D.A. Smuga et al. / Steroids 75 (2010) 1146–1152
1149
CH2Cl2:benzene (1:1, v/v, 3× 5 ml). The dried residue was dis-
solved in dry pyridine (1.5 ml) and DCC (49 mg, 0.24 mmol) in
500 ml of dry pyridine was added. After stirring for 48 h, the reac-
tion was quenched with addition of H2O (100 l) and then filtered
and concentrated in vacuo. The oily residue was chromatographed
on a silica-gel column with increasing amounts (from 2% to 10%)
of CH3OH in CHCl3. The corresponding fractions (checked on TLC
with the primuline test) were collected and evaporated to give the
appropriate phosphatide derivative of DHEA. Purity of compounds
was determined by HPLC (>98%).
30.89, 31.33 (C-2), 32.17, 34.35 (C-2 from acid), 34.50(C-2 from
acid), 36.13 (C-16), 37.02 (C-1), 37.62 (C-8, C-10), 40.45 (C-4), 42.60
(C-9), 45.25 (C-14), 47.61 (C-13), 62.82 (C-1ꢀ), 63.81 (C-3ꢀ), 63.92 (C-
7), 70.77 (C-2ꢀ), 75.73 (C-3), 124.24 (C-6), 145.32 (C-5), 173.88 (C-1
from acids), 174.25 (C-1 from acids), 221.0 (C-17).
31P NMR (243 MHz, CDCl3:MeOD, 2:1, v/v) ı: −2.08.
2.11.4. 3ˇ-O-(1,2-Diacyl-sn-glycero-3-phospho)-7˛
-hydroxy-androst-5-en-17-one (P-7˛-OH-DHEA) (6b)
Yield 59%, light yellow solid.
TLC Rf: 0.23 (CHCl3:MeOH:NH3 aq., 85:15:2, v/v/v), HPLC
Rt = 13.6 min.
2.11.1. 3ˇ-O-(1,2-Dipalmitoyl-sn-glycero-3-phospho)
-androst-5-en-17-one (DPP-DHEA) (5a)
Yield 74%, white solid.
1H NMR (600 MHz, CDCl3:MeOD, 2:1, v/v) ı: 0.89 (t, J = 7.2 Hz,
6H, 2× CH3– from acids), 0.90 (s, 3H, CH3-18), 1.03 (s, 3H, CH3-19),
1.21–1.40 (m, 46H, 22× CH2 from acids), 1.57–1.65 (m, 4H, 2× CH2-
3 from acids), 2.28–2.36 (m, 4H, 2× CH2-COO from acids), 2.74–2.90
(m, 1H, CH-CH2-CH from acids), 3.91 (m, 1H, H-7), 3.94 – 4.02
(m, 3H, CH2-3ꢀ, H-3), 4.18 (m, 1H, one of CH2-1ꢀ), 4.42 (m, 1H, one of
CH2-1ꢀ), 5.23 (m, 1H, H-2ꢀ), 5.29–5.45 (m, 2H, –CH CH– from acids),
5.62 (dd, J = 5.3, 1.5 Hz, 1H, H-6).
TLC Rf: 0.29 (CHCl3:MeOH:NH3 aq., 85:15:2, v/v/v), HPLC
Rt = 11.6 min.
+
−
917.7.
ESI-MS m/z: [M−NH4
]
1H NMR (600 MHz, CDCl3:MeOD, 2:1, v/v) ı: 0.89 (t, J = 7.0 Hz,
6H, 2× CH3-16 from acids), 0.91 (s, 3H, CH3-18), 1.06 (s, 3H, CH3-
19), 1.19–1.44 (m, 48H, 2 × 12× CH2 from acids), 1.57–1.65 (m, 4H,
2× CH2-3 from acids), 2.25–2.40 (m, 4H, 2× CH2-COO from acids),
3.94–4.03 (m, 3H, H-3, CH2-3ꢀ), 4.19 (dd, J = 12.0, 6.6 Hz, 1H, one of
CH2-1ꢀ), 4.40 (m, 1H, one of CH2-1ꢀ), 5.23 (m, 1H, H-2ꢀ), 5.40 (m, 1H,
H-6).
31P NMR (243 MHz, CDCl3:MeOD, 2:1, v/v) ı: −1.90.
2.12. Conversion of phosphatides to ammonium salt
13C NMR (150 MHz, CDCl3:MeOD, 2:1, v/v) ı: 13.76 (C-18), 14.22
(2× C-16 from acids), 19.56 (C-19), 20.65 (C-11), 22.17 (C-15), 22.97
(2× C-15 from acids), 25.22 (2× C-3 from acids), 29.50, 29.67, 30.00,
31.11 (C-7), 31.68 (C-8), 31.82 (C-12), 32.24, 34.43 (C-2 from acid),
34.60 (C-2 from acid), 36.21 (C-16), 36.96 (C-10), 37.41 (C-1), 40.55
(C-4), 48.07 (C-13), 50.57 (C-9), 52.11 (C-14), 62.87 (C-1ꢀ), 63.80 (C-
3ꢀ), 70.82 (C-2ꢀ), 76.18 (C-3), 121.79 (C-6), 140.85 (C-5), 173.86 (C-1
from acids), 174.25 (C-1 from acids), 221.2 (C-17).
Phosphatide was dissolved in CHCl3:MeOH:H2O (5:4:1, v/v/v)
and applied to a column DOWEX 50W X8 (NH4 form) resin. The
required product was eluted with the same solvent, the solution
was evaporated in vacuo and the residue was co-evaporated several
times from CHCl3:MeOH (2:1, v/v). The solid was lyophilised from
benzene to give a solid residue.
+
3. Results and discussion
31P NMR (243 MHz, CDCl3:MeOD, 2:1, v/v) ı: −2.54.
cation. This situation is most crucial after oral administration [28].
In a similar way, DHEA has many diverse physiological, biologi-
cal and biochemical effects in vitro, but oral DHEA administration
produces far more limited clinical effects [4,10,11]. One of the pos-
sible solutions to this problem is the conversion of DHEA to active
metabolites and/or its transformation into prodrug form.
In this article, the synthesis of DHEA analogues modified with
tive (7␣-OH DHEA), two different phosphatidic acids, DPPA (2a)
and 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA: 2b), were used.
These acids were obtained by the enzymatic hydrolysis of DPPC (1a)
and PC (1b) from egg yolk, respectively (Scheme 1).
was due to the fact that PC obtained from egg yolk contains polyun-
saturated fatty acids (PUFAs), that is, linoleic acid, arachidonic
human nutrition and show many biological and medical properties
[29–31].
DPPC (1a) was obtained according to well-known procedures
[32,33] in the reaction of cadmium salt of sn-glycero-3-
phosphocholine with palmitic acid using as a coupling agent DCC
was isolated from egg yolk according to the method described by
Palacios and Wang [23].
2.11.2. 3ˇ-O-(1,2-Diacyl-sn-glycero-3-phospho)
-androst-5-en-17-one (P-DHEA) (5b)
Yield 75%, light yellow solid.
TLC Rf: 0.29 (CHCl3:MeOH:NH3 aq., 85:15:2, v/v/v), HPLC
Rt = 11.7 min.
1H NMR (600 MHz, CDCl3:MeOD, 2:1, v/v) ı: 0.89 (t, J = 7.2 Hz,
6H, 2× CH3-16 from acids), 0.91 (s, 3H, CH3-18), 1.02 (m, 1H, H-
9), 1.05 (s, 3H, CH3-19), 1.22–1.42 (m, 44H, 22× CH2 from acids),
1.57–1.65 (m, 4H, 2× CH2-3 from acids), 1.94–2.17 (m, 4H, 2× CH2-
CH CH- from acids), 2.27–2.39 (m, 4H, 2× CH2-COO from acids),
2.78 (m, 1H, CH-CH2-CH from acids), 3.92–4.03 (m, 3H, H-3, CH2-
3ꢀ), 4.19 (dd, J = 12.0, 6.6 Hz, 1H, one of CH2-1ꢀ), 4.41 (dd, J = 12.0,
3.4 Hz, 1H, one of CH2-1ꢀ), 5.23 (m, 1H, H-2ꢀ), 5.27–5.37 (m, 2H,
–CH CH– from acids), 5.40 (m, 1H, H-6).
31P NMR (243 MHz, CDCl3:MeOD, 2:1, v/v) ı: −2.18.
2.11.3. 3ˇ-O-(1,2-Dipalmitoyl-sn-glycero-3-phospho)-7˛
-hydroxy-androst-5-en-17-one (DPP-7˛-OH-DHEA) (6a)
Yield 57%, white solid.
TLC Rf: 0.23 (CHCl3:MeOH:NH3 aq., 85:15:2, v/v/v), HPLC
Rt = 13.7 min.
+
−
933.7.
ESI-MS m/z: [M−NH4
]
1H NMR (600 MHz, CDCl3:MeOD, 2:1, v/v) ı: 0.89 (t, J = 7.0 Hz,
6H, 2× CH3-16 from acids), 0.90 (s, 3H, CH3-18), 1.02 (s, 3H, CH3-
19), 1.21–1.35 (m, 48H, 24× CH2 from acids), 1.55–1.64 (m, 4H,
2× CH2-3 from acids), 2.28–2.36 (m, 4H, 2× CH2-COO from acids),
3.92 (m, 1H, H-7), 3.95–4.01 (m, 3H, CH2-3ꢀ, H-3), 4.18 (m, 1H, one
of CH2-1ꢀ), 4.40 (dd, J = 12.0, 3.4 Hz, 1H, one of CH2-1ꢀ), 5.23 (m, 1H,
H-2ꢀ), 5.62 (dd, J = 5.3, 1.6 Hz, 1H, H-6).
The enzymatic hydrolysis of PCs (1a) and (1b) were carried out
in a biphasic system by phospholipase D from S. chromofuscus with
very good yields (89% and 82%, respectively) (Scheme 1).
The key reactions of DHEA and 7␣-OH-DHEA with PAs were
carried out by the phosphodiester approach, which was often
used in the synthesis of phospholipids modifications [15,28] or
13C NMR (151 MHz, CDCl3:MeOD, 2:1, v/v) ı: 13.41 (C-18), 14.12
(2× C-16 from acids), 18.35 (C-19), 20.33 (C-11), 22.03 (C-15), 22.90
(2× C-15 from acids), 25.15 (2× C-3 from acids), 29.40, 29.60, 29.92,