Specific deuteration in patuletin and related flavonoids via keto - Enol tautomerism: Solvent- and temperature-dependent 1H-NMR studies

Article abstract of DOI:10.1002/hlca.200900249

An H/D exchange process in patuletin (1) and its derivatives in D-donor solvents (e.g., CF₃COOD), which occurs regioselectively at C(8) was observed for the first time during NMR studies. The effect of substituents and temperature on the deuteration of various flavonoids (see Fig. 1) which include apigenin, chrysin, galangin, kaempferol, luteolin, morin, myricetin, patuletin, patulitrin, and quercetin, as well as derivatives of patuletin was examined extensively under NMR conditions. The rate constant of deuteration at C(8) of patuletin (1) and two flavones, luteolin (3) and apigenin (12), was also determined in CF₃COOD. The D-atom was introduced into the flavonoids via a keto - enol tautomerism (Scheme 1). During these studies, monodeuterated patuletin was also obtained as a new compound. The examined flavonoids have been reported to possess significant pharmacological activities, and their deuterated derivatives would be of importance for the identification and quantification of these compounds in biological matrices.

Full text of DOI:10.1002/hlca.200900249

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Helvetica Chimica Acta – Vol. 93 (2010)
Specific Deuteration in Patuletin and Related Flavonoids via Keto – Enol
Tautomerism: Solvent- and Temperature-Dependent 1H-NMR Studies
by Shaheen Faizi*, Humaira Siddiqi, Aneela Naz, Samina Bano, and Lubna
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
(phone: þ 92-21-34824912; fax þ 92-21-34819018 and þ 92-21-34819019;
e-mail: shaheenfaizi@hotmail.com)
Dedicated to the fond memory of Prof. Salimuzzman Siddiqui FRS (1897 – 1994), the founding director
of H.E.J. Research Institute of Chemistry, University of Karachi
An H/D exchange process in patuletin (1) and its derivatives in D-donor solvents (e.g., CF3COOD),
which occurs regioselectively at C(8) was observed for the first time during NMR studies. The effect of
substituents and temperature on the deuteration of various flavonoids (see Fig. 1) which include
apigenin, chrysin, galangin, kaempferol, luteolin, morin, myricetin, patuletin, patulitrin, and quercetin, as
well as derivatives of patuletin was examined extensively under NMR conditions. The rate constant of
deuteration at C(8) of patuletin (1) and two flavones, luteolin (3) and apigenin (12), was also determined
in CF3COOD. The D-atom was introduced into the flavonoids via a keto – enol tautomerism (Scheme 1).
During these studies, monodeuterated patuletin was also obtained as a new compound. The examined
flavonoids have been reported to possess significant pharmacological activities, and their deuterated
derivatives would be of importance for the identification and quantification of these compounds in
biological matrices.
Introduction. – Flavonoids are polyphenol compounds that are diverse in both
chemical structure and chemical properties. Since flavonoids are naturally present in
fruits, vegetables, and tea, they are an integral part of the human diet. It is generally
accepted that dietary flavonoids have important biological effects such as decreasing
the risk of death from coronary heart disease and cancer [1a][1b][2]. Most of the
beneficial health effects of flavonoids are attributed to their antioxidant and chelating
abilities. The antioxidant activity of flavonoids and their metabolites in vitro depends
upon the arrangement of functional groups at the molecular-skeleton parent [1 – 5].
Increasingly, the flavonoids are becoming the subject of medical research. They have
been reported to possess many useful properties, including antiallergic, anti-HIV
integrase, anti-inflammatory, antimicrobial, antioxidant, antitumor, cytotoxic, enzyme-
inhibition, and oestrogenic activities [1 – 6].
Plants belonging to the genus Tagetes have been found to possess various biological
properties, e.g., antimicrobial, anti-inflammatory, antioxidant, and antiviral activities
[7 – 10]. Two species of the genus, Tagetes erecta and Tagetes patula, are highly reputed
in traditional medicine for their therapeutic uses. They are also the source of pesticidal
agents [8 – 10]. In pursuance of our endeavor [11 – 13] for isolation of bioactive
chemical constituents from Tagetes plants, we have recently obtained patuletin (¼2-
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 93 (2010)
467
(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-6-methoxy-4H-1-benzopyran-4-one; 1), a rare
6-methoxy flavonol, and its 7-glucoside patulitrin (¼2-(3,4-dihydroxyphenyl)-7-(b-d-
glucopyranosyloxy)-3,5-dihydroxy-6-methoxy-4H-1-benzopyran-4-one; 2) [14 – 17] in
large quantity from the flowers of Tagetes patula, along with luteolin (¼2-(3,4-
dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one; 3), a flavonoid which has
earlier been isolated from Tagetes rupestris [17] (Fig. 1). Bioassay-guided isolation
studies revealed that patuletin (1) is the active antibacterial and antioxidant principle of
the plant, while 2 is less active in both assays [18a][18b]. The tetra-O-benzoyl, tetra-O-
cinnamoyl, and tetra-O-methyl derivatives 4 – 6 of 1 have also been prepared for the
establishment of structure – activity relationships [18a][18b]. During these studies, it
1
has been observed that the H-NMR spectrum of 1 in CF3COOD has a very weak
signal for HꢀC(8), due to H/D exchange at C(8) of ring A, revealing the keto – enol
tautomerism between OHꢀC(7) and HꢀC(8).
Fig. 1. Flavonoids used in this study
For a more comprehensive study and to establish the positional and substitutional
effect of OH groups on H/D-exchange processes in different flavonoids, five flavonols
(see 7 – 11), three flavones (see 3, 12, and 13), one flavonol glucoside (see 2), as well as
the benzoyl, cinnamoyl, and methyl derivatives 4 – 6 of 1 were now examined by means
of solvent- and temperature-dependent 1H-NMR experiments. The 8-deuterated
patuletin, (8-D)patuletin (1a), was isolated as a new compound during these
investigations. Moreover, flavonols which are more active as antioxidant agents than
flavones [3][4][19] showed more reactivity towards H/D exchange as compared to
flavones. Rate constants of the H/D exchange of selected flavonols and flavones were

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Helvetica Chimica Acta – Vol. 93 (2010)
also determined by the 1H-NMR studies. This is the first time that H/D exchange during
NMR studies was observed in patuletin (1) and patulitrin (2); previously this
phenomenon was witnessed in the case of mangiferin (¼2-b-d-glucopyranosyl-1,3,6,7-
tetrahydroxy-9H-xanthen-9-one), a xanthone C-glucoside, which is more related to
flavonols than to xanthones [20]. Furthermore, the preparation of polydeuterated
flavonoids and isoflavonoids was documented to allow identification and quantitation
of these compounds in biological matrices [21a][21b]; moreover, deuterated flavonoids
have also been used for biosynthetic and metabolic study of flavonoids [21c].
Results and Discussion. – The 1H-NMR spectra of patuletin (1) recorded in
(D6)acetone, (D6)DMSO, and (D5)pyridine (Table 1) exhibited no abnormal integra-
tion of H-atoms, whereas the spectrum run in (D5)pyridine þ D2O showed the low
intensity of HꢀC(8) as compared to other methine H-atoms at ring B. This revealed an
H/D exchange possibly due to the formation of the conjugate acid (D-donor) of
1
(D5)pyridine and D2O. On the other hand, the H-NMR spectrum of 1 measured in
CF3COOD exhibited a very low intensity for HꢀC(8) (d 7.95, 0.29 H; Table 2),
revealing the higher reactivity of HꢀC(8) towards H/D exchange in a D-donor acidic
solvent.
Table 1. 1H-NMR Data of Patuletin (1) in Different Solvents and of (8-D)Patuletin (1a) in (D6)Acetone.
d in ppm, J in Hz.
1a)
1ab)
(D6)acetone
(D5)pyridine
(D6)DMSO
(D6)acetone
HꢀC(8)
HꢀC(2’)
HꢀC(5’)
HꢀC(6’)
6.59 (s)
6.82 (s)c)
8.59 (d, J ¼ 2.00)
7.38 (d, J ¼ 8.42)
8.11 (dd,
6.50 (s)
–
7.82 (d, J ¼ 2.08)
6.98 (d, J ¼ 8.47)
7.69 (dd,
7.65 (d, J ¼ 2.10)
6.88 (d, J ¼ 8.46)
7.52 (dd,
7.82 (d, J ¼ 1.96)
6.98 (d, J ¼ 8.48)
7.70 (dd,
J ¼ 8.47, 2.08)
J ¼ 8.42, 2.00)
J ¼ 8.46, 2.10)
J ¼ 8.48, 1.96)
MeOꢀC(6)
OHꢀC(5)
ArꢀOH
3.87 (s)
3.94 (s)
3.74 (s)
3.87 (s)
12.31 (s)d)
9.12 (s)d)
13.39 (s)d)
12.55 (s)d)
12.30 (s)d)
7.92 (s)d)
8.30 (s)d)
8.52 (s)d)
9.13 (s)d)
11.61 – 12.08 (hump)d)
9.20 – 9.41 (s, hump)d)
8.48 (s)d)
8.27 (s)d)
7.94 (s)d)
a) 400 MHz. b) 500 MHz. c) Integration 0.85 H in (D5)pyridine þ D2O. d) Disappeared on shaking with
D2O.
1
To investigate this phenomenon further, the H-NMR spectra of 1 were taken in
different mixtures (D6)DMSO/CF3COOD (Table 3). The spectral data revealed that in
pure (D6)DMSO and up to (D6)DMSO/CF3COOD 1:1.5 the integration of HꢀC(8)
was the same as those of other H-atoms of the molecule; however, at the ratio of 1:2
the integration of HꢀC(8) decreased due to the initiation of the H/D exchange.
Moreover, the effect of temperature on the rate of the H/D exchange in 1 was also
1
studied by variable-temperature H-NMR spectroscopy in CF3COOD (Table 4) over
the range of 308 (303 K) to 708 (343 K). At 308, 29% of the patuletin molecules were
undeuterated at C(8). On increasing the temperature, as expected, the H/D-exchange

Helvetica Chimica Acta – Vol. 93 (2010)
469
Table 2. 1H-NMR Data of Patuletin (1), Patulitrin (2), and 3,3’,4’,7-Tetra-O-methylpatuletin (6) in
CF3COOD. d in ppm, J in Hz.
1a)
2b)
6b)
HꢀC(8)
HꢀC(2’)
HꢀC(5’)
HꢀC(6’)
7.95 (s, 0.29 H)
8.99 (br. s, 1.00 H)
8.20 (s, 0.88 H)
9.07 (br. s, 1.00 H)
7.92 (s, 0.75 H)
8.99 (br. s, 1.00 H)
8.04 (d, J ¼ 8.59, 1.00 H) 8.12 (d, J ¼ 8.10, 1.00 H) 8.04 (d, J ¼ 8.64, 1.00 H)
8.93 (br. d,
8.98 (br. d,
8.97 (br. d,
J ¼ 8.64, 1.00 H)
4.88 (s, 3.00 H)
4.97 (s, 3.00 H)
4.90 (s, 3.00 H)
4.88 (s, 3.00 H)
4.83 (s, 3.00 H)
–
J ¼ 8.59, 1.00 H)
J ¼ 8.10, 1.00 H)
MeOꢀC(6)
4.96 (s, 3.00 H)
5.09 (s, 3.00 H)
MeOꢀC(3)c)
MeOꢀC(3’)c)
MeOꢀC(4’)c)
MeOꢀC(7)c)
HꢀC(1’’)
–
–
–
–
–
–
–
–
–
–
6.54 (d, J ¼ 6.71, 1.00 H)
HꢀC(2’’) to CH2(6’’)
4.81 – 5.23 (m, 6.00 H)
–
c
a) 300 MHz. b) 400 MHz. ) 1H-NMR Values are interchangeable.
rate increased, and then, at 608, 100% of the molecules of 1 were deuterated only at
C(8). It is known that the bond-dissociation energy of CꢀD is higher than that of CꢀH
[22]; thus, the H/D exchange led to the formation of a stable 8-deuterated patuletin i.e.,
(8-D)patuletin (1a). Interestingly, when 1 was heated with CF3COOD in a boiling
water bath, almost complete H/D exchange took place, and after evaporation of the
1
CF3COOD at room temp., the H-NMR spectrum in (D6)acetone (Table 1) revealed
the exclusive formation of 1a, as well as its stability under the above experimental
1
conditions. The H- and 13C-NMR spectra in (D6)acetone (Tables 1 and 5, resp.)
showed that 1 and 1a were present in a proportion of 1:4. This regioselectivity and
reactivity at C(8) towards deuteration is in agreement with the fact that the
nucleophilicity of C(8) is much higher than that of other CH moieties, i.e., HꢀC(2’),
HꢀC(5’), and HꢀC(6’) in patuletin (1), as supported by its upfield 13C-NMR chemical-
shift value (d 94.61) compared with those of other C-atoms (CH) of the molecule
((D5)pyridine, Table 5). Moreover, the 13C-NMR spectra of 1a did not contain the
resonance for the deuterated C(8), due to its increased spin – lattice relaxation time
(T1) and also increased complexity and decreased intensity from 13C,D splitting and a
decreased nuclear Overhauser effect [23]. The d(C) of all the undeuterated C-atoms of
1a are similar to those of 1, except for the C-atoms adjacent to C(8), i.e., C(7) and
C(8a) which appeared upfield [23] as compared to those of 1 due to deuteration at
C(8). In the EI-MS, the molecular-ion peak of 1a was observed at m/z 333.
For the H/D-exchange process in 1, two plausible mechanisms (Scheme 1) are
suggested, where mechanism A is a keto – enol tautomerism and would be the
predominant one provided that there is a free OH group at C(7). It is known that
aromatic compounds including flavonoids carrying OH groups react like enols with
electrophiles, except that the final product is also an enol because of the aromaticity of
the compound [24]. On the other hand, mechanism B is a simple electrophilic-
substitution reaction. The effect of substitution at C(7) on the reactivity of C(8) was
nicely shown by patulitrin (2) and the patuletin derivatives, the O-benzoyl (see 4), O-

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Table 4. 1H-NMR Data of Patuletin (1), Quercetin (11), and Apigenin (12) at Different Temperatures
from 308 to 708 in CF3COOD at 400 MHz. d in ppm.
T
1
11
12
HꢀC(8)
HꢀC(6)
HꢀC(8)
HꢀC(6)
HꢀC(8)
308 (303 K) 7.79 (s, 0.29 H) 7.60 (s, 0.66 H)
408 (313 K) 7.92 (s, 0.16 H) 7.68 (s, 0.60 H)
508 (323 K) 8.03 (s, 0.10 H) 7.78 (s, 0.40 H)
disappeared 7.58 (s, 0.60 H) 7.62 (s, 0.28 H)
disappeared 7.67 (s, 0.60 H) 7.90 (s, 0.14 H)
disappeared 7.80 (s, 0.45 H) disappeared
608 (333 K) disappeared
708 (343 K) disappeared
7.85 (s, negligible) disappeared 7.90 (s, 0.40 H) disappeared
disappeared disappeared 8.01 (s, 0.20 H) disappeared
Table 5. 13C-NMR Data of Patuletin (1), (8-D)Palutetin (1a), and Quercetin (11). d in ppm.
1
1a
11
(D5)pyridinea)
(D6)acetonea)
(D5)pyridineb)
(D6)acetonea)
(D6)acetonec)
C(2)
C(3)
C(4)
C(4a)
C(5)
C(6)
C(7)
C(8)
C(8a)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
6-MeO
147.92
137.53
177.51
104.89
153.07
132.01
158.61
94.61
152.82
121.11
116.65
147.09
149.35
116.65
121.11
60.34
147.14
136.40
176.80
104.58
153.12
131.72
157.79
94.53
152.39
123.86
115.82
145.75
148.30
116.22
121.55
60.79
147.89
137.53
177.49
104.86
153.04
132.02
158.57
–
152.75
121.08
116.63
147.08
149.37
116.63
121.13
60.33
147.14
136.40
176.80
104.58
153.12
131.72
157.76
–
152.34
123.86
115.82
145.75
148.30
116.22
121.55
60.79
146.94
136.73
176.55
104.12
162.31
99.15
164.98
94.44
157.76
123.77
115.76
145.81
148.33
116.19
121.46
–
a) 100 MHz. b) 75 MHz. c) 125 MHz.
cinnamoyl (see 5), and O-methyl derivatives (see 6) [25], of which 4 and 5 are new
synthetic compounds [18a]. The structures of all these compounds were determined
through spectral studies including 1D- and 2D-NMR spectroscopy (Exper. Part,
1
Table 2). In the H-NMR spectra (CF3COOD, Exper. Part, Table 2) of patulitrin (2)
and 4 – 6, very minor differences in the intensity of HꢀC(8) relative to that of other H-
atoms of the respective molecules were observed; this might be due to the absence of a
free OH group at C(7) preventing the keto – enol tautomerism, and thus the exchange
reaction proceeds through a simple electrophilic-substitution reaction as outlined in
Scheme 1 (Mechanism B). The above observations suggested that the contribution of
keto – enol tautomerism (Mechanism A) to the exchange process is prevailing over the
electrophilic substitution (Mechanism B) in molecules having a free OH group at C(7).
To study the positional effect of OH groups on deuteration of flavonoids, the
1H-NMR spectra in CF3COOD of five flavonols, i.e., galangin (7), kaempferol (8),

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Helvetica Chimica Acta – Vol. 93 (2010)
Scheme 1. Mechanism A: Keto – enol Tautomerism for Deuteration at C(8) of Patuletin (1) and
Formation of 1a; Mechanism B: Electrophilic-Substitution Reaction at C(8) of Patuletin (1), Patulitrin
(2), and Compounds 4 to 6

Helvetica Chimica Acta – Vol. 93 (2010)
473
morin (9), myricetin (10), and quercetin (11), and of three flavones, i.e., apigenin (12),
chrysin (13), and luteolin (3) (Fig. 1), were also examined (Table 6). These data
revealed that the signal of HꢀC(8) vanished in flavonols 7 – 11, while in flavones 3, 12,
and 13 it appeared but with reduced intensity.
Table 6. 1H-NMR Data of 3 and 7 – 13 in CF3COOD. d in ppm.
HꢀC(6)
HꢀC(8)
Galangin (7)
Kaempferol (8)
Morin (9)
7.58 (br. s, 0.31 H)
7.53 (br. s, 0.83 H)
7.55 (br. s)a)
7.53 (br. s, 0.80 H)
7.61 (br. s, 0.12 H)
7.63 (br. s, 0.75 H)
7.68 (br. s, 0.75 H)
7.54 (br. s, 0.91 H)
disappeared
disappeared
disappeared
disappeared
Myricetin (10)b)
Quercetin (11)
Luteolin (3)
disappeared
7.80 (br. s, 0.40 H)
7.87 (br. s, 0.27 H)
7.74 (br. s, 0.64 H)
Apigenin (12)
Chrysin (13)
a) Integration could not be measured due to overlapping. b) In myricetin (10), the integration of
HꢀC(2’) and HꢀC(6’) also decreases in CF3COOD.
The rate constant of deuteration at C(8) were calculated for patuletin (1) and the
two flavones luteolin (3) and apigenin (12) in CF3COOD. The progress of the reaction
1
was monitored by H-NMR spectroscopy, and the concentration of deuterated and
nondeuterated flavonoids was determined by integrating the residual signal of HꢀC(8)
relative to the nonexchangable H-atom signals in the spectra obtained at time intervals
of ca. 5 – 10 min. The change in the peak integration with time in CF3COOD appeared
at d 7.92, 7.87, and 7.86 in 1, 3, and 12, respectively (Table 7). The deuteration rate of
these flavonoids follows the general rate expression given by rate ¼ ꢀd[flavonoid]/dt ¼
k [flavonoid], where k is the rate constant. The deuteration kinetics of 1, 3, and 12 were
those of first-order reactions, as obtained by the least-squares fitting method, with the
slope values of 6 · 10ꢀ4 sꢀ1, 3.3 · 10ꢀ4 sꢀ1, and 3.3 · 10ꢀ4 sꢀ1, respectively, as shown in
Fig. 2; a plot of ln[flavonoid] vs. time for 1 and 3 gave linear fits over most of the range
for first-order reactions. The k value for quercetin (11) could not be determined since
the rate of H/D exchange at C(8) was very fast under the experimental conditions. The
order of reactivity of these compounds is 11 > 1 > 3 ¼ 12.
On comparing the integration of HꢀC(8) in the 1H-NMR spectra of patuletin (1),
quercetin (11), and apigenin (12) in CF3COOD at variable temperatures (þ 308 to
þ 708; Table 4) and at different ratios (D6)DMSO/CF3COOD (Table 3), the reactivity
order observed was 1 < 11 > 12. In quercetin (11), the rate of H/D exchange at C(8) was
very high, and HꢀC(8) had almost disappeared even at 308; however, in the case of the
MeOꢀC(6) derivative patuletin (1), the complete exchange occurred at relatively
higher temperature (608). It might be due to the negative inductive effect of
MeOꢀC(6) of 1 which decreases the electron density at C(8), as also revealed by the
downfield 13C-NMR chemical-shift value (d 94.53) of C(8) in contrast to the value for
quercetin (d 94.44) (in (D6)acetone; Table 5). Thus, the absence of MeOꢀC(6) is
necessary for a greater nucleophilicity at C(8) or for faster keto – enol tautomerism. On
the other hand, the presence of an OHꢀC(3) enhances the nucleophilicity at C(8) as

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Helvetica Chimica Acta – Vol. 93 (2010)
Table 7. Integration of 1H-NMR (300 MHz) Peaks during Deuteration of Flavonoids 1, 11, 3, and 12 with
CF3COOD at Different Time Intervals
Time
1
11
3
12
[s] (min)
HꢀC(8) (d 7.92) HꢀC(8) (d 7.89) HꢀC(6) (d 7.70) HꢀC(8) (d 7.87) HꢀC(8) (d 7.86)
300 (5)
540 (9)
–
–
0.30 H
–
5.30 H
–
–
–
3.35 H
3.05 H
720 (12) 1.00 H
–
–
–
–
960 (16)
1020 (17)
–
–
0.30 H
–
4.45 H
–
–
–
2.85 H
2.05 H
1260 (21) 0.60 H
–
–
–
–
1440 (24)
1500 (25)
–
–
0.30 H
–
3.80 H
–
2.35 H
–
–
2.05 H
1800 (30) 0.45 H
–
–
–
–
1860 (31)
1980 (33)
2100 (35)
2280 (38)
–
–
–
–
–
–
2.05 H
–
–
–
–
0.30 H
–
–
3.10 H
–
–
1.70 H
–
1.75 H
2340 (39) 0.35 H
–
–
–
–
2520 (42)
2700 (45)
3000 (50)
3120 (52)
3180 (53)
–
–
–
–
–
–
–
–
1.55 H
–
1.35 H
–
0.30 H
–
–
2.55 H
–
–
1.50 H
–
1.35 H
–
0.30 H
2.30 H
–
3420 (57) 0.25 H
–
–
–
3480 (58)
3540 (59)
3840 (64)
3960 (66)
4380 (73)
4440 (74)
4800 (80)
4920 (82)
5520 (92)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.15 H
–
–
1.00 H
–
0.80 H
–
0.75 H
–
–
1.15 H
–
1.05 H
0.90 H
–
–
–
–
–
0.25 H
–
–
–
0.25 H
–
0.25 H
0.25 H
1.95 H
–
–
–
1.50 H
–
1.20 H
1.00 H
6420 (107) –
the 3-hydroxy derivative quercetin (11) is more reactive than the 3-deoxy derivative
apigenin (12) towards C(8) deuteration.
Moreover, the H/D-exchange process was also observed at C(6) of compounds 3
and 7 – 13 (Tables 3, 4, and 6). However, in these compounds, the electron density at
C(6) is lower than that at C(8) as revealed by the comparison of their 13C-NMR data
[5b][26a][26b]. In quercetin (11), C(6) resonated at d 99.15, which is downfield with
1
respect to the signal of C(8) (d 94.44, Table 5). In the H-NMR spectrum of 11 in
CF3COOD at 308, the integration of HꢀC(6) was slightly reduced as compared to
other H-atoms of the molecule, while HꢀC(8) had disappeared (Table 4). However,
with the increase in temperature, the exchange process at C(6) was accelerated, and at
708, HꢀC(6) was completely replaced by a D-atom. In the light of these observations, it
may be concluded that C(8) is more nucleophilic than C(6), and therefore, deuteration

Helvetica Chimica Acta – Vol. 93 (2010)
475
Fig. 2. Plots of ln[flavonoid] vs. time for the first-order deuteration reaction of patuletin (1) and luteolin
(3) with CF3COOD
at C(8) is much faster as compared to C(6). It is noteworthy that C(6) has two OH
groups at the adjacent C(7) and C(5); however, the contribution of OHꢀC(5) in keto –
enol tautomerism was not pronounced, which might be due to its chelation with the
keto group at C(4) (Scheme 2).
Scheme 2. Keto – Enol Tautomerism for Deuteration at C(6) of Flavonoids 3 and 7 – 13
On treatment of quercetin (11) with CF3COOD at high temperatures, a mixture of
two deuterated compounds (shown by NMR and MS) was obtained, in which the major
one was deuterated both at C(8) and C(6), and the minor one was deuterated only at
C(8). The 5’-hydroxy derivative myricetin (10) of quercetin exhibited deuteration also
at ring B as revealed by its 1H-NMR spectrum in CF3COOD which contained only two
resonances of very low integration, one for HꢀC(6) and the other for both HꢀC(2’)
and HꢀC(6’), while that of HꢀC(8) had vanished.

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Helvetica Chimica Acta – Vol. 93 (2010)
It is interesting to note that during spectral studies in CF3COOD, morin (9), a
regioisomer of querectin with an OH group at C(2’), converted into the 2’-deoxy
derivative kaempferol (8). Moreover, the authentic sample of 9 also contained trace
1
amounts of 8 as an impurity, as shown by comparison of its TLC and H-NMR data
with that of an authentic sample of 8. It may be suggested that OHꢀC(3) at ring C is
responsible for the removal of OHꢀC(2’) in morin (9) by a photochemical reaction.
In conclusion, the 1H-NMR studies revealed that patuletin (1) and related
flavonoids underwent H/D-exchange processes. Extensive NMR investigation demon-
strated the regioselective deuteration at C(8) of 1 while other flavonoids which are
devoid of a substituent at C(6) showed deuteration at C(8) as well as at C(6). Flavonols
were more reactive than flavones towards H/D exchange. The kinetic study established
the reactivity order of flavonols 1 and 11 and flavones 3 and 12 as 11 > 1 > 3 ¼ 12. The
reactivity order of H/D exchange at ring-A C-atoms in these compound was C(8) >
C(6). It is important to note that there are a number of reports in the literature
dealing with the deuteration studies of various natural products, e.g., estrogens [27a],
flavanones [21c], isoflavones [21a], isoflavanones [21a], lignans [27b], and resorcinols
[27c]; however, there is very little previous work on the controlled synthesis of
polydeuterated polyhydroxy-substituted flavones and flavonols [21a][21b]. It has been
reported that H/D exchange occurred at highly activated sites ortho to two OH or MeO
groups in flavonoids. No exchange occurred at the aromatic ring containing only one
OH or two adjacent MeO groups [21a]. Our results which correlate to the flavonoid
behavior described in [21a][21b] are as follows: a) owing to the highest nucleophilicity
of C(8) in the flavonols examined, this position is regioselectively deuterated, and the
presence of RO (R ¼ Me, Glc) at C(6) and C(7) greatly reduces the reactivity of
HꢀC(8) in ring A; b) the presence of an OHꢀC(3) enhances the nucleophilicity of
C(8) and C(6), cf. quercetin (11) with apigenin (12) or luteolin (3); c) H-atoms of
aromatic rings containing one OH or two adjacent OH groups at the ring do not
exchange, e.g., H-atoms in ring B of apigenin (12), luteolin (3), patuletin (1), and
quercetin (11), etc.; d) meta-oriented OH groups activated the H-atoms ortho and para
to them for exchange such as in ring A of all the above flavonoids, and in rings A and B
of morin (9) and myricetin (10) in which two OH groups are meta-oriented; e) the rate
of the exchange process increases as the temperature increases resulting in the
complete replacement of HꢀC(8); f) the free OH group at C(7) facilitates the keto –
enol tautomerism and thus the H/D exchange.
Various biological activities attributed to flavonoids, e.g., antimicrobial
[2][6a][18a][25], anticancer [28], anti-HIV-1 integrase [6b], as well as platelet-
aggregation inhibitory [29a][29b][30], and UV-protection properties [2][31] depend
on the structure of these compounds, particularly on the number of free OH groups at
the molecular-skeleton parent. There are also reports of flavonoids inhibiting the
activities of an array of enzymes [29b]. These biological effects are believed to come
from the antioxidant properties of the related flavonoids [2 – 4][19][29a][29b]
[32a][32b], which are powerful antioxidants against free radicals because they act as
ꢂradical scavengersꢃ. This activity is attributed to their H-donating ability, which
depends on the position and degree of hydroxylation. It has been suggested that the
OHꢀC(3) group is a significant contributor to high antiradical activity as flavonols are
more potent antioxidants than the corresponding flavones. Glycosylation of this group,

Helvetica Chimica Acta – Vol. 93 (2010)
477
as in rutin, reduces the radical-scavenging capacity. In general, aglycones are more
potent antioxidant than their corresponding glycosides. An A-ring sugar results in a
greater activity decrease than glycosylation at C(3) of the heterocycle moiety.
Methylation of OH groups also suppresses the antioxidant activity [3][4][19]
[29a][29b].
The keto – enol tautomerism has been suggested as necessary for the ꢂscavengerꢃ
activity of the flavonoids [29a]. Moreover, in case of flavanones, it has been reported
that D-atoms are incorporated into these compounds via enolization [21c]. The present
studies also showed similar trends between the process of deuteration and antioxidant
activity of the flavonoids. Thus, flavonols (see 1 and 11) which exhibited more reactivity
towards H/D exchange as compared to flavones (see 3 and 12) were also more potent
antioxidants [4][16a][18b][19]. Furthermore, a free OH group at C(7) enhances the
H/D exchange as observed in 1 and 11, while in case of compound 2 the reaction was
slower due to the substitution of OHꢀC(7) OH by a sugar moiety. Interestingly, 1 was a
more powerful antioxidant than its 7-glucoside 2 [16a][18b], and 11 was also more
active than its 7-glucoside [4][29b]. Substitution at both OHꢀC(3) and OHꢀC(7) has
a greater influence in the decrease of the H/D exchange as well as of the antioxidant
activity, as observed in the cases of 4 – 6 [18b]. On the other hand, quercetin (11) is
itself a more powerful antioxidant than patuletin (1) [18b], and its H/D exchange at
C(8) was also very efficient as compared to that of 1 which contained a MeO group at
C(6).
Moreover, in the current studies, (8-D)patuletin (1a) was also prepared as a new
compound which could be used as a reference compound to understand diverse
pharmacological properties of patuletin, as labeled rutin has been used for drug-
metabolism studies [21a]. This investigation described the significance of H/D
exchange for obtaining deuterated compounds by a simple method. H/D Exchange is
considered as a powerful technique for gaining structural information about stereo-
isomers, reaction mechanistic studies, structure elucidation, and analytical purposes. It
has also been used for the preparation of polydeuterated polyphenol isoflavonoids as
internal standard in quantifying these compounds in biological fluids, and for profiling
estrogens [21a][27a]. More recently, synthesis of deuterated oxidative metabolites of
acrylamide and acrylonitrile has been reported for the quantification of their toxicities
in humans [33].
One of the authors, H. S., acknowledges the enabling role of the Higher Education Commission
Islamabad, Pakistan, and appreciates its financial support through ꢂMerit Scholarship Scheme for Ph.D.
Studies in Science & Technology (200 Scholarships)ꢃ.
Experimental Part
1. General. All the chemicals and solvents used were of anal. grade. The flavonoids 7 – 13 were from
Merck (Hohenbrunn, Germany). TLC: precoated silica-gel plates (SiO2; Merck KGaA, 64271
Darmstadt, Germany; HF 60254). Flash column chromatography (FC): SiO2 9385. UV Spectra: U-3200
(Hitachi, Japan); lmax (log e) in nm. IR Spectra: FT-IR-8900 (Shimadzu, Japan); in CHCl3 or KBr disc; ˜n
in cmꢀ1. EI- and HR-EI-MS: MAT-312 (Finnigan, Germany) and JMS-HX-110 spectrometer (Jeol,
Japan); in m/z (rel. %).
2. 1H- and 13C-NMR Measurements. 2.1. General Procedure. NMR Spectra: Bruker Aspect AV 300,
AV 400, and AV 500; at 300, 400, and 500 MHz for 1H; in (D6)DMSO, (D6)acetone, (D5)pyridine, CDCl3,

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Helvetica Chimica Acta – Vol. 93 (2010)
and CF3COOD; the 1H-NMR of 1, 11, and 12 at different temperatures (þ 308 to þ 708) in CF3COOD at
400 MHz. For the individual measurement, the following standard parameters were used: 1H-NMR,
KFID (1 – 13) spectral width 5 – 7 kHz (1), 7 kHz (1a, 2, and 4), 5.6 kHz (5), 5.7 kHz (6), 8 – 10 kHz (11),
6 kHz (9 and 10), 5 kHz (8, 7, 3, and 13), 6 – 10 kHz (12).
13C-NMR Measurement in (D5)pyridine (1 and 1a), (D6)acetone (1a and 4), and CDCl3 (5) at 75,
100, and 125 MHz, resp. 2D-COSY, HMQC, and HMBC with usual pulse programs and acquisition
parameter. The chemical shifts were referenced to the central peak of (D6)DMSO (d(H) 2.50 and d(C)
39.50), CDCl3 (d(H) 7.25 and d(C) 77.00), CF3COOD (d(H) 11.30 and d(C) 113.18 and 161.66),
(D5)pyridine (d(H) 7.19, 7.55, and 8.71 and d(C) 123.50, 135.50, and 149.90), and (D6)acetone (d(H) 2.05
and d(C) 30.0 and 206.29).
2.2. Reaction Kinetics. Kinetic measurements of the flavonoids 1, 3, and 12 were carried out in
CF3COOD, and progress of the reaction was monitored with the Avance-AV-300 spectrometer. The
NMR spectra were recorded after time intervals of ca. 5 – 10 min until no changes in peak heights were
observed over the defined time periods. The peak integration of nonexchangable H-atoms in the
flavonoid molecules were used to calculate the concentration of the deuterated products, and the first-
order rate constant was obtained by the least-squares fitting method.
3. Plant Material. Flowers of Tagetes patula were collected in 2000 and 2003 on the Karachi
University Campus. T. patula was identified by Dr. Rubina Dawar of the Department of Botany,
University of Karachi, and the voucher specimen (No. 67280) was deposited with the herbarium of the
same department.
4. Extraction and Isolation. The dried orange red flowers of Tagetes patula (181 g) were extracted
twice with MeOH. The combined MeOH extract was concentrated in vacuo and kept overnight at r.t.
when brownish insoluble matter (¼ JFR) settled down which was filtered. It turned black on exposure to
light and air. The filtrate on evaporation of the solvent furnished a residue JF (17 g) that was partitioned
between dist. H2O and petroleum ether. The aq. phase was extracted successively with CHCl3 (3ꢁ),
AcOEt (6ꢁ), and BuOH (3ꢁ), and each phase (petroleum ether, CHCl3, AcOEt, and BuOH) was
washed with H2O, dried (anh. Na2SO4), and concentrated providing respective residues, JF-P, JF-C, JF-
EA, and JF-BuOH. Since the first three AcOEt phases (JFEA, 1 – 3) showed a single spot on TLC, they
were pooled in a fraction JF-EA1 (1.2 g) which was found to be patuletin (1, 0.55%). Since the remaining
three AcOEt phases and H2O washings of all the six AcOEt phases had the same TLC profile showing a
single spot, they were combined to yield the fraction JF-EA2 (0.27 g), spectral studies of which
elucidated its structure as patulitrin (2; 0.11%). The TLC of the first BuOH phase (JF-BuOH1) also
showed the presence of 2, while BuOH phases 2 and 3 (JF-BuOH2) demonstrated different TLC profile.
In another work, the fresh, undried, and uncrushed red flowers (1160 g) of T. patula were extracted
with petroleum ether (3 ꢁ 10 l) followed by MeOH at r.t. The extracts were combined separately and the
solvents evaporated to give a concentrate (JFP) and a gummy material (JFM), resp. The methanol
extract JFM was divided into a MeOH-soluble and -insoluble (JFMI) portion, and the former was
concentrated to give a residue (JFMM). A portion (6.9 g) of JFMM was subjected to FC (SiO2 9385,
petroleum ether, AcOEt, MeOH, and H2O of increasing polarity): Fractions 1 – 142. Fr. 20b was a pure
compound (single spot on TLC (petroleum ether/AcOEt 7.5 :2.5)) and identified as luteolin (3) by
spectral studies.
5. (8-D)Patuletin (¼2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-6-methoxy-(8-2H)-4H-1-benzopyran-
4-one; 1a). Patuletin (1; 0.030 g) was converted into 1a when treated with CF3COOD (10 ml) on a water
bath for 2 h. UV (MeOH): 257.4, 371.0. IR (KBr): 3408.1, 3285.8, 2924.6, 1662.0, 1597.1. 1H-and 13C-NMR
((D6)acetone): Tables 1 and 5. EI-MS: 333.07 (70.6), 332.07 (100.0), 289.05 (66.5).
6. 3,3’,4’,7-Tetra-O-benzoylpatuletin (¼ 3,7-Bis(benzoyloxy)-2-[3,4-bis(benzoyloxy)phenyl]-5-hy-
droxy-6-methoxy-4H-1-benzopyran-4-one; 4). Patuletin (1; 0.1 g) in pyridine (0.5 ml) was treated with
benzoic anhydride (0.2078 g), and the mixture was kept at r.t. After 3 d, it was freed of pyridine and
divided into petroleum ether-soluble and -insoluble portions. The latter was kept for crystallization with
CHCl3/MeOH 1:1 furnishing yellow shiny crystals of 4 (85 mg; m.p. 2048) producing a single spot on
TLC (SiO2, petroleum ether/AcOEt 6.7:3.3, Rf 0.54). Yield 0.085 g (37.8%) of 4 [18a]. UV (MeOH):
330, 273 – 232, (MeONa) 445, 272, 228. IR (CHCl3): 3662.1, 3460.6, 2924.6, 1604.06, 1748.9, 1251.1.
1H-NMR (300 MHz, CF3COOD): 8.09 (s, 0.66 H, HꢀC(8)); 8.95 (d, J ¼ 1.89, 1.00 H, HꢀC(2’)); 8.38 (d,

Helvetica Chimica Acta – Vol. 93 (2010)
479
J ¼ 7.44, 1.00 H, HꢀC(5’)); 8.92 (dd, J ¼ 1.90, 8.63, HꢀC(6’)); 4.87 (s, MeOꢀC(6)); 8.76, 8.81, 9.02, 9.06
(br. d, J ¼ 7.75, 7.72, 7.89, 7.72, HꢀC(2’’,6’’) of 4 Bz); 8.17, 8.17 (br. t, J ¼ 7.50, HꢀC(3’’,5’’) of 2 Bz); 8.39,
8.39 (m, HꢀC(3’’,5’’) of 2 Bz); 8.30, 8.37, 8.49, 8.54 (br. t, J ¼ 7.24, 7.65, 7.46, 7.52, HꢀC(4’’) of 4 Bz).
1H-NMR (300 MHz, (D6)acetone): 7.32 (s, HꢀC(8)); 8.28 (d, J ¼ 2.10, HꢀC(2’)); 7.74 (d, J ¼ 8.60,
HꢀC(5’)); 8.17 (dd, J ¼ 2.02, 8.60, HꢀC(6’); 3.91 (s, MeOꢀC(6)); 12.30 (s, OHꢀC(5)); 7.98, 8.00, 8.21,
8.21 (dd, J ¼ 1.51, 8.50; 1.30, 8.41; 1.50, 8.91; 1.51, 8.91, HꢀC(2’’,6’’) of 4 Bz); 7.46, 7.46, 7.61, 7.61 (td, J ¼
8.02, 1.51; 8.00, 1.51; 7.50, 1.51; 7.51, 1.50, HꢀC(3’’,5’’) of 4 Bz); 7.64, 7.64 (br. t, J ¼ 7.50, HꢀC(4’’) of
2 Bz); 7.76, 7.76 (m, HꢀC(4’’) of 2 Bz). 13C-NMR (125 MHz, (D6)acetone): 151.29 (C(2)); 137.07 (C(3));
177.64 (C(4)); 154.16 (C(5)); 132.80 (C(6)); 156.94 (C(7)); 103.32 (C(8)); 152.03 (C(8a)); 110.52 (C(4a));
129.57 (C(1’)); 125.08 (C(2’)); 143.98 (C(3’)); 146.44 (C(4’)); 125.39 (C(5’)); 128.03 (C(6’)); 60.83
(MeOꢀC(6)); 128.74, 129.08, 129.24, 129.25 (C(1’’) of Bz); 130.72, 130.74, 130.97, 131.19 (C(2’’,6’’) of Bz);
128.03, 129.57, 129.57, 129.84 (C(3’’,5’’) of Bz); 134.96, 135.00, 135.07, 135.24 (C(4’’) of Bz); 164.12, 164.21,
164.36, 164.64 (CO of Bz). EI-MS: 748 (2.3), 644 (7.2), 540 (5.0), 105 (100), 183.1 (21). HR-EI-MS:
748.1561 (Mþ, C44H28O1þ2 ; calc. 748.1581).
7. 3,3’,4’,7-Tetra-O-cinnamoylpatuletin (¼(2E)-3-Phenylprop-2-enoic Acid 1,1’-{2-{3,4-bis{[(2E)-1-
oxo-3-phenylprop-2-en-1-yl]oxy}phenyl}-5-hydroxy-6-methoxy-4-oxo-4H-1-benzopyran-3,7-diyl} Ester;
5). Patuletin (1; 0.1 g) in pyridine (0.95 ml) was treated with cinnamoyl chloride (0.508 g), and the
mixture was kept for 10 d at r.t. The residue obtained on concentration of the mixture was treated with
MeOH yielding a MeOH-soluble and -insoluble off-white powdery compound (18.2 mg) demonstrating a
single spot on TLC (SiO2, petroleum ether/AcOEt 6.7:3.3, Rf 0.44). Yield 0.125 g (57.6%) of 5 [18a].
UV (MeOH): 286, 219 (MeONa), 405.4, 390, 275. IR (CHCl3): 3661.6, 3062.9, 1738.2, 163.91, 1601.27,
1120.5. 1H-NMR (300 MHz, CF3COOD): 7.99 (s, 1.00 H, HꢀC(8)); 8.81 (d, J ¼ 2.30, 1.00 H, HꢀC(2’));
8.38 (d, J ¼ 6.93, 1.00 H, HꢀC(5’)); 8.88 (m, 1.00 H, HꢀC(6’)); 4.85 (s, MeOꢀC(6)); 8.21, 8.21, 8.36, 8.36
(br. d, J ¼ 9.21, 9.21, 7.89, 7.89, HꢀC(2’’,6’’) of 4 Cinn); 8.03, 8.03 (td, J ¼ 7.82, 2.63, HꢀC(3’’,5’’) of
2 Cinn); 8.12, 8.12 (m, HꢀC(3’’,5’’) of 2 Cinn); 8.15, 8.15 (m, HꢀC(4’’) of 2 Cinn); 8.31, 8.31 (br. t, J ¼
9.21, HꢀC(4’’) of 2 Cinn); 7.29, 7.38, 7.51, 7.53 (d, J ¼ 15.98, HꢀC(8’’) of 4 Cinn); 8.57, 8.73, 8.85, 8.87 (d,
J ¼ 15.99, HꢀC(9’’) of 4 Cinn). 1H-NMR (400 MHz, CDCl3): 6.92 (s, HꢀC(8)); 7.89 (d, J ¼ 1.87,
HꢀC(2’)); 7.46 (d, J ¼ 7.00, HꢀC(5’)); 7.83 (dd, J ¼ 1.91, 8.71, HꢀC(6’)); 3.95 (s, MeOꢀC(6)); 7.47, 7.47
(br. d, J ¼ 6.50, HꢀC(2’’,6’’) of 2 Cinn); 7.60, 7.60 (m, HꢀC(2’’,6’’) of 2 Cinn); 7.32, 7.32 (br. t, J ¼ 6.81,
HꢀC(3’’,5’’) of 2 Cinn); 7.40, 7.40 (m, HꢀC(3’’,5’’) of 2 Cinn); 7.35, 7.35 (br. t, J ¼ 6.91, HꢀC(4’’) of
2 Cinn); 7.43, 7.43 (m, HꢀC(4’’) of 2 Cinn); 6.53, 6.55, 6.67, 6.70 (d, J ¼ 15.91, HꢀC(8’’) of 4 Cinn); 7.77,
7.83, 7.92, 7.94 (d, J ¼ 15.91, HꢀC(9’’) of 4 Cinn). 13C-NMR (100 MHz, CDCl3): 149.93 (C(2)); 136.25
(C(3)); 176.78 (C(4)); 153.97 (C(5)); 131.87 (C(6)); 155.64 (C(7)); 101.74 (C(8)), 150.97 (C(8a)); 109.86
(C(4a)); 127.71 (C(1’)); 123.98 (C(2’)); 142.74 (C(3’)); 145.16 (C(4’)); 124.10 (C(5’)), 126.68 (C(6’)); 60.73
(MeOꢀC(6)); 133.80, 133.81, 133.99, 134.03 (C(1’’) of Cinn); 128.42, 128.42, 128.42, 128.48 (C(2’’,6’’) of
Cinn); 128.95, 128.97, 128.98, 129.05 (C(3’’,5’’) of Cinn); 130.95, 130.95, 131.00, 131.00 (C(4’’) of Cinn);
163.75, 163.75, 163.83, 164.26 (C(7’’) of Cinn); 115.61, 115.96, 115.96, 116.15 (C(8’’) of Cinn); 147.80,
147.80, 147.92, 148.57 (C(9’’) of Cinn). EI-MS: 852.3 (3), 592.1 (4), 332.0 (5), 289.2 (50). HR-EI-MS:
852.21868 (Mþ, C52H36O1þ2 ; calc. 852.22068).
8. 3,3’,4’,7-Tetra-O-methylpatuletin (¼2-(3,4-Dimethoxyphenyl)-5-hydroxy-3,6,7-trimethoxy-4H-1-
benzopyran-4-one; 6). Patuletin (1; 0.1 g) was treated with K2CO3 (1 g) and dimethyl sulfate (0.14 ml)
in dried acetone at r.t. After 4 d (TLC monitoring), the mixture was concentrated and the residue treated
with petroleum ether, petroleum ether/CHCl3 4 :1, 1:1, and 1:4, CHCl3, and CHCl3/MeOH 1:1, and
finally with MeOH. Since all the petroleum ether and petroleum ether/CHCl3 fractions had the same
TLC profile, they were combined and subjected to prep. TLC (SiO2, petroleum ether/CHCl3 9.8 :0.2)
affording two bands, a pink and a blue one. The pink band was extracted to yield 6 (20 mg). TLC (SiO2,
1
petroleum ether/CHCl3 9.8 :0.2): Rf 0.47. UV, IR, H- and 2D-NMR, and EI-MS: in agreement with
structure. 1H-NMR (CF3COOD): Table 2.

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Received June 30, 2009