Direct metallation of thienopyrimidines using a mixed lithium-cadmium base and antitumor activity of functionalized derivatives

Article abstract of DOI:10.1039/b915274a

A series of thieno[2,3-d]- and thieno[3,2-d]pyrimidines have been easily synthesized using as key step a deproto-cadmiation-trapping sequence. Some of the compounds thus synthesized were screened for anticancer (cytotoxic) activities, and (S)-2-(6-iodo-2-

Full text of DOI:10.1039/b915274a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Direct metallation of thienopyrimidines using a mixed lithium–cadmium base
and antitumor activity of functionalized derivatives†
Katia Sne´garoff,a Fre´de´ric Lassagne,a Ghenia Bentabed-Ababsa,a,b Ekhlass Nassar,c Sidaty Cheikh Sid Ely,a
Ste´phanie Hesse,d Enrico Perspicace,d A¨ıcha Derdourb and Florence Mongin*a
Received 27th July 2009, Accepted 24th August 2009
First published as an Advance Article on the web 22nd September 2009
DOI: 10.1039/b915274a
A series of thieno[2,3-d]- and thieno[3,2-d]pyrimidines have been easily synthesized using as key step a
deproto-cadmiation–trapping sequence. Some of the compounds thus synthesized were screened for
anticancer (cytotoxic) activities, and (S)-2-(6-iodo-2-phenylthieno[2,3-d]pyrimidin-4-ylamino)-3-
phenylpropanoic acid proved to have a significant activity towards liver, human breast and cervix
carcinoma cell lines.
More recently, RnMLi type compounds where M is not an alkali
Introduction
metal have also been described for their unprecedented metallation
Thienopyrimidine derivatives, which are structural analogues of
ability. These species display a large range of reactivities, depend-
purines, have been drawn attention due to their large range of
ing on both the metal M and the groups connected to it.6 By
pharmacological activities.1 Numerous thieno[2,3-d]pyrimidines
have been proved to be phosphodiesterase inhibitors and var-
combining soft organometallic compounds with lithium additives,
bases allowing both efficient and chemoselective reactions have
ious receptor antagonists; compounds were designed to act as
been prepared and used to generate functionalized aromatic
immunomodulators or to be used in case of cerebral ischemia,
compounds.7
malaria, tuberculosis, Alzheimer’s and Parkinson’s diseases.1
We recently accomplished the deproto-metallation of a large
Many thieno[2,3-d]- and thieno[3,2-d]pyrimidines exhibit antitu-
range of aromatics including heterocycles using a newly de-
mor and radioprotective activities.1
veloped lithium–cadmium base, (TMP)3CdLi (TMP = 2,2,6,6-
Lithium bases have been largely employed for the deproto-
tetramethylpiperidino),8 and we here describe its use for the
metallation of aromatic rings.2 Nevertheless, for aromatics bearing
reactive functions (e.g., ester or cyano groups) or sensitive
p-deficient heterocycles, either extremely low reaction tempera-
tures or in situ electrophilic trapping are required due to the high
functionalization of thieno[2,3-d]- and thieno[3,2-d]pyrimidines.
Some intermediates and target compounds were evaluated for their
cytotoxic activity.
reactivity of the corresponding (hetero)aryllithiums.
The use of metal additives in order to get more efficient or
more chemoselective bases is a challenging field, and various
Results and discussions
RnMLi type compounds already prepared behave as superbases
since such species exhibit behaviours that cannot be reproduced
by the monometallic compounds on their own. Among them, the
RnMLi mixtures of organolithiums and M alkali metal alkoxides
described by Schlosser,3 Lochmann,4 and Caube`re5 are powerful
reagents.
Synthetic aspects
To this purpose, 4-chlorothieno[2,3-d]pyrimidine (1) was
synthesized as described recently under microwave irradiation.9
4-Chloro-2-phenylthieno[3,2-d]pyrimidine (2) was prepared
(as shown in Scheme 1) from commercially available
methyl 3-aminothiophene-2-carboxylate by adapting
described procedure.10 Isomeric 4-chloro-2-phenylthieno[2,3-
d]pyrimidine (3) was similarly synthesized, but from
a
aChimie et Photonique Mole´culaires, UMR 6510 CNRS, Universite´
de Rennes 1, Baˆtiment 10A, Case 1003, Campus Scientifique de
Beaulieu, 35042, Rennes, France. E-mail: florence.mongin@univ-rennes1.fr;
Fax: +33-2-2323-6955
a
known 2-aminothiophene-3-carboxamide.11 Starting from 4-
chlorothieno[2,3-d]pyrimidine (1), a subsequent functionalization
at the 4 position was easily achieved either by nucleophilic
substitution using sodium methoxide to afford the ether 4,
or by copper-catalyzed N-arylation of pyrazole by adapting
described conditions12 to furnish the derivative 5. 4-Chloro-
2-phenylthieno[2,3-d]pyrimidine (3) was readily converted by
reaction with sodium methoxide or morpholine to the compounds
6 and 7, respectively (Scheme 1).
In contrast to thiophene compounds which can be easily
metallated using lithium bases,13 pyrimidines are much more
prone to nucleophilic attacks,14 and their metallation can only be
achieved at room temperature when softer bases are employed.8,15
The lithium–cadmium base (TMP)3CdLi was chosen for the
bLaboratoire de Synthe`se Organique Applique´e, Faculte´ des Sciences de
l’Universite´, BP 1524 Es-Senia, Oran, 31000, Algeria
cDepartment of Chemistry, Faculty of Women for Arts, Science and
Education, Ain Shams University, Asma Fahmy Street, Heleopolis
(El-Margany), Cairo, Egypt
dLaboratoire d’Inge´nie´rie Mole´culaire et Biochimie Pharmacologique, In-
stitut Jean Barriol, FR CNRS 2843, Universite´ Paul Verlaine-Metz, 1
Boulevard Arago, 57070, Metz Technopoˆle, France
† Electronic supplementary information (ESI) available: 1H and 13C NMR
data for methyl-3-benzoylaminothiophene-2-carboxylate, 2-phenyl-3H-
thieno[3,2-d]pyrimidin-4-one, and compounds 2–22, and copies of 1H and
13C NMR spectra for methyl-3-benzoylaminothiophene-2-carboxylate,
2-phenyl-3H-thieno[3,2-d]pyrimidin-4-one, and compounds 3, 14–22.
CCDC reference number 729714. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/b915274a
4782 | Org. Biomol. Chem., 2009, 7, 4782–4788
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Scheme 1 Synthesis of the substrates 1–7.
Table 1 Deproto-cadmiation (using (TMP)3CdLi)-iodination
metallation of the thienopyrimidine substrates because of its high
compatibility with sensitive heterocycles.8
Conducting the reaction from 4-chloro- (1) and 4-meth-
oxythieno[2,3-d]pyrimidine (4) using 0.5 equivalent8 of base in
tetrahydrofuran (THF) at room temperature for 2 h resulted, after
quenching with iodine, in the formation of the iodides 8 and 9,
respectively (48 and 90% yield). Unlike five-membered heterocy-
cles, for which protons adjacent to heteroatoms have the strongest
acidity, six-membered heterocycles have the weakest acidic protons
adjacent to nitrogens, a result of the more important repulsion
between the lone electron pair of nitrogen and the negative charge
of the carbanion for the latter, due to the smaller angle between the
two electron clouds.16 As a consequence, the substrates 1 and 4 are
regioselectively deprotonated at the most acidic 6 position (Table 1,
entries 1,2). Upon treatment with 0.5 equiv of (TMP)3CdLi
under the same reaction conditions, 4-(pyrazol-1-yl)thieno[2,3-
d]pyrimidine (5) did not undergo regioselective deprotonation,
and only a mixture of several inseparable derivatives was obtained
(entry 3). Starting from 4-chloro-2-phenylthieno[3,2-d]pyrimidine
(2), metallation still took place next to the sulfur atom, as
evidenced by trapping with iodine to afford the 6-iodo derivative
10a in 70% yield (compound 10a was identified unequivocally
by X-ray structure analysis).† Turning to 1 equivalent of base
(instead of 0.5) allowed a slight yield improvement (76%) but
also, more importantly, the formation of the diiodide 10b in 18%
yield. The latter could result from a dideprotonation due to the
activation of the site at the 7 position by the close nitrogen atom
(entries 4,5). Isomeric 4-chloro-2-phenylthieno[2,3-d]pyrimidine
(3) was similarly functionalized in an excellent yield (entry 6).
By replacing the chloro group with a methoxy or a morpholino
group17 (substrates 6 and 7), the conditions were optimized,
and the best yields were obtained using 1 equivalent of base
(entries 7,8).
Entry
1
Substrate
x
Product
Yield (%)
48
1
0.5
8
9
2
4
0.5
90
a
3
4
5
2
0.5
0.5
Mixture
—
10a
10a
70
5
2
1
76
+10b
18
97
6
7
8
3
6
7
0.5
1
11
12
13
81
83
1
It also proved possible to trap the metallated 4-(morpholin-4-
yl)-2-phenylthieno[2,3-d]pyrimidine prepared from the substrate
7 in a palladium-catalyzed cross-coupling reaction with 2-
bromopyridine. Indeed, by using catalytic amounts of palladium
acetate and 1,1¢-bis(diphenylphosphino)ferrocene (dppf), the ex-
pected product 14 was isolated in 70% yield (Scheme 2).
In order to progress towards molecules with potential antitumor
activities, we decided to prepare thiophene analogues of 2-
(6-iodo-2-phenylquinazolin-4-ylamino)-3-phenylpropanoic acid,
a Metallation took place but not regioselectively, and the products formed
could not be separated.
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Table 2 Nucleophilic substitution of the 4-chlorothienopyrimidines by
an amino acid or derivative
Entry Substrate RR¢NH
Product
Yield (%)
66
Scheme
2
Deproto-cadmiation (using (TMP)3CdLi) followed by
1
2
3
2
L-Phenylalanine
L-Phenylalanine
L-Phenylalanine
15
cross-coupling.
10a
10b
16 55
Scheme
3
Cytotoxic activity of 2-(6-iodo-2-phenylquinazolin-4-
ylamino)-3-phenylpropanoic acid.
17 48
Table 3 aIn vitro cytotoxic activity (IC50) bof the newly synthesized
compounds and doxorubicin against a liver carcinoma cell line (HEPG2),
a human breast carcinoma cell line (MCF7), and a cervix carcinoma cell
line (HELA)c
HEPG2/mg
HELA/mg
Entry
Compound
mL-1
MCF7/mg mL-1
mL-1
4
5
6
7
8
3
L-Phenylalanine
L-Phenylalanine
18
53
19 80
20 74
21 40
1
2
3
4
5
6
7
8
6
3.4
2.5
2.6
1.2
4.3
2.7
0.40
1.6
1.7
0.60
1.6
1.3
2.1
2.7
2.2
1.5
0.94
1.6
2.4
0.70
2.8
4.4
3.5
4.0
4.7
5.0
1.3
3.0
5.6
0.85
7
12
13
14
16
19
11
11
11
11
20
9
10
21
Doxorubicin
a The cadmium presence in the tested compounds was not evaluated since
CdCl2·TMEDA was used at the last-step-but-one, but in case of further
development these compounds would have to be synthesized using a safe
base and tested again. b IC50 is defined as the concentration which results
in a 50% decrease in cell number as compared with that of the control
structures in the absence of an inhibitor. c Pharmacological tests have also
been performed for compounds 15, 17, 18 and 22, but they failed to give
logical results.
N-Methyl-L-
phenylalanine
L-Phenylalaninol
reaction of the chloride by L-phenylalanine, under the conditions
described in the quinazoline series18 to afford the analogues 15–19
(Table 2, entries 1-5). N-methyl-L-phenylalanine, L-phenylalaninol
and L-proline were then used in order to evidence the importance
of the secondary amine and carboxylic acid functions (entries 6–8,
compounds 20–22).
L-Proline
22 38
Pharmacology
The compounds 6, 7, 12–14, 16, 19, 20 and 21 were tested against a
liver carcinoma cell line (HEPG2), a human breast carcinoma cell
line (MCF7), and a cervix carcinoma cell line (HELA) (Table 3).
The compounds 6, 7, 12, 14 and 16 showed a low activity towards
the liver carcinoma cell line HEPG2 compared to a reference drug
(doxorubicin); the compounds 13, 20 and 21 showed a moderate
activity whereas the compound 19 was found to have a very potent
activity towards HEPG2 with a IC50 of 0.40 mg mL-1 (the IC50 value
which proved to exhibit anticancer activity (cytotoxic) against
U937 leukemia cell lines (Scheme 3).18
To this purpose, different 2-phenyl-4-chlorothienopyrimidines,
iodinated or not, were involved in the nucleophilic substitution
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for the reference drug, doxorubicin, is 0.70 mg mL-1). Concerning
the activity towards the human breast carcinoma cell line MCF7,
all the compounds showed moderate activities compared to the
reference drug doxorubicin except the compound 19 which turned
out to be promising. For the cervix carcinoma cell line HELA, all
the compounds have low activities compared to the reference drug
doxorubicin, except the compounds 6 and 19 which were found to
be more effective towards this kind of carcinoma cell line (HELA)
than other compounds.
3-benzoylaminothiophene-2-carboxylate (3.7 g, 98%) as a white
solid: mp 98 ◦C (lit.21 98–99 ◦C).
2-Phenyl-3H-thieno[3,2-d]pyrimidin-4-one22 was prepared by
adapting a described procedure.10 To a solution of methyl-3-
benzoylaminothiophene-2-carboxylate (2.0 g, 7.6 mmol) in MeOH
(80 mL) was added a 25% aqueous NH3 solution (200 mL). After
stirring at 50 ◦C for 17 h, the reaction mixture was concentrated to
one-half of its initial volume and the resulting solid was collected to
give a mixture of 3-benzoylamino-thiophene-2-carboxamide and
2-phenyl-3H-thieno[3,2-d]pyrimidin-4-one (0.84 g). To this mix-
ture in MeOH (34 mL) was added a 2 N aqueous NaOH solution
(12 mL). After stirring at reflux for 17 h, the reaction mixture was
acidified with 1 M HCl until pH 4 and the resulting precipitate
was collected to give 2-phenyl-3H-thieno[3,2-d]pyrimidin-4-one
(0.80 g, 46%) as a white solid: mp > 260 ◦C. HRMS: calcd for
C12H8N2OS (M+) 228.0357, found 228.0348.
Conclusions
A series of thieno[2,3-d]- and thieno[3,2-d]pyrimidines have been
easily synthesized. The key step is a deprotonation–trapping
sequence using a mixed lithium–cadmium base. Most of the
compounds were screened in order to evidence an anticancer
activity. Some of them such as (S)-2-(6-iodo-2-phenylthieno[2,3-
d]pyrimidin-4-ylamino)-3-phenylpropanoic acid (19) showed sig-
nificant cytotoxic activities towards the tested cell lines, in
particular with the liver and human breast carcinoma cell lines.
4-Chloro-2-phenylthieno[3,2-d]pyrimidine (2)22 was prepared by
adapting a described procedure.10 A mixture of 2-phenyl-3H-
thieno[3,2-d]pyrimidin-4-one (1.6 g, 7.0 mmol) and POCl3 (16 mL,
0.17 mol) was heated at reflux for 4 h. POCl3 was then distilled
under reduced pressure. The residue was diluted in CH2Cl2,
and neutralized with a cooled saturated NaHCO3 solution. The
aqueous layer was extracted with CH2Cl2 and the combined
organic layers were washed with brine, dried over Na2SO4 and
concentrated. The resulting solid was purified by column chro-
matography on silica gel (eluent: heptane–CH2Cl2 100 : 0 to 0 : 100)
to give 2 (1.5 g, 90%) as a white solid: mp 118–119 ◦C. Anal. calcd
for C12H7N2ClS: C, 58.42; H, 2.86; N, 11.35; S, 13.00. Found: C,
58.08; H, 3.00; N, 11.28; S, 12.98%.
Experimental
Syntheses: general methods
Metallation reactions were performed under argon atmosphere.
THF was distilled over sodium/benzophenone. Column chro-
matography separations were achieved on silica gel (40–63 mm).
Melting points were measured on a Kofler apparatus. 1H and 13
C
nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker ARX-200 spectrometer at 200 and 50 MHz, respectively,
on a Bruker Avance III spectrometer at 300 and 75 MHz,
respectively, or on a Bruker AC-400 spectrometer at 300 and
4-Chloro-2-phenylthieno[2,3-d]pyrimidine (3) was prepared as
before but starting from 2-phenyl-3H-thieno[2,3-d]pyrimidin-4-
one (1.6 g), and was obtained after recrystallization from AcOEt
in a quantitative yield as a white solid: mp 148 ◦C (lit.23 146 ◦C).
1
75 MHz, respectively. H chemical shifts (d) are given in ppm
relative to the solvent residual peak, and 13C chemical shifts
relative to the central peak of the solvent signal.19 Mass spectra
(HRMS) measurements were performed at the CRMPO (Centre
Re´gional de Mesures Physiques de l’Ouest) of Rennes using either
a Waters Q-TOF 2 or a Bruker micrOTOF Q II instrument
in positive or negative elestrospray CI mode, respectively, or
a Micromass MS/MS ZABSpec TOF instrument in EI mode.
Elemental analyses were performed at the CRMPO using a
Thermo-Finnigan Flash EA 1112 CHNS analyzer.
4-Methoxythieno[2,3-d]pyrimidine (4)24. To a solution of Me-
ONa in MeOH (0.18 g, 8.0 mmol of Na in 40 mL of MeOH)
was added 4-chlorothieno[2,3-d]pyrimidine (1, 0.68 g, 4.0 mmol).
After stirring at reflux for 1.5 h, the reaction mixture was
quenched with water, extracted with CH2Cl2, dried over Na2SO4
and concentrated. The resulting solid was purified by column
chromatography on silica gel (eluent: heptane–AcOEt 70 : 30) to
4-Chlorothieno[2,3-d]pyrimidine (1)9 and 2-aminothiophene-
3-carboxamide11 were prepared according to described proce-
dures. 2-Benzoylaminothiophene-3-carboxamide and 2-phenyl-
3H-thieno[2,3-d]pyrimidin-4-one20 were prepared by adapting a
described procedure.10
◦
give 4 (0.45 g, 67%) as a white solid: mp 51 C. Anal. calcd for
C7H6N2OS: C, 50.59; H, 3.64; N, 16.86; S, 19.29. Found: C, 50.89;
H, 3.73; N, 16.46; S, 19.21%.
4-Methoxy-2-phenylthieno[2,3-d]pyrimidine (6) was prepared as
before from 4-chloro-2-phenylthieno[2,3-d]pyrimidine (3, 0.98 g).
Column chromatography on silica gel (eluent: CH2Cl2–heptane
50 : 50) afforded 6 (2.1 g, 91%) as a pale yellow solid: mp 90–
92 ◦C. Anal. calcd for C13H10N2OS: C, 64.44; H, 4.16; N, 11.56; S,
13.23. Found: C, 64.53; H, 4.26; N, 11.36; S, 12.74%.
Methyl-3-benzoylaminothiophene-2-carboxylate was prepared
by adapting a described procedure.10 To a mixture of methyl-
3-aminothiophene-2-carboxylate (2.2 g, 14 mmol) and Et3N
(2.4 mL, 17 mmol) in CH2Cl2 (18 mL) at 0 ◦C was added dropwise
benzoyl chloride (2.0 mL, 17 mmol). After stirring at room
temperature for 3 h, the reaction mixture was diluted with CH2Cl2
and washed with saturated NaHCO3 and brine. The organic layer
was dried over Na2SO4 and concentrated under reduced pressure.
The resulting solid was purified by column chromatography on
silica gel (eluent: heptane–Et2O 96 : 4 to 80 : 20) to give methyl-
4-(Pyrazol-1-yl)thieno[2,3-d]pyrimidine (5) was prepared from 4-
chlorothieno[2,3-d]pyrimidine (1, 0.68 g) by adapting a described
procedure.12 Purification by column chromatography on silica
gel (eluent: heptane/Et2O 80 : 20) afforded 5 (0.61 g, 75%) as
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a white solid: mp 115 ◦C. Anal. calcd for C9H6N4S: C, 53.45;
H, 2.99; N, 27.70; S, 15.86. Found: C, 53.77; H, 2.99; N, 27.52;
S, 15.77%.
4-Chloro-6-iodo-2-phenylthieno[2,3-d]pyrimidine (11) was pre-
pared from 4-chloro-2-phenylthieno[2,3-d]pyrimidine (3, 0.25 g)
following the general procedure. After purification by column
chromatography on silica gel (eluent: CH2Cl2–heptane 50 : 50),
compound 11 was obtained as a white solid (0.36 g, 97%): mp
148 ◦C. Anal. calcd for C12H6ClIN2S: C, 38.68; H, 1.62; N, 7.52;
S, 8.61. Found: C, 39.01; H, 1.92; N, 7.37; S, 8.70%.
4-(Morpholin-4-yl)-2-phenylthieno[2,3-d]pyrimidine (7). To a
solution of morpholine (0.38 g, 4.4 mmol) and 4-chloro-2-
phenylthieno[2,3-d]pyrimidine (3, 1.0 g, 4.0 mmol) in dry toluene
(25 mL) was added K2CO3 (1.1 g, 8.0 mmol). After stirring at
reflux for 1 h, one more equivalent of morpholine was added
(0.35 g, 4.0 mmol). The mixture was stirred again for 2 h at
General procedure for the deproto-cadmiation (using 1 equiv.
(TMP)3CdLi)–iodination
R
reflux, filtered through celiteꢀ, and the filtrate was concentrated
under reduced pressure. The resulting solid was purified by column
chromatography on silica gel (eluent: CH2Cl2) to give 7 (1.0 g,
89%) as a yellow solid: mp 140 ◦C (lit.23 139 ◦C). Anal. calcd for
C16H15N3OS: C, 64.62; H, 5.08; N, 14.13; S, 10.78. Found: C, 64.79;
H, 5.15; N, 14.08; S, 10.43%.
To
a
stirred, cooled (0 ◦C) solution of 2,2,6,6-
tetramethylpiperidine (0.25 mL, 1.5 mmol) in THF (5 mL)
was added BuLi (about 1.6 M hexanes solution, 1.5 mmol). After
15 min at 0 ◦C, CdCl2·TMEDA (0.15 g, 0.5 mmol) was added,
and the mixture was stirred for 15 min at this temperature before
introduction of the substrate (0.5 mmol). After 2 h at room
temperature, a solution of I2 (0.37 g, 1.5 mmol) in THF (5 mL)
was added. The mixture was stirred overnight before addition
of an aqueous saturated solution of Na2S2O3 (10 mL) and
extraction with Et2O (3 ¥ 20 mL). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure before purification by
column chromatography on silica gel (the eluent is given in the
product description).
General procedure for the deproto-cadmiation (using 0.5 equiv
(TMP)3CdLi)–iodination
To
a
stirred, cooled (0 ◦C) solution of 2,2,6,6-
tetramethylpiperidine (0.25 mL, 1.5 mmol) in THF (5 mL)
was added BuLi (about 1.6 M hexanes solution, 1.5 mmol). After
15 min at 0 ◦C, CdCl2·TMEDA (0.15 g, 0.5 mmol) was added,
and the mixture was stirred for 15 min at this temperature before
introduction of the substrate (1.0 mmol). After 2 h at room
temperature, a solution of I2 (0.37 g, 1.5 mmol) in THF (5 mL)
was added. The mixture was stirred overnight before addition
of an aqueous saturated solution of Na2S2O3 (10 mL) and
extraction with Et2O (3 ¥ 20 mL). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure before purification by
column chromatography on silica gel (the eluent is given in the
product description).
4-Chloro-6,7-diiodo-2-phenylthieno[3,2-d]pyrimidine (10b) was
prepared from 4-chloro-2-phenylthieno[3,2-d]pyrimidine (2, 0.12
g) following the general procedure. After purification by column
chromatography on silica gel (eluent: heptane–CH2Cl2 98 : 2 to
74 : 26), compound 10b was obtained as a white solid (45 mg, 18%):
◦
mp 176 C. Anal. calcd for C12H5ClI2N2S: C, 28.91; H, 1.01; N,
5.62; S, 6.43. Found: C, 33.23; H, 2.12; N, 5.21; S, 6.13% (partial
loss of iodine was observed upon storage). Note that compound
10a was also obtained in 76% yield.
4-Chloro-6-iodothieno[2,3-d]pyrimidine (8) was prepared from
4-chlorothieno[2,3-d]pyrimidine (1, 0.17 g) following the general
procedure. After purification by column chromatography on silica
gel (eluent: CH2Cl2), compound 8 was obtained as a white solid
(0.14 g, 48%): mp 168 ◦C. Anal. calcd for C6H2ClIN2S: C, 24.30;
H, 0.68; N, 9.45; S, 10.81. Found: C, 24.68; H, 1.06; N, 9.12; S,
10.73%.
6-Iodo-4-methoxy-2-phenylthieno[2,3-d]pyrimidine (12) was pre-
pared from 4-methoxy-2-phenylthieno[2,3-d]pyrimidine (6, 0.12
g) following the general procedure. After purification by column
chromatography on silica gel (eluent: CH2Cl2–heptane 50 : 50),
compound 12 was obtained as a white solid (0.15 g, 81%): mp
144–146 ◦C. Anal. calcd for C13H9IN2OS: C, 42.41; H, 2.46; N,
7.61; S, 8.71. Found: C, 42.44; H, 2.42; N, 7.95; S, 8.43%.
6-Iodo-4-methoxythieno[2,3-d]pyrimidine (9) was prepared from
4-methoxythieno[2,3-d]pyrimidine (4, 0.17 g) following the general
procedure. After purification by column chromatography on silica
gel (eluent: heptane–AcOEt 90 : 10), compound 9 was obtained as
a white solid (0.26 g, 90%): mp 152 ◦C. Anal. calcd for C7H5IN2OS:
C, 28.78; H, 1.73; N, 9.59; S, 10.98. Found: C, 29.09; H, 1.82; N,
9.43; S, 11.43%.
6 - Iodo - 4 - (morpholin - 4 - yl) - 2 - phenylthieno[2,3-d]pyrimidine
(13) was prepared from 4-(morpholin-4-yl)-2-phenylthieno[2,3-
d]pyrimidine (7, 0.15 g) following the general procedure. After
purification by column chromatography on silica gel (eluent:
CH2Cl2), compound 13 was obtained as a yellow solid (0.18 g,
83%): mp 206–208 ◦C. Anal. calcd for C16H14IN3OS: C, 45.40; H,
3.33; N, 9.93; S, 7.85. Found: C, 45.11; H, 3.46; N, 9.57; S, 7.36%.
4-Chloro-6-iodo-2-phenylthieno[3,2-d]pyrimidine (10a) was pre-
pared from 4-chloro-2-phenylthieno[3,2-d]pyrimidine (2, 0.25 g)
following the general procedure. After purification by column
chromatography on silica gel (eluent: heptane–CH2Cl2 98 : 2 to
74 : 26), compound 10a was obtained as a white solid (0.26 g,
70%): mp 138 ◦C. Anal. calcd for C12H6ClIN2S: C, 38.68; H,
1.62; N, 7.52; S, 8.61. Found: C, 38.69; H, 1.73; N, 7.71;
S, 8.32%.
4-(Morpholin-4-yl)-2-phenyl-6-(pyridin-2-yl)thieno[2,3-d]-
pyrimidine (14). To a stirred, cooled (0 ◦C) solution of 2,2,6,6-
tetramethylpiperidine (0.26 mL, 1.5 mmol) in THF (2 mL) was
added BuLi (about 1.6 M hexanes solution, 1.5 mmol). After
15 min at 0 ◦C, CdCl2·TMEDA (0.15 g, 0.5 mmol) was added, and
the mixture was stirred for 15 min at this temperature before intro-
duction of 4-(morpholin-4-yl)-2-phenylthieno[2,3-d]pyrimidine
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(7, 0.15 g, 0.5 mmol). After 2 h at room temperature, Pd(OAc)2
(3.4 mg, 15 mmol), 1,1¢-bis(diphenylphosphinoferrocene) (9 mg,
15 mmol) and 2-bromopyridine (0.48 mL, 0.5 mmol) were added
successively, and the mixture was heated at reflux overnight. The
N-methyl-L-phenylalanine (25 mg) following the general proce-
dure, and was obtained as a pale yellow solid (50 mg, 74%): mp >
◦
240 C. HRMS: calcd for C22H17IN3O2S ([M - H]-) 514.0092,
found 514.0085.
R
mixture was filtered through celiteꢀ, and the cake was washed
with CH2Cl2. After purification by column chromatography on
silica gel (eluent: CH2Cl2–AcOEt 100 : 0 to 90 : 10), and crystal-
lization from acetonitrile, compound 14 was obtained as a yellow
solid (0.13 g, 70%): mp > 260 ◦C. HRMS: calcd for C21H19N4OS
([M+H]+) 375.1280, found 375.1282.
(S)-2-(6-Iodo-2-phenylthieno[2,3-d]pyrimidin-4-ylamino)-3-
phenylpropan-1-ol (21) was prepared from 4-chloro-6-iodo-
2-phenylthieno[2,3-d]pyrimidine (11, 48 mg) and using L-
phenylalaninol (21 mg) following the general procedure, and was
obtained as a pale yellow solid (25 mg, 40%): mp > 240 ◦C. HRMS:
calcd for C14H11IN3OS ([M-C7H7]+) 395.9668, found: 395.9626.
General procedure for the nucleophilic substitution of
4-chlorothienopyrimidines by an amino acid or derivative
(S)-1-(6-Iodo-2-phenylthieno[2,3-d]pyrimidin-4-yl)pyrrolidine-
2-carboxylic acid (22) was prepared from 4-chloro-6-iodo-2-
phenylthieno[2,3-d]pyrimidine (11, 48 mg) and using L-proline
(16 mg) following the general procedure, and was obtained as
a pale yellow solid (22 mg, 38%): mp > 240 ◦C. HRMS: calcd for
C16H14IN3S ([M-CO2]+) 406.9953, found: 406.9925.
A previously described procedure was adapted.18 A mixture of the
required 4-chlorothienopyrimidine (0.13 mmol) and amino acid or
derivative (0.14 mmol), and potassium carbonate (0.59 mmol) in
DMSO (0.6 mL) was heated overnight at 100 ◦C. After cooling to
room temperature, 1 N HCl was added until pH 4. The resulting
solid was collected and purified by column chromatography on
silica gel (AcOEt–MeOH 80 : 20).
Pharmacology
The method applied is similar to that reported by Skehan et al.25
using 20 Sulfo-Rhodamine-B stain (SRB). Cells were plated in 96-
multiwell plate (104 cells/well) for 24 h before treatment with the
test compound to allow attachment of cell to the wall of the plate.
Different concentrations of the compound under test (0, 1.0, 2.5,
5.0, and 10 mg ml-1) were added to the cell monolayer in triplicate
wells individual dose, and monolayer cells were incubated with
the compounds for 48 h at 37 ◦C and in atmosphere of 5% CO2.
After 48 h, cells were fixed, washed and stained with SRB stain,
excess stain was washed with acetic acid and attached stain was
recovered with Tris–EDTA buffer. Color intensity was measured
in an ELISA reader, and the relation between surviving fraction
and drug concentration is plotted to get the survival curve of each
tumor cell line after the specified compound, and the IC50 was
calculated.
(S)-3-Phenyl-2-(2-phenylthieno[3,2-d]pyrimidin-4-ylamino)pro
panoic acid (15) was prepared from 4-chloro-2-phenylthieno[3,2-
d]pyrimidine (2, 32 mg) and using L-phenylalanine (23 mg)
following the general procedure, and was obtained as a pale yellow
solid (32 mg, 66%): mp > 240 ◦C. HRMS: calcd for C21H16N3O2S
([M - H]-) 374.0969, found 374.0971.
(S)-2-(6-Iodo-2-phenylthieno[3,2-d]pyrimidin-4-ylamino)-3-
phenylpropanoic acid (16) was prepared from 4-chloro-6-iodo-
2-phenylthieno[3,2-d]pyrimidine (10a, 48 mg) and using L-
phenylalanine (23 mg) following the general procedure, and was
obtained as a pale yellow solid (36 mg, 55%): mp > 240 ◦C. HRMS:
calcd for C21H15IN3O2S ([M - H]-) 499.9935, found 499.9947.
(S)-2-(6,7-Diiodo-2-phenylthieno[3,2-d]pyrimidin-4-ylamino)-3-
phenylpropanoic acid (17) was prepared from 4-chloro-6,7-diiodo-
2-phenylthieno[3,2-d]pyrimidine (10b, 65 mg) and using L-
phenylalanine (23 mg) following the general procedure, and was
obtained as a pale yellow solid (39 mg, 48%): mp > 240 ◦C. HRMS:
calcd for C21H14I2N3O2S ([M - H]-) 625.8902, found 625.8916.
Acknowledgements
We are grateful to Re´gion Bretagne for financial support to KS
and GBA. We thank Thierry Roisnel for his contribution to this
study.
(S)-3-Phenyl-2-(2-phenylthieno[2,3-d]pyrimidin-4-ylamino)pro-
panoic acid (18) was prepared from 4-chloro-2-phenylthieno[2,3-
d]pyrimidine (3, 32 mg) and using L-phenylalanine (23 mg)
following the general procedure, and was obtained as a pale yellow
solid (31 mg, 53%): mp > 240 ◦C. HRMS: calcd for C21H16N3O2S
([M - H]-) 374.0969, found 374.0968.
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The Royal Society of Chemistry 2009
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