Antibacterial and?antifungal activity of?acyclic and?macrocyclic uracil derivatives with quaternized nitrogen atoms in?spacers

Article abstract of DOI:10.1016/j.ejmech.2006.03.030

The reactions of 1,3-bis(α,ω-bromoalkyl)-6-methyluracils with 1,3-bis(α,ω-ethylaminoalkyl)-6-methyluracils or 1,3-bis(bromopentyl)thymine with butylamine afforded pyrimidinophanes containing one or two uracil units and nitrogen atoms in bridging polymethy

Full text of DOI:10.1016/j.ejmech.2006.03.030

European Journal of Medicinal Chemistry 41 (2006) 1093–1101
http://france.elsevier.com/direct/ejmech
Preliminary communication
Antibacterial and antifungal activity of acyclic and macrocyclic uracil
derivatives with quaternized nitrogen atoms in spacers
V.E. Semenov *, A.D. Voloshina, E.M. Toroptzova, N.V. Kulik, V.V. Zobov, R.K. Giniyatullin,
A.S. Mikhailov, A.E. Nikolaev, V.D. Akamsin, V.S. Reznik
A.E. Arbuzov Institute of Organic and Physical Chemistry of the Russian Academy of Sciences, Kazan, 420088, Arbuzov str. 8, Russian Federation
Received 27 July 2005; received in revised form 23 March 2006; accepted 27 March 2006
Available online 09 June 2006
Abstract
The reactions of 1,3-bis(α,ω-bromoalkyl)-6-methyluracils with 1,3-bis(α,ω-ethylaminoalkyl)-6-methyluracils or 1,3-bis(bromopentyl)thymine
with butylamine afforded pyrimidinophanes containing one or two uracil units and nitrogen atoms in bridging polymethylene chains. In some
cases individual geometric isomers of pyrimidinophanes differing in the mutual arrangement of the carbonyl and methyl groups at different
pyrimidine rings were isolated. Quaternization of the bridging nitrogen atom with o-nitrobenzyl bromide, benzyl bromide, n-decyl bromide gave
rise to water-soluble pyrimidinophanes which were evaluated for their antibacterial and antifungal activity. The arrangement of the carbonyl
groups in macrocycles doesn’t affect the activity. Antibacterial and antifungal activity of pyrimidinophanes increases with the increase of poly-
methylene N(pyr)-N-chain length and dramatically increases upon the introduction of n-decyl substituent at nitrogen atoms in spacers. Pyrimidi-
nophanes with 5 and 6 methylene groups in N(pyr)-N-chain and n-decyl substituent showed significant bacteriostatic, fungistatic, bactericidal,
fungicidal activity which comparable with standard antibacterial and antifungal drugs. Acyclic counterpart demonstrated the highest activity
against fungi. Toxicity of more effective pyrimidinophanes was determined for mice and Daphnia magna Straus.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Pyrimidinophanes; Uracils; Quaternization; Antibacterial activity; Antifungal activity; Toxicity
1. Introduction
It was supposed that pyrimidine derivative (a building unit
of some coenzymes) being contained in a molecule which also
includes a fragment exhibiting a specific activity with respect
to certain biotarget, would increase the specificity of the mole-
cule relative to the target. This approach was successfully ap-
plied to development of adrenolytics containing pyrimidine
unit [14] and a new class of cholinesterase inhibitors based
on uracil derivatives [15–20]. Unique specificity of acyclic 3-
mono- or 1,3-bis[(trialkylamino)alkyl]uracil derivatives with
respect to acetylcholinesterase is explained by “appropriate”
mutual arrangement of uracil and tetralkylammonium frag-
ments. It is obvious that this “appropriate” arrangement of ur-
acil unit allow the inhibitor to bind more strongly with the mo-
lecule of acetylcholinesterase.
At present time the synthesis of macrocyclic compounds
containing pyrimidine rings, pyrimidinophanes, is rapidly de-
veloping field of pyrimidines chemistry. Since first reports
concerning pyrimidinophanes synthesis [1,2] diverse macro-
cyclic structures have been prepared. These macrocycles can
contain different number of pyrimidine units linked to each
other by hydrocarbon or polyether spacers through either the
N(1) and N(3) atoms or carbon atoms of pyrimidine rings or
substituents at pyrimidine rings [3–13]. In most of known pyr-
imidinophanes uracil derivatives fragments were introduced as
pyrimidine units because the chemical features of uracils allow
to vary the structure of the macrocyclic compounds synthe-
sized.
We proposed that an efficacy of specific fragment connect-
ing with pyrimidinophane consisting of uracil units would dra-
matically increase due to additional binding with biotarget. It
can be caused by more rigid framework of macrocyclic com-
pounds in comparison with their acyclic counterparts and pro-
* Corresponding author.
E-mail address: sve@iopc.kcn.ru (V.E. Semenov).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.030

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V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
vided that the macrocycle have a complementarity with biotar-
get particularly with enzyme. It is possible that the pyrimidino-
phanes can exhibit activity by other mechanism due to macro-
cyclic framework as specific fragment.
matography. The isomeric pyrimidinophanes differ one from
another by the mutual arrangement of the carbonyl and methyl
groups at different pyrimidine rings— cis- and trans-isomers
1a and 1b, 2a and 2b. The structure of these macrocycles
was confirmed by X-ray [13]. As the result the formulas with
cis- (1a and 2a) or trans- (1b and 2b) arrangement of the car-
bonyl groups were unambiguously assigned to concrete iso-
lated macrocycles (Fig. 1).
The same synthetic protocol was used for the preparation of
pyrimidinophanes with 6 methylene groups in N(pyr)-N-chain.
Interaction of 1,3-bis(6-bromohexyl)-6-methyluracil (4) with
1,3-bis(6-ethylaminohexyl)-6-methyluracil (5), the latter being
obtained from 4 and ethylamine, gave isomeric pyrimidino-
phanes 3a and 3b (Scheme 1). However, in this case we didn’t
succeed to isolate individual geometric isomers of macrocycles
and obtained a mixture of macrocycles 3a and 3b with trans-
and cis- arrangement of the carbonyl and methyl groups.
Water-soluble pyrimidinophanes 6 [13], 7a, 7b, 8 [13] and 9
were prepared by quaternization of the nitrogen atoms in
bridges with o-nitrobenzyl, benzyl or n-decyl bromides in acet-
onitrile according to established procedure. The reaction of 3a
and 3b mixture with benzyl bromide or n-decyl bromide af-
forded the mixture of isomers 10a and 10b, 11a and 11b, re-
spectively. Synthesized isomeric pyrimidinophanes with qua-
ternary ammonium in spacers are presented in Fig. 2. These
macrocycles are well soluble in water in wide region of con-
centrations enough for screening.
Acyclic compounds 12–14 were studied for comparison.
These reference compounds may be considered as acyclic
counterparts of pyrimidinophanes. Ammonium salt (15) is used
for simulating of cationic head in acyclic and macrocyclic
compounds (Fig. 3). Synthetic procedures of 12–14 are the
same as that for pyrimidinophanes and Scheme 2 shows the
preparation of compounds 12 and 13 which can be considered
as acyclic “half” of the macrocycles. In all cases quaternization
of the nitrogen atoms with benzyl or n-decyl bromides is fol-
lowed by the preparation of neutral ligands 18a, 18b and 19.
Neutral ligands in their turn were afforded by the reactions of
corresponding bromides with corresponding amines.
To our knowledge, in spite of advances in pyrimidino-
phanes chemistry biological activity data for these compounds
absolutely have not been disclosed so far. The possible expla-
nation is insolubility of almost all known pyrimidinophanes in
polar solvents, particularly, in water, which prevents their bio-
logical properties study. Earlier we have reported the synthesis
of first pyrimidinophanes which contain two 6-methyluracil
fragments linked to each other by polymethylene chains with
nitrogen atom [13]. We isolated individual geometric isomers
with cis- or trans-arrangement of the carbonyl and methyl
groups at different pyrimidine rings. Reaction of the isomeric
macrocycles with substituted benzyl bromide gave rise to
water-soluble pyrimidinophanes with quaternary ammonium
in bridges. For macrocyclic compounds of this type a wide
screening of various kinds of biological activity is possible.
It is widely known that quaternary ammonium salts in com-
bination with lipophilic residues display unspecific activity
through interaction with cell walls and cell membranes of mi-
croorganisms. Use of the organic cations as disinfectants in
agriculture, the food processing industry and clinics is particu-
larly important because they possess a high antibacterial activ-
ity and a broad spectrum of antimicrobial activity. Taking into
account the mentioned above proposals and considering the
pyrimidinophane framework as specific fragment available to
interfere in main biochemical pathways (cell wall synthesis,
DNA and RNA synthesis, protein formation) we have prepared
a series of pyrimidinophanes as individual isomers and couple
of isomers, quaternized them by benzyl and alkyl bromides,
and this preliminary communication demonstrates the in vitro
antibacterial and antifungal activity of the pyrimidinophanes,
their toxicity, which have not been studied so far. Also we
have synthesized and tested new type of pyrimidinophanes
consisting of one uracil unit and the spacer with nitrogen atom
which connects N1 and N3 of pyrimidine ring. Moreover, we
tested the acyclic compounds having structural features of the
examined pyrimidinophanes. The acyclic compounds are intro-
duced in the screening for attempt to determine fragments re-
sponsible for antimicrobial activity and evaluate importance of
ring-closure for efficacy. The objective of our study is to gen-
erate novel leads and novel active compounds and to optimize
the structure to display the potent efficacy. At present time
when new infectious diseases appear it becomes especially im-
portant and the search of quiet new agents is necessary.
1-Mono- or 1,3-bis(aminoalkyl)uracils were prepared by
amination of 1-mono- or 1,3-bis(bromoalkyl)uracils with a
2. Chemistry
Recently we reported the synthesis of pyrimidinophanes
with nitrogen atom in spacers by reactions of 1,3-bis(α,ω-bro-
mobutyl or pentyl)-6-methyluracils with 1,3-bis(α,ω-ethylami-
nobutyl or pentyl)-6-methyluracils [13]. These reactions imply
the formation of isomers and we succeeded in isolating indivi-
dual geometric isomers of pyrimidinophanes by column chro-
Fig. 1. Isomeric pyrimidinophanes with cis- (1a, 2a, 3a) and trans- (1b, 2b, 3b)
arrangement of the carbonyl groups.

V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
1095
Scheme 1. Synthesis of 3a and 3b, reagents and conditions: (i) Na, n-BuOH, reflux, 15 h; (ii) Br(CH2)6Br, DMF, 50–60 °C, 10 h; (iii) 40-fold excess of NH2Et, i-
PrOH, room temperature, 7 days; (iiii) K2CO3, CH3CN, 60–70 °C, 12 h.
pentyl)thymine (20) and the following quaternization with n-
decyl bromide gave macrocycle 22. These pyrimidinocyclo-
phanes have interesting conformational and spectral features,
which will be discussed elsewhere.
3. Biological results and discussion
The in vitro antibacterial and antifungal activity of the pyr-
imidinophanes 6, 7a,b, 8, 9, 10a,b, 11a,b, 22 was investigated
against several pathogenic representative Gram-negative bacter-
ia (Pseudomonas aeruginosa 9027 and Escherichia coli F-50),
Gram-positive bacteria (Staphylococcus aureus 209p, Bacillus
subtilis 6633 and Enterococcus faecalis ATCC 8043), patho-
genic fungi (Aspergillus niger BKMF-1119, Trichophyton men-
tagrophytes var. gypseum 1773 and Aspergillus fumigatus AF-
27) and yeast (Candida Albicans 885-653). Results are pre-
sented in Tables 1 and 2. It is necessary to note that couples
of isomeric macrocycles 10a and 10b, 11a and 11b were
Fig. 2. Isomeric pyrimidinophanes with quaternized N in spacers.
screened as mixtures because we didn’t succeed in isolating of
individual geometric isomers of macrocycles 3a and 3b and in-
troduced the mixture of them in the reaction of quaternization
by benzyl bromide or n-decyl bromide. Data of the tables can
be related to structural features of the compounds, particularly
trans- or cis-arrangement of the carbonyl groups, aliphatic or
aromatic substituent at the nitrogen atoms in bridges, length of
spacer, cyclic or acyclic geometry. The screened compounds
exhibit bacteriostatic and fungistatic activity in the regions of
concentrations 0.98–5000 μg/ml and 30–2500 μg/ml, respec-
tively. As evident from antibacterial data pyrimidinophanes
1a, 6, 7a,b, 8 are showing a slight bacteriostatic activity against
Gram-positive bacteria S. aureus and B. subtilis but absolutely
are not active against Gram-negative bacteria E. coli and P. aer-
uginosa and moulds A. niger and T. mentagrophytes. Quaterni-
zation of macrocycle 1a by o-nitrobenzyl bromide don’t affect
the activity of the resulting pyrimidinophane 6. However, qua-
Fig. 3. Acyclic counterparts of pyrimidinophanes 12–14 and model compound
15.
ternization of macrocycle 1a by benzyl bromide which hasn’t
electron withdrawing substituent gave macrocycle 7a with dra-
matic decrease of MIC values. Pyrimidinophanes 7a and 7b
considerable excess of appropriate amine (Schemes 1 and 2).
When 1,3-bis(bromoalkyl)uracil/amine ratio is 1:1–1:3 as 1(3)-
mono- or 1,3-bis(aminoalkyl)uracils as macrocyclic product
can be formed. Particularly preparation of macrocycle 21 is
depicted in Scheme 3. We isolated pyrimidinocyclophane 21
from the reaction mixture of n-butylamine and 1,3-bis(bromo-
with cis- and trans-arrangement of the carbonyl groups at dif-
ferent pyrimidine rings, respectively, exhibit the same antibac-
terial activity and it shows that the arrangement of the carbonyl
groups doesn’t affect the activity. Also in this case cyclic struc-
ture of the isomers 7a and 7b doesn’t influence their activity
because the acyclic counterpart 12 with one nitrogen atom

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V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
Scheme 2. Synthesis of 12 and 13, reagents and conditions: (i) 40-fold excess of NH2Et, i-PrOH, room temperature, 3 days; (ii) 16a or 16b, K2CO3, CH3CN, reflux,
12 h; (iii) 1.2-fold excess of benzyl bromide or twofold excess of n-decyl bromide, CH3CN, reflux, 20 h.
Scheme 3. Synthesis of pyrimidinocyclophanes 21 and 22, reagents and conditions: (i) threefold excess of n-BuNH2, K2CO3, TBA. HSO4, n-BuOH, 70–75 °C, 11 h;
(ii) fourfold excess of n-decyl bromide, CH3CN, reflux, 20 h.
Table 1
In vitro antibacterial and antifungal activity of pyrimidinophanes and acyclic counterpartsa
Minimal Inhibitory Concentration (MICs) μg/ml
No
1a
6
7a
7b
8
9
Sa
Bs
Ec
–
–
–
–
–
62.5
>1000
62.5
–
Pa
–
–
–
–
–
>1000
–
500
–
–
–
–
n.d.
Ef
An
–
–
–
–
–
250
–
250
–
> 1000
62.5
10
n.d.
Tm
–
–
–
–
–
125
–
125
–
625
31.3
500
n.d.
Af
n.d.
n.d.
n.d.
n.d.
n.d.
> 1000
n.d.
250
n.d.
n.d.
125
> 1000
n.d.
Ca
> 1000
> 1000
312
312
125
0.98
50
0.98
625
78
> 1000
> 1000
625
625
500
7.8
100
1.95
> 1000
156
n.d.
n.d.
n.d.
n.d.
n.d.
500
n.d.
156
n.d.
n.d
n.d.
n.d.
n.d.
n.d.
n.d.
250
n.d.
250
n.d.
n.d.
62.5
500
n.d.
10a and 10b
11a and 11b
12
13
14
15
22
> 1000
12.5
1.56
2.44
78
7.8
50
625
625
312
625
n.d.
156
> 1000
3.13
Ampicillin
0.13
Clo
Cip
Amph
a
3.13
0.19
0.39
0.12
3.9
20
The tests were performed in duplicate and repeated twice; n.d., not done; -, no activity was showed; Pa, Pseudomonas aeruginosa; Ec, Escherichia coli; Sa,
Staphylococcus aureus; Ba, Bacillus subtilis; Ef, Enterococcus faecalis; An, Aspergillus niger; Af, Aspergillus fumigatus; Tm, Trichophyton mentagrophytes;
Ca, Candida albicans; Clo, Clotrimazole; Cip, Ciprofloxacin; Amph, Amphotericin B.
which is quaternized by benzyl bromide shows MICs precisely
twice as large as MICs of these pyrimidinophanes. Increase of
the polymethylene N(pyr)-N-chain length up to 5 methylene
groups in macrocycle 8 and up to 6 methylene groups in mix-
ture of isomeric pyrimidinophanes 10a and 10b dramatically
increases antibacterial activity: MICs of pyrimidinophane 8
and mixture of the pyrimidinophanes 10a and 10b are threefold
and sixfold, respectively, less than MICs of 7a or 7b with 4
methylene groups in N(pyr)-N-chain.
It is obvious from the structure–activity profile of pyrimidi-
nophanes that lipophilic n-decyl substituent at nitrogen atom in
spacer greatly influence the antibacterial and antifungal activ-
ity. Macrocycle 9 and isomeric pyrimidinophanes 11a and 11b
mixture show high activity against bacteria and fungi, and in
this case there is the trend of an increase of the efficacy of the
compounds with an increase of the polymethylene spacer
length. In particular pyrimidinophanes 11a and 11b mixture
exhibit the dramatic increase of the efficacy against Bacillus

V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
1097
Table 2
Table 4
Bactericidal activity of pyrimidinophanes 9, 11a,b and acyclic compounds 13–
Toxicity of pyrimidinophanes 6, 8, 9, 11a and 11b on mice and Daphnia
15a
Number
LC50, Daphnia magna, mg/l LD50, mice, mg/kg
Number
S. aureus
MBC, Time,
B. subtilis
MBC, Time,
E. aecalis
MBC Time,
6
8
9
110.6 (94.5 ÷ 37.4)
54.3 (46.8 ÷ 62.9)
14.3 (12.1 ÷ 16.9)
5.1 (4.4 ÷ 6.0)
13,6 (11.9 ÷ 15.5)
9.5 (8.2 ÷ 11.0)
23.4 (19.6 ÷ 25.1)
18.8 (16.3 ÷ 21.6)
μg/ml
500
500
104
min
30
10
μg/ml
4000
990
min
120
120
–
μg/ml
500
min
240
15
11a and 11b
9
11a and 11b
13
500
–
104
104
–
exhibit the bactericidal and fungicidal properties anyway. The
antibacterial and antifungal efficacy of the screened com-
pounds were compared with that showed by the standard anti-
bacterial drugs ampicillin and ciprofloxacin, and antifungal
drugs clotrimazole and amphotericin B. Pyrimidinophanes 9,
11a and 11b display bacteriostatic activity against E. coli and
S. aureus comparable to the reference drug ampicillin while
their activity against B. subtilis is 300-fold more than that of
ampicillin. The screening data against fungi revealed that pyr-
imidinophanes 9, 11a and 11b, acyclic compound 14 show
modest activity compared with that of the reference drugs.
We have evaluated the toxicity of the pyrimidinophanes 6,
8, 9 and the mixture of isomeric pyrimidinophanes 11a and
11b in terms of lethal doses (LD50) for mice and lethal concen-
trations (LC50) for Daphnia magna Straus. Toxicity data are
presented in Table 4. According to levels of ecological safety
for Daphnia magna [22] pyrimidinophanes 6, 8, 9 can be con-
sidered as slightly toxic or practically non-toxic, and isomeric
pyrimidinophanes 11a and 11b as moderately toxic com-
pounds. According to levels of acute toxicity for mammals
the pyrimidinophanes can be considered as slightly toxic and
moderately toxic compounds. Toxicity data show that lethal
doses for Daphnia magna dramatically decrease with the in-
crease of activity of the pyrimidinophanes. On the contrary,
for mammals lethal concentrations increase with the increase
of activity.
14
15
800
1250
0.5
240
240
240
4000
104
6250
240
–
240
300
1000
240
240
Ampicillin
Cip
50
15
a
MBC, minimum bactericidal concentration; Time, time of total cell death; -
, no activity was showed at the concentration; Cip, Ciprofloxacin.
subtilis, Enterococcus faecalis and Aspergillus fumigatus com-
pared with pyrimidinophane 9.
Acyclic compound 14 which has the structure similar to
pyrimidinophane 9 exhibits bacteriostatic activity lower than
the macrocyclic counterpart but demonstrates the highest effi-
cacy against fungi and yeast. Compound 13 is “half” of pyri-
midinophane 9; however, its activity is significantly lower than
activity of macrocyclic and acyclic counterparts 9 and 14. Im-
portance of long alkyl chain substituent at nitrogen atom in
spacer is confirmed by MICs values of the reference compound
15. In fact, a long alkyl chain suggest detergent-like properties
and unspecific mechanism of action. However, if discussed
compounds simply acted by a non-specific detergent-like me-
chanism, much lipophilic pyrimidinocyclophane 22 would be
the most active compound. Contrary, MICs of the macrocycle
are the lowest for acyclic and macrocyclic uracil derivatives 9,
11a and 11b, 13, 14 with n-decyl substituent. It is evident that
in vitro antibacterial and antifungal activity of the compounds
depends not only on substituents at the cationic head but also
the mutual arrangement of these substituents and uracil unit.
Macrocyclic and acyclic compounds with the highest bac-
teriostatic and fungistatic efficacy were further screened for
their bactericidal and fungicidal activity (Tables 2 and 3). The
screening data against Gram-positive bacteria and pathogenic
fungi revealed that pyrimidinophane 9, mixture of the isomeric
pyrimidinophanes 11a and 11b have the best bactericidal prop-
erties and acyclic compound 14 have the best fungicidal prop-
erties. Meanwhile, model detergent-like compound 15 doesn’t
4. Conclusions
We have described the antibacterial and antifungal activity
of a series of pyrimidinophanes with one and two uracil units,
cis- or trans-arrangement of carbonyl groups at different pyri-
midine rings, with quaternized or not quaternized nitrogen
atoms in the bridges and their acyclic counterparts. The screen-
ing data show that the activity depends on different structural
features, particularly, lipophily of substituent at nitrogen atom
in spacer, length of N(pyr)-N-chain and relative orientation of
the cationic head and uracil fragment. In some cases cyclic
structure of the screened compounds increases the activity with
respect to acyclic counterparts. However, acyclic compound 14
is more active against fungi than isostructural pyrimidino-
phanes. The mode of action of the screened compounds is
not clear. Direct data show that a specific mechanism of action
is lacking. It is obvious that uracil units are not responsible for
the antibacterial and antifungal effect of the pyrimidinophanes
and their acyclic counterparts; however, the appropriate ar-
rangement of uracil unit relative to quaternary ammonium in
spacer can significantly increase antibacterial and antifungal
efficacy.
Table 3
Fungicidal activity of pyrimidinophanes 9, 11a,b and acyclic compounds 13–
15a
Number
A. niger
T. mentagrophytes
C. albicans
MFC
μg/ml
500
Time
min
180
90
MFC
μg/ml
250
Time
min
180
120
360
180
–
MFC
μg/ml
250
250
n.d.
65
500
31.25
Time
min
180
180
n.d.
180
240
360
9
11a and 11b 500
13
14
15
Clo
Amph
a
250
104
125
104
103
250
–
2500
62.5
104
180
–
360
120
12.5
360
MFC, minimum fungicidal concentration; Time, time of total cell death; n.
d., not done; -, no activity was showed at the concentration; Clo, Clotrimazole;
Amph, Amphotericin B.

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V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
5. Experimental protocols
eluent); IR (KBr) cm−1: 1680, 1720 (CO), 3550–3200 (NH);
1H NMR (CDCl3): δ 1.12 (m, 6H, CH3), 1.36–1.63 (m, 16H,
CH2), 2.26 (s, 3H, C6(pyr)CH3), 2.57–2.68 (m, 10H, NHCH2),
3.80 (t, J = 8 Hz 2H, N(pyr)CH2), 3.90 (t, J = 7.6 Hz, 2H,
5.1. Chemistry
N(pyr)CH2), 5.57 (s, C5(pyr)H); Anal. calcd for C21H40N4O2:
C, 66.28; H, 10.59; N, 14.72; found: C, 66.35; H, 10.66; N,
14.66.
5.1.1. Materials
1
The H NMR spectra were recorded on Bruker MSL-400
spectrometer with Me4Si as the internal standard. The IR spec-
trum (in KBr) was recorded on a Vector 22 Fourier spectro-
meter (Bruker) under standard conditions. The mass spectra
(EI) were obtained on a Finnigan MAT-212 mass spectrometer
(70 eV). The melting points were measured on a Boetius hot-
stage apparatus or in open capillary tubes. Thin layer chroma-
tography was performed on Silufol-254 plates; visualization
was carried out with UV light. For column chromatography,
silica gel of 60 mesh from Fluka and neutral Al2O3 (activity
II) were used. All solvents were dried according to standard
protocols. Pyrimidinophanes 1a, 1b, 2a, 2b, 6, 8 were prepared
by known procedures [13]. Also known protocol was applied
to synthesis of compounds 16a [11] and 19 [15]. Compounds
16b, 17a, 17b were obtained using established synthetic routes.
n-Decyltriethylammonium bromide 15 was obtained with 87%
yield and m.p. 124–127 °C by the reaction of NEt3 with n-
decyl bromide in CH3CN (106–107 °C [22]).
5.1.2.3. 18,
34-Dimethyl-8,26-diethyl-1,8,15,19,26,33-hexa-
azatricyclo[31,3,1,115,19]-octatriaconta-17,34-diene-
16,36,37,38-tetraone (3a) and 18,36-dimethyl-8,26-diethyl-
1,8,15,19,26,33-hexaazatricyclo[31,3,1,115,19]-octatriaconta-
17,35-diene-16,34,37,38-tetraone (3b). Potassium carbonate
(4.0 g, 30.0 mmol) was added to a solution of dibromide 4
(4.88 g, 10.8 mmol) and diamine 5 (4.0 g, 10.5 mmol) in acet-
onitrile (100 ml) and the reaction mixture was stirred at
60–70 °C for 12 h. The precipitate was filtered off. The solu-
tion was concentrated to 10–15 ml and transferred to a column
with SiO2. The column was successively washed with ethyl
ether, ethyl acetate and a 20:1 ethyl acetate/diethyl amine mix-
ture. From the ethyl acetate/diethyl amine fractions the mixture
of pyrimidinophanes 3a and 3b was obtained as oil in a yield
of 1.2 g (17%); Rf 0.48 and 0.54 (6:6:1 ethyl acetate/ethyl
1
ether/diethyl amine as the eluent; H NMR (CDCl3) δ 1.01
(m, 6H, CH3), 1.35–1.63 (м, 32H, CH2), 2.24 (s, 6H,
C6(pyr)CH3), 2.38–2.51 (m, 12H, NCH2), 3.79 (м, 4H,
5.1.2. Synthesis of pyrimidinophanes 3a and 3b
N
(пиp)CH2), 3.91 (м, 4H, 2N(пиp)CH2), 5.56 (s, 2H, C5(pyr)H);
EIMS m/z (%rel. int.): 671 (21) [M + 1]+, 670 (50) [M]+, 656
(15), 655 (31), 642 (41), 641 (100), 628 (11), 627 (24), 348
(21), 335 (28), 266 (20), 203 (59), 127 (18); HRMS: calcd for
C38H66N6O4: M = 670.5146, found: m/z 670.515 [M]+.
5.1.2.1. 1,3-Bis(6-bromohexyl)-6-methyluracil (4). 1,6-Dibro-
mohexane (300 g, 1.23 mol) was added with stirring to a sus-
pension of disodium salt of 6-methyluracil (27 g, 0.159 mol) in
DMF (250 ml) and the mixture was stirred at 55–65 °C for 8 h.
The mixture was evaporated in a vacuum, and the residue was
treated with chloroform to extract the reaction products. The
precipitate was filtered off and the filtrate was concentrated to
10–20 ml and transferred to a column with Al2O3. The column
was successively washed with petroleum ether and a 1:1 petro-
leum ether-ethyl ether mixture. From petroleum ether-ethyl
ether fractions compound 4 was obtained in a yield of 11.2 g
(16%) as oil; Rf 0.41 (5:1 ethyl ether-hexane as the eluent); 1H
NMR (CDCl3); δ 1.34–1.90 (m, 16H, CH2), 2.27 (s, 3H,
5.1.3. General procedure for obtaining acyclic compounds 18a
and 18b
A mixture of corresponding ω-bromoalkyl-3,6-dimethylura-
cil 16a or 16b (10 mmol) and ω-ethylaminoalkyl-3,6-dimethy-
luracil 17a or 17b (10 mmol) in the presence of K2CO3
(50 mmol) in acetonitrile (100 ml) was refluxed for 12 h. The
mixture was evaporated in a vacuum, and the residue was trea-
ted with chloroform to extract the reaction products. The pre-
cipitate that formed was filtered off, the filtrate was concen-
trated to 10–20 ml and submitted to chromatography over
Al2O3. The column was washed with ethyl acetate in the case
of 18a and with chloroform in the case of 18b to give desired
compounds.
C
6(pyr)CH3), 3.41 (m, 4H, CH2Br), 3.79 (t, J = 8 Hz, 2H,
N(pyr)CH2), 3.94 (t, J = 7.6 Hz, 2H, N(pyr)CH2,), 5.56 (s,
5(pyr)H); Anal. calcd for C17H28Br2N2O2: C, 45.15; H, 6.24;
C
Br 35.34; N, 6.19; found: C, 45.22; H, 6.26; Br 35.44; N, 6.36.
5.1.2.2. 1,3-Bis(6-ethylaminohexyl)-6-methyluracil (5). Com-
pound 4 (5 g, 11.1 mmol) was added to a 20% EtNH2 solution
in i-PrOH (100 ml ). The reaction mixture was kept at room
temperature for 7 days and then concentrated in vacuo. A solu-
tion of MeONa, which was prepared from sodium (0.51 g,
22.2 mmol) in methanol (30 ml), was added to the residue.
The solvent was evaporated in vacuo and the reaction product
was extracted with ethyl ether (2 × 50 ml). The ether was dis-
tilled off to obtain compound 5 in a yield of 4 g (97%) as oil;
Rf 0.09 (6:6:1 ethyl acetate/ethyl ether/diethyl amine as the
5.1.3.1. Bis[4-(3,6-dimethyluracil-1-yl)butyl]ethylamine (18a).
Yield 23%; m.p. 88 °C; Rf 0.27 (10:1 ethyl acetate/diethyl
1
amine as the eluent); H NMR (CDCl3): δ 1.03 (t, J = 7.0 Hz,
3H, CH3); 1.66–1.53 (m, 8H, CH2), 2.26 (s, 6H, C6(pyr)CH3),
2.48 (m, 6H, NCH2), 3.32 (s, 6H, N(pyr)CH3), 3.83 (t, J = 7.3
Hz, 4H, N(pyr)CH2), 5.60 (2H, s, C5(pyr)H); Anal. calcd for
C22H35N5O4: C, 60.95; H, 8.14; N, 16.15; found: C, 61.10;
H, 8.03; N, 16.13.

V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
1099
5.1.3.2. Bis[5-(3,6-dimethyluracil-1-yl)pentyl]ethylamine
5.1.5. General procedure for the quaternization
of pyrimidinophanes 1a, 1b, 2b, 3a and 3b, 21, acyclic
compounds 18a, 18b and 19
(18b). Yield 81% as oil; Rf 0.32 (10:10:1 ethyl acetate/ethyl
1
ether/diethyl amine 10:10:1 as the eluent); H-NMR (CDCl3):
δ 1.00 (t, J = 7.2 Hz, 3H, CH3); 1.35 (m, 4H, CH2), 1.47 (m,
4H, CH2), 1.66 (m, 4H, CH2), 2.25 (s, 6H, C6(pyr)CH3), 2.41 (t,
J = 7.5 Hz, 4H, NCH2), 2.50 (q, J = 7.2, 2H, NCH2,), 3.32 (s,
6H, N(pyr)CH3), 3.80 (t, J = 7.9 Hz, 4H, N(pyr)CH2); 5.60 (s,
2H, C5(pyr)H); Anal. calcd for C24H39N5O4: C, 62.45; H,
8.52; N, 15.17; found: C, 62.40; H, 8.43; N, 15.19.
A solution of macrocyclic or acyclic compound (0.30 mmol)
and 2–4-fold excess of n-decyl bromide or 1.2-fold excess of
benzyl bromide in acetonitrile (30 ml) was refluxed for 20 h.
The solvent was distilled off. The residue was thoroughly tri-
turated in ethyl ether (5 × 30 ml), each time decantated and
finally the solvent was evaporated.
5.1.4. Synthesis of pyrimidinocyclophane (21)
5.1.5.1. 14,26-Dimethyl-6,20-diethyl-6,20-dibenzyl-
1,6,11,15,20,25-hexaazatricyclo-[23,3,1,111,15]-triaconta-
13,26-diene-12,28,29,30-tetraone dibromide (7a). Yield 75%;
1
5.1.4.1. 1,3-Bis(5-bromopentyl)thymine (20). A solution of 1,5-
dibromopentane (155.9 g, 677.8 mmol) in DMF (90 ml) was
added dropwise with stirring to a suspension of 14.4 g
(84.7 mmol) of disodium salt of thymine in DMF (150 ml).
The mixture was stirred for 5 h at 50–60 °C, after which it
was evaporated in a vacuum and the residue was treated with
150 ml of CHCl3. The precipitate that formed was filtered off.
The solution was concentrated and submitted to chromatogra-
phy over Al2O3. The column was successively washed with
petroleum ether and a 2:1 ether/petroleum ether mixture. From
ether/petroleum ether mixture fractions compound 6 was ob-
m.p. 100–110 °C (dec.); H NMR (CD3CN).
δ 1.39 (м, 6H, CH3), 1.66–1.77 (м, 16H, CH2), 2.34 (c, 6H,
C6(pyr)CH3), 3.19 (м, 12H, NCH2), 3.88 (м, 8H, N(pyr)CH2),
4.42 (br.s, 2H, CH2Ph), 4.50 (br.s, 2H, CH2Ph), 5.59 (c, 2H,
C5(pyr)H), 7.52 (м, 10H, Ar-H); Anal. calcd for
C44H64Br2N6O4: C, 58.67; H, 7.16; Br 17.74; N, 9.33; found:
C, 58.62; H, 7.10; Br 17.82; N, 9.41.
5.1.5.2. 14,28-Dimethyl-6,20-diethyl-6,20-dibenzyl-
1,6,11,15,20,25-hexaazatricyclo-[23,3,1,111,15]-triaconta-
13,27-diene-12,26,29,30-tetraone dibromide (7b). Yield 62%;
1
tained as oil in a yield of 15.9 g (45%); H NMR (CDCl3): δ
1
1.50 (m, 4H, CH2), 1.76–1.58 (m, 4H, CH2), 1.89 (m, 4H,
CH2), 1.93 (c, 3H, C5(pyr)CH3), 3.42 (м, 4H, CH2Br), 3.73 (t,
J = 7 Hz, 2H, N(pyr)CH2), 3.95 (t, J = 7 Hz, 2H, N(pyr)CH2),
7.01 (s, 1H, C6(pyr)H); EIMS (EI) m/z (%rel. int.): 426 (9) [M]
+, 424 (22) [M]+, 422 (10) [M]+, 346 (28), 345 (88), 344 (28),
343 (88), 275 (75), 195 (86), 140 (100); Anal. Calcd. for
C15H24Br2N2O2: C, 42.47; H, 5.70; N, 6.60; Br, 37.68. Found:
C, 42.48; H, 5.81; N, 6.53; Br, 37.75.
m.p. 100–110 °C (dec.); H NMR (CD3CN).
δ 1.33 (м, 6H, CH3), 1.66–1.77 (м, 16H, CH2), 2.25 (c, 6H,
C6(pyr)CH3), 3.13 (м, 12H, NCH2), 3.83 (м, 8H, N(pyr)CH2),
4.40 (br. m, 4H, CH2Ph,), 5.52 (c, 2H, C5(pyr)H), 7.53 (м,
10H, Ar-H); Anal. calcd for C44H64Br2N6O4: C, 58.67; H,
7.16; Br 17.74; N, 9.33; found: C, 58.60; H, 7.14; Br 17.77;
N, 9.39.
5.1.5.3. 16,30-Dimethyl-7,23-diethy-7,23-di-n-decyl-
1,7,13,17,23,29-hexaazatricyclo-[27,3,1,113,17]-tetratriaconta-
15,30-diene-14,32,33,34-tetraone dibromide (9). Yield 90%;
5.1.4.2. 15-Methyl-7-butyl-1,7,13-triazabicyclo[11.3.1]hepta-
deca-15-en-14,17-dione (21). At 70 °C to a stirred mixture of
n-butylamine (2.07 g, 28.4 mmol), K2CO3 (4.00 g, 29.0 mmol)
1
m.p. 75–100 °C (dec.); H NMR (CD3CN).
.
and catalytic amount TBA HSO4 in n-BuOH (250 ml) com-
δ 1.24–1.70 (m, 68H, CH2, CH3), 2.25 (s, 6H, C6(pyr)CH3),
3.07–3.23 (m, 16H, NCH2), 3.76–3.86 (m, 8H, N(pyr)CH2),
5.50 (s, 2H, C5(pyr)H). Anal. calcd for C54H100Br2N6O4: C,
61.35; H, 9.53; Br 15.12; N, 7.95; found: C, 61.12; H, 9.36;
Br 15.34; N, 7.86; Anal. calcd for C54H100Br2N6O4: C, 61.35;
H, 9.53; Br 15.12; N, 7.95; found: C, 61.33; H, 9.54; Br 15.02;
N, 8.07.
pound 20 (3.0 g, 7.08 mmol) in n-BuOH solution (100 ml)was
added and stirring was continued for 11.5 h at 70–75 °C. After
evaporating of the solvent, treating by CHCl3, filtering and
concentration of CHCl3 solution residue was eluated through
column with Al2O3 by 2:1 ether/petroleum ether mixture. From
the fractions of the eluent pyrimidinocyclophane 21 was ob-
1
tained in a yield of 0.45 g (19%); m.p. 44–45 °C; H NMR
(CDCl3) δ (ppm): 0.85 (t, J = 7.4 Hz, 3H, CH3), 1.12 (m,
J = 7 Hz, 2H), 1.15–1.28 (m, 9H, CH2), 1.34(m, J = 7.4 Hz,
2H, CH2), 1.42 (m, 1H, CH), 1.57 (m, 1H, CH), 1.75 (m,
1H, CH), 1.86 (s, 3H, C5(pyr)CH3), 2.11–2.25 (m, 4H, NCH2),
2.32 (m, 2H, NCH2), 3.09 (m, 1H, N(pyr)CH), 3.92 (m, 1H,
5.1.5.4. 18,34-Dimethyl-8,26-diethyl-8,26-dibenzyl-
1,8,15,19,26,33-hexaazatricyclo-[31,3,1,115,19]-octatriaconta-
17,34-diene-16,36,37,38-tetraone dibromide (10a) and 18,36-
dimethyl-8,26-diethyl-8,26-dibenzyl-11,8,15,19,26,33-hexaaza-
tricyclo-[31,3,1,115,19]-octatriaconta-17,35-diene-16,34,37,38-
N
(pyr)CH), 4.21 (m, 1H, N(pyr)CH), 4.42 (m, 1H, N(pyr)CH),
1
6.83 (s, 1H, C6(pyr)H); EIMS m/z (%rel. int.): 335 (13) [M]+,
292 (100) [M – 43]+, 278 (8) [M – 57]+; HRMS: calcd for
C19H33N3O2: M = 335.2573, found m/z 335.257 [M]+; calcd
for C16H26N3O2: M = 292.2025, found m/z 292.202
[M – 43]+.
tetraone dibromide (10b). Yield 77%; m.p. > 70 °C (dec.); H
NMR (CD3CN).
δ 1.79–1.37 (м, 38H, CH2, CH3), 2.28 (c, 6H, C6(pyr)CH3),
3.09–3.34 (м, 12H, CH2), 3.80(м, 8H, N(pyr)CH2), 4.50 (м, 4H,
CH2Ph), 5.62 (s, 2H, C5(pyr)H), 7.52 (м, 10H, Ar-H); Anal.

1100
V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
calcd for C52H80Br2N6O4: C, 61.65; H, 7.96; Br 15.78; N,
8.30; found: C, 61.73; H, 8.04; Br 15.66; N, 8.27.
5.2. Biological evaluation
5.2.1. Antibacterial and antifungal activity
The in vitro antibacterial and antifungal activity of the
macrocyclic and acyclic synthesized compounds were investi-
gated against several pathogenic representative Gram-negative
bacteria (Pseudomonas aeruginosa 9027, Escherichia coli F-
50), Gram-positive bacteria (Staphylococcus aureus 209p, Ba-
cillus subtilis 6633, Enterococcus faecalis ATCC 8043),
moulds (Aspergillus niger BKMF-1119, Aspergillus fumigatus
AF-27, Trichophyton mentagrophytes var. gypseum 1773) and
yeast (Candida Albicans 885–653). Minimal inhibitory con-
centrations (MICs) were estimated by conventional dilution
methods for bacteria [23] and fungi [24]. The antibacterial
and antifungal assays were performed in nutrient broth (bacter-
ia 3 × 105 cfu/ml) and Sabouraud dextrose broth (fungi 2 ×
103–4 cfu/ml). Ampicillin, ciprofloxacin, amphotericin B and
clotrimazole were used as standard drugs. Positive growth con-
trol and standard drug controls were also run simultaneously.
The MICs were defined as the lowest concentrations that
showed no growth and recorded by visual observation in every
24 h during 5 days for bacteria and after incubation during
14 days for fungi.
The bactericidal and fungicidal activity was determined as
follows. Assay tubes were filled with 1 ml of test compound
solution in nutrient agar. Concentrations of test compounds
were varied from 12.5 to 104 μg/ml. Normal saline broth (bac-
teria 3 × 105 cfu/ml), 1 ml was added to the tubes and for
5 min, 10 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h the inocula
were prepared by transferring the broth onto Petri plates con-
taining meal-peptone agar. Petri plates were incubated at 37 °C
and minimum bactericidal concentration (MBC) recorded as
the test compound dilution affecting total cell death. For fungi-
cidal activity determination the tubes with the test compounds
and fungi were incubated at 26 °C. For 10 min, 20 min,
30 min, 1 h, 1.5 h, 2 h, 3 h, 6 h the inocula were prepared in
Sabouraud dextrose broth and incubated at 26 °C.
5.1.5.5. 18,34-Dimethyl-8,26-diethyl-8,26-di-n-decyl-
1,8,15,19,26,33-hexaazatricyclo-[31,3,1,115,19]-octatriaconta-
17,34-diene-16,36,37,38-tetraone dibromide (11a) and 18,36-
dimethyl-8,26-diethyl-8,26-di-n-decyl-11,8,15,19,26,33-hexaa-
zatricyclo-[31,3,1,115,19]-octatriaconta-17,35-diene-
16,34,37,38-tetraone dibromide (11b). Yield 71%; m.p.
1
60–75 °C (dec.); H NMR (CD3CN).
δ 1.29–1.82 (m, 76H, CH2, CH3), 2.23 (s, 6H, C6(pyr)CH3),
3.08–3.20 (m, 16H, NCH2), 3.81–3.87 (m, 8H, N(pyr)CH2),
5.51 (s, 2H, C5(pyr)H); Anal. calcd for C58H108Br2N6O4: C,
62.57; H, 9.78; Br 14.35; N, 7.55; found: C, 62.68; H, 9.87;
Br 14.24; N, 7.49.
5.1.5.6. Bis[4-(3,6-dimethyluracil-1-yl)butyl]benzylethylammo-
nium bromide (12). Yield 71%; m.p. > 110 °C (dec.); 1H NMR
(CDCl3).
δ 1.38 (t, J = 7.3 Hz, 3H, CH3), 1.64 (m, 4H, CH2), 1.82 (m,
4H, CH2), 2.26 (s, 6H, C6(pyr)CH3), 3.16–3.24 (m, 6H, NCH2),
3.19 (s, 6H, N(pyr)CH3); 3.86 (t, J = 7.6 Hz, 4H, N(pyr)CH2,),
4.53 (s, 2H, CH2Ph,), 5.55 (2H, s, C5(pyr)H), 7.42 (м, 5H, Ar-
H); Anal. calcd for C29H42BrN5O4: C, 57.61; H, 7.00; Br,
13.22; N, 11.58; found: C, 57.73; H, 7.09; Br, 13.34; N, 11.51.
5.1.5.7. Bis[5-(3,6-dimethyluracil-1-yl)pentyl]-n-decylethylam-
monium bromide (13). Yield 79%; m.p. > 70 °C (dec.); H-
NMR (CDCl3).
1
δ 0.88 (t, J = 6.9 Hz, 3H, CH3), 1.29 (m, 19H, CH3, CH2);
1.66 (m, 12H, CH2), 2.25 (s, 6H, C6(pyr)CH3), 3.06 (m, 8H,
NCH2,), 3.19 (s, 6H, N(pyr)CH3), 3.81 (t, J = 7.6 Hz, 4H,
N
(pyr)CH2,); 5.54 (s, 2H, C5(pyr)H); Anal. calcd for
C34H60BrN5O4: C, 59.81; H, 8.86; Br, 11.70; N, 10.26; found:
C, 59.90; H, 8.85; Br, 11.74; N, 10.31.
5.2.2. Toxicity
5.1.5.8. 1,3-Bis[5-(n-decyldiethylammonium)pentyl]-6-methy-
luracil dibromide (14). Yield 52%; m.p. > 80 °C (dec.); H
1
Toxicity tests were carried out via: (1) Single peroral (per
os) introductions of pyrimidinophanes aqueous solutions in
acute tests on white outbreed mice of both sexes with the mass
of 17–21 g. The observation period was 72 hours. As the cri-
teria of toxicity, the average lethal doses were used—LD50. To
measure these values each compound was introduced to five
groups of mice (10 mice per dose; N = 50); (2) Using labora-
tory partenogenetic culture of Daphnia magna Straus (water
solutions of compounds were used) at the age of 18 6 hours,
NMR (CDCl3).
δ 0.88 (m, 6H, CH3), 1.26 (m, 12H, CH3), 1.38–1.52 (m,
32H, CH2), 1.71–1.92 (m, 12H, CH2), 2.34 (s, 6H, C6(pyr)CH3),
3.28–3.32 (m, 8H, NCH2), 3.49–3.53 (m, 8H, NCH2), 3.90 (м,
4H, N(пиp)CH2), 5.55 (s, 1H, C5(pyr)H): Anal. calcd for
C43H86Br2N4O2: C, 60.69; H, 10.19; Br 18.78; N, 6.58; found:
C, 60.79; H, 10.01; Br 18.82; N, 6.55.
kept in standard conditions. Sensitivity of daphnia culture was
24 h
5.1.5.9. 15-Methyl-7-butyl-7-n-decyl-1,7,13-triazabicyclo
normal, i.e. 0.9–2.0 mg/l, as estimated via LC50
of potas-
[11.3.1]heptadeca-15-en-14,17-dione bromide (22). Yield
sium bichromate. Observation period was equal 48 hours. As
1
33%; m.p. > 100 °C (dec.); H NMR (CDCl3).
the criteria of toxicity, the average lethal concentrations were
48 h.
used—LC5048 h. To determine LC50
each compound was
δ 0.95 (m, 6H, CH3), 1.34–1.85 (m, 32H, CH2), 1.97 (s, 3H,
C5(pyr)CH3), 2.88–3.01 (m, 6H, NCH2), 3.72–4.10 (m, 6H,
applied for three groups of daphnia (30 daphnia per each con-
centration; N = 90). The results were processed using the pro-
gram ToxCalc™ v.5.0.23E (Tidepool Scientific Software;
USA) [21].
N
(пиp)CH2, NCH2), 7.00 (s, 1H, C6(pyr)H); Anal. calcd for
C29H54BrN3O2: C, 62.57; H, 9.78; Br, 14.35; N, 7.55; found:
C, 62.44; H, 9.71; Br, 14.23; N, 7.62.

V.E. Semenov et al. / European Journal of Medicinal Chemistry 41 (2006) 1093–1101
1101
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