New silybin scaffold for chemical diversification: Synthesis of novel 23-phosphodiester silybin conjugates

Article abstract of DOI:10.1055/s-0032-1317688

Silybin is the major component (ca. 30%) of the silymarin complex extracted from the seeds of Silybum marianum, with multiple biological activities operating at various cell levels. As an ongoing effort toward the exploitation of natural products as scaffolds for chemical diversification at readily accessible positions, we present here an efficient synthetic procedure to obtain new 23-phosphodiester silybin conjugates with different labels. A key point in our approach is the new 3,5,7,20-tetra-O-acetylsilybin-23-phosphoramidite, useful for a variety of derivatizations following a reliable and well-known chemistry. The feasibility of the procedure has been demonstrated by preparing new 23-silybin conjugates, exploiting standard phosphoramidite chemistry. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0032-1317688

LETTER
▌45
lNetteerw Silybin Scaffold for Chemical Diversification: Synthesis of Novel
23-Phosphodiester Silybin Conjugates
Efficient Synthesis of Novel 23-Phosphodiester Silybin Analogues
Armando Zarrelli, Valeria Romanucci, Marina Della Greca, Lorenzo De Napoli, Lucio Previtera, Giovanni Di Fabio*
Department of Chemical Sciences, University of Napoli 'Federico II', via Cintia 4, 80126 Napoli, Italy
Fax +39(081)674393; E-mail: difabio@unina.it
Received: 01.10.2012; Accepted after revision: 02.11.2012
bioavailability. In an attempt to improve its biological
Abstract: Silybin is the major component (ca. 30%) of the silyma-
properties and facilitate in vivo applications of silybin,
rin complex extracted from the seeds of Silybum marianum, with
only limited structural modifications have been
multiple biological activities operating at various cell levels. As an
ongoing effort toward the exploitation of natural products as scaf-
proposed3,8–12 and the available analogues are still unsat-
folds for chemical diversification at readily accessible positions, we isfactory. Therefore new synthetic approaches for selec-
present here an efficient synthetic procedure to obtain new 23-phos-
phodiester silybin conjugates with different labels. A key point in
our approach is the new 3,5,7,20-tetra-O-acetylsilybin-23-phos-
phoramidite, useful for a variety of derivatizations following a reli-
tively modifying silybin are of interest.
As a part of our continuing research effort towards the
synthesis of new natural product analogues,13,14 we pres-
ent here the preliminary results of a efficient synthetic
procedure to obtain new 23-phosphodiester silybin conju-
gates with different labels.
able and well-known chemistry. The feasibility of the procedure has
been demonstrated by preparing new 23-silybin conjugates, exploit-
ing standard phosphoramidite chemistry.
Key words: natural products, flavonolignans, silybin, phosphory-
lation, drugs
The introduction of a phosphate group may bring pharma-
ceutical and pharmacokinetic benefits.15 Conjugation is
usually considered as an efficient route in drug discovery
to improve the biological properties of a large number of
drugs and can improve the bioavailability and delivery as
well as the biological activity.
Flavonoids and flavonolignans are widely distributed
among various citrus plants and are frequently found in
the human diet. They may act as enzyme inhibitors, free-
radical scavengers, antitumor agents, antibacterial agents,
anti-inflammatory agents, and antioxidants.1–3
We chose to start from the new 23-phosphoramidite build-
ing block 3 (Scheme 1) which could be transformed into a
series of conjugates using a solution-phase parallel array
protocol, exploiting standard and reliable phosphorami-
dite chemistry.15 We initially converted silybin (1) into its
23-ODMT ether by a reaction with DMT-chloride in pyr-
idine at 50 °C. After exhaustive acetylation with an excess
of acetic anhydride in pyridine, subsequent treatment with
5% formic acid in dichloromethane allowed the removal
of the DMT protecting group to give 2 in 75% yield and
this could be converted into the corresponding phosphor-
amidite derivatives 3. Thus intermediate 2 was reacted
Silybin (Figure 1) is the major component (ca. 30%) of the
silymarin complex extracted from the seeds of Silybum
marianum consisting of two diastereomers A and B in a
ratio of approximately 1:1.
23
16
O
O
10
11
15
OH
8
HO
O
17
2
3
21
13
6
18
OH
OH
4
5
with
2-cyanoethyl-N,N-diisopropylamino-chloro-
OH
O
OMe
B (2R,3R,10S,11S)
phosphoramidite and DIPEA in anhydrous dichlorometh-
ane. In these preliminary studies, the silybin used was a
mixture of diastereomers, and the derivatives 3 were ob-
tained as a mixture of inseparable diastereomers, although
the 1H NMR and 31P NMR spectra appeared to be of a sin-
gle compound. After purification the identities of com-
pounds 3, obtained in good yields (65%), were confirmed
by NMR (1H, 13C, and 31P) and ESI-HRMS analysis.16
A (2R,3R,10R,11R)
Figure 1 Chemical structure of silybin A and silybin B
Silybin is a natural compound with multiple biological ac-
tivities operating at various cell levels most of them relat-
ed to its radical-scavenging activity. Along with the
beneficial activities resulting from the antioxidant and
radical-scavenging properties,3,4 silybin has recently re- Subsequently we selected a group of model molecules
ceived attention due to its anticancer and chemopreven- having a free hydroxyl group (A–E, Scheme 1), useful for
tive actions,5,6 as well as hypocholesterolemic, coupling with the key intermediate 3.17 In particular, we
cardioprotective, and neuroprotective activities.3,7 In vivo selected molecules known for their ability to act as molec-
applications of silybin are rather hampered by its very low ular carriers (steroids, bile acids),18–21 to improve water
solubility (polyethers)22 and as radical scavengers (nucle-
osides).23,24 While A, B, and C are commercially avail-
able, D and E were efficiently obtained starting from 2′-
deoxyadenosine and 3α,7α,12α,24-tetrahydroxycholane,
SYNLETT 2013, 24, 0045–0048
Advanced online publication: 04.12.2012
DOI: 10.1055/s-0032-1317688; Art ID: ST-2012-D0837-L
© Georg Thieme Verlag Stuttgart · New York
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6

46
A. Zarrelli et al.
LETTER
OH
OH
O
O
O
O
a) DMTCl, DMAP, 50 °C, 87%
b) Ac2O, r.t., 92%
HO
O
OMe
OH
AcO
O
O
OMe
OAc
c) formic acid (5%), r.t., 75%
OH
OAc
OH
O
OAc
2
silybin (1)
CEO
N(i-Pr)2
DIPEA, 20 min, r.t., 65%
P
d)
O
P
Cl
O
R
O
OCE
O
O
OR2
O
P
R
OH
R1O
O
O
N(i-Pr)2
O
O
e) tetrazole, 30 min, r.t.
f) 5.5 M t-BuOOH
AcO
O
OMe
OR1
OR1
OR1
OMe
OAc
OAc
4a–e R1 = Ac; R2 = CE = 2-cyanoethyl
5a–e R1 = R2 = H
OAc
O
NH4OH, MeOH
1 h, r.t.
3
5a = 68%; 5b = 55%; 5c = 72%; 5d = 70%; 5e = 65%*
NHR1
N
N
O
R1O
N
NH
O
OH
N
H
HO
HO
N
O
R
OH
O
O
HO
OMe
H
OR1
H
4
R1O
H
OR1
R1O
R1O
HO
A
B
D
C
E
Scheme 1 Synthesis of 23-phosphodiester silybin conjugates 5a–e. *Yields calculated from starting product 3; CE = cyanoethyl
respectively. Exploiting the different reactivity of hydrox- by chromatography, although the NMR spectra of many
yl groups our studies were carried out using TBDMSCl or of them appeared to indicate a single compound.
DMTCl for the transient protection of the primary OH
In conclusion a facile and efficient protocol for the syn-
moiety while all other groups were protected by acetyla-
thesis of a broad array of new 23-phosphodiester silybin
tion. 2′-Deoxyguanosine and 3α,7α,12α,24-tetrahydroxy-
structured conjugates has been achieved. The feasibility
of this procedure has been demonstrated by preparing new
cholane were protected by reaction with TBDMSCl and
DMTCl, respectively.25,26 The products were acetylated
23-silybin conjugates, exploiting standard phosphorami-
with acetic anhydride to yield fully protected products. Fi-
dite chemistry. A key point in our strategy is the new sily-
nally, the TBDMS group was cleaved by Et3N·3HF and
bin building block 3, useful for a variety of derivatizations
following established chemistry. In principle, this meth-
DCA to yield N2-acetyl-2′,3′-O-diacetyl-deoxyguanosine
(D) and 3α,7α,12α-O-triacetyl-24-hydroxycholane (E) in
overall yields of 80 and 75%, respectively.
odology can be readily extended to other molecules which
have a free hydroxyl group.15,28
The coupling of 3 with A–E (Scheme 1) was carried out
by using the classic coupling reagent (0.45 M tetrazole in
MeCN), and then treatment with 5.5 M tert-butyl hydro-
Acknowledgment
This study was supported by AIPRAS Onlus (Associazione Italiana
per la Promozione delle Ricerche sull’Ambiente e la Saluta umana).
We also thank Dr. Vincenzo Perino of the CIMCF, Università degli
Studi di Napoli ‘Federico II’, for access to NMR facilities.
peroxide solution in decane led to phosphotriesters 4a–e.
After purification by flash chromatography, the deriva-
tives 4 were treated with concemtrated aqueous ammonia
and MeOH (1:1, v:v) at room temperature, allowing full
deprotection, leading to the desired phosphodiester deriv-
atives 5a–e in good yields (Scheme 1).27 All final deriva- Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnuIpofoiprmntgirStanoIuifpog
r
t
iornat
tives 5a–e were purified by flash chromatography and
then characterized by NMR (1H and 31P) and MS analysis.
All the intermediates and final derivatives were obtained
as mixtures of two diastereomers, that were inseparable
References and Notes
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Synlett 2013, 24, 45–48
© Georg Thieme Verlag Stuttgart · New York

LETTER
Efficient Synthesis of Novel 23-Phosphodiester Silybin Analogues
47
(2) Ferrazzano, G. F.; Amato, I.; Ingenito, A.; Zarrelli, A.; Pinto,
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62.6, 62.2 (C-23, C-23′), 58.8, 58.5 (OCH2CH2CN), 56.0
(OCH3), 43.3, 43.1 {N[CH(CH3)2]2}, 24.5 {N[CH(CH3)2]2},
21.1, 20.9, 20.7, 20.4 (CH3 of acetyl groups), 19.9
(OCH2CH2CN) ppm. 31P NMR (161.98 MHz, CDCl3): δ =
152.9, 150.2 ppm. ESI-HRMS (positive ions): m/z calcd for
C42H47N2O15P: 850.2714; found: [MH]+: 851.2788; [MNa]+
= 873.2606.
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Bioconjugate Chem. 1997, 8, 520.
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(25) Wolf, S.; Zismann, T.; Lunau, N.; Meier, C. Chem.–Eur. J.
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Chem. Commun. 2005, 2586; and references cited therein.
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(27) General Procedure for the Synthesis of Conjugates 5 (A–
E)
Derivative 3 (150 mg, 0.22 mmol) and the requisite
compound A–E (0.21 mmol, previously dried and kept
under reduced pressure, were reacted with a 0.45 M tetrazole
solution in anhydrous MeCN (1.0 mL, 0.45 mmol). The
reaction was left under stirring at r.t. and monitored by TLC
with an eluent system n-hexane–EtOAc = 1:1 (v/v). After 1
h, a 5.5 M t-BuOOH solution in decane (100 μL) was added
to the mixture and left stirring at r.t. After 30 min the reaction
mixture was diluted with CHCl3, transferred into a
separatory funnel, washed three times with H2O,
concentrated under reduced pressure, and purified by flash
chromatography, eluting with n-hexane–EtOAc = 7:3 (v/v),
to afford pure 4a–e as a yellow-brown amorphous powder.
Treatment with concd aq NH3 in MeOH (1:1, v/v) for 1 h at
r.t., led to full removal of the acetyl and 2-cyanoethyl
groups. The concentrated mixture was then purified on a
silica gel column, eluting with CHCl3–MeOH containing
increasing proportions of MeOH. Compounds 5a–e thus
obtained were converted into the corresponding sodium salts
by cation exchange on a DOWEX (Na+ form) resin to obtain
homogeneous samples in good yield (55–72%). See the
Supporting Information.
Compound 5a: NMR spectra of this compound showed
dramatic line broadening, diagnostic of a slow equilibrium
on the NMR time scale, which could suggest a strong
propensity toward aggregation in CDCl3. 1H NMR (500
MHz, CDCl3, r.t., mixture of diastereomers): δ = 7.02–6.60
(6 H, complex signals), 5.85 (2 H, br s), 5.59 (1 H, dd, J =
11.9, 11.7 Hz), 5.11 (2 H, complex signals), 4.78 (1 H, br s),
4.04 (1 H, br signal), 3.95–3.63 (6 H, complex signals), 2.05
(2 H, br signal), 1.92–0.99 (26 H, complex signals), 0.85 (3
H, br s), 0.79 (3 H, d, J = 4.8 Hz), 0.74 (6 H, br s), 0.55 (3 H,
s) ppm. 31P NMR (161.98 MHz, CDCl3): δ = –0.83 ppm.
HRMS (MALDI-TOF, negative ions): m/z calcd for
C52H66O13P: 929.4246; found: 929.4246 [M – H]–.
Compound 5b: 1H NMR (500 MHz, CD3OD, r.t., mixture of
diastereomers): δ = 7.11–6.72 (6 H, complex signals), 5.91
(1 H, d, J = 1.5 Hz), 5.85 (1 H, m), 4.98–4.75 (2 H, complex
signal), 4.41 (1 H, d, J = 11.5 Hz), 4.21–4.05 (5 H, m), 3.88–
3.68 (24 H, complex signals) ppm. 31P NMR (161.98 MHz,
(16) Natarajan, V.; Jeong, K. S.; Byeang, K. Curr. Med. Chem.
2003, 10, 1973.
(17) General Procedure for the Preparation of
Phosphoramidite 3
To 3,5,7,20-tetra-O-acetylsilybin (2, 240.0 mg, 0.37 mmol)
dissolved in anhydrous CH2Cl2 (5 mL), DIEA (390 μL,
2.22 mmol), and 2-cyanoethyl-N,N-diisopropylamino-
chlorophosphoramidite (107 μL, 0.48 mmol) were mixed
under argon. After 30 min the solution was diluted with
EtOAc, and the organic phase was washed twice with brine
and then concentrated. Silica gel chromatography of the
residue (eluent n-hexane–EtOAc = 3:7, v/v, in the presence
of 1% of Et3N), afforded desired compound 3 (205.0 mg,
0.24 mmol) in a 65% yield. Rf = 0.8 (n-hexane–EtOAc = 3:7,
v/v).
1H NMR (500 MHz, CDCl3, r.t., mixture of diastereomers):
δ = 7.11–6.90 (6 H, overlapped signals, H-13, H-15, H-16,
H-18, H-21, H-22), 6.81 (1 H, s, H-8), 6.58 (1 H, s, H-6),
5.66 (1 H, d, J = 11.6 Hz, H-3), 5.37 (1 H, d, J = 11.6 Hz, H-
2), 5.03 (1 H, d, J = 7.6 Hz, H-11), 4.13 (1 H, m, H-10),
3.90–3.60 {9 H, complex signals, OCH3, H-23,
OCH2CH2CN, N[CH(CH3)2]2}, 2.65 (2 H, complex signals,
OCH2CH2CN), 2.37, 2.32, 2.29, 2.04 (12 H, s for each, CH3
of acetyl groups), 1.25–1.15 {12 H, complex signals,
N[CH(CH3)2]2} ppm. 13C NMR (125 MHz, CDCl3, r.t.,
mixture of diastereomers): δ = 185.3 (C-4), 169.1 (C-7),
168.7, 167.8 (CO of acetyl groups), 162.5 (C-5), 156.3 (C-
8a), 151.4 (C-19), 144.3 (C-20), 143.6 (C-16a), 140.2 (C-
12a), 134.8 (C-14), 123.0 (C-17), 120.8 (C-15), 119.9 (C-
22), 119.8, 117.6, 117.2 (C-13, C-16, OCH2CH2CN), 116.2
(C-21), 111.4, 111.2, 110.6, 108.9 (C-6, C-8, C-18, C-4a),
81.0 (C-2), 76.2 (C-10), 75.8 (C-11), 73.3, 73.1 (C-3, C-3′),
© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 45–48

48
A. Zarrelli et al.
LETTER
CDCl3): δ = 3.1 ppm. HRMS (MALDI-TOF, negative ions):
m/z calcd for C36H44O18P: 795.2271; found: 795.2271 [M –
H]–.
(1 H, m), 4.54 (1 H, m), 4.41 (1 H, d, J = 11.5 Hz), 4.35 (1
H, m), 4.20 (2 H, m), 4.21–4.05 (3 H, m), 3.88–3.68 (3 H, s)
ppm. 31P NMR (161.98 MHz, CDCl3): δ = 3.1 ppm. HRMS
(MALDI-TOF, negative ions): m/z calcd for C35H33N5O16P:
810.1665; found: 810.1666 [M – H]–.
Compound 5c: 1H NMR (500 MHz, CD3OD, r.t., mixture of
diastereomers): δ = 7.79 (1 H, s), 7.11–6.72 (6 H, complex
signals), 6.41 (1 H, dd, J = 6.8, 6.8 Hz), 5.91 (1 H, d, J = 1.5
Hz), 5.85 (1 H, m), 4.97–4.74 (2 H, complex signal), 4.65 (1
H, m), 4.41 (1 H, d, J = 11.5 Hz), 4.22–4.10 (4 H, m), 4.00–
3.85 (5 H, complex signals), 2.44 (2 H, m), 1.99 (3 H, s)
ppm. 31P NMR (161.98 MHz, CDCl3): δ = 3.3 ppm. HRMS
(MALDI-TOF, negative ions): m/z calcd for C35H34N2O17P:
785.1600; found: 785.1602 [M – H]–.
Compound 5e: 1H NMR (500 MHz, CD3OD, r.t., mixture of
diastereomers): δ = 7.08–6.81 (6 H, complex signals), 5.95–
5.89 (2 H, m), 5.07 (1 H, m), 4.97 (1 H, m), 4.52 (2 H, m),
4.24 (1 H, m), 4.06 (1 H, m), 3.95–3.68 (7 H, complex
signals), 3.30 (1 H, m), 2.01–0.70 (33 H, complex signals)
ppm. 31P NMR (161.98 MHz, CDCl3): δ = 4.3 ppm. ESI-MS
(positive ions): m/z calcd for C49H63O16P: 938.39; found:
939.48 [MH]+; [MNa]+ = 961.37. HRMS (MALDI-TOF,
negative ions): m/z calcd for C49H62O16P: 937.3781; found:
937.3782 [M – H]–.
Compound 5d: 1H NMR (500 MHz, CD3OD, r.t., mixture of
diastereomers): δ = 8.56 (1 H, s), 8.29 (1 H, s), 7.11–6.72 (6
H, complex signals), 6.05 (1 H,d, J = 6.0 Hz), 5.91 (1 H, d,
J = 1.5 Hz), 5.85 (1 H, m), 4.90 (2 H, complex signals), 4.63
(28) Stawinski, J.; Kraszewski, A. Acc. Chem Res. 2002, 35, 952.
Synlett 2013, 24, 45–48
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