Synthesis and cytotoxic evaluation of new terpenylpurines

Article abstract of DOI:10.1039/c6ra24254e

Several new terpenylpurine derivatives were prepared through alkylation of different purines with halogenated reagents derived from natural terpenoids, commercially available or isolated from cones of C. sempervirens L. and further transformed into approp

Full text of DOI:10.1039/c6ra24254e

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Garcia, J. M. Miguel del Corral, M. Pérez, I. C. F. R. Ferreira, R. C. Calhelha, A. San Feliciano and A. Castro,
RSC Adv., 2016, DOI: 10.1039/C6RA24254E.
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Page 1 of 19
RSC Advances
Synthesis and cytotoxic evaluation of new terpenylpurines
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DOI: 10.1039/C6RA24254E
Elena Valles,a Pablo A. García,a José Mª Miguel del Corral,a Marta Pérez,a Isabel C.F.R. Ferreira,b,*
Ricardo C. Calhelha,b Arturo San Feliciano,a Mª Ángeles Castroa,*
a
Departamento de Ciencias Farmacéuticas, Sección de Química Farmacéutica, Facultad de Farmacia, CIETUS-
IBSAL. Universidad de Salamanca. Campus Miguel de Unamuno. E-37007-Salamanca. Spain.
b
Centro de Investigação de Montanha (CIMO), Escola Superior Agrária, Instituto Politécnico de Bragança, Campus de
Santa Apolónia, 1172, 5300-253 Bragança, Portugal.
*Corresponding authors:
Mª Ángeles Castro
Departamento de Química Farmacéutica. Facultad de Farmacia.
Campus Miguel de Unamuno. Universidad de Salamanca.
37007 Salamanca. SPAIN
Ph. +34 923 294528
Fax +34 923 294515
E-mail: macg@usal.es
Isabel C.F.R. Ferreira
Centro de Investigação de Montanha, ESA, Instituto Politécnico de Bragança.
Campus de Santa Apolónia, 1172, 5300-253 Bragança. PORTUGAL
Ph. +351 273303219
Fax +351 273325405
E-mail: iferreira@ipb.pt

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Page 2 of 19
Abstract.- Several new terpenylpurine derivatives were prepared through alkylation of different
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DOI: 10.1039/C6RA24254E
purines with halogenated reagents derived from natural terpenoids, commercially available or
isolated from cones of C. sempervirens L. and further transformed into appropriate alkylated agents.
Alkylation of the purines gave mixtures of 9- and 7- alkylpurines, being the 9-alkylpurines the
major regioisomers. The presence of the terpenyl residue induced cytotoxicity on simple purines
and, in general, that activity improved as the substituent was larger. The 7-diterpenyl-6-
chloropurine E-21b was the most cytotoxic in the series and it can be considered an analogue of the
marine natural compounds agelasines and agelasimines, which were taken as models for this work.
Key words.- Terpenoids; cupressic acid; purines; 9-terpenylpurines; 7-terpenylpurines;
cytotoxicity.

Page 3 of 19
RSC Advances
1. Introduction
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DOI: 10.1039/C6RA24254E
The purine heterocycle is one of the most widely distributed in Nature since it can be found in the
nucleic acids and in other important primary metabolites as NADP or ATP.1 Although unsubstituted
purine does not exist in Nature, there are a great number of purine derivatives, particularly adenine
derivatives, that are involved in numerous metabolic processes. Many structurally modified purine
nucleosides and nucleotides are biologically significant with activities ranging from antineoplastic
and antiviral to antihypertensive, antiasthmatic or antituberculosis among others2 and the scope of
therapeutic applications seems to be far from being completed.
Among the purine derivatives, nucleosides and nucleotides, either from natural or synthetic origin,
are the best known, but we have put our attention on other natural N-alkylpurines,3 in which the
alkyl chains can be found on any nitrogen atom at the purine moiety. Those alkyl chains can go
from a simple methyl group in xanthines to large diterpenoids in agelasines or asmarines.4
Some representative examples of natural N-alkylpurines are shown in Fig. 1 and usually those
natural products have served as models for the design and synthesis of a great number of derivatives
and analogues for which a variety of biological activities have been described.2,3
NH2
O
HN
N
N
N
N
7
9
N
N
N
N
N
O
N
OH
COOH
3
N
N
NH2
Caffeine
Discadenine
Eritadenine
OH
HOOC
N
HO
N
N
N
N
NH2
H
H
7
N
N
+
N
H
N
7
N
N
N
Cl-
N
N
OH
Agelasine A
Agelasimine A
Asmarine B
Figure 1. Chemical structure of several natural alkylpurines
In the course of our research towards new antitumour cytotoxics based on natural products, we were
particularly interested on those secondary metabolites of marine origin formed by a diterpenoid
attached to the 7-nitrogen atom of an adenine derivative as asmarines or agelasimines, some
examples are shown in Fig. 1.3 Those derivatives can be called terpene-adenine hybrids or
terpenylpurines. These compounds have attracted the scientific interest because of their biological
properties,3 such as cytotoxic, antimicrobial, antiprotozoal, antifungal, antifouling, etc.
Our interest in this type of compounds is related mainly with the potent cytotoxicity described for
some of them. Our research group has been involved for years in the design, synthesis and
biological evaluation of cytotoxic compounds related to natural products. We have obtained very

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interesting results on the chemomodulation of cytotoxicity in podophyllotoxin related lignans,5 and
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6
DOI: 10.1039/C6RA24254E
also in the chemoinduction of bioactivity on inactive terpenoids such as communic acids. In this
sense, we have synthesized a large number of derivatives, named terpenylquinones that showed
very interesting cytotoxicity.6 These terpenylquinones can be considered hybrids of a terpenoid rest
and a quinone moiety and can also be considered analogues of other cytotoxic marine natural
products such as avarone.7 Recently, we have also described the synthesis and cytotoxicity of a new
family of hybrids, lignopurines, between lignans and purines8 that revealed the importance of the
purine core on their cytotoxicity.
This background, and the fact that the natural alkylpurines are usually isolated in very small
quantities which limited their structure-activity relationship studies,3 prompted us to design and
prepare new terpenylpurine derivatives starting from natural monoterpenoids and diterpenoids,
either commercially available or isolated by us from their natural sources, and to evaluate the
influence of the terpenoid size on their cytotoxic properties.
2. Results and discussion
2.1. Chemistry
The N-alkylpurines described in this work have been prepared by the classical procedure of
alkylation of purines with alkyl halides.9 As starting materials for the synthesis of terpenyl bromides
we used the commercial monoterpenoid myrtenal and the diterpenoids trans-communic and
cupressic acids isolated from their natural sources. We also used other commercially available alkyl
halides such as 1-bromopentane, cynnamyl bromide, isoprenyl chloride and geranyl bromide.
Myrtenal was easily transformed into the myrtenyl bromide 1 through NaBH4 reduction followed
by substitution of the hydroxyl group with CBr4,10 in this way, compound 1 was obtained in 87%
overall yield from myrtenal (Scheme 1). Trans-communic and cupressic acids were isolated from
the acid fraction of the n-hexane extract of Cupressus sempervirens L. cones (Cupressaceae). Both
acids were quantitatively transformed into their corresponding methyl esters by treatment with
trimethylsilyldiazomethane11 and then transformed into the terpenyl bromides 3, 3’ and 4 as shown
in Scheme 1. Epoxidation of the trisubstituted double bond in methyl trans-communate, followed
by oxidation with periodic acid12 yielded the tetranorditerpenic aldehyde 2, which was transformed
into the bromide 3 by reduction and substitution as described for myrtenal. Bromide 3’ was
prepared following the same procedure, previous isomerization of the exocyclic double bond.6b
Diterpenyl bromide 4 was obtained in 77% yield by treatment of methyl cupressate with PBr3 at -35
ºC.13 Nucleophilic substitution and allylic isomerization took place at once and compound 4 was
obtained as an unresoluble mixture of the E and Z isomers in a 5:1 ratio that was used for the
alkylation step.

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10
Br
CHO
2
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DOI: 10.1039/C6RA24254E
1
1) NaBH4
2) CBr4
3
6
5
8
4
7
9
(-)-Myrtenal
1
17
11
20
13
16
CHO
15
1
17
1) MCPBA
2) HIO4
10
8
7
5
3
COOCH3
COOR
18
19
2
R
H
trans-Communic acid
CH3 Methyl trans-communate
1) NaBH4
2) CBr4
HI 57 %
C6H6
14
13
R
Br
11
4
5
12
4a
8a
6
1
COOCH3
10
COOCH3
R
9
3
2'
3'
CHO
1) NaBH4
2) CBr4
CH2Br
OH
12
14
11
16
15
Br
13
15
16
PBr3
COOR
COOCH3
4 (E/Z : 5/1)
R
H
Cupressic acid
CH3 Methyl cupressate
Scheme 1. Preparation of the bromo-derivatives,
used as electrophiles, from natural terpenoids.
The alkylation of purines was performed in DMF, using potassium carbonate or cessium carbonate
as a base14 (Scheme 2). In general, the reaction products were mixtures of alkylpurines, in which the
major regioisomer was the corresponding 9-alkylated product (isomer “a”). In most of the cases, the
minor regioisomer, 7-alkylated purine (isomer “b”), was also separated by chromatographic
procedures (Scheme 2, entries 1-9). When the diterpenyl bromide 4 was used (entries 16-19), not
only isomers “a” and “b” were detected but also the 3-alkylated products (isomers “c”) were
isolated and even the Z and E stereoisomers were also separated and characterized on the bases of

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the chemical shift (δ) observed in 13C NMR spectra for the C-16’ methyl group on the terpenyl
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DOI: 10.1039/C6RA24254E
moiety. In the Z isomers, this methyl signal appeared at ≈23 ppm whereas in the E isomers, δ was at
≈16 ppm.15 For the assignments of the NMR data in the synthesized alkylpurines, the purine
positions were numbered from 1 to 9, whereas prime numbers were used for the alkyl side chain,
keeping the terpenoid numbering system stated in Scheme 1: from 1’ to 10’ in those alkylpurines
derived from monoterpenes and from 1’ to 20’ in those derived from the diterpenes. In the case of
the terpenylpurines obtained from the tetranor-derivatives 3 and 3’, the numbering shown in
Scheme 1 with primes was used.
Scheme 2. Preparation of the alkylpurines 5-23 by treatment with alkyl halides.
R
X
X
X
X
1) K2CO3 (Cs2CO3)
2) R-Br(Cl)
7
9
5
1
N
N
7
N
N
N
N
N
N
N
N
8
+
+
2
N
9
N
H
N
N
N
4
N
3
X
H
R
R
isomer "c"
isomer "a"
isomer "b"
Purine
Cl
6-Chloropurine
6-Methoxypurine
Adenine
9-Alkyl purines
7-Alkyl purines
3-Alkyl purines
(major regioisomer)
(minor regioisomers)
OCH3
NH2
5-23
Entry
R-Br(Cl)
Starting Purine
Purine
6-Chloropurine
6-Methoxypurine
Products
5a + 5b
6a + 6b
7a + 7b
1
2
3
1'
Br
1'
Br
6-Chloropurine
8a + 8b
9a + 9b
4
1'
5
6
6-Chloropurine
6-Chloropurine
Cl
1'
10a + 10b
Br
7
Purine
11a
10'
8
9
6-Chloropurine
6-Methoxypurine
Adenine
12a + 12b
13a + 13b
14a
15a
16a
Br
1
10
11
12
13
14
Br
14'
Purine
6-Chloropurine
6-Methoxypurine
Adenine
17a
18a
COOCH3
3
Br
14'
15
6-Methoxypurine
19a
COOCH3
3’
Br
Purine
6-Chloropurine
6-Methoxypurine
(Z)-20a+(E)-20a+(Z)-20b+(E)-20b+(E)-20c
(E)-21a+(Z)-21b+(E)-21b+(E)-21c
(Z)-22a+(E)-22a+(Z)-22b+(E)-22b+(Z)-22c+(E)-22c
16
17
18
15'
COOCH3
19
Adenine
(Z)-23a+(E)-23a+(Z)-23c+(E)-23c
4
All the alkylpurines isolated are shown in Scheme 2. Some of the regioisomeric 7- and 9- pairs of
1
alkylated purines showed characteristic chemical shift differences in their H and 13C spectra as
those described for similar alkylpurines,16 however we had several compounds in which the

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differences were not so evident and so that, the position of the radical was unequivocal assigned by
View Article Online
DOI: 10.1039/C6RA24254E
two-dimensional HMBC correlations obtained for purines 6a, 7a, 7b, 13a, 13b, 17a, Z-20b, E-20b,
E-20c, E-21a, E-21b, E-21c, E-22a, E-22b, E-22c, Z-23a, E-23a, Z-23c and E-23c. Correlations
observed between 2-H, 8-H and the CH2 group of the side chain attached to N-7(N-9), with the
quaternary carbons C-4 (C-5) in the purine ring were the most determinant. NMR spectra and
1
13
complete H and C NMR data assignments are included in the ESI. As an example of the three
isolated regioisomers, representative correlations experimentally observed for 13a, 13b and E-22c
are shown in Figure 2.
OCH3
H
H
N
N
N
8
5
CH3O
10'
H
4
2
N
N
N
H
N
2
5
10'
H
4
8
H
N
H
H
13a
13b
OCH3
N
H
2
N
4
15'
5
N
8
H
N
H
H
COOCH3
E-22c
Figure 2. Selected long-range H/C
connectivities found for some regioisomers.
2.2. Cytotoxicity on tumour and normal cell lines
Most of the compounds prepared were evaluated in vitro, using the sulforhodamine B colorimetric
assay,17 to establish their cytotoxicity against the following human tumoural cell lines: NCI-H460
(non-small cell lung carcinoma), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma) and
MCF-7 (breast adenocarcinoma). The toxicity on non-tumour cell lines was also evaluated using a
porcine liver primary cell culture designated as PLP2. The results were expressed as GI50,
compound concentration in µM that inhibited 50% of the net cell growth and they are shown in
Table 1. Only those derivatives that showed GI50 values lower that 125 µM against one or more cell
lines were considered active. In general, those compounds with a GI50 value under 20 µM were
considered good cytotoxic, while values over 70 µM were considered slightly cytotoxic. The
starting purines (purine, 6-chloropurine, 6-methoxypurine and adenine) together with the anticancer
drugs 6-mercaptopurine and ellipticine were included in the assays as references.
As it can be seen in Table 1, it is possible to establish some general considerations of structure-
activity relationship. First, it could be said that the presence of alkyl groups in a purine system

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promotes cytotoxicity compared to simple analogues as 6-chloropurine and adenine, which were
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inactive in the tests, and even better than 6-mercaptopurine which was only active in DthOeI: 1H0.1e0p39G/C26RcAe24ll254E
line.
Table 1. Cytotoxicity data (GI50 in µM)a for the synthesized alkylpurines.
Compound
NCI-H460
HeLa
HepG2
MCF-7
PLP2
>125
5a
>125
>125
>125
>125
5b
>125
>125
>125
>125
>125
6a
6b
84.9±8.1
>125
65.8±6.8
>125
19.1±0.8
>125
90.3±0.9
>125
120.2±2.6
>125
8a
8b
9a
9b
10a
10b
11a
33.4±0.3
38.4±2.9
119±3
32.0± 0.3
22.5 ±1.9
90.6 ±9.2
40.0 ±3.9
28.9 ±1.0
29.1 ±0.7
90.8±6.2
>125
37.7±2.8
64.1±5.8
>125
15.3±1.4
60.3±0.8
>125
13.6±0.8
40.2±2.7
36.5±3.1
109±7
>125
44.5±3.2
>125
96±10
>125
>125
32.1±1.1
27.2±0.5
102±5
32.0±1.0
39.3±0.6
>125
70.8±3.7
75.2±5.1
>125
12b
>125
119±3
>125
>125
13b
15a
>125
>125
97.3±9.1
>125
>125
>125
>125
>125
>125
>125
17a
18a
61.1±2.9
>125
26.6±0.1
97.0±3.1
28.0±2.0
>125
38.9±3.8
3.98±0.41
7.58±0.89
24.4±2.0
3.06±0.42
>125
41.4±1.7
>125
>125
>125
80.8±6.4
>125
23.3±0.5
35.9±1.4
30.1±3.1
70.8±6.0
>125
>125
>125
>125
7.96±0.25
86.9±3.6
103±10
32.3±3.3
52.6±2.7
40.1±2.5
>125
61.4±0.1
12.8 ±1.3
3.30± 0.19
118±3
16.0 ±0.3
>125
31.8±2.0
>125
>125
100±8
64.1±6.1
>125
13.5±1.1
20.4±1.6
29.6±1.7
>125
>125
>125
>125
>125
21.7±0.9
>125
37.6±2.1
48.9±2.6
35.3±1.5
>125
33.8±0.9
11.2±0.3
11.0±0.4
>125
15.2±0.4
>125
43.4±2.5
>125
>125
20.2±1.5
105±4
82.4±6.1
76.2±5.0
34.7±2.7
32.7±0.8
37.4±0.5
>125
43.4±0.1
13.7±0.2
10.7±0.9
39.7±0.6
27.6±2.0
>125
35.5±1.8
>125
>125
72.0±7.4
>125
>125
>125
(E)-20a
(Z)-20a
(E)-20b
(Z)-20b
(E)-20c
(E)-21a
(E)-21b
(Z)-21b
(E)-21c
(Z)-22a
(E)-22a
(E)-22b
(Z)-22b
(E)-22c
(E/Z)-23a
(Z)-23a
(E/Z)-23b
(E)-23c
(Z)-23c
purine
chloropurine
methoxypurine
adenine
mercaptopurine
ellipticine
60.0±5.0
72.8±1.8
63.9±3.9
>125
106±6
30.2±2.0
55.9±1.0
>125
86.9±2.3
>125
51.5±2.7
>125
>125
>125
>125
>125
>125
>125
34.5±1.5
41.1±3.5
26.4±2.6
29.3±0.6
>125
>125
>125
28.2±1.9
13.1±0.5
34.9±2.5
34.1±1.8
35.4±0.4
58.2±1.1
>125
>125
>125
>125
3.69±0.16
79.1±1.6
70.9±6.6
68.2±2.9
>125
>125
>125
>125
>125
8.57±0.13
4.75±0.55
aGI50 values are expressed as mean ± standard deviation of two independent experiments, each carried out in duplicate.
The increasing of chain size led to a better cytotoxic activity. Thus, purines with linear and shorter
side chains were inactive (5a, 5b and 6b) or slightly active (6a, 9a, and 9b) against any of the four
tumour cell lines tested, only purines 6a and 9b showed an interesting cytotoxicity against HepG2

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(19.1 µM) and MCF-7 cells (13.6 µM) respectively. The presence of a phenyl group improved the
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DOI: 10.1039/C6RA24254E
cytotoxicity as happened with 8a and 8b, which had a cinnamyl residue and showed moderate
activity.
Among the monoterpenyl substituents, a linear geranyl substituent was better than the bulky pinene
moiety (10a, 10b vs 11a, 12b, 13b) with similar GI50 values for all the tumour cell lines and the
same applied to the evaluated tetranorditerpenyl derivatives 15a, 17a and 18a. It is interesting to
note the activity of the purine 8a against MCF-7 line (15.3 µM) and the purine 17a against HepG2
(21.7 µM) without showing toxicity in the primary line PLP2.
Purines with a diterpenoid moiety included the most cytotoxic derivatives obtained, although the
terpenoid is not as determinant as the substitution on the C-6 position of the purine ring: 6-
chloropurine derivatives (21) were most potent than purine (20), 6-methoxypurine (22) and adenine
(23) derivatives, independently if the terpenoid was at N-9, N-7 or N-3. Exceptionally some
differences were observed between several Z / E isomers at the diterpenoid moiety, being more
potent the E-isomers (E-20a, E-20b, E-21b and E-22a vs Z-20a, Z-20b, Z-21b and Z-22a).
The most cytotoxic of all compounds tested on the different tumour cell lines were
diterpenylpurines E-21a and E-21b (3.30-13.7 µM), being several times more potent against tumour
lines than against non-tumour line PLP2. Particularly, compound E-21a was the most potent against
NCI-H460 line (3.98 µM) and E-21b presented the best value cytotoxicity against HeLa cell line
(3.30 µM). Both compounds improved cytotoxicity values of ellipticine in NCI-H460 and HepG2
tumour lines and besides showed less toxicity to non-tumour cells.
3. Conclusions
As a conclusion, several new terpenylpurine derivatives were efficiently prepared through
alkylation of different purines with halogenated reagents derived from natural terpenoids,
commercially available or isolated from their natural sources. Thus, cupressic acid was easily
isolated in a good yield from cones of C. sempervirens L. and further transformed into appropriate
alkylated agents. Alkylation of the purines gave mixtures of 9- and 7-alkylpurines, being the 9-
alkylpurines the major regioisomers. Sometimes the 3-alkylpurine derivatives were also isolated in
low yield from the reaction product. The presence of the terpenyl residue induced cytotoxicity on
simple purines and, in general, that activity improved as the substituent was larger, like those
present in the marine terpenylpurines. Although more derivatives are necessary to obtain more
significant conclusions on structure-activity relationship, the fact that derivative E-21b was the
most cytotoxic in the series, encourage us to continuous with our research towards the selective
preparation of 7-alkylated purines, which could be considered analogues of agelasines and
agelasimines, which were the marine natural terpenylpurines taken as models for this work.

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View Article Online
DOI: 10.1039/C6RA24254E
4. Experimental section
4.1. Chemistry.
1H and 13C NMR experiments were recorded on a Bruker AC 200 (200 or 50.3 MHz, respectively)
or Bruker Avance 400DRX (400 or 100 MHz) spectrometers in CDCl3 or CD3OD using the residual
solvent signal as reference. Chemical shift (δ) values are expressed in ppm followed by multiplicity
1
and coupling constants (J) in Hz. Only representative chemical shift values of H NMR data are
1
described and assigned in this section for the new compounds and complete H and 13C NMR
assignments are included in the ESI.
Optical rotations were recorded on a Perkin-Elmer 241 polarimeter in CHCl3 solution and
[α]D values are given in 10-1 deg cm2 g−1. UV spectra were obtained on a Hitachi 100-60
spectrophotometer. IR spectra were obtained on a Nicolet Impact 410 spectrophotometer in NaCl
film. HRMS were run in a QSTAR XL Q-TOF (Applied Biosystems) using electrospray ionization
(ES) at 5500 V with an HPLC Agilent 1100 chromatograph. Solvents and reagents were purified by
standard procedures as necessary and the chlorinated solvents, including CDCl3, were filtered
through NaHCO3 prior its use, in order to eliminate acid traces. DMF was dried over molecular
sieves and treated with K2CO3 for the same reason. Column chromatography (CC) purifications
were performed using silica gel 60 (40-63 µm, 230-400 mesh, Merck).
4.1.1. Starting materials.
Myrtenal, 1-bromopentane, cynnamyl bromide, isoprenyl chloride and geranyl bromide were
commercially available and trans-communic and cupressic acids were isolated from cones of
Cupressus sempervirens L. (Cupressaceae) as follows. Dry cones (2.3 kg) were extracted with n-
hexane in a Soxhlet over 10 h. After cooling overnight, the insoluble part was separated, the solvent
was evaporated and the residue was redissolved in Et2O and extracted with 4% NaOH yielding an
acid part (7.4 g). CC of this fraction, using hexane/EtOAc 8:2 as eluent, afforded trans-communic
acid18 (4.8 g, 64%) and cupressic acid19 (1.5 g, 20%). Both acids were quantitatively transformed
into their methyl esters by treatment with trimethylsilyldiazomethane11 in C6H6/MeOH (1:1).
4.1.2. 10-Bromo-2-pinene 1. To a solution of myrtenal (500 mg, 3.3 mmol) in THF (8 mL) NaBH4
(400 mg, 10.5 mmol) was added and kept stirring at room temperature for 8 h. Then, the reaction
mixture was quenched with a saturated aqueous solution of NH4Cl and extracted with EtOAc. The
combined organic layers were washed with brine and dried over anhydrous Na2SO4. Removal of the
solvent gave a reaction product that was dissolved in CH2Cl2 (10 mL); then, CBr4 (1.58 g, 4.78
mmol) and PPh3 (1.25 g, 4.78 mmol) were added at 0 ºC and stirred at this temperature for 30 min.
The solvent was evaporated and filtered over celite and chromatographed on silica gel to give 1
(600 mg, 84%).

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4.1.3. Bromoderivative 3. Methyl trans-communate was transformed into the aldehyde 2 (55%) by
View Article Online
DOI: 10.1039/C6RA24254E
12
a described procedure . Treatment of 2 with NaBH4 first, and then with CBr4 and PPh3 following
the same procedure described above for 1 afforded 3 (60%).
4.1.4. Bromoderivative 3’. To a solution of 2 (50 mg, 0.18 mmol) in C6H6 (18 mL) was added HI
57% (0.40 mL, 0.18 mmol). The mixture was stirred at 80 ºC for 10 min. Then, EtOAc was added
and the organic layer was washed with aq satd NaHCO3, brine, dried over Na2SO4, filtered and
evaporated off yielding the isomerised product 2’ (35 mg, 70%). Treatment of 2’ with NaBH4 first,
and then with CBr4 and PPh3 following the same procedure described above for 1 afforded 3’
(50%).
4.1.5. Bromoderivative 4. To a solution of methyl cupressate (687 mg, 2.06 mmol) and pyridine
(166 µL, 2.06 mmol) in dry Et2O (10 mL) at -35 ºC, phosphorous tribromide in dry Et2O (5 mL)
was added dropwise and stirred under inert atmosphere at the same temperature for 1 h. The
reaction mixture was diluted with EtOAc and the organic layer was washed with 2 N HCl, aq satd
1
NaHCO3, brine, dried over Na2SO4, filtered and evaporated off to give 4 (634 mg, 77%). H NMR
(CDCl3) δ: 1.65 (3 H, s, 16’-H), 3.55 (3 H, s, 19’-OCH3), 3.95 (2 H, d, 8.4, 15’-H), 4.43 (1 H, s,
17’-Ha), 4.78 (1 H, s, 17’-Hb), 5.42 (1 H, t, 8.4, 14’-H).
4.1.6. General Method for the Synthesis of Alkylated Purines 5-23.
A solution of purine and K2CO3 or Cs2CO3 in dry DMF was stirred at room temperature for 15 min.
The alkylating agent was then added and the mixture stirred for a specified time and temperature
under inert atmosphere. The crude product was diluted with water, extracted with EtOAc, and
washed with sat aq NaCl, dried over Na2SO4 and filtered. The solvent was removed under vacuum
to give the alkylated purines 5-23.
9(7)-Pentylpurine 5. From purine (150 mg, 1.25 mmol), K2CO3 (173 mg, 1.25 mmol) and 1-
bromopentane (160 µL, 1.25 mmol) in dry DMF (3 mL), at 80 ºC for 24 h. The reaction product
was chromatographed on silica gel, eluting with CH2Cl2/EtOH 97:3 to give: (a) 5a (114 mg, 48%).
1H NMR (CDCl3) δ: 4.26 (2 H, t, 7.4, 1’-H), 8.07 (1 H, s, 8-H), 8.95 (1 H, s, 2-H), 9.11 (1 H, s, 6-
1
H). HRMS (ES, M+Na): m/z 213.1110 (calc. for C10H14N4Na: 213.1111). (b) 5b (16 mg, 7%). H
NMR (CDCl3) δ: 4.22 (2 H, t, 7.0, 1’-H), 8.16 (1 H, s, 8-H), 8.91 (1 H, s, 6-H), 9.09 (1 H, s, 2-H).
HRMS (ES, M+H): m/z 191.1287 (calc. for C10H15N4: 191.1291).
6-Chloro-9(7)-pentylpurine 6. From 6-chloropurine (150 mg, 0.97 mmol), Cs2CO3 (125 mg) and
1-bromopentane (100 µL, 0.81 mmol) in dry DMF (5 mL) at 100 ºC for 24 h. The reaction product
1
was chromatographed on silica gel (Et2O/acetone 95:5) to give: (a) 6a (80 mg, 44%). H NMR
(CDCl3) δ: 4.28 (2 H, t, 7.2, 1’-H), 8.11 (1 H, s, 8-H), 8.74 (1 H, s, 2-H). HRMS (ES, M+H): m/z
1
225.0897 (calc. for C10H14N4Cl: 225.0901). (b) 6b (17 mg, 9%). H NMR (CDCl3) δ: 4.46 (2 H, t,
7.2, 1’-H), 8.21 (1 H, s, 8-H), 8.88 (1 H, s, 2-H). HRMS (ES, M+H): m/z 225.0905 (calc. for

RSC Advances
Page 12 of 19
C10H14N4Cl: 225.0901).
View Article Online
6-Methoxy-9(7)-pentylpurine 7. From 6-methoxypurine (100 mg, 0.66 mmol), Cs2DCOIO: 103.1(01392/5C6RmA2g4)254E
and 1-bromopentane (100 µL, 0.80 mmol) in dry DMF (5 mL) at 100 ºC for 24 h. The reaction
1
product was chromatographed on silica gel to give: (a) 7a (Et2O/EtOAc 1:1) (75 mg, 52%). H
NMR (CDCl3) δ: 4.20 (2 H, t, 7.2, 1’-H), 4.15 (3 H, s, 6-OCH3), 7.87 (1 H, s, 8-H), 8.50 (1 H, s, 2-
H). HRMS (ES, M+Na): m/z 243.1217 (calc. for C11H16N4ONa: 243.1216). (b) 7b (Et2O/EtOAc
3:7) (15 mg, 10%). 1H NMR (CDCl3) δ: 4.30 (2 H, t, 7.6, 1’-H), 4.15 (3 H, s, 6-OCH3), 7.97 (1 H, s,
8-H), 8.62 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 243.1215 (calc. for C11H16N4ONa: 243.1216).
6-Chloro-9(7)-(3-phenyl-2-propenyl)purine 8. From 6-chloropurine (300 mg, 1.94 mmol), K2CO3
(268 mg, 1.94 mmol) and cynnamyl bromide (395 mg, 1.94 mmol) in dry DMF (32 mL) at 80 ºC
for 20 h. The reaction product was chromatographed on silica gel (CH2Cl2/EtOH 97:3) to give: (a)
8a (100 mg, 20%). 1H NMR (CD3OD) δ: 4.93 (2 H, d, 6.1, 1’-H), 8.43 (1 H, s, 2-H), 8.54 (1 H, s, 8-
1
H). HRMS (ES, M+H): m/z 271.0750 (calc. for C14H12N4Cl: 271.0745). (b) 8b (20 mg, 4%). H
NMR (CD3OD) δ: 5.22 (2 H, d, 4.6, 1’-H), 8.62 (1 H, s, 2-H), 8.67 (1 H, s, 8-H). HRMS (ES,
M+H): m/z 271.0741 (calc. for C14H12N4Cl: 271.0745).
6-Chloro-9(7)-(3-methyl-2-butenyl)purine 9. From 6-chloropurine (300 mg, 1.94 mmol), K2CO3
(268 mg, 1.94 mmol) and isoprenyl chloride (0.22 mL, 1.9 mmol) in dry DMF (32 mL) at 80 ºC for
20 h. The reaction product was chromatographed on silica gel (CH2Cl2/EtOH 95:5) to give: (a) 9a
(56 mg, 13%). 1H NMR (CD3OD) δ: 4.84 (2 H, d, 7.2, 1’-H), 8.42 (1 H, s, 2-H), 8.63 (1 H, s, 8-H).
1
HRMS (ES, M+H): m/z 223.0740 (calc. for C10H12N4Cl: 223.0745). (b) 9b (14 mg, 3%). H NMR
(CD3OD) δ: 5.13 (2 H, d, 6.7, 1’-H), 8.60 (1 H, s, 2-H), 8.70 (1 H, s, 8-H). HRMS (ES, M+H): m/z
223.0743 (calc. for C10H12N4Cl: 223.0745).
6-Chloro-9(7)-geranylpurine 10. From 6-chloropurine (200 mg, 1.29 mmol), K2CO3 (178 mg,
1.29 mmol) and geranyl bromide (0.26 mL, 1.3 mmol) in dry DMF (7 mL) at 80 ºC for 20 h. The
reaction product was chromatographed on silica gel (CH2Cl2/EtOH 98:2) to give: (a) 10a (157 mg,
1
42%). IR νmax/cm-1: 3050, 2973, 1592, 1563, 1490, 1440, 933, 856. H NMR (CD3OD) δ: 4.92 (2
H, d, 7.2, 1’-H), 8.48 (1 H, s, 8-H), 8.70 (1 H, s, 2-H). HRMS (ES, M+H): m/z 291.1378 (calc. for
C15H20N4Cl: 291.1371). (b) 10b (50 mg, 13%). IR νmax/cm-1: 3050, 2980, 1599, 1536, 1476, 1446,
1
973, 840. H NMR (CD3OD) δ: 5.18 (2 H, d, 6.8, 1’-H), 8.62 (1 H, s, 8-H), 8.77 (1 H, s, 2-H).
HRMS (ES, M+H): m/z 291.1369 (calc. for C15H20N4Cl: 291.1371).
9-(2-Pinen-10-yl)purine 11. From purine (61 mg, 0.51 mmol), K2CO3 (70 mg, 0.51 mmol) and 10-
bromo-2-pinene 1 (110 mg, 0.51 mmol) in dry DMF (15 mL) at 50 ºC for 5 h. The reaction product
was chromatographed on silica gel (hexane/acetone 1:1) to give 11a (45 mg, 36%). IR νmax/cm-1:
2985, 1595, 1578, 1501, 1407, 1347. 1H NMR (CDCl3) δ: 4.76 (2 H, ABq, 15.4, 10’-H), 8.09 (1 H,

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RSC Advances
s, 8-H), 9.00 (1 H, s, 2-H), 9.15 (1 H, s, 6-H). HRMS (ES, M+H): m/z 255.1607 (calc. for C15H19N4:
View Article Online
DOI: 10.1039/C6RA24254E
255.1604).
6-Chloro-9(7)-(2-pinen-10-yl)purine 12. From 6-chloropurine (65 mg, 0.42 mmol), K2CO3 (58
mg, 0.42 mmol) and 10-bromo-2-pinene 1 (90 mg, 0.42 mmol) in dry DMF (15 mL) at 50 ºC for 5
h. The reaction product was chromatographed on silica gel (hexane/acetone 7:3) to give: (a) 12a (40
mg, 33%). IR νmax/cm-1: 3060, 2983, 1591, 1557, 1495, 1333, 1185. 1H NMR (CDCl3) δ: 4.80 (2 H,
ABq, 15.0, 10’-H), 8.11 (1 H, s, 8-H), 8.76 (1 H, s, 2-H). HRMS (ES, M+H): m/z 289.1221 (calc.
for C15H18N4Cl: 289.1214). (b) 12b (20 mg, 17%). IR νmax/cm-1: 3080, 2987, 1599, 1537, 1472,
1
1365. H NMR (CDCl3) δ: 4.98 (2 H, ABq, 15.4, 10’-H), 8.21 (1 H, s, 8-H), 8.89 (1 H, s, 2-H).
HRMS (ES, M+H): m/z 289.1215 (calc. for C15H18N4Cl: 289.1214).
6-Methoxy-9(7)-(2-pinen-10-yl)purine 13. From 6-methoxypurine (49 mg, 0.32 mmol), K2CO3
(44 mg, 0.32 mmol) and 10-bromo-2-pinene 1 (70 mg, 0.32 mmol) in dry DMF (15 mL) at 50 ºC
for 5 h. The reaction product was chromatographed on silica gel to give: (a) 13a (hexane/acetone
1
8:2) (50 mg, 71%). IR νmax/cm-1: 3033, 2985, 1598, 1574, 1477, 1404, 1312. H NMR (CDCl3) δ:
4.19 (3 H, s, 6-OCH3), 4.73 (2 H, ABq, 15.2, 10’-H), 7.88 (1 H, s, 8-H), 8.54 (1 H, s, 2-H). (b) 13b
1
(hexane/acetone 6:4) (15 mg, 21%). IR νmax/cm-1: 3071, 2984, 1616, 1558, 1483, 1402, 1354. H
NMR (CDCl3) δ: 4.13 (3 H, s, 6-OCH3), 4.82 (2 H, ABq, 15.2, 10’-H), 7.98 (1 H, s, 8-H), 8.63 (1
H, s, 2-H). HRMS (ES, M+H): m/z 285.1718 (calc. for C16H21N4O: 285.1710).
9-(2-Pinen-10-yl)adenine 14. From adenine (57 mg, 0.42 mmol), K2CO3 (58 mg, 0.42 mmol) and
10-bromo-2-pinene 1 (90 mg, 0.42 mmol) in dry DMF (15 mL) at 50 ºC for 5 h. The reaction
product was chromatographed on reverse phase-silica gel (LiChroprep RP-18, 40-63 µm, Merck),
eluting with MeOH/H2O 8:2 to give 14a (25 mg, 22%). IR νmax/cm-1: 3308, 3140, 2985, 1676,
1
1654, 1623, 1560, 1480, 1418. H NMR (CD3OD) δ: 4.63 (2 H, s, 10’-H), 7.97 (1 H, s, 8-H), 8.10
(1 H, s, 2-H). HRMS (ES, M+H): m/z 270.1708 (calc. for C15H20N5: 270.1713).
Tetranorterpenylpurine 15. From purine (46 mg, 0.39 mmol), K2CO3 (43 mg, 0.39 mmol) and
terpenylbromide 3 (133 mg, 0.39 mmol) in dry DMF (15 mL) at reflux for 22 h. The reaction
product was chromatographed on silica gel, eluting with Et2O/acetone 8:2 to give 15a (53 mg,
1
35%). IR νmax/cm-1: 3077, 2873, 1723, 1683, 1595, 1503, 1407, 1095, 898. H NMR (CDCl3) δ:
4.21 (1 H, m, 14’-Ha), 4.41 (1 H, m, 14’-Hb), 8.03 (1 H, s, 8-H), 9.00 (1 H, s, 2-H), 9.15 (1 H, s, 6-
H). HRMS (ES, M+H): m/z 383.1448 (calc. for C22H31N4O2: 383.2441).
Tetranorterpenyl-6-chloropurine 16. From 6-chloropurine (70 mg, 0.46 mmol), K2CO3 (63 mg,
0.57 mmol) and terpenylbromide 3 (157 mg, 0.46 mmol) in dry DMF (15 mL) at reflux for 7 h. The
reaction product was chromatographed on silica gel, eluting with CH2Cl2/acetone 9:1 to give 16a
1
(18 mg, 10%). IR νmax/cm-1: 2850, 1724, 1698, 1592, 1508, 1409, 1225, 1153, 887. H NMR

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(CDCl3) δ: 4.26 (1 H, m, 14’-Ha), 4.41 (1 H, m, 14’-Hb), 8.06 (1 H, s, 8-H), 8.77 (1 H, s, 2-H).
View Article Online
Tetranorterpenyl-6-methoxypurine 17. From 6-methoxypurine (106 mg, 0.67 mmDoOl)I:,10K.102C39O/C63R(A9243254E
mg, 0.67 mmol) and terpenylbromide 3 (230 mg, 0.67 mmol) in dry DMF (15 mL) at reflux for 7 h.
The reaction product was chromatographed on silica gel, eluting with CH2Cl2/acetone 9:1 to give
17a (78 mg, 30%). IR νmax/cm-1: 2872, 1722, 1598, 1574, 1477, 1405, 1153, 887. 1H NMR (CDCl3)
δ: 4.18 (3 H, s, 6-OCH3), 4.12 (1 H, m, 14’-Ha), 4.36 (1 H, m, 14’-Hb), 7.82 (1 H, s, 8-H), 8.53 (1
H, s, 2-H). HRMS (ES, M+H): m/z 413.2540 (calc. for C23H33N4O3: 413.2547).
Tetranorterpenyladenine 18. From adenine (84 mg, 0.63 mmol), K2CO3 (86 mg, 0.63 mmol) and
terpenylbromide 3 (215 mg, 0.63 mmol) in dry DMF (15 mL) at reflux for 23 h. The reaction
product was chromatographed on silica gel, eluting with Et2O/EtOH 9:1 to give 18a (76 mg, 31%).
IR νmax/cm-1: 3317, 1722, 1650, 1597, 1416, 1151, 889. 1H NMR (CDCl3) δ: 4.13 (1 H, m, 14’-Ha),
4.32 (1 H, m, 14’-Hb), 7.74 (1 H, s, 8-H), 8.37 (1 H, s, 2-H). HRMS (ES, M+H): m/z 398.2547
(calc. for C22H32N5O2: 398.2550).
Tetranorterpenyl-6-methoxypurine 19. From 6-methoxypurine (30 mg, 0.22 mmol), K2CO3 (26
mg, 0.20 mmol) and terpenylbromide 3’ (67 mg, 0.20 mmol) in dry DMF (15 mL) at reflux for 18
h. The reaction product was chromatographed on silica gel, eluting with CH2Cl2/acetone 9:1 to give
19a (18 mg, 23%). IR νmax/cm-1: 2872, 1723, 1598, 1574, 1477, 1160. 1H NMR (CDCl3) δ: 4.18 (3
H, s, 6-OCH3), 4.18 (2 H, m, 14’-H), 7.88 (1 H, s, 8-H), 8.55 (1 H, s, 2-H). HRMS (ES, M+H): m/z
413.2544 (calc. for C23H33N4O3: 413.2547).
Diterpenylpurines 20. From purine (41 mg, 0.34 mmol), K2CO3 (47 mg, 0.34 mmol) and
terpenylbromide 4 (136 mg, 0.34 mmol) in dry DMF (10 mL) at 80 ºC for 20 h. The reaction
product was chromatographed on silica gel to give the following compounds:
(a) (Z)-20a (12 mg, 9%), (CH2Cl2/EtOH 97:3). [α]D22 +24.9 (c 0.43 in CHCl3). UV: λmax(EtOH)/nm
266 (ε/dm3 mol-1 cm-1 970). IR νmax/cm-1: 2928, 1723, 1643, 1597, 1451, 1127. 1H NMR (CDCl3) δ:
4.48 (2 H, d, 7.0, 15’-H), 8.09 (1 H, s, 8-H), 8.99 (1 H, s, 6-H), 9.02 (1 H, s, 2-H). HRMS (ES,
M+Na): m/z 459.2734 (calc. for C26H36N4O2Na: 459.2730).
1
(b) (E)-20a (16 mg, 12%), (CH2Cl2/EtOH 97:3). IR νmax/cm-1: 2925, 1723, 1595, 1453, 1123. H
NMR (CDCl3) δ: 4.85 (2 H, d, 7.0, 15’-H), 8.10 (1 H, s, 8-H), 9.00 (1 H, s, 6-H), 9.14 (1 H, s, 2-H).
HRMS (ES, M+Na): m/z 459.2734 (calc. for C26H36N4O2Na: 459.2730).
(c) (Z)-20b (7 mg, 6%), (CH2Cl2/EtOH 95:5). IR νmax/cm-1: 2924, 1723, 1601, 1451, 1418, 1045. 1H
NMR (CDCl3) δ: 4.75 (2 H, m, 15’-H), 8.18 (1 H, s, 8-H), 8.88 (1 H, s, 6-H), 9.10 (1 H, s, 2-H).
(d) (E)-20b (6 mg, 4%), (CH2Cl2/EtOH 95:5). IR νmax/cm-1: 3080, 2923, 1724, 1609, 1449, 1154,
880. 1H NMR (CDCl3) δ: 4.85 (2 H, d, 7.0, 15’-H), 8.22 (1 H, s, 8-H), 8.92 (1 H, s, 6-H), 9.13 (1 H,
s, 2-H).

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(e) (E)-20c (6 mg, 4%), (CH2Cl2/EtOH 95:5). IR νmax/cm-1: 3080, 2939, 1721, 1650, 1620, 1444,
View Article Online
1
DOI: 10.1039/C6RA24254E
1161, 880. H NMR (CDCl3) δ: 4.99 (2 H, d, 7.0, 15’-H), 8.64 (1 H, s, 2-H), 8.68 (1 H, s, 6-H), 8.77
(1 H, s, 8-H). HRMS (ES, M+Na): m/z 459.2752 (calc. for C26H36N4O2Na: 459.2730).
Diterpenyl-6-chloropurines 21. From 6-chloropurine (188 mg, 1.21 mmol), K2CO3 (168 mg, 1.21
mmol) and terpenylbromide 4 (481 mg, 1.21 mmol) in dry DMF (10 mL) at 80 ºC for 20 h. The
reaction product was chromatographed on silica gel (CH2Cl2/EtOH) to give the following
compounds:
(a) (E)-21a (65 mg, 11%), (CH2Cl2/EtOH 99:1). [α]D22 +25.1 (c 1.3 in CHCl3). UV λmax(EtOH)/nm:
266 (ε/dm3 mol-1 cm-1 2380). IR νmax/cm-1: 2924, 1724, 1633, 1420, 1066, 878. 1H NMR (CDCl3) δ:
4.87 (2 H, m, 15’-H), 8.10 (1 H, s, 8-H), 8.75 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 493.2644
(calc. for C26H35N4O2ClNa: 493.2341).
(b) (Z)-21b (7 mg, 1%), (CH2Cl2/EtOH 99:1). IR νmax/cm-1: 2924, 1725, 1640, 1153, 895. 1H NMR
(CDCl3) δ: 5.00 (2 H, d, 7.0, 15’-H), 8.22 (1 H, s, 8-H), 8.87 (1 H, s, 2-H). HRMS (ES, M+Na): m/z
493.2339 (calc. for C26H35N4O2ClNa: 493.2341).
(c) (E)-21b (10 mg, 2%), (CH2Cl2/EtOH 99:1). IR νmax/cm-1: 3081, 2935, 1722, 1642, 1597, 1448,
1155, 975, 889. 1H NMR (CDCl3) δ: 5.07 (2 H, d, 7.0, 15’-H), 8.24 (1 H, s, 8-H), 8.87 (1 H, s, 2-H).
HRMS (ES, M+Na): m/z 493.2334 (calc. for C26H36N4O2ClNa: 493.2341).
(d) (E)-21c (14 mg, 3%), (CH2Cl2/EtOH 97:3). [α]D22 +19.3 (c 0.51 in CHCl3). IR νmax/cm-1: 2925,
1
1722, 1597, 1420, 1114, 1049. H NMR (CDCl3) δ: 4.75 (2 H, d, 7.0, 15’-H), 7.71 (1 H, s, 8-H),
8.36 (1 H, s, 2-H).
Diterpenyl-6-methoxypurines 22. From 6-methoxypurine (101 mg, 0.68 mmol), K2CO3 (93 mg,
0.68 mmol) and terpenylbromide 4 (268 mg, 0.68 mmol) in dry DMF (10 mL) at 80 ºC for 20 h.
The reaction product was chromatographed on silica gel (CH2Cl2/EtOH) to give the following
compounds:
(a) (Z)-22a (6 mg, 2%), (CH2Cl2/EtOH 99:1). IR νmax/cm-1: 3080, 2933, 2851, 1722, 1620, 1453,
1155, 889. 1H NMR (CDCl3) δ: 4.18 (3 H, s, 6-OCH3), 4.75 (2 H, d, 7.0, 15’-H), 7.87 (1 H, s, 8-H),
8.54 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 489.2850 (calc. for C27H38N4O3Na: 489.2836).
(b) (E)-22a (23 mg, 7%), (CH2Cl2/EtOH 99:1). [α]D22 +33.7 (c 0.54 in CHCl3). IR νmax/cm-1: 3078,
2928, 2851, 1722, 1620, 1454, 1155, 889. 1H NMR (CDCl3) δ: 4.18 (3 H, s, 6-OCH3), 4.82 (2 H, m,
15’-H), 7.90 (1 H, s, 8-H), 8.55 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 489.2860 (calc. for
C27H38N4O3Na: 489.2836).
(c) (Z)-22b (3 mg, 1%), (CH2Cl2/EtOH 98:2). IR νmax/cm-1: 3080, 2939, 2850, 1723, 1643, 1596,
1471, 1156, 888. 1H NMR (CDCl3) δ: 4.16 (3 H, s, 6-OCH3), 4.93 (2 H, d, 7.0, 15’-H), 7.98 (1 H, s,
8-H), 8.63 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 489.2855 (calc. for C27H38N4O3Na: 489.2836).

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(d) (E)-22b (14 mg, 2%), (CH2Cl2/EtOH 97:3). [α]D22 +26.1 (c 0.39 in CHCl3). IR νmax/cm-1: 3081,
View Article Online
2932, 2850, 1722, 1641, 1597, 1473, 1156, 889. 1H NMR (CDCl3) δ: 4.16 (3 H, s, 6-DOOCI: 1H0.310),394/C.96R3A2(42254E
H, d, 7.0, 15’-H), 8.02 (1 H, s, 8-H), 8.64 (1 H, s, 2-H). HRMS (ES, M+Na): m/z 489.2844 (calc.
for C27H38N4O3Na: 489.2836).
1
(e) (Z)-22c (2 mg, 1%), (CH2Cl2/EtOH 95:5). H NMR (CDCl3) δ: 4.30 (3 H, s, 6-OCH3), 5.09 (2
H, d, 7.0, 15’-H), 8.18 (1 H, s, 2-H), 8.25 (1 H, s, 8-H). HRMS (ES, M+Na): m/z 489.2834 (calc.
for C27H38N4O3Na: 489.2836).
1
(f) (E)-22c (7 mg, 2%), (CH2Cl2/EtOH 95:5). H NMR (CDCl3) δ: 4.30 (3 H, s, 6-OCH3), 5.15 (2
H, m, 15’-H), 8.23 (1 H, s, 2-H), 8.26 (1 H, s, 8-H). HRMS (ES, M+Na): m/z 489.2840 (calc. for
C27H38N4O3Na: 489.2836).
Diterpenyladenines 23. From adenine (124 mg, 0.90 mmol), K2CO3 (122 mg, 0.90 mmol) and
terpenylbromide 4 (357 mg, 0.90 mmol) in dry DMF (15 mL) at 110 ºC for 22 h. The reaction
product was chromatographed on silica gel (CH2Cl2/EtOH) to give the following compounds:
(a) (Z)-23a (6 mg, 1%), (CH2Cl2/EtOH 95:5). IR νmax/cm-1: 3334, 3083, 2927, 1722, 1649, 1617,
1446, 1160, 848. 1H NMR (CDCl3) δ: 4.41 (2 H, d, 7.0, 15’-H), 7.77 (1 H, s, 8-H), 8.36 (1 H, s, 2-
H). HRMS (ES, M+H): m/z 452.3040 (calc. for C26H38N5O2: 452.3020).
(b) (E)-23a (17 mg, 4%), (CH2Cl2/EtOH 9:1). IR νmax/cm-1: 3357, 2923, 1724, 1595, 1451, 1119,
1
889. H NMR (CDCl3) δ: 4.77 (2 H, t, 7.0, 15’-H), 7.80 (1 H, s, 8-H), 8.36 (1 H, s, 2-H). HRMS
(ES, M+H): m/z 452.3032 (calc. for C26H38N5O2: 452.3020).
(c) (Z)-23c (11 mg, 2%), (CH2Cl2/EtOH 9:1). IR νmax/cm-1: 3372, 3086, 2927, 1723, 1642, 1613,
1453, 1116, 889. 1H NMR (CDCl3) δ: 4.94 (2 H, m, 15’-H), 8.00 (1 H, s, 2-H), 8.08 (1 H, s, 8-H).
HRMS (ES, M+H): m/z 452.3034 (calc. for C26H38N5O2: 452.3020).
(d) (E)-23c (3 mg, 0,5%), (CH2Cl2/EtOH 8:2). [α]D22 +24.9 (c 0.62 in CHCl3). IR νmax/cm-1: 3358,
3083, 2930, 1723, 1640, 1613, 1478, 1116, 889. 1H NMR (CDCl3) δ: 5.01 (2 H, t, 7.0, 15’-H), 8.03
(1 H, s, 2-H), 8.07 (1 H, s, 8-H). HRMS (ES, M+H): m/z 452.3029 (calc. for C26H38N5O2:
452.3020).
4.2. Cytotoxicity assays.
Four human tumour cell lines were used: NCI-H460 (non-small cell lung cancer), HeLa (cervical
carcinoma), HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) from DSMZ
(Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH).
Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10%
FBS and 2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, at 37 ºC, in a
humidified air incubator containing 5% CO2. The cell line was plated at an appropriate density (1.0

Page 17 of 19
RSC Advances
× 104 cells/well) in 96-well plates and allowed to attach for 24 h. Cells were then treated for 48 h
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17a
DOI: 10.1039/C6RA24254E
with the different diluted compound solutions. Afterwards, sulforhodamine B assay
was
performed as follows: cold trichloroacetic acid (TCA 10%, 100 µL) was used in order to bind the
adherent cells and further incubated for 60 min at 4 °C. After the incubation period, the plates were
washed with deionised water and dried and sulforhodamine B solution (SRB 0.1% in 1% acetic
acid, 100 µL) was then added to each plate well and incubated for 30 min at room temperature. The
plates were washed with acetic acid (1%) in order to remove the unbound SRB and then air dried;
the bound SRB was solubilised with Tris (10 mM, 200 µL) and the absorbance was measured at
540 nm using an ELX800 microplate reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA). The
results were expressed as GI50 values; compound concentration that inhibited 50% of the net cell
growth. Ellipticine was used as positive control.
For hepatotoxicity evaluation, a cell culture was prepared from a freshly harvested porcine liver
obtained from a local slaughterhouse, according to an established procedure17b and it was designed
as PLP2. Cultivation of the cells was continued with direct monitoring every two to three days
using a phase contrast microscope. Before confluence was reached, cells were subcultured and
plated in 96-well plates at a density of 1.0×104 cells/well, and cultivated in DMEM medium with
10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were treated with different
concentrations of the compounds and SRB assay was performed as previously described. The
results were expressed as GI50 values; sample concentration that inhibited 50% of the net cell
growth. Ellipticine was used as positive control.
For each compound, two independent experiments were performed, each one carried out in
duplicate. The results are expressed as mean values and standard deviation (SD).
Acknowledgments.
This work is supported by: Spanish Junta de Castilla y León co-funded by FSE (BIO/SA59/15,
SA028A10-2 and pre-doctoral fellowship to E. Valles), Spanish MINECO (CTQ2015-68175-R)
and Portuguese Foundation for Science and Technology (FCT) through CIMO (Pest-
OE/AGR/UI0690/2014) and R. C. Calhelha (SFRH/BPD/68344/2010) grant.
Supplementary data (ESI)
1
13
Complete H and C NMR data and assignments for the new compounds are included, together
with HMQC and HMBC spectra for several separated regioisomers.
References

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View Article Online
DOI: 10.1039/C6RA24254E
Table of contents
New 7/9-terpenylpurines were synthesized and evaluated as cytotoxics. Those derived
from the natural compounds trans.communic and cupressic acids, showed GI50 at the
µM level.
R
X
7
R
N
N
N
N
9
X
H
Cl
OCH3
NH2
COOCH3
COOCH3