration
under vacuum of the reaction media, the crude residue was recrystallized from
ethanol to give pure activated ester 17, 20 or 22.
1-{[(2E)-3-(1H-imidazol-1-yl)prop-2-enoyl]oxy}pyrrolidine-2, 5-dione (17) The
general procedure was used with CDI (1,10-Carbonyldiimidazole) (0.50 g,
3.05 mmol), N-hydroxysuccinimide (0.35 g, 3.06 mmol) and propiolic acid 10
(0.25 g, 2.55 mmol) in dichloromethane (35 mL). After complete homogenization
of the reaction medium, a white precipitate was formed. This was filtered and
123

L. Lucescu et al.
washed with CH2Cl2 to afford pure compound 17 as a white solid in 39 % yield
(0.23 g); mp (CH2Cl2) 209–211 °C. IR m cm-1: 1770, 1727, 1651, 1495, 1305,
1
1223, 1188. H NMR (DMSO-d6, 400 MHz): d (ppm) 2.87 (s, 4H, 2CH2), 6.80 (d,
J = 14.0 Hz, 1H, CH), 7.16 (s, 1H, ArH), 7.93 (s, 1H, ArH), 8.28 (s, 1H, ArH), 8.51
(d, J = 14.0 Hz, 1H, CH). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 25.5 (2CH2),
99.5 (CH), 117.3 (CH), 131.4 (CH), 139.6 (CH), 141.9 (CH), 162.3 (C), 170.3 (2C).
MS (EI) m/z (%): 235 (5) [M?], 138 (4), 121 (100), 93 (22).
1-[(2-Pyridinylcarbonyl)oxy]-2,5-pyrrolidinedione (20) The general procedure
was used with N-hydroxysuccinimide (1.12 g, 9.76 mmol), CDI (carbonyldiimida-
zole) (1.58 g, 9.76 mmol) and picolinic acid 19 (1.0 g, 8.13 mmol) in dichlor-
omethane (50 mL). After concentration of the reaction medium under vacuum, the
crude residue was recrystallized from ethanol to give pure activated ester 20 as a
white solid in 17 % yield (0.3 g) with the same physico-chemical properties as
described in the literature [59]; mp (EtOH) 172–175 °C. IR m cm-1: 1796, 1775,
1737, 1439, 1357, 1284, 1255, 1196. 1H NMR (DMSO-d6, 400 MHz): d (ppm) 2.93
(s, 4H, 2CH2), 7.84 (t, J = 7.6 Hz, 1H, ArH), 8.14 (t, J = 7.6 Hz, 1H, ArH), 8.24
(d, J = 7.6 Hz, 1H, ArH), 8.86 (d, J = 7.6 Hz, 1H, ArH). 13C NMR (DMSO-d6,
100 MHz): d (ppm) 25.6 (2CH2), 126.6 (CH), 128.4 (CH), 137.3 (CH), 144.0 (C),
150.4 (CH), 160.4 (C), 168.8 (2C).
1-{[3-(10H-Phenothiazin-10-yl)propanoyl]oxy}pyrrolidine-2,5-dione
(22) The
general procedure was used with N-hydroxysuccinimide (1.02 g, 8.82 mmol),
EDCI (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (1.69 g,
8.82 mmol) and carboxylic acid 21 (2.0 g, 7.38 mmol) in dichloromethane
(30 mL). After concentration of the reaction medium under vacuum, the crude
residue was recrystallized from ethanol to give pure activated ester 22 as a white
solid in 90 % yield (2.44 g) with the same physico-chemical characteristics as
described in the literature [45, 60, 61]; mp (EtOH) 159–160 °C. IR m cm-1: 1814,
1
1779, 1735, 1456, 1363, 1323, 1200, 1124, 1069, 997, 931, 838, 746. H NMR
(DMSO-d6, 400 MHz) d (ppm) 2.81 (s, 4H, 2CH2), 3.14 (t, J = 6.4 Hz, 2H, CH2),
4.28 (t, J = 6.4 Hz, 2H, CH2N), 7.0 (t, J = 7.6 Hz, 2H, ArH), 7.06 (d, J = 8.0 Hz,
2H, ArH), 7.18–7.26 (m, 4H, ArH). 13C NMR (DMSO-d6, 100 MHz) d 25.4
(2CH2), 28.9 (CH2), 41.5 (CH2), 115.5 (2CH), 123.0 (2CH), 124.0 (2C), 127.3
(2CH), 127.7 (2CH), 144.1 (2C), 167.4 (C), 170.0 (2C).
(E)-N,N-Diethyl-2-(4,6-dimethoxy-1,3,5-triazin-2-yl)ethenamine (18)
To a solution of 2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1 (0.58 g, 3.52 mmol) in
CH2Cl2 (25 mL), diethylamine (0.36 mL, 3.42 mmol) was added. The obtained
solution was stirred at room temperature for 15 h. The residue obtained upon
evaporation was purified by flash chromatography (EtOAc/n-heptane) to provide
pure (E)-N,N-diethyl-2-(4,6-dimethoxy-1,3,5-triazin-2-yl)ethenamine 18 as an yel-
low oil in 70 % yield (0.58 g); Rf = 0.18 (EtOAc:n-heptane, 4:6). IR m cm-1: 2974,
1
2941, 2874, 1629, 1539, 1512, 1445, 1404, 1338, 1255, 1193, 1115, 971. H NMR
123

Synthesis and biological evaluation of a new class of…
(CDCl3, 400 MHz): d (ppm) 1.21 (t, J = 7.1 Hz, 6H, 2CH3), 3.30 (q, J = 6.7 Hz,
4H, 2CH2), 3.97 (s, 6H, 2OCH3), 5.13 (dd, J = 13.0, 1.6 Hz, 1H, CH), 8.03 (dd,
J = 13.0, 1.6 Hz, 1H, CH). 13C NMR (CDCl3, 100 MHz): d (ppm) 11.4 (CH3), 14.6
(CH3), 42.2 (CH2), 50.2 (CH2), 54.0 (2CH3), 91.8 (CH), 149.8 (CH), 171.2 (C),
176.1 (2C). Elemental analysis calcd (%) for C11H18N4O2: C 55.44, H 7.61, N
23.51; found: C 55.73, H 7.44, N 23.79.
Biological evaluation
Farnesyltransferase assay Assays were conducted in 96-well plates, prepared with a
Biomek NKMC and a Biomek 3000 from Beckman Coulter and read on a Wallac
Victor fluorimeter from Perkin–Elmer. Per well, 20 lL of farnesylpyrophosphate
(10 lM) was added to 180 lL of a solution containing 2 lL of varied concentra-
tions of potential inhibitors (dissolved in DMSO) and 178 lL of a solution
composed by 10 lL of partially purified recombinant human FTase (1.5 mg/mL)
and 1.0 mL of Dansyl-GCVLS peptide [in the following buffer: 5.6 mM DTT,
5.6 mM MgCl2, 12 lM ZnCl2 and 0.2 % (w/v) octyl-b-D-glucopyranoside, 52 mM
Tris/HCl, pH 7.5]. Fluorescence development was recorded for 15 min (0.7 s per
well, 20 repeats) at 30 °C with an excitation filter to 340 nm and an emission filter
of 486 nm. Each measurement was realized twice, in duplicate or in triplicate. The
kinetic experiments were realized under the same conditions, either with FPP as
varied substrate with a constant concentration of Dns-GCVLS of 2.5 lM, or with
Dns-GCVLS as varied substrate with a constant concentration of FPP of 10 lM.
Nonlinear regressions were performed by KaleidaGraph 4.03 software [62].
Cell proliferation assay Compounds 2a–i have been tested on a panel of 60 human
cancer cell lines at the National Cancer Institute, Rockville, MD. The cytotoxicity
studies were conducted using a 48 h exposure protocol using the sulforhodamine B
assay [63, 64].
Results and discussion
The activity of newly triazin–triazoles 2a–i was evaluated on human farnesyltrans-
ferase (FTase) [62] and on the NCI-60 human tumor cell lines [63, 64]. The results
are summarized in Tables 2 and 3. Interestingly, triazin–triazole 2c, substituted by a
para-bromophenyl unit, showed the best inhibitory activity on farnesyltransferase
(IC50 = 38.62 1.7 lM) in the current study (Table 3). The para-chloro analogue,
compound 2b, showed decreased biological potential (IC50 = 72.05 6.9 lM),
confirming the structure–activity relationships previously established [45]. The
other investigated substituents (F, CN, Me or OMe) at the para-position of the
phenyl ring in compounds 2a, 2d–f or the replacement of the phenyl unit by a
phenothiazine or an indazole moiety in compounds 2g–i abolished the inhibitory
activity toward FTase. In conclusion, the investigation of triazin–triazoles 2a–i as
potential inhibitors of human FTase furnished compounds with decreased inhibitory
potency relative to our previously described triazoles [45].
123

L. Lucescu et al.
Table 3 Results of the human farnesyltransferase assay
Compound % Inh (FTase)a,b IC50 (lM) SD Compound % Inh (FTase)a,b IC50 (lM) SD
2a
2b
2c
2d
2e
19
59
73
37
48
–
2f
0
–
–
–
–
72.05 6.9
2g
2h
2i
0
38.62 1.7
35
24
–
–
SD standard deviation
a
Inhibition ratio of protein farnesyltransferase at a 100 lM concentration
b
Values represent mean of two experiments
Table 4 Results of the NCI cell growth inhibition assay. In vitro percentage growth inhibition (GI%)
caused by the selected compounds against some tumor cell lines in the single-dose assaya
Compound
Leukemia
K-562
Non-small cell lung cancer
Renal cancer
MOLT-4
SR
HOP-62
HOP-92
NCI-H522
A498
UO-31
2a
2b
2c
2d
2e
2f
2g
2h
2i
21
7
17
6
12
6
13
n.a.
12
13
13
7
11
n.a.
20
21
16
13
21
14
28
26
28
26
n.a.b
n.a.
n.a.
n.a.
10
n.a.
n.a.
5
n.a.
n.a.
n.a.
n.a.
n.a.
11
10
9
3
18
6
19
n.a.
10
13
13
6
8
5
18
16
14
14
14
12
n.a.
n.a.
10
n.a
26
9
20
24
n.a.
13
11
25
n.a.
6
n.a.
a
Data obtained from NCI’s in vitro disease-oriented human tumor cell screen at 10 lM concentration
b
No inhibitory effect
The results of the NCI single-dose assay at a 10 lM concentration of each
compound indicated that triazin–triazoles 2a–i have low cytostatic effects (Table 4).
The most interesting growth inhibition potentials have been registered for
compounds 2f and 2h on UO-31 renal cancer cell lines (28 % inhibition).
Conclusion
The investigation of the triazin–triazole unit as a potential chelating unit of the zinc
atom of human farnesyltransferase resulted in the discovery of a new series of
compounds 2a–i with unprecedented biological activity. The best compound in the
current study showed an IC50 value against FTase of 38.6 lM. This activity is
123

Synthesis and biological evaluation of a new class of…
moderate comparing to known families of inhibitors of protein farnesyltransferase.
However, this new class of triazin–triazoles deserves further chemical modulations
on different points of the skeleton that could valuably lead to significant biological
results.
From a synthetic strategy point of view, 2-ethynyl-4,6-dimethoxy-1,3,5-triazine 1
proved to be a new heterocycle with interesting properties. Indeed, in this
compound, the triazine group, which is substituted by two strongly electro-donating
methoxy groups proves to be strongly withdrawing,3 able to activate the triple bond
toward nucleophile addition (Cl-, imidazole or diethylamine). Ethynyldimethoxy-
triazine 1 is a significant platform-compound for developing new chemical entities
for anticancer research and for other biological applications. It is recognized that
alkynes constitute privileged structural units in medicinal chemistry and chemical
biology [67].4 Consequently, further chemical and biological studies are necessary
to highlight the great importance of molecule 1.
Acknowledgments This work was supported by the strategic Grant POSDRU/159/1.5/S/137750,
Project ‘‘Doctoral and Postdoctoral programs support for increased competitiveness in Exact Sciences
research,’’ co-financed by the European Social Fund within the Sectorial Operational Program Human
Resources Development 2007–2013 (Ph. D. scholarship L. L.). The authors acknowledge the National
Cancer Institute (NCI) for biological evaluation of compounds on their 60-cell panel: the testing was
performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis
(the URL to the Program’s website: http://dtp.cancer.gov).
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