Synthesis of bis(trifluoromethyl)pyrimido[4,5-d]pyrimidine-2,4-diones and evaluation of their antibacterial and antifungal activities

Article abstract of DOI:10.1016/j.jfluchem.2016.06.019

A series of 5,5-bis(trifluoromethyl)-5,6-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-diones has been prepared by the cyclocondensation of N-acylimines of hexafluoroacetone and 6-aminouracils. The obtained compounds were screened for their activities against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus anthracis) and Gram-negative (Escherichia coli) bacteria, as well as fungus Candida albicans. A few of the title compounds showed promising antimicrobial activity.

Full text of DOI:10.1016/j.jfluchem.2016.06.019

Accepted Manuscript
Title: Synthesis of
bis(trifluoromethyl)pyrimido[4,5-d]pyrimidine-2,4-diones and
evaluation of their antibacterial and antifungal activities
Author: Alexey Yu. Aksinenko Tatyana V. Goreva Tatyana A.
Epishina Sergey V. Trepalin Vladimir B. Sokolov
PII:
S0022-1139(16)30165-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jfluchem.2016.06.019
FLUOR 8804
To appear in:
FLUOR
Received date:
Revised date:
Accepted date:
21-4-2016
27-6-2016
28-6-2016
Please cite this article as: Alexey Yu.Aksinenko, Tatyana V.Goreva, Tatyana A.Epishina,
Sergey V.Trepalin, Vladimir B.Sokolov, Synthesis of bis(trifluoromethyl)pyrimido[4,5-
d]pyrimidine-2,4-diones and evaluation of their antibacterial and antifungal activities,
Journal of Fluorine Chemistry http://dx.doi.org/10.1016/j.jfluchem.2016.06.019
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Synthesis of bis(trifluoromethyl)pyrimido[4,5-d]pyrimidine-2,4-diones and evaluation of
their antibacterial and antifungal activities
Alexey Yu. Aksinenko*, Tatyana V. Goreva, Tatyana A. Epishina, Sergey V. Trepalin, and
Vladimir B. Sokolov
Institute of Physiologically Active Compounds of Russian Academy of Sciences, Severnyi pr. 1,
Chernogolovka, Moscow region, 142432, Russia, e-mail: alaks@ipac.ac.ru
Graphical abstract
O
O
F3C CF3
O
R
O
R
O
F3C
F3C
Et3N
-H2O
N
N
N
N
R2
+
NH2
R2
N
N
H
N
R1
R1
70-93%
Highlights
 Fluorinated pyrimido[4,5-d]pyrimidine-2,4-diones have been prepared.
 Antibacterial and antifungal activities of synthesized compounds were evaluated.
 The obtained heterocycles are more active against the Gram-positive rather than the
Gram-negative bacteria.

ABSTRACT
A series of 5,5-bis(trifluoromethyl)-5,6-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-
diones has been prepared by the cyclocondensation of N-acylimines of hexafluoroacetone and 6-
aminouracils. The obtained compounds were screened for their activities against Gram-positive
(Staphylococcus aureus, Staphylococcus epidermidis and Bacillus anthracis) and Gram-negative
(Escherichia coli) bacteria, as well as fungus Candida albicans. A few of the title compounds
showed promising antimicrobial activity.
Key words: fluorinated heterocycles, pyrimido[4,5-d]pyrimidines, cyclocondensation.
1. Introduction
Pyrimidines represent one of the important classes of heterocyclic compounds occurring
widely in living organisms. Thus, pyrimidine fragment is present in uracil, thymine and cytosine
that are three important constituents of nucleic acids [1]. The synthesis of new uracil-annelated
heterocycles has received considerable attention in the field of drug discovery since many
monocyclic uracils (for example, 5-fluorouracil 1 and sulfonamides 2), as well as their fused
derivatives (for examples, acyclovir 3, methotrexate 4) have found wide clinical applications
(Figure 1).
Among them, the pyrimido[4,5-d]pyrimidines that have close resemblance with purine and
pteridine systems, are of high interest as potential biologically active compounds. However, their
synthesis and biological effects, mainly antimicrobial and antifungal, have been described only
in a few works.

The pyrimido[4,5-d]pyrimidine bicycle was formed by the Biginelli reaction [2] from
aldehydes, barbituric acid/thiobarbituric acid and urea/thiourea [3,4] or a variation of this
reaction: a) from pyrimidine-5-carboxylate and thioureas[5], b) from 6-aminouracils,
araldehydes and isothioureas [6], c) from pyrrole-2-carboxaldehyde, 2-aminobenzimidazole and
1,3-dimethylbarbituric acid [7], d) by a three-component reaction of 6-[(dimethylamino)-
methylene]-1,3-dimethylaminouracil, terephthalaldehyde and amine derivatives [8], e) by the
reaction between 2-pyrrolidones and 6-aminopyrimidines followed by the cyclocondensation
with benzoyl chloride [9]. Some of the described compounds showed good antibacterial and
antifungal activities.
One of the modern synthetic approaches used in drug discovery to increase
pharmaceutical effectiveness, biological half-life, and bioabsorption consists in an incorporation
of fluorine atom or fluoroalkyl group into a molecule of the potential drug candidate (for
example, into an aromatic or heterocyclic system) or synthesis of new drug candidates from
highly reactive fluorinated building blocks [10-17]. As of now, about 36% of approved drugs
and 40% of drug candidates that are currently in phase II–III clinical trials contain at least one
fluorine atom [18].
In our previous works, a new approach to the construction of trifluoromethylated
pyrimido[4,5-d]pyrimidine ring systems by the cyclocondensation of N-acylimines of
hexafluoroacetone or methyl trifluoropyruvate with 6-aminouracils/thiouracils was reported [19-
22]. Thus, in continuation of our research in the field of bioactive heterocyclic compounds, a
number of unexplored fluorinated pyrimido[4,5-d]pyrimidines were synthesized and their
antibacterial and antifungal activities were evaluated.
2. Results and discussion.
2.1. Chemistry.
The synthesis of titled compounds was performed through the C-alkylation of 6-
aminouracils 1a-h with N-acylimines of hexafluoroacetone 2a-e. The subsequent

cyclocondensation of the in situ generated intermediates 3 in the presence of Et3N afforded
pyrimido[4,5-d]pyrimidines 4a-k. It should be mentioned that 19F NMR spectrum of the reaction
mixture containing uracil 1g and imine 2g that were kept in DMF for 0.5 h at room temperature
in the absence of Et3N showed two compounds – intermediate 3j and product 4j. Broad singlets
at -57.9 and -70.0 ppm could be assigned to the intermediate 3j that is a similar product of C-
alkylation of 6-amino-1,3-dimethyluracil with benzoylimine of hexafluoroacetone [19], while a
narrow singlet at -70.8 ppm corresponds to the product 4j. Thus, it demonstrates that the
cyclocondensation of the intermediates 3 to form target compounds 4 occurs easily. The
structures of compounds 4a-k were confirmed by 1H and 19F NMR, MS spectroscopies and
elemental analysis.
In the 13C NMR spectra, the signals of carbon atoms of pyrimido[4,5-d]pyrimidin-2,4-
dione skeleton were assigned using “nmrshiftdb2” – a free NMR database [23]. The signals of
carbon atoms of uracil ring are in the expected region of 149-155 ppm for C-2 and 159-161 ppm
for C-4, 78-80 ppm for C-4a, and 150 ppm for C-8a, correspondingly. The signals of C-5 carbon
atoms that are connected with two CF3-groups appeared as septets at 64 - 66 ppm with the
coupling constants 2JCF 30 - 32 Hz. Finally, the signals of C-7 carbon atoms of second
pyrimidine ring are at 158 - 160 ppm. It should be noted that chemical shifts of C-4, C-4a, C-5,
and, especially, C-8a, as well as CF3 carbon atoms do not sufficiently depend on substituents at
1- and 7-positions, the deviations do not exceed ±1.0 ppm: the Δδ = (δmax - δmin) is 1.3 ppm for C-
4, 1.7 ppm for C-4a, 1.9 ppm for C-5, 0.8 ppm for C-8a (see Table 1). The signals of carbon
atoms of substituents at 1- and 7-positions are in accordance with their structures.
In the 19F NMR spectra, the signals corresponding to CF3 groups appeared as singlets at 4
- 6 ppm (4a-g and 4i-k) in the range that is characteristic for this type of pyrimido[4,5-
d]pyrimidines [19]. However, CF3 groups in 4h are nonequivalent and they appeared as two
quartets at -70.3 and -71.1 ppm with the coupling constant of JFF = 7 Hz. The signals of fluorine

atoms that are directly connected with a phenyl ring (compounds 4a, 4e and 4g) appeared in the
corresponding region.
2.2. Antimicrobial and antifungal activity.
The newly synthesized compounds 4a-k were evaluated for their in vitro antibacterial
activity by well diffusion method expressed by zone of inhibition (mm in diameter) against
Gram-positive, namely, Staphylococcus aureus (SA), Staphylococcus epidermidis (SE) and
Bacillus anthracis(BA),and Gram-negative - Escherichia coli (EC) bacterial strains. In addition,
they were tested for their in vitro antifungal activity against Candida albicans (CA). Streptocide
and tetracycline were used as a reference to estimate the potency of the testing compounds under
the same conditions. The results are presented in Table 2.
Most of the tested compounds revealed better activity against the Gram-positive rather
than the Gram-negative bacteria. Heterocycles 4a-c derived from dimethyluracil 1a are active
against almost all tested microorganisms. Compounds 4f,i exhibited similar activity against
Gram-positive bacteria. All compounds, excluding 4a,c, demonstrated low antifungal activity
against Candida albicans.
3. Conclusion
In the present work, a novel series of fluorinated pyrimido[4,5-d]pyrimidine-2,4-diones
was prepared by the reaction of 6-aminouracils with N-acylimines of hexafluoroacetone. The
obtained compounds were screened for their potential antimicrobial and antifungal activity. Most
of the tested compounds revealed better activity against the Gram-positive rather than the Gram-
negative bacteria. Synthesized compounds has low, except 4a,c antifungal activity.
4. Experimental
4.1. General
4.1.1 Chemistry

The 1H, 13C, and 19F NMR spectra were recorded on Bruker DXP at 200, 50, and 188
MHz, respectively, in CDCl3 and Me2SO-d6 using tetramethylsilane (TMS) as an internal
standard and CFCl3 as an external standard. Chemical shifts are reported in ppm units with the
use of δ scale. Mass-spectra were recorded on a Finnigan 4021 spectrometer. Melting points
were measured in open capillary tubes and are uncorrected.
4.1.2. Agar well diffusion method.
The assay was carried out by using 1 ml of inoculum (1x109 colony forming units)
prepared from an overnight culture for given test microorganisms. 1 ml of the resultant spore/cell
suspension was poured in the petri plate and the plates were poured with respective medium to
seed each prepared plate. The medium was allowed to solidify. Using a sterilized cork-borer,
wells of 5mm diameter were made in the solidified inoculated medium and the plate area
uniformly. The wells were filled with 3 drop (0.1 mL) of compound solution. Plates were then
incubated aerobically at 37± 2°C and the zone of inhibition if any was then measured in mm for
the particular compound and specific organism after 24 hr. The efficiency of the tested
compounds was compared to that of streptocide and tetracycline.
4.2. General procedure for preparation of 5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-diones 4a-k.
To a solution of 5 mmol of uracil 1 in 10 ml of DMF while stirring at 20ºC was added 5
mmol of imine 2. The reaction mixture was stirred for 1 h at 20ºC, then 0.1 g Et3N was added.
The reaction mixture was heated at 80-90 ºC during 2 h and poured into 50 ml of H2O, the
precipitate formed was separated and crystallized from 50% EtOH.
4.2.1. 1,3- Dimethyl-7-(4-fluorophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4a.
Yield 91%; mp 246-248 C. 1H NMR (200 MHz, Me2SO-d6, ): 3.20 (s, 3H, CH3), 3.50
(s, 3H, CH3), 7.42 (t, 2H, CHAr, 3JHH =3JHF= 8.9 Hz), 8.03 (dd, 2H, CHAr, 3JHH = 8.9 Hz, 4JHF=
5.5 Hz), 10.10 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 27.8 (CH3N), 29.9 (CH3N), 65.9

(septet, 2JCF = 32 Hz, C-5), 78.7 (C-4a), 115.5 (d, 2JCF = 22 Hz, 3,5-CPhF), 122.5 (q, 1JCF = 291
Hz, CF3), 128.3 (d, 4JCF = 2 Hz, 1-CPhF),131.8 (d, 3JCF = 9 Hz, 2,6-CPhF), 150.8 (C-8a), 152.9 (C-
2), 159.0 (C-7), 160.2 (C-4), 164.9 (d, 1JCF = 251 Hz, 4-CPhF). 19F NMR (188.29 MHz, Me2SO-
d6, ): -105.1 (tt, 1F, 3JFH = 9.0 Hz, 4JFH 5.5 Hz), -70.8 (s, 6F). EI-MS (m/z): 424 [M]+. Calc. for
C16H11F7N4O2: C, 45.29, H, 2.61, N, 13.21. Found: C, 45.48, H, 2.42, N, 13.27.
4.2.2. 1,3- Dimethyl-7-(4-cyanophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4b.
Yield 93%; mp 283-285 C. 1H NMR (200 MHz, Me2SO-d6, ): 3.21 (s, 3H, CH3), 3.50
(s, 3H, CH3), 8.05 (s, 4H, CHAr), 10.38 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 27.9
(CH3N), 30.0 (CH3N), 66.0 (septet, 2JCF = 30 Hz, C-5), 79.1 (C-4a), 114.9 (4-CPh), 118.1 (C≡N),
122.6 (q, 1JCF = 290 Hz, CF3), 129.6 (2,6-CPh), 132.3 (3,5-CPh), 136.0 (1-CPh), 150.7 (C-8a),
152.6 (C-2), 159.0 (C-7), 160.0 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -72.5 s. EI-MS
(m/z): 431 [M]+. Calc. for C17H11F6N5O2: C, 47.34, H, 2.57, N, 16.24. Found: C, 47.52, H, 2.44,
N, 13.42.
4.2.3. 1,3- Dimethyl-7-(3-nitrophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4c.
Yield 86%; mp 240-242 C. 1H NMR (200 MHz, Me2SO-d6, ): 3.22 (s, 3H, CH3), 3.52
(s, 3H, CH3), 7.72 (t, 1H, 3JHH = 8.2 Hz),8.31 (d, 1H, CHAr, 3JHH = 7.6 Hz), 8.40 (d, 1H, CHAr,
3JHH = 7.6 Hz), 8.75 (s, 1H, CHAr), 10.08 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 27.9
(CH3N), 30.0 (CH3N), 65.8 (septet, 2JCF = 30 Hz, CCF3), 79.2 (C-4a), 122.6 (q, 1JCF = 291 Hz,
CF3), 123.5 (2-CPhNit), 127.2 (4-CPh), 130.3 (5-CPh), 133.2 (1-CPh), 135.3 (6-CPh), 147.5 (3-CPh),
150.8 (C-8a), 152.7 (C-2), 159.0 (C-7), 159.4 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -
70.4 s. EI-MS (m/z): 451 [M]+. Calc. for C16H11F6N5O4: C, 42.58, H, 2.46, N, 15.52. Found: C,
42.52, H, 2.40, N, 15.62.
4.2.4. 1- Phenyl-7-(3-nitrophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4d.

Yield 88%; mp 294-295 C. 1H NMR (200 MHz, Me2SO-d6, ): 7.30 – 7.60 (m, 5H,
CHAr), 7.73 (t, 1H, 3JHH = 8.2 Hz), 7.99 (d, 1H, CHAr, 3JHH = 7.6 Hz), 8.38 (s, 1H, CHAr), 8.41 (d,
1H, CHAr, 3JHH = 7.9 Hz), 10.30 (s, 1H, NH), 11.64 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6,
): 64.4 (septet, 2JCF = 32 Hz, C-5), 80.4 (C-4a), 122.9 (q, 1JCF = 291 Hz, CF3), 124.2 (2-CPhNit),
127.8 (4-CPhNit), 128.8 (4-CPh), 129.1 (3-CPh), 130.0 (2-CPh), 130.6 (5-CPhNit), 133.3 (1-CPhNit),
135,3 (6-CPhNit), 136.2 (1-CPh), 147.9 (3-CPhNit), 150.6 (C-8a), 155.1 (C-2), 159.0 (C-7), 160.5
(C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -70.7 s. EI-MS (m/z): 499 [M]+. Calc. for
C20H11F6N5O4: C, 48.11, H, 2.22, N, 14.03. Found: C, 48.32, H, 2.04, N, 14.16.
4.2.5. 1- (4-Fluorophenyl)-7-(3-nitrophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4e.
Yield 87%; mp 286-288 C. 1H NMR (200 MHz, Me2SO-d6, ): 7.40 – 7.60 (m, 2H,
CHAr), 7.76 (t, 1H, 3JHH = 8.2 Hz), 8.01 (d, 1H, CHAr, 3JHH = 7.9 Hz), 8.39 (s, 1H, CHAr), 8.42 (d,
1H, CHAr, 3JHH = 8.2 Hz), 10.31 (s, 1H, NH), 11.64 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6,
): 65.9 (septet, 2JCF = 30 Hz, C-5), 79.9 (br. s, C-4a), 115.3 (d, 2JCF = 22 Hz, 3,5-CPhF), 122.5 (q,
1JCF = 290 Hz, CF3), 123.6 (2-CPhNit), 127.2 (4-CPhNit), 130.1 (5-CPhNit), 131.6 (d, 3JCF = 8 Hz,
2,6-CPhF), 131.7 (1-CPhF), 132.8 (1-CPhNit), 134.7 (6-CPhNit), 147.4 (3-CPhNit ), 150.1 (C-8a), 154.5
(C-2), 158.5 (C-7), 160.0 (C-4), 161.7 (d, 1JCF = 245 Hz, 4-CPhF). 19F NMR (188.29 MHz,
Me2SO-d6, ): -112.7 (s, 1F), -70.7 (s, 6F). EI-MS (m/z): 517 [M]+. Calc. for C20H10F7N5O4: C,
46.43, H, 1.95, N, 13.54. Found: C, 46.33, H, 2.06, N, 13.38.
4.2.6. 1- (4-Methylphenyl)-7-(3-nitrophenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4f.
Yield 83%; mp 272-274 C. 1H NMR (200 MHz, Me2SO-d6, ): 2.42 (s, 3H, CH3), 7.28
(d, 2H, CHAr, J = 8.2 Hz), 7.76 (t, 1H, 3JHH = 7.6 Hz), 7.86 (d, 2H, CHAr, 3JHH = 8.2 Hz), 8.02 (d,
1H, CHAr, 3JHH = 7.6 Hz), 8.38 (s, 1H, CHAr), 8.41 (d, 1H, CHAr, 3JHH = 7.6 Hz), 9.82 (s, 1H,
NH), 11.56 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 20.4 (CH3), 65.6 (septet, 2JCF = 32
Hz, C-5), 79.8 (C-4a), 117.5 (2-CTol), 122.4 (q, 1JCF = 290 Hz, CF3), 124.0 (2-CPhNit), 127.4 (4-

CPhNit), 128.9 (3-CTol), 130.4 (5-CPhNit), 131, 2 (4-CTol), 132.9 (1-CPhNit), 135,1 (6-CPhNit), 137.8
(1-CTol), 147.5 (3-CPhNit), 150.8 (C-8a), 152.4 (C-2), 158.7 (C-7), 160.1 (C-4). 19F NMR (188.29
MHz, Me2SO-d6, ): -70.6 s. EI-MS (m/z): 513 [M]+. Calc. for C21H13F6N5O4: C, 49.13, H, 2.55,
N, 13.64. Found: C, 49.23, H, 2.48, N, 13.53.
4.2.7. 1- (4-Fluorophenyl)-7-(3-methylphenyl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4g.
Yield 87%; mp 297-299 C. 1H NMR (200 MHz, Me2SO-d6, ): 2.38 (s, 3H, CH3), 7.05 –
7.32 (m, 6H, CHAr), 7.38 (d, 1H, CHAr, 3JHH = 7.6 Hz), 7.46 (s, 1H, CHAr), 9.40 (s, 1H, NH),
11.30 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 21.4 (CH3), 66.3 (septet, 2JCF = 32 Hz, C-
5), 80.0 (C-4a), 115.8 (d, 2JCF = 23 Hz, 3,5-CPhF), 122.9 (q, 1JCF = 291 Hz, CF3), 126.3 (6-CTol),
128.8 (5-CTol), 129.6 (2-CTol), 132.0 (4-CTol), 132.2 (d, 3JCF = 9 Hz, 2,6-CPhF), 132.6 (d, 4JCF = 3
Hz, 1-CPhF), 134.1 (1-CTol), 138.2 (3-CTol), 150.7 (C-8a), 155.5 (C-2), 160.6 ( C-7), 161.1 (C-4),
162.1 (d, 1JCF = 245 Hz, 4-CPhF). 19F NMR (188.29 MHz, Me2SO-d6, ): -111.9 (tt, 1F, 3JFH = 9.2
Hz, 4JFH 5.7 Hz), -69.8 (s, 6F). EI-MS (m/z): 486 [M]+. Calc. for C21H13F7N4O2: C, 51.86, H,
2.69, N, 11.52. Found: C, 51.73, H, 2.78, N, 11.63.
4.2.8. 1- (2-Fluorophenyl)-7-(furan-2-yl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4h.
Yield 78%; mp 261-264 C. 1H NMR (200 MHz, Me2SO-d6,): 6.71 (dd, 1H, CHFur,
3JHH = 3.4 Hz, 3JHH 1.5 Hz), 7.18 (d, 1H, CHFur, 3JHH = 3.7 Hz), 7.26 – 7.45 (m, 2H, CHAr), 7.46
– 7.61 (m, 2H, CHAr), 8.02 (br. s, 1H, CHFur), 9.88 (s, 1H, NH), 11.68 (s, 1H, NH). 13C NMR (50
MHz, Me2SO-d6, ): 65.3 (septet, 2JCF = 32 Hz, C-5), 79.2 (C-4a), 113.1 (3-CFur), 122.2 (q, 1JCF
= 290 Hz, CF3), 122.3 (q, 1JCF = 290 Hz, CF3), 115.7 (d, 2JCF = 19 Hz, 3-CPhF), 119.0 (4-CFur),
123.2 (d, 3JCF = 13 Hz, 1-CPhF), 124.5 (d, 4JCF = 3 Hz, 6-CPhF), 130.8 (d, 3JCF = 8 Hz, 4-CPhF),
131.7 (5-CPhF), 144.5 (5-CFur), 148.6 (2-CFur), 149.4 (C-2), 150.7 (C-8a), 154.7 (C-7), 157.6 (d,
1JCF = 247 Hz, 2-CPhF), 159.8 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -121.3 (m, 1F), -70.3

(q, 3F, 4JFF = 7.0 Hz), -71.15 (q, 3F, 4JFF = 7.0 Hz). EI-MS (m/z): 462 [M]+. Calc. for
C18H9F7N4O3: C, 46.77, H, 1.96, N, 12.12. Found: C, 46.92, H, 2.07, N, 12.33.
4.2.10. 1- (4-Chlorophenyl)-7-(furan-2-yl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4i.
Yield 81%; mp 274-276 C. 1H NMR (200 MHz, Me2SO-d6,): 6.68 (dd, 1H, CHFur,
3JHH = 3.4 Hz, 3JHH 1.7 Hz), 7.18 (d, 1H, CHFur, 3JHH = 3.2 Hz), 7.37 (d, 2H, CHAr, 3JHH = 8.5
Hz), 7.51 (d, 2H, CHAr, 3JHH = 8.5 Hz), 7.99 (d, 1H, CHFur, 3JHH = 1.7 Hz), 9.73 (s, 1H, NH),
11.51 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 65.2 (septet, 2JCF = 32 Hz, C-5), 79.3 (C-
4a), 113.0 (3-CFur), 118.8 (4-CFur), 122.4 (q, 1JCF = 291 Hz, CF3), 128.4 (2,6-CPhCl), 131.5 (3,5-
CPhCl), 132.7 (1-CPhCl), 134.7 (4-CPhCl), 144.6 (5-CFur), 148.3 (2-CFur), 149.9 (C-2), 150.3 (C-8a),
154.8 (C-7), 159.8 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -72.4 s. EI-MS (m/z): 478
[M]+. Calc. for C18H9ClF6N4O3: C, 45.16, H, 1.89, N, 11.70. Found: C, 45.02, H, 1.77, N, 11.63.
4.2.11. 1-Benzyl-7-(thiophen-2-yl)-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-
d]pyrimidine-2,4(1H,3H)-dione 4j.
Yield 72%; mp 270-272 C. 1H NMR (200 MHz, Me2SO-d6,): 5.24 (s, 2H, CH2), 7.17 –
7.41 (m, 6H, CHAr), 8.03 (d, 1H, CHAr, 3JHH = 4.6 Hz), 8.47 (d, 1H, CHAr, 3JHH = 3.4 Hz), 9.78
(s, 1H, NH), 11.48 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 45.1 (CH2), 66.2 (septet, 2JCF
= 32 Hz, C-5), 79.6 (C-4a), 122.9 (q, 1JCF = 290 Hz, CF3), 127.8 (4-CPh), 127.9 (2-CPh), 129.0 (3-
CPh), 129.5 (3-CTh), 133.6 (4-CTh), 135.9 (2-CTh), 136.3 (1-CPh), 138.2 (1-CTh), 150.9 (C-8a),
154.6 (C-2), 155.4 (C-7), 160.0 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -72.4 s. EI-MS
(m/z): 474 [M]+. Calc. for C19H12F6N4O2S: C, 48.11, H, 2.55, N, 11.81. Found: C, 48.03, H,
2.37, N, 11.68.
4.2.12. 1-[2-(3,4-Dimethoxyphenyl)ethyl]-7-(4-methylphenyl)-5,5-bis(trifluoromethyl)-5,8-
dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione 4k.
Yield 69%; mp 223-225 ºC. 1H NMR (200 MHz, Me2SO-d6, δ): 2.38 (s, 3H, CH3), 2.83
(t, 2H, CH2,3JHH = 6.6 Hz), 3.66 (s, 3H, CH3O), 3.70 (s, 3H, CH3O), 4.06 (t, 2H, CH2, 3JHH
=

6.6Hz), 6.75 (dd, 1H, CHAr, 3J = 7.9 Hz, 4JHH 1.2 Hz), 6.81 (d, 1H, CHAr, 4JHH = 1.2 Hz), 6.86 (d,
1H, CHAr, 3JHH = 7.9 Hz), 7.30 (d, 2H, CHAr, 3JHH = 8.2 Hz), 7.82 (d, 2H, CHAr, 3JHH = 8.2 Hz),
9.83 (s, 1H, NH), 11.08 (s, 1H, NH). 13C NMR (50 MHz, Me2SO-d6, ): 39.2 (CCH2C), 42.2
(CH2N), 55.5 (CH3O), 55.7 (CH3O), 65.8 (septet, 2JCF = 30 Hz, C-5), 80.1 (C-4a), 111.8 (2,5-
CPh), 120.8 (6-CPh), 122.6 (q, 1JCF = 290 Hz, CF3), 125.5 (2-CTol), 130.1 (3-CTol), 130.5 (1-CTol),
131.8 (1-CPh), 140.2 (4-CTol), 147.4 (4-CPh), 148.7 (3-CPh), 150.6 (C-2), 150.7 (C-8a), 158.8 (C-
7), 159.9 (C-4). 19F NMR (188.29 MHz, Me2SO-d6, ): -71.6 s. EI-MS (m/z): 556 [M]+. Calc. for
C25H22F6N4O4: C, 53.96, H, 3.98, N, 10.07. Found: C, 54.06, H, 3.77, N, 10.24.
4.3. General procedure for preparation of imines 2b-d.
To mixture of 0.1 mol of benzamide in 50 mL of benzene, 0.2 mol of pyridine, 0.11 mol
of hexafluoroacetone was bubbled at stirring at 20°C during 30 min. Then, 0.1 mol of SOCl2
was added, and the mixture was stirred during 2 h. The formed precipitate was filtered off. After
the solvent removal, the residue was fractionated for 2b,d or crystallized from hexane for 2c.
4.3.1. 4-Cyano-N-(1,1,1,3,3,3-hexafluoropropan-2-ylidene)benzamide 2b.
Yield 82%; bp 81-82 C (1 Torr). 1H NMR (200 MHz, CDCl3,): 7.86 (d, 2H, CHAr,
3JHH = 8.0 Hz), 8.05 (d, 2H, CHAr, 3JHH = 8.0 Hz). 19F NMR (188.29 MHz, CDCl3, ): -68.3 s.
Calc. for C11H4F6N2O: C, 44.91, H, 1.37, N, 9.52. Found: C, 44.76, H, 1.26, N, 9.44.
4.3.2. N-(1,1,1,3,3,3-Hexafluoropropan-2-ylidene)-3-nitrobenzamide 2c.
Yield 71%; mp 83-84 C. 1H NMR (200 MHz, CDCl3,): 7.74 (t, 1H, 3JHH = 8.2 Hz),
8.32 (d, 1H, CHAr, 3JHH = 7.6 Hz), 8.38 (d, 1H, CHAr, 3JHH = 7.6 Hz), 8.65 (s, 1H, CHAr). 19F
NMR (188.29 MHz, CDCl3, ): -67.45 s. Calc. for C10H4F6N2O3: C, 38.23, H, 1.28, N, 8.92.
Found: C, 38.56, H, 1.46, N, 8.76.
4.3.3. N-(1,1,1,3,3,3-Hexafluoropropan-2-ylidene)-3-methylbenzamide 2d.
Yield 80%; bp 101-102 C (20 Torr). 1H NMR (200 MHz, CDCl3,): 7.08 (d, 1H, CHAr,
3JHH = 7.6 Hz), 7.26 (t, 1H, CHAr, 3JHH = 7.6 Hz), 7.38 (d, 1H, CHAr, 3JHH = 7.6 Hz), 7.46 (s, 1H,

CHAr). 19F NMR (188.29 MHz, CDCl3, ): -69.53 s. Calc. for C11H7F6NO: C, 46.66, H, 2.49, N,
4.95. Found: C, 46.52, H, 2.65, N, 4.73.
Acknowledgements
This publication was supported in the part by the Russian Foundation for Basic Research
(project number 16-03-00696).
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O
O
H N
S NH
2
F
HN
N
O
N
R
O
N
H
O
OH
2
1
O
R
R
1: 5-Fluorouracil
2: Sulfonamides
O
NH
N
NH
2
N
N
OH
N
HN
H N
N
N
O
N
N
H N
2
2
OH
4: Methotrexate
O
3: Acyclovir
Figure 1. Pyrimidine-containing drugs.

Scheme 1
O
F3C CF3
O
O
F3C CF3
O
NH R2
O
R
N
R
O
F3C
F3C
R
O
4
5
Et3N
-H2O
N
N
4a
8a
N
N
R2
2
+
+
7
N
R2
O
N
NH2
N
H
NH2
N
R1
R1
R1
4a-k
70-93%
2a-e
3
1à-h
O
F3C CF3
O
O
F3C CF3
O
NH R2
O
R
O
R
O
F3C
F3C
R
O
4
5
Et3N
-H2O
N
N
N
7
4a
8a
N
N
R2
2
N
R2
N
NH2
N
H
NH2
N
R1
R1
R1
4a-k
70-93%
2a-e
3
1à-h
R
R1
R2
4-FC6H4
4-NCC6H4
3-NO2C6H4 1f
3-NO2C6H4 1g
3-NO2C6H4 1h
3-NO2C6H4
R
H
H
H
H
H
R1
4-FC6H4
2-FC6H4
4-ClC6H4
CH2Ph
R2
1a
4a
Me Me
Me Me
Me Me
H
H
H
4g
4h
4i
4j
4k
3-MeC6H4
2-Furyl
2-Furyl
4b
4c
4d
4e
4f
1e
1b
1c
1d
Ph
4-FC6H4
4-MeC6H4
2-Thienyl
3,4-(MeO)2C6H3CH2CH2 4-MeC6H4
Imines 2b-d which were used in the synthesis of heterocycles 4 were synthesized for the first
time according to the method described in [24] by a successive addition of hexafluoroacetone to
a solution (or suspension) of equimolar amounts of benzamide, pyridine, and SOCl2 in benzene
at 20 °C; the reaction was completed within 1.5-2 h.

Scheme 2
Py, SOCl2
O
F3C
O
F3C
F3C
F3C
O
R2
N
R2
+
H2N
70-80%
2a-e
R2 = 4-FC6H4 (2a), 4-CN-C6H4 (2b), 3-NO2C6H4 (2c), 3-MeC6H4 (2d), 4-MeC6H4 (2e), 2-Furyl
(2f), 2-Thienyl (2g)
Table 1.
Chemical shifts (δ) of carbon atoms of pyrimido[4,5-d]pyrimidin-2,4-dione skeleton for
compounds 4a-k
Comp.
4a
C-2
C-4
C-4a
78.7
79.1
79.2
80.4
79.9
79.8
80.0
79.2
79.3
79.6
80.1
1.7
C-5
C-7
C-8a
152.9
152.6
152.7
155.1
154.5
152.4
155.5
149.4
149.9
154.6
150.6
5.7
160.2
160.0
159.4
160.5
160.0
160.1
161.1
159.8
159.8
160.0
159.9
1.3
65.9
66.0
65.8
64.4
65.9
65.6
66.3
65.3
65.2
66.2
65.8
1.9
159.0
159.0
159.0
159.0
158.5
158.7
160.6
154.7
154.8
155.4
158.8
5.9
150.8
150.7
150.8
150.6
150.1
150.8
150.7
150.7
150.3
150.9
150.7
0.8
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
*Δδ
* Δδ = (δmax - δmin)

Table 2.
Inhibition zones (in millimeters) of the synthesized compounds 4a-k.
Compounds
Microorganisms and inhibition zone (mm)
Gram-positive
Gram-
Fungi
negative
EC
17
17
16
11
11
0
0
0
0
0
0
0
0
0
0
0
0
0
12
11
0
0
0
0
12
0
0
0
%w
1.0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
1.0
SE
18
21
25
14
12
11
26
23
0
13
0
0
15
11
0
15
11
0
12
0
0
11
0
0
20
11
0
11
0
SA
19
22
26
17
18
18
29
24
0
BA
20
33
36
22
21
20
25
21
0
CA
20
25
27
14
13
12
30
25
0
12
0
0
12
11
0
13
11
0
13
12
0
0
0
0
11
0
4a
4b
4c
4d
4e
4f
16
0
0
15
0
0
19
14
0
21
17
14
13
0
19
12
0
20
16
14
13
12
0
17
11
0
19
12
12
13
11
0
4g
4h
4i
0
12
0
0.1
0.01
1.0
0.1
0.01
1.0
0.1
0.01
0
18
12
11
11
0
0
13
11
0
4j
0
0
0
1.0
0.1
0.01
13
0
0
51
35
22
25
23
16
16
0
0
58
29
18
38
36
30
16
13
12
11
0
0
0
0
38
30
20
32
29
23
14
12
0
11
0
4k
Streptocide 1.0
0.1
0.01
Tetracycline 10
0
0
1.0
0.1
SA: Staphylococcus aureus, SE: Staphylococcus epidermidis, BA: Bacillus anthracis, EC:
Escherichia coli, CA: Candida albicans