Synthesis, and structure-activity relationship for C(4) and/or C(5) thienyl substituted pyrimidines, as a new family of antimycobacterial compounds

Article abstract of DOI:10.1016/j.ejmech.2015.05.007

Abstract Combination of the Suzuki cross-coupling and nucleophilic aromatic substitution of hydrogen (SNH) reactions proved to be a convenient method for the synthesis of C(4) and/or C(5) mono(thienyl) and di(thienyl) substituted pyrimidines from commerci

Full text of DOI:10.1016/j.ejmech.2015.05.007

Accepted Manuscript
Synthesis, and structure–activity relationship for C(4) and/or C(5) thienyl substituted
pyrimidines, as a new family of antimycobacterial compounds
Egor V. Verbitskiy, Ekaterina M. Cheprakova, Pavel A. Slepukhin, Marionella A.
Kravchenko, Sergey N. Skornyakov, Gennady L. Rusinov, Oleg N. Chupakhin, Valery
N. Charushin
PII:
S0223-5234(15)30033-7
10.1016/j.ejmech.2015.05.007
EJMECH 7886
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 April 2015
Revised Date: 3 May 2015
Accepted Date: 5 May 2015
Please cite this article as: E.V. Verbitskiy, E.M. Cheprakova, P.A. Slepukhin, M.A. Kravchenko,
S.N. Skornyakov, G.L. Rusinov, O.N. Chupakhin, V.N. Charushin, Synthesis, and structure–activity
relationship for C(4) and/or C(5) thienyl substituted pyrimidines, as a new family of antimycobacterial
compounds, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.007.
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Synthesis, and structure–activity relationship
for C(4) and/or C(5) thienyl substituted
pyrimidines, as a new family of
Leave this area blank for abstract info.
antimycobacterial compounds
E. V. Verbitskiy, E. M. Cheprakova, P. A. Slepukhin, M. A. Kravchenko, S. N. Skornyakov, G. L. Rusinov,
O. N. Chupakhin, V. N. Charushin
I. Postovsky Institute of Organic Synthesis, S. Kovalevskoy Str., 22, 620137 Ekaterinburg, Russia
R'
N
H- reaction
S
N
S
Pd-Catalyzed
cross-coupling
N
R'
Br
N
N
N
S
S
N
R
Br
H- reaction
N
Pd-Catalyzed
cross-coupling
S
N
S
N
R
MIC = 0.15 - 12.5µ g/mL
Mycobacterium tuberculosis (H37Rv, MDR),
Mycobacterium avium and terrae

ACCEPTED MANUSCRIPT
Synthesis, and structure–activity relationship for C(4) and/or C(5) thienyl
substituted pyrimidines, as a new family of antimycobacterial compounds
a,b
a,b
a,b
Egor V. Verbitskiy, * Ekaterina M. Cheprakova, Pavel A. Slepukhin,
c
c
a,b
Marionella A. Kravchenko, Sergey N. Skornyakov, Gennady L. Rusinov,
a,b
a,b
Oleg N. Chupakhin, Valery N. Charushin
a
I. Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences,S. Kovalevskoy Str., 22, Ekaterinburg, 620137, Russia
Ural Federal University, Mira St. 19, Ekaterinburg, 620002, Russia
b
c
Ural Research Institute for Phthisiopulmonology, 22 Parts’ezda St., 50, Ekaterinburg, 620039, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Combination of the Suzuki cross-coupling and nucleophilic aromatic substitution of
hydrogen (SN ) reactions proved to be a convenient method for the synthesis of C(4)
and/or C(5) mono(thienyl) and di(thienyl) substituted pyrimidines from commercially
available 5-bromopyrimidine. All new pyrimidines were found to be active in micromolar
H
concentrations in vitro against H Rv, avium, terrae, rifampicin and isoniazid-resistance
strains of Mycobacterium tuberculosis. The data for acute in vivo toxicity in mice have
been obtained for these compounds which appear to be promising antitubercular agents.
Keywords:
Pyrimidine
Antimicobacterial
Tuberculosis
37
Cross-coupling
Nucleophilic aromatic substitution of
hydrogen
2
015 Elsevier Ltd. All rights reserved
.
*
1
. Introduction
Tuberculosis, a lung infection mostly caused by Mycobacterium tuberculosis (MTB), is
considered to be one of the most threatening diseases for public health. During the last decade a
number of incidences of tuberculosis have been increasing rapidly worldwide due to diffusion of
multi-drug resistant strains of Mycobacterium tuberculosis. In only 2012, more than eight millions
of people have got tuberculosis and more than one million died from this disease, developed mainly
in the poor developed countries [1]. Therefore, new effective and safety drugs against tuberculosis,
acting through novel mechanisms, are urgently needed for a combination chemotherapy, capable of
improving efficacy of anti-tuberculosis agents, and avoiding or delay with the resistance
phenomena.
The nitrogen-containing heterocycles in general, and pyrimidines in particular, have been
found in a great number of biologically active compounds of natural origin, thus showing their
considerable therapeutic potential [2]. Since a wide spectrum of biological activities, such as anti-
allergic [3], antitumor [4], antipyretic [5], anti-inflammatory [5] and antiparasitic [6] ones, exhibited
by synthetic pyrimidines, compounds of this family have gained a considerable attention. Also, it
has been established that many pyrimidines [7-12], and purine derivatives [13-20] possess a high
*
Corresponding author. Tel./fax: +7 343 3693058; e-mail address: Verbitsky@ios.uran.ru (E.V. Verbitskiy).
1

ACCEPTED MANUSCRIPT
antituberculosis activity. Screening of the series of 5-(hetero)aryl- and 4,5-di(hetero)aryl-2-
thio)morpholinopyrimidines revealed several promising compounds with a good level of activity in
vitro against Mycobacterium tuberculosis H Rv [21].
(
3
7
Very recently, we reported the synthesis a new series of 5-arylethenyl-4-(het)aryl-
pyrimidines (Fig. 1), which exhibit a good level of activity against multidrug-resistant strain of
Mycobacterium tuberculosis, and a low acute toxicity on white mice [22]. On the other hand, it has
been established that variation of thienyl fragments at C(5) and C(6) positions in 5-aryl-6-hetaryl
substituted 1,6-dihydropyrazine derivatives (Fig. 2) strongly affects their antitubercular activity
[
23].
Figure 1.
Figure 2.
In continuation of our interest in designing of pyrimidines with a plausible antituberculosis
activity, we have obtained a new series of C(4) and/or C(5) mono(thienyl) and di(thienyl)
substituted pyrimidine derivatives in order to study their structure-activity relationship.
2
. Results and discussion
2
.1. Chemistry
The desired 4,5-di(thienyl) substituted pyrimidines have been obtained via combination of
the palladium-catalyzed Suzuki–Miyaura cross-coupling reaction and nucleophilic aromatic
H
substitution of hydrogen (the SN reaction) [24].
As the first step we have synthesized C(4) and C(5) mono(thienyl) substituted pyrimidines.
5-(Hetero)arylpyrimidines 6-9 were obtained from 5-bromopyrimidine and 2-thienylboronic (2) [3-
thienylboronic (3), benzo[b]thien-2-ylboronic (4) or benzo[b]thien-3-ylboronic (5)] acid by using
the aerobic Suzuki cross-coupling reaction with
bis(dicyclohexylamine)palladium(II) acetate (DAPCy) [25].
a
new catalyst, namely trans-
Scheme 1.
The further bromination of 5-(thien-2-yl)pyrimidine (6) using N-bromosuccinimide (NBS)
in DMF at room temperature gave 5-(5-bromo-thiophen-2-yl)pyrimidine (10) in good 74% yield
(Scheme 2).
Scheme 2.
Earlier, we have established that the reaction of 5-bromopyrimidine with 2 equiv. of 2,2'-
bithiophene (11a) or 2-phenylthiophene (11b) in DMF under microwave irradiation at 180 ºC in the
presence of a mixture of 10 mol% Pd(OAc) and 20 mol% PCy – tricyclohexylphosphine (catalyst)
2
3
2

ACCEPTED MANUSCRIPT
with 3 equiv. K CO (base) provides the best yields (49-52%) of 5-[2,2']bithiopen-5-yl-pyrimidine
2
3
(12a) and 5-(5-phenylthiophen-2-yl)-pyrimidine (3b) (Scheme 3) [26].
Scheme 3.
On the other hand, the synthesis of 5-unsubsituted 4-(hetero)arylpyrimidines 14a-d by using
H
N
the S -reactions of pyrimidine (13) with thiophene derivatives 11a-d has been reported in the
literature [24a, 24c, 26].
Scheme 4.
Similarly, 5-bromo-4-thienyl substituted pyrimidines 15a-d have been synthesized by
reacting 5-bromopyrimidine (1) with 2-substituted thiophene 11a-d in CF COOH or methanol in
3
the presence of boron trifluoride diethyl etherate (BF ×Et O) under the same reaction conditions
3
2
(Scheme 5) [24a, 26].
Scheme 5.
The target 4,5-di(thienyl) substituted pyrimidines 16-19 have been obtained in high yields
through the reaction route described in Scheme 6. The starting materials 15a, 15b and 15с were
coupled with the corresponding (hetero)arylboronic acid 2 (3, 4 or 5) under microwave irradiation,
by using 1,4-dioxane-H O as solvent, K CO as base, and Pd(PPh ) as catalyst. All the reactions
2
2
3
3 4,
studied proved to afford the corresponding hetaryl-hetaryl coupling products 16-19a-c in 56-91%
yields within the reaction time not exceeding 20 min (Scheme 6). Unfortunately, use of DAPCy
under conditions of the aerobic Suzuki cross-coupling reaction of compounds 15a-c with
(hetero)arylboronic acids 2-5 was not effective, thus giving 4,5-di(thienyl) substituted pyrimidines
in rather low yields (~18-37%). The structures of 18b,c and 19b,c have been established
unequivocally by the X-ray diffraction analysis (see Figures 3, 4, S1 and S2 in Supplementary
material.). According to the XRD data, compounds 19b and 19c are isostructural, with standard
bonds length and angles. Both pyrimidine and thiophene rings in these molecules are placed into the
same plane, while the benzothiophenyl system and the terminal thiophenyl substituent in 8b are
disordered relative this plane. No shortened intermolecular contacts in the crystals of 19b have been
observed.
Scheme 6.
Figure 3.
Figure 4.
H
N
Finally, we prepared 4-(bromothiophen-2-yl)-5-(hetero)arylpyrimidines by using the S -
reactions 5-(hetero)arylpyrimidines 6-9 with 2-bromothiophene (11d) in CF COOH, followed by
3
the subsequent oxidation of intermediates (Scheme 7). The crystal structure of 5-benzo[b]thiophen-
3

ACCEPTED MANUSCRIPT
3
5
-yl-4-(5-bromothiophen-2-yl)pyrimidine (19d) was also confirmed by X-ray diffraction (see Fig.
).
Scheme 7.
Figure 5.
2
.2. Antimycobacterial in vitro activity and in vivo toxicity evaluation
3
2 Compounds were screened for their activity in vitro against Mycobacterium tuberculosis
H Rv, avium, terrae, and multi-drug-resistant strains, and the data on the minimum inhibitory
3
7
concentrations (MICs) of these compounds are presented in Table 1. All new pyrimidines were
compared with the commercially available drugs Isoniazid and Pyrazinamide, tested under the same
experimental conditions.
The parent 5-bromopyrimidine (1) has a low MIC (12.5 ꢀg/mL). Besides that, we have
observed that both 4-unsubsituted (6-8, 10 and 12a,b), and 5-unsubsituted 4-thienylpyrimidines
14a-d have demonstrated a low level of tuberculostatic activity (MIC: 6.25-12.5 ꢀg/mL). The only
exception was 5-benzo[b]thiophen-3-pyrimidune (9), demonstrating a high value of
antimycobacterial activity (see Table1, entry 5). On the contrary, 5-bromo substituted pyrimidines
15a-d proved to exhibit a significant level of inhibitory properties against Mycobacterium
tuberculosis H Rv (MIC from 6.25 to 0.7 ꢀg/mL).
3
7
As a whole, substitution of bromo atom at C(5) with the thienyl fragment results in a
decrease of tuberculostatic concentrations to 6.25-12.5 ꢀg/mL. It is interesting to point out that
combinations of 2-thienyl-, 5-bromo-2-thienyl- and 5-phenylthienyl- substituents at C(4) lead to
increase of the level of antimycobacterial activity in case of compounds 16a, 18c and 19d (MIC: 3.1
ꢀ
g/mL). Introduction of [2,2′]bithiophenyl substituent into C(4) position is accompanied with a
considerable increase in anti-TB activities of pyrimidines (for example, see Table 1, entries 8, 14,
2 and 30) reaching the maximum MIC in the case of pyrimidine 16b (Table 1, entry 18). The best
2
result has been found for 5-benzo[b]thiophen-3-yl-4-(5-phenyl-thiophen-2-yl)-pyrimidine (19c). Its
MIC (0.15 ꢀg/mL) is comparable with the activity of Isoniazid (0.1 ꢀg/mL).
In summary, we can state that small substituents, such as bromine, thienyl, 5-phenylthienyl
or [2,2′]bithiophenyl at C(4) / C(5) positions of the pyrimidine ring promote a good activity
Mycobacterium tuberculosis, including multidrug-resistant strains.
A good level of antimycobacterial activity of the whole number of compounds of the
pyrimidine family gave us a reason for further detailed evaluation of the toxic data for these
compounds. For the most active compounds 9, 15a, 15c, 16b, 19b and 19c with MIC ≤ 1.5 ꢀg/mL
against Mycobacterium tuberculosis H Rv, the data on their acute toxicities on white mice have
3
7
been determined (Table 1). A number of compounds proved to be less toxic for white mice than
4

ACCEPTED MANUSCRIPT
Isoniazid. It should be noted that in case of 5-bromo substituted compounds 15a,b the acute oral
toxicities were almost two times lower than that for compound 16b. Unfortunately, the highly active
4,5-di(hetero)arylpyrimidines 19b,c proved to be more toxic, than the reference drugs. And it is
clear that these substances require further substantial modifications in order to reduce toxicity with
retention of the existing level of activity.
Table 1.
3
. Conclusions
An efficient way for the synthesis of new family of C(4) and C(5) mono(thienyl) and
di(thienyl) substituted pyrimidines has been realized. Among novel pyrimidine derivatives obtained
there are compounds with antimycobacterial activity, which can possibly be further modified to
reach a better potency than the reference drugs. Also it has been found that the various thienyl
fragments are supposed to be in the structure of pyrimidines, in particular at C(4) position, to
exhibit anti-TB activity. The results evidenced that some C(4) and/or C(5) mono(thienyl) and
di(thienyl) substituted pyrimidine derivatives, among the compounds presented here, can be
considered as promising anti-M. tuberculosis agents, since one of them has MIC of 0.15 µg/mL and
other five have MICs ranging from 0.3 to 1.5 µg/mL. Further studies to get more data concerning
quantitative structure – activity relationships (SQAR) are in progress in our laboratory.
4
. Experimental section
4
.1. General information
All reagents and solvents were obtained from commercial sources and dried by using the
standard procedures before use. 5-Bromopyrimidine (1) and all (hetero)arylboronic acids were
purchased from Alfa Aesar. 5-[2,2']Bithiophenyl-5-yl-pyrimidine (12a) [26], 5-(5-phenylthiophen-
2-yl)-pyrimidine (12b) [26], 4-thiophen-2-yl-pyrimidine (14a) [24a], 4-[2,2']-bithiophenyl-5-yl-
pyrimidine (14b) [24c], 4-(5-phenylthiophen-2-yl)-pyrimidine (14c) [26], 4-(5-bromothiophen-2-
yl)-pyrimidine (14d) [24a], 5-bromo-4-thiophen-2-yl-pyrimidine (15a) [24a], 4-[2,2']bithiophenyl-
5
-yl-5-bromopyrimidine (15b) [26], 5-bromo-4-(5-phenyl-thiophen-2-yl)-pyrimidine (15c) [26], 5-
bromo-4-(5-bromothiophen-2-yl)-pyrimidine (15d) [24a], 4,5-dithiophen-2-yl-pyrimidine (16a)
24a], 4-[2,2']-bithiophenyl-5-yl-5-thiophen-2-ylpyrimidine (16b) [24a], 4-(5-bromothiophen-2-yl)-
-thiophen-2-yl-pyrimidine (16d) [24a], 5-(thien-3-yl)-4-(thien-2-yl)pyrimidine (17a) [28], 4-[5-
bromo(thien-2-yl)]-5-(thien-3-yl)pyrimidine (17d) [28], 5-benzo[b]thien-2-yl-4-(thien-2-
[
5
yl)pyrimidine (18a) [28], 5-(benzo[b]thien-3-yl)-4-(thien-2-yl)pyrimidine (19a) [28] were
synthesized as described previously. Solvents (1,4-dioxane and H O) for the microwave-assisted
2
Suzuki cross-coupling reaction were deoxygenated by bubbling argon for 1h.
5

ACCEPTED MANUSCRIPT
1
13
The H and C NMR spectra were recorded on a Bruker DRX-400 and AVANCE-500
instruments, using Me Si as an internal standard. Elemental analysis was carried on a Eurovector
4
EA 3000 automated analyzer. Melting points were determined on Boetius combined heating stages
and were not corrected.
The GC-MS analysis of all samples was carried out using an Agilent GC 7890A MS 5975C
Inert XL EI/CI GC-MS spectrometer with a quadrupole mass-spectrometric detector with electron
ionization (70 eV) and scan over the total ionic current in the range m/z 20–1000 and a quartz
capillary column HP-5MS (30 m × 0.25 mm, film thickness 0.25 mm). Helium served as a carrier
–
gas, the split ratio of the flow was 1 : 50, and the consumption through the column was 1.0 mL min
1
;
the initial temperature of the column was 40 °C (storage 3 min), programming rate was 10 °C
–1
min to 290 °C (storage 20 min), the temperature of the evaporator was 250 °C, the temperature of
the source was 230 °C, the temperature of the quadrupole was 150 °C, and the temperature of the
–
1
transition chamber was 280 °C. Solutions of the samples with a concentration of 3-4 mg mL were
prepared in THF. Samples of 1 mL of the obtained solutions were analyzed.
Column chromatography was carried out using Alfa Aesar silica gel 0.040-0.063 mm (230–
400 mesh), eluting with ethyl acetate-hexane, 1:1 or 1:3. The progress of reactions and the purity of
compounds were checked by TLC on Sorbfil plates (Russia), in which the spots were visualized
with UV light (λ 254 or 365 nm).
Microwave experiments were carried out in a Discover unimodal microwave system (CEM,
USA) with a working frequency of 2.45 GHz and the power of microwave radiation ranged from 0
to 300 W. The reactions were carried out in a 10 mL reaction tube with the hermetic Teflon cork.
The reaction temperature was monitored, using an inserted IR sensor by the external surface of the
reaction vessel.
The X-ray diffraction analysis of single crystals of compounds 18b,c and 19b,c,d was
accomplished on a Xcalibur S diffractometer on standard procedure (graphite-monochromated
o
MoK-irradiation, T= 295(2) K, ω-scanning with step 1 ). Analytical absorption correction was
applied [29] Using Olex2 [30], the structure was solved with the ShelXS [31] structure solution
program using direct method and refined with the ShelXL [31] refinement package using Least
Squares minimisation. The X-ray crystallography data for structures 18b,c and 19b,c,d reported in
this paper have been deposited with Cambridge Crystallography Data Centre as supplementary
publications no CCDC 1058040 for 18b, CCDC 1058038 for 18c, CCDC 1058041 for 19b, CCDC
1058042 for 19c, and CCDC 1058039 for 19d. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
6

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.2. General procedure for the room temperature aerobic Suzuki cross-coupling
4
reactions
K PO (531 mg, 2.5 mmol) was added to a solution of 5-bromopyrimidine (1) (159 mg, 1.0
3
4
mmol), 2-thienylboronic acid (2) [3-thienylboronic acid (3), benzo[b]thien-2-ylboronic acid (4) or
benzo[b]thien-3-ylboronic acid (5)] (1.5 mmol) and trans-bis(dicyclohexylamine)palladium(II)
acetate (29 mg, 0.05 mmol) in EtOH (10 mL). The resulting suspension was stirred at ambient
temperature for 24 h. EtOH was evaporated under reduced pressure and the residue was suspended
in CH Cl (20 mL) and filtered from inorganic salts. After that solvent was distilled off under
2
2
reduced pressure, and the residue was purified by flash column chromatography on silica gel
hexane/ethyl acetate, 1:3) to afford the desired cross-coupling products (6, 7, 8 or 9).
(
4
.2.1. 5-Thiophen-2-yl-pyrimidine (6) [24a].
4'
3'
6
5'
5
1
2'
S1'
N
2
4
N
3
6
It has been obtained by reaction 1 with 2-thienylboronic (2) acid. Yield (96 mg, 59%), pale
1
yellow solid; mp 77-78 °C. H NMR (400 MHz, CDCl ): δ = 7.18 (dd, 1H, H-4', J = 5.0, 3.6 Hz),
3
7
9
.44 (dd, 1H, H-3', J = 3.6, 1.1 Hz), 7.47 (dd, 1H, H-5', J = 5.0, 1.1 Hz), 8.98 (s, 2H, H-4 and H-6),
+
.13 (s, 1H, H-2) ppm. GC t 16.04 min; MS m/z (rel intensity) 162 (M , 100). Anal. Calcd for
R
C H N S (162.21): C, 59.24; H, 3.73; N, 17.27. Found: C, 59.18; H, 3.63; N, 17.55.
8
6
2
4
.2.2. 5-Thiophen-3-yl-pyrimidine (7) [24b].
5
'
4
'
S1'
6
3
'
1
N
5
2'
2
N
4
3
7
It has been obtained by reaction 1 with 3-thienylboronic (3) acid. Yield 128 mg (79%), pale
1
beige solid; m.p. 102-103 °C. H NMR (500 MHz, CDCl ): δ = 7.41 (dd, 1H, J = 5.0, 1.4 Hz, H-4'),
3
7
1
1
.51 (dd, 1H, J = 5.0, 3.0 Hz, H-5'), 7.61 (dd, 1H, J = 3.0, 1.4 Hz, H-2'), 8.96 (s, 2H, H-4,6), 9.15 (s,
1
3
H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 122.51 (C-2'), 125.34 (C-4'), 127.77 (C-5'),
3
29.49 (C-5), 135.06 (C-3'), 154.13 (C-4,6), 157.15 (C-2) ppm. GC t 16.40 min; MS m/z (rel
R
+
intensity): 162 (М , 100). Anal. Calcd for C H N S (162.21): C 59.24, H 3.73, N, 17.27, S 19.77.
8
6
2
Found: C 59.28; H, 3.59; N, 17.47, S 19.66.
7

ACCEPTED MANUSCRIPT
.2.3. 5-Benzo[b]thiophen-2-yl-pyrimidine (8) [32].
4
4'
5'
3'
6'
6
7'
5
1
2'
S1'
N
2
4
N
3
8
It has been obtained by reaction 1 with benzo[b]thien-3-ylboronic acid (5). Yield (146 mg,
1
6
9%), off-white solid; mp 129-130 °C. H NMR (500 MHz, CDCl ): δ = 7.36-7.44 (m, 2H,
3
benzo[b]thienyl), 7.66 (s, 1H, H-3'), 7.82-7.90 (m, 2H, benzo[b]thienyl), 9.05 (s, 2H, H-4 and H-6),
1
3
9
1
.18 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 121.92, 122.44, 124.13, 125.09, 125.46,
3
28.67, 136.10, 139.92, 140.09, 153.93 (C-4,6), 157.82 (C-2) ppm. GC t 22.16 min; MS m/z (rel
R
+
intensity) 212 (M , 100). Anal. Calcd for C H N S (212.27): C, 67.90; H, 3.80; N, 13.20. Found:
1
2
8
2
C, 67.84; H, 3.69; N, 13.19.
4
.2.4. 5-Benzo[b]thiophen-3-yl-pyrimidine (9) [33].
6
'
5'
7
'
4'
S1'
6
3'
1
N
5
2'
2
N
4
3
9
It has been obtained by reaction 1 with benzo[b]thien-3-yl boronic acid (5). Yield 161 mg
1
(
76%), off-white solid; m.p. 89-90 °C. H NMR (500 MHz, CDCl ): δ = 7.42-7.50 (m, 2H,
3
benzo[b]thienyl), 7.56 (s, 1H, H-2'), 7.80-7.85 (m, 1H, benzo[b]thienyl), 7.93-8.00 (m, 1H,
1
3
benzo[b]thienyl), 8.98 (s, 2H, H-4 and H-6), 9.27 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ
3
=
121.85, 123.19, 125.06, 125.10, 125.87, 130.02, 130.57, 136.99, 140.71, 156.09 (C-4,6), 157.67
+
(
C-2) ppm. GC t 21.78 min; MS m/z (rel intensity): 212 (М , 100). Anal. Calcd for C H N S
R
12
8
2
(212.27): C, 67.90; H, 3.80; N, 13.20. Found: C 68.07; H, 3.72; N, 13.19.
4
.3. Synthesis of 5-(5-bromothiophen-2-yl)-pyrimidine (10) [34].
4'
3'
5
'
6
5
Br
1
2
'
S1'
N
2
4
N
3
10
NBS (267 mg, 1.5 mmol) was added to a solution of 5-thiophen-2-yl-pyrimidine (6) (162
mg, 1.0 mmol) in DMF (5 mL) at room temperature. Then the mixture was stirred for 24 h at room
8

ACCEPTED MANUSCRIPT
temperature. The reaction mixture was diluted with H O and the precipitate formed was filtered,
2
washed with H O, and air-dried. The residue was purified by flash column chromatography
2
(hexane/ethyl acetate, 1:1). The product 10 was isolated as a beige solid (178 mg, 74%), m.p. 80–81
1
°
C. H NMR (500 MHz, DMSO-d ): δ = 7.38 (d, 1H, H-3'or H-4', J = 3.9 Hz), 7.63 (d, 1H, J = 3.9
6
1
3
Hz, H-3'or H-4' ), 9.10 (s, 2H, H-4 and H-6), 9.14 (s, 1H, H-2) ppm. C NMR (126 MHz, DMSO-
d ): δ = 113.23, 127.19, 127.23, 131.99, 137.50, 153.07 (C-4,6), 157.25 (C-2) ppm. GC t 18.97
6
R
+
79
+
81
min; MS m/z (rel intensity) 240 (M , 100) for Br, 242 (M , 100) for Br. Anal. Calcd for
C H BrN S (241.11): C, 39.85; H, 2.09; N, 11.62. Found: C, 39.84; H, 2.27; N, 11.59.
8
5
2
4
.4. General procedure for the microwave-assisted Suzuki cross-coupling reactions
A solution of K CO (173 mg, 1.25 mmol) in H O (3 mL) was added to a mixture of а 5-
2
3
2
bromo substituted 4-(hetero)arylpyrimidine (15a, 15b or 15c) (0.5 mmol), the corresponding
hetero)arylboronic acid (1) (0.6 mmol) and Pd(PPh ) (29 mg, 5 mol %) in 1,4-dioxane (4 mL).
(
3
4
The resulting mixture was irradiated in a microwave apparatus at 160 °C (250 W) for 20 min. After
that solvent was distilled off in vacuo, and the residue was purified by flash column
chromatography (hexane/ethyl acetate, 1:3) to afford the desired cross-coupling products (16c, 17b,
17c, 18b, 18c, 19b and 19c).
4
.4.1. 4-(5-Phenyl-thiophen-2-yl)-5-thiophen-2-yl-pyrimidine (16c).
4'
3'
6
5'
5
1
2'
S1'
N
4
1"
S
2
N
5"
2
"
3
3"
4"
16c
It has been obtained by reaction 15c with 2-thienylboronic acid (2). Yield 90 mg (56%),
1
yellow solid; m.p. 153-156 °C. H NMR (500 MHz, CDCl ): δ = 6.97 (d, 1H, J = 4.0 Hz, H-3''or H-
3
4''), 7.14 (d, 1H, J = 4.0 Hz, H-3''or H-4''), 7.17 (dd, 1H, J = 3.5, 1.2 Hz, H-3'), 7.20 (dd, J = 5.1, 3.5
Hz, 1 H, H-4'), 7.29-7.34 (m, 1H, Ph), 7.36-7.40 (m, 2H, Ph), 7.54 (dd, 1H, J = 5.1, 1.2 Hz, H-5'),
1
3
7
1
1
8
.60-7.62 (m, 2H, Ph), 8.62 (s, 1H, H-6), 9.11 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ =
3
23.96, 124.19, 126.00, 127.77, 127.96, 128.44, 128.47, 128.99, 131.99, 133.62, 136.38, 140.11,
49.43, 157.38, 157.86, 159.10 ppm. Anal. Calcd for C H N S (320.44): C, 67.47; H, 3.77; N,
1
8
12
2 2
.74. Found: C 67.66; H, 3.73; N, 8.58.
4
.3.2. 4-[2,2']Bithiophen-5-yl-5-thiophen-3-yl-pyrimidine (17b).
9

ACCEPTED MANUSCRIPT
5'
4'
6
S1'
5
1
3'
N
2'
4
1"
S
2
N
5"
2
"
3
S
3"
4"
17b
It has been obtained by reaction 15b with 3-thienylboronic acid (3). Yield 98 mg (60%),
1
yellow solid; m.p. 91-94 °C. H NMR (500 MHz, CDCl ): δ = 6.78 (d, 1H, J = 4.0 Hz, H-3''or H-
3
4
4
3
1
1
8
''), 6.98 (d, 1H, J = 4.0 Hz, H-3''or H-4''), 7.03 (dd, J = 4.6, 4.1 Hz, 1 H, thienyl), 7.10 (d, 1H, J =
.9 Hz, thienyl), 7.23-7.27 (m, 2H, thienyl), 7.39 (d, 1H, J = 2.4 Hz, thienyl), 7.49 (dd, 1H, J = 4.9,
13
.0 Hz, thienyl), 8.56 (s, 1H, H-6), 9.08 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ =
3
24.51, 124.64, 124.79, 125.58, 126.02, 127.15, 128.06, 128.16, 131.44, 136.17, 136.72, 139.93,
42.18, 156.36, 157.38, 158.23 ppm. Anal. Calcd for C H N S (326.46): C, 58.87; H, 3.09; N,
1
6
10
2 3
.58. Found: C 58.80; H, 3.25; N, 8.64.
4
.3.3. 4-(5-Phenyl-thiophen-2-yl)-5-thiophen-2-yl-pyrimidine (17c).
5'
4'
6
S1'
5
1
3'
N
2'
4
1"
S
2
N
5"
2
"
3
3"
4"
17c
It has been obtained by reaction 15c with 3-thienylboronic acid (3). Yield 107 mg (65%),
1
yellow solid; m.p. 131-132°C. H NMR (500 MHz, CDCl ): δ = 6.88 (d, 1H, J = 4.0 Hz, H-3''or H-
3
4''), 7.11 (dd, 1H, J = 4.9, 0.8 Hz, thienyl), 7.13 (d, 1H, J = 4.0 Hz, H-3''or H-4''), 7.30-7.33 (m, 1H,
Ph), 7.37-7.40 (m, 3H, Ph and thienyl), 7.49 (dd, 1H, J = 4.9, 3.0 Hz, thienyl), 7.61-7.62 (m, 2H,
1
3
Ph), 8.57 (s, 1H, H-6), 9.10 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 124.13, 124.63,
3
1
1
6
25.95, 126.07, 127.10, 128.22, 128.38, 128.98, 131.65, 133.63, 136.27, 140.53, 149.05, 156.56,
57.42, 158.28 ppm. Anal. Calcd for C H N S (320.44): C, 67.47; H, 3.77; N, 8.74. Found: C
1
8
12
2 2
7.27; H, 3.64; N, 8.66.
4
.3.4. 5-Benzo[b]thiophen-2-yl-4-[2,2']bithiophen-5-yl-pyrimidine (18b).
4'
5'
6'
3'
6
7'
5
1
2'
S 1'
N
4
1"
S
2
N
5"
2
"
3
S
3"
4"
18b
1
0

ACCEPTED MANUSCRIPT
It has been obtained by reaction 15b with benzo[b]thien-2-ylboronic acid (4). Yield 141 mg
1
(
75%), yellow solid; m.p. 189-191 °C. H NMR (500 MHz, CDCl ): δ = 6.94 (d, 1H, J = 4.1 Hz, H-
3
3''or H-4''), 7.00 (d, 1H, J = 4.1 Hz, H-3''or H-4''), 7.01-7.03 (m, 1H, thienyl), 7.23-7.25 (m, 2H,
thienyl), 7.42 (s, 1H, H-3'), 7.43-7.46 (m, 2H, benzo[b]thienyl), 7.86 (dd, 1H, J = 6.8, 1.8 Hz,
1
3
benzo[b]thienyl), 7.89-7.91 (m, 1H, benzo[b]thienyl), 8.68 (s, 1H, H-6), 9.13 (s, 1H, H-2) ppm. C
NMR (126 MHz, CDCl ): δ = 122.50, 123.90, 124.12, 124.79, 124.89, 124.97, 125.14, 125.16,
3
125.75, 128.10, 132.25, 136.63, 137.04, 139.26, 139.76, 140.80, 142.70, 156.86, 158.09, 159.03
+
ppm. GC t 38.48 min; MS m/z (rel intensity): 376 (М , 100). Anal. Calcd for C H N S (376.52):
R
20 12
2 3
C, 63.80; H, 3.21; N, 7.44. Found: C 63.93; H, 3.13; N, 7.28.
4
.4.5. 5-Benzo[b]thiophen-2-yl-4-(5-phenyl-thiophen-2-yl)-pyrimidine (18c).
4'
5'
6'
3'
6
7'
5
1
2'
S 1'
N
4
1"
S
2
N
5"
2
"
3
3"
4"
18c
It has been obtained by reaction 15c with benzo[b]thien-2-ylboronic acid (4). Yield 159 mg
1
(
86%), yellow solid; m.p. 190-192 ºC. H NMR (500 MHz, CDCl3): δ = 7.09 (m, 2H, thienyl), 7.31
(dd, 1H, J = 5.8, 3.7 Hz, thienyl), 7.36-7.39 (m, 2H, Ar), 7.43 (s, 1H, H-3'), 7.44-7.46 (m, 2H, Ar),
7.58-7.62 (m, 2H, Ar), 7.87 (dd, 1H, J = 6.8, 1.9 Hz, benzo[b]thienyl), 7.89-7.91 (m, 1H,
1
3
benzo[b]thienyl), 8.69 (s, 1H, H-6), 9.15 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ =
3
1
1
22.53, 123.98, 124.15, 124.44, 124.90, 125.14, 125.19, 126.06, 128.51, 129.02, 132.45, 133.61,
37.23, 139.84, 139.97, 140.88, 149.65, 157.10, 158.16, 159.11 ppm. Anal. Calcd for C H N S
2 2
22
14
(370.50): C, 71.32; H, 3.81; N, 7.56. Found: C 71.27; H, 3.94; N, 7.47.
4
.4.6. 5-Benzo[b]thiophen-3-yl-4-[2,2']bithiophen-5-yl-pyrimidine (19b).
6'
5'
7'
4'
6
5
S1'
1
3'
N
2
'
4
1"
2
S
N
5"
2
"
3
S
3"
4"
19b
It has been obtained by reaction 15b with benzo[b]thien-3-ylboronic acid (5). Yield 147 mg
1
(
78%), yellow solid; m.p. 139-140 °C. H NMR (500 MHz, CDCl ): δ = 6.54 (d, 1H, J = 4.1 Hz, H-
3
3''or H-4''), 7.06 (d, 1H, J = 4.1 Hz, H-3''or H-4''), 7.09 (dd, 1H, J = 5.0, 3.7 Hz, thienyl), 7.32 (d,
1
1

ACCEPTED MANUSCRIPT
1
3
8
H, J = 0.7 Hz, benzo[b]thienyl), 7.33 (dd, 1H, J = 2.2, 0.9 Hz, benzo[b]thienyl), 7.35 (dd, 1H, J =
.7, 1.1 Hz, thienyl), 7.39-7.48 (m, 1H, benzo[b]thienyl), 7.57 (dd, 1H, J = 5.1, 1.1 Hz, thienyl),
.08 (s, 1H, H-2'), 8.15 (d, 1H, J = 8.2 Hz, benzo[b]thienyl), 8.75 (s, 1H, H-6), 9.22 (s, 1H, H-2)
1
3
ppm. C NMR (126 MHz, CDCl ): δ = 122.21, 123.29, 123.98, 124.92, 124.98, 125.01, 125.36,
3
1
1
26.92, 127.81, 128.56, 130.85, 131.11, 135.55, 137.28, 139.38, 139.59, 141.20, 155.72, 157.74,
+
59.38 ppm. GC t 36.77 min; MS m/z (rel intensity): 376 (М , 100). Anal. Calcd for C H N S
R
20 12
2
3
(376.52): C, 63.80; H, 3.21; N, 7.44. Found: C 63.60; H, 3.27; N, 7.43.
4
.4.7. 5-Benzo[b]thiophen-3-yl-4-(5-phenyl-thiophen-2-yl)-pyrimidine (19c).
6'
5'
7'
4'
6
S
1'
5
1
3'
N
2'
4
1"
S
2
N
5"
2
"
3
3"
4"
19c
It has been obtained by reaction 15c with benzo[b]thien-3-ylboronic acid (5). Yield 169 mg
1
(
91%), yellow solid; m.p. 151-152°C. H NMR (500 MHz, CDCl ): δ = 6.60 (d, 1H, J = 4.1 Hz, H-
3
3
''or H-4''), 7.28 (d, 1H, J = 4.1 Hz, H-3''or H-4''), 7.31-7.36 (m, 3H, Ar), 7.41-7.45 (m, 3H, Ar),
.59-7.62 (m, 2H, Ar), 8.08 (s, 1H, H-2'), 8.15 (d, 1H, J = 8.1 Hz, benzo[b]thienyl), 8.76 (s, 2H, H-
7
13
H-6), 9.24 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 122.22, 123.29, 124.02, 124.91,
3
1
1
7
24.98, 125.01, 125.51, 127.76, 128.62, 129.19, 130.96, 131.25, 132.78, 137.37, 139.57, 140.09,
47.96, 155.92, 157.77, 159.39 ppm. Anal. Calcd for C H N S (370.50): C, 71.32; H, 3.81; N,
2
2
14
2 2
.56. Found: C 72.12; H, 3.61; N, 7.52.
4
.5. General procedure for the synthesis of 5-(hetero)aryl-4-(5-bromo-thiophen-2-yl)-
pyrimidine
2
-Bromothiophene (11d) (193 ꢀL, 2.0 mmol) was added to a solution of 5-
benzo[b]thiophen-2-yl-pyrimidine (8) [or 5-benzo[b]thiophen-3-yl-pyrimidine (9)] (212 mg, 1.0
mmol) in CF COOH (5 mL). The reaction mixture was stirred at room temperature for 24 h and the
3
solvents evaporated. A solution of KOH (224 mg, 4.0 mmol, 4 equiv.) and K [Fe(CN) ] (658 mg,
3
6
2.0 mmol, 2 equiv.) in water (10 mL) was added to the residue. The resulting mixture was stirred
for 6 h at room temperature, and the precipitate formed was filtered, washed with H O, and air-
2
dried. The residue was purified by flash column chromatography (hexane/ethyl acetate, 1:1) to
H
N
afford the desired S -products (18d or 19d).
1
2

ACCEPTED MANUSCRIPT
.5.1. 5-Benzo[b]thiophen-2-yl-4-(5-bromo-thiophen-2-yl)-pyrimidine (18d).
4
4'
5'
6'
3'
6
7'
5
1
2'
S 1'
N
4
1"
S
2
N
5"
2
"
Br
3
3"
4"
18d
It has been obtained by reaction 8 with 2-bromothiophene (11d). Yield 172 mg (46%), off-
1
white solid; m.p. 187-189°C. H NMR (500 MHz, CDCl ): δ = 6.85 (d, 1H, J = 4.1 Hz, H-3''or H-
3
4
7
6
1
''), 6.92 (d, 1H, J = 4.1 Hz, H-3''or H-4''), 7.40 (s, 1H, H-3'), 7.41-7.49 (m, 2H, benzo[b]thienyl),
.86 (dd, 1H, J = 6.6, 2.0 Hz, benzo[b]thienyl), 7.89-7.91 (m, 1H, benzo[b]thienyl), 8.70 (s, 1H, H-
1
3
), 9.27 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 119.51, 122.52, 123.76, 124.18,
3
24.96, 125.26, 125.37, 131.37, 131.61, 136.45, 139.70, 140.82, 142.47, 156.21, 158.10, 159.27
+
79
+
81
ppm. GC t 29.64 min; MS m/z (rel intensity) 372 (M , 100) for Br, 374 (M , 100) for Br. Anal.
R
Calcd for C H BrN S (373.30): C, 51.48; H, 2.43; N, 7.50. Found: C 51.67; H, 2.53; N, 7.34.
1
6
9
2 2
4
.5.2. 5-Benzo[b]thiophen-3-yl-4-(5-bromo-thiophen-2-yl)-pyrimidine (19d).
6'
5'
7'
4'
6
S
1'
5
1
3'
N
2'
4
1"
S
2
N
5"
2
"
Br
3
3"
4"
19d
It has been obtained by reaction 9 with 2-bromothiophene (11d). Yield 231 mg (62%), off-
1
white solid; m.p. 167-169°C. H NMR (500 MHz, CDCl ): δ = 6.54 (d, 1H, J = 4.1 Hz, H-3''or H-
3
4''), 6.73 (d, 1H, J = 4.1 Hz, H-3''or H-4''), 7.30-7.34 (m, 2H, benzo[b]thienyl), 7.38-7.45 (m, 1H,
benzo[b]thienyl), 7.52 (s, 1H, H-2'), 7.97 (d, 1H, J = 8.1 Hz, benzo[b]thienyl), 8.63 (s, 1H, H-6),
1
3
9
1
2
.16 (s, 1H, H-2) ppm. C NMR (126 MHz, CDCl ): δ = 119.16, 122.51, 123.07, 124.20, 124.97,
3
25.22, 126.41, 130.96, 131.10, 131.27, 137.43, 140.17, 142.71, 156.41, 157.90, 159.40 ppm. GC tR
+
79
+
81
8.93 min; MS m/z (rel intensity) 372 (M , 100) for Br, 374 (M , 100) for Br. Anal. Calcd for
C H BrN S (373.30): C, 51.48; H, 2.43; N, 7.50. Found: C 51.34; H, 2.49; N, 7.69.
16
9
2 2
4
.6. Antimycobacterial assay
To evaluate the inhibitory efficiency of molecules on Mycobacterium tuberculosis (MTB),
M. tuberculosis H Rv, which is susceptible to all classical antituberculosis drugs, was used. The
3
7
1
3

ACCEPTED MANUSCRIPT
minimal inhibitory concentration (MIC) for M. tuberculosis H Rv for each compound was
3
7
determined by a micro broth dilution method. All molecules tested were dissolved in
dimethylsulfoxide and their 1/2 dilutions were prepared in 5 mL tubes using Löwenstein-Jensen
medium. A few colonies from freshly grown M. tuberculosis H Rv were suspended in Löwenstein-
3
7
Jensen medium to obtain 1.0 McFarland turbidity and diluted ten times using the same medium and
the tubes were incubated at 37 °C medium with a different concentration of the tested molecule and
to a positive control tube containing only clear growth medium. After 24 hours the tubes were
placed in a vertical position and the free edge of the buried 0.3 mL of the substance in the test
compounds concentrations: 12.5, 6.25, 3.1, 1.5, 0.7, 0.37, 0.15 ꢀg/mL. The tubes were then placed
in an thermostat at a temperature of 37 °C and incubated for 10 days. Growth estimate for the MTB
were determined by standard methods, where the appearance of zones of growth retardation MTB
(over 10 mm) indicated the presence of tuberculostatic properties in concentration of the
compounds under study. Penetration size stunting MTB (in mm) is proportional to the degree of
tuberculostatic activity. Growth delay of 100 mm or more is considered as a complete growth
inhibition MTB. The multi-drug-resistant (MDR) tuberculosis strain have been isolated from
tuberculosis patients in Ural Research Institute for Phthisiopulmonology (Russia). The minimal
inhibitory concentrations against Mycobacterium tuberculosis avium, Mycobacterium tuberculosis
terrae, and MDR tuberculosis strains were evaluated similarly.
4
.7. Evaluation of acute toxicity in vivo.
All experimental protocols were conducted in accordance to the standard protocol approved by
the Committee of the Ethical Use of Animals of the Ural Research Institute for Phthisiopulmonology
CEUA/URIP). The synthesized compounds were evaluated for their approximate LD50 in white
(
healthy mice (17 - 20 g body weight) divided into 3 groups of 5 animals each for testing of one
compound [35,36]. Toxicity tests were carried out via a single peroral introduction of compound in a
1% starch aqueous solution. The volume introduced did not exceed 0.5 mL for mice. The observation
period was 14 days. The median lethal doses LD were used as the criteria of toxicity.
5
0
Acknowledgments
The research was financially supported by the Russian Science Foundation (Project No. 15-13-
0077).
0
1
4

ACCEPTED MANUSCRIPT
References and notes
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. D.T. Hurst, Introduction to the Chemistry and Biochemistry of Pyrimidines, Purines and Pteridines.
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. M. Ban, H. Taquchi, T. Katsushima, S. Akoki, A. Wantanbe, Bioorg. Med. Chem. 6 (1998) 1057-
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5
6
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1
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1
1
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685-1695.
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1
(
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3897.
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0. A.D. Khoje, A. Kulendrn, C. Charnock, B. Wan, S. Franzblau, L.-L. Gundersen, Bioorg. Med.
Chem. 18 (2010) 7274-7282.
2
1. E.M. Cheprakova, E.V. Verbitskiy, M.A. Ezhikova, M.I. Kodess, M.G. Pervova, P.A. Slepukhin,
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1
6

Supplementary Material
ACCEPTED MANUSCRIPT
1
13
Supplementary material ( H and C NMR spectra of the new compounds, crystal structural data for
compounds 18b, 18c, 19b, 19c and 19d.) associated with this article can be found, in the online version.
1
7

ACCEPTED MANUSCRIPT
Table 1. In vitro antituberculosis activity of C(4) and C(5) mono(thienyl) (6-10, 12a,b, 14a-d and 15a-d) and di(thienyl) (from 16a-d to 19a-d)
substituted pyrimidines and their acute in vivo toxicities in mice
2
Entry
R
R
Antimycobacterial activity against different strains of Mycobacterium
MIC)
N
LD50
mouse,
mg/kg
(
Molecular
Mass
1
a
N
H37Rv
M. avium
M. terrae
MDR
1
2
Compound R
R
µg/mL
µM
µg/mL
µM
µg/mL
µM
µg/mL
µM
1
2
1
H
Br
158.99
162.21
12.5
6.25
78.62
38.53
12.5
78.62
19.11
12.5
3.1
78.62
12.5
78.62
38.53
n.d.
n.d.
6
H
3.1
19.11
77.06
58.89
6.25
12.5
12.5
S
3
4
162.21
212.27
12.5
12.5
77.06
58.89
12.5
12.5
77.06
58.89
12.5
12.5
77.06
58.89
n.d.
n.d.
7
8
H
H
S
S
5
9
H
212.27
1.5
7.07
0.7
3.30
0.7
3.30
1.5
7.07
700
S
6
7
10
H
H
241.11
244.34
12.5
6.25
51.84
25.58
12.5
6.25
51.84
25.58
12.5
6.25
51.84
25.58
12.5
6.25
51.84
25.58
n.d.
n.d.
Br
S
S
12a
S
8
9
12b
H
S
238.31
6.25
26.23
6.25
26.23
6.25
26.23
6.25
26.23
n.d.
d
14a
H
H
162.21
244.34
12.5
12.5
77.06
51.16
12.5
12.5
77.06
51.16
12.5
12.5
77.06
51.16
12.5
12.5
77.06
51.16
n.d.
n.d.
S
S
1
1
0
1
14b
S
d
14c
S
H
238.31
12.5
52.45
12.5
52.45
12.5
52.45
12.5
52.45
n.d.

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1
1
1
2
3
4
d
241.11
241.11
323.23
12.5
0.7
51.84
2.90
12.5
0.3
51.84
1.24
12.5
0.3
51.84
1.24
12.5
0.7
51.84
2.90
n.d.
315
n.d.
1
4d
H
Br
S
d
Br
1
5a
S
d
S
6.25
19.34
6.25
19.34
6.25
19.34
6.25
19.34
1
5b
Br
S
d
1
5
15c
15d
6a
6b
S
Br
Br
317.21
1.5
4.73
0.7
2.21
0.7
2.21
1.5
4.73
225
1
1
6
7
320.01
244.34
3.1
3.1
9.69
3.1
3.1
9.69
3.1
3.1
9.69
3.1
3.1
9.69
n.d.
n.d.
Br
S
S
S
S
12.69
12.69
12.69
12.69
1
S
S
S
1
1
8
9
326.46
320.44
0.7
2.14
0.3
0.92
0.3
0.92
0.7
2.14
160
n.d.
1
S
16c
S
S
S
12.5
39.01
12.5
39.01
12.5
39.01
12.5
39.01
2
2
0
1
16d
17a
323.23
244.34
12.5
6.25
38.67
25.58
12.5
6.25
38.67
25.58
12.5
6.25
38.67
25.58
12.5
6.25
38.67
25.58
n.d.
n.d.
Br
S
S
S
S
2
2
2
3
17b
17c
326.46
320.44
6.25
6.25
19.14
19.50
6.25
6.25
19.14
19.50
6.25
6.25
19.14
19.50
6.25
6.25
19.14
19.50
n.d.
n.d.
S
S
S
S
2
2
4
5
17d
18a
323.23
294.40
6.25
6.25
19.34
21.23
6.25
6.25
19.34
21.23
6.25
6.25
19.34
21.23
6.25
6.25
19.34
21.23
n.d.
n.d.
Br
S
S
S
S
26
18b
376.52
12.5
33.20
12.5
33.20
12.5
33.20
12.5
33.20
n.d.
S

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2
2
7
8
18c
18d
S
370.50
373.30
3.1
8.37
3.1
8.37
1.5
4.05
3.1
8.37
n.d.
n.d.
S
S
12.5
33.49
12.5
33.49
12.5
33.49
12.5
33.49
Br
S
2
3
3
3
9
0
1
2
19a
19b
19c
19d
294.40
376.52
370.50
373.30
12.5
0.37
0.15
3.1
42.46
0.98
0.40
8.30
12.5
0.37
0.15
3.1
42.46
0.98
0.40
8.30
12.5
0.15
0.15
1.5
42.46
0.40
0.40
4.02
12.5
0.37
0.15
3.1
42.46
0.98
0.40
8.30
n.d.
90
S
S
S
S
S
S
S
S
45
n.d.
Br
S
b
n.a
PZA
INH
123.12
137.14
12.5
0.1
101.53
0.73
n.d.
0.1
n.d.
n.d.
0.1
n.d.
-
-
-
-
c
1
1
1
680
33
51
b
c
0.73
0.73
n.d. - not determined; n.a. - data not available; INH – Isoniazid; PZA – Pyrazinamide;
a
MDR (multi-drug-resistant tuberculosis strain) – Rifampin and Isoniazid resistant Mycobacterium tuberculosis strain having Beijing genotype with a
combination of mutations Ser 531 - Leu 315 and Ser-Thr in rpoB and katG genes, respectively.
b
LD Oral mouse [27]
5
0
c
LD Intraperitoneal mouse [27]
5
0
d
Data from previous study [22]

ACCEPTED MANUSCRIPT
R'
N
X
N
R
X= S or O; R= H, Ph, 2-thienyl; R'= H, Ph
Antimycobacterial activity - MIC, 1.5-12.5 µg/mL
Fig. 1. The structures of antitubercular 5-arylethenyl-4-(hetero)arylpyrimidines

ACCEPTED MANUSCRIPT
NC
NC
N
Ar
NC
N
Ar
N
NC
N
H
H
Et
X
Et
S
X= S or O; Ar= Ph, thiophen-2-yl, thiophen-3-yl
µ
Antimycobacterial activity - MIC, 0.3-1.5 g/mL
Fig. 2. 1-Ethyl-2,3-dicyano-5-(hetero)aryl-6-heteroaryl-1,6-dihydropyrazines,
exhibiting a good level of antituberculosis activity

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Fig. 3. X-ray structure of 19b.

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Fig. 4. X-ray structure of 19c.

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Fig. 5. X-ray structure of 19d.

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DAPCy (5 mol%), K PO , r.t.
Br
+
Het
N
3
4
N
Het B(OH)2
EtOH
N
2-5
N
H
Suzuki cross-coupling
1
6-9
2
3
4
5
, 6: Ar= 2-thienyl;
, 7: Ar= 3-thienyl;
, 8: Ar= benzo[b]thien-2-yl;
, 9: Ar= benzo[b]thien-3-yl.
Yields 59-79 %
Scheme 1. Synthesis of 5-(hetero)arylpyrimidines 6-9 through Suzuki cross-coupling.

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Br
S
N
NBS
DMF
S
N
N
N
6
10
Yield 74 %
Scheme 2. Synthesis of 5-(5-bromo-thiophen-2-yl)pyrimidine (10).

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Ar
Br
O
S
Pd(OAc ), PCy K CO , MW, 180 C
N
N
2
3
2
3
+
Ar
DMF
S
N
N
Pd-Catalyzed aryl-aryl coupling
1
1a,b
12a,b
Yields 52-56 %
1
a: Ar=
; b: Ar =
S
Scheme 3. Synthesis of 5-(hetero)arylpyrimidines through microwave-assisted palladium-
catalyzed C-C cross-coupling reaction [26].