Unexpected B-ring regioselective di-nitration of diosmetin, a Citrus flavonoid

Article abstract of DOI:10.1016/j.tetlet.2009.12.062

Nitration in position C-8 of diosmetin, an easily available citroflavonoid, was studied in order to gain access to original analogs. The one-step nitration proved impossible, as mono or di-nitration on C-2′ and C-6′ positions on the lateral B ring of the molecule was exclusively observed. This surprisingly straightforward di-nitration of ring B, showing a lack of reactivity of ring A despite its high activation, has never been mentioned to date. Nitration in position C-8 was therefore performed in five steps, requiring selective deactivation of ring B.

Full text of DOI:10.1016/j.tetlet.2009.12.062

Tetrahedron Letters 51 (2010) 1145–1148
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Unexpected B-ring regioselective di-nitration of diosmetin, a Citrus flavonoid
a
b
Guy Lewin a, , Jean-Christophe Jullian , Jordi Rodrigo
*
a Laboratoire de Pharmacognosie, (BIOCIS, UPRES-A 8076 CNRS), Faculté de Pharmacie, av. J.B. Clément, 92296 Châtenay-Malabry Cedex, France
b Laboratoire de Chimie Thérapeutique (BIOCIS, UPRES-A 8076 CNRS), Faculté de Pharmacie, av. J.B. Clément, 92296 Châtenay-Malabry Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitration in position C-8 of diosmetin, an easily available citroflavonoid, was studied in order to gain
access to original analogs. The one-step nitration proved impossible, as mono or di-nitration on C-20
and C-60 positions on the lateral B ring of the molecule was exclusively observed. This surprisingly
straightforward di-nitration of ring B, showing a lack of reactivity of ring A despite its high activation,
has never been mentioned to date. Nitration in position C-8 was therefore performed in five steps, requir-
ing selective deactivation of ring B.
Received 16 October 2009
Revised 11 December 2009
Accepted 11 December 2009
Available online 16 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Numerous publications describe the preparation of nitroflav-
ones. Some of these compounds have been synthesized for their
pharmacological interest, with anxiolytic,1 cytotoxic,2 and anti-
allergic3 properties. Others, without remarkable properties, are
intermediates toward aminoflavones which have an interest in
the fields of cancer,4 diabetes,5 and neuroprotection.6 Different
strategies have been described, in which nitro groups are intro-
duced either at the early stages of synthesis, or during the late
steps, on the fully constituted flavone skeleton itself. We focused
on the latter approach, using diosmetin 1, a natural citroflavonoid
readily accessible from hesperidin or diosmin as a starting mate-
rial. Our aim was to study conditions of nitration so as to obtain
original analogs bearing a substituent in position 8 on the A ring.
A thorough examination of the literature concerning the nitration
of natural 5,7-dioxygenated flavones showed that the site of
nitration (A or B ring) is strongly dependent on the nature of
substituents. When the B ring is unsubstituted (5,7-dihydroxyflav-
one = chrysin 2), nitration occurs only, as expected, on the A ring,
primarily in position 8 then, under stronger conditions, in position
6.3,5,7 For a monosubstituted B ring, with a hydroxyl group in posi-
TFA (1 equiv, HNO3, 0 °C, 30 min), the reaction medium was
homogeneous and yielded, after treatment and flash chromatog-
raphy, a major product (42%). This compound displayed a molec-
ular peak at m/z 345 in EIMS, accordingly to a mono-nitration
process. However, 1H NMR revealed split signals in a 53:47 ratio,
in favor of a mixture of two isomers. In particular, the occurrence
of two doublets (J = 8.2 Hz) at 7.30 and 7.48 ppm, assigned to H-
50 and H-60 and of two singlets at 7.14 and 7.79 ppm correspond-
ing to H-20 and H-50 proved them to be 20-nitrodiosmetin 5a
(major) and 60-nitrodiosmetin 5b (minor).9 In CH3COOH (HNO3
1 equiv, 60 °C, 1 h), the reaction medium never cleared. TLC on
silica gel showed, in addition to residual diosmetin and monon-
itrated products, the presence of a bright yellow, highly polar
compound. The latter became the largely major product of the
reaction performed with 2 equiv of HNO3 in CH3COOH (60 °C,
1 h) or in TFA (0 °C, 30 min), while diosmetin and mononitrated
compounds were no longer observable. The CH3COOH reaction,
cleaner than that in TFA, allowed isolation of this major com-
pound (58%), identified as 20,60-dinitrodiosmetin 6. The dinitro-
substitution was evidenced by EIMS with a molecular peak at
m/z 390. Di-substitution of the B ring was deduced from 1H
NMR spectra: H-6 and H-8 signals were still observed at 6.26
and 6.33 ppm (J = 2 Hz). An Overhauser effect between signals
of H-50 and methoxyl group allowed positioning of nitro groups
at positions 20 and 60. Lastly, 2D NMR provided full structural
confirmation for 6.10
tion 40 (apigenin
3 and its 7-O-neohesperidoside = rhoifolin),
mono-nitration is mainly observed at position 30 of the B ring.8
However, surprisingly, for a methoxy substitution in 40 (acacetin
4), nitration exclusively occurs on the A ring, even after deactiva-
tion (acacetin-7-O-benzyl-sulfonate leading to a mixture of 6 and
8 mononitrated derivatives).8
Nitration of diosmetin with nitric acid (1 or 2 equiv) was con-
ducted in acetic acid (CH3COOH) or in trifluoracetic acid (TFA). In
MMono-nitration at C-20 or C-60, the two activated positions
of the B ring, is not surprising in itself. However, mono-nitration
would be expected at the A ring for iodination, the only electro-
philic substitution described so far on diosmetin, occurred
exclusively in positions 6 and 8.11 In this case, the iodination re-
agent (BTMA.ICl2) was used in a CH2Cl2/MeOH/CaCO3 system.
We therefore decided to study the behavior of diosmetin toward
* Corresponding author. Tel.: +33 146835593; fax: +33 146835399.
E-mail address: guy.lewin@u-psud.fr (G. Lewin).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.062

1146
G. Lewin et al. / Tetrahedron Letters 51 (2010) 1145–1148
a first nitro substitution is well known to deactivate an aromatic
5'
B
2'
R1
R2
4'
3'
6'
1'
ring in regard to a second nitration (flavone itself leads, with ex-
cess HNO3, to a mixture of two dinitroflavones substituted at C-6
on the A ring, and at C-30 or C-40 on ring B).1 We thus wondered
whether the exclusive nitration at C-20 and/or C-60 of the B ring
could result (when 20 and 60 are activated as in 1) from a priv-
ileged reactivity of these positions on the B ring. However, that
hypothesis was not supported by the nitration reaction of 30-
hydroxyflavone 8 (1 equiv HNO3, TFA, 0 °C, 30 min) which led
to the isolation and the identification of 9a (23%), 9b (23%),
and 9c (18%), the three mononitro isomers at 20, 40, and 60,
respectively.13 Looking for the proof of a possible interaction of
the first nitro substituent with ring A, we compared A ring pro-
ton chemical shifts in mononitro analogs 5a, 5b, and 9a–c and in
their respective precursors, 1 and 8. Significant d variations
(shielding) were observed for the sole H-8 signal and only with
20 or 60 nitroflavones [ꢀ0.23 ppm for 5a and 5b vs 1; ꢀ0.30 and
ꢀ0.19 ppm for 9a and 9c vs 8 (but +0.01 ppm for 9b)], which
can be indicative of a diamagnetic anisotropy effect of the 20
or 60 nitro group on H-8. In order to verify this possible aniso-
tropic effect of the 20 or 60 nitro group on H-8, we performed
8
7
6
HO
O 2
9
A
3
10
4
O
5
OH
R1
R2
1
OMe
H
OH
H
2
3
OH
H
4
OMe
H
R3
OMe
OH
R1
HO
R1
O
O
a
GAUSSIAN 0314 optimization calculation HF/6-31G(d,p) over 1,
R2
5a, and 5b compounds. According to Gaussian results and con-
sidering charge distribution we did not find a plausible explana-
tion for these privileged nitrations in positions 20 or 60 belonging
to ring B. In fact, Gaussian results for compound 1 clearly dem-
onstrate that position 8 should be the first nitration site. How-
ever, the distance and position of the nitro groups in respect
to H-8 in the optimized structures of 5a and 5b shows that
nitration at position 20 or 60 could have an anisotropic effect
on H-8. A similar result showing this anisotropic effect of a nitro
group was published by Martin and Nance.15 So we think that
this observed then confirmed through-space effect of the first
20 or 60 nitro group on H-8 could be related to the absence of
the second nitration at this position.
OH
R1
R2
R3
H
H
H
NO2
H
H
5a
5b
6
NO2
NO2
NO2
Br
H
H
7
This attack of diosmetin, at C-20 and C-60 only, indicates that
nitration on the A ring at C-8 requires a previous deactivation of
the B ring. Therefore, our initial objective was reached from dios-
metin by the following five-step sequence (Scheme 1): (a) 7-O ben-
zylation to 10;16 (b) 30-O tosylation to 11 (mp 208–211 °C); (c)
debenzylation to 12;17 (d) nitration at C-8 to 13;18 (e) hydrolysis
to 8-nitrodiosmetin 14.19 The nitration at C-8 was unambiguously
proved by comparison of 1H and 13C NMR spectra of 1 and 14: in
14, deshielding of H-6 (+0.18 ppm) and shielding of C-7
(ꢀ6.4 ppm) and C-9 (ꢀ7.5 ppm) signals (carbons ortho to the nitro
group) versus diosmetin.20
As a conclusion, this nitro-functionalization at C-8, originally
supposed to be a straightforward reaction, appears to be more
complex, and highlights an unusual and unknown reactivity
within this already well known and well-explored series of
flavonoids.
R3
R1
O
OH
R2
O
R1
R2
H
R3
H
H
H
H
8
H
NO2
H
9a
9b
NO2
H
H
NO2 9c
Acknowledgments
another electrophilic reagent, N-bromosuccinimide, under similar
conditions as those described for nitration (NBS 1 or 2 equiv,
TFA, rt, 1 h). One equivalent of NBS provided a mixture of four
compounds: the remaining diosmetin, 6- and 8-bromodiosmetin,
and 6,8-dibromodiosmetin 7,12 the latter being quantitatively ob-
tained with 2 equiv NBS. Di-nitration at C-20 and C-60 appears
much more surprising for several reasons: (a) formation of 6
was possible under mild conditions while, in comparison, prepa-
ration of 6,8-dinitrochrysin requires 10 h at 65 °C;5 (b) two nitro
groups were introduced on the B ring, despite the presence of a
strongly activating 5,7-diphenolic system on the A ring, although
We thank Pierre Champy for fruitful discussions and Andrew
Pearson for English corrections of the Letter.
Supplementary data
Supplementary data (1H NMR spectrum of 5a/5b, 1H and 13C
NMR spectra of 6, 12, 13 and 14) associated with this article
can be found, in the online version, at doi:10.1016/
j.tetlet.2009.12.062.

G. Lewin et al. / Tetrahedron Letters 51 (2010) 1145–1148
1147
diosmetin 1
a
OMe
OH
OMe
OH
NO2
BnO
O
HO
O
OH
O
OH
O
10
14
e
b
OMe
OTs
OMe
NO2
HO
O
BnO
O
OTs
OH
O
OH
O
11
13
c
d
OMe
HO
O
OTs
OH
O
12
Scheme 1. Reagents and conditions: (a) KHCO3 1.1 equiv, benzyl chloride 1.2 equiv, DMF, 120 °C, 2 h, 66%; (b) tosyl chloride 5 equiv, pyridine, rt, 1 h, 93%; (c) H2, Pd–C 10%,
DMF, rt, 2 h, 94%; (d) HNO3 1 equiv, CH3COOH, 120 °C, 0.25 h then 60 °C, 1.5 h, 45%; (e) 0.5 N aqueous KOH/MeOH 2/1, 120 °C, 0.5 h, 75%.
150.8 (C-40), 157.8 (C-2 and C-9), 161.5 (C-5), 165.0 (C-7), 180.9 (C-4). HREIMS
References and notes
(+) m/z [M]+ 390.0342 (calcd for C16H10N2O10, 390.0336).
11. Quintin, J.; Lewin, G. Tetrahedron Lett. 2004, 45, 3635–3638.
1. (a) Marder, M.; Viola, H.; Wasowski, C.; Wolfman, C.; Waterman, P. G.; Medina,
12. Quintin, J.; Lewin, G. Tetrahedron Lett. 2005, 46, 4341–4343.
J. H.; Paladini, A. C. Bioorg. Med. Chem. Lett. 1995, 5, 2717–2720; (b) Marder, M.;
13. The position of the nitro group in each isomer was proved by comparative 1H
Zinczuk, J.; Colombo, M. I.; Wasowski, C.; Viola, H.; Wolfman, C.; Medina, J. H.;
NMR experiments (NOESY and COSY). The conclusions were based on: (a) the
Rúveda, E. A.; Paladini, A. C. Bioorg. Med. Chem. Lett. 1997, 7, 2003–2008; (c)
presence of Overhauser effects between H-3 and H-20/H-60 (two NOE
Patent WO 9714414, 1997.
observed for 9b, only one for 9a and 9c); (b) the strong deshielding of H-50
2. (a) Zheng, X.; Meng, W.-D.; Xu, Y.-Y.; Cao, J.-G.; Qing, F.-L. Bioorg. Med. Chem.
signal by the ortho nitro group at 40 for 9b and 60 for 9c. Compound 9a. White
Lett. 2003, 13, 881–884; (b) Cardenas, M.; Marder, M.; Blank, V. C.; Roguin, L. P.
crystals: mp: 231–233 °C (MeOH); 1H NMR (DMSO-d6) d ppm 6.78 (s, 1H, H-
Bioorg. Med. Chem. 2006, 14, 2966–2971.
3), 7.29 (d, J = 8.4 Hz, 1H, H-40), 7.36 (d, J = 7.7 Hz, 1H, H-60), 7.45 (d,
3. Japanese Patent JP 61 68484, 1986.
J = 8.4 Hz, 1H, H-8), 7.50–7.58 (m, 2H, H-6 and H-50), 7.83 (br t, 1H, H-7), 8.05
4. (a) Cushman, M.; Nagarathnam, D.; Burg, L. D.; Geahlen, R. L. J. Med. Chem.
(d, J = 7.9 Hz, 1H, H-5). Compound 9b. White crystals: mp: 238–240 °C
1991, 34, 798–806; (b) Cushman, M.; Zhu, H.; Geahlen, R. L.; Kraker, A. J. J. Med.
(MeOH); 1H NMR (DMSO-d6) d ppm 7.08 (s, 1H, H-3), 7.53 (br t, J = 7.5 Hz,
Chem. 1994, 37, 3353–3362; (c) Quintin, J.; Buisson, D.; Thoret, S.; Cresteil, T.;
1H, H-6), 7.68 (br d, J = 8.7 Hz, 1H, H-60), 7.75–7.79 (m, 2H, H-8 and H-20),
Lewin, G. Bioorg. Med. Chem. Lett. 2009, 19, 3502–3506.
7.86 (br t, 1H, H-7), 8.03 (d, J = 8.7 Hz, 1H, H-50), 8.06 (d, J = 7.9 Hz, 1H, H-5).
5. Gao, H.; Kawabata, J. Bioorg. Med. Chem. 2005, 13, 1661–1671.
Compound 9c. White crystals: mp: 279–282 °C (MeOH); 1H NMR (DMSO-d6) d
6. Larget, R.; Lockhart, B.; Renard, P.; Largeron, M. Bioorg. Med. Chem. Lett. 2000,
ppm 6.63 (s, 1H, H-3), 7.08–7.13 (m, 2H, H-40 and H-20), 7.52 (t, J = 7.7 Hz, 1H,
10, 835–838.
H-6), 7.56 (d, J = 8.5 Hz, 1H, H-8), 7.81 (br t, 1H, H-7), 8.08 (d, J = 7.8 Hz, 1H,
7. Hu, C.-Q.; Chen, K.; Shi, Q.; Kilkuskie, R. E.; Cheng, Y.-C.; Lee, K.-H. J. Nat. Prod.
H-5), 8.16 (d, J = 8.4 Hz, 1H, H-50).
1994, 57, 42–51.
14. Frisch, M. J. et al GAUSSIAN 03; Gaussian: Wallingford, CT, 2004.
8. Quintin, J.; Roullier, C.; Thoret, S.; Lewin, G. Tetrahedron 2006, 62, 4038–
15. Martin, N. H.; Nance, K. H. J. Mol. Graphics Modell. 2002, 21, 51–56.
4051.
16. Quintin, J.; Lewin, G. J. Nat. Prod. 2004, 67, 1624–1627.
9. Mixture of 5a (53%) and 5b (47%). Yellow microcrystalline powder. Compound
17. Compound 12. Yellowish powder: mp: 244–246 °C (MeOH); 1H NMR (DMSO-
5a: 1H NMR ((DMSO-d6)) d ppm 4.00 (s, 3H, OMe-40), 6.19 (d, J = 2.0 Hz, 1H, H-
d6) d ppm flavone moiety: 3.62 (s, 3H, OMe-40), 6.20 (d, J = 2.0 Hz, 1H, H-6),
6), 6.23 (d, J = 2.0 Hz, 1H, H-8), 6.72 (s, H-3), 7.30 (d, J = 8.2 Hz, 1H, H-50), 7.48
6.37 (d, J = 2.0 Hz, 1H, H-8), 6.80 (s, 1H, H-3), 7.21 (d, J = 8.9 Hz, 1H, H-50), 7.54
(d, J = 8.2 Hz, 1H, H-60), 12.64 (s, 1H, OH-5); compound 5b: 3.98 (s, 3H, OMe),
(d, J = 2.0 Hz, 1H, H-20), 8.00 (dd, J = 8.9 and 2.0 Hz, 1H, H-60), 10.9 (br s, 1H, OH-
6.19 (d, J = 2.0 Hz, 1H, H-6), 6.23 (d, J = 2.0 Hz, 1H, H-8), 6.54 (s, H-3), 7.14 (s, H-
7), 12.80 (s, 1H, OH-5); tosyl moiety: 2.45 (s, 3H, Me-400), 7.49 (d, J = 8.9 Hz, 2H,
20), 7.79 (s, H-50), 12.70 (s, 1H, OH-5). HREIMS (+) m/z [M]+ 345.0489 (calcd for
H-300 and 500), 7.75 (d, J = 8.9 Hz, 2H, H-200 and 600). 13C NMR (DMSO-d6) d ppm
C16H11NO8, 345.0485).
flavone moiety: 56.1 (OMe-40), 93.9 (C-8), 98.9 (C-6), 103.7 (C-10), 104.3 (C-3),
10. Compound 6. Beige yellowish powder: mp: 275–277 °C (MeOH); 1H NMR
113.8 (C-50), 121.4 (C-20), 122.9 (C-10), 127.0 (C-60), 145.8 (C-30), 154.3 (C-40),
(DMSO-d6) d ppm 4.05 (s, 3H, OMe-40), 6.26 (d, J = 2.0 Hz, 1H, H-6), 6.33 (d,
157.1 (C-9), 161.4 (C-2), 161.5 (C-5), 164.3 (C-7), 181.6 (C-4); tosyl moiety:
J = 2.0 Hz, 1H, H-8), 6.45 (s, 1H, H-3), 8.00 (s, 1H, H-50), 12.4 (br s, 1H, OH-5).
21.1 (Me), 128.4 (C-200 and 600), 129.9 (C-300 and 500), 131.8 (C-100), 137.7 (C-400).
13C NMR (DMSO-d6) d ppm 57.4 (OMe-40), 94.0 (C-8), 99.5 (C-6), 103.7 (C-10),
EIMS (+) m/z [M]+ 454.
109.7 (C-50), 110.3 (C-3), 114.0 (C-10), 136.7 (C-60), 139.1 (C-20), 146.2 (C-30),

1148
G. Lewin et al. / Tetrahedron Letters 51 (2010) 1145–1148
18. Compound 13. Yellowish powder: mp: 266–267 °C (MeOH); 1H NMR (DMSO-
d6) d ppm flavone moiety: 3.60 (s, 3H, OMe-40), 6.37 (s, 1H, H-6), 7.07 (s, 1H, H-
3), 7.27 (d, J = 8.9 Hz, 1H, H-50), 7.68 (d, J = 2.0 Hz, 1H, H-20), 7.95 (dd, J = 8.9
and 2.0 Hz, 1H, H-60), 13.32 (s, 1H, OH-5); tosyl moiety: 2.45 (s, 3H, Me-400),
7.48 (d, J = 8.9 Hz, 2H, H-300 and 500), 7.74 (d, J = 8.9 Hz, 2H, H-200 and 600). 13C
NMR (DMSO-d6) d ppm flavone moiety: 56.1 (OMe-40), 98.9 (C-6), 102.9 (C-10),
105.1 (C-3), 114.0 (C-50), 121.3 (C-8), 121.7 (C-20), 122.2 (C-10), 127.2 (C-60),
145.8 (C-30), 149.8 (C-9), 154.7 (C-40), 157.9 (C-7), 161.8 (C-2), 162.7 (C-5),
181.1 (C-4); tosyl moiety: 21.1 (Me), 128.2 (C-200 and 600), 129.9 (C-300 and 500),
131.8 (C-100), 137.8 (C-400). EIMS (+) m/z [M]+ 499.
19. Compound 14. Yellow crystals: mp: 303–306 °C (MeOH); 1H NMR
(DMSO-d6)
ppm 3.88 (s, 3H, OMe-40), 6.37 (s, 1H, H-6), 6.96 (s, 1H,
H-3), 7.10 (d, J = 8.9 Hz, 1H, H-50), 7.37 (d, J = 2.0 Hz, 1H, H-20), 7.48 (dd,
d
J = 8.9 and 2.0 Hz, 1H, H-60), 13.40 (s, 1H, OH-5). 13C NMR (DMSO-d6)
d
ppm 55.9 (OMe-40), 98.8 (C-6), 102.9 (C-10), 104.2 (C-3), 112.3 (C-50),
113.1 (C-20), 119.0 (C-60), 121.5 (C-8), 122.2 (C-10), 146.9 (C-30), 149.8
(C-9), 151.7 (C-40), 157.8 (C-7), 162.7 (C-5), 163.7 (C-2), 181.1 (C-4).
EIMS (+) m/z [M]+ 345.
20. Park, Y.; Moon, B.-H.; Yang, H.; Lee, Y.; Lee, E.; Lim, Y. Magn. Reson. Chem. 2007,
45, 1072–1075.