Discovery of new hit-molecules targeting Plasmodium falciparum through a global SAR study of the 4-substituted-2-trichloromethylquinazoline antiplasmodial scaffold

Article abstract of DOI:10.1016/j.ejmech.2016.09.029

From 4 antiplasmodial hit-molecules identified in 2-trichloromethylquinazoline series, we conducted a global Structure-Activity relationship (SAR) study involving 26 compounds and covering 5 molecular regions (I – V), aiming at defining the corresponding

Full text of DOI:10.1016/j.ejmech.2016.09.029

Accepted Manuscript
Discovery of new hit-molecules targeting Plasmodium falciparum through a global
SAR study of the 4-substituted-2-trichloromethylquinazoline antiplasmodial scaffold
Justine Desroches, Charline Kieffer, Nicolas Primas, Sébastien Hutter, Armand Gellis,
Hussein El-Kashef, Pascal Rathelot, Pierre Verhaeghe, Nadine Azas, Patrice Vanelle
PII:
S0223-5234(16)30759-0
10.1016/j.ejmech.2016.09.029
EJMECH 8895
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 July 2016
Revised Date: 8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: J. Desroches, C. Kieffer, N. Primas, S. Hutter, A. Gellis, H. El-Kashef, P.
Rathelot, P. Verhaeghe, N. Azas, P. Vanelle, Discovery of new hit-molecules targeting Plasmodium
falciparum through a global SAR study of the 4-substituted-2-trichloromethylquinazoline antiplasmodial
scaffold, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.029.
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ACCEPTED MANUSCRIPT
Graphical Abstract:

ACCEPTED MANUSCRIPT
Discovery of new hit-molecules targeting Plasmodium falciparum through a
global SAR study of the 4-substituted-2-trichloromethylquinazoline
antiplasmodial scaffold
1
#
1#
1
2
1
Justine Desroches , Charline Kieffer , Nicolas Primas , Sébastien Hutter , Armand Gellis ,
3
1
4*
2
1*
Hussein El-Kashef , Pascal Rathelot , Pierre Verhaeghe , Nadine Azas and Patrice Vanelle
1
Aix-Marseille Université, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de
Pharmacie, 27 Boulevard Jean Moulin – CS30064, 13385 Marseille cedex 05, France.
2
Aix-Marseille Université, UMR MD3, Infections Parasitaires, Transmission et Thérapeutique, Faculté
de Pharmacie, 27 Boulevard Jean Moulin – CS30064, 13385 Marseille cedex 05, France.
3
Department of Chemistry, Faculty of Science, Assiut University, 71516 Assiut, Egypt.
4
Université Paul Sabatier, Faculté des Sciences Pharmaceutiques − CNRS UPR 8241, Laboratoire de
Chimie de Coordination, 205 Route de Narbonne, 31077 Toulouse cedex 04, France.
#
*
Co-first authors. Corresponding authors: E-mail addresses: pierre.verhaeghe@univ-tlse3.fr (P.
Verhaeghe), patrice.vanelle@univ-amu.fr (P. Vanelle).
Abstract:
From 4 antiplasmodial hit-molecules identified in 2-trichloromethylquinazoline series, we
conducted a global Structure-Activity relationship (SAR) study involving 26 compounds and
covering 5 molecular regions (I – V), aiming at defining the corresponding pharmacophore
and identifying new bioactive derivatives. Thus, after studying the aniline moiety in detail,
thienopyrimidine, quinoline and quinoxaline bio-isosters were synthesized and tested on the
K1 multi-resistant P. falciparum strain, along with a cytotoxicity evaluation on the human
HepG2 cell line, to define selectivity indecies. SARs first showed that thienopyrimidines and
quinolines were globally more cytotoxic, while quinoxaline analogs appeared as active as-
and less cytotoxic than their quinazoline counterparts. Such pharmacomodulation in
quinoxaline series not only provided a new antiplasmodial reference hit-molecule (IC = 0.4
5
0
µM, selectivity index = 100), but also highlighted an active (IC50 = 0.4 µM) and quite
selective (SI = 265) synthesis intermediate.
Highlights:
►
Antiplasmodial pharmacomodulation of CCl -substituted-nitrogen containing heterocycles
3
was made.► Thienopyrimidine derivatives appeared more cytotoxic. ► Original 3-
substituted-2-trichloromethylquinoxaline analogs were prepared. ►Two quinoxaline
derivatives displayed in vitro IC values of 0.4 and 0.5 µM on the K1 multi-resistant P.
5
0
falciparum strain. ► Cytotoxicity was assessed on the human HepG2 cell line showing low
cytotoxicity (CC ~ 40 µM) and improved selectivity indecies (77-100).
5
0

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Keywords: Quinazoline; Quinoline; Quinoxaline; Thienopyrimidine; Trichloromethyl goup;
Plasmodium falciparum; In vitro antiplasmodial activity; In vitro HepG2 cytotoxicity,
Structure-Activity Relationships.
1
. Introduction
Cerebral malaria, caused by Plasmodium falciparum, is the leading cause of death among
parasitic infections worldwide. According to the 2015 World Malaria Report [1], 214 million
people were infected by Plasmodium in 2014, leading to 438.000 deaths out of which about
8
8% occurred in Africa, mainly in children under five. It is to note that a significant
improvement of the situation has been noted since the beginning of the 21st century, thanks to
the action of the WHO and the financial involvement of several non-governmental
organizations.
However, the control of the infection is facing worldwide the emergence of drug-resistant
strains of the parasite which turns into a major concern for the medical and scientific
community. Indeed, the WHO recommended the treatment of P. falciparum malaria is based
on an artemisinin-combination therapy (ACT), but resistances to artemisinin derivatives are
emerging in Asia [2], and it was demonstrated that they were responsible for therapeutic
failures in several infected patients [3,4]. Moreover, it was also highlighted that the African
Anopheles gambiae mosquito could transmit such Asian resistant parasites [5], indicating a
major worldwide spreading risk. Thus, research efforts have to be developed, in order to
discover new chemical entities presenting novel mechanisms of action, to complete and
guaranty the durable efficiency of ACTs.
Several research teams working in the field of antimalarial agents previously reported
novel quinazoline derivatives displaying significant antiplasmodial activities, in particular
when bearing an amino- [6], alkylamino- [7,8] or an aniline- substituent [9] at position 4 of
the quinazoline ring.
The research activity of our group is focused on the synthesis and anti-infective evaluation
of new nitrogen-containing heterocycles [10]. We have intensively studied a large series of
antiplasmodial derivatives based on the original 2-trichloromethylquinazoline scaffold. Thus,
the synthesis and in vitro biological evaluation of a large quinazoline chemical library bearing
an arylamino- [11,12], aryl- [13], phenoxy- [14], phenylthio- [15], sulfonamido- [16],
alkynyl- [17], heteroarylamino- [18], benzyloxy- or alkoxy- [19] substituent at position 4 of
the quinazoline ring, revealed several hit-molecules (A-D) which are presented in Figure 1.

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Figure 1. Antiplasmodial hit-molecules identified in 2-trichloromethylquinazoline series.
In the light of these hit-molecules, within the aim of defining the antiplasmodial Structure
Activity Relationships (SARs) of the corresponding series, 5 key-regions (I-V) were chosen
and modulated, as summarized in Figure 2. Thus, for region I, we first investigated the role of
the –CCl group by replacing it by various substituents including closely related halogenated
3
groups. For region II, we studied the influence of the combination of the halogen atoms on the
aniline moiety of hit A. The effect of the methylation of the secondary amine nitrogen of hit A
was then investigated in region III. Further modulations in region IV were made by replacing
the quinazoline ring of hits A-D by a bio-isosteric thienopyrimidine. Indeed, starting from the
natural quinazoline-based antimalarial molecule Febrifugine, such ring replacement was
successfully carried out [20]. Moreover, some recent thienopyrimidine derivatives
demonstrated promising antiplasmodial activities [21,22]. Finally, in region V, we looked for
novel analogs, centered either on the quinoline nucleus, as this heterocycle is encountered in
numerous antimalarial drugs such as quinine, chloroquine, mefloquine, amodiaquine,
piperaquine and primaquine, or on the quinoxaline nucleus, taking advantage of previously
reported results obtained in our team in the pyrrolo[1,2-a]quinoxaline series [23]. The
synthesis of all new derivatives, their in vitro biological evaluation and the resulting SARs is
presented and discussed herein.
Figure 2. Antiplasmodial SAR study involving 5 key-regions (I-V) from the quinazoline hits

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2
. Results and discussion
.1. Synthesis of hit A analogues
2
2
.1.1. Region I: Variation of the nature of the substituent at position 2 of the
quinazoline ring
The nature of the substituent at position 2 of hit A was first studied. Thus, the synthesis of
the 2-methyl- 1, 2-chloromethyl- 2, 2-dichloromethyl- 3, 2-chloro-2-difluoro-methyl- 7, 2-
trifluoromethyl- 8 and the 2-unsubstituted- 9 quinazoline analogs were operated, starting from
appropriate 4-chloroquinazolines (Scheme 1). Compound 1 [11] was already synthesized by
our team via an Aromatic Nucleophilic Substitution (S Ar) reaction, starting from 4-chloro-2-
N
trichloromethylquinazoline E [24]. Derivatives 2 and 3 were prepared by S Ar reactions
N
starting
from
4-chloro-2-chloromethylquinazoline
F
[25]
and
4-chloro-2-
dichloromethylquinazoline G [26]. The synthesis of the parent 2-unsubstituted compound 9
was already reported in the literature, but to the best of our knowledge, it was never studied
for its antiplasmodial potential [27]. The biological evaluation of the above prepared
quinazolines is summed up in Table 1.
Cl
Cl
Cl
N
HN
i
N
N
R
N
R
1-3, 7-9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
HN
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
CH3
N
CH Cl
N
CHCl2
N
CF Cl
N
CF3
N
H
2
2
[
11]
1
71%
2
42%
3
30%
7
37%
8
[27] 9
Scheme 1. SAR modulation of region I: modification of the substituent at position 2 of the
quinazoline ring.
Reagents and conditions : (i) 2,4-dichloroaniline 0.8 equiv, conc. HCl cat., iPrOH, 70 °C, 2 h.
For the synthesis of derivative 7, the corresponding 4-chloroquinazoline intermediate was
not reported in the literature. Thus, starting from 2-aminobenzonitrile, acylation of the amine
was made by reacting with 2-chloro-2-difluoroacetic acid in the presence of the coupling
agent N-dimethylaminopropyl-N’-ethylcarbodiimide (EDCI) and dimethylaminopyridine

ACCEPTED MANUSCRIPT
(DMAP), affording 4 in low yield (15%). Then, a smooth oxidative hydration of nitrile 4 was
operated with H O , followed by a cyclization in alkaline medium [17,28], leading to
2
2
quinazolinone 5. This last was further chlorodehydroxylated using POCl , affording the
3
expected intermediate 6 (Scheme 2).
Scheme 2. Variation of the region I: Synthesis of intermediate 6.
Reagents and conditions : (i) 2-chloro-2-difluoroacetic acid 1 equiv, EDCI.HCl 1 equiv,
DMAP 0.8 equiv, CH Cl , 0 °C then rt, 24 h; (ii) H O 30%, NaOH, EtOH/H O, 0 °C then rt,
2
2
2
2
2
9
0 min; (iii) POCl , 140 °C, MW, 10 min, 800 W.
3
2
.1.2. Region II: Variation of the halogen atoms at position 2’ and 4’ of the 4-
anilino-substituent
The synthesis of the anilino derivatives 10-13 was easily performed under classical S Ar
N
reaction conditions by reacting the appropriate 2,4-dihaloaniline with 4-chloro-2-
trichloromethylquinazoline E in refluxing isopropanol, in 55-70 % range yield (Scheme 3).
The results of their in vitro biological evaluation are presented in Table 1.
Scheme 3. Chemical modulations at regions II and III of the pharmacophore
Reagents and conditions: (i) 2,4-dihaloaniline (2 equiv) or 2,4-dichloro-N-methylaniline (1.1
equiv), iPrOH, reflux, 2 h.

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2
.1.3. Region III: N-methylation of the 4-(2’,4’-dichlorophenyl)amine substituent
The influence of the hydrogen atom on the secondary amine, in region III, was also
studied. For this purpose, a S Ar reaction was performed between E and N-methyl-2,4-
N
dichloroaniline, affording the expected methylated compound 14 in low yield (Scheme 3).
The biological evaluation of compound 14 is summarized in Table 1.
2
.2. Biological evaluation of the series belonging to regions I, II and III
These new derivatives were evaluated in vitro toward both their antiplasmodial activity
against the K1 multi-resistant P. falciparum strain (determination of the IC = inhibitory
5
0
concentration 50%) and their cytotoxicity (determination of the CC50 = cytotoxic
concentration 50%) on the HepG2 human cell line. The results were compared with three
commercial antimalarial reference-drugs (atovaquone, chloroquine and doxycycline) and a
cytotoxic reference drug (doxorubicine). For all tested compounds, the corresponding
selectivity indecies (SI) were calculated (SI = CC / IC ). The results are presented in Table
50
50
1
.
The aim of the modulation of region I was to confirm that the trichloromethyl group, at
position 2 of the quinazoline ring, was the only substituent providing the antiplasmodial
activity. Indeed, the antiplasmodial activity was totally lost when replacing it with other
groups, including closely related ones such as CF or CHCl . Looking at the combination of
3
2
the halogen substituents on the aniline ring at position 4 (region II), the antiplasmodial
activity was maintained for brominated compounds 10-12, in comparison with hit A
(respectively 0.3, 0.5 and 0.6 vs 0.4 µM), but the cytotoxicity was significantly increased,
leading to less selective molecules. Contrary to the results obtained with brominated
analogues, fluorinated analogue 13 presented the same cytotoxicity profile as hit A but was
less active (IC50 = 3.1 µM). Finally in region III, the methylation of the aniline moiety
(compound 14) significantly impaired both antiplasmodial activity and cytotoxicity.

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Table 1. In vitro antiplasmodial and cytotoxicity evaluation of compounds 1-3 and 7-14:
Modulations at regions I, II and III.
R4
N
N
R2
HepG2 CC50 K1 P. falciparum
Selectivity
Index
Molecule
-R2
-R4
c
(µM)
IC (µM)
5
0
1
2
3
-CH
3
15
85
0.18
< 0.04
< 0.09
0.14
-
d
-CH
Cl
0.5
4.7
7
>12.5
>50
48.4
>50
>50
0.3
2
-CHCl
2
7
8
9
-CF Cl
2
-CF
-H
>50
35
2
3
<0.7
6.7
10
11
12
13
14
-CCl
-CCl
-CCl
-CCl
-CCl
3
3
3
3
3
5
0.5
10
2.9
0.6
4.8
19.6
10
3.1
6.3
3.4
2.9
Hit A
-CCl3
16
0.4
-
0.001
0.6
40
a
Doxorubicine
Atovaquone
Chloroquine
Doxycycline
0.2
> 15.6
30
-
15600
50
b
d
b
b
20
6.0
3.3
a
b
Doxorubicine was used as a cytotoxic reference-drug; Atovaquone, Chloroquinine and doxycycline were used
as antimalarial reference-drugs, Selectivity indecies were calculated according to the formula : SI = HepG2
c
d
CC / K1 IC . Highest concentration tested due to a lack of solubility.
5
0
50

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2
.3. Region IV: Synthesis of thieno[3,2-d]pyrimidine series.
The replacement of the phenyl ring of the quinazoline nucleus, by a thiophene nucleus as a
bio-isostere, was next studied. We first tried to synthesize 4-substituted-2-
(
trichloromethyl)thieno[3,2-d]pyrimidine derivatives by a S Ar reaction between the key
N
chlorinated intermediate 20 and various nucleophilic species, corresponding to the
substituents borne by hits A-D, respectively 2,4-dichloroaniline a, 3-trifluoromethylaniline b,
4
-chlorophenol c and 4-chlorothiophenol d (Figure 3).
Figure 3. Thieno[3,2-d]pyrimidines initial retrosynthesis pathway.
Starting from the commercially available 3-aminothiophene-2-carboxylate, we first
acetylated the amino group in presence of Ac O, leading to 15 in 78% yield [29]. Then, the
2
cyclization was made by heating 15 in presence of 25% ammonia in a sealed vial, affording
thienopyrimidinone 16 [30] in 63% yield. Unfortunately, in our hands, the chlorination
reaction of 16, to access 20, did not succeed. Thus, we decided to realize the chlorination
reaction in 2 steps. We first synthesized 4-chloro-2-methylthieno[3,2-d]pyrimidine 17 [30].
Then, 17 was engaged in a second chlorination reaction with PCl /POCl , under classical
5
3
heating or microwave activation to convert the 2-methyl group into a 2-trichloromethyl
substituent. Unfortunately, here again, no trace of the key intermediate 20 was formed, and
only inseparable mixtures of polychlorinated compounds were observed by LC-MS analysis
(Scheme 4).

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iii
x
O
S
O
OH
Cl
N
Cl
N
iii
S
OMe
i
ii
iv
S
S
S
OCH3
N
N
N
x
NH
7
8%
NH2
N
CH3
CH3
17
CCl3
O
15
CH3
63% 16
97%
99% 20
v
iv
O
OH
N
S
ii
S
OMe
x
NH
N
CCl3
9
9% 18 O
63%
19
CCl3
vi
Scheme 4. Synthesis of key intermediate 20.
Reagents and conditions: (i) Ac O, AcOH cat., 120 °C, MW, 10 min; (ii) NH OH 25%, 105
2
4
°
C, 3 h, sealed vial; (iii) PCl 6 equiv, POCl as solvent, 100 °C, MW, 20 min, 800 W; (iv)
5 3
POCl , pyridine cat., reflux 24 h (molecule 17) or 1 h (molecule 20); (v) Et N, trichloroacetyl
3
3
chloride, 5 °C, 30 min; (vi) AcOH saturated with gaseous HCl, trichloroacetonitrile, 100 °C,
1
8 h, evaporated then refluxed in iPrOH, 5 min.
To introduce the –CCl group, we chose to start again from 3-aminothiophene-2-
3
carboxylate and carried out the acylation reaction, using trichloroacetyl chloride in presence
of Et N. The corresponding product 18 was obtained in quantitative yield. Unfortunately, the
3
following cyclization step to lactam 19 was unsuccessful under the reaction conditions used
(
either NH OH at 105 °C in sealed vial or NH in MeOH at 120 °C under MW). Finally, we
4
3
succeeded in preparing lactam 19 by reacting 3-aminothiophene-2-carboxylate with
trichloroacetonitrile in AcOH, saturated with gaseous HCl [31]. This was a similar approach
to the one of Ried [32] who used a more expensive reagent: methyl 2,2,2-trichloroacetimidate.
Then, the chlorodehydroxylation of lactam 19 in refluxing POCl in the presence of a catalytic
3
amount of pyridine, furnished the key intermediate 20 in almost quantitative yield. S Ar
N
reactions between 20 and some N, O or S centered nucleophiles were then performed in a
similar way to those operated in quinazoline series. The target 4-substituted-2-
(trichloromethyl)thieno[3,2-d]pyrimidine derivatives 21a-d were obtained, however, in low to
moderate yields (7-55%) (Scheme 5). The results of the biological evaluation of this series are
presented in Table 2.

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Scheme 5. Synthesis of 4-substituted-2-(trichloromethyl)thieno[3,2-d]pyrimidine.
Reagents and conditions: (i) aniline derivative a or b 1 equiv, conc. HCl cat., EtOH, 100 °C,
1
8 h, sealed vial; (ii) NaH 2 equiv, 4-chlorophenol c or 4-chlorothiophenol d 1 equiv, DMSO,
rt or 50 °C, 24 h.
2
.4. Region IV: Synthesis of the thieno[2,3-d]pyrimidine series.
To complete this SAR study with more thiophene-containing bio-isosteres, we decided to
synthesize the position isomer analogues in thieno[2,3-d]pyrimidine series. This could be
performed by a S Ar reaction between the desired nucleophiles a-d and a key chlorinated
N
intermediate 25, obtained from 2-aminothiophene-3-carbonitrile (Figure 4).
Figure 4. Thieno[2,3-d]pyrimidines initial retrosynthesis pathway.
Thus, 2-aminothiophene-3-carbonitrile was acetylated with Ac O to give 22 [33]. Then, the
2
cyclization was made in the presence of H O 30% in alkaline medium (NaOH) [17,28]
2
2
leading to thienopyrimidinone 23 [34] which was then chlorodehydroxylated into 24.
Unfortunately, as previously observed in the thieno[3,2-d]pyrimidine series, we did not
succeed in synthesizing 25, neither by the gem-trichloromethylation of 24 [35], nor by the
tetrachlorination of 23 with in a PCl /POCl mixture. (Scheme 6). Acylation of 2-
5
3
aminothiophene-3-carbonitrile was then realized with trichloroacetyl chloride, to give 26.
Cyclization into lactam 27 was next conducted with a mixture of polyphosphoric acid (PPA)

ACCEPTED MANUSCRIPT
and phosphoric acid. Nevertheless, once again, it remained impossible to isolate key
intermediate 25 via a chlorodehydroxylation reaction. It seemed that 25 was too unstable to be
isolated, indeed some traces were detected by LC-MS, but direct engagement of crude 25 in
SNAr reaction did not afforded target compounds.
Scheme 6. Various synthesis routes investigated for the synthesis of key intermediate 25.
Reagents and conditions: (i) Ac O, AcOH cat., 120 °C, MW, 10 min; (ii) H O 35%, NaOH,
2
2
2
EtOH/H O, 0 to 55 °C, 15 min; (iii) PCl 6 equiv, POCl as solvent, 100 °C, MW, 20 min,
2
5
3
8
00 W; (iv) POCl , pyridine cat., reflux 24 h; (v) Et N, trichloroacetyl chloride, rt, 36 h; (vi)
3 3
H PO , polyphosphoric acid, 70 °C, 3 h; (vii) POCl or PCl or oxalyl chloride or
3
4
3
5
P O /tetrabutylammonium chloride (TBACl) or SOCl .
2
5
2
Facing the difficulty to prepare the intermediate 25 and in order to access to the target
thieno[2,3-d]pyrimidine derivatives, we tried another synthesis pathway based on the
cyclization of thiophene derivatives which could be cyclized into thieno[2,3-d]pyrimidine
rings bearing already both the CCl group and an aniline (or phenol or thiophenol) moiety
3
(Scheme 7). Thus the synthesis of amidines 28a and 28b was conducted by reacting nitrile 26
with appropriate anilines in presence of AlCl at room temperature in CH Cl [36]. The crudes
3
2
2
residues obtained were a mixture of unreacted starting material 26 (even after 72 h at rt), the
expected amidines 28a-b and cyclized target derivatives 29a and 29b. When the temperature
was increased to improve the conversion, undesirable side products appeared. Refluxing the
obtained amidines 28a-b in ethanol led to the target compounds 29a and 29b, respectively in
3
6 and 15% yields [37]. For phenol c and thiophenol d, the same conditions using AlCl did
3
not afford the expected derivatives. We then considered the synthesis work of Baati and al.,

ACCEPTED MANUSCRIPT
describing the addition of thiols onto nitrile groups in presence of gaseous HBr in ether at 0
°
C [38]. Indeed, to our delight, by using these reaction conditions, thioimidate 28d was
produced, and further cyclization in refluxing ethanol afforded compound 29d in low yield
Scheme 7). Unfortunately, the same reaction conditions were inefficient to obtain
(
compounds 28c and 29c. Other conditions were attempted in order to produce the imidate 28c
via the addition of 4-chlorophenol into the nitrile group, in the presence of NaH [39], Na [40],
K CO [41] or gaseous HCl [42], but all of them conducted to the same disappointing result.
2
3
The results of the biological evaluation are presented in Table 2.
Scheme 7. Synthesis of 4-substituted-2-(trichloromethyl)thieno[2,3-d]pyrimidine 29a-d.
Reagents and conditions: (i) anilines a or b, AlCl , CH Cl , rt, 72 h; (ii) 4-chlorothiophenol,
3
2
2
gaseous HBr, Et O, - 20 °C, 30 min then 18 h at rt; (iii) iPrOH, reflux, 5 min.
2
2
.5. Modulation of region IV: Biological evaluation
In region IV, the replacement of the quinazoline ring by thienopyrimidine scaffolds led to
molecules displaying similar antiplasmodial activity as hit-molecules A-D, with IC values in
5
0
the submicromolar range (0.4 to 0.9 µM) (Table 2). However, all the thienopyrimidine
derivatives, belonging either to the [3,2-d] or [2,3-d] series, appeared more cytotoxic (CC =
50
0
.7 to 13.3 µM) than the corresponding hit-molecules, leading to lower selectivity indecies.
Globally, SARs indicate that there is no significant difference between the two
thienopyrimidine series, regarding both activity and cytotoxicity.

ACCEPTED MANUSCRIPT
Table 2. In vitro antiplasmodial and cytotoxicity evaluation of compounds 21a-d and 29a-d:
modulations at region IV
HepG2 CC50 K1 P.falciparum Selectivity
Molecule
Structure
c
(µM)
IC (µM)
Index
5
0
2
1a
3.2
0.6
5.3
2
1b
0.7
4.3
6.9
6.2
4.0
0.9
0.6
0.4
0.8
7.2
2
1c
2
1d
17.2
2
9a
0.5
0.6
0.8
12.4
6.7
2
2
9b
9d
13.3
16.6
Hit A
Hit B
Hit C
Hit D
16
150
50
0.4
1.8
1.1
0.9
-
0.001
0.6
6.0
40
83
45
>28
-
15600
50
3.3
d
>25
a
Doxorubicine
Atovaquone
Chloroquine
Doxycycline
0.2
> 15.6
30
b
d
b
b
20
a
b
Doxorubicine was used as a cytotoxic reference-drug; Atovaquone, Chloroquinine and doxycycline were used
as antimalarial reference-drugs, Selectivity indecies were calculated according to the formula : SI = HepG2
CC / K1 IC . Highest concentration tested due to a lack of solubility.
c
d
5
0
50

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2
.6. Region V: Synthesis of quinoline series.
In order to evaluate the influence of the pyrimidine moiety of the quinazoline ring onto the
biological profile, we chose to synthesize some analogs in 4-substituted-2-
trichloromethylquinoline series bearing, at position 4, the same substituents a-d as the hit-
molecules. Thus, starting from 4-chloro-2-trichloromethyquinoline H obtained by
chlorination of 2-methylquinolin-4(1H)-one with PCl and POCl [42], we first tried solvent-
5
3
free S Ar reaction conditions [11] with anilines but without any conversion. Compounds 30a
N
and 30b were finally obtained by using a catalytic amount of conc. HCl in refluxing EtOH to
obtain the desired compounds in 45 and 55% yields respectively. For the phenol c and
thiophenol d, we generated the corresponding anions using NaH before reacting H. Thus,
phenol derivative 30c and thiophenol derivative 30d were obtained, respectively, in 72 and
3
2% yields (Scheme 8). The biological evaluation of these molecules is summarized in Table
3
.
Scheme 8. Synthesis of 4-substituted-2-trichloromethylquinoline series.
Reagents and conditions : (i) aniline derivative 1 equiv, conc. HCl cat., EtOH, 100 °C, 18 h,
sealed vial; (ii) NaH 2 equiv, 4-chlorophenol or 4-chlorothiophenol reagent 1 equiv, DMSO,
rt or 50 °C, 24 h.
2
.7. Region V: Synthesis of quinoxaline derivatives.
Previously, we reported that 2-trichloromethylquinoxaline I displayed an in vitro
antiplasmodial activity (IC = 1.5 µM), hindered by a significant in vitro cytotoxicity (CC =
50
50
3
.1 µM) [23]. Moreover, the introduction of a phenyl substituent at position 3 (compound J)
gave a more selective derivative J (SI = 17.5 versus 2.1, respectively), thanks to a better

ACCEPTED MANUSCRIPT
antiplasmodial IC50 value (0.2 µM) [23]. Such interesting results noted with compound J
prompted us to carry on the investigation of the antiplasmodial potential of the 3-substituted-
2
-trichloromethylquinoxaline scaffold, in continuation of the pharmacomodulation of the
quinazoline moiety in region V (Figure 5).
Figure 5. Rational for the synthesis of 2-substituted-3-trichloromethylquinoxaline derivatives.
The first attempted synthetic route involved the reaction of nucleophiles a-d with 2-chloro-
3
-trichloromethylquinoxaline K [44] under S Ar conditions, as previously reported in
N
quinazoline series. Unfortunately, all tested conditions failed to afford the expected
derivatives 32a-d, where no conversion or degradation of starting material could occur.
To solve this problem, we next investigated another pathway by reacting 2-chloro-3-
methylquinoxaline L [45] with the nucleophiles a-d via a S Ar reaction, before operating the
N
gem-trichloromethylation reaction (Scheme 9). The best reaction conditions found were to
carry out the S Ar reaction in DMF, under moderate heating, in the presence of Cs CO
3
N
2
leading to 31a, 31b, 31c and 31d in, respectively, 19%, 5%, 85% and 83% respectively.
Another strategy to improve these low reaction yields was successful only with 3-
trifluoromethylaniline b, by reacting L in aniline b (without any solvent) under microwave
heating in a sealed vial (69%). Then, from intermediates 31a-d, a final chlorination step was
needed to transform the methyl group into a –CCl one. This step was achieved using the
3
classical route elaborated by our team using a PCl /POCl mixture, under microwave heating.
5
3
Unfortunately, only the ether and thioether derivatives 32c and 32d were obtained in good
yields (70-72%) (Scheme 9). It seemed that the presence of the amine function of 31a and
3
1b inhibited the chlorination reaction. The results of the biological assays of this series are
presented in Table 3.

ACCEPTED MANUSCRIPT
Scheme 9. Synthesis of 2-substituted-3-trichloromethylquinoxaline series.
Reagents and conditions: (i) 2,4-dichloroaniline, 4-chlorophenol or 4-chlorothiophenol 1
equiv, Cs CO 1 equiv, anh. DMF, 70 °C, 12 h, sealed vial, N ; (ii) 3-trifluoromethylaniline 7
2
3
2
equiv, 140 °C, MW, 45 min, sealed vial; (iii) PCl 6 equiv, POCl as solvent, 100 °C, MW, 20
5
3
min, 800 W.
2
.8. Modulation of region V: biological evaluation.
Thus, the ring variation of region V afforded two series of compounds: a quinoline and a
quinoxaline series.
Among the four tested compounds in quinoline series, it appeared that all exerted an
antiplasmodial activity globally similar to the ones of hits A-D. However, the cytotoxicity
toward the HepG2 human cell line was increased for all of the quinoline series, in comparison
to the Hits A-D and particularly for aniline b (6.5 µM vs 150 µM for hit B). It must be
pointed out that for quinoline 3d, a lack of solubility in the biological media was observed, as
previously reported for hit D. Thus, the substitution of the quinazoline ring by a quinoline
ring was globally detrimental.
Contrary to the quinoline series, the quinoxaline series appeared very promising. Thus,
derivative 32c showed both a better antiplasmodial activity than its analog hit C (0.4 µM vs
1
.1 µM) and a great improvement in the cytotoxic profile, its SI reaching 100, compared to 45
for hit C. Similarly, the same profile was observed for compound 32d with a 2-fold activity
increase compared to hit D (0.45 µM vs 0.9 µM) and a SI of 77 without any solubility
impairment, as observed for hit D.

ACCEPTED MANUSCRIPT
To conclude, we noted that for the region V, the suppression of nitrogen N-3 of the
quinazoline led to generally more cytotoxic compounds while the replacement of the
quinazoline moiety by a quinoxaline one was a significant improvement of the biological
profile, compound 32c reaching a SI of 100.
Table 3. In vitro antiplasmodial and cytotoxicity evaluation of compounds 30a-d, and 32c-d:
modulations at region V.
K1
HepG2
Selectivity
Index
Molecule
Structure
P.falciparum
c
CC (µM)
5
0
IC (µM)
5
0
3
0a
11.7
6.5
1.3
9.0
5.0
3
0b
1.3
1.5
1.1
3
0c
31
20.7
>14.2
d
3
0d
>15.6
3
2c
40.2
38.6
0.4
0.5
100.5
77.2
3
2d
Hit A
Hit B
Hit C
16
150
50
>25
0.2
0.4
1.8
1.1
0.9
-
0.001
0.6
6.0
40
83
45
>28
-
15600
50
d
Hit D
a
Doxorubicine
Atovaquone
Chloroquine
Doxycycline
b
d
> 15.6
30
b
b
20
3.3
a
b
Doxorubicine was used as a cytotoxic reference-drug; Atovaquone, Chloroquinine and doxycycline were used
c
as antimalarial reference-drugs, Selectivity indecies were calculated according to the formula : SI = HepG2
CC / K1 IC . Highest concentration tested due to a lack of solubility.
d
5
0
50

ACCEPTED MANUSCRIPT
Finally, when comparing the biological activities of the synthetic intermediates prepared
for studying region V, it was interesting to note that the intermediate E was not active (IC =
5
0
5
4.5 µM) while intermediates H and K displayed a significant IC50 (0.8 and 0.4 µM
respectively). Noticeably, the intermediate K possessed a very good SI (265) thanks to a
moderate value of its CC . Moreover, the presence of a substituent at position 3 appeared to
5
0
be mandatory for displaying a good antiplasmodial profile by comparison of compound I
which displayed a SI of only 2 (Figure 6). It is to note that both derivatives 32c (IC = 0.15
5
0
µg/mL) and K (IC50 = 0.11 µg/mL) meet the hit to lead in vitro TDR criteria [46] for
selectively active antiplasmodial agents”: IC <0.2 µg/mL, SI>100.
“
5
0
Figure 6. Biological assessment of synthetic intermediates.
3
. Conclusion
From previously identified antiplasmodial hits A-D, in 2-trichloromethyl-4-substituted-
quinazoline series, new derivatives were synthesized in order to study the SARs in 5 precisely
defined regions (I-V). The modulation of region I showed that the –CCl group was
3
mandatory for providing antiplasmodial activity in quinazoline series. In the same series, the
study of regions II and III demonstrated that the optimal substitution of the aniline moiety by
halogens atoms was 2,4-dichloro and that the N-methylation was detrimental toward activity .
Then, by modulating regions IV and V, it respectively appeared that the replacement of the
quinazoline ring by a quinoline ring led to activity levels similar to the ones of Hits A-D but
also to increased cytotoxicities values, while the replacement of the quinazoline ring by a
quinoxaline ring highlighted derivatives 32c-d, displaying both a preserved activity and a
reduced cytotoxicity, in comparison with hits A-D, as summarized in Table 4. These
molecules now appear as new reference antiplasmodial hits. Finally, the great antiplasmodial
potential of intermediate K was revealed. Both derivatives 32c (IC = 0.15 µg/mL) and K
5
0
(
IC = 0.11 µg/mL) meet the hit to lead in vitro TDR criteria [46] for “selectively active
50
antiplasmodial agents”: IC <0.2 µg/mL, SI>100, opening the way to the synthesis of novel
50
lead-compounds.

ACCEPTED MANUSCRIPT
Table 4. Summary of SAR data at regions IV and V.
R
IC50
CC50
SI
0,4
16
0.6
3.2
5.3
0.9
0.7
0.8
0.6
4.3
7.2
0.4
6.9
17.2
0.5
6.2
12.4
0.6
4.0
6.7
-
1.3
11.7
9.0
-
-
40
-
IC50
CC50
SI
1,8
150
83
1.3
-
6.5
-
5.0
-
IC50
CC50
SI
1.1
50
1.5
0.4
40.2
100.5
0.5
38.6
77.2
-
31
45
-
20.7
1.1
IC50
CC50
SI
0.9
>25
>28
0.8
13.3
16.6
*
>15.6
>14.2
*Highest concentration tested due to a lack of solubility.
In bold, newly identified hit-molecules
4
. Experimental
.1. Chemistry
4
Commercial reagents were used as received without additional purification. Melting points
were determined on a Kofler bench and are uncorrected. Elemental analysis and HRMS were
carried out at the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Marseille,
France. NMR spectra were recorded on a Bruker ARX 200 spectrometer or a Bruker AV 250
spectrometer at the Faculté de Pharmacie de Marseille or a BRUKER Avance III nanobay 400
1
at the the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Marseille ( H-
1
3
NMR: 200, 250 or 400 MHz, C-NMR: 50, 63 or 100 MHz). NMR references were the
1
13
following: H: CHCl δ = 7.26, DMSO-d δ = 2.50 and C: CHCl δ = 76.9, DMSO-d δ =
3
6
3
6
3
9.5. Solvents were dried by conventional methods. The following adsorbent was used for
column chromatography: silica gel 60 (Merck, particle size 0.063–0.200 mm, 70–230 mesh
ASTM). TLC was performed on 5 cm × 10 cm aluminium plates coated with silica gel 60F-

ACCEPTED MANUSCRIPT
2
54 (Merck) in an appropriate eluent. Visualization was made with ultraviolet light (234 nm).
HRMS spectra were recorded on QStar Elite (Applied Biosystems SCIEX) spectrometer. PEG
was the matrix for HRMS. The experimental exact mass was given for the ion which has the
maximum isotopic abundance. Purity of synthetized compounds was checked with LC-MS
analyses which were realized at the Faculté de Pharmacie de Marseille with a Thermo
®
Scientific Accela High Speed LC System coupled with a single quadrupole mass
®
®
spectrometer Thermo MSQ Plus . The RP-HPLC column used is a Thermo Hypersil Gold
0 × 2.1 mm (C18 bounded), with particles of 1.9 µm diameter. The volume of sample
5
injected on the column was 1 µL. The chromatographic analysis, total duration of 8 min, is
made with the gradient of following solvents: t = 0 min, water/methanol 50/50; 0 < t < 4 min,
linear increase in the proportion of water to a ratio water/methanol 95/5; 4 < t < 6 min,
water/methanol 95/5; 6 < t < 7 min, linear decrease in the proportion of water to return to a
ratio 50/50 water/methanol; 6 < t < 7 min, water/methanol 50/50. The water used was
buffered with 5 mM ammonium acetate. The retention times (t ) of the molecules analyzed
R
are indicated in min. The preparation of 4-chloro-2-trichloromethylquinazoline E [24], 2-
methyl-N-(2,4-dichlorophenyl)quinazolin-4-amine 1 [10], N-(2,4-dichlorophenyl)quinazolin-
4
-amine 9 [27], 4-chloro-2-trichloromethyquinoline H [43], methyl 3-acetamidothiophene-2-
carboxylate 15 [28], 4-chloro-2-chloromethylquinazoline [24], 4-chloro-2-
F
dichloromethylquinazoline G [26], 4-chloro-2-trifluoromethylquinazoline [47], 2-chloro-3-
trichloromethylquinoxaline K [44] and 2-chloro-3-methylquinoxaline L [45] was achieved as
described in the literature and characterization were consistent as reported in literature.
4
.1.1. General procedure for the preparation of N-aryl-2-
trichloromethylquinazolin-4-amine (2, 3, 7, 8 and 14)
To a mixture of appropriate 4-chloroquinazoline (1 equiv) in isopropanol (10 mL) and
appropriate aniline (0.8 equiv) were added a few drops of concentrated HCl. The vial was
then sealed and heated at 70 °C for 2 h. The mixture was allowed to cool to room temperature
and was poured into 20 mL of iced water. The mixture was then extracted twice with CH Cl .
2
2
The combined organic phases were washed twice with water, then dried (Na SO ) and the
2
4
solvent was evaporated under reduced pressure. The residue was then purified by silica gel
column chromatography (Petroleum Ether/CH Cl 1/1).
2
2
4.1.1.1. 2-Chloromethyl-N-(2,4-dichlorophenyl)quinazolin-4-amine (2)
Starting from 4-chloro-2-chloromethylquinazoline [25] (500 mg, 2.35 mmol) and 2,4-
dichloroaniline (304 mg, 1.88 mmol).

ACCEPTED MANUSCRIPT
1
Yield 71%. White powder. mp 171 °C. H NMR (200 MHz, CDCl ) δ = 8.97 (d, J =
3
8
2
1
.9 Hz, 1H), 8.21 (bs, 1H), 7.99-7.82 (m, 3H), 7.68-7.60 (m, 1H), 7.47-7.35 (m, 2H), 4.75 (s,
1
3
H). C NMR (50 MHz, CDCl ) δ =160.9, 157.1, 149.9, 133.6, 128.9, 128.7, 128.0, 127.6,
3
27.5, 123.9, 122.9, 120.1, 113.9, 47.6. A quaternary C was not observed under these
experimental conditions. Anal. Calcd for C H Cl N : C, 53.20; H, 2.98; N, 12.41. Found: C,
1
5
10
3
3
+
5
3.60; H, 3.12; N, 11.61. LC-MS (ESI, 35 eV): t = 4.21 min, m/z 338 [M+H] .
R
4.1.1.2. 2-Dichloromethyl-N-(2,4-dichlorophenyl)quinazolin-4-amine (3)
Starting from 4-chloro-2-dichloromethylquinazoline G [26] (500 mg, 2.02 mmol) and 2,4-
dichloroaniline (259 mg, 1.62 mmol).
1
Yield 42%. White powder. mp 203 °C. H NMR (200 MHz, CDCl ) δ = 9.11 (d, J =
3
8
1
1
.9 Hz, 1H), 8.30 (bs, 1H), 8.02-7.85 (m, 3H), 7.73-7.66 (m, 1H), 7.49-7.39 (m, 2H), 6.78 (s,
1
3
H). C NMR (50 MHz, CDCl ) δ =160.6, 157.6, 149.6, 133.9, 133.6, 129.5, 128.7, 128.3,
3
28.2, 123.7, 122.9, 120.1, 114.5, 71.8. A quaternary C was not observed under these
experimental conditions. Anal. Calcd for C H Cl N : C, 48.29; H, 2.43; N, 11.26. Found: C,
1
5
9
4
3
+
4
8.41; H, 2.29; N, 11.03. LC-MS (ESI, 35 eV): t = 4.71 min, m/z 372 [M+H] .
R
4.1.1.3. 2-(chlorodifluoromethyl)-N-(2,4-dichlorophenyl)quinazolin-4-
amine (7)
Starting from 4-chloro-2-chlorodifluoromethylquinazoline 6 (500 mg, 2.01 mmol) and 2,4-
dichloroaniline (293 mg, 1.8 mmol).
1
Yield 30%. White powder. mp 195 °C. H NMR (400 MHz, CDCl ) δ = 8.93 (d, J =
3
9
7
.0 Hz, 1H), 8.38 (bs, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.00-7.92 (m, 2H), 7.79-7.74 (m, 1H),
1
3
.48 (d, J = 2.5 Hz, 1H), 7.42-7.35 (m, 1H). C NMR (100 MHz, CDCl ) δ =157.3, 155.6 (t,
3
J = 29 Hz), 149.4, 134.2, 133.2, 130.0, 129.2, 129.0, 128.8, 128.2, 123.9, 122.9 (t, J = 292
+
Hz), 122.8, 120.1, 114.9. HRMS (ESI): m/z calcd. for C H Cl F N [M+H] : 374.9908.
1
5
8
3
2
3
+
Found: 374.9909. LC-MS (ESI, 35 eV): t = 4.58 min, m/z 376 [M+H] .
R
4.1.1.4. N-(2,4-dichlorophenyl)-2-(trifluoromethyl)quinazolin-4-amine (8)
Starting from 4-chloro-2-trifluoromethylquinazoline [45] (500 mg, 2.15 mmol) and 2,4-
dichloroaniline (279 mg, 1.72 mmol).
1
Yield 37%. White powder. mp 198 °C. H NMR (200 MHz, CDCl ) δ = 8.88 (d, J =
3
9
7
1
.0 Hz, 1H), 8.29 (bs, 1H), 8.11-8.07 (m, 1H), 7.97-7.89 (m, 2H), 7.78-7.70 (m, 1H), 7.47-
1
3
.35 (m, 2H). C NMR (50 MHz, CDCl ) δ =157.3, 152.1 (q, J = 36 Hz), 149.5, 134.2, 133.2,
3
30.1, 129.2, 129.1, 128.8, 128.2, 123.9, 122.8, 120.1, 119.8 (q, J = 275 Hz), 115.2. Anal.
Calcd for C H Cl F N : C, 50.30; H, 2.25; N, 11.73. Found: C, 50.39; H, 2.21; N, 11.80.
1
5
8
2
3
3

ACCEPTED MANUSCRIPT
4.1.1.5. N-(2,4-dichlorophenyl)-N-methyl-2-trichloromethylquinazolin-4-
amine (14)
Starting from 4-chloro-2-(trichloromethyl)quinazoline E [24] (500 mg, 1.77 mmol) and N-
methyl-2,4-dichloroaniline (188 µL, 1.95 mmol).
1
Yield 15%. Colorless oil. H NMR (200 MHz, CDCl ) δ = 8.12-8.08 (m, 1H), 7.61-
3
1
3
7
.60 (m, 2H), 7.35-7.17 (m, 3H), 6.93-6.89 (m, 1H), 3.63 (s, 3H). C NMR (50 MHz, CDCl )
3
δ =161.8, 160.1, 150.9, 143.1, 134.3, 133.1, 133.0, 131.3, 129.9, 129.5, 129.1, 127.1, 124.4,
+
1
4
14.9, 97.4, 41.3. HRMS (ESI): m/z calcd. for C H Cl N [M+H] : 418.9317. Found:
1
6
10
5
3
+
18.9319. LC-MS (ESI, 35 eV): t = 5.18 min, m/z 419.7 [M+H] .
R
4
.1.2. Synthesis of 4-chloro-2-chlorodifluoromethylquinazoline (6)
.1.2.1. 2-Chloro-N-(2-cyanophenyl)-2,2-difluoroacetamide (4)
4
In a 250 mL round-bottom flask was successively introduced 2-aminobenzonitrile (2.0 g, 16.9
mmol), 4-(N,N-dimethylamino)pyridine (DMAP) (1.7 g, 13.3 mmol) and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI.HCl) (3.4 g, 17.6 mmol) in
CH Cl (50 mL). Then, 2-chloro-2,2-difluoroacetic acid (1.4 mL, 16.9 mmol) was added into
2
2
the mixture and it was stirred for 24 h at rt. The solvent was evaporated under reduced
pressure and water (200 mL) was added to the residue. The mixture was gentle heated at 40
°
C until a homogeneous solution was obtained. After cooloing down to rt, conc. HCl was
added to adjust the pH = 1. The resulting precipitate was filtered, thoroughly washed with
water and dried in an oven leading 2-chloro-N-(2-cyanophenyl)-2,2-difluoroacetamide.
1
Yield 15%. White powder. mp 84 °C. H NMR (400 MHz, CDCl ) δ = 8.37 (d, J = 8.5
3
1
3
Hz, 2H), 7.71 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.6 Hz, 1H). C NMR (100 MHz, CDCl ) δ
3
=157.1 (t, J = 30 Hz), 137.6, 134.5, 132.7, 126.3, 121.6, 121.5, (t, J = 300 Hz), 115.3, 103.7.
Anal. Calcd for C H ClF N O: C, 46.88; H, 2.19; N, 12.15. Found: C, 46.98; H, 2.13; N,
9
5
2
2
-
1
2.35. LC-MS (ESI, 35 eV): t = 0.85 min, m/z 229 [M-H] .
R
4
.1.2.2. 2-Chlorodifluoromethylquinazolin-4(3H)-one (5)
In a 100 mL round-bottom flask was successively introduced 2-chloro-N-(2-cyanophenyl)-
,2-difluoroacetamide 4 (500 mg, 2.17 mmol), 30% H O (2 mL) and a mixture of EtOH (6
2
2
2
mL) and water (8 mL). Then, NaOH (140 mg, 3.47 mmol) was added at 0 °C. The mixture
was stirred at rt for 90 min and the volatiles were removed under vacuum. Water (100 mL)
was added to the residue and the resulting mixture was acidified to pH = 4 with conc. HCl.

ACCEPTED MANUSCRIPT
The precipitate obtained was filtered, thoroughly washed with water and dried in an oven
gave the 2-chlorodifluoromethylquinazolin-4(3H)-one (5).
1
Yield 80%. Beige powder. mp 246 °C. H NMR (400 MHz, CDCl ) δ = 10.44 (bs,
3
1
3
1
H), 8.37 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 3.7 Hz, 2H), 7.67-7.61 (m, 1H). C NMR (100
MHz, CDCl ) δ =135.6, 129.2, 128.7, 126.9. Five quaternary C were not observed under these
3
-
experimental conditions. LC-MS (ESI, 35 eV): t = 0.66 min, m/z 229 [M-H] .
R
4
.1.2.3. 4-Chloro-2-chlorodifluoromethylquinazoline (6)
In a 20 mL vial was successively introduced 2-chlorodifluoromethylquinazolin-4(3H)-one 5
500 mg, 2.17 mmol) and POCl (1 mL, 10.8 mmol). The mixture was stirred at 140 °C for 10
(
3
min under microwave irradiation (800 W). After cooling at rt, the crude was purified by silica
gel column chromatography (CH Cl ) to afford 4-chloro-2-chlorodifluoromethylquinazoline.
2
2
1
Yield 75%. Beige powder. mp 90 °C. H NMR (400 MHz, CDCl ) δ = 8.38 (d, J = 8.3
3
Hz, 1H), 8.23 (d, J = 8.3 Hz, 1H), 8.12-8.08 (dt, J = 8.2, 1.2 Hz, 1H), 7.89 (dt, J = 8.2, 1.2 Hz,
1
3
1
1
H). C NMR (100 MHz, CDCl ) δ =164.2, 155.0 (t, J = 30 Hz), 150.4, 136.2, 130.9, 129.6,
3
26.1, 123.8, 122.0 (t, J = 292 Hz). Anal. Calcd for C H Cl F N : C, 43.40; H, 1.62; N, 11.25.
9
4
2
2
2
+
Found: C, 43.29; H, 1.56; N, 11.42. LC-MS (ESI, 35 eV): t = 3.42 min, m/z 249 [M+H] .
R
4
.1.3. Representative procedure for the preparation of N-(2,4-
dihalogenophenyl)-2-trichloromethylquinazolin-4-amine (10-13)
A 100 mL round-bottomed single-neck flask equipped with a condenser and a magnetic stir
bar was charged with 4-chloro-2-trichloromethylquinazoline E [24] (500 mg, 1.77 mmol), 2
equiv. of the appropriated dihalogeno-aniline (3.54 mmol) and 30 mL of isopropanol. The
mixture was refluxed for 48 h. After cooling, water was added (50 mL), and the resulting
solution was extracted three times with CH Cl . Then, the combined organic layers were
2
2
washed with water and dried (Na SO ). Finally, the solvent was evaporated under reduced
2
4
pressure. The residue was purified by trituration with CH Cl and filtration yielding the N-
2
2
(2,4-dihalogenophenyl)-2-trichloromethylquinazolin-4-amines (10-13).
4
.1.3.1. N-(2,4-dibromophenyl)-2-trichloromethylquinazolin-4-amine (10)
1
Yield 55%. White powder. mp 243 °C. H NMR (200 MHz, DMSO-d ) δ = 10.32 (bs,
6
1
3
1
H), 8.55 (d, J = 8.2 Hz, 1H), 8.02- 7.91 (m, 3H), 7.82-7.59 (m, 3H). C NMR (50 MHz,
DMSO-d ) δ =160.0, 159.5, 148.9, 136.3, 134.7, 134.4, 131.1, 130.8, 128.5, 128.3, 123.1,
6
1
22.5, 119.5, 113.5, 97.7. Anal. Calcd for C H Br Cl N : C, 36.59; H, 1.62; N, 8.46. Found:
15 8 2 3 3
+
C, 36.16; H, 1.53; N, 8.50. LC-MS (ESI, 35 eV): t = 5.41 min, m/z 494 [M+H] .
R

ACCEPTED MANUSCRIPT
4.1.3.2. N-(4-bromo-2-chlorophenyl)-2-trichloromethylquinazolin-4-amine
(
11)
1
Yield 63%. White powder. mp 222 °C. H NMR (200 MHz, DMSO-d ) δ = 10.38 (bs,
6
1
H), 8.62 (d, J = 8.3 Hz, 1H), 8.07-7.97 (m, 2H), 7.93 (s, 1H), 7.87-7.80 (m, 1H), 7.75 - 7.65
1
3
(
m, 2H). C NMR (50 MHz, DMSO-d ) δ =160.2, 159.7, 149.1, 135.0, 134.6, 132.1, 131.8,
6
1
3
30.8, 130.6, 128.7, 128.5, 123.4, 119.3, 113.7, 97.9. Anal. Calcd for C H BrCl N : C,
15 8 4 3
9.86; H, 1.78; N, 9.30. Found: C, 39.83; H, 1.70; N, 9.45. LC-MS (ESI, 35 eV): t = 5.41
R
+
min, m/z 450 [M+H] .
4.1.3.3. N-(2-bromo-4-chlorophenyl)-2-trichloromethylquinazolin-4-amine
(
12)
1
Yield 70%. White powder. mp 223 °C. H NMR (200 MHz, DMSO-d ) δ = 9.09 (d, J
6
=
9.0 Hz, 1H), 8.38 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.00-7.89 (m, 2H), 7.77-7.70 (m, 1H),
1
3
7
.63 (d, J = 2.3 Hz, 1H), 7.44 (dd, J = 9.0, 2.3 Hz, 1H). C NMR (50 MHz, DMSO-d ) δ
6
=160.6, 157.2, 149.6, 134.5, 134.0, 131.7, 130.3, 129.2, 128.8, 128.7, 122.8, 120.1, 120.0,
1
1
14.1, 97.5. Anal. Calcd for C H BrCl N : C, 39.86; H, 1.78; N, 9.30. Found: C, 40.01; H,
1
5
8
4
3
+
.69; N, 9.26. LC-MS (ESI, 35 eV): t = 4.82 min, m/z 450 [M+H] .
R
4
.1.3.4. N-(2,4-difluorophenyl)-2-trichloromethylquinazolin-4-amine (13)
1
Yield 70%. White powder. mp 165 °C. H NMR (200 MHz, DMSO-d ) δ = 9.00-8.87
6
13
(
m, 1H), 8.12-8.08 (m, 1H), 7.95-7.66 (m, 4H), 7.07-6.92 (m, 2H). C NMR (50 MHz,
DMSO-d ) δ =160.7, 157.4, 153.2 (d, J = 260 Hz), 152.9 (d, J = 260 Hz), 149.6, 133.9, 130.2,
6
1
28.5, 123.5 (d, J = 8.8 Hz), 123.1 (dd, J = 9.5, 4.1 Hz), 120.1, 113.8, 111.4 (dd, J = 21.6, 3.6
Hz), 104.3-103 (m, 1CH), 97.6. Anal. Calcd for C H Cl F N : C, 48.09; H, 2.15; N, 11.22.
15
8
3
2
3
+
Found: C, 48.45; H, 1.99; N, 11.25. LC-MS (ESI, 35 eV): t = 4.58 min, m/z 374 [M+H] .
R
4
.1.4. 2-Methylthieno[3,2-d]pyrimidin-4(3H)-one (16)
A solution of methyl 3-acetamidothiophene-2-carboxylate 15 [29] (760 mg, 3.81 mmol) in
NH OH 25% (9.5 mL) was heated, in a sealed vial, at 105 °C for 3 h. After cooling to room
4
temperature, concentrated HCl was added until pH 8. The precipitate thus formed was
filtered, washed with water and dried in a vacuum oven at 40 °C to afford 2-methylthieno[3,2-
d]pyrimidin-4(3H)-one. No further purification necessary.
1
Yield 63%. White solid. mp 91°C. H NMR (200 MHz, DMSO-d ) δ = 12.31 (bs, 1 H,
6
13
NH), 8.12 (d, J = 5.2 Hz, 1 H), 7.29 (d, J = 5.2 Hz, 1 H), 2.36 (s, 3 H). C NMR (63 MHz,
DMSO-d ) δ = 181.0, 158.8, 158.0, 156.3, 133.9, 124.5, 21.1. LC-MS (ESI, 35eV): t = 0.64
6
R
+
min, m/z 167 [M+H] .

ACCEPTED MANUSCRIPT
4
.1.5. 4-Chloro-2-methylthieno[3,2-d]pyrimidine (17) [30]
To a solution of compound 16 (200 mg, 1.20 mmol) in POCl (5.0 mL) were added a few
3
drops of pyridine. The mixture was then refluxed for 24 h. After cooling down to room
temperature, the solution was poured on ice and a saturated Na CO solution was added until
2
3
the excess of POCl was neutralized. The aqueous phase was then extracted with EtOAc, the
3
combined organic phases were washed with brine, dried (Na SO ) and the solvent was
2
4
evaporated under reduced pressure. The residue was then purified by silica gel column
chromatography (eluent: Petroleum Ether/EtOAc 8/2) to afford 4-chloro-2-methylthieno[3,2-
d]pyrimidine.
1
Yield 97%. White solid. mp 84 °C. H NMR (250 MHz, CDCl ) δ = 7.96 (d, J = 5.5
3
1
3
Hz, 1H), 7.45 (d, J = 5.5 Hz, 1H), 2.78 (s, 3H). C NMR (63 MHz, CDCl ) δ = 164.7, 162.6,
3
+
1
54.7, 136.7, 127.9, 124.8, 25.5. LC-MS (ESI, 35 eV): t = 1.74 min, m/z 185 [M+H] .
R
4
.1.6. Methyl 3-(2,2,2-trichloroacetamido)thiophene-2-carboxylate (18)
To a solution of methyl 3-aminothiophene-2-carboxylate (500 mg, 3.18 mmol) and
triethylamine (440 µL, 3.18 mmol) in tetrahydrofuran (4.0 mL) at 5 °C was added
trichloroacetyl chloride (350 µL, 3.18 mmol). The reaction was stirred at 5 °C for 30 min.
Water (5.0 mL) was then added and the aqueous phase was extracted with EtOAc. The
organic phase was then washed, dried (Na SO ) and the solvent was evaporated to afford
2
4
methyl 3-(2,2,2-trichloroacetamido)thiophene-2-carboxylate. No further purification
necessary.
1
Yield 99%. White solid. mp 244 °C. H NMR (200 MHz, DMSO-d ) δ = 11.42 (bs, 1
6
13
H), 8.06 (d, J = 5.4 Hz, 1 H), 7.89 (d, J = 5.4 Hz, 1 H), 3.88 (s, 3 H). C NMR (63 MHz,
DMSO-d ) δ = 163.4, 158.1, 140.9, 133.5, 121.0, 113.6, 91.8, 52.2. LC-MS (ESI, 35 eV): t =
6
R
+
4
.11 min, m/z 300 [M+H] .
4
.1.7. 2-(Trichloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one (19)
HCl(g) was bubbled into acetic acid (1.23 mL) for 30 minutes. Then, nitrogen was swept
across the solution. This solution was added to a mixture of methyl 3-aminothiophene-2-
carboxylate (3.0 g, 19.08 mmol) and trichloroacetonitrile (0.85 mL, 8.48 mmol) in acetic acid
(1.86 mL). The reaction was then heated at 100 °C for 18 h. After cooling down to rt, the
acetic acid was evaporated under reduced pressure and isopropanol (4.5 mL) was added. This
solution was refluxed for 5 min, then refrigerated. The precipitate thus formed was filtered,
and then purified by silica gel column chromatography (eluent: Petroleum Ether 100% + 10

ACCEPTED MANUSCRIPT
mL of EtOAc every 100 mL of eluent) to afford 2-(trichloromethyl)thieno[3,2-d]pyrimidin-
4
(3H)-one.
Yield 63%. White solid. mp 237 °C. H NMR (200 MHz, CDCl ) δ = 10.50 (bs, 1 H),
1
3
1
3
7
.93 (d, J = 5.3 Hz, 1 H), 7.51 (d, J = 5.3 Hz, 1 H). C NMR (50 MHz, CDCl ) δ = 157.7,
3
-
1
55.7, 151.8, 135.7, 126.1, 123.7, 92.0. LC-MS (ESI, 35 eV): t = 0.78 min, m/z 267 [M-H] .
R
4
.1.8. 4-Chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine (20)
To a solution of 2-(trichloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one 19 (200 mg, 0.74
mmol) in POCl (3.0 mL) were added a few drops of pyridine. The reaction was refluxed for 1
3
h. After cooling to room temperature, the solution was poured on ice and the aqueous phase
was extracted with EtOAc. The combined organic phases were washed with brine, dried
(
Na SO ) and the solvent was evaporated under reduced pressure. The residue was then
2 4
purified by silica gel column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to yield 4-
chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine.
1
Yield 99%. White solid. mp 99 °C. H NMR (200 MHZ, CDCl ) δ = 8.21 (d, J = 5.5
3
13
Hz, 1 H), 7.74 (d, J = 5.5 Hz, 1 H). C NMR (50 MHZ, CDCl ) δ = 161.9, 161.7, 155.6,
3
1
39.0, 130.7, 125.6, 96.0. LC-MS (ESI, 35 eV): t = 4.05 min (no ionization).
R
4
.1.9. N-(2,4-Dichlorophenyl)-2-(trichloromethyl)thieno[3,2-d]pyrimidin-4-
amine (21a)
To a solution of 4-chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (190 mg, 0.66 mmol)
and 2,4-dichloroaniline (106 mg, 0.66 mmol) in ethanol (3.0 mL) were added a few drops of
concentrated HCl. The vial was sealed and the reaction was heated at 100 °C for 24 h. The
solvent was evaporated under reduced pressure and the residue was purified by silica gel
column chromatography (eluent: Petroleum Ether/CH Cl 8/2 + 10 mL of CH Cl every 100
2
2
2
2
mL of eluent) to afford N-(2,4-dichlorophenyl)-2-(trichloromethyl)thieno[3,2-d]pyrimidin-4-
amine.
1
Yield 45%. White solid. mp 197 °C. H NMR (200 MHz, CDCl ) δ = 8.72 (d, J = 8.9
3
Hz, 1 H), 7.96 (d, J = 5.4 Hz, 1 H), 7.66 (d, J = 5.4 Hz, 1 H), 7.61 (s, 1 H), 7.48 (d, J = 2.3
1
3
Hz, 1 H), 7.37 (dd, J = 8.9, 2.3 Hz, 1 H). C NMR (200 MHz, CDCl ) δ = 161.6, 161.0,
3
1
54.6, 134.1, 133.2, 130.0, 129.1, 128.3, 126.2, 125.1, 124.0, 116.2, 97.1. LC-MS (ESI, 35
-
eV): t = 4.90 min, m/z 410 [M-H] . Anal. Calcd for C H Cl N S: C, 37.76; H, 1.46; N,
R
13
6
5
3
1
0.16. Found: C, 38.03; H, 1.26; N, 10.30.
4
.1.10. 2-(Trichloromethyl)-N-[3-(trifluoromethyl)phenyl]thieno[3,2-d]pyrimidin-
-amine (21b)
4

ACCEPTED MANUSCRIPT
To a solution of 4-chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (150 mg, 0.52 mmol)
and 3-(trifluoromethyl)aniline (87 µL, 0.52 mmol) in ethanol (2.5 mL) were added a few
drops of concentrated HCl. The vial was sealed and the reaction was heated at 100°C for 24 h.
The solvent was evaporated under reduced pressure and the residue was purified by silica gel
column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to yield 2-(trichloromethyl)-N-
[
3-(trifluoromethyl)phenyl]thieno[3,2-d]pyrimidin-4-amine.
1
Yield 55%. White solid. mp 141 °C. H NMR (200MHz, CDCl ) δ = 8.42 (s, 1 H),
3
1
3
7
1
.88-7.83 (m, 2 H), 7.58-7.39 (m, 4 H). C NMR (50 MHZ, CDCl ) δ = 161.9, 161.0, 155.0,
3
38.3, 133.9, 131.9, 131.3, 129.7, 125.7, 124.3, 121.3, 118.6, 115.7, 97.4. Anal. Calcd for
C H Cl F N S: C, 40.75; H, 1.71; N, 10.18. Found: C, 40.99; H, 1.80; N, 10.39. LC-MS
14
7
3
3
3
+
(
ESI, 35 eV): t = 4.87 min, m/z 412 [M+H]
R
4
.1.11. 4-(4-Chlorophenoxy)-2-(trichloromethyl)thieno[3,2-d]pyrimidine (21c)
To a solution of 4-chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (210 mg, 0.73 mmol)
and 4-chlorophenol (281 mg, 2.19 mmol) in acetonitrile (5.0 mL) was added K CO (302 mg,
2
3
2
.19 mmol). The reaction was heated at 160 °C for 1h10 under microwave irradiation. The
mixture was poured onto a NaOH 10% solution. The aqueous phase was extracted with
EtOAc. The organic phases were combined, washed with brine, dried (Na SO ) and the
2
4
solvent was evaporated under reduced pressure. The residue was then purified by silica gel
column chromatography (eluent: Petroleum Ether/CH Cl 8/2 + 10 mL of CH Cl every 100
2
2
2
2
mL of eluent) to yield 4-(4-chlorophenoxy)-2-(trichloromethyl)thieno[3,2-d]pyrimidine.
1
Yield 39%. White solid. mp 161 °C. H NMR (200 MHz, CDCl ) δ = 8.09 (d, J = 5.4
3
1
3
Hz, 1 H), 7.70 (d, J = 5.4 Hz, 1 H), 7.45-7.31 (m, 4 H). C NMR (50 MHz, CDCl ) δ =
3
1
63.5, 163.1, 161.5, 150.3, 136.9, 131.4, 129.9, 129.5, 125.2, 122.9, 117.7, 116.4, 96.4. Anal.
Calcd for C H Cl N OS: C, 41.08; H, 1.59; N, 7.37. Found: C, 41.28; H, 1.72; N, 7.45. LC-
1
3
6
4
2
+
MS (ESI, 35 eV): t = 5.08 min, m/z 379 [M+H] .
R
4
.1.12. 4-[(4-Chlorophenyl)thio]-2-(trichloromethyl)thieno[3,2-d]pyrimidine (21d)
To a solution of 4-chlorothiophenol (100 mg, 0.69 mmol) in DMSO (0.5mL), NaH (33 mg,
.39 mmol) was added portionwise under inert atmosphere. The mixture was stirred at room
1
temperature for 20 mi. 4-Chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (200 mg, 0.69
mmol) was then added portionwise and the reaction was stirred for 24 h at rt. Water (1.0 mL)
was added and the aqueous phase was then extracted with CH Cl . The organic phase was
2
2
washed with water, brine and then dried (Na SO ). The solvent was evaporated and the
2
4
residue was purified by silica gel column chromatography (eluent: Petroleum Ether 100% +

ACCEPTED MANUSCRIPT
1
0 mL of CH Cl2 every 100 mL of eluent) to afford 4-[(4-chlorophenyl)thio]-2-
2
(trichloromethyl)thieno[3,2-d]pyrimidine.
1
Yield 7%. White solid. mp not determined. H NMR (200 MHz, CDCl ) δ = 8.02 (d, J
3
13
=
5.5 Hz, 1 H), 7.67-6.64 (m, 3 H), 7.45 (d, J = 8.8 Hz, 2 H). C NMR (50 MHz, CDCl ) δ =
3
1
64.5, 161.1, 159.9, 137.0, 136.6, 136.4, 129.5, 127.5, 125.0, 124.7, 96.8. Anal. Calcd for
C H Cl N S : C, 39.41; H, 1.53; N, 7.07. Found: C, 39.75; H, 1.72; N, 7.20. LC-MS (ESI, 35
13
6
4
2 2
+
eV): t = 5.36 min, m/z 395 [M+H] .
R
4
.1.13. N-(3-Cyanothiophen-2-yl)acetamide (22)
To a solution of 2-aminothiophene-3-carbonitrile (1.020 g, 8.21 mmol) in acetic anhydride
3.0 mL) were added a few drops of acetic acid. The reaction was heated at 120 °C for 10 min
(
under microwave irradiation. The brown solid thus obtained was filtered, washed with water
and dried in a vacuum oven at 40 °C to afford N-(3-cyanothiophen-2-yl)acetamide. No further
purification necessary.
1
Yield 90%. Brown solid. mp 210 °C (litt. 210-211 °C). H NMR (200 MHz, DMSO-
1
3
d ) δ = 11.70 (bs, 1 H), 7.13-7.12 (m, 2 H), 2.20 (s, 3 H). C NMR (50 MHz, DMSO-d ) δ =
6
6
1
68.4, 149.4, 124.7, 118.7, 114.9, 91.8, 22.5. LC-MS (ESI, 35 eV): t = 0.81 min, m/z 165
R
-
[
M-H] .
4
.1.14. 2-Methylthieno[2,3-d]pyrimidin-4(3H)-one (23)
To a solution of N-(3-cyanothiophen-2-yl)acetamide 22 (1.232 g, 7.41 mmol) and H O 30%
2
2
(6.5mL) in ethanol (22 mL) and water (4 mL), at 0 °C, was added NaOH in pellets (0.495 g,
1
2.4 mmol). The reaction was stirred at room temperature for 15 min and then heated at 55 °C
until a homogeneous solution was obtained. After cooling to rt, the solvent was evaporated
under reduced pressure. Water (10 mL) was added and the mixture was heated to 50 °C. After
cooling to rt, a HCl 1N solution was added until pH 4. The precipitate was filtered, washed
with water and dried in a vacuum oven at 40°C to afford 2-methylthieno[2,3-d]pyrimidin-
4
(3H)-one. No further purification necessary.
1
Yield 72%. White solid. mp 210 °C. H NMR (200 MHz, DMSO-d ) δ = 7.90 (s, 1 H),
6
13
7
.39 (d, J = 5.8 Hz, 1 H), 6.93 (d, J = 5.8 Hz, 1 H), 2.19 (s, 3 H). C NMR (50 MHz, DMSO-
d ) δ = 166.9, 166.8, 145.8, 123.0, 115.8, 114.8, 23.3. LC-MS (ESI, 35 eV): t = 0.65 min,
6
R
+
m/z 167 [M+H] .
4
.1.15. 4-Chloro-2-methylthieno[2,3-d]pyrimidine (24)
To a solution of 2-methylthieno[2,3-d]pyrimidin-4(3H)-one 23 (300 mg, 1.80 mmol) in POCl3
5.0 mL) were added a few drops of pyridine. The mixture was refluxed for 24 h and, after
(