Synthesis and in vitro activity of limonene derivatives against Leishmania and Trypanosoma

Article abstract of DOI:10.1016/j.ejmech.2009.12.061

The synthesis and in vitro activity of R(+)-Limonene derivatives against Leishmania and Trypanosoma cruzi strains are reported. Seven compounds have shown better in vitro activity against Leishmania (V.)braziliensis than the standard drug pentamidine. Add

Full text of DOI:10.1016/j.ejmech.2009.12.061

European Journal of Medicinal Chemistry 45 (2010) 1524–1528
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro activity of limonene derivatives against Leishmania
and Trypanosoma
Cedric S. Graebin a, Maria de F. Madeira b, Jenicer K.U. Yokoyama-Yasunaka c, Danilo C. Miguel c,
c
d
d
d
e
´
Silvia R.B. Uliana , Diego Benitez , Hugo Cerecetto , Mercedes Gonzalez , Ricardo Gomes da Rosa ,
,
Vera Lucia Eifler-Lima a
*
a
´
ˆ
´
Lab. de Sıntese Organica Medicinal (LaSOM), Faculdade de Farmacia, UFRGS, Avenida Ipiranga, 2752, sala 705, 90610-000, Porto Alegre, RS, Brazil
Serviço de Parasitologia, Instituto de Pesquisa Clınica Evandro Chagas, Fundaçao Oswaldo Cruz (Fiocruz), Rio de Janeiro/RJ, Brazil
Departamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo (USP), Sao Paulo/SP, Brazil
b
c
´
˜
ˆ
´
˜
˜
d
e
ˆ
´
´
Departamento de Qu´ımica Organica, Facultad de Quımica-Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay
´
˜
´
Lab. de Catalise por Metais de Transiçao, Instituto de Quımica, UFRGS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and in vitro activity of R(þ)-Limonene derivatives against Leishmania and Trypanosoma
cruzi strains are reported. Seven compounds have shown better in vitro activity against Leishmania
(V.)braziliensis than the standard drug pentamidine. Additionally, we have identified two promising new
anti-T. cruzi limonene derivatives.
Received 22 June 2009
Received in revised form
16 December 2009
Accepted 23 December 2009
Available online 14 January 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Limonene
Leishmania
Trypanosoma cruzi
Anti-parasitic activity
Hydroaminomethylation
1. Introduction
United States, being considered one of the most serious parasite
diseases in tropical regions [1,2]. There are currently 8 to 10 million
Parasitic diseases are the cause of much suffering and deaths
throughout the world, mainly in developing areas like Africa, Asia
and Latin America. In these regions the economic and social impact
caused by these illnesses are very high. Protozoan parasites of the
Trypanosoma and Leishmania genera are amongst the main
morbidity and mortality causes in Latin America. Leishmaniasis is
a parasitic disease affecting around 12 million people in 80 coun-
tries of the world, endangering about 350 million people living in
endemic areas, particularly in the African continent, India, Middle
East and Latin America [1]. Clinical manifestations include the
cutaneous, mucocutaneous and visceral forms, the latter leading to
a very high incidence of fatalities if the disease is left untreated. On
the other hand, Chagas’ disease, a chronic systemic parasitic
infection caused by the protozoan parasite Trypanosoma cruzi, is
endemic in South and Central America, Mexico and the southern
people infected and a further 28 million, mainly in Latin America,
are at risk of infection [3,4]. Typically, 30–40% of the chagasic
individuals show clinical symptoms of the chronic phase associated
with neuronal, cardiac and digestive dysfunctions, and at least
12,500 die every year [4,5].
In spite of their clinical importance, the therapeutic arsenal
available to treat these illnesses is deficient, with most of the
drugs presenting several side effects. Antimonial compounds
such as meglumine antimoniate or sodium stibogluconate are
still the first-choice drugs for Leishmaniasis. Second line drugs
include amphotericin B and pentamidine. All these drugs share
a high incidence of undesirable and potentially serious side
effects. For the treatment of Chagas’ disease only nifurtimox and
benznidazole, both developed about 30 years ago, are available.
Both of them are active against the acute stage of illness and
present strong side effects.
Unfortunately, all of these treatments have significant draw-
backs in terms of route of administration, length of treatment,
* Corresponding author. Tel.: þ55 51 3308 5362; fax: þ55 51 3308 5407.
E-mail address: veraeifler@ufrgs.br (V.L. Eifler-Lima).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.061

C.S. Graebin et al. / European Journal of Medicinal Chemistry 45 (2010) 1524–1528
1525
toxicity and cost, which limit their use in endemic areas. For all
2. Chemistry
these reasons, the search for new leishmanicidal and trypanoso-
micidal agents are a priority [6].
Using the same protocol from the previous report [18],
extending only the hydrogenation step time (24–60 h, depending
on the amine used as substrate) we were able to synthesize new
aromatic derivatives (9–14, see Scheme 1). For this purpose, we
have selected aniline derivatives, furfurylamine and 2-amino-
pyridine as amine substrates. All synthesized compounds
(including the previously synthesized ones 2–8) were obtained as
a 50:50 mixture of diastereomers, according to the performed GC-
FID and NMR analyses [18].
Ethanolamine derivative 16 was obtained by the use of a classic
reductive amination protocol, employing the aldehyde derivative
15, ethanolamine and NaCNBH3 as reducing agent. The sulphona-
mide 17 was obtained from a direct tosylation [19] of n-propyl
derivative 2 (Scheme 2).
Limonene (1, Scheme 1) is a monoterpene and a main constit-
uent of the essential oil of citrical plants, available in both enan-
tiomeric forms: R(þ) (1) and S(ꢀ)-limonene. This terpene has been
used as a building block for the organic synthesis of several
compounds [7–9] as well as a chiral auxiliary in asymmetric
syntheses [10]. It is well known that essential oils containing this
terpene can present anti-microbial [11], anti-fungal [12], anti-
malarial [13] and anti-tumoral action [14]. The action mechanisms
involved are unclear, but for P. falciparum it was associated with
inhibition of dolichol and ubiquinone biosynthesis and reduction in
protein isoprenylation [13]. Limonene has two double bounds and
one of them or even the two insaturations can be responsible for
the interaction with receptor. In a recent work, Arruda et al. [15]
showed that limonene was effective in in vitro and in vivo assays
against Leishmania species with an IC50 of 185
Ferrarini et al. have stereoselectively synthesized several limonene
-aminoalcohol derivatives at the cyclic double bond and evaluated
mM in the MTT test.
3. Results and discussion
b
3.1. L. (V.) braziliensis (promastigote) assay
its leishmanicidal activity against isolated parasites. They observed
that the inhibitory action against promastigotes is greater than of
the standard drug pentamidine, and two of the synthesized
compounds were found to be 100-fold more potent. In this test,
limonene has not shown any activity [16]. This same library showed
a good activity against both larvae and eggs of Rhipicephalus
(Boophilus) microplus [17], indicating the high potentiality of these
compounds. These results have encouraged new investigations
involving limonene derivatives with an amino group at isoprene
(exocyclic double bond) group. Our group has recently published
the synthesis of some limonene derivatives from one-pot tandem
hydroformylation/reductive amination (also known as hydro-
aminomethylation) [18]. The hydroaminomethylation is a versatile
reaction presenting a very high atom economy. In this process three
reactions (hydroformylation, imine/enamine formation and its
reduction) occur in a tandem form, where the reduction (hydro-
genation) step is catalyzed by the hydroformylation catalyst. In our
previous work [18] we have obtained the amine derivatives 2–8
(Scheme 1) in about 10–29 h of reaction time, depending of the
amine used as substrate. In this present work, we synthesized
seven new compounds in order to evaluate if the structural modi-
fications at isoprenyl chain contribute to the pharmacological
effect; so, we examined this series searching for anti-leishmania
and anti-trypanosoma activities.
In order to determine the leishmanicidal activity of the
synthesized compounds, they were tested against in vitro cultures
of the promastigotes of Leishmania (Viannia) braziliensis [20]
employing pentamidine as a standard drug. The results are gath-
ered in Table 1 and they show that, except for compound 5, the
introduction of amine groups in the limonene (1) scaffold have led
to more active compounds. Seven of the fifteen compounds (2, 7,10,
11, 14, 16 and 17) tested in this assay presented a lower IC50 value
than pentamidine. Compounds 3, 4 and 6 showed low activity
while 1 was found to be inactive against the parasite, as well as the
morpholine derivative 5.
Comparing 2 with 3, it seems that increasing the volume of the
alkyl group R decreases the activity of the compound. The good
activity of the aminoalcohol 16 indicates that polarity is not
a relevant factor to the activity of the compounds. Six-member
cycles like the ones in 5 (morpholine) and 6 (piperazine) are not
good for the activity of the compounds, probably due to the same
steric factors affecting the activity of 3.
As we have previously observed for aminoalcohols limonene
derivatives [16], in the present work the aromatic derivative 7 is the
most active compound of the series. Variations at the benzene ring
of this compound have lead to 8, 9, 10, 12, 13 and 14 with lower
activities. The more bulky benzyl derivative 4 is about 22 fold less
Scheme 1. Conditions: (i) HRhCO(PPh3)3 (cat.), CO (20 bar), H2 (20 bar), amine substrate, THF, 100 ꢁC, 5–24 h, then H2 (40 bar), 5–48 h; (ii) HRhCO(PPh3)3 (cat.), CO (20 bar), H2
(20 bar), THF, 100 ꢁC, 5 h; (iii) ethanolamine (4 equiv.), AcOH (cat.), CH2Cl2, r. t., 5 h, then NaCNBH3 (3 equiv), 24 h, r. t.

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C.S. Graebin et al. / European Journal of Medicinal Chemistry 45 (2010) 1524–1528
Table 1 (continued)
Product
R
IC50 (m
M)a
10
11
28.0 ꢂ 1.2
35.6 ꢂ 1.6
Scheme 2. Synthesis of derivative 17.
active than 7, indicating that the volume of the compounds is
important to the activity, although replacing the phenyl ring of 4 for
a furane ring in 11 results in a more active compound. The sul-
phonamide 17 seems to be an exception to this volume/activity
‘‘rule’’, presenting an activity similar to 7.
12
84.6 ꢂ 2.6
13
14
54.5 ꢂ 14.8
Table 1
L. braziliensis IC50 of the synthesized products 1–14, 16 and 17.
27.0 ꢂ 2.9
23.2 ꢂ 1.9
16
17
Product
R
–
IC50 (m
M)a
12.1 ꢂ 0.3
1 (limonene)
876.2 ꢂ 216
Pentamidine
–
48.5 ꢂ 28.7
a
IC50 Values are means ꢂ standard deviation of three experiments.
2
3
17.2 ꢂ 0.9
3.2. L. amazonensis and cytotoxicity assay
The four most active compounds against L. braziliensis pro-
mastigotes (2, 7, 16 and 17) were also tested against L. amazonensis
promastigotes and their cytotoxicity was assayed against the Rhe-
sus monkey kidney cell line (LLCMK2) [21]. These results are
detailed in Table 2 and show that derivative 2 was the most active
compound against L. amazonensis. Additionally, all of the deriva-
tives studied presented low toxicity against LLCMK2 cell line (at
least 3 times greater IC50 values for mammalian cells compared to
L. amazonensis, see selectivity indexes in Table 2) and 10–11 times
greater than L. (V.) braziliensis IC50 values. When compared to the
anti-T. cruzi activity, the selectivity indexes, specially for derivative
7 (SI ¼ 10.4) were also high.
253.6 ꢂ 33.9
257.9 ꢂ 22.5
4
5
6
7
1027 ꢂ 138.6
269.5 ꢂ 38.9
11.5 ꢂ 0.8
3.3. Anti-Trypanosoma cruzi assay
Selected compounds, 2, 4–7, 11, 13 and 16, were also tested
against in vitro cultures of T. cruzi epimastigotes (Tulahuen 2 strain)
[22]. The results (Table 3) show that, compounds 6 and 7 are
excellent hits as anti-T. cruzi agents for further structural
Table 2
In vitro L. amazonensis (promastigote form) and LLCMK2 IC50 assay results.
Product
L. amazonensis
IC50 48 h ( M)
LLCMK2
IC50 48 h (mM)
SIa
m
8
9
58.4 ꢂ 5.1
57.6 ꢂ 7.3
2
7
16
17
53.54 ꢂ 6.88
89.75 ꢂ 22.32
173.97 ꢂ 33.02
195.75 ꢂ 51.73
192.06 ꢂ 72.99
312.43 ꢂ 134.98
586.86 ꢂ 341.84
3.6
3.5 (10.4b)
3.4
>1012
>5.2
a
SI: selectivity index: IC50,LLCMK2/IC50,L. amazonensis
b
IC50,LLCMK2/IC50,T. cruzi
.

C.S. Graebin et al. / European Journal of Medicinal Chemistry 45 (2010) 1524–1528
1527
Table 3
In vitro activity of limonene derivatives against T. cruzi Tulahuen 2 strain.
5.2. Synthetic protocols
,
b
GI (%)a
IC50 (m
M)b
The procedure for the synthesis of compounds 2–7 was reported
on ref. 18. Protocols for compounds 8–17 are reported below.
Product
1 (limonene)
2
4
5
6
7
11
13
0.0
21.0
53.5
20.7
82.4
70.5
51.4
0.0
41.8
>50.0
>50.0
50.0
>50.0
19.2
29.5
50.0
>50.0
>50.0
7.7
5.2.1. General method for the synthesis of amines 8–14
A stainless steel autoclave reactor was charged with a mixture of
limonene 1 (1.0 mL, 1.19 g, 8.73 mmol), HRh(CO)(PPh3)3 (11.48 mg,
0.0125 mmol, 0.143 mol%),THF (10 mL) and amine substrate under
argon atmosphere. The reactor was purged and charged with 20 bar
of H2 and 20 bar of CO (CAUTION! Carbon monoxide is a very toxic gas
and its handling must be done in a well-ventilated hood) and heated
over a heating plate with magnetic stirring and a silicon oil bath at
100 ꢁC for 5 h. After this time the reactor was cooled, depressurized,
purged and pressurized with 40 bar of H2, and heated in the same
conditions as above for some time (required time for each amine is
detailed below). The reactor was cooled, depressurized and the
reaction mixture was eluted in a tiny silica-gel column for the
remotion of the catalyst. The solvent and the lightweight compo-
nents of the mixture (limonene, isomerizated products) were
removed under reduced pressure.
16
Nifurtimox
>100.0
a
Growth inhibition (%) of the given parasite at 50
Values are means ꢂ standard deviation of three experiments.
m
M.
b
modifications, showing IC50 values in the order of the standard
drug Nifurtimox. Contrasting to the anti-L. braziliensis assay find-
ings, compounds 2 and 11–16 are inactive against T. cruzi, being 6
the most active of them. In a structural point of view, chemical
changes on the parent compound conducted to active compounds
(comparing activities of parent compound 1 with the synthesized
derivatives). Unfortunately, we were unable to establish a struc-
ture–activity relationship with this library.
5.2.2. 4-Chloro-N-[3-(4-methylcyclohexen-3-yl)-butyl]-aniline 8
Amine substrate: 4-chloroaniline (1.114 g, 8.73 mmol). Hydroge-
nation step time: 20 h. The product was isolated by frac. distillation.
Yield: 23%. Mass spectrometry: 277.20 (Mþ, 8.27). 1H NMR (300 MHz,
CDCl3): 0.9 (3H, m),1.3 (4H, m),1.65 (3H, s),1.75 (3H, m),1.98 (3H, m),
3.08 (2H), 5.38 (1H, s), 6.49 (2H, d, J ¼ 8.6 Hz), 7.09 (2H, d, J ¼ 8.6 Hz).
13C NMR (75 MHz, CDCl3): 15.7, 16.2, 23.4, 25.2, 26.9, 27.3, 29.2, 30.7,
30.8, 35.0, 35.1, 38.1, 38.3, 42.8, 113.6, 120.7, 120.8, 128.9, 133.9, 147.
4. Conclusion
In conclusion, we have synthesized seven new compounds
with good in vitro activity against L. (V.) braziliensis, in an easy, fast
and scalable one-pot three step synthetic protocol, using a natural
and easily available starting material. The four most active
compounds in this assay also presented activity against the pro-
mastigote form of L. amazonensis and low toxicity against LLCMK2
cell line. Moreover, new anti-T. cruzi limonene derivatives with
adequate selectivity indexes (compounds 6 and 7) have been
identified.
5.2.3. 4-Methoxy-N-[3-(4-methylcyclohexen-3-yl)-butyl]-aniline 9
Amine substrate: anisidine (1.183 g, 9.6 mmol). Hydrogenation
step time: 24 h. The product was isolated by frac. distillation followed
by acid-base extraction. Yield: 24%. Mass spectrometry: 273.40 (Mþ,
9.12).1H NMR: (300 MHz, CDCl3): 0.9 (3H, m),1.3 (4H, m),1.65 (3H, s),
1.75 (3H),1.98 (3H, m), 3.08 (2H, m), 3.65 (3H, s), 5.38 (1H, s), 6.5 (2H,
d, J ¼ 8.8 Hz), 6.73 (2H, d, J ¼ 8.8 Hz). 13C NMR: (75 MHz, CDCl3): 15.7,
16.2, 23.4, 25.2, 26.9, 27.3, 29.2, 30.7, 30.8, 35.0, 35.1, 38.1, 38.3, 42.8,
48.0, 114.9, 115.0, 115.5, 115.8, 119.8, 134.0, 142.8, 152.0.
New assays aiming at elucidating these compounds anti-para-
sitic mechanisms of action are being prepared and will be reported
elsewhere.
5. Experimental protocols
5.2.4. 4-Methyl-N-[3-(4-methylcyclohexen-3-yl)-butyl]-aniline 10
Amine substrate: 4 toluidine (1.029 g, 9.6 mmol). Hydrogenation
step time: 48 h. The product was isolated by frac. dstillation. Yield:
70%. Mass spectrometry: 257.30 (Mþ, 13.48). 1H NMR: (300 MHz,
CDCl3): 0.9 (3H, m), 1.3 (4H, m), 1.65 (3H, s), 1.75 (3H, m), 1.98 (3H,
m), 2.18 (3H, s), 2.42 (1H, s), 3.08 (2H, m), 5.39 (1H, s), 6.45 (2H, d,
J ¼ 7.0 Hz), 6.9 (2H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3): 15.7,
16.2, 20.3, 23.4, 25.2, 26.9, 27.3, 29.2, 30.7, 30.8, 35.1, 35.2, 38.1, 38.3,
42.8, 113.0, 121.0, 126.0, 129.8, 134.0, 146.4.
5.1. General experimental procedures
1H and 13C NMR spectra were obtained in a INOVA-300 spec-
trophotometer with standard pulse sequences operating at
300 MHz in 1H NMR and 75 MHz in 13C NMR, using CDCl3 as
solvent. Chemical shifts are reported as
d values (ppm) relative to
TMS (0.0 ppm). Gas chromatography was performed in a Shimadzu
model GC-17A instrument with FID detector and equipped with
a DB-5 (30 m ꢃ 0.25 mm) column. The carrier gas was hydrogen
with a flux of 1.1 mL/min. The following method was used: sample
5.2.5. 3-(4-Methylcyclohexen-3-yl)-N-furfuryl-1-butanamine 11
Amine substrate: furfurylamine (0.9 mL, 9.6 mmol). Hydroge-
nation step time: 22 h. The product was isolated by frac. distillation.
Yield: 75%. Mass spectrometry: 247.25 (Mþ, 6.28). 1H NMR
(300 MHz, CDCl3): 0.9 (3H, m), 1.3 (4H, m), 1.65 (3H, s), 1.75 (3H, m),
1.98 (3H, m), 2.55 (3H, s), 3.7 (2H, s), 5.39 (1H, s), 6.1 (1H, s), 6.23
(1H, s), 7.25 (1H, s). 13C NMR (75 MHz, CDCl3): 15.7, 16.2, 23.4, 25.2,
26.9, 27.3, 29.2, 30.7, 30.8, 35.1, 35.2, 38.1, 38.3, 46.0, 47.5, 108.5,
110.0, 121.0, 134.0, 141.7, 154.1.
injection ¼ 0.5
m
L; initial column temperature ¼ 50 ꢁC; heating
rate ¼ 10 ꢁC/min; final temperature ¼ 250 ꢁC; final temperature
hold ¼ 10 min (total method time ¼ 30 min). Mass spectrometry
was performed in a Shimadzu model CGMS-QP5050 with resolu-
tion range from 45 to 400 Da, in SCAN mode (70 eV), coupled with
a Shimadzu model GC-17A gas chromatograph equipped with a DB-
17MS (30 m ꢃ 0.25 mm) column. The carrier gas was Helium with
a flux of 1.4 mL/min. The method was the same as detailed in the
gas chromatography. The mass spectrometry results are reported as
the m/z ratio with the respective relative abundance in parenthesis.
The Mþ symbol indicates that the peak is the molecular ion of the
compound.
5.2.6. 3-Trifluoromethyl-N-[3-(4-methylcyclohexen-3-yl)-butyl]-
aniline 12
Amine substrate: 3-trifluoromethylaniline (1.2 mL, 9.6 mmol).
Hydrogenation step time: 48 h. The product was isolated by frac.

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C.S. Graebin et al. / European Journal of Medicinal Chemistry 45 (2010) 1524–1528
distillation. Yield: 21%. Mass spectrometry: 311.20 (Mþ, 7.75). 1H
NMR (300 MHz, CDCl3): 0.9 (3H, m), 1.3 (4H, m), 1.65 (3H, s), 1.75
(3H, m), 1.98 (3H, m), 3.05 (2H, m), 3.65 (1H, s), 5.38 (1H, s), 6.65
(1H, m), 6.81 (2H, m), 7.18 (1H, m). 13C NMR: (75 MHz, CDCl3): 15.7,
16.2, 23.4, 25.2, 26.9, 27.3, 29.2, 30.7, 30.8, 35.0, 35.1, 38.1, 38.3, 42.0,
108.5, 113.5, 115.8, 120.8, 120.9, 129.9, 132.0, 134.0, 148.5.
filtered and the solvent was removed under reduced pressure. The
product was isolated by frac. distillation. Yield: 44%. 1H NMR
(300 MHz, CDCl3): 0.85 (3H, m), 0.92 (3H, t, J ¼ 7.48 Hz), 1.35 (4H,
m), 1.5 (4H, m, J ¼ 7.5 Hz), 1.65 (3H, s), 1.95 (4H, s), 2.4 (3H, s), 3.1
(4H, m), 5.38 (1H, s), 7.3 (2H, m), 7.7 (2H, m). 13C NMR (75 MHz,
CDCl3): 11.2, 15.7, 16.1, 21.4, 21.9, 23.4, 25.3, 26.9, 27.4, 29.2, 30.7,
30.8, 34.8, 35.0, 38.1, 38.3, 46.6, 49.8, 120.8, 127.1, 129.5, 134.0, 137.1,
142.0.
5.2.7. N-methyl-N-[3-(4-methylcyclohexen-3-yl)-butyl]-aniline 13
Amine substrate: N-methylaniline (1.05 mL, 9.6 mmol). Hydro-
genation step time: 22 h. The product was isolated by frac. distil-
lation. Yield: 40%. Mass spectrometry: 257.35 (Mþ, 13.67). 1H NMR:
(300 MHz, CDCl3): 0.9 (3H, m), 1.3 (4H, m), 1.65 (3H, s), 1.75 (3H, m),
1.98 (3H, m), 2.85 (3H, s), 3.15 (2H, m), 5.38 (1H, s), 6.6 (3H, m), 7.15
(2H, m). 13C NMR: (75 MHz, CDCl3): 15.7, 16.2, 23.4, 25.2, 26.9, 27.3,
29.2, 30.7, 30.8, 35.0, 35.1, 38.1, 38.3, 39.0, 51.5, 112.0, 115.7, 120.9,
129.0, 134.0, 149.2.
Acknowledgements
C. S. G. would like to thank CAPES/MEC for scholarship, V. L.
Eifler-Lima to CNPq, MCT/CNPq 02/2006 – Universal (Proc. 483674/
2006-0) for funding; R. G. da Rosa thanks CNPq, MCT/CNPq
´
14/2008 – Universal and INCT-Catalise for financial support; S.
Uliana, thanks CNPq and FAPESP.
5.2.8. 2-{N-[3-(4-methylcyclohexen-3-yl)-butyl]}-aminopyridine 14
Amine substrate: 2-aminopyridine (0.904 g, 9.6 mmol). Hydro-
genation step time: 24 h. The product was isolated by frac. distil-
lation. Yield: 24%. Mass spectrometry: 244.30 (Mþ, 24.52). 1H NMR
(300 MHz, CDCl3): 0.9 (3H, m), 1.3 (4H, m), 1.65 (3H, s), 1.75 (3H, m),
1.98 (3H, m), 3.2 (2H, m), 4.45 (1H, s), 5.38 (1H, s), 6.4 (2H, m), 7.35
(1H, m), 8.0 (1H, m). 13C NMR (75 MHz, CDCl3, ppm): 15.7, 16.2,
23.4, 25.2, 26.9, 27.3, 29.2, 30.7, 30.8, 35.0, 35.1, 38.1, 38.3, 40.5,
106.2, 112.3, 121.0, 134.0, 137.7, 150.0, 158.8.
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5.2.9. 2-{[3-(4-Methylcyclohexen-3-yl)-butyl]-amino}-ethanol 16
A two-neck rounded flask was charged, under argon, with
aldehyde 15 (300 mg, 1.804 mmol), ethanolamine (0.17 mL,
2.71 mmol), one drop of conc. sulfuric acid and 5 mL of ethanol. The
flask was kept at room temperature, under vigorous stirring, for 4 h,
when sodium cyanoborohydride (340 mg, 5.412 mmol) was added.
The stirring was maintained for more 22 h. The solvent was
removed under reduced pressure and the crude reside was filtered
and washed with cold Et2O. The filtrate was dried over sodium
sulfate and the solvent was removed under reduced pressure. The
product was isolated by frac. distillation. Yield: 57%. Mass spec-
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1.3 (4H, m), 1.65 (3H, s), 1.75 (3H, m), 1.98 (3H, m), 3.05 (4H, m), 3.8
(2H, m), 4.9 (2H, s), 5.38 (1H, s). 13C NMR: (75 MHz, CDCl3): 15.7,
16.2, 23.4, 25.2, 26.9, 27.3, 29.2, 30.7, 30.8, 35.0, 35.1, 38.1, 38.3, 47.0,
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5.2.10. N-propyl-N-[3-(4-methylcyclohexen-3-yl)-butyl]-4-
metylbenzene-1-sulphonamide 17
A two-neck rounded flask was charged, under argon, with 3 mL
of CH2Cl2, 150 mg of amine 2 (0.717 mmol) and Et3N (146 mg,
1.434 mmol) in an ice bath, when p-toluenesulphonyl chloride
(302 mg, 1.58 mmol) was added in small portions, over a 10 min
time span. Thirty minutes later, the ice bath was removed and the
reaction was carried for additional 4 h. After this time the solvent
was removed in a rotatory evaporator and the crude residue was
dissolved in ethyl acetate and washed with 12 portions of water
(10 mL each). The organic layer was dried over sodium sulfate,
´
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