Synthesis and evaluation of some lipidic aminoalcohols and diamines as immunomodulators

Article abstract of DOI:10.1016/j.bmcl.2006.08.113

Lymphoproliferation inhibition and cytotoxicity of a number of lipidic aminoacids, aminoalcohols and diamines were evaluated as a preliminary screening to select potential immunomodulators. The four most potent/less toxic compounds were submitted to delayed hypersensibility (DTH) assays to define the best to be evaluated further Graft-vs-Host, NO production and other immunoevaluation (CD4⁺, CD45, CD8, CD11b, I-Ek, and NK cells) assays, to establish their immunomodulation potential for being further considered as auxiliary agents for vaccination against some parasitic infections. Compounds 5d, 6d, 6f, 7a, and 9a, fairly inhibited the lymphoproliferation (71.6-79.5%, at 3.2-2.4 nM), while the aminoalcohol derivative 6f and the diamine 7a gave the most promising results in the DTH assays. Diamine derivative 8b induced nitrite production on normal macrophages, whereas compounds 6f and 7a induced nitrite production on LPS pre-stimulated macrophages. These two last compounds have been selected to follow in vivo vaccination assays.

Full text of DOI:10.1016/j.bmcl.2006.08.113

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6091–6095
Synthesis and evaluation of some lipidic aminoalcohols
and diamines as immunomodulators
b
b,
c
Esther del Olmo,a, Alvaro Plaza, Antonio Muro, Antonio R. Martınez-Fernandez,
*
*
´
´
c
a
a
´
´
´
Juan J. Nogal-Ruiz, Jose L. Lopez-Perez and Arturo San Feliciano
aDepartamento de Quı´mica Farmace´utica, Facultad de Farmacia, CIETUS, Universidad de Salamanca, 37007 Salamanca, Spain
bLaboratorio de Parasitologı´a, Facultad de Farmacia, CIETUS, Universidad de Salamanca, 37007 Salamanca, Spain
cDepartamento de Parasitologı´a, Facultad de Farmacia, Universidad Complutense, 47007 Madrid, Spain
Received 13 July 2006; revised 25 August 2006; accepted 28 August 2006
Available online 26 September 2006
Abstract—Lymphoproliferation inhibition and cytotoxicity of a number of lipidic aminoacids, aminoalcohols and diamines were
evaluated as a preliminary screening to select potential immunomodulators. The four most potent/less toxic compounds were sub-
mitted to delayed hypersensibility (DTH) assays to define the best to be evaluated further Graft-vs-Host, NO production and other
immunoevaluation (CD4+, CD45, CD8, CD11b, I-Ek, and NK cells) assays, to establish their immunomodulation potential for
being further considered as auxiliary agents for vaccination against some parasitic infections. Compounds 5d, 6d, 6f, 7a, and 9a,
fairly inhibited the lymphoproliferation (71.6–79.5%, at 3.2–2.4 nM), while the aminoalcohol derivative 6f and the diamine 7a gave
the most promising results in the DTH assays. Diamine derivative 8b induced nitrite production on normal macrophages, whereas
compounds 6f and 7a induced nitrite production on LPS pre-stimulated macrophages. These two last compounds have been selected
to follow in vivo vaccination assays.
Ó 2006 Elsevier Ltd. All rights reserved.
Immunosuppressants play important clinical roles in
organ transplantation and in the treatment of autoim-
mune diseases such as rheumatoid arthritis, psoriasis
and systemic lupus erythematosus. Cyclosporin A
(CsA)1 and tacrolimus (FK506)2 have made great con-
tributions to the prevention of acute rejection in hu-
man organ transplantation. Both drugs have similar
mechanisms of action and proved their immunosup-
pressant activity by inhibiting the production of inter-
leukin-2 (IL-2) in antigen-stimulated helper T-cells,3–5
but they also have severe side effects,6 such as a high
cytotoxicity and renal and liver toxicities. Therefore,
less toxic drugs for the prevention of graft rejection
are needed.
nosuppressant activity,8 that was nearly five times more
potent than that of CsA, with lower toxicity. They also
worked on the identification of the minimal structure
requirements for the activity and concluded that a 2-al-
kyl-2-aminoethanol moiety was needed.9 They also
optimised the alkyl chain length, the functions and
their locations on the alkyl chain to develop compound
FTY720 (IC50 6.1 nM), as the best drug candidate.10 In
the course of these studies, compound OA-12 displayed
an intermedium immunosuppressant activity (IC50
98.3 nM), nearly seven times lesser potent than CsA
in the allogeneic mouse mixed lymphocyte reaction
(MLR) assay.11
Our research group has been working on lipidic ethy-
lenediamine and b-aminoalcohol derivatives displaying
different biochemical, pharmacological and antimicrobi-
al properties.12–16 The close structural relationship
between the above-mentioned drugs and compound
OA-12 with some related compounds prepared by us
suggested their evaluation as immunomodulators
through lymphoproliferation inhibition, delayed hyper-
sensitivity and other studies.
ISP-1 (myriocin, thermozymocidin) was isolated in
1989 from the culture broth of Isaria sinclairii by Fuj-
ita et al.7 These authors also described the ISP-1 immu-
Keywords: Synthesis; Evaluation; Lipidic aminoalcohols; Lipidic dia-
mines; Immunomodulators.
*
Corresponding authors. Fax: +34 923294515; e-mail: olmo@usal.es
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.113

6092
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6091–6095
COOH
equivalent of glutaric anhydride gave 8a and 8b,
NH2
H2N
HO
OH
O
HO
NH2
respectively. Boc removal with HCl(g) in THF gave,
respectively, compounds 9a and 9b. Similarly the free
aminoalcohol 2b and the diamine 7b were obtained
from compounds 2a and 7a, respectively, by treatment
with acid.
HO
OH
FTY 720
5
6
13
7
OH
OA-12
ISP-1
(myriocin, thermozymocidin)
A faster procedure leading to aminoalcohols of type 6
was also applied to obtain another group of aminoalco-
hol derivatives. Starting from 2-bromohexadecanoic
acid and the proper amines, the intermediate aminoacids
of type 10 were prepared, from which LiAlH4 reduction
yielded the corresponding aminoalcohols 6a and 6d–h
(Scheme 2).
The lipidic compounds being tested in this multi-screen-
ing research were obtained as described previously
(Scheme 1).12 The starting aminoacid derivative 1 was
prepared17 from the intermediate diethyl 2-tetradecyl-
acetamidomalonate (obtained by alkylation of diethyl
2-acetamidomalonate in the presence of NaOEt), after
refluxing in concd HCl and treatment with Boc anhy-
dride. Compound 1, transformed into the mixed anhy-
dride by reaction with ethyl chloroformate,18 followed
by reduction with NaBH4, provided the alcohol 2a.
Compound 2a was transformed into the benzyl ether 3,
then by acid hydrolysis of the Boc-protecting group, into
the benzyloxy amine 4. Amine 4 was transformed into
the secondary amine (5a) or the tertiary amine (5d) by
alkylation with ethyl bromide or into the amide deriva-
tive 5b, by acylation with the chloride methylester of glu-
taric acid. The free acid 5c was obtained after treatment
of 5b with aq KOH (10%). Hydrogenolysis of the benzyl
group of compounds 5a–d with H2/Pd–C led to the free
alcohols 6a–d, respectively.
Lymphoproliferation inhibition was assessed on
splenocyte cultures stimulated with concanavalin-A
(Con-A)19 and evaluated through the cellular respira-
tion levels determined by a colourimetric MTT as-
say.20 Cytotoxicity was determined in parallel.
AnapsosÒ, a hydroalcoholic extract obtained from
the rhizome of the American fern Phlebodium pseud-
oaureum (synom.: Polypodium leucotomos P., Polypo-
diaceae), was included in the tests as reference
standard. This drug, reported to be clinically useful
against neoplasms21 and autoimmune diseases such
as atopic dermatitis,22 psoriasis23,24 and vitiligo,25
might also have some utility in the treatment of aller-
gic disorders26 and as immunomodulator in vaccine
formulation against parasites.27
Diamine derivatives 7–9 were obtained from com-
pound 2a. The primary alcohol was first converted
into its mesylate, then into the azide and then reduced
with H2/Pd–C, NaBH4 to give the diamine 7a, which
treated with an excess of ethyl bromide or with one
The results of the assays, shown in Table 1, were calcu-
lated from colourimetric measures according to the
formulas:
R2
Boc-HN
O
CH3
N
R1
CH3
Boc-HN
2a
CH3
Boc-HN
CH3
H2N
CH3
13
13
13
13
13
iv (v)
i
ii
iii
OH
OBn
OH
OBn
OBn
1
3
4
5
iii
vii
60%
vi
5, 6
R1
Et
R2
H
H
H
Et
70%
H2N
H2N
CH3
13
a
b
c
d
R2
N
Boc-HN
7a
CH3
13
OH
2b
MeOOC(CH2)3CO
HOOC(CH2)3CO
Et
CH3
R1
13
NH2
OH
iii
6
CH3
iv (viii)
90-100%
13
NH2
7b
R1
Et
R2
Et
H
Boc-HN
CH3
13
H2N
CH3
R2
8, 9
a
b
iii
90%
13
R2
N
N
HOOC(CH2)3CO
R1
R1
9
8
Scheme 1. Synthesis of lipidic aminoalcohol and diamine derivatives. Reagents and conditions: (i) a—EtOOCCl/N-methylmorpholine/THF;
b—NaBH4/MeOH; (ii) BnCl/NaH/DMF; (iii) HCl(g)/THF; (iv) EtBr/Et3N/DMF; (v) glutaric acid monomethyl ester chloride/ether; (vi)
H2/Pd–C/AcOH; (vii) a—MsCl/Et3N/CH2Cl2; b—NaN3/DMF; Pd–C/HCCl3; (viii) glutaric anhydride/CH2Cl2.
6, 10 R1
R2
H
Et
H
H
R2
N
R2
N
a
d
e
f
Et
Et
n-Bu
n-Hex
n-Dec
n-Bu
Br
O
CH3
CH3
CH3
R1
R1
R1R2NH
80-100%
13
13
LiAlH4
30-70%
13
OH
O
OH
10
OH
6
g
h
H
n-Bu
Scheme 2. Synthesis of aminoalcohols of type 6 from 2-bromopalmitic acid.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6091–6095
6093
Table 1. Lymphoproliferation inhibition, cytotoxicity and nitrite production by lipidic aminoacid, aminoalcohol and diamine derivatives
Compound
Lymphoproliferation inhibition
Nitrite production increase
10 lg/mL
1 lg/mL
Cytotoxcity (%)
Inhibition (%)
Cytotoxcity (%)
Inhibition (%)
Normal
machrophages
LPS pre-stimulated
machrophages
1
32.9
72.4
79.1
49.8
3.4
5.1
20.2
12.2
11.9
2.5
nd
nd
15.1
0
nd
nd
ni
nd
nd
ni
2b
4
68.7
0
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
6g
6h
nd
nd
nd
nd
0
nd
ni
nd
ni
65.4
72.6
72.9
50.2
49.1
79.5
49.1
78.4
59.8
50.3
6.4
27.8
71.6
68.4
nd
ni
ni
15.6
17.1
18.9
14.5
12.8
16.4
14.6
12.6
11.7
14.1
10.5
nd
nd
7.5
nd
5.0
6.6
nd
nd
nd
nd
nd
nd
nd
ni
nd
nd
nd
nd
nd
nd
nd
74.8
nd
74.1
12.0
nd
25% (at 100 lg/mL)*
nd
nd
nd
nd
7a
81.2
4.3
73.4
4.2
ni
20% (at 10 lg/mL)
29% (at 100 lg/mL)*
7b
8a
8b
9a
9b
50.1
89.5
80.8
84.5
51.0
2.5
7.8
nd
nd
6.5
0
nd
nd
nd
nd
ni
65.2
19.3
74.2
nd
*
1.2
+
24.2
12.8
1.7
nd
ni
ni
ni
ni
10d
10e
43.1
32.9
47.8
nd
12.1
5.1
nd
nd
nd
nd
nd
nd
nd
ni
nd
nd
nd
ni
10h
AnapsosÒ
12.5
nd
nd
66.3
nd
23.2
nd, no determined; ni, no increase of NO production.
* Statistically significant increase, p < 0.05 (ANOVA Fisher test).
the potency, but the effect is also dependent on the size
of the alkyl chains. Thus, dialkylation of aminoalcohols
seems to increase the potency with the number of ethyl
groups: 2b (NH2) ꢃ 6a (NHEt) < 6d (NEt2); whereas in
the case of butyl derivatives the alkylation reduces con-
siderably the potency: 2b (NH2) ꢄ 6e (NHBu) ꢃ 6h
(NBu2). For monoalkylamino derivatives there is an opti-
mum C6-size of the chain: [2b < 6a(Et) > 6e(Bu) ꢅ 6f
(hex) ꢄ 6g (dec)]. Thus, within this series of aminoalco-
hol derivatives, compound 6d became the most potent
at those concentrations tested. In other sense, the pres-
ence of a tertiary amine seems to be better for the activity
than that of a secondary one 6d/6a (79.5/72.9), 6h/6e
(50.3/49.1) and 10h/10e (47.8/32.9).
Proliferation inhibition ð%Þ
¼ ½1 ꢀ ðODcellsþConAþcompdÞ=ðODcellsþConAÞꢁ ꢂ 100;
Cytotoxicityð%Þ ¼ ½1 ꢀ ðODcellsþcompdÞ=ðODcellsÞꢁ ꢂ 100;
ðOD ¼ Optical densityÞ
Twenty-four compounds, four aminoacids, nine
aminoalcohols, five benzyloxyamines and six diamines,
were tested in the lymphoproliferation inhibition assay
at a 10 lg/mL concentration initially. Those displaying
inhibition values higher than 50% were then tested at
1 lg/mL (Table 1).
Within the small number of 1,2-diamine derivatives test-
ed, 7–9, four compounds displayed lymphoproliferation
inhibition values over 80% at 10 lg/mL and two of them
over 70% at 1 lg/mL, compound 8a (79.5% at 10 lg/
mL) being the most potent inhibitor. The Boc group
on the 2-amino function increases the inhibitory activity,
as it can be observed through comparison of the pairs
7a/7b (81.2/50.1%), 8a/9a (89.5/84,5%) and 8b/9b (80.8/
51.0%) though the last pair contains another structure
change that could invalidate this comparison.
Thus, the starting Boc-aminoacid 1, and the N-alkyl- or
N,N-dialkyl-aminoacids 10d, 10e or 10h did not show
strong inhibition at 10 lg/mL and were not further tested.
Within the group of 2-aminohexadecanol derivatives
those having the free primary hydroxyl function, com-
pounds 2b and 6a–h, displayed fair proliferation inhibito-
ry effects, attaining up to almost 75% inhibition, in the
case of compound 6d. Benzyl ethers 4 and 5 showed great-
ly reduced potencies in general, as it can be seen for the
pairs of compounds 2b/4; 6a/5a; 6b/5b; 6d/5d, with a not
totally clear exception, for the pair 6c/5c. The number
of alkyl groups attached to the amino group influences
Taking into consideration the results of proliferation
inhibition and cytotoxicity, compounds 4, 6f, 7a, and

6094
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6091–6095
9a were selected for evaluating their response in the as-
say of delayed type of hypersensivity (DTH) induced by
the crude antigen of Trichinella spiralis (TSA).28 The
modulation of the inflammatory response was tested
after 24 and 48 h of administration and revealed that
compounds 6f (at 24 h) and 7a (at 48) increased, but
not significantly, the TSA response, while compounds
Acknowledgments
Financial support came from Spanish MCyT (Projects
AGL-2000-0039 and AGL-2005-02168 GAN) and
´
´
‘Redes Tematicas de Investigacion Cooperativa de
Centros de Enfermedades Tropicales’. RICET (FIS,
ref: CO3-C04 and ref.: PI05552). AP thanks the
USAL-CIETUS fellowship during this research.
4 and 9a decreased it, also with no statistical
significance.
In another experiment, some selected four aminoalco-
hol and four diamine derivatives were evaluated for
their ability for influencing nitric oxide production
on normal or pre-stimulated rat alveolar macrophages
in vitro.29 As it can be seen (Table 1), the diamine 8b
induced nitrite production in normal macrophage
References and notes
1. Borel, J. F. Pharmacol. Rev. 1990, 41, 259.
2. Peters, D. H.; Fitton, A.; Ploster, G. L.; Fanolds, D.
Drugs 1993, 46, 746.
3. Screiber, S. L. Science 1991, 251, 283.
4. Liu, J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.;
Weissman, I.; Schreiber, S. L. Cell 1991, 66, 807.
5. Screiber, S. L. Cell 1992, 70, 365.
6. European FK506 Multicentre Liver Study Group; Lancet
1994, 344, 423.
7. Fujita, T.; Toyama, R.; Sasaki, S.; Okumoto, T.; Chiba,
K.; Japan Patent (Japan Kokai), Heisei 01-104087, 1989;
Chem. Abstr. 1990, 112, 19950n.
cultures in
a dose-dependent manner (data not
shown), whereas compounds 6f30 and 7a31 increased
NO production significantly in the LPS pre-stimulated
macrophages culture. In those experiments we did not
find any increase of nitrite production in normal or
pre-stimulated macrophage cultures treated with
AnapsosÒ.
8. Fujita, T.; Inoue, K.; Yamamoto, T.; Ikumoto, S.; Sasaki,
S.; Toyama, R.; Chiba, K.; Hoshino, Y.; Okumoto, T.
J. Antibiot. 1994, 208, 47.
9. Fujita, T.; Hirose, R.; Hamamichi, N.; Kitao, Y.; Sasaki,
S.; Yoneta, M.; Chiba, K. Bioorg. Med. Chem. Lett. 1995,
5, 1875.
10. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.;
Mishina, T.; Sasaki, S.; Fujita, T. Bioorg. Med. Chem.
Lett. 1995, 5, 853.
11. Hamamichi, N.; Kitao, Y.; Matsuzaki, T.; Chiba, K.;
Fujita, T. Bioorg. Med. Chem. Lett. 1996, 6, 2647.
12. Lucas, R.; Ubeda, A.; Paya, M.; Alves, M.; Olmo, E.;
Lopez, J. L.; San Feliciano, A. Bioorg. Med. Chem. Lett.
2000, 10, 285.
13. Olmo, E.; Alves, M.; Lopez, J. L.; Inchaustti, A.; Yaluff,
G.; Rojas de Arias, A.; San Feliciano, A. Bioorg. Med.
Chem. Lett. 2002, 12, 659.
14. Olmo, E.; Macho, M.; Alves, M.; Lopez, J. L.; Banaua, F.;
Mun˜oz, E.; San Feliciano, A. Bioorg. Med. Chem. Lett.
2002, 12, 2621.
15. Lucas, R.; Alves, M.; Olmo, E.; San Feliciano, A.; Paya,
M. Biochem. Pharmacol. 2003, 65, 1539.
16. Lucas, R.; Villamon, E.; Paya, M.; Alves, M.; Olmo, E.;
Gozalbo, D.; Gil, M. L. FEMS Immunol. Med. Chem.
2004, 40, 239.
As an additional proof for establishing their immuno-
modulatory potential, the two selected compounds, 6f
and 7a, were evaluated through a Graft-vs-Host
assay32 to define their immunosuppresant ability in
comparison with cyclophosphamide (Cy, 100 mg/kg/
day), taken as the reference drug. The aminoalcohol
derivative 6f significantly reduced the lymphocyte
stimulation by some 63%, close to the Cy immunosup-
pression value of 71%. In the same experimental con-
ditions the diamine 7a attained also to reduce
lymphocyte stimulation by around 32%, though with
no statistical significance.
Other in vitro experiments carried out with the select-
ed compounds were focused on immunophenotype
characterisation, through analysing changes in the
production of a number of immunomodulation rele-
vant cells by flux cytometry. Both compounds 6f
and 7a increased the percentage of T helper lympho-
cytes CD4+ or CD8 and I-Ek cells of exposure to
antigen, though with no statistical significance (data
not shown).
17. Gibbons, W. A.; Hughes, R. A.; Charalanibous, M.;
Christodoulou, M.; Szeto, A.; Aulabauh, A. E.; Mascagni,
P.; Toth, I. Liebigs Ann. Chem. 1990, 1175.
18. Kokotos, G. Synthesis 1990, 299.
In summary, eight out of those twenty-four com-
pounds evaluated were better than the reference drug
AnapsosÒ, and some of them also more potent than
those literature compounds OA-12 and FTY720, for
inhibiting lymphoproliferation, with low toxicity. After
several immunoevaluation assays, compounds 6f and
7a have proven their good qualities, and have been
selected to follow further in vivo evaluations as poten-
tial adjuvants for vaccination. In order to progress in
this research, the racemic resolution of these com-
pounds 6f and 7a is being carried out. Additionally,
taking into account the reduced number of com-
pounds included in the initial prospective screening,
the synthesis and evaluation of larger aminoalcohol
an diamine libraries of compounds are currently being
considered.
19. Perteguer, M. J.; Rodero, M.; Flores, J. M.; Dorea, R.
´
C. C.; Cuellar del Hoyo, C. Parasitol. Res. 2001, 87,
396.
20. Tada, H.; Shiho, O.; Kuroshima, K.; Koyama, M.;
Tsukamoto, K. J. Immunol. Methods 1986, 93, 157.
21. Horvath, A.; Alvarado, E.; Szocs, J.; Alvarado, Z. N.;
Padilla, G. Nature 1967, 277, 67.
´
22. Jimenez, D.; Naranjo, R.; Doblare, E.; Mun˜oz, C.;
Vargas, J. F. Allergy Immunopathol. 1987, 15, 185.
´
´
23. Hernan, P.; Hector, N.; Pacheco, J. Int. J. Dermatol. 1974,
13, 276.
24. Thomas-Barry, A. Am. Fern. J. 1999, 89, 101.
25. Mohammad, A. Int. J. Dermatol. 1989, 28, 479.
26. Yssel, H.; Faster, S.; de Vries, J.; Waal, R. Int. Immunol.
1994, 6, 1091.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6091–6095
6095
´
´
´
27. Martınez-Fernandez, A. R.; Nogal, J. J.; Lopez, J.;
Ramajo, V.; Oleaga, A.; Monge, Y.; Hillyer, G. V.; Muro,
A. Vet. Parasitol. 2004, 126, 287.
C22H47NO: C, 77.35; H, 13.87; N, 4.10. Found: C,
77.05; H, 13.52; N, 4.07.
31. Analytical data for compound 7a. mp: 56 °C. IR
28. Asano, K.; Matsuo, S.; Okamoto, K. Immunol. Lett. 1995,
46, 195.
29. Andrade, M. A.; Siles, M.; Espinoza, E.; Perez Arellano,
J.; Gottstein, B.; Muro, A. Nitric Oxide 2004, 10, 150.
mmax: 3349, 2918, 2850, 1687, 1531, 1365, 1349 and
1174 cmꢀ1
4.48 (1H, sa, NH-Boc), 3.51 (1H, m, H-2), 2.77 (1H,
. d (ppm)
1H RMN (200 MHz, CDCl3):
´
dd, J1 = 12.8, J2 = 7.0 Hz, H-1A); 2.61 (1H, dd,
J1 = 12.8,
30. Analytical data for compound 6f. IR mmax: 3600, 3050,
J2 = 4.0 Hz,
H-1B);
1.25
[26H,
m,
2927, 1522, 1220, 1046 and 928 cmꢀ1
.
1H RMN
(CH2)13]; 1.49, [9H, s, (CH3)3], 0.88, (3H, t, CH3).
13C RMN (50.3 MHz, CDCl3): d (ppm) 155.3, 54.7,
50.4, 33.0, 32.0, 29.7, 29.4, 28.5, 26.1, 22.8,
14.2 ppm. MS (FAB) m/z = 357,37. Anal. Calcd for
C21H44N2O2: C, 70.73; H, 12.44; N, 7.86. Found: C,
70.65; H, 12.38; N, 7.81.
(200 MHz, CDCl3): d (ppm) 3.62 (1H, dd, J1 = 10.2,
J2 = 6.9 Hz, H-1A); 3.25 (1H, dd, J1 = 10.2, J2 = 6.5 Hz,
H-1B); 2.60 (3H, m, H-2 +N-CH2); 1.25 [34H, m,
(CH2)13 + (CH2)4]; 0.91, (3H, t, CH3), 0.87, (3H, t,
CH3). 13C RMN (50.3 MHz, CDCl3): d (ppm) 62.8, 59.2,
46.9, 31.9, 31.7, 31.3, 30.1, 29.7, 29.4, 27.0, 26.2, 22.7,
14.1 ppm. MS (CI) m/z = 341,37. Anal. Calcd for
32. Ford, W. L.; Burr, W.; Simonsen, M. Transplantation
1970, 10, 258.