Design, synthesis, and antimicrobial activity of some novel homodrimane sesquiterpenoids with diazine skeleton

Article abstract of DOI:10.1007/s00044-013-0720-3

Herein we report a feasible study concerning the design, synthesis, and in vitro antimicrobial activity of some novel homodrimane sesquiterpenoids with diazine skeleton. The reaction pathway is efficient and straight, involving the direct N-acylation of diazine with homodrimane sesquiterpenoids bearing acyl chlorides or organic acids functionality. A reliable explication and a feasible reaction mechanism for the obtained compounds are presented. The in vitro antimicrobial activity of the homodrimane sesquiterpenoids with and without diazine skeleton has been evaluated. All the tested compounds have an excellent antibacterial activity against Gram-positive strains S. aureus and B. cereus. SAR correlations concerning antimicrobial activity are reported. Graphical Abstract: The design, synthesis, and in vitro antimicrobial activity of some novel homodrimane sesquiterpenoids with diazine skeleton are described. Antimicrobial tests prove that some homodrimane sesquiterpenoids have an excellent biological activity.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0720-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1559–1568
DOI 10.1007/s00044-013-0720-3
ORIGINAL RESEARCH
Design, synthesis, and antimicrobial activity of some novel
homodrimane sesquiterpenoids with diazine skeleton
•
Kaleria Kuchkova Aculina Aricu Elena Secara Alic Barba
•
•
•
•
•
•
Pavel Vlad Nicon Ungur Cristina Tuchilus Sergiu Shova
•
•
Gheorghita Zbancioc Ionel I. Mangalagiu
Received: 9 May 2013 / Accepted: 13 August 2013 / Published online: 4 September 2013
Ó Springer Science+Business Media New York 2013
Abstract Herein we report a feasible study concerning the
design, synthesis, and in vitro antimicrobial activity of some
novel homodrimane sesquiterpenoids with diazine skeleton.
The reaction pathway is efficient and straight, involving the
direct N-acylation of diazine with homodrimane sesquiterp-
enoids bearing acyl chlorides or organic acids functionality. A
reliable explication and a feasible reaction mechanism for the
obtained compounds are presented. The in vitro antimicrobial
activity of the homodrimane sesquiterpenoids with and
without diazine skeleton has been evaluated. All the tested
compounds have an excellent antibacterial activity against
Gram-positive strains S. aureus and B. cereus. SAR correla-
tions concerning antimicrobial activity are reported.
Keywords Sesquiterpenoids ꢀ Homodrimane ꢀ Diazine ꢀ
Antimicrobial ꢀ Design and synthesis
Introduction
Infectious diseases caused by bacteria and fungi have sub-
stantially increased over the last few years, becoming amajor
concern in many countries around the world (Grahman,
2001; Greenwood et al., 2007). In particular, the emergences
of drug resistance and multidrug resistance have caused life-
threatening infectious diseases. Although new generations of
antimicrobial drugs have been introduced, bacterial and
fungal infections remain a major problem in modern therapy.
There is, therefore, a clear need to discover and develop
novel structures with antimicrobial properties, which could
lead to the development of new useful agents for the man-
agement of bacterial and fungal infections.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0720-3) contains supplementary
material, which is available to authorized users.
Natural products have been found to be a relevant source of
novel and potent bioactive compounds with a good activity,
well tolerated by the organism, with low or no toxicity. One of
the promising classes within this area is sesquiterpenoids,
which have a wide application in medicine, pharmaceutics,
cosmetics, and agriculture (Jansen and de Groot, 2004). Ses-
quiterpenoids, especially those with a drimane skeleton, are
natural, semisynthetic, or synthetic compounds possessing a
wide variety of biological activities, including antimalarial
K. Kuchkova ꢀ A. Aricu ꢀ E. Secara ꢀ A. Barba ꢀ P. Vlad ꢀ
N. Ungur
Institute of Chemistry of the Academy of Sciences of Moldova,
˘
Academy 3, MD-2028 Chis¸inau, Republic of Moldova
C. Tuchilus
‘‘Grigore T. Popa’’ University of Medicine and Pharmacy of Iasi,
Str. Universitatii 16, 700115 Iasi, Romania
S. Shova
‘‘P. Poni’’ Institute of Macromolecular Chemistry, Aleea Grigore
Ghica Voda 41-A, 700487 Iasi, Romania
´
(Lhinhatrakool et al., 2011; do Ceu de Madureira et al., 2002),
antidiabetic (Zhao et al., 2012; Miura et al., 2005), anti-
inflammatory (Owoyele et al., 2004; Ru¨ngeler et al., 1998),
antimicrobial (Komala et al., 2010), and anticancer (Komala
et al., 2010; Allouche et al., 2009) ones.
G. Zbancioc ꢀ I. I. Mangalagiu
Organic Chemistry Department, ‘‘Al. I. Cuza’’ University of Iasi,
Bd. Carol 11, 700506 Iasi, Romania
I. I. Mangalagiu (&)
The majority of pharmaceutical products that mimic
natural products with biological activity are heterocycles.
Diazines heterocycles (in particular, pyrimidine and
Department of Organic and Biochemistry, ‘‘Al. I. Cuza’’
University of Iasi, 11 Carol I, 700506 Iasi, Romania
e-mail: ionelm@chem.uaic.ro
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Med Chem Res (2014) 23:1559–1568
pyrazine) have a wide range of biological properties,
mixture of unsaturated isomers 3–5 in a ratio of 21:14:65
(from 1H-NMR spectral data); further epoxidation with
monoperftalic acid (MPFA) of 3–5 gave a mixture of
compounds 6–8. Acetate 6 was separated from this mixture
by flash chromatography. The saponification of 6 led to
D8,13-bicyclohomofarnezene-12-ol 9, which was subse-
quently oxidized with Jones reagent into the key interme-
diary, D8,13-bicyclohomofarnezenic acid 10.
including antibacterial and antifungal (Ungureanu et al.,
´
´
2006; Kratky et al., 2012), antituberculosis (Ballell et al.,
2007; Abdel-Aziz and Bdel-Rahman, 2010), anti-inflam-
matory (Amr et al., 2007; da Silva et al., 2010), anti-HIV
(Fujiwara et al., 2008; de Castro et al., 2011), cardiotonic
(Kostapanos et al., 2008; Masereel et al., 2003), anticancer
´
(Wagner et al., 2008; Racane et al., 2012), analgesic (Giles
et al., 2012; Ferreira and Kaiser, 2012) ones, etc.
Once farnezenic acid 10 was obtained, two strategies
were used to obtain HSDS derivatives.
As part of our ongoing research in the field of biologically
active sesquiterpenes ( Kuchkova et al., 2009, 2010, 2011;
Vlad et al., 1997) and diazines (Ungureanu et al., 2006; Luca
et al., 2010; Balan et al., 2009; Mangalagiu et al., 2001), we
decided to synthesize new homodrimane sesquiterpenoids
with a diazine skeleton (HSDS) in order to combine their
respective biological potentials, our attention being focused
on antibacterial and antifungal activities.
The former strategy (Method I, Scheme 2) involved the
N-acylation of aminodiazine with acyl chloride. Using this
method, the desired HSDS derivatives 13a–c were obtained
by treating D8,13-bicyclohomofarnezenic acid chloride 11
[generated in situ from the corresponding acid 10] with
aminopyrazine 12b, 2-aminopyrimidine 12c, and 4-ami-
nopyrimidine 12a.
As indicated in Scheme 2, in the case of aminopyrimi-
dines the N-acylation reactions occur in a different way
according to the relative position of the amino group on the
pyrimidine ring. In the case of 4-aminopyrimidine 12a the
mono-acyl amide 13a was obtained in good yield in the
N-acylation reaction. In the case of 2-aminopyrimidine
12c, besides the desired mono-N-acylation, which leads to
mono-acyl amide 13c, the bis-N-acylation reaction occurs
unexpectedly, leading to the bis-acyl amide 14 as a prin-
cipal product.
Results and discussion
In a preliminary communication (Kuchkova et al., 2013),
we designed an efficient route for the obtaining of HSDS.
The strategies adopted for the construction of novel ho-
modrimane sesquiterpenoids with diazine skeleton involve
several steps and two different methods of synthesis, having
D8,13-bicyclohomofarnezenic acid 10 as the key interme-
diary. The starting material for the D8,13-bicyclohomo-
farnezenic acid synthesis (Scheme 1) was commercially
available norambreinolide I, which was converted into bi-
cyclohomofarnezane-8a,12-diol monoacetate 2 in two steps
following the previously described procedure (Vlad et al.,
1997; Martres et al., 1993). The dehydration of 2 led to a
The latter strategy used (Method II, Scheme 2) had
several aims: to ascertain if the bis-N-acylation reaction of
2-aminopyrimidine could take place in any conditions, to
observe if the method could be applied to the other amin-
odiazines, and also to increase the reaction yields. For this
purpose, we perform the N-acylation of aminodiazines
O
OH
OAc
OH
OAc
O
c
a
d
b
OH
H
1
H
I
H
2
H
Δ7,8
Δ
Δ
3.
4.
5.
8,9
OAc
OAc
OAc
8,13
O
12
COOH
H
O
+
+
OH
11
16
13
H
7
1
9
6
f
H
8
H
2
3
10
8
7
H
6
5
4
15
e
H
H
9
10
14
(a) LiAlH4, Et2O, Δ , 2h (98%); (b) Ac2O, Py, 20°C, 2h (100%); (c) POCl3, Py, 0°C, 2h, 24°C, 3h, (96%); (d)
MPFA, Et2O, 0°C, 5h, (65%); (e) KOH, MeOH, 20°C, 2h (98%); (f) Jones reagent, (CH3)2CO, 20°C, 3h (98%).
Scheme 1 Reaction pathway for the preparation of D8,13-bicyclohomofarnezenic acid
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Med Chem Res (2014) 23:1559–1568
1561
19
18
20
20
N
N
19
18
N
O
O
17
O
12
R
17
11
11'
16'
9'
12'
N
11
N
N
+
N
12
O
16
16
H
H
1
13'
N
9
9
17
18
13
20
13
1'
13a R=
1
H
H
H
H
3'
15'
5'
7'
5
5
3
N
7
7
3
19
H
H
6
H
H
15
15
17
N
14
14'
14
20
13b R=
13c
14
13a,b
12a,b
12c
18
19
N
R-NH2
12a-c
COOH
COCl
(COCl)2
Method I
H
H
H
H
25
27
10
11
+
24
HN
R-NH2
12a-c
O
23
29
O
11
N
22
12
9
DCC
4-DMAP
17
18
16
1
Method II
13
12c
H
12a,b
20
5
3
7
6
H
15
14
13a,b
15
13c + 14 + 15
+ 15
Scheme 2 Reaction pathway for the preparation of HSDS
12a–c directly with D8,13-bicyclohomofarnezenic acid 10
[in classical conditions: dicyclocarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP)]. Again, besides the
desired HSDS derivatives 13a–c, in the case of 2-amino-
pyrimidine the bis-acyl amide 14 was obtained. In this
synthetic pathway, besides the desired amides, another
reaction product was coming up, N-D8,13-bicyclohomo-
farnezenoyl-N,N0-dicyclohexylurea 15, as an unwanted by-
product.
nitrogen atom in the neighboring area and, from sterically
point of view hydrogen bonds with nitrogen would not be
possible). Consequently, the second electrophilic attack of
the acyl group to the amide nitrogen will possibly be rather
smooth (Scheme 3).
The structures of all new compounds were proved by the
1
elemental and spectral analysis (IR, H-NMR, 13C NMR),
two-dimensional experiments 2D-COSY, 2D-HETCOR
(HMQC), long range 2D-HETCOR (HMBC), MS, and
finally, in the case of the bis-acyl amide 14, by the single
crystal X-ray structure determination (Fig. 1).
Having in view the above considerations, it is clear that
the N-acylation of aminodiazines with acyl chloride is
more efficient in terms of yields and selectivity than the
direct N-acylation with organic acids.
The compound crystallizes in the orthorhombic
˚
system, space group P212121 with a = 6.9890(6) A, b =
˚
˚
As far as, the bis-N-acylation reaction of 2-aminopy-
rimidine is concerned, a feasible explication could be
related to the placement of the amino group between the
two nitrogen atoms from the pyrimidine ring. We presume
that after the first acylation, the remaining hydrogen from
the amide nitrogen atom became very acidic as a result of
two factors: the powerful electron withdrawing effect
exerted by the two nitrogen atoms and a hydrogen bond
with the nonbonding electrons from one nitrogen pyrimi-
dine ring. In the case of 4-aminopyrimidine and 2-amino-
pyrazine these factors could not take place (there is only a
14.1283(7) A, c = 33.8288(17) A, a = b = c = 90.0°,
3
V = 3340.3(4) A , and Z = 4. Full information concerning
˚
X-ray structure could be found in the Cambridge Crystal-
lographic Data Centre, the CCDC-893824 structure.
Design and biological activity
Among many other biological activities, natural, semisyn-
thetic, or synthetic sesquiterpenoids were proved to have a
123

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Med Chem Res (2014) 23:1559–1568
Scheme 3 The proposed
reaction mechanism for the
obtaining of the bis-acyl amide
14
N
O
N
N
O
N
O
O
N
N
R
H
H
H
O
- H
H
H
H
14
H
13.c
O
Y-NH2
R
N
H
X
X
=
R
N
O
H
H
H
R
H
13.a. (pyrimidine)
13.b. (pyrazine)
Fig. 1 X-ray molecular structure of 14. Thermal ellipsoids are drawn at 40 % probability level
good antimicrobial activity (Komala et al., 2010). Recently,
we have reported successful results on the identification of
new antimicrobial (Ungureanu et al., 2006; Balan et al., 2009;
Mangalagiu et al., 2001) compounds which contain a diazine
skeleton as pharmacophoric moiety. As a consequence, in an
attempt to increase the potential of pharmacophoric models,
we decided to combine their respective biological potentials,
intending to obtain the compounds with better activity, well
123

Med Chem Res (2014) 23:1559–1568
1563
tolerated by the organism, having low or no toxicity, and with
minimal or no side effects (Scheme 4).
homodrimane sesquiterpenoids without a diazine skeleton
(15), have an activity against only two Gram-positive
bacteria (S. aureus and B. cereus). In the HSDS (13a–c, 14)
series, we could notice that pyrimidine derivatives (13a,
13c) have a better activity than pyrazine derivative (13b)
and that the mono-acyl amides (13a–c) have also a better
activity compared with the bis-acyl amide (14).
As shown in Table 1, no compounds have an antifungal
activity.
For this purpose, five of the newly synthesized com-
pounds were preliminary screened for their in vitro anti-
microbial activity against six different strains of Gram-
positive (Staphylococcus aureus ATCC 25923, Sarcina
lutea ATCC 9341, Bacillus cereus ATCC 14579, Bacillus
subtilis) and Gram-negative (Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853) bacteria,
respective to three fungal strains (Candida albicans ATCC
10231, Candida glabrata ATCC MYA 2950, Candida
sake). The preliminary screening was carried out by agar
diffusion assay (Villamizar et al., 2003), using nutrient
agar medium (Mueller–Hinton agar for antibacterial tests
and Sabouraud agar for antifungal tests). Ampicillin,
chloramphenicol, and nystatin were used as reference
drugs. Table 1 summarizes the antimicrobial activity of the
compounds and reference drugs. The results are expressed
as diameters of inhibition zones (mm).
Conclusion
In conclusion, we reported herein an efficient and general
method for the preparation of new homodrimane sesquit-
erpenoids with diazine skeleton. The reaction pathway
involves the direct N-acylation of diazines with homodri-
mane sesquiterpenoids bearing acyl chlorides or organic
acids functionality. A reliable explication and a feasible
reaction mechanism for the obtained compounds are pre-
sented. The structure of compounds was proven unambig-
uously by elemental and spectral analyses, in the case of
the bis-acyl amide 14, by a single crystal X-ray structure
determination. The in vitro antimicrobial activities of the
homodrimane sesquiterpenoids with and without diazine
skeleton have been evaluated. The tested compounds have
an excellent antibacterial activity against Gram-positive
strains S. aureus and B. cereus. SAR correlation reveals
that homodrimane sesquiterpenoids with diazine skeleton
As shown in Table 1, all the tested compounds have a
good antibacterial activity against Gram-positive strains S.
aureus and B. cereus. If we compare the activity of ho-
modrimane sesquiterpenoids with diazine skeleton (13a–
c) with that of homodrimane sesquiterpenoids without
diazine skeleton (15), we may notice a spectacular increase
of antibacterial activity of HSDS compounds, the influence
of a diazine skeleton being certain. While HSDS com-
pounds possess a good activity against five bacterial strains
(S. aureus, S. lutea, B. cereus, B. subtilis, E. coli), the
Scheme 4 Design in the class
of HSDS derivatives
N
X
X
O
N
X
X
COOH
N
H2N
H
X= C, N
H
H
H
Antimicrobial
H
Table 1 Antimicrobial activity of the tested compounds 7–9
Diameter of inhibition zone (mm)
S. aureus S. lutea B. cereus B. subtilis E. coli P. aeruginosa C. albicans C. glabrata C. sake
ATCC ATCC 27853 ATCC
ATCC
25923
ATCC
9341
ATCC
14579
ATCC
MYA 2950
25922
10231
13a
15
15
14
10
19
25
18
0
10
10
12
0
7
0
12
0
0
0
0
0
0
0
0
0
13b
13c
19
0
7
10
0
0
0
0
0
14
0
10
0
0
0
0
15
0
11
0
0
0
0
0
0
Ampicillin (25 lg/disk)
30
30
–
25
31
–
20
29
–
0
–
–
–
Chloramphenicol (30 lg/disk) 25
Nystatin (100 mg/disk)
28
–
19
–
–
–
–
–
29
26
31
123

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Med Chem Res (2014) 23:1559–1568
possess a better antibacterial activity compared with ho-
modrimane sesquiterpenoids without diazine skeleton.
Also, in the HSDS series, the pyrimidine derivatives have a
better activity than the pyrazine ones and, the mono-acyl
amide has a better activity than the bis-acyl amide. No
tested compounds have an antifungal activity.
acetate in petroleum ether afforded 3.21 g [65 % from
monoacetate (2)] of 12-acetoxy-D8,13-bicyclohomofarne-
zen-12-ol (6). A solution of KOH (3.37 g, 60 mmol) in
85 mL of methanol was added to acetate (6) (3.16 g,
11.3 mmol), and the reaction mixture was stirred at room
temperature for 2 h. Methanol was evaporated under the
reduced pressure to approximately 10, 100 mL of water
and 100 mL of ether were added to the reaction mixture,
shacked vigorously, and the organic layer separated. The
aqueous phase was extracted again with ether. The com-
bined ether extracts were washed with water, dried, and
Experimental
Melting points were determined on a Boetius heating stage
and are uncorrected. IR spectra were recorded on a Perkin-
Elmer Spectrum 100 FT-IR spectrometer. The 1H, 13C, and
15N NMR spectra were recorded in CDCl3 on Avance III
Bruker 400 spectrometer (400 and 100 MHz). Chemical
shifts are given on the d scale in ppm relative to CHCl3
resonances as an internal standard (d 7.24 and 77.00 ppm
for H and C, respectively) and TMS for 15N spectra. Res-
onances in 13C NMR spectra were assigned using DEPT,
evaporated to dryness to give 2.66 g (98 %) of D8,13
bicyclohomofarnezen-12-ol 9.
-
Preparation of D8,13-bicyclohomofarnezenic acid (10)
1.34 M solution of Jones reagent (5 mL) was added
dropwise to a stirred solution of alcohol 9 (1.0 g,
4.23 mmol) in 25 mL acetone at 20 °C. After being stirred
for 3 h at room temperature, water was added, and the
mixture was extracted with ether. The extract was washed
with water, dried, and evaporated to give a colorless low-
melting solid (1.04 g, 98 %), of D8,13-bicyclohomofarn-
ezenic acid, 10. Spectral data of alcohol 9 and acid 10
completely correspond to literature data (Villamizar et al.,
2003).
1
1H–1H COSY-45, H–13C HMQC, HMBC, NOESY pro-
grams and in 15N NMR spectra, a 1H–15N HMBC program.
The following abbreviations were used to designate
chemical shift multiplicities: s = singlet, d = doublet,
t = triplet, m = multiplet, br = broad. Chemical shifts are
given in ppm (d-scale), coupling constants (J) in Hz.
Optical rotations were determined on a Perkin-Elmer 241
polarimeter with a 1 dm microcell, using CHCl3 as solvent.
The X-ray analyses were recorded with an Agilent Xcali-
bur Eos diffractometer. CCDC-893824 contains supple-
mentary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html [or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK: fax: (internat.) ?44-1223/336-033; Email: depos-
it@ccdc.cam.ac.uk]. The course of reactions was moni-
tored by TLC on Silufol plates with the detection by I2
vapor. Column chromatography used L 100/400 lm silica
gel and petroleum ether (bp 30–60 °C). Ether extracts were
dried over anhydrous MgSO4.
Reaction of D8,13-bicyclohomofarnezenic acid (10)
and 2-aminopyrimidine (12c)
Method A
A solution of (COCl)2 (0.8 mL, 1.16 g, 9.17 mmol) in
2 mL of dry benzene was added to a solution of acid 10
(200 mg, 0.80 mmol) in 4 mL of dry benzene. Then the
reaction mixture was stirred at room temperature for 1 h
and was refluxed for 1 h. Benzene and an excess of
(COCl)2 were evaporated under the reduced pressure.
Dichloromethane (8 mL) and 2-aminopyrimidine 12c
(120 mg, 1.26 mmol) were added to the residue, and the
resulting mixture was refluxed with stirring for 15 h. A
precipitate was filtered off, washed with dichloromethane,
and the filtrate was concentrated to dryness. The residue
(279 mg) was dissolved in 3 mL of chloroform and chro-
matographed on silica gel (8.4 g). The elution with chlo-
Preparation of D8,13-bicyclohomofarnezen-12-ol (9)
A solution of MPFA (35 mL, 3.57 g, 19.6 mmol) in ether
was added dropwise at 0 °C to a solution of the mixture of
unsaturated isomers 3–5 (4.77 g, 17.1 mmol) [obtained
from monoacetate of diol (2) as described previously (Vlad
et al., 1997)] in 30 mL of ether, and the reaction mixture
was stirred at the same temperature for 5 h. The obtained
precipitate was filtered off and washed successively with
ether. The filtrate was washed with 10 % NaOH solution
and water, dried, and evaporated under the reduced pres-
sure. The crude product (4.86 g) was chromatographed
over silica gel (146 g), and the elution with 2 % ethyl
roform afforded gradually 120 mg (54 %) of 2-di-D8,13
-
bicyclohomofarnezenoylaminopyrimidine 14, 80 mg of the
two-component mixture, and 10 mg of amide 13c. This
mixture was rechromatographed on silica gel (4 g). The
elution with 20 % ether in petroleum ether gave 9 mg
(4.5 %) of the unreacted acid 10. Elution with 60 % ether
in petroleum ether afforded 32 mg of amide 13c. An
overall yield of amide 13c is 42 mg (16 %).
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Med Chem Res (2014) 23:1559–1568
1565
Method B
2-Bis-D8,13-bicyclohomofarnezenoylaminopyrimidine
(14) White crystals; mp 205–206 °C; [a]2D6 27.78° (c
0.33, CHCl3); IR (KBr): m/cm-1: 3088, 897 (semicyclic
methylene), 1704 (C=O, amide), 1644, 1567, 1460, 1410,
A solution of DCC (215 mg, 1.04 mmol), 4-DMAP
(100 mg, 0.82 mmol), 2-aminopyrimidine 12c (80 mg,
0.84 mmol), and acid 10 (100 mg, 0.40 mmol) in 4 mL of
dichloromethane was stirred at room temperature for 5 h
and refluxed with stirring for 15 h. A precipitate was fil-
tered off, washed with dichloromethane, and the filtrate
was evaporated under the reduced pressure. The residue
(440 mg) was dissolved in 5 mL of chloroform and chro-
matographed over silica gel (13.2 g). The elution with
chloroform gave first 85 mg of the mixture of aminopy-
rimidine 14 and urea 15. For the separation of these
compounds, the mixture was extracted with petroleum
ether. The undissolved precipitate of aminopyrimidine 14
(37 mg, 33 %) was filtered off and washed with petroleum
ether. The crystalline residue after the evaporation of the
filtrate was recrystallized from acetonitrile to give 40 mg
(22 %) of urea 15. The following elution with chloroform
afforded 75 mg of the mixture of amide 13c and DCC. This
mixture was extracted with ether. The undissolved pre-
cipitate of DCC (30 mg) was separated, and the ether fil-
trate was concentrated to dryness. The obtained crystalline
residue was recrystallized from acetonitrile to give 30 mg
(22 %) of amide 13c.
1
1162 (pyrimidine cycle); H NMR (400 MHz, CDCl3): d
8.87 (2H, d, J = 4.9 Hz, H-18, H-20), 7.34 (1H, t,
J = 4.9 Hz, H-19), 4.80 (2H, s, Ha-13, Ha-130), 4.52 (2H,
s, Hb-13, Hb-130), 2.72 (2H, dd, J = 16.9, 2.6 Hz, Ha-11,
Ha-110), 2.56 (2H, dd, 16.9, 10.2 Hz, Hb-11, Hb-110), 2.49
(2H, dd, J = 10.0, 2.0 Hz, H-9, H-90), 2.39 (2H, ddd,
J = 13.0, 4.0, 2.3 Hz, Ha-7, Ha-70), 2.10 (2H, td, J = 13.0,
5.0, Hz, Hb-7, Hb-70), 1.18 (2H, dd, J = 12.5, 2.5 Hz, H-5,
H-50), 1.80–1.03 (16H, m), 0.87 (6H, s, H-14, H-140), 0.79
(6H, s, H-15, H-150), 0.59 (6H, s, H-16, H-160); 13C NMR
(100 MHz, CDCl3): d 175.22 (s, C-12), 159.95 (s, C-17),
159.34 (d, C-18), 159.34 (d, C-20), 148.92 (s, C-8), 120.20
(d, C-19), 106.41 (t, C-13), 55.12 (d, C-5), 51.90 (d, C-9),
42.05 (t, C-3), 39.03 (t, C-1), 38.90 (s, C-10), 37.54 (t,
C-7), 34.38 (t, C-11), 33.56 (q, C-14), 33.49 (s, C-4), 23.97
(t, C-6), 21.73 (q, C-15), 19.25 (t, C-2), 14.63 (q, C-16);
15N NMR (40.5 MHz, CDCl3): d 293 (N of pyrimidine
cycle); EI-MS: 560 (3 %, M?), 328 (100 %, BP), 545
(2 %), 531 (1 %), 356 (6 %), 342 (2 %), 329 (18 %), 327
(39 %), 312 (13 %), 294 (3 %), 232 (16 %), 217 (2 %),
204 (2 %), 191 (13 %), 137 (14 %), 124 (6 %), 122 (6 %),
96 (14 %), 81 (4 %), 69 (4 %).
D8,13-Bicyclohomofarnezenic acid N-(pyrimidin-2-yl)amide
(13c) White crystals; mp: 149–150 °C; [a]2D6 -14.8° (c
0.6, CHCl3); IR (KBr): m/cm-1: 3262, 3189, 3111 (NH
amide), 3078, 892 (semicyclic methylene), 1725 (C=O,
amide), 1649, 1574, 1435, 1155 (pyrimidine cycle); 1H
NMR (400 MHz, CDCl3): dppm: 8.60 (2H, d, J = 8.0 Hz,
H-18, H-20), 8.51 (1H, br. s, NH), 6.99 (1H, t, J = 4.9 Hz,
H-19), 4.79 (1H, s, Ha-13), 4.54 (1H, s, Hb-13), 2.95 (1H,
dd, J = 16.6, 10.1 Hz, Ha-11), 2.79 (1H, dd, J = 16.6,
3.7 Hz, Hb-11), 2.56 (1H, dd, J = 10.1, 3.7 Hz, H-9), 2.41
(1H, ddd, J = 13.0, 4.0, 2.4 Hz, Ha-7), 2.15 (1H, td,
J = 13.0, 4.0 Hz, Hb-7), 1.26 (1H, dd, J = 12.6, 2.1 Hz,
H-5), 1.80–1.10 (8H, m), 0.90 (3H, s, H-14), 0.83 (3H, s,
H-15), 0.76 (3H, s, H-16); 13C NMR (100 MHz, CDCl3):
N-D8,13-Bicyclohomofarnezenoyl-N,N0-dicyclohexylurea
(15) White crystals; mp 166–167 °C; [a]2D6 11.9° (c 1.34,
CHCl3); IR (KBr): m/cm-1: 3266 (NH amide), 3074, 879
(semicyclic methylene), 1698 (C=O amide), 1658 (C=O
1
amide); H NMR (400 MHz, CDCl3): d 6.47 (1H, br. s,
NH), 4.75 (1H, s, Ha-13), 4.43 (1H, s, Hb-13), 4.02 (1H, m,
H-17), 3.72 (1H, m, H-24), 2.49 (3H, m, Ha-11, Hb-11,
H-9), 2.38 (1H, ddd, J = 12.9, 3.8, 2.2 Hz, Ha-7), 2.13
(1H, td, J = 12.9, 4.9 Hz, Hb-7), 2.05–1.05 (28H, m), 1.22
(1H, dd, J = 12.7, 2.1 Hz, H-5), 0.90 (3H, s, H-14), 0.82
(3H, s, H-15), 0.71 (3H, s, H-16); 13C NMR (100 MHz,
CDCl3): d 172.51 (s, C-12), 154.32 (s, C-23), 149.45 (s,
C-8), 105.97 (t, C-13), 55.15 (d, C-5), 52.09 (d, C-9), 49.86
(d, C-17), 49.13(d, C-24), 42.04 (t, C-3), 39.09 (t, C-1),
39.02 (s, C-10), 37.68 (t, C-7), 33.56 (q, C-14), 33.50 (s,
C-4), 32.80 (t, C-25 and C-29), 31.55 (t, C-11), 31.22 (t,
C-22), 30.69 (t, C-18), 26.25 (t, C-19 and C-21), 25.48 (t,
C-27), 25.40 (t, C-20), 24.73 (t, C-26 and C-28), 24.05 (t,
C-6), 21.73 (q, C-15), 19.29 (t, C-2), 14.81 (q, C-16); EI-
MS: 457 (38 %, M?), 332 (100 %, BP), 458 (9 %), 456
(24 %), 441 (27 %), 375 (4 %), 360 (13 %), 346 (3 %),
331 (25 %), 316 (40 %), 232 (28 %), 225 (17 %), 223
(8 %), 196 (11 %), 191 (14 %), 175 (3 %), 154 (4 %), 141
(12 %), 126 (8 %), 121 (2 %), 98 (16 %), 97 (6 %), 83
(36 %), 69 (8 %).
d
ppm: 172.64 (s, C-12), 158.32 (d, C-20), 158.32 (d, C-18),
157.70 (s, C-17), 149.27 (s, C-8), 116.14 (d, C-19), 106.51
(t, C-13), 55.21 (d, C-5), 52.27 (d, C-9), 42.12 (t, C-3),
39.24 (s, C-10), 39.09 (t, C-1), 37.67 (t, C-7), 33.57 (q,
C-14), 33.56 (t, C-11), 33.53 (s, C-4), 24.13 (t, C-6), 21.77
(q, C-15), 19.35 (t, C-2), 14.75 (q, C-16); 15N NMR
(40.5 MHz, CDCl3): d 263 (N of pyrimidine cycle), 146
(NH amide); EI-MS: 328 (48 %, M?), 96 (100 %, BP), 329
(7 %), 327 (79 %), 312 (22 %), 232 (57 %), 217 (14 %),
204 (12 %), 191 (82 %), 175 (6 %), 163 (7 %), 150
(11 %), 137 (58 %), 124 (14 %), 122 (16 %), 81 (8 %), 69
(8 %).
123

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Med Chem Res (2014) 23:1559–1568
Reaction of D8,13-bicyclohomofarnezenic acid (10)
and 4-aminopyrimidine (12a)
Method B
Amide (13b) (68 mg, 52 %) and urea (15) (31 mg, 17 %)
were prepared in the reaction of acid (10) (100 mg) and
aminopyrazine (12b) (80 mg), which was realized follow-
ing the procedure described for this reaction of acid (10)
with 2-aminopyrimidine (12c).
Method A
Following the same procedure described for the reaction of
acid (10) and 2-aminopyrimidine (12c), amide (13a)
(156 mg, 60 %) was synthesized from acid (10) (200 mg)
and amine (12a) (120 mg).
D8,13-Bicyclohomofarnezenic acid N-(pyrazinil-3-yl)amide
(13b) White crystals; mp: 265–266 °C; [a]2D7 -22.5° (c
0.87, CHCl3); IR (KBr): m/cm-1: 3253, 3094 (NH amide),
3043, 894 (semicyclic methylene), 1719 (C=O, amide),
Method B
1
Amide (13a) (73 mg, 53 %), and urea (15) (39 mg, 20 %)
were prepared in the reaction of acid (10) (105 mg) and
4-aminopyrimidine (12a) (86 mg), which was realized
following the procedure described for this reaction with
2-aminopyrimidine (12c).
1697, 1544, 1417 (pyrazine cycle); H NMR (400 MHz,
CDCl3): dppm: 9.53 (1H, s, H-18), 8.33 (1H, d, J = 4.0 Hz,
H-20), 8.22 (1H, d, J = 4.0 Hz, H-19), 8.06 (1H, br. s,
NH), 4.84 (1H, s, Ha-13), 4.56 (1H, s, Hb-13), 2.68 (1H, dd,
J = 21.0, 8.8 Hz, Ha-11), 2.50 (1H, dd, J = 21.0, 10.6 Hz,
Hb-11). 2.50 (1H, dd, J = 10.6, 8.8 Hz, H-9), 2.43 (1H,
ddd, J = 13.1, 4.2, 2.3 Hz, Ha-7), 2.16 (1H, td, J = 13.1,
5.2 Hz, Hb-7), 1.25 (1H, dd, J = 12.7, 2.6 Hz, H-5),
1.80–1.15 (8H, m), 0.90 (3H, s, H-14), 0.83 (3H, s, H-15),
D8,13-Bicyclohomofarnezenic acid N-(pyrimidin-4-yl)amide
(13a) White crystals; mp: 132–133 °C; [a]2D7 -32.7° (c
3.6, CHCl3); IR (KBr): m/cm-1: 3210, 3147 (NH amide),
3072, 993 (semicyclic methylene), 1708 (C=O amide),
1645, 1572, 1510, 1390, 1160 (pyrimidine cycle); 1H NMR
(400 MHz, CDCl3): dppm: 8.85 (1H, s, H-20), 8.61 (1H, d,
J = 4.0 Hz, H-18), 8.34 (1H, br. s, NH), 8.16 (1H, d,
J = 8.0, H-19), 4.83 (1H, s, Ha-13), 4.52 (1H, s, Hb-13),
2.66 (1H, dd, J = 22.5, 10.0 Hz, Ha-11), 2.48 (1H, dd,
J = 22.5, 10.3 Hz, Hb-11), 2.48 (1H, dd, J = 10.3,
10.0 Hz, H-9), 2.43 (1H, ddd, J = 13.0, 4.2, 2.3 Hz, Ha-7),
2.15 (1H, td, J = 13.0, 5.2, Hb-7), 1.85–1.1 (8H, m), 0.90
(3H, s, H-14), 0.82 (3H, s, H-15), 0.73 (3H, s, H-16); 13C
NMR (100 MHz, CDCl3): dppm: 172.49 (s, C-12), 158.40
(d, C-18), 158.19 (d, C-20), 157.03 (s, C-17), 148.91 (s,
C-8), 110.29 (d, C-19), 106.70 (t, C-13), 55.14 (d, C-5),
52.18 (d, C-9), 41.91 (t, C-3), 39.16 (s, C-10), 39.08 (t,
C-1), 37.55 (t, C-7), 33.87 (t, C-11), 33.55 (q, C-14), 33.53
(s, C-4), 23.98 (t, C-6), 21.71 (q, C-15), 19.24 (t, C-2),
14.65 (q, C-16); 15N NMR (40.5 MHz, CDCl3): d 280 and
260 (N(3) and N(1) of pyrimidine cycle), 142 (NH amide);
EI-MS: 328 (100 %, M?, BP), 329 (23 %), 327 (13 %),
312 (9 %), 232 (7 %), 217 (2 %), 204 (4 %), 191 (16 %),
175 (2 %), 163 (2 %), 150 (3 %), 137 (9 %), 124 (11 %),
122 (3 %), 96 (23 %), 95 (3 %), 81 (2 %), 69 (1 %).
0.74 (3H, s, H-16); 13C NMR (100 MHz, CDCl3): dppm
:
171.49 (s, C-12), 149.01 (s, C-8), 148.12 (s, C-17), 141.87
(d, C-19), 140.06 (d, C-20), 137.10 (d, C-18), 106.71 (t,
C-13), 55.14 (d, C-5), 52.31 (d, C-9), 41.93 (t, C-3), 39.19
(s, C-10), 39.06 (t, C-1), 37.59 (t, C-7), 33.56 (q, C-14),
33.56 (s, C-4), 33.47 (t, C-11), 24.00 (t, C-6), 21.71 (q,
C-15), 19.25 (t, C-2), 14.65 (q, C-16); 15N NMR
(40.5 MHz, CDCl3): d 338 and 294 (N(4) and N(1) of pyr-
azine cycle), 138 (NH amide); EI-MS: 328 (100 %, M?,
BP), 329 (12 %), 327 (97 %), 312 (28 %), 294 (4 %), 232
(66 %), 217 (17 %), 204 (17 %), 191 (70 %), 176 (4 %),
163 (5 %), 150 (18 %), 124 (20 %), 122 (22 %), 96
(58 %), 81 (12 %), 69 (9 %).
Biological activity
Agar diffusion assay
Antibacterial and antifungal activities of the compounds
were evaluated by agar diffusion assay (Reeves et al.,
1978). The microbial suspension (0.5 McFarland turbidity
standard) was mixed (1/10 ratio) with agar nutrient (Mu-
eller–Hinton agar for antibacterial tests, respectively Sab-
ouraud agar for antifungal tests). Sterile stainless steel
cylinders (5 mm internal diameter; 10 mm height) were
applied on the agar surface in Petri plates. Each compound
(10 mg/mL in dimethylsulfoxide; 0.1 mL) was added to
another cylinder. After 24 h of incubation at 30 °C for
bacteria and 48 h of incubation at 28 °C for fungi, the
diameter of inhibition zone (mm) was measured (Table 1).
Ampicillin (25 lg/disk), Chloramphenicol (30 lg/disk),
and Nystatin (100 lg/disk) were purchased from Himedia
Reaction of D8,13-bicyclohomofarnezenic acid (10)
and aminopyrazine (12b)
Method A
Amide (13b) (10 mg, 15 %) was obtained in the reaction of
acid (10) (50 mg) and aminopyrazine (12b) (30 mg),
which was carried out as described above for this reaction
with 2-aminopyrimidine (12c).
123

Med Chem Res (2014) 23:1559–1568
1567
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extra-lipid effects of rosuvastatin. J Cardiovasc Pharmacol Ther
13:157–174
(Mumbai, India) and were used as reference drugs for
antibacterial and antifungal assays. All the experiments
were performed in triplicate; the results were expressed as
diameter of inhibition zones (mm).
´
´
ˇ
´
Kratky M, Vinsova J, Buchta V (2012) In vitro antibacterial and
antifungal activity of salicylanilide pyrazine-2-carboxylates.
Med Chem 8:732–741
Kuchkova KI, Aricu AN, Vlad PF (2009) Synthesis of 11-aminodrim-
7-ene from drimenol. Chem Nat Compd 45:367–370
Acknowledgments The work was supported by the Grant Roma-
nia–Moldova 418/2010 (10.820.05.19.RoF), which is highly appre-
ciated by the authors.
Kuchkova KI, Aricu AN, Vlad PF, Deleanu C, Nicolescu A (2010)
Synthesis of N-containing drimane sesquiterpenoids from 11-di-
homodriman-8a-ol-12-one. Chem Nat Compd 46:539–544
Kuchkova KI, Aricu AN, Barba AN, Vlad PF, Lipkovskii J, Simonov
YS, Kravtov VK (2011) Synthesis of nitrogen-containing
drimane sesquiterpenoids from 11-dihomodrim-8(9)-en-12-one.
Chem Nat Compd 47:223–228
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