Design, synthesis, binding, and molecular modeling studies of new potent ligands of cannabinoid receptors

Article abstract of DOI:10.1016/j.bmc.2007.05.060

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel cannabinoid receptor ligands derived from olivetol and hexyl-resorcinol, we have designed a structural model for new derivatives on the basis of a previous study. He

Full text of DOI:10.1016/j.bmc.2007.05.060

Bioorganic & Medicinal Chemistry 15 (2007) 5406–5416
Design, synthesis, binding, and molecular modeling studies
of new potent ligands of cannabinoid receptors
Antonella Brizzi,a,* Maria Grazia Cascio,b,c Vittorio Brizzi,a Tiziana Bisogno,b
Maria Teresa Dinatolo,a Adriano Martinelli,d
Tiziano Tuccinardid and Vincenzo Di Marzob
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via A. Moro 2, 53100 Siena, Italy
bEndocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche,
Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
c
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy
d
`
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy
Received 7 February 2007; revised 24 May 2007; accepted 25 May 2007
Available online 31 May 2007
Abstract—In our ongoing program aimed at the design, synthesis, and biological evaluation of novel cannabinoid receptor ligands
derived from olivetol and hexyl-resorcinol, we have designed a structural model for new derivatives on the basis of a previous study.
Here we report the synthesis, binding, and molecular modeling studies of new potent compounds with high affinity toward CB1 and
CB2 receptors. Compounds with amidic ‘heads’ with alkyloxy chains varying in length from 8 to 12 carbon atoms showed nanomo-
lar affinity for both receptors, depending on the type of aromatic backbone. Two of the new compounds, although not very potent,
exhibit selectivity for CB1 receptors (CB1/CB2 = 0.07 and 0.08, respectively). Molecular modeling studies fitted this new class of can-
nabinoid ligands into a CB1 receptor model, and the qualitative analysis of the results was in general agreement with the CB1 affinity
constants observed experimentally for these derivatives.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
is, the endocannabinoids anandamide (AEA)7 and
2-arachidonoylglycerol (2-AG) (Chart 1),8–10 as well as
the pharmacological characterization of the mechanisms
responsible for endocannabinoid cellular uptake and
inactivation, that is, the putative anandamide membrane
transporter (AMT)11 and the fatty acid amide hydrolase
(FAAH)12 or the monoacylglycerol lipase, in the case of
2-AG, offer areas for the development of new therapeu-
tic interventions.13 Despite their different chemical struc-
tures, THC, a very rigid molecule, and AEA, a very
flexible14 and easily oxidized compound, have several
common pharmacological properties. Bearing in mind
the pharmacophore requirements15 of both AEA and
THC, necessary for the binding to cannabinoid CB1
and CB2 receptors, in a previous paper16 we have
described a series of metabolically stable and potent
ligands of cannabinoid receptors, which consist of both
a rigid aromatic portion, as in THC, and a flexible
chain, as in AEA. Furthermore, our recent studies17
indicate that our compounds act as partial CB1 agonists
and CB2 neutral antagonists. The binding assay results
of those derivatives, which can be considered as a new
In the last few years the therapeutic potential of Canna-
bis sativa L. for the treatment of many pathologies has
been more and more appreciated.1 Its most important
component, (ꢀ)-trans-D9-tetrahydrocannabinol (THC,
Chart 1), mainly exerts its pharmacological properties
by binding to two G-protein-coupled receptors, CB1,2
expressed at high levels in several brain areas but also
present in peripheral tissues,3 and CB2, predominantly
found in the periphery,4 mainly in the immune system
(spleen, tonsils, immune cells), but recently also in the
brain.5,6
The discovery and characterization of CB1 and CB2
receptors and of their major endogenous ligands, that
Keywords: Anandamide; Endocannabinoids; Cannabinoid receptors;
Docking.
*
Corresponding author. Tel.: +39 577 234327; fax: +39 577 234333;
e-mail: brizzi3@unisi.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.060

A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
5407
O
1'
8
5
OH
N
H
11
6
2'
9
11
14
20
OH
1
AEA
7
OH
OH
3
O
O
4
1'
5'
5
O
THC
2-AG
Chart 1.
class of compounds with a great affinity for cannabinoid
receptors,18 supplied us with useful information on their
structure–activity relationships. The most important
critical points for the observation of high affinity ap-
peared to be: (a) the presence of a free phenolic hydroxyl
group; (b) the presence of an aliphatic chain on the aro-
matic ring and its length; (c) the length of the alkyloxy
chain carrying the amidic ‘head’; (d) the nature of the
amidic ‘head’ (Fig. 1). Thanks to the information gained
in our previous study it has now been possible to design
a basic structure for the development of new compounds
of this class with high affinity for cannabinoid receptors.
atoms. Moreover, for the amidic ‘head’, besides ethanol-
amine and cyclopropylamine, three new amines, that is,
methylcyclopropylamine,
3-hydroxytyramine
(3,4-
dihydroxyphenethylamine), and 3-methoxy-4-hydroxy-
benzylamine, were introduced (Fig. 2).
Finally a molecular modeling and docking study was
carried out in order to explain the structure–activity
relationships experimentally observed. For this purpose
a previously developed model of the CB1 receptor in its
activated form19 was used. A series of compounds were
docked into this receptor model and the observed li-
gand–receptor interactions were discussed also taking
into account the site-directed mutagenesis data available
for this receptor subtype.
With the aim to investigate these features in more detail,
in the present study we have designed and synthesized 21
new derivatives which maintained a free phenolic
hydroxyl group and a linear aliphatic chain of five or
six carbon atoms in the C portion, but with some mod-
ifications in the A and B portions (Fig. 1). In our previ-
ous study,16 we found that the length of the alkyloxy
chain plays a very important role in the affinity of this
class of compounds for cannabinoid receptors: in fact,
the 15-carbon atom derivatives were inactive and the
majority of the five-carbon atom derivatives were also
inactive, with some exceptions. Therefore, the length
of the alkyloxy chain was kept at 8, 11, and 12 carbon
2. Chemistry
As shown in Scheme 1, the synthesis of the new com-
pounds starts from olivetol or 4-hexylresorcinol, which
are reacted with a bromoalkylmethylester (8-bromo-
hexanoic acid methylester or 11-bromo-undecanoic acid
methylester or 12-bromo-dodecanoic acid methylester)
in dry acetone in presence of anhydrous K2CO3 and
KF, yielding esters 1–9. The final amides are obtained
by either of two simple methods. Method A: esters were
refluxed with methanolic/aqueous sodium hydroxide
solution to give the corresponding acids, which were
used as such and reacted with the amines in presence
of 1-hydroxybenzotriazole (HOBt) and 1-cyclohexyl-3-
(2-morpholinoethyl)carbodiimide methyl-p-toluenesulf-
onate (CMC) in dichloromethane or acetonitrile.20
Method B: esters were warmed up with ethanolamine
as solvent.
a free phenolic group
OH
A: the nature of
the amidic "head"
C: an aliphatic chain on
the aromatic backbone
O(CH2)nCONHR
R'
B: the length of the alkyloxy chain
The O-alkylation of phenols afforded all possible alkyl-
ated products at the two free hydroxyl groups; more-
over, O-alkylation of 4-hexylresorcinol afforded two
Figure 1. Proposed pharmacophoric model of this new class of
cannabinoid receptor ligands.
OH
n = 10;
n = 7, 11;
R = nC5H11, nC6H13
;
R = nC5H11, nC6H13
;
R1 = methylcyclopropyl, 3,4-dihydroxyphenethyl,
3-methoxy-4-hydroxy-benzyl.
R1 = CH2CH2OH, cyclopropyl.
R
O(CH2)nCONHR1
Figure 2. Structures of the synthesized compounds 10–30.

5408
A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
OH
OH
OH
ii; iii
i
R
R
R
O(CH2)nCONHR1
O(CH2)nCOOCH3
OH
10-30
1-9
Scheme 1. Reagents and conditions: (i) Br-(CH2)nCOOCH3, acetone, K2CO3, KF, reflux, 48–72 h; (ii) method A: MeOH/NaOH, reflux, 3 h; amine,
HOBt, CMC, rt, overnight; (iii) method B: H2NCH2CH2OH, 120–130 °C, 5 h.
different monoalkylated regioisomers. The reaction oc-
curred mainly on the hydroxyl in C-1, the less hindered
position (Scheme 2), irrespectively of the bromomethy-
lester used, and independently from the length of its
chain. As described in our previous paper,16 the struc-
ture of two regioisomers has been definitively assigned
by NOESY experiments.
duced from [14C]anandamide hydrolysis was measured
by scintillation counting of the aqueous phase after
extraction of the incubation mixture with 2 volumes of
CHCl3/CH3OH (2:1 by volume). In most cases, only
the most potent compounds in the binding assays were
subjected to this assay.
4. Results and discussion
3. Pharmacological evaluation
3.1. Binding assays
All the newly synthesized compounds that exhibited
IC50 6 10 lM in the preliminary screening for cannabi-
noid receptor binding activity were evaluated in radioli-
gand binding assays for affinity at recombinant human
CB1, CB2 over-expressed in COS cells, or for their inhib-
itory action on FAAH. The results are summarized in
Table 1. Analysis of the binding assay results of the
compounds described here allows us to assess with more
accuracy the structure–activity relationships in this class
of compounds, summarized as follows: (a) in accordance
with data from our previous study16 and from the liter-
ature23 cyclopropylamides (11, 18, 23, 25, 30) were more
potent than the respective ethanolamides (10, 17, 22, 24,
29) in the binding assays (with the exception of com-
pound 15 that was more potent than 16); (b) the amidic
‘head’ must be of little steric hindrance. Amine size in-
crease always causes a decrease (13, 14, 20, 21) or the
loss (27 and 28) of receptor affinity; (c) the length of
the alkyloxy chain carrying the amidic ‘head’ can vary
from 8 to 12 carbon atoms, leading to compounds with
nanomolar affinity, depending on the type of aromatic
backbone (11, n = 7; 15, n = 11; 23, n = 11; 25, n = 7),
even though the best ligand was still CB 25 (n = 10);
(d) two compounds (17 and 24), although not very po-
tent in the binding assays, show selectivity for CB1
receptors (CB1/CB2 = 0.07 and 0.08, respectively).
Worth of note is that these compounds are the two
regioisomers of 4-hexlresorcinol with n = 7, and both
are ethanolamides; (e) 4-hexylresorcinol derivatives with
alkylic chain ortho to the hydroxyl group (24–30) show
generally less receptor affinity, suggesting that the
For CB1 and CB2 receptor binding assays, the new com-
pounds were first subjected to a preliminary screening
carried out using three concentrations of the compounds
(5, 10, and 25 lM), membranes from HEK cells trans-
fected with either the human CB1 or CB2 receptor,
and [3H]-(ꢀ)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-
phenyl]-trans-4-(3-hydroxy-propyl)-cyclohexanol
([3H]CP-55,940) (Kd = 0.31 nM for CB2 and 0.18 nM for
CB1 receptors) as the high affinity ligand as described by
the manufacturer (Perkin-Elmer, Italia).21 Compounds
that displaced [3H]CP-55,940 by more than 50% at
10 lM were further analyzed by carrying out a complete
dose–response curve. Displacement curves were gener-
ated by incubating drugs with [3H]CP-55,940 (0.084
for CB2 and 0.14 nM for CB1 binding assay). In all
cases, Ki values were calculated by applying the
Cheng–Prusoff equation22 to the IC50 values (obtained
by GraphPad) for the displacement of the bound radio-
ligand by increasing concentrations of the test
compounds.
3.2. Fatty acid amide hydrolase assays
The effect of compounds on the enzymatic hydrolysis of
[14C]anandamide (6 lM) was studied by using mem-
branes prepared from rat brain incubated with increas-
ing concentrations of compounds in 50 mM Tris–HCl,
pH 9, for 30 min at 37 °C.20 [14C]ethanolamine pro-
R
CH2
O
OH
1
OH
H
H
H
3
OH
C6H13
OH
OCH2R
C6H13
C6H13
alkylation OH in C-3
alkylation OH in C-1
Scheme 2.

A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
5409
Table 1. Radioligand CB1 and CB2 binding assays and selectivity over FAAH of the synthetized compoundsa
OH
R
1
O(CH2)nCONHR1
Compound
R
N
R1
CB1
0.13
CB2
0.39
CB1/CB2
FAAH
10
11
5-n-Pentyl
5-n-Pentyl
5-n-Pentyl
5-n-Pentyl
5-n-Pentyl
5-n-Pentyl
5-n-Pentyl
2-n-Hexyl
2-n-Hexyl
2-n-Hexyl
2-n-Hexyl
2-n-Hexyl
2-n-Hexyl
2-n-Hexyl
4-n-Hexyl
4-n-Hexyl
4-n-Hexyl
4-n-Hexyl
4-n-Hexyl
4-n-Hexyl
4-n-Hexyl
5-n-Pentyl
7
CH2CH2OH
c-C3H5
1.35
0.33
6.4
>50
>50
>50
nt
7
10
10
10
11
11
7
0.016
0.51
0.56
2.1
0.039
0.08
12
13
CH2-c-C3H5
3,4-OH-Phenylethyl
3-OH-4-OCH3-benzyl
CH2CH2OH
c-C3H5
na
14
15
1.65
0.039
0.31
3.94
1.8
1.3
1.5
=10
>50
=50
=25
=50
>50
nt
0.06
0.75
0.26
0.31
0.75
2.1
16
17
2.4
CH2CH2OH
c-C3H5
0.07
0.17
1.6
18
19
7
10
10
10
11
11
7
CH2-c-C3H5
3,4-OH-Phenylethyl
3-OH-4-OCH3-Benzyl
CH2CH2OH
c-C3H5
0.47
7.9
20
21
2.82
0.17
0.013
0.19
0.056
2.1
=50
=50
>50
=50
>50
>50
nt
22
23
1.42
0.039
2.4
0.12
0.33
0.08
0.2
24
25
CH2CH2OH
c-C3H5
7
0.28
2.9
26
27
10
10
10
11
11
10
CH2-c-C3H5
3,4-OH-Phenylethyl
3-OH-4-OCH3-benzyl
CH2CH2OH
c-C3H5
na
na
na
na
na
na
28
29
na
na
1.3
0.4
>50
=10
>50
>50
30
CB 25
3.94
0.013
c-C3H5
0.0052
0.072
0.021
Anandamide
WIN 55,212-2
HU-210
0.0021
0.15 10ꢀ3
a Data are means of n = 3 separate experiments and are expressed as Ki (lM), for CB1 and CB2 binding assays, and in IC50 (lM) for FAAH assays.
Reference compounds were tested under the same conditions in this study. Anandamide was tested in the presence of PMSF (100 nM). na,
IC50 > 10 lM in the preliminary screening carried out with rat brain and spleen membranes; nt, not tested. Binding affinity constants of the most
potent compounds (Ki 6 1 lM) are highlighted in bold as well as the most selective compounds for CB1 and CB2. Standard errors are not shown for
the sake of simplicity and were never higher than 10% of the means.
phenolic group must be not hindered; (g) regarding the
interaction of the most potent cannabinoid receptor li-
gands with FAAH, the data obtained here confirm the
results of the previous paper16 that all such compounds
are essentially unable to inhibit the enzyme.
atom of the ligand interacted with K3.28, in agreement
with site-directed mutagenesis studies,24 and the hydro-
xy group formed a H bond with S7.39 (see Fig. 3a).
The docking of compound CB 25 (Ki = 5.2 nM, Fig. 4),
which is the most CB1 active compound among those
tested here and previously,16 revealed a similar occupa-
tion of the binding site. However, as shown in Figure
3b and differently from AEA, the carboxamide system
was directed toward TM1 and TM2 and formed two
H bonds with T1.46 and S2.54, while K3.28 formed a
H bond with the phenolic substituent.
4.1. Molecular modeling
As previously reported, the docking of AEA into our
CB1 receptor model suggested that it was placed among
TM2-3-6-7 with the aliphatic chain directed toward the
intracellular side of the receptor. The amide oxygen
Figure 3. AEA (a) and compound CB 25 (b) docked into the CB1 receptor (extracellular point of view).

5410
A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
O(CH2)10CONH
OH
OH
OH
C6H13
C6H13
O(CH2)10CONH
C5H11
O(CH2)10CONHR1
CB 43
CB 52
R1 = CH2CH2OH CB 24
R
1 = cyclopropyl CB 25
Figure 4. Previously published compounds14 docked into CB1 receptor.
˚
hydrogen of the threonine was 2.8 A while for com-
With regard to lipophilic interactions, the n-pentyl sub-
stituent was stabilized by F3.25, V6.59, and F7.35, the
decyl-alkyloxy chain was stabilized through the interac-
tion with F2.57, V3.32, F3.36, and L7.43, whereas the
cyclopropyl substituent was inserted in a lipophilic
pocket principally delimited by V2.58, L7.43 and the
backbone of TM1 (see Fig. 5a).
˚
pound CB 25 it was 1.8 A (see Fig. 5b).
The increase of the length of the alkyloxy chain, add-
ing only one C to the 10 C atoms of compound CB
25, determined a 144-fold decrease of affinity (16,
Ki = 750 nM). As shown in Figure 5c the docking of
compound 16 into our CB1 receptor model revealed
a different rearrangement of the lipophilic alkyloxy
chain with the maintenance of the H bond with
T1.46 and S2.54. However with this disposition the
ligand was not able to interact with K3.28, whose
mutagenesis data have suggested to be a fundamental
residue for ligand recognition, thus explaining its low
CB1 affinity.
The reduction of the alkyloxy chain from ten to seven C
atoms (compound 11) determined only a threefold
reduction of CB1 affinity and in fact the docking studies
revealed that this compound showed all the H bonds ob-
served for compound CB 25, even though the H bond
with T1.46 was weaker since the distance between the
amide oxygen atom of the ligand and the polar
Figure 5. Compounds CB 25 (a), 11 (b), 16 (c), CB 24 (d), CB 52 (e), 23 (f), 14 (g), CB 43 (h) docked into the CB1 receptor model.

A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
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A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
The substitution of the cyclopropyl substituent with
ethanol determined a 152-fold decrease of affinity, as
compound CB 24 (Fig. 4) showed a 800 nM CB1 affin-
ity.16 The docking of this compound highlighted a very
similar disposition to the one observed for compound
CB 25. However, as shown in Figure 5d, the ethanol/
cyclopropyl substitution determined the loss of the lipo-
philic interaction with V2.58 and L7.43. Furthermore,
the hydroxy group of the ethanol substituent did not
seem to form any electrostatic interaction, thus explain-
ing the strong decrease of affinity.
5. Conclusions
In this study, we have designed and synthetized 21 new
olivetol and hexylresorcinol-derived compounds with
the aim to identify the structural modifications that de-
fine affinity for cannabinoid receptors in this class of
compounds, which are obtained by linking a stable
and rigid structure, such as THC, to a flexible chain car-
rying an amidic ‘head’, as in AEA. The range of carbon
atom length in the alkyloxy chain and the structural fea-
tures of the amidic ‘head’ necessary to observe high
affinity have been characterized. In fact, although com-
pound CB 25 still shows the lowest Ki values, many of
the newly synthesized compounds exhibited strong affin-
ity for CB1 and CB2 receptors and two of them exhibited
also a reasonable selectivity for CB1 versus CB2 recep-
tors. Moreover, molecular modeling studies fitted this
new class of cannabinoid ligands into a CB1 receptor
model, and the qualitative analysis of the results was
in general agreement with the CB1 affinity constants ob-
served experimentally for these derivatives.
The displacement of the alkyl chain from the 50 to the
20 position determined a general decrease of the CB1
affinity, as typified by compound CB 5216 (Fig. 4),
which presented a n-hexyl chain in 20 position and dis-
played
a
40-fold decrease of CB1 affinity
(Ki = 210 nM) with respect to CB 25. The docking
of compound CB 52 revealed that, as for CB 25,
the carboxamide group formed two H bonds with
T1.46 and S2.54, whereas the n-hexyl chain interacted
with F3.25, V6.59, and F7.35. However, with this dis-
position of the substituents, the phenolic oxygen did
not interact with K3.28 (see Fig. 5e).
6. Experimental
Very interestingly, differently from the 30,50 disubsti-
tuted derivatives where the increase of the length of
the alkyloxy chain from ten to eleven carbons deter-
mined a decrease of affinity, for the 20,50 disubstituted
compounds this change determined an increase of
affinity.
6.1. General information
Melting points were determined on a Kofler hot stage
apparatus (K) or using a Mettler FPI apparatus (2 °C/
min) (M) and are uncorrected. Elemental analyses of
all synthesized compounds were performed by our ana-
lytical laboratory in a Perkin-Elmer elemental apparatus
Mod. 240 for C, H, N and the data are within 0.4% of
Docking studies of compound 23, which possesses an
alkyloxy chain of eleven carbons, highlighted not
only all the main interactions of compound CB 52,
but also the H bond between K3.28 and the phenolic
oxygen of the ligand (see Fig. 5f), as for CB 25.
These results were in agreement with the CB1 binding
affinity of this compound, which showed a Ki of
13 nM.
1
the theoretical values. H NMR spectra were recorded
at 25 °C on a Bruker AC200F employing TMS as inter-
nal standard and chemical shifts are expressed as d
(ppm). Mass spectral data were determined by direct
insertion at 70 eV with a VG70 spectrometer. All com-
pounds were checked for purity by T.L.C. on Merck
60 F254 silica plates. For column chromatography,
Merck 60 silica gel, 230–400 mesh, was used. Final prod-
ucts were purified by a Biotage flash chromatography
system with columns 12.25 mm, packed with KP-Sil,
60A, 32–63 lM. Reagents were purchased from Sig-
ma–Aldrich Srl (Italy) and used as received unless other-
wise stated.
With regard to the substitution of the cyclopropyl group
with the 4-hydroxy-3-methoxy-benzyl, in one compound
(14) this determined a great decrease of CB1 affinity
(Ki = 2100 nM). The reason could be the high dimension
of the aromatic substituent, compared with the cyclo-
propyl group. In fact, as shown in Figure 5g, the inter-
actions of the aromatic group inside the lipophilic
pocket principally constituted of V2.58 and L7.43 deter-
mined a different rearrangement of the decyl alkyloxy
chain with the consequent loss of the H bond between
the phenolic substituent and K3.28.
6.2. General method for esters
A mixture of phenolic compound (5.0 mmol), anhy-
drous potassium carbonate (2.5 mmol), and potassium
fluoride (5.0 mmol) in dry acetone was refluxed under
nitrogen atmosphere and continuous stirring for half
an hour, and then a solution of the corresponding bro-
moalkylmethylester (5.0 mmol) in dry acetone was
added, refluxing for another 48–72 h and checking the
reaction by TLC. Afterwards the reaction mixture was
concentrated, diluted with water, and extracted with
chloroform. The extracts were collected, dried (MgSO4),
evaporated under reduced pressure, and the residue
purified by column chromatography on silica gel.
Chemical and physical data of all synthetized esters
are reported in Table 2.
Finally the displacement of the alkyl chain from the 50
(compound CB 25, Ki = 0.0052) to the 40 position (com-
pound CB 43,16 Fig. 4) determined a complete loss of
affinity (Ki > 10 lM). The docking analysis revealed that
the n-hexyl chain was unable to interact with F3.25,
V6.59, and F7.35, and the disposition of the ligand
was completely overturned: the phenolic system was di-
rected toward the TM2 and the carboxamide group was
directed toward the extracellular side of the receptor (see
Fig. 5h).

A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
5413
6.3. General methods for the final products
(t, 2H, J = 7.7 Hz), 2.18 (t, 2H, J = 7.5 Hz), 1.74–1.52
(mm, 8H), 1.48–1.26 (mm, 14H), 0.96–0.92 (m, 1H),
0.86 (t, 3H, J = 6.3 Hz), 0.53–0.44 (m, 2H), 0.21–0.16
(m, 2H). MS m/z: 418 [M+1]+ (100). Anal. Calcd for
C26H43NO3: C, 74.77; H, 10.38; N, 3.35. Found: C,
74.52; H, 10.57; N, 3.18.
6.3.1. Method A. Each ester (1.0 mmol) was refluxed in a
methanolic/aqueous sodium hydroxide solution (0.1 M,
3.0 equiv) for 3 h. Then the reaction mixture was allowed
to get to room temperature, made acidic (pH 3–4) by add-
ing diluted HCl, and finally extracted with ethyl acetate;
the organic layer was dried and the solvent evaporated
to yield the crude acid, which was thoroughly dried under
vacuum before being subjected to the subsequent reaction
without further manipulation. To a mixture of the crude
acid (1.0 mmol), the appropriate amine (1.5 mmol), and
1-hydroxybenzotriazole (HOBt, 1.2 mmol) in dry dichlo-
romethane or in dry acetonitrile, kept in an ice bath, a
solution, in the same solvent, of 1-cyclohexyl-3-(2-mor-
6.3.6. 11-(3-Hydroxy-5-pentyl-phenoxy)-undecanoic acid
[2-(3,4-dihydroxy-phenyl)-ethyl]-amide (13). Yellow oil
1
(CHCl3/MeOH = 50:1) (70% yield). H NMR (CDCl3)
d (ppm): 6.77 (d, 1H, J = 7.9 Hz), 6.71 (d, 1H,
J = 1.3 Hz), 6.53 (d, 1H, J = 8.0 Hz), 6.28–6.22 (m,
3H), 5.70 (t br, 1H), 3.90 (t, 2H, J = 6.4 Hz), 3.60–3.41
(m, 2H), 2.66 (t, 2H, J = 6.8 Hz), 2.48 (t, 2H,
J = 7.6 Hz), 2.12 (t, 2H, J = 7.5 Hz), 1.71–1.56 (mm,
8H), 1.32–1.03 (mm, 14H), 0.87 (t, 3H, J = 6.5 Hz).
MS m/z: 522 [M+Na]+ (100). Anal. Calcd for
C30H45NO5: C, 72.11; H, 9.08; N, 2.80. Found: C,
71.88; H, 9.12; N, 2.75.
pholinoethyl)carbodiimide
methyl-p-toluensulfonate
(CMC, 1.5 mmol) was added dropwise under nitrogen
atmosphere and continuous stirring. The reaction mix-
ture was left to room temperature and stirring was contin-
ued for 24 h. The solvent was removed under reduced
pressure and the residue was diluted with chloroform;
the organic layer was washed with 5% aqueous NaHCO3,
then with 1N HCl and dried (MgSO4). After drying and
evaporation of the solvent, a crude residue was obtained
and purified by column chromatography on silica gel.
6.3.7. 11-(3-Hydroxy-5-pentyl-phenoxy)-undecanoic acid
(3-methoxy-4-hydroxy-phenyl)-amide (14). Pale yellow
oil (CHCl3/MeOH = 50:1) (88% yield). 1H NMR
(CDCl3) d (ppm): 6.86–6.76 (m, 3H), 6.26–6.24 (m,
3H), 6.17 (s, 1H), 5.72 (t br, 1H), 5.63 (s, 1H), 4.34 (d,
2H, J = 5.7 Hz), 3.88 (t, 2H, J = 6.6 Hz), 3.84 (s, 3H),
2.47 (t, 2H, J = 7.5 Hz), 2.19 (t, 2H, J = 7.3 Hz), 1.75–
1.52 (mm, 8H), 1.49–1.26 (mm, 14H), 0.86 (t, 3H,
J = 6.2 Hz). MS m/z: 522 [M+Na]+ (100). Anal. Calcd
for C30H45NO5: C, 72.11; H, 9.08; N, 2.80. Found: C,
71.92; H, 9.03; N, 2.69.
6.3.2. Method B. Each ester (0.150 g) was dissolved, un-
der nitrogen atmosphere and stirring, in redistilled etha-
nolamine (4 mL) and warmed at 120–130 °C for 4–6 h.
The resulting mixture was diluted with water and ex-
tracted with chloroform; the extracts were washed with
a solution of ammonium chloride, dried (MgSO4), evap-
orated, and the raw material purified by column chro-
matography on silica gel.
6.3.8. 12-(3-Hydroxy-5-pentyl-phenoxy)-dodecanoic acid
(2-hydroxy-ethyl)-amide (15). Transparent oil (ethyl ace-
1
tate) (66% yield). H NMR (CDCl3) d (ppm): 6.27–6.24
6.3.3. 8-(3-Hydroxy-5-pentyl-phenoxy)-octanoic acid (2-
hydroxy-ethyl)-amide (10). Pale pink oil (ethyl acetate)
(65% yield). H NMR (CDCl3) d (ppm): 6.32–6.25 (m,
3H), 3.87 (t, 2H, J = 6.4 Hz), 3.69 (t, 2H, J = 4.9 Hz),
3.43–3.35 (m, 2H), 2.47 (t, 2H, J = 7.3 Hz), 2.18 (t,
2H, J = 7.3 Hz), 1.73–1.56 (mm, 6H), 1.48–1.28 (mm,
10H), 0.86 (t, 3H, J = 6.1 Hz). MS m/z: 388 [M+Na]+
(100), 753 [2M+Na]+. Anal. Calcd for C21H35NO4: C,
69.01; H, 9.65; N, 3.83. Found: C, 68.80; H, 9.98; N,
3.75.
(m, 3H), 5.98 (s br, 1H), 3.89 (t, 2H, J = 6.4 Hz), 3.71 (t,
2H, J = 4.9 Hz), 3.45–3.37 (m, 2H), 2.48 (t, 2H,
J = 7.5 Hz), 2.19 (t, 2H, J = 7.4 Hz), 1.69–1.79 (m,
2H), 1.66–1.53 (m, 4H), 1.49–1.26 (mm, 18H), 0.86 (t,
3H, J = 6.6 Hz). MS m/z: 444 [M+Na]+ (100). Anal.
Calcd for C25H43NO4: C, 71.22; H, 10.28; N, 3.32.
Found: C, 71.08; H, 10.41; N, 3.23.
1
6.3.9. 12-(3-Hydroxy-5-pentyl-phenoxy)-dodecanoic acid
solid
cyclopropylamide
(16).
White
(CHCl3/
1
MeOH = 50:1) (91% yield): mp 75.5 °C (M). H NMR
(CDCl3) d (ppm): 6.27–6.26 (m, 3H), 5.78 (s br, 1H),
3.88 (t, 2H, J = 6.5 Hz), 2.73–2.62 (m, 1H), 2.47 (t,
2H, J = 7.6 Hz), 2.12 (t, 2H, J = 7.5 Hz), 1.75–1.49
(mm, 6H), 1.32–1.25 (mm, 18H), 0.86 (t, 3H,
J = 6.6 Hz), 0.79–0.70 (m, 2H), 0.50–0.42 (m, 2H). MS
m/z: 418 [M+1]+ (100). Anal. Calcd for C26H43NO3: C,
74.77; H, 10.38; N, 3.35. Found: C, 74.56; H, 10.22;
N, 3.24.
6.3.4. 8-(3-Hydroxy-5-pentyl-phenoxy)-octanoic acid
cyclopropylamide (11). Pale yellow oil (CHCl3/
MeOH = 48:2) (60% yield). 1H NMR (CDCl3) d
(ppm): 6.79 (s, 1H), 6.27–6.25 (m, 3H), 5.74 (s br, 1H),
3.87 (t, 2H, J = 6.6 Hz), 2.72–2.64 (m, 1H), 2.46 (t,
2H, J = 7.4 Hz), 2.12 (t, 2H, J = 7.2 Hz), 1.73–1.55
(mm, 6H), 1.34–1.23 (mm, 10H), 0.85 (t, 3H,
J = 6.6 Hz), 0.80–0.70 (m, 2H), 0.49–0.44 (m, 2H). MS
m/z: 362 [M+1]+ (100). Anal. Calcd for C22H35NO3: C,
73.09; H, 9.76; N, 3.87. Found: C, 72.91; H, 9.58; N,
3.90.
6.3.10. 8-(2-Hexyl-5-hydroxy-phenoxy)-octanoic acid (2-
hydroxy-ethyl)-amide (17). Pale yellow oil (ethyl acetate)
1
(45% yield). H NMR (CDCl3) d (ppm): 6.90 (d, 1H,
6.3.5. 11-(3-Hydroxy-5-pentyl-phenoxy)-undecanoic acid
cyclopropylmethyl-amide (12). Pasty pale yellow solid
(CHCl3/MeOH = 50:1) (96% yield): mp 54–55 °C (K).
1H NMR (CDCl3) d (ppm): 6.26 (s, 3H), 5.72 (s br,
1H), 3.88 (t, 2H, J = 6.3 Hz), 3.13–3.07 (m, 2H), 2.46
J = 8.1 Hz), 6.37 (d, 1H, J = 2.1 Hz), 6.24 (dd, 1H,
J = 2.2 Hz, J = 8.0 Hz), 6.13 (s br, 1H), 3.87 (t, 2H,
J = 6.5 Hz), 3.69 (t, 2H, J = 4.9 Hz), 3.43–3.35 (m,
2H), 2.54–2.43 (m, 2H), 2.18 (t, 2H, J = 7.3 Hz), 1.76–
1.58 (m, 4H), 1.1.46–1.28 (mm, 14H), 0.85 (t, 3H,

5414
A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
J = 6.3 Hz). MS m/z: 402 [M+Na]+ (100), 781
[2M+Na]+. Anal. Calcd for C22H37NO4: C, 69.62; H,
9.83; N, 3.69. Found: C, 69.81; H, 9.90; N, 3.57.
J = 4.9 Hz), 3.45–3.37 (m, 2H), 2.50 (t, 2 H,
J = 7.4 Hz), 2.19 (t, 2H, J = 7.5 Hz), 1.79–1.69 (m,
2H), 1.65–1.44 (m, 4H), 1.41–1.27 (mm, 20H), 0.87 (t,
3H, J = 6.1 Hz). MS m/z: 458 [M+Na]+ (100). Anal.
Calcd for C26H45NO4: C, 71.68; H, 10.41; N, 3.22.
Found: C, 71.52; H, 10.56; N, 3.37.
6.3.11. 8-(2-Hexyl-5-hydroxy-phenoxy)-octanoic acid
cyclopropylamide
(18).
White
solid
(CHCl3/
MeOH = 48:2) (75% yield): mp 53–55 °C (K). 1H
NMR (CDCl3) d (ppm): 6.90 (d, 1H, J = 8.0 Hz), 6.40
(s, 1H), 6.33 (dd, 1H, J = 2.4 Hz, J = 8.1 Hz), 5.64 (s
br, 1H), 3.89 (t, 2H, J = 6.6 Hz), 2.71–2.66 (m, 1H),
2.48 (t, 2H, J = 7.0 Hz), 2.13 (t, 2H, J = 7.2 Hz), 1.77–
1.53 (mm, 6H), 1.50–1.37 (mm, 6H), 1.36–1.25 (mm,
6H), 0.85 (t, 3H, J = 6.4 Hz), 0.80–0.70 (m, 2H), 0.49–
0.41 (m, 2H). MS m/z: 376 [M+1]+ (100). Anal. Calcd
for C23H37NO3: C, 73.56; H, 9.93; N, 3.73. Found: C,
73.72; H, 9.81; N, 3.62.
6.3.16. 12-(2-Hexyl-5-hydroxy-phenoxy)-dodecanoic acid
cyclopropylamide (23). White bright solid (CHCl3/
MeOH = 50:1) (90% yield): mp 53–55 °C (K). 1H
NMR (CDCl3) d (ppm): 6.91 (d, 1H, J = 8.0 Hz),
6.39–6.30 (m, 2H), 5.57 (s br, 1H), 3.88 (t, 2H,
J = 6.3 Hz), 2.70–2.66 (m, 1H, CH), 2.49 (t, 2H,
J = 7.1 Hz), 2.11 (t, 2H, J = 7.2 Hz), 1.79–1.51 (mm,
6H), 1.47–1.26 (mm, 20H), 0.86 (t, 3H, J = 6.2 Hz),
0.77–0.71 (m, 2H), 0.50–0.42 (m, 2H). MS m/z: 432
[M+1]+ (100). Anal. Calcd for C27H45NO3: C, 75.13;
H, 10.51; N, 3.24. Found: C, 74.95; H, 10.66; N, 3.41.
6.3.12. 11-(2-Hexyl-5-hydroxy-phenoxy)-undecanoic acid
cyclopropylmethyl-amide (19). Pasty pale yellow solid
(CHCl3/MeOH = 48:2) (96% yield): mp 49–50 °C (K).
1H NMR (CDCl3) d (ppm): 6.90 (d, 1H, J = 7.9 Hz),
6.70 (s br, 1H), 6.40–6.31 (m, 2H), 5.68 (s br, 1H),
3.86 (t, 2H, J = 6.3 Hz), 3.13–3.07 (m, 2H), 2.48 (t,
2H, J = 7.5 Hz), 2.17 (t, 2H, J = 7.5 Hz), 1.77–1.43
(mm, 8H), 1.39–1.02 (m, 16H), 0.98–0.92 (m, 1H), 0.85
(t, 3H, J = 6.0 Hz), 0.53–0.40 (m, 2H), 0.21–0.13 (m,
2H). MS m/z: 432 [M+1]+ (100). Anal. Calcd for
C27H45NO3: C, 75.13; H, 10.51; N, 3.24. Found: C,
74.95; H, 10.73; N, 3.40.
6.3.17. 8-(4-Hexyl-3-hydroxy-phenoxy)-octanoic acid (2-
hydroxy-ethyl)-amide (24). White solid (CHCl3/
MeOH = 47:3) (88% yield): mp 63–65 °C (K). 1H
NMR (CDCl3) d (ppm): 7.80 (s br, 1H), 6.93 (d, 1H,
J = 8.1 Hz), 6.66 (s br, 1H), 6.38–6.31 (m, 2H), 3.82 (t,
2H, J = 5.8 Hz), 3.66–3.64 (m, 2H), 3.43–3.35 (m, 2H),
2.50 (t, 2H, J = 6.7 Hz), 2.15 (t, 2H, J = 6.3 Hz), 1.66–
1.54 (m, 6H), 1.45–1.27 (mm, 12H), 0.85 (t, 3H,
J = 6.3 Hz). MS m/z: 402 [M+Na]+ (100). Anal. Calcd
for C22H37NO4: C, 69.62; H, 9.83; N, 3.69. Found: C,
69.78; H, 9.94; N, 3.55.
6.3.13. 11-(2-Hexyl-5-hydroxy-phenoxy)-undecanoic acid
[2-(3,4-dihydroxy-phenyl)-ethyl]-amide (20). Pale yellow
oil (CHCl3/MeOH = 47:3) (70% yield). 1H NMR
(CDCl3) d (ppm): 6.91 (d, 1H, J = 7.9 Hz), 6.78 (d,
1H, J = 8.0 Hz), 6.71 (d, 1H, J = 2.1 Hz), 6.55 (dd, 1H,
J = 2.1, J = 8.1 Hz), 6.37–6.31 (m, 2H), 5.65 (t br, 1H),
3.85 (t, 2H, J = 6.3 Hz), 3.46–3.42 (m, 2H), 2.65 (t,
2H, J = 6.9 Hz), 2.49 (t, 2H, J = 7.4 Hz), 2.12 (t, 2H,
J = 7.5 Hz), 1.74–1.70 (m, 2H), 1.58–1.47 (m, 4H,),
1.42–1.24 (mm, 18H), 0.85 (t, 3H, J = 6.3 Hz). MS m/
z: 514 [M+1]+ (100). Anal. Calcd for C31H47NO5: C,
72.48; H, 9.22; N, 2.73. Found: C, 72.25; H, 9.48; N,
2.89.
6.3.18. 8-(4-Hexyl-3-hydroxy-phenoxy)-octanoic acid
cyclopropylamide (25). White bright solid (CHCl3/
MeOH = 48:2; recrystallized n-hexane/ethyl acetate)
(81% yield): mp 123.4 °C (M). 1H NMR (CDCl3) d
(ppm): 9.50 (s, 1H), 8.15 (s br, 1H), 6.84 (d, 1H,
J = 8.1 Hz), 6.29 (d, 1H, J = 2.5 Hz), 6.23 (dd, 1H,
J = 2.4 Hz, J = 8.4 Hz), 3.80 (t, 2H, J = 6.5 Hz), 2.59–
2.50 (m, 1H), 2.38 (t, 2H, J = 7.0 Hz), 1.96 (t, 2H,
J = 7.1 Hz), 1.62–1.41 (m, 6H), 1.37–1.22 (mm, 12H),
0.82 (t, 3H, J = 6.4 Hz), 0.59–0.49 (m, 2H), 0.35–0.30
(m, 2H). MS m/z: 376 [M+1]+ (100). Anal. Calcd for
C23H37NO3: C, 73.56; H, 9.93; N, 3.73. Found: C,
73.41; H, 9.98; N, 3.80.
6.3.14. 11-(2-Hexyl-5-hydroxy-phenoxy)-undecanoic acid
(3-methoxy-4-hydroxy-phenyl)-amide (21). Pale pink oil
6.3.19. 11-(4-Hexyl-3-hydroxy-phenoxy)-undecanoic acid
cyclopropylmethyl-amide (26). White solid (CHCl3/
MeOH = 50:1) (83.0% yield): mp 87.3 °C (M). 1H
NMR (CDCl3) d (ppm): 8.90 (s, 1H), 7.63 (s br, 1H),
6.80 (d, 1H, J = 8.4 Hz), 6.26 (d, 1H, J = 2.3 Hz), 6.16
(dd, 1H, J = 2.1 Hz, J = 8.3 Hz), 3.76 (t, 2H,
J = 6.2 Hz), 2.90 (t, 2H, J = 6.1 Hz), 2.36 (t, 2H,
J = 7.3 Hz), 2.00 (t, 2H, J = 7.3 Hz), 1.62–1.42 (mm,
6H), 1.40–1.20 (mm, 18H), 0.83–0.77 (m, 4H), 0.38–
0.31 (m, 2H), 0.11–0.04 (m, 2H). MS m/z: 432 [M+1]+
(100). Anal. Calcd for C27H45NO3: C, 75.13; H, 10.51;
N, 3.24. Found: C, 74.98; H, 10.69; N, 3.38.
1
(CHCl3/MeOH = 50:2) (50% yield). H NMR (CDCl3)
d (ppm): 6.92–6.71 (m, 4H), 6.38 (d, 1H, J = 2.3 Hz),
6.32 (dd, 1H, J = 2.3 Hz, J = 8.0 Hz), 5.75 (t br, 1H),
5.67 (s, 1H), 4.34 (d, 2H, J = 5.5 Hz), 3.87 (t, 2H,
J = 6.3 Hz), 3.84 (s, 3H), 2.49 (t, 2H, J = 7.1 Hz), 2.19
(t, 2H, J = 7.3 Hz), 1.81–1.63 (mm, 6H), 1.59–1.43 (m,
4 H), 1.39–1.27 (mm, 14H), 0.86 (t, 3H, J = 6.5 Hz).
MS m/z: 514 [M+1]+ (100). Anal. Calcd for
C31H47NO5: C, 72.48; H, 9.22; N, 2.73. Found: C,
72.33; H, 9.40; N, 2.84.
6.3.15. 12-(2-Hexyl-5-hydroxy-phenoxy)-dodecanoic acid
(2-hydroxy-ethyl)-amide (22). Pasty white solid (CHCl3/
MeOH = 50:1) (50% yield): mp 57–58 °C (K). 1H
NMR (CDCl3) d (ppm): 6.91 (d, 1H, J = 8.00 Hz),
6.37 (s, 1H), 6.32 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz),
6.08 (s br, 1H), 3.88 (t, 2H, J = 6.3 Hz), 3.72 (t, 2H,
6.3.20. 11-(4-Hexyl-3-hydroxy-phenoxy)-undecanoic acid
[2-(3,4-dihydroxy-phenyl)-ethyl]-amide (27). Pale yellow
bright solid (CHCl3/MeOH = 50:1) (70.0% yield): mp
97–100 °C (K). 1H NMR (CDCl3) d (ppm): 6.96 (d,
1H, J = 8.9 Hz), 6.83–6.66 (m, 3H), 6.54 (d, 1H,

A. Brizzi et al. / Bioorg. Med. Chem. 15 (2007) 5406–5416
5415
J = 8.2 Hz), 6.40–6.36 (m, 1H), 5.63 (t br, 1H), 3.88 (t,
2H, J = 6.3 Hz), 3.52–3.42 (m, 2H), 2.67 (t, 2H,
J = 6.9 Hz), 2.51 (t, 2H, J = 7.6 Hz), 2.13 (t, 2H,
J = 7.5 Hz), 1.74–1.68 (m, 2H), 1.60–1.52 (m, 4H),
1.40–1.24 (mm, 18H), 0.86 (t, 3H, J = 6.6 Hz). MS m/
z: 514 [M+1]+ (100). Anal. Calcd for C31H47NO5: C,
72.48; H, 9.22; N, 2.73. Found: C, 72.30; H, 9.45; N,
2.86.
Using the Lamarckian Genetic Algorithm, the com-
pound was subjected to 250 runs of the AUTODOCK
search, in which the default values of the other parame-
ters were used. Cluster analysis was performed on the
˚
docked results using an RMS tolerance of 1.0 A.
Acknowledgments
6.3.21. 11-(4-Hexyl-3-hydroxy-phenoxy)-undecanoic acid
(3-methoxy-4-hydroxy-phenyl)-amide (28). White solid
(CHCl3/MeOH = 50:1) (84.0% yield): mp 103–104 °C
(K). 1H NMR (CDCl3) d (ppm): 7.00–6.71 (m, 3H),
6.42–6.34 (m, 2H), 6.19 (s, 1H), 5.76 (t br, 1H), 5.67
(s, 1H), 4.34 (d, 2H, J = 5.7 Hz), 3.87 (t, 2H,
J = 6.5 Hz), 3.84 (s, 3H), 2.51 (t, 2H, J = 7.1 Hz), 2.19
(t, 2H, J = 7.1 Hz), 1.74–1.51 (mm, 8H), 1.48–1.07
(mm, 16H), 0.86 (t, 3H, J = 6.4 Hz). MS m/z: 514
[M+1]+ (100). Anal. Calcd for C31H47NO5: C, 72.48;
H, 9.22; N, 2.73. Found: C, 72.28; H, 9.46; N, 2.89.
We thank MIUR (PRIN 2004037521) and the Univer-
`
sita degli Studi di Siena, Italy, for financial support.
We thank Mr. Marco Allara for technical assistance
`
with assays.
References and notes
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6.3.22. 12-(4-Hexyl-3-hydroxy-phenoxy)-dodecanoic acid
(2-hydroxy-ethyl)-amide (29). White solid (ethyl acetate)
(40.0% yield): mp 80.5 °C (M). 1H NMR (CDCl3) d
(ppm): 6.95 (d, 1H, J = 8.9 Hz), 6.42–6.36 (m, 2H),
5.96 (s br, 1H), 3.88 (t, 2H, J = 6.3 Hz), 3.72 (t, 2H,
J = 4.4 Hz), 3.45–3.38 (m, 2H), 2.50 (t, 2H,
J = 7.2 Hz), 2.20 (t, 2H, J = 7.3 Hz), 1.76–1.51 (mm,
6H), 1.48–1.25 (mm, 20H), 0.86 (t, 3H, J = 6.5 Hz).
MS m/z: 436 [M+1]+, 458 [M+Na]+ (100). Anal. Calcd
for C26H45NO4: C, 71.68; H, 10.41; N, 3.22. Found:
C, 71.49; H, 10.58; N, 3.40.
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6.3.23. 12-(4-Hexyl-3-hydroxy-phenoxy)-dodecanoic acid
cyclopropylamide (30). White bright solid (CHCl3/
MeOH = 50:1) (90.0% yield): mp 115.9 °C (M). 1H
NMR (CDCl3) d (ppm): 6.96 (d, 1H, J = 7.9 Hz),
6.41–6.36 (m, 2H), 5.57 (s br, 1H), 3.90 (t, 2H,
J = 6.4 Hz), 2.73–2.69 (m, 1H), 2.52 (t, 2H,
J = 7.2 Hz), 2.12 (t, 2H, J = 7.4 Hz), 1.77–1.61 (mm,
6H), 1.57–1.28 (mm, 20H), 0.87 (t, 3H, J = 6.5 Hz),
0.81–0.72 (m, 2H), 0.51–0.43 (m, 2H). MS m/z: 432
[M+1]+ (100). Anal. Calcd for C27H45NO3: C, 75.13;
H, 10.51; N, 3.24. Found: C, 74.98; H, 10.63; N, 3.38.
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Shinoda, A.; Itoh, K.; Yamashita, A. Biochem. Biophys.
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