The fungal phytotoxin alternariol 9-methyl ether and some of its synthetic analogues inhibit the photosynthetic electron transport chain

Article abstract of DOI:10.1021/np4005882

Alternariol and monomethylalternariol are natural phytotoxins produced by some fungal strains, such as Nimbya and Alternaria. These substances confer virulence to phytopathogens, yet no information is available concerning their mode of action. Here we show that in the micromolar range alternariol 9-methyl ether is able to inhibit the electron transport chain (IC₅₀ = 29.1 ± 6.5 μM) in isolated spinach chloroplasts. Since its effectiveness is limited by poor solubility in water, several alternariol analogues were synthesized using different aromatic aldehydes. The synthesized 6H-benzo[c]cromen-6-ones, 5H-chromene[4,3-b]pyridin-5-one, and 5H-chromene[4,3-c]pyridin-5-one also showed inhibitory properties, and three 6H-benzo[c]cromen-6-ones were more effective (IC₅₀ = 12.8-22.8 μM) than the lead compound. Their addition to the culture medium of a cyanobacterial model strain was found to inhibit algal growth, with a relative effectiveness that was consistent with their activity in vitro. In contrast, the growth of a nonphotosynthetic plant cell culture was poorly affected. These compounds may represent a novel lead for the development of new active principles targeting photosynthesis.

Full text of DOI:10.1021/np4005882

Article
pubs.acs.org/jnp
The Fungal Phytotoxin Alternariol 9‑Methyl Ether and Some of Its
Synthetic Analogues Inhibit the Photosynthetic Electron Transport
Chain
Antonio Jacinto Demuner,† Luiz Claudio Almeida Barbosa,*,†,‡ Ana Cristina Mendes Miranda,‡
́
Guilherme Carvalho Geraldo,† Cleiton Moreira da Silva,† Samuele Giberti,§ Michele Bertazzini,§
and Giuseppe Forlani*,§
†Department of Chemistry, Federal University of Vico
̧
sa, Av. P.H. Rolfs, Vico
̧
sa, Minas Geraís, 36570-000, Brazil
nio Carlos 6627, Campus Pampulha, Belo Horizonte,
‡Department of Chemistry, Federal University of Minas Geraís, Av. Pres. Anto
̂
Minas Geraís, 31270-901, Brazil
§Department of Life Science and Biotechnology, University of Ferrara, Via L. Borsari 46, I-44121 Ferrara, Italy
S
* Supporting Information
ABSTRACT: Alternariol and monomethylalternariol are natural phytotoxins produced by some fungal strains, such as Nimbya
and Alternaria. These substances confer virulence to phytopathogens, yet no information is available concerning their mode of
action. Here we show that in the micromolar range alternariol 9-methyl ether is able to inhibit the electron transport chain (IC50
= 29.1 6.5 μM) in isolated spinach chloroplasts. Since its effectiveness is limited by poor solubility in water, several alternariol
analogues were synthesized using different aromatic aldehydes. The synthesized 6H-benzo[c]cromen-6-ones, 5H-chromene[4,3-
b]pyridin-5-one, and 5H-chromene[4,3-c]pyridin-5-one also showed inhibitory properties, and three 6H-benzo[c]cromen-6-ones
were more effective (IC50 = 12.8−22.8 μM) than the lead compound. Their addition to the culture medium of a cyanobacterial
model strain was found to inhibit algal growth, with a relative effectiveness that was consistent with their activity in vitro. In
contrast, the growth of a nonphotosynthetic plant cell culture was poorly affected. These compounds may represent a novel lead
for the development of new active principles targeting photosynthesis.
icrobial phytotoxins and their synthetic analogues are a
potential source of new bioactive compounds for
plant.12,13 All pathogenic species of Alternaria produce a series
of mycotoxins14,15 that have been found in many different
foodstuffs including fruits, vegetables, cereals, nuts, and oil
seeds.2,16−21 Several of these metabolites have been identified
as phenolic compounds with a coumarin-like structure.
Considering the interest in bioactive metabolites produced
by weed pathogens as sources of novel natural herbicides, the
production of toxins by Alternaria species was thoroughly
investigated,22 leading to the discovery of alternariol (1),
alternariol 9-methyl ether (2), and alternuisol (3) (Figure
1),15,23,24 for which total syntheses have also been
reported.22−29 Among Alternaria species, A. alternata is the
M
agriculture, medicine, and the food industry.1−7 Since the
discovery of paclitaxel production by the endophytic fungus
Taxomyces andreanae in 1993, the interest of many scientists in
fungal endophytes as potential producers of bioactive
compounds has increased even more, and over the past two
decades many valuable substances endowed with antimicrobial,
insecticidal, cytotoxic, anticancer, and other activities have been
isolated from these organisms. Such bioactive compounds
include alkaloids, terpenoids, steroids, quinones, isocoumarins,
lignans, phenylpropanoids, phenols, and lactones.6,8,9
Endophytic fungi spend all or part of their life cycle inter- or
intracellularly colonizing healthy tissues of their host plants.10,11
The endophytic fungus Alternaria sp. causes lesions in young
leaves, fruits, and stems. Depending on the intensity of the
disease, the lesions can lead to a total defoliation of the
Received: July 19, 2013
Published: November 18, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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true uncoupler, ammonium, at a level high enough to
completely dissipate the proton gradient (uncoupling con-
dition). Results (Figure 2) showed that neither condition
significantly changed the inhibition pattern, suggesting that
alternariol 9-methyl ether (2) directly interacts with one or
more of the electron carriers involved in the electron transport
chain. However, when the level of the toxin was raised above 20
μM, the resulting inhibitory effect reached a plateau and did not
increase further (Figure 2). The lack of a concentration−effect
relationship at high rates is likely due to the poor solubility of
alternariol in water.
Figure 1. Structure of alternariol (1), alternariol 9-methyl ether (2),
and alternuisol (3).
main producer of alternariol, alternariol 9-methyl ether, and
some structurally related dibenzopyrone compounds.30
Although there are reports of genotoxic, estrogenic, and
mutagenic effects in cell culture or in laboratory animals,31 the
toxicity of alternariols for humans and animals is low. Because
of such low toxicity, these compounds constitute a new lead
structure for the development of new herbicides for weed
control. During our research on the use of natural products as
models for the development of new agrochemicals,32−35 we
investigated the ability of alternariol 9-methyl ether (2) to
inhibit the chloroplastic electron transport chain. Some
alternariol analogues were also synthesized, several of which
were found to share the same target. Results strengthen the
hypothesis that the photosynthetic machinery represents a
target of Alternaria mycotoxins.
Twenty Alternariol Analogues were Synthesized
through Suzuki Condensation, Oxidation of Aldehyde,
and Cyclization Reaction. To overcome this issue and to
gain insight into the structure−activity relationship, some
analogues were designed and synthesized. The starting
aldehyde was prepared through Vilsmeyer formylation.25,36 In
this synthesis, a key stage is the formation of a C−C bond,
promoting the connection of two aryl compounds by the
Suzuki methodology37,38 resulting in the formation of 5a−s,
which were then oxidized to carboxylic acids 6a−j and 6p−s
using as the oxidizing agent sodium chlorite25,39 or to 6k−o
using potassium permanganate40 (Figure 3). The biaryl
compounds were submitted to a further reaction for the
elimination of methyl groups and formation of the lactone
compounds 7a−t (Figure 4). In some cases, low yields were
obtained (5f (37.6%), 5n (42.1%), 5o (40.9%), 5p (54.1%),
and 5s (37.6%)). However, at this stage of the research, no
effort was made to improve yields since our major interest was
the preparation of several analogues for biological evaluation.
No clear structural aspect was evident that could account for
such results, although the presence of a nitrogen atom at the
meta position in relation to the Br moiety corresponded in
most cases to a low yield at the biarylation step. This nitrogen
atom might in fact interfere with the palladium oxidative
addition of compound 3d, a key step on the Suzuki coupling
reaction.37,38 The biaryl compounds were submitted to a
further reaction for the elimination of methyl groups and the
formation of the lactone compounds 7a−t (Figure 4) in good
yields. All compounds were fully characterized by spectroscopic
RESULTS AND DISCUSSION
■
Alternariol 9-Methyl Ether Interferes with the Photo-
synthetic Process by Inhibiting the Electron Transport
Chain. When increasing levels of alternariol 9-methyl ether (2)
were added to the reaction mixture in the range from 1 to 20
μM, the light-dependent ferricyanide reduction by isolated
spinach chloroplasts was progressively reduced (Figure 2), with
1
data (IR, MS, H NMR, and 13C NMR).
Some Analogues Also Inhibit the Photosynthetic
Electron Transport Chain and Affect the Growth of a
Photoautotrophic Microalgae Culture but Not That of
Heterotrophic Cultured Plant Cells. The products obtained
were evaluated for their ability to interfere with the Hill
reaction. The results, summarized in Table 1, showed that some
of them (namely, 7a−d and 7l−m) are endowed with an
excellent inhibitory activity, whereas others are substantially
ineffective. The inhibitory potential seems related to the
presence of two hydroxyl groups at positions 4 and 6 (or 5) of
the first phenyl ring. Indeed, compounds sharing the whole
structure but lacking these substituents showed IC50 values 1−2
orders of magnitude higher. The effect could be at least in part
related to the greater polarity of the resulting analogues,
reflecting in turn an increased solubility. Indeed, although the
concentration−activity relationship profiles obtained for some
of the most active compounds (7c, 7d) were suggestive of a still
incomplete solubilization (Figure 5), the water solubility of the
synthesized analogues was in general much greater than that of
alternariol 9-methyl ether. In fact, although a certain degree of
lipophilicity is required to gain accessibility to the target site
inside the thylakoid membranes,41 an inadequate water
Figure 2. Effect of increasing concentrations of alternariol 9-methyl
ether (2) on the photosynthetic electron transport chain in isolated
spinach thylakoids. The Hill reaction was measured under basal,
uncoupling, and phosphorylating conditions. Results were expressed as
percent of untreated controls (82.1 1.8, 191.9 6.3, and 116.5 1.9
nmol s−1 (mg chlorophyll)−1, respectively) and are mean SE over
four replicates.
an estimated concentration causing 50% inhibition (IC50) of
29.1 6.5 μM. This effect could result from an interaction with
either the electron transport chain or the ATP synthase
coupling factor, or be a consequence of uncoupling properties
preventing the formation of a proton gradient across the
thylakoid membrane. To discriminate among these possibilities,
the activity in the presence of the same concentrations of the
mycotoxin was measured under conditions that allow ATP
synthesis (phosphorylating condition) and in the presence of a
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Figure 3. Synthesis of biaryl acids 6 as precursors of alternariol analogues.
solubility can prevent both inhibitor translocation inside the
plants and achieving high local concentrations.
all alternariols able to inhibit the electron transport chain are
also capable of reducing cyanobacterial growth, whereas the
negative controls were inactive. Interestingly, when the active
analogues were added even at high concentration to the
medium of heterotrophically growing plant cells, in most cases
no inhibition took place. Significant phytotoxic effects were
evident only for compound 7b. A significant (P = 0.0013)
correlation was found (Figure 6) between the concentrations
able to inhibit by 50% the photosynthetic electron transport
chain and those inhibiting the cyanobacterial growth.
Conclusions. The mycotoxin alternariol 9-methyl ether (2)
was found to inhibit the photosynthetic electron transport
chain in isolated spinach chloroplasts at the same concen-
trations at which its presence reduced the growth constant of a
cyanobacterial model strain. The possible occurrence of other
Substances able to interfere with the photosynthetic electron
transport chain are potentially active against photosynthetic
organisms. However, several factors, including differential
membrane permeability or solubility, subcellular compartmen-
talization, and the occurrence of metabolization or detox-
ification reactions, can completely abolish the inhibitory
potential found in vitro for a given compound. To verify such
a possibility, alternariol 9-methyl ether (2) and its analogues
that had previously shown efficacy against the Hill reaction
were evaluated for their ability to inhibit the photoautotrophic
growth of the blue-green alga Synechococcus. Three inactive
analogues were included in the experimental plan as negative
controls. Results, presented in Table 2, clearly pointed out that
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Figure 4. Final stage in the synthesis of alternariol analogues 7a−t.
Table 1. Concentrations of Alternariol 9-Methyl Ether (2)
and of Its Analogues (7a−7t) Able to Inhibit by 50% the
Basal Rate of the Photosynthetic Electron Transport in
a
Spinach (Spinacia oleracea L.) Thylakoids
compd
ID50 (μM)
2
29.1 6.5
22.8 8.8
12.8 3.0
19.5 8.7
35.7 14.9
>500
7a
7b
7c
7d
7e
7f
435 60
>500
7g,h,i,j,k
7l
Figure 5. Effect of increasing concentrations of two active alternariol
analogues on the basal rate of electron transport in spinach thylakoids.
Data concerning the commercial herbicide diuron are also included.
39.7 16.7
31.5 6.3
>500
7m
7n,o,p,q,r,s,t
diuron
0.27 0.02
0.11 0.02
0.08 0.02
part to higher water solubility. Our data suggests that
interference with the photosynthetic apparatus may contribute
to the phytotoxic activity of Alternaria toxins. Interestingly, the
most active alternariol analogues showed IC50 values only 1−2
orders of magnitude higher than those of commercial
herbicides targeting photosynthesis.42 Ineffective analogues
did not exert any toxic effect on the model cyanobacterial
strain. Moreover, the heterotrophic growth of suspension-
cultured plant cells was affected only in part, suggesting that the
interaction with the photosynthetic apparatus may be their
main mode of action inside the plant cell. The alternariol
scaffold may thus represent a new lead for the design of active
hexazinone
lenacil
a
Data obtained with some commercial herbicides targeting the
photosynthetic apparatus are also reported. Values for untreated
controls were 78.7 2.3 nmol s−1 (mg chlorophyll)−1.
targets cannot be ruled out, since at high levels it slightly
inhibited the proliferation of dark-grown cultured plant cells.
However, the effect was higher against the photosynthetically
active cultures, and similar effects were shown by some
alternariol analogues, whose superior activity may be related in
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Table 2. Autotrophic Growth of the Cyanobacterium Synechococcus elongatus and Heterotrophic Growth of Arabidopsis thaliana
Suspension Cultured Cells in the Presence of Micromolar Levels of Alternariol 9-Methyl Ether (2) and Selected Alternariol
a
Analogues
S. elongatus
compd
20 μM
50 μM
100 μM
200 μM
A. thaliana 200 μM
2
94.9 0.9
94.1 0.5
77.5 0.7
96.2 0.8
104.9 1.7
106.2 1.6
97.3 0.4
101.3 2.2
99.1 0.8
95.1 1.1
107.7 1.6
96.8 2.7
86.9 1.9
71.8 2.5
91.5 1.3
96.5 1.5
103.5 5.2
97.5 1.4
107.6 1.0
93.5 1.1
97.9 2.5
100.2 0.9
82.3 6.6
72.6 1.9
0.0 0.0
67.9 9.9
56.4 2.8
0.0 0.0
83.2 7.0
93.8 3.3
0.0 0.0
95.0 4.7
92.2 3.0
88.3 2.4
b
7a
7b
7c
7d
7f
94.3 3.0
90.4 3.1
111.5 0.2
108.9 2.9
93.4 1.3
66.1 1.1
80.4 0.5
100.4 2.2
30.8 5.3
71.9 6.2
108.1 1.5
97.7 1.5
89.4 0.9
20.1 0.8
63.7 0.8
110.6 1.0
7h
7k
7l
b
86.9 2.2
91.0 3.5
b
7m
7q
a
Growth was expressed as percentage of untreated controls (growth constant of 0.387 0.005 for cyanobacteria, dry weight increase of 5.25 0.03
b
mg d.w. mL−1 for plant cells). Data are means SE over at least three replications. Not determined.
(0.115 g, 0.100 mmol) were added. The reaction mixture was stirred
for 24 h at 80 °C. After this time, the mixture was quenched in NH4Cl
(20 mL) and extracted with ethyl acetate (3 × 30 mL). The organic
phase was washed with water (5 × 30 mL), dried over MgSO4, and
concentrated under reduced pressure to yield the required aldehyde
(5a) as a yellow solid (193.9 mg, 0.68 mmol). Yield: 68%; mp 119.5−
120.3 °C; IR (νmax) 3005, 2922, 2853, 2771, 1677, 1599, 1571, 1499,
1
1470, 1425, 1235, 1220, 1147, 872, 816 cm−1; H NMR (300 MHz,
CDCl3) δ 2.32 (t, J = 0.6 Hz, 3H, 5′-CH3), 3.70 (s, 3H, 2′-OCH3),
3.87 (s, 3H, 6-OCH3), 3.93 (s, 3H, 4-OCH3), 6.39 (d, J = 2.4 Hz, 1H,
H5), 6.45 (d, J = 2.4 Hz, 1H, H3), 6.81 (d, J = 8.4 Hz, 1H, H3′), 7.02
(dd, J = 2.4, 0.6 Hz, 1H, H6′), 7.16 (ddd, J = 8.4, 2.4, 0.6 Hz, 1H,
H4′), 9.85 (s, 1H, CHO); 13C NMR (75 MHz, CDCl3) δ 20.7 (5′-
CH3), 55.8 (6-OCH3 and 2′-OCH3), 56.1 (4-OCH3), 98.1 (C3),
108.2 (C5), 110.6 (C1 and C3′), 128.3 (C5′), 130.1 (C4′ and C1′),
131.3 (C6′), 146.1 (C2), 154.4 (C2′), 162.3 (C6), 164.4 (C4) and
Figure 6. Relationship between the ability of alternariol analogues to
inhibit the photosynthetic electron transport chain in spinach
chloroplasts and their toxicity against the photosynthetic cyanobacte-
rium Synechococcus elongatus.
+
190.7 (CHO); HRMS (ESI TOF-MS) calcd for C17H18NaO4
309.1097; found 309.1112.
The same procedure was used for the synthesis of biaryls (5b−s),
whose yields are presented in Figure 3.
principles capable of interfering with the photosynthetic
electron transport.
2,4-Dimethoxy-6-(5-chloro-2-methoxyphenyl)benzaldehyde
(5b). Yellow solid; mp 127.7−128.7 °C; IR (νmax) 3005, 2940, 2840,
1
2769, 1684, 1594, 1570, 1492, 1450, 1205, 1030, 809 cm−1; H NMR
EXPERIMENTAL SECTION
(300 MHz, CDCl3) δ 3.70 (s, 3H, 2′-OCH3), 3.87 (s, 3H, 6-OCH3),
3.93 (s, 3H, 4-OCH3), 6.34 (d, 1H, J = 2.4 Hz, H5), 6.48 (d, 1H, J =
2.4 Hz, H3). 6.83 (d, 1H, J = 8.7 Hz, H3′), 7.17 (d, 1H, J = 2.4 Hz,
H6′), 7.30 (dd, 1H, J = 8.7, 2.4 Hz, H4′), 9.97 (s, 1H, CHO); 13C
NMR (75 MHz, CDCl3) δ 55.8 (2′-OCH3), 56.1 (6-OCH3), 56.2 (4-
OCH3), 98.3 (C3), 108.4 (C5), 111.8 (C3′), 117.5 (C1), 125.7 (C5′),
129.1 (C4′), 130.0 (C6′), 130.9 (C1′), 143.5 (C2), 155.3 (C2′), 162.9
(C6), 164.6 (C4), 189.7 (CHO); HRMS (ESI TOF-MS) calcd for
■
General Experimental Procedures. Reagents and solvents were
purified as described by Perrin and Armarego (1988).43 1H and 13C
NMR spectra were recorded on a Varian Mercury 300 spectrometer at
300 and 75 MHz using CDCl3 or DMSO-d6 or CD3OD as solvent and
TMS as internal standard. Mass spectra were recorded using a
Shimadzu GCMS-QP5050A (70 eV). Infrared spectra were recorded
on a Perkin Elmer Paragon 1000 FTIR spectrophotometer, with the
samples prepared as a thin film on a NaCl plate, scanning from 635 to
4000 cm−1. Accurate mass (HRMS) data were recorded under ESI
conditions in Bruker MicroToF spectrometer (resolution = 10000
fwhm) using a lock-spray source. The lock-mass used for calibration
was tetraoctylammonium bromide in the positive ion mode. Analytical
thin layer chromatography analyses were conducted on aluminum-
packed precoated silica gel plates. Flash chromatography was
performed over silica gel (0.035−0.070 mm). Melting points were
recorded in MQAPF-301 (Microquimica, Brazil). 2-Bromo-4,6-
dimethoxybenzaldehyde 3a was synthesized by Vilsmeyer formyla-
tion25,36 of 1-bromo-3,5-dimethoxybenzene, purchased from Sigma-
Aldrich (St. Louis, MO).
+
C16H15ClNaO4 329.0551; found 329.0560.
2,4-Dimethoxy-6-(5-fluoro-2-methoxyphenyl)benzaldehyde (5d).
Yellow solid; mp 105.8−106.6 °C; IR (νmax) 3002, 2941, 2839, 2772,
1686, 1595, 1499, 1459, 1210, 1159, 1033 cm−1; 1H NMR (300 MHz,
CDCl3) δ 3.69 (s, 3H, 2′-OCH3), 3.87 (s, 3H, 6-OCH3), 3.93 (s, 3H,
4-OCH3), 6.35 (d, 1H, J = 2.4 Hz, H5), 6.50 (d, 1H, J = 2.4 Hz, H3),
6.83 (dd, 1H, J = 9.0, 4.5 Hz, H3′), 6.93 (dd, 1H, J = 8.4, 3.0 Hz, H6′),
7.04 (ddd, 1H, J = 9.0, 8.1, 3.0 Hz, H4′), 9.95 (s, 1H, CHO); 13C
NMR (75 MHz, CDCl3) δ 55.8 (6-OCH3), 56.2 (4-OCH3), 56.2 (2′-
OCH3), 98.2 (C3), 108.3 (C5), 111.5 (d, J = 8.3 Hz, C3′), 115.3 (d, J
= 22.4 Hz, C4′), 117.3 (d, J = 23.8 Hz, C6′), 117.5 (C2), 130.4 (d, J =
7.7 Hz, C1′), 143.8 (C1), 152.6 (d, J = 2.2 Hz, C2′), 157.0 (d, J = 238
Hz, C5′), 162.8 (C6), 164.6 (C4), 189.9 (CHO); HRMS (ESI TOF-
Synthesis. 2,4-Dimethoxy-6-(5-methyl-2-methoxyphenyl)-
benzaldehyde (5a). To a 25 mL two neck round-bottomed flask,
equipped for reflux, 2-bromo-4,6-dimethoxybenzaldehyde (0.245 g,
1.00 mmol) in DMF (15 mL), 5-methyl-2-methoxyphenylboronic acid
(0.498 g, 3.00 mmol), K2CO3 (0.198 g, 2.00 mmol), and Pd(PPh3)4
+
MS) calcd for C16H15FNaO4 313.0847; found 313.0870.
2-(2-Methoxy-5-methylphenyl)benzaldehyde (5e). Colorless oil;
IR (νmax) 3061, 3008, 2926, 2839, 2750, 1694, 1598, 1503, 1254, 1235,
1
1035, 1023, 809, 772, and 744 cm−1; H NMR (300 MHz, CDCl3) δ
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2.36 (t, 3H, J = 0.6 Hz, 5′-CH3), 3.71 (s, 3H, 2′-OCH3), 6.87 (d, 1H, J
= 8.1 Hz, H3′), 7.11 (dd, 1H, J = 2.1, 0.6 Hz, H6′), 7.21 (ddd, 1H, J =
8.1, 2.1, 0.6 Hz, H4′), 7.36 (ddd, 1H, J = 7.5, 1.5, 0.6 Hz, H6), 7.47
(ddd, 1H, J = 7.8, 7.5, 1.5 Hz, H4), 7.64 (td, 1H, J = 7.5, 1.5 Hz, H5),
7.99 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz, H3), 9.80 (d, 1H, J = 0.6 Hz,
CHO); 13C NMR (75 MHz, CDCl3) δ 20.7 (5′-CH3), 55.7 (2′-
OCH3), 110.8 (C3′), 126.7 (C5′), 126.8 (C3), 127.9 (C5), 130.5
(C4′), 131.4 (C6′), 132.3 (C6), 133.9 (C4), 134.2 (C2), 142.2 (C1),
154.7 (C2′), 193.0 (CHO); HRMS (ESI TOF-MS) calcd for
127.7 (d, J = 8.0 Hz, C1′), 135.5 (C1), 148.8 (C5), 152.8 (d, J = 1.8
Hz, C2′), 153.5 (C4), 156.8 (d, J = 240.2 Hz, C5′), 191.0 (CHO);
+
HRMS (ESI TOF-MS) calcd for C16H15FNaO4 313.0847; found
313.0882.
3,4-Dimethoxy-6-(4-fluoro-2-methoxyphenyl)benzaldehyde
(5m). Colorless solid; mp 117.7−118.2 °C; IR (νmax) 3085, 3025,
2974, 2956, 2915, 2876, 1675, 1592, 1497, 1441, 1260, 1212, 1134,
1017, 946, 838, 728 cm−1; 1H NMR (300 MHz, CDCl3) δ 3.74 (3H, s,
OCH3), 3.95 (3H, s, OCH3), 3.97 (3H, s, OCH3), 6.71 (1H, dd, J =
10.0, 2.4 Hz, H3′), 6.76 (1H, s, H3), 6.77 (1H, td, J = 8.2, 2.4 Hz,
H5′), 7.21 (1H, dd, J = 8.2, 6.7 Hz, H6′), 7.50 (1H, s, H6), 9.61 (1H,
s, CHO); 13C NMR (75 MHz, CDCl3) δ 56.2 (OCH3), 56.3 (OCH3),
56.5 (OCH3), 99.1 (d, J = 26.0 Hz, C3′), 107.3 (d, J = 24.0 Hz, C5′),
108.2 (C6), 113.2 (C3), 122.4 (d, J = 2.5 Hz, C1′), 127.3 (C2), 132.3
(d, J = 9.8 Hz, C6′), 136.1 (C1), 148.7 (C5), 153.5 (C4), 158.0 (d, J =
9.9 Hz, C2′), 163.7 (d, J = 247.2 Hz, C4′), 191.1 (CHO); HRMS (ESI
+
C15H14NaO2 249.0886; found 249.0872.
2-(5-Chloro-2-methoxyphenyl)benzaldehyde (5f). White solid;
mp 95.1−96.3 °C; IR (νmax) 3065, 3008, 2941, 2842, 2750, 1697,
1
1597, 1495, 1255, 1025, 811, 771, 646 cm−1; H NMR (300 MHz,
CDCl3) δ 3.72 (s, 3H, 2′-OCH3), 6.90 (d, 1H, J = 9.0 Hz, H3′), 7.28
(d, 1H, J = 2.7 Hz, H6′), 7.33 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz, H6), 7.37
(dd, 1H, J = 9.0, 2.7 Hz, H4′), 7.50 (ddd, 1H, J = 7.8, 7.5, 1.5 Hz, H5),
7.65 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.99 (ddd, 1H, J = 7.5, 1.5, 0.6 Hz,
H3), 9.78 (d, 1H, J = 0.6 Hz, CHO); 13C NMR (75 MHz, CDCl3) δ
56.0 (2′-OCH3), 112.1 (C3′), 126.2 (C5′), 127.1 (C3), 128.4 (C5),
128.7 (C1′), 129.7 (C4′), 131.2 (C6′), 131.2 (C6), 134.0 (C4), 134.2
(C2), 140.4 (C1), 155.4 (C2′), 192.2 (CHO); HRMS (ESI TOF-MS)
+
TOF-MS) calcd for C16H15FNaO4 313.0847; found 313.0870.
3-(5-Fluoro-2-methoxyphenyl)isonicotinaldehyde (5n). White
solid; mp 105.8−106.9 °C; IR (νmax) 3036, 2996, 2953, 2913, 2846,
1700, 1581, 1493, 1400, 1212, 1191, 1177, 1022, 880, 749, 714 cm−1;
1H NMR (300 MHz, CDCl3) δ 3.69 (3H, s, 2′-OCH3), 6.92 (1H, dd, J
= 9.0, 4.3 Hz, H3′), 7.06 (1H, dd, J = 8.3, 3.1 Hz, H6′), 7.13 (1H, ddd,
J = 9.0, 7.9, 3.1 Hz, H4′), 7.72 (1H, dd, J = 5.0, 0.6 Hz, H5), 8.66 (3H,
s, H2), 8.77 (1H, d, J = 5.0 Hz, H6), 9.79 (1H, d, J = 0.6 Hz, CHO);
13C NMR (75 MHz, CDCl3) δ 56.1 (2′-OCH3), 112.1 (d, J = 8.2 Hz,
C3′), 117.0 (d, J = 23.0 Hz C4′), 118.1 (d, J = 24.0 Hz, C6′), 119.2
(C5), 124.5 (d, J = 7.5 Hz, C1′), 133.5 (C3), 139.5 (C4), 150.2 (C6),
152.2 (C2), 153.0 (C2′), 157.3 (d, J = 241.0 Hz, C5′), 191,3 (CHO);
+
calcd for C14H11ClNaO2 269.0340; found 269.0298.
2-(4-Fluoro-2-methoxyphenyl)benzaldehyde (5g). White solid;
mp 68.7−70.0 °C; IR (νmax) 3066, 3008, 2940, 2839, 2749, 1690,
1
1597, 1507, 1449, 1279, 1192, 1151, 830, 765 cm−1; H NMR (300
MHz, CDCl3) δ 3.73 (s, 3H, 2′-OCH3), 6.71 (dd, 1H, J = 10.8, 2.4 Hz,
H3′), 6.80 (td, 1H, J = 8.4, 2.4 Hz, H5′), 7.24 (dd, 1H, J = 8.4, 6.6 Hz,
H6′), 7.32 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz, H6), 7.49 (ddd, 1H, J = 7.8,
7.5, 1.5 Hz, H5), 7.64 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.99 (ddd, 1H, J =
7.5, 1.5, 0.6 Hz, H3), 9.77 (d, 1H, J = 0.6 Hz, CHO); 13C NMR (75
MHz, CDCl3) δ 55.9 (2′-OCH3), 99.3 (d, J = 26.3 Hz, C3′), 107.7 (d,
J = 21.2 Hz, C5′), 122.9 (d, J = 3.6 Hz, C1′), 127.1 (C3), 128.1 (C5),
131.5 (C6),132.3 (d, J = 10.4 Hz, C6′), 134.0 (C4), 134.3 (C2), 141.1
(C1), 157.9 (d, J = 9.7 Hz, C2′), 164.1 (d, J = 246 Hz, C4′), 192.6
+
HRMS (ESI TOF-MS) calcd for C13H10FNNaO2 254.0588; found
254.0561.
3-(5-Chloro-2-methoxyphenyl)isonicotinaldehyde (5o). Colorless
solid; mp 112.7−113.5 °C; IR (νmax) 3046, 2979, 2946, 2874, 2762,
1
1695, 1600, 1504, 1469, 1399, 1276, 1150, 1028, 840 cm−1; H NMR
(300 MHz, CDCl3) δ 3.73 (3H, s, 2′-OCH3), 6.93 (1H, d, J = 8.8 Hz,
H3′), 7.33 (1H, d, J = 2,6 Hz, H6′), 7.43 (1H, dd, J = 8.8, 2.6 Hz,
H4′), 7.75 (1H, dd, J = 5.0, 0.7 Hz, H5), 8.68 (1H, s, H2), 8.80 (1H,
d, J = 5.0 Hz, H6), 9.80 (d, J = 0.7 Hz, 1H, CHO); 13C NMR (75
MHz, CDCl3) δ 55.7 (2′-OCH3), 112.0 (C3′), 119.0 (C5), 124.6
(C1′), 126.3 (C5′), 130.4 (C4′), 130.7 (C6′), 133.0 (C3), 139.2 (C4),
149.9 (C6), 151.9 (C2), 155.2 (C2′), 190.9 (CHO); HRMS (ESI
+
(CHO); HRMS (ESI TOF-MS) calcd for C14H11FNaO2 253.0635;
found 253.0634.
2-(3-Bromo-5-methyl-2-methoxyphenyl)benzaldehyde (5j). Yel-
low oil; IR (νmax) 3060, 2996, 2927, 2849, 2750, 1693, 1595, 1469,
1
1419, 1266, 1237, 1196, 998, and 858 cm−1; H NMR (300 MHz,
CDCl3) δ 2.35 (3H, s, 5′-CH3), 3.32 (3H, s, 2′-OCH3), 7.07−7.05
(1H, m, H4′), 7.40 (1H, dd, J = 7.5, 0.6 Hz, H6), 7.47−7.45 (1H, m,
H6′), 7.56−7.49 (1H, m, H5), 7.65 (1H, td, J = 7.6, 7.6, 1.5 Hz, H4),
8.03 (1H, dd, J = 7.6, 7.6, 1.1 Hz, H3), 9.83 (1H, d, J = 0.6 Hz, CHO);
13C NMR (75 MHz, CDCl3) δ 20.4 (5′-CH3), 60.1 (2′-OCH3), 117.3
(C3′) 127.1 (C3), 128.2 (C5), 130.9 (C6), 131.3 (C4′), 132.7 (C5′),
133.5 (C4), 134.1 (C6′), 135.4 (C2), 140.6 (C1), 152.1 (C2′), 191.6
+
TOF-MS) calcd for C13H10ClNNaO2 270.0292; found 270.0312.
3-(3-Bromo-5-fluoro-2-methoxyphenyl)isonicotinaldehyde (5p).
White solid; mp 138.5−139.6 °C; IR (νmax) 3075, 2939, 2857, 2750,
1709, 1595, 1572, 1498, 1463, 1416, 1317, 1280, 1257, 1233, 1209,
1
1178, 1029, 1001, 871, 839, 798, 739 cm−1; H NMR (300 MHz,
CDCl3) δ 3.34 (s, 3H, 2′-OCH3), 7.10 (dd, 1H, J = 7.8, 3.0 Hz, H4′),
7.45 (dd, 1H, J = 7.5, 3.0 Hz, H6′), 7.82 (d, 1H, J = 5.0 Hz, H5), 8.75
(s, 1H, H2), 8.88 (d, 1H, J = 5.0 Hz, H6), 9.86 (d, 1H, J = 0.9 Hz,
CHO); 13C NMR (75 MHz, CDCl3) δ 60.7 (2′-OCH3), 117.4 (d, J =
23.5 Hz, C6′), 118.5 (d, J = 10.4 Hz, C3′), 119.9 (C5), 121.9 (d, J =
25.2 Hz, C4′), 130.8 (d, J = 8.0 Hz, C1′), 132.4 (C3), 139.1 (C4),
151.1 (C6), 151.3 (C2′), 151.8 (C2), 158.8 (d, J = 247.8 Hz, C5′),
190.3 (CHO); HRMS (ESI TOF-MS) calcd for C13H9BrFNNaO2
331.9693; found 331.9706.
+
(CHO); HRMS (ESI TOF-MS) calcd for C15H13BrNaO2 326.9991;
found 326.9888.
2,4-Dimethoxy-6-(5-bromo-2-methoxyphenyl)benzaldehyde
(5k). Yellow solid; mp 124.8−125.3 °C; IR (νmax) 3054, 3005, 2938,
2849, 2772, 1680, 1589, 1489, 1326, 1202, 1118, 1160, 1200, 803, 731
cm−1; 1H NMR (300 MHz, CDCl3) δ 3.70 (3H, s, OCH3), 3.87 (3H,
s, OCH3), 3.93 (3H, s, OCH3), 6.34 (1H, d, J = 2.3 Hz, H5), 6.50
(1H, d, J = 2.3 Hz, H3), 6.78 (1H, d, J = 8,8 Hz, H3′), 7.31 (1H, d, J =
2.5 Hz, H6′), 7.45 (1H, dd, J = 8.8, 2.5 Hz, H4′), 9.96 (1H, s, CHO);
13C NMR (75 MHz, CDCl3) δ 55.6 (OCH3), 55.7 (OCH3), 55.9
2-(5-Fluoro-2-methoxyphenyl)nicotinaldehyde (5q). White solid;
mp 130.1−131.0 °C; IR (νmax) 3070, 2998, 2968, 2908, 2840, 1694,
1
(OCH3), 98.1 (C3), 108.1 (C5), 112.0 (C3′), 112.7 (C1), 117.2
(C5′), 131.0 (C1′), 131.8 (C4′), 132.5 (C6′), 143.1 (C2), 155.5
(C2′), 162.6 (C6), 164.3 (C4), 189.5 (CHO); HRMS (ESI TOF-MS)
1580, 1566, 1495, 1435, 1251, 1191, 1169, 809, 749, 716 cm−1; H
NMR (300 MHz, CDCl3), δ 3.72 (s, 3H, 2′-OCH3), 6.93 (dd, 1H, J =
9.0, 4.2 Hz, H3′), 7.17 (ddd, 1H, J = 9.0, 7.8, 3.3 Hz, H4′), 7.33 (dd,
1H, J = 8.6, 3.3 Hz, H6′), 7.45 (ddd, 1H, J = 7.8, 4.8, 0.9 Hz, H5), 8.26
(dd, 1H, J = 7.8, 1.8 Hz, H4), 8.89 (dd, 1H, J = 4.8, 1.8 Hz, H6), 9.78
(d, 1H, J = 0.9 Hz, CHO); 13C NMR (75 MHz, CDCl3), δ 56.2 (2′-
OCH3), 112.2 (d, J = 8.1 Hz, C3′), 117.6 (d, J = 22.8 Hz, C4′), 118.4
(d, J = 24.0 Hz, C6′), 123.2 (C5), 127.7 (d, J = 7.4 Hz, C1′), 130.3
(C3), 134.9 (C4), 152.8 (C2), 154.0 (C6), 157.5 (d, J = 238.6 Hz,
C5′), 157.6 (C2′), 191.4 (CHO); HRMS (ESI TOF-MS) calcd for
+
calcd for C16H15BrNaO4 373.0046; found 373.0126.
3,4-Dimethoxy-6-(5-fluoro-2-methoxyphenyl)benzaldehyde (5l).
Colorless solid; mp 114.4−115.2 °C; IR (νmax) 3077, 3063, 3023,
3008, 2997, 2939, 2886, 2858, 1677, 1593, 1511, 1496, 1460, 1263,
1
1251, 1176, 1023, 731 cm−1; H NMR (300 MHz, CDCl3) δ 3.71
(3H, s, OCH3), 3.95 (3H, s, OCH3), 3.97 (3H, s, OCH3), 6.76 (1H, s,
H3), 6.90 (1H, dd, J = 8.7, 4.4, Hz, H3′), 7.20 (1H, dd, J = 8.5, 3.1 Hz,
H6′), 7.10 (1H, ddd, J = 8.7, 3.1 Hz, H4′), 7.50 (1H, s, H6), 9.62 (1H,
s, CHO); 13C NMR (75 MHz, CDCl3) δ 56.2 (OCH3), 56.3 (OCH3),
56.5 (OCH3), 108.1 (C6), 111.5 (d, J = 8.3 Hz, C3′), 112.8 (C3),
115.6 (d, J = 22.5 Hz, C4′), 118.2 (d, J = 23.5 Hz, C6′), 127.0 (C2),
+
C13H10FNNaO2 254.0588; found 254.0521.
2-(2-Methoxy-5-methylphenyl)nicotinaldehyde (5r). White solid;
mp 63.4−64.1 °C; IR (νmax) 2948, 2838, 1699, 1580, 1565, 1501,
1
1438, 1381, 1277, 1251, 1237, 1147, 1025, 835, 808 cm−1; H NMR
2239
dx.doi.org/10.1021/np4005882 | J. Nat. Prod. 2013, 76, 2234−2245

Journal of Natural Products
Article
(300 MHz, CDCl3) δ 2.37 (s, 3H, 5′-CH3), 3.71 (s, 3H, 2′-OCH3),
6.88 (d, 1H, J = 8.4 Hz, H3′), 7.27 (ddd, 1H, J = 8.4, 2.4, 0.6 Hz, H4′),
7.38−7.40 (m, 1H, H6′), 7.41 (ddd, 1H, J = 7.8, 4.8, 0.8 Hz, H5), 8.24
(dd, 1H, J = 7.8, 1.8 Hz, H4), 8.88 (dd, 1H, J = 4.8, 1..8 Hz, H6), 9.78
(d, 1H, J = 0.8 Hz, CHO); 13C NMR (75 MHz, CDCl3) δ 20.6 (5′-
CH3), 55.6 (2′-OCH3), 111.0 (C3′), 122.7 (C5), 126.0 (C1′), 130.3
(C3), 131.1 (C5′), 132.0 (C4′), 132.1 (C6′), 134.7 (C4), 153.8 (C6),
154.6 (C2′), 159.0 (C2), 192.0 (CHO); HRMS (ESI TOF-MS) calcd
141.4 (C2), 152.5 (d, J = 2.5 Hz, C2′), 157.1 (d, J = 238 Hz, C5′),
159.1 (C6), 162.5 (C4), 168.7 (COOH); HRMS (ESI TOF-MS)
+
calcd for C16H15FNaO5 329.0796; found 329.0778.
2-(5-Methyl-2-methoxyphenyl)benzoic Acid (6e). White solid; mp
162.9−165.0 °C; IR (νmax) 3500−2500, 3060, 3002, 2924, 2834, 1690,
1599, 1503, 1296, 1279, 1251, 1237 cm−1; 1H NMR (300 MHz,
CDCl3) δ 2.35 (t, 3H, J = 0.6 Hz, 5′-CH3), 3.68 (s, 3H, 2′-OCH3),
6.77 (d, 1H, J = 8.1 Hz, H3′), 7.09 (dd, 1H, J = 2.1, 0.6 Hz, H6′), 7.13
(ddd, 1H, J = 8.1, 2.1, 0.6 Hz, H4′), 7.34 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz,
H6), 7.40 (td, 1H, J = 7.8, 1.5 Hz, H5), 7.57 (td, 1H, J = 7.8, 1.5 Hz,
H4), 7.92 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz, H3); 13C NMR (75 MHz,
CDCl3) δ 20.8 (5′-CH3), 55.3 (2′-OCH3), 110.7 (C3′), 127.3 (C3),
129.6 (C5), 130.0 (C4′/C5′), 130.3 (C1′), 130.8 (C6), 130.9 (C2),
131.8 (C6′), 132.5 (C4), 139.3 (C1), 154.3 (C2′), 173.7 (COOH);
+
for C14H13NNaO2 250.0838; found 250.0861.
2-(3-Bromo-5-fluoro-2-methoxyphenyl)nicotinaldehyde (5s).
White solid; mp 88.5−89.9 °C; IR (νmax) 3075, 3003, 2939, 2865,
2824, 2744, 1702, 1579, 1567, 1467, 1449, 1419, 1383, 1265, 1257,
1233, 1196, 996, 931, 803, 741 cm−1, 1H NMR (300 MHz, CDCl3) δ
3.34 (s, 3H, 2′-OCH3), 7.32 (dd, 1H, J = 8.1, 3.0 Hz, H4′), 7.45 (dd,
1H, J = 7.5, 3.0 Hz, H6′), 7.52 (ddd, 1H, J = 8.0, 4.8, 0.6 Hz, H5), 8.33
(dd, 1H, J = 8.0, 1.8 Hz, H4), 8.90 (dd, 1H, J = 4.8, 1.8 Hz, H6), 9.80
(d, 1H, J = 0.6 Hz, CHO); 13C NMR (75 MHz, CDCl3) δ 61.2 (2′-
OCH3), 112.2 (d, J = 8.3 Hz, C3′), 117.6 (d, J = 23.7 Hz, C6′), 122.2
(d, J = 25.2 Hz, C4′), 123.8 (C5), 129.9 (C3), 133.7 (d, J = 8.6 Hz,
C1′), 135.6 (C4), 153.8 (C6), 153.9 (C2), 156.6 (C2′), 158.9 (d, J =
247.6 Hz, C5′), 190.3 (CHO); HRMS (ESI TOF-MS) calcd for
+
HRMS (ESI TOF-MS) calcd for C15H14NaO3 265.0835; found
265.0859.
2-(5-Chloro-2-methoxyphenyl)benzoic Acid (6f). White solid; mp
195.9−197.5 °C; IR (νmax) 3500−2500, 3067, 3008, 2974, 1691, 1596,
1
1496, 1027, 813, 769 cm−1; H NMR (300 MHz, CD3OD) δ 3.69 (s,
3H, 2′-OCH3), 6.93 (d, 1H, J = 9.0 Hz, H3′), 7.18 (d, 1H, J = 2.7 Hz,
H6′), 7.27 (ddd, 1H, J = 7.5, 1.5, 0.6 Hz, H6), 7.28 (dd, 1H, J = 9.0,
2.7 Hz, H4′), 7.42 (td, 1H, J = 7.5, 1.5 Hz, H5), 7.57 (td, J = 7.5, 1.5
Hz, H4), 7.85 (ddd, 1H, J = 7.5, 1.5, 0.6 Hz, H3), 13C NMR (75 MHz,
CD3OD) δ 54.7 (2′-OCH3), 111.8 (C3′), 125.2 (C5′), 127.4 (C3),
128.1 (C5), 129.2 (C6′), 129.3 (C6), 131.0 (C4′), 131.5 (C4), 132.4
(C1′), 132.7 (C2), 137.7 (C1), 155.4 (C2′), 170.2 (COOH); HRMS
+
C13H9BrFNNaO2 331.9693; found 331.9732.
2,4-Dimethoxy-6-(5-methyl-2-methoxyphenyl)benzoic Acid (6a).
To a round bottomed flask (50 mL), a solution of 2,4-dimethoxy-6-(5-
methyl-2-methoxyphenyl)benzaldehyde (0.170 g, 0.594 mmol) in t-
BuOH/water (15 mL, 5:1 v/v) was added. Then NaH2PO4 (0.214 g,
1.78 mmol), NaClO2 (0.430 g, 0.594 mmol), and THF (0.50 mL, 5.35
mmol) were added, and the mixture was stirred for 3 h at room
temperature. The solvents were evaporated, and the residue was
extracted with DCM (3 × 10 mL). The organic extracts were
combined and the organic phase was dried with anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The acid obtained
was used in the subsequent stage of reaction.
+
(ESI TOF-MS) calcd for C14H11ClNaO3 285.0289; found 285.0310.
2-(4-Fluoro-2-methoxyphenyl)benzoic Acid (6g). White solid; mp
151.4−152.3 °C; IR (νmax) 3500−2500, 3084, 3006, 2980, 2944, 1689,
1
1603, 1506, 1449, 1153, 1031, 947, 840, 764 cm−1; H NMR (300
MHz, CDCl3) δ 3.68 (s, 3H, 2′-OCH3), 6.61 (dd, 1H, J = 10.8, 2.4 Hz,
H3′), 6.74 (td, 1H, J = 8.4, 2.4 Hz, H5′), 7.19 (dd, 1H, J = 8.4, 6.9 Hz,
H6′), 7.30 (ddd, 1H, J = 7.5, 1.5, 0.3 Hz, H6), 7.42 (td, 1H, J = 7.5, 1.5
Hz, H5), 7.61 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.95 (ddd, 1H, J = 7.5, 1.5,
0.3 Hz, H3); 13C NMR (75 MHz, CDCl3) δ 55.5 (2′-OCH3), 99.2 (d,
J = 25.7 Hz, C3′), 107.3 (d, J = 21.2 Hz, C5′), 126.4 (d, J = 3.5 Hz,
C1′), 127.5 (C5), 130.3 (C3), 130.6 (d, J = 9.8 Hz, C6′), 130.6 (C2),
131.8 (C6), 132.7 (C4) 138.6 (C1), 157.5 (d, J = 9.8 Hz, C2′), 163.6
(d, J = 245 Hz, C4′), 173.5 (COOH); HRMS (ESI TOF-MS) calcd
The same procedure was used for the synthesis of acids (6b−j and
6p−s), whose yield is presented in Figure 3.
2,4-Dimethoxy-6-(5-chloro-2-methoxyphenyl)benzoic Acid (6b).
White solid; mp 123.2−123.9 °C; IR (νmax) 3300−2500, 3005, 2940,
2841, 1692, 1599, 1493, 1249, 1031 cm−1; 1H NMR (300 MHz,
CDCl3) δ 3.68 (s, 3H, 2′-OCH3), 3.84 (s, 3H, 6-OCH3), 3.92 (s, 3H,
4-OCH3), 6.41 (d, 1H, J = 2.4 Hz, H5), 6.52 (d, 1H, J = 2.4 Hz, H3),
6.79 (d, 1H, J = 8.7 Hz, H3′), 7.18 (d, 1H, J = 2.7 Hz, H6′), 7.26 (dd,
1H, J = 8.7, 2.7 Hz, H4′); 13C NMR (75 MHz, CDCl3) δ 55.8 (2′-
OCH3), 55.8 (6-OCH3), 56.6 (4-OCH3), 98.5 (C3), 108.6 (C5),
111.9 (C3′), 114.0 (C1), 125.6 (C5′), 128.8 (C6′), 129.7 (C4′), 132.0
(C1′), 141.1 (C2), 155.1 (C2′), 159.1 (C6), 162.5 (C4), 169.5
+
for C14H11FNaO3 269.0584; found 269.0602.
2-(5-Fluoro-2-methoxyphenyl)benzoic Acid (6h). White solid; mp
141.7−142.9 °C; IR (νmax) 3500−2500, 3068, 3002, 2941, 2837, 1691,
1
1596, 1499, 1251, 1179, 1033, 807, 731 cm−1; H NMR (300 MHz,
CDCl3) δ 3.67 (s, 3H, 2′-OCH3), 6.79 (dd, 1H, J = 9.3, 4.5 Hz, H3′),
7.00 (dd, 1H, J = 8.1, 3.3 Hz, H6′), 7.01 (ddd, 1H, J = 9.3, 8.1, 3.3 Hz,
H4′), 7.32 (ddd, 1H, J = 7.5, 1.5, 0.6 Hz, H6), 7.44 (td, 1H, J = 7.5, 1.5
Hz, H5), 7.60 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.85 (ddd, 1H, J = 7.5, 1.5,
0.6 Hz, H3); 13C NMR (75 MHz, CDCl3) δ 55.8 (2′-OCH3), 111.5
(d, J = 8.6 Hz, C3′), 114.9 (d, J = 22.3 Hz, C4′), 116.6 (d, J = 24.0 Hz,
C6′), 127.9 (C5), 130.3 (C3), 130.6 (C2), 131.6 (C6), 131.7 (C1),
132.8 (C4), 138.3 (d, J = 3.3 Hz, C1′), 152.5 (d, J = 1.7 Hz, C2′),
157.3 (d, J = 237 Hz, C5′), 173.5 (COOH); HRMS (ESI TOF-MS)
+
(COOH); HRMS (ESI TOF-MS) calcd for C16H15ClNaO5
345.0500; found 345.0524.
2,4-Dimethoxy-6-(4-fluoro-2-methoxyphenyl)benzoic Acid (6c).
White solid; mp 172.0−173.2 °C; IR (νmax) 3500−2500, 3012, 2976,
1
2940, 2844, 1695, 1601, 1508, 1280, 1022, 829 cm−1; H NMR (300
MHz, CDCl3) δ 3.68 (s, 3H, 2′-OCH3), 3.84 (s, 3H, 6-OCH3), 3.92
(s, 3H, 4-OCH3), 6.42 (d, 1H, J = 2.4 Hz, H5), 6.51 (d, 1H, J = 2.4
Hz, H3), 6.61 (dd, 1H, J = 10.8, 2.4 Hz, H3′), 6.69 (td, 1H, J = 8.4, 2.4
Hz, H5′), 7.15 (dd, J = 8.4, 6.9 Hz, H6′); 13C NMR (75 MHz, CDCl3)
δ 55.7 (2′-OCH3), 55.7 (6-OCH3), 56.5 (4-OCH3), 98.2 (C3), 99.3
(d, J = 25.7 Hz, C3′), 107.1 (d, J = 21.2 Hz, C5′), 108.8 (C5), 114.4
(C1), 126.2 (d, J = 3.4 Hz, C1′), 130.7 (d, J = 9.8 Hz, C6′), 141.3
(C2), 157.5 (d, J = 9.8 Hz, C2′), 159.0 (C6), 162.3 (C4), 164.0 (d, J =
244 Hz, C4′), 169.8 (COOH); HRMS (ESI TOF-MS) calcd for
+
calcd for C14H11FNaO3 269.0584; found 269.0613.
2-(5-Bromo-2-methoxyphenyl)benzoic Acid (6i). Colorless solid;
mp 211.6−212.0 °C; IR (νmax) 3150−2250, 3070, 2998, 2973, 2940,
1
2814, 1680, 1594, 1482, 1286, 1277, 1246, 1224, 1023, 810 cm−1; H
NMR (300 MHz, CDCl3) δ 3.64 (3H, s, OCH3), 6.96 (1H, d, J = 8.8
Hz, H3′), 7.28 (1H, d, J = 7.6, 1.3 Hz, H6), 7.30 (1H, d, J = 2.5 Hz,
H6′), 7.44 (1H, dt, J = 7.6, 7.6, 1.2 Hz, H5), 7.48 (1H, dd, J = 8.8, 2.5
Hz, H4′), 7.57 (1H, dt, J = 7.6, 7.6, 1.3 Hz, H4), 7.77 (1H, dd, J = 7.6,
1.2 Hz, H3); 13C NMR (75 MHz, CDCl3) δ 56.0 (2′-OCH3), 112.3
(C5′), 113.5 (C3′), 128.2 (C5), 129.6 (C3), 131.7 (C4′), 131.7 (C1′),
132.0 (C6′), 132.2 (C6), 133.0 (C4), 133.3 (C2), 137.1 (C1), 156.0
(C2′), 169.1 (COOH); HRMS (ESI TOF-MS) calcd for
+
C16H15FNaO5 329.0796; found 329.0784.
2,4-Dimethoxy-6-(5-fluoro-2-methoxyphenyl)benzoic Acid (6d).
White solid; mp 117.6−118.8 °C; IR (νmax) 3500−2500, 3072, 3005,
2942, 2840, 1693, 1600, 1500, 1461, 1209, 1164, 1034; 1H NMR (300
MHz, CDCl3) δ 3.68 (s, 3H, 2′-OCH3), 3.85 (s, 3H, 6-OCH3), 3.93
(s, 3H, 4-OCH3), 6.43 (d, 1H, J = 2.4 Hz, H5), 6.52 (d, 1H, J = 2.4
Hz, H3), 6.80 (dd, 1H, J = 8.7, 4.5 Hz, H3′), 6.96 (td, 1H, J = 8.7, 3.0
Hz, H4′), 7.01 (dd, 1H, J = 8.1, 3.0 Hz, H6′); 13C NMR (75 MHz,
CDCl3) δ 55.8 (6-OCH3), 56.1 (2′-OCH3), 56.6 (4-OCH3), 96.1
(C1), 98.5 (C3), 108.7 (C5), 111.7 (d, J = 8.6 Hz, C3′), 114.9 (d, J =
22.4 Hz, C6′), 116.9 (d, J = 24.0 Hz, C4′), 131.8 (d, J = 7.6 Hz, C1′),
+
C14H11BrNaO3 328.9784; found 328.9813.
2-(3-Bromo-5-methyl-2-methoxyphenyl)benzoic Acid (6j). Color-
less solid; mp 184.9−186.4 °C; IR (νmax) 3200−2250, 3028, 2992,
2926, 2853, 2821, 2646, 1696, 1597, 1470, 1449, 1409, 1296, 1232,
1002, 928, 751, 736 cm−1; 1H NMR (300 MHz, CDCl3) δ 2.30 (3H, s,
5′-CH3), 3.25 (3H, s, 2′-OCH3), 7.03 (1H, s, H4′), 7.34 (1H, d, J =
2240
dx.doi.org/10.1021/np4005882 | J. Nat. Prod. 2013, 76, 2234−2245

Journal of Natural Products
Article
7.5 Hz, H6), 7.41 (1H, s, H6′), 7.49 (1H, t, J = 7.5, 7.5 Hz, H5), 7.60
(1H, t, J = 7.5, 7.5 Hz, H4), 7.85 (1H, d, J = 7.5 Hz, H3); 13C NMR
(75 MHz, CDCl3) δ 20.6 (5′-CH3), 60.5 (2′-OCH3), 116.2 (C3′),
128.5 (C5), 130.1 (C3), 131.0 (C4′), 131.7 (C6), 131.9 (C4), 132.6
(C5′), 133.0 (C6′), 135.5 (C2), 137.2 (C1′), 138.0 (C1), 152.0 (C2′),
destroyed with saturated sodium sulfite (Na2SO3), the pH of the
mixture was adjusted to 3 using HCl solution (2 mol L−1) so as to
solubilize the MnO2 derived from the destruction of KMnO4, and the
mixture was subjected to extraction with ethyl acetate (3 × 30 mL).
The organic phases were combined and dried with anhydrous
magnesium sulfate (MgSO4), filtered, and concentrated under reduced
pressure to yield the required aldehyde (6k) as a white solid (113.0
mg; 0.31 mmol). Yield: 91%; mp 182.4−183.2 °C; IR (νmax) 3213−
2400, 3020, 3005, 2917, 2848, 2834, 1680, 1593, 1567, 1488, 1332,
+
169.1 (COOH); HRMS (ESI TOF-MS) calcd for C15H13BrNaO3
342.9940; found 342.9889.
2-(3-Bromo-5-fluoro-2-methoxyphenyl)isonicotinic Acid (6p).
White solid; mp 232.3−234.2 °C; IR (νmax) 3446, 3078, 3034, 2948,
2862, 2420, 1916, 1721, 1602, 1573, 1465, 1423, 1394, 1283, 1218,
1194, 1174, 1103, 1073, 1044, 994, 927, 871, 851, 775, 743, 707, 670,
605, 494, 431 cm−1; 1H NMR (300 MHz, CD3OD) δ 3.26 (s, 3H, 2′-
OCH3), 7.32 (dd, 1H, J = 8.7, 3.0 Hz, H4′), 7.64 (dd, 1H, J = 8.1, 3.0
Hz, H6′), 7.75 (d, 1H, J = 5.0 Hz, H5), 8.64 (s, 1H, H2), 8.76 (d, 1H,
J = 5.0 Hz, H6); 13C NMR (75 MHz, CD3OD) δ 60.9 (2′-OCH3),
117.3 (C3′), 117.4 (d, JC,F = 23.8 Hz, C6′), 120.3 (d, J 25.4 Hz, C4′),
123.2 (C5), 130.8 (C1′), 135.0 (C3), 139.7 (C4), 150.9 (C6), 151.5
(d, J = 243.5 Hz, C2′), 151.8 (C2), 158.4 (d, J = 243.5 Hz, C5′), 167.6
1
1285, 1266, 1200, 1026, 803, 799, 621 cm−1; H NMR (300 MHz,
CDCl3) δ 3.67 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.91 (3H, s,
OCH3), 6.41 (1H, d, J = 2.3 Hz, H5), 6.52 (1H, d, J = 2.3 Hz, H3),
6.75 (1H, d, J = 8.8 Hz, H3′), 7.32 (1H, d, J = 2.5 Hz, H6′), 7.45 (1H,
dd, J = 8.7, 2.7 Hz, H4′); 13C NMR (75 MHz, CDCl3) δ 55.7
(OCH3), 55.8 (OCH3), 56.6 (OCH3), 98.5 (C3), 108.5 (C5), 112.5
(C3′), 112.9 (C1), 113.2 (C5′), 131.8 (C4′), 132.5 (C1′), 132.6
(C6′), 140.8 (C2), 155.6 (C2′), 159.1 (C6), 162.5 (C4) and 170.0
+
(COOH); HRMS (ESI TOF-MS) calcd for C16H15BrNaO5
+
(COOH); HRMS (ESI TOF-MS) calcd for C13H9BrFNNaO3
388.9995; found 388.9989.
347.9642; found 347.9587.
The same procedure was used for the synthesis of acids (6l and 6o),
2-(5-Fluoro-2-methoxyphenyl)nicotinic Acid (6q). White solid; mp
133.8−135.2 °C; IR (νmax) 3493, 3084, 2998, 2986, 2938, 2837, 2478
1743, 1684, 1578, 1498, 1459, 1438, 1285, 1247, 1185, 1179, 1090,
1042, 1025, 907, 884, 844, 813, 809, 799, 744, 713, 643, 604, 561
whose yield is presented in Figure 3.
3,4-Dimethoxy-6-(5-fluoro-2-methoxyphenyl)benzoic Acid (6l).
Colorless solid; mp 215.2−216.2 °C; IR (νmax) 3250−2500, 3068,
2998, 2964, 2936, 2837, 1675, 1596, 1518, 1499, 1460, 1417, 1347,
1262, 1212, 1170, 1097, 1025, 740 cm−1; 1H NMR (300 MHz,
CDCl3) δ 3.71 (3H, s, OCH3), 3.95 (3H, s, OCH3), 3.97 (3H, s,
OCH3), 6.79 (1H, s, H3), 6.93 (1H, dd, J = 8.5, 4.6 Hz, H3′), 7.04
(1H, dd, J = 7.5, 3.0 Hz, H6′), 7.09 (1H, td, J = 8.5, 8.5, 3.0 Hz, H4′),
7.35 (1H, s, H6); 13C NMR (75 MHz, CDCl3) δ 56.2 (OCH3), 56.3
(OCH3), 56.5 (OCH3), 112.2 (d, J = 8.4 Hz, C3′), 113.0 (C6), 114.3
(d, J = 22.4 Hz, C4′), 114.8 (C3), 118.2 (d, J = 23.5 Hz, C6′), 124.4
(C1′), 131.4 (C1), 132.8 (d, J = 8.1 Hz, C2′), 147.8 (C5), 151.3 (C2),
153.2 (C4), 156.7 (d, J = 235.3 Hz, C5′), 168.5 (COOH); HRMS
1
cm−1; H NMR (300 MHz, CD3OD) δ 3.71 (s, 3H, 2′-OCH3), 6.98
(dd, 1H, J = 9.0, 4.2 Hz, H3′), 7.12 (ddd, 1H, J = 9.0, 8.1, 3.3 Hz,
H4′), 7.18 (ddd, 1H, J = 8.7, 3.3, 0.3 Hz, H6), 7.50 (dd, 1H, J = 7.8,
5.0 Hz, H5), 8.26 (dd, 1H, J = 7.8, 1.8 Hz, H4), 8.70 (dd, 1H, J = 5.0,
1.8 Hz, H6); 13C NMR (75 MHz, CD3OD) δ 54.8 (2′-OCH3), 111.6
(d, J = 8.1 Hz, C3′), 115.7 (d, J = 22.9 Hz, C4′), 116.6 (d, J = 24.5 Hz,
C6′), 122.6 (C5), 129.8 (C3), 130.5 (d, J = 7.5 Hz, C1′), 138.0 (C4),
150.8 (C6), 153.2 (C2), 154.7 (C2′), 157.2 (d, J = 235.8 Hz, C5′),
+
167.6 (COOH); HRMS (ESI TOF-MS) calcd for C13H10FNNaO3
+
270.0537; found 270.0588.
(ESI TOF-MS) calcd for C16H15FNaO5 329.0796; found 329.0884.
2-(2-Methoxy-5-methylphenyl)nicotinic Acid (6r). White solid; mp
63.4−64.1 °C; IR (νmax) 3538, 3003, 2968, 2835, 2626, 2493, 1868,
1738, 1679, 1615, 1587, 1570, 1503, 1467, 1441, 1396, 1293, 1275,
1246, 1217, 1197, 1182, 1143, 1099, 1070, 1047, 1029, 952, 883, 829,
805, 790, 741, 710, 671, 639, 607, 583, 547, 517, 483 cm−1; 1H NMR
(300 MHz, CD3OD) δ 2.34 (s, 3H, 5′-CH3), 3.70 (s, 3H, 2′-OCH3),
6.89 (d, 1H, J = 8.4 Hz, H3′), 7.20 (ddd, 1H, J = 8.4, 2.4, 0.6 Hz, H4′),
7.23−7.26 (m, 1H, H6′), 7.46 (dd, 1H, J = 7.8, 5.0 Hz, H5), 8.22 (dd,
1H, J = 7.8, 1.8 Hz, H4), 8.67 (dd, 1H, J = 5.0, 1.8 Hz, H6); 13C NMR
(75 MHz, CD3OD) δ 19.4 (5′-CH3), 54.3 (2′-OCH3), 110.4 (C3′),
122.1 (C5), 128.6 (C1′), 129.8 (C3), 130.1 (C5′), 130.5 (C4′), 130.6
(C6′), 137.9 (C4), 150.3 (C6), 154.8 (C2′), 155.9 (C2), 169.1
(COOH); HRMS (ESI TOF-MS) calcd for C14H13NNaO3+ 266.0788;
found 266.0762.
3,4-Dimethoxy-6-(4-fluoro-2-methoxyphenyl)benzoic Acid (6m).
Colorless solid; mp 217.5−218.3 °C; IR (νmax) 3250−2400, 3062,
2999, 2954, 2920, 2845, 1670, 1595, 1498, 1455, 1256, 1207, 1148,
1020 cm−1; 1H NMR (300 MHz, CDCl3) δ 3.65 (3H, s, OCH3), 3.80
(3H, s, OCH3), 3.82 (3H, s, OCH3), 6.74 (1H, s, H3), 6.78 (1H, ddd,
J = 8.5, 8.5, 2.5 Hz, H5′), 6.87 (1H, dd, J = 11.5, 2.5 Hz, H3′), 7.18
(1H, dd, J = 8.3, 7.0 Hz, H6′), 7.35 (1H, s, H6); 13C NMR (75 MHz,
CDCl3) δ 56.1 (2xOCH3), 56.2 (OCH3), 99.5 (d, J = 25.7 Hz, C3′),
106.7 (d, J = 21.0 Hz, C5′), 113.1 (C6), 114.9 (C3), 124.4 (C2), 127.6
(d, J = 3.2 Hz, C1′), 131.0 (d, J = 9.9 Hz, C6′), 131.9 (C1), 147.7
(C5), 151.3 (C4), 158.1 (d, J = 10.2 Hz, C2′), 162.9 (d, J = 242.3 Hz,
C4′), 168.6 (COOH); HRMS (ESI TOF-MS) calcd for
+
C16H15FNaO5 329.0796; found 329.0824.
3-(5-Fluoro-2-methoxyphenyl)isonicotinic Acid (6n). Colorless
solid; mp 248.3−250.2 °C; IR: 3022, 2973, 2939, 2834, 1704, 1595,
1494, 1477, 1284, 1252, 1217, 1032, 950, 905, 867, 821, 724 cm−1; 1H
NMR (300 MHz, DMSO-d6) δ 3.65 (s, 3H, 2′-OCH3), 7.03 (dd, 1H, J
= 9.6, 4.5 Hz, H3′), 7.22−7.15 (m, 2H, H6′ and H4′), 7.66 (d, 1H, J =
5.0 Hz, H5), 8.56 (s, 1H, H2) 8.69 (d, 1H, J = 5.0 Hz, H6); 13C NMR
(75 MHz, DMSO-d6) δ 56.3 (2′-OCH3), 112.6 (d, J = 8.4 Hz, C3′),
115.8 (d, J = 22.5 Hz, C4′), 117.1 (d, J = 24.1 Hz, C6′), 122.6 (C5),
128.2 (d, J = 8.1 Hz, C1′), 131.2 (C3), 140.4 (C4), 149.9 (C6), 151.7
(C2), 153.2 (C2′), 156.8 (d, J = 236.1 Hz, C5′), 167.9 (COOH);
2-(3-Bromo-5-fluoro-2-methoxyphenyl)nicotinic Acid (6s). White
solid; mp 192.9−194.7 °C; IR (νmax) 3445, 3091, 3070, 2955, 2927,
2826, 2476, 1870, 1729, 1701, 1572, 1469, 1420, 1299, 1268, 1143,
1094, 1063, 999, 933, 875, 835, 799, 776, 766, 742, 679, 655, 647, 609,
1
599, 457 cm−1; H NMR (300 MHz, CD3OD) δ 3.36 (s, 3H, 2′-
OCH3), 7.19 (dd, 1H, J = 8.4, 3.0 Hz, H4′), 7.46 (dd, 1H, J = 8.0, 3.0
Hz, H6′), 7.59 (dd, 1H, J = 8.0, 4.9 Hz, H5), 8.38 (dd, 1H, J = 8.0, 1.8
Hz, H4), 8.76 (dd, 1H, J = 4.9, 1.8 Hz, H6); 13C NMR (75 MHz,
CD3OD) δ 60.4 (2′-OCH3), 116.2 (d, J = 24.1 Hz, C6′), 116.8 (d, J =
10.3 Hz, C3′), 120.2 (d, J = 25.8 Hz, C4′), 123.4 (C5), 129.0 (C3),
137.2 (d, J = 8.6 Hz, C1′), 138.7 (C4), 151.2 (C6), 151.4 (C2), 154.5
(C2′), 158.6 (d, J = 244.4 Hz, C5′), 167.9 (COOH); HRMS (ESI
+
HRMS (ESI TOF-MS) calcd for C13H10FNNaO3 270.0537; found
270.0498.
3-(5-Chloro-2-methoxyphenyl)isonicotinic Acid (6o). White solid;
+
TOF-MS) calcd for C13H9BrFNNaO3 347.9642; found 347.9612.
mp 253.2−254.1 °C; IR: 3083, 3027, 2985, 2942, 2843, 2361, 1702,
1
2,4-Dimethoxy-6-(5-bromo-2-methoxyphenyl)benzoic Acid (6k).
To a round bottomed flask (25 mL) a solution of 2,4-dimethoxy-6-(5-
bromo-2-methoxyphenyl)benzaldehyde (0.120 g, 0.34 mmol) in 2 mL
of t-BuOH was added. Then to the mixture, 2 mL of sodium hydrogen
phosphate (NaH2PO4), pH 4.4, was added and then the solution of
potassium permanganate (0.5 mol L−1) (0.320 g; 2.00 mmol) under
vigorous stirring. The reaction mixture was kept at room temperature
for about 2 h, with the consumption of the aldehyde followed by thin
layer chromatography. At the end of reaction, excess KMnO4 was
1596, 1472, 1397, 1281, 1251, 1219, 1186, 1031, 820 cm−1; H NMR
(300 MHz, DMSO-d6) δ 3.67 (s, 3H, 2′-OCH3), 7.06 (d, 1H, J = 8.0
Hz, H3′), 7.35 (d, 1H, J = 2.6 Hz, H6′), 7.41 (dd, 1H, J = 8.7, 2.7 Hz,
H4′), 7.66 (d, 1H, J = 5.0 Hz, H5), 8.55 (s, 1H, H2), 8.68 (d, 1H, J =
5.0 Hz, H6); 13C NMR (75 MHz, DMSO-d6) δ 56.9 (2′-OCH3),
100.1 (C3′), 100.5 (C5), 102.8 (C1′), 118.0 (C5′), 120.2 (C4′), 127.3
(C6′), 134.2 (C3), 135.9 (C4), 149.8 (C6), 163.8 (C2), 164.2 (C2′),
+
167.2 (COOH); HRMS (ESI TOF-MS) calcd for C13H10ClNNaO3
286.0241; found 286.0202.
2241
dx.doi.org/10.1021/np4005882 | J. Nat. Prod. 2013, 76, 2234−2245

Journal of Natural Products
Article
+
7,9-Dihydroxy-2-methyl-6H-benzo[c]chromen-6-one (7a). To a
round bottomed flask (20 mL), under an atmosphere of nitrogen and
at the temperature of 0 °C, a solution of 2,4-dimethoxy-6-(5-methyl-2-
methoxyphenyl)benzoic acid (0.145 g, 0.480 mmol) in anhydrous
DCM (5 mL) was added. To this mixture a 0.5 mol L−1 solution of
BBr3 (3.84 mL, 1.92 mmol) in anhydrous DCM was added slowly.
After the addition, the ice bath was removed and the system was
maintained under magnetic agitation for a period of 24 h at room
temperature. Then, methanol (0.5 mL) was added slowly (approx-
imately 3 min) to the system until all gas had been released and all
precipitate formed had been dissolved. The solvent was evaporated,
and the residue was extracted with ethyl acetate (3 × 15 mL). The
organic extracts were combined, and the resulting organic phase was
dried with anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue obtained was submitted to separation
in column (hexane/ethyl acetate 7:1 v/v), resulting in the chromen-6-
one (7a) (38.7 mg, 0.160 mmol) with 34.5% yield. Mp 281.8−283.1
°C; IR (νmax) 3222, 3130, 2925, 1641, 1587, 1510, 1456, 1209, 1172
cm−1; 1H NMR (300 MHz, DMSO-d6) δ 2.39 (s, 3H, 2-CH3), 6.44 (d,
1H, J = 2.4 Hz, H8), 7.13 (d, 1H, J = 2.4 Hz, H10), 7.28 (d, 1H, J =
8.4 Hz, H4), 7.35 (dd, 1H, J = 8.4, 2.1 Hz, H3), 7.92 (d, 1H, J = 2.1
Hz, H1), 11.34 (s, 2H, 7-OH/9-OH); 13C NMR (75 MHz, DMSO-d6)
δ 20.0 (2-CH3), 97.7 (C12), 100.1 (C10), 102.1 (C8), 116.5 (C4),
116.8 (C13), 123.2 (C1), 131.3 (C3), 134.0 (C2), 136.1 (C11), 147.7
(C14), 163.1 (C7), 163.9 (C6), 165.4 (C9); HRMS (ESI TOF-MS)
HRMS (ESI TOF-MS) calcd for C14H10NaO2 233.0573; found
233.0581.
2-Chloro-6H-benzo[c]chromen-6-one (7f). White solid; mp
174.8−176.4 °C; IR (νmax) 3084, 3068, 1747, 1604, 1484, 1265,
1067, 1035, 811, 766, 712, 680 cm−1; 1H NMR (300 MHz, CDCl3) δ
7.32 (d, 1H, J = 8.7 Hz, H4), 7.44 (dd, 1H, J = 8.7, 2.4 Hz, H3), 7.64
(td, 1H, J = 7.8, 1.5 Hz, H8), 7.86 (td, 1H, J = 7.8, 1.5 Hz, H9), 8.03
(d, 1H, J = 2.4 Hz, H1), 8.07 (d, 1H, J = 7.8 Hz, H7), 8.42 (dd, 1H, J =
7.8, 1.5 Hz, H10); 13C NMR (75 MHz, CDCl3) δ 119.4 (C4), 119.6
(C2), 121.5 (C13), 122.0 (C7), 122.9 (C1), 129.9 (C8), 130.3 (C11),
130.6 (C3), 131.0 (C10), 133.8 (C12), 135.3 (C9), 149.9 (C14),
+
160.8 (C6); HRMS (ESI TOF-MS) calcd for C13H7ClNaO2
253.0027; found 253.0018.
3-Fluoro-6H-benzo[c]chromen-6-one (7g). White solid; mp
150.5−152.0 °C; IR (νmax) 3072, 1752, 1607, 1456, 1274, 1155,
1098, 875, 763, 712, 680 cm−1; H NMR (300 MHz, CDCl3) δ 7.08
1
(td, 1H, J = 8.4, 2.4 Hz, H2), 7.09 (dd, 1H, J = 8.4, 2.4 Hz, H4), 7.58
(ddd, 1H, J = 8.1, 7.5, 1.5 Hz, H8), 7.83 (ddd, 1H, J = 8.1, 7.5, 1.5 Hz,
H9), 8.04 (ddd, 1H, J = 8.1, 1.5, 0.6 Hz, H7), 8.05 (dd, 1H, J = 8.4, 7.2
Hz, H1), 8.39 (ddd, 1H, J = 8.1, 1.5, 0.6 Hz, H10); 13C NMR (75
MHz, CDCl3), δ 105.4 (d, J = 25.1 Hz, C4), 112.7 (d, J = 22.3 Hz,
C2), 114.9 (d, J = 2.9 Hz, C13), 120.7 (C11), 121.7 (C7), 124.6 (d, J
= 9.8 Hz, C1), 129.0 (C8), 130.9 (C10), 134.5 (C12), 135.3 (C9),
152.4 (d, J = 12.0 Hz, C14), 161.0 (C6), 163.7 (d, J = 250 Hz, C3);
+
HRMS (ESI TOF-MS) calcd for C13H7FNaO2 237.0322; found
237.0314.
+
calcd for C14H10NaO4 265.0471; found 265.0477.
2-Fluoro-6H-benzo[c]chromen-6-one (7h). White solid; mp
The compounds (7b−n) were prepared using the same procedure.
2-Chloro-7,9-dihydroxy-6H-benzo[c]chromen-6-one (7b). White
solid; mp >295 °C; IR (νmax) 3395, 3086, 1678, 1570, 1457, 1410,
1090 cm−1; 1H NMR (300 MHz, DMSO-d6) δ 6.45 (d, 1H, J = 2.1 Hz,
H8), 7.13 (d, 1H, J = 2.1 Hz, H10), 7.37 (d, 1H, J = 9.0 Hz, H4), 7.53
(dd, 1H, J = 9.0, 2.4 Hz, H3), 8.14 (d, 1H, J = 2.4 Hz, H1), 11.17 (s,
2H, 7-OH/9-OH); 13C NMR (75 MHz, DMSO-d6), δ 98.8 (C12),
102.1 (C8), 103.9 (C1), 119.9 (C4), 120.1 (C2), 124.1 (C10), 130.0
(C13), 131.3 (C3), 136.1 (C11), 149.4 (C14), 164.1 (C9), 164.4
148.3−149.4 °C; IR (νmax) 3062, 1724, 1600, 1496, 1182, 1073,
873, 817, 764, 712, 681 cm−1; H NMR (300 MHz, CDCl3) δ 7.19
1
(ddd, 1H, J = 9.0, 7.5, 3.0 Hz, H3), 7.35 (dd, 1H, J = 9.0, 4.5 Hz, H4),
7.63 (ddd, 1H, J = 8.1, 7.5, 1.5 Hz, H8), 7.72 (dd, 1H, J = 9.0, 3.0 Hz,
H1), 7.85 (ddd, 1H, J = 8.1, 7.5, 1.5 Hz, H9), 8.03 (ddd, 1H, J = 8.1,
1.5, 0.6 Hz, H7), 8.42 (ddd, 1H, J = 8.1, 1.5, 0.6 Hz, H10); 13C NMR
(75 MHz, CDCl3) δ 109.0 (d, J = 24.6 Hz, C1), 118.0 (d, J = 24.1 Hz,
C3), 119.5 (d, J = 8.6 Hz, C4), 121.5 (C11), 122.1 (C7), 129.8 (C8/
C12), 131.0 (C10), 134.2 (d, J = 2.9 Hz, C13), 135.2 (C9), 147.6 (d, J
= 2.7 Hz, C14), 159.5 (d, J = 245 Hz, C2), 161.0 (C6); HRMS (ESI
+
(C7), 166.6 (C6); HRMS (ESI TOF-MS) calcd for C13H7ClNaO4
284.9925; found 284.9943.
+
TOF-MS) calcd for C13H7FNaO2 237.0322; found 237.0336.
3-Fluoro-7,9-dihydroxy-6H-benzo[c]chromen-6-one (7c). White
solid; mp 281.8−283.1 °C; IR (νmax) 3450, 3239, 1673, 1607, 1465,
1293, 1207, 1192, 6 cm−1; 1H NMR (300 MHz, DMSO-d6) δ 6.42 (d,
1H, J = 2.1 Hz, H8), 7.08 (d, 1H, J = 2.1 Hz, H10), 7.23 (td, 1H, J =
8.7, 2.7 Hz, H2), 7.34 (dd, 1H, J = 9.3, 2.7 Hz, H4), 8.16 (dd, 1H, J =
8.7, 6.0 Hz, H1), 11.14 (s, 2H, 7-OH/9-OH); 13C NMR (75 MHz,
DMSO-d6) δ 98.4 (C12), 101.4 (C10), 103.2 (C8), 105.3 (d, J = 25.7
Hz, C4), 113.5 (d, J = 22.3 Hz, C2), 115.4 (C11), 126.6 (d, J = 9.8 Hz,
C1), 136.8 (d, J = 2.8 Hz, C13), 151.7 (d, J = 12.6 Hz, C14), 163.6 (d,
J = 247 Hz, C3), 164.2 (C9), 164.5 (C7), 166.7 (C6); HRMS (ESI
2-Bromo-6H-benzo[c]chromen-6-one (7i). White solid; mp
195.8−197.1 °C; IR (νmax) 3065, 1733, 1717, 1599, 1480, 1437,
1287, 1399, 1260, 1212, 1062, 1033, 1017, 872, 805, 763, 709, 677
1
cm−1; H NMR (300 MHz, CDCl3) δ 7.24 (1H, d, J = 8.8 Hz, H4),
7.69 (1H, dd, J = 8.8, 2.3 Hz, H3), 7.70 (1H, ddd, J = 8.0, 7.4, 1.2 Hz,
H9), 7.94 (1H, ddd, J = 8.0, 7.4, 1.4 Hz, H8), 8.23 (1H, dd, J = 8.0, 1.2
Hz, H7), 8.48 (1H, brd, J = 8.0 Hz, H10), 8.55 (1H, d, J = 2.3 Hz, H-
1); 13C NMR (75 MHz, CDCl3) δ 117.6 (C2), 120.2 (C10), 120.5
(C11), 121.3 (C8), 123.8 (C4), 126.8 (C12), 130.4 (C7), 130.6 (C3),
133.8 (C13), 133.9 (C1), 136.1 (C9), 150.5 (C14), 160.5 (C6);
+
+
TOF-MS) calcd for C13H7FNaO4 269.0221; found 269.0218.
HRMS (ESI TOF-MS) calcd for C13H7BrNaO2 296.9522; found
2-Fluoro-7,9-dihydroxy-6H-benzo[c]chromen-6-one (7d). White
solid; mp 268.3−270.1 °C; IR (νmax) 3445, 3301, 1676, 1579, 1467,
1185, 1102, 691 cm−1; 1H NMR (300 MHz, DMSO-d6) δ 6.48 (d, 1H,
J = 1.8 Hz, H8), 7.17 (d, 1H, J = 1.8 Hz, H10), 7.36−7.52 (m, 2H,
H4/H3), 8.02 (dd, 1H, J = 9.6, 2.4 Hz, H1), 11.08 (s, 1H, 9-OH),
11.27 (s, 1H, 7-OH); 13C NMR (75 MHz, DMSO-d6) δ 98.8 (C12),
102.3 (C10), 103.9 (C8), 110.7 (d, J = 24.6 Hz, C1), 118.8 (d, J = 24.6
Hz, C3), 120.0 (d, J = 7.4 Hz, C4/C11), 136.5 (d, J = 4.1 Hz, C13),
147.1 (d, J = 2.0 Hz, C14), 159.6 (d, J = 239 Hz, C2), 164.2 (C9),
164.7 (C7), 166. Five (C6); HRMS (ESI TOF-MS) calcd for
296.9511.
4-Bromo-2-methyl-6H-benzo[c]chromen-6-one (7j). White solid;
mp 169.4−170.9 °C; IR (νmax) 3077, 2956, 2921, 2851, 1718, 1602,
1561, 1491, 1478, 1421, 1271, 1240, 1214, 1097, 1069, 1056, 1028,
846, 798, 768 cm−1; H NMR (300 MHz, CDCl3) δ 2.39 (3H, s, 2-
1
CH3), 7.63 (1H, d, J = 1.2 Hz, H3), 7.68 (1H, ddd, J = 8.0, 7.8, 0.8 Hz,
H8), 7.93 (1H, ddd, J = 8.0, 7.8, 1.0 Hz, H9), 8.12 (1H, s, H1), 8.22
(1H, dd, J = 8.0, 1.0 Hz, H7), 8.36 (1H, d, J = 8.0 Hz, H10); 13C NMR
(75 MHz, CDCl3) δ 20.8 (2-CH3), 110.3 (C4), 119.7 (C10), 121.2
(C11), 123.6 (C8), 123.9 (C12), 130.5 (C1), 134.5 (C7), 135.0 (C3),
136.0 (C9), 136.1 (C13), 135.3 (C2), 145.9 (C14), 160.3 (C6);
+
C13H7FNaO4 269.0221; found 269.0231.
+
2-Methyl-6H-benzo[c]chromen-6-one (7e). White solid; mp
125.2−126.5 °C; IR (νmax) 3030, 2954, 2920, 2852, 1730, 1606,
HRMS (ESI TOF-MS) calcd for C14H9BrNaO2 310.9678; found
310.9670.
1
1495, 1266, 1072, 1037, 807, 768 cm−1; H NMR (CDCl3) δ 2.45 (s,
2-Bromo-7-hydroxy-9-methoxy-6H-benzo[c]chromen-6-one (7k).
White solid; mp 294.5−295.7 °C; IR (νmax) 3097, 3004, 2935, 1665,
1631, 1565, 1401, 1270, 1200, 1163, 1082, 1045, 989, 813, 752 cm−1;
1H NMR (300 MHz, CDCl3) δ 3.94 (3H, s, 9-OCH3), 6.64 (1H, d, J =
2.2 Hz, H8), 7,37 (1H, d, J = 8.8 Hz, H4), 7.43 (1H, d, J = 2.2 Hz,
H10), 7.71 (1H, dd, J = 8.8, 2.3 Hz, H3), 8.57 (1H, d, J = 2.3 Hz, H1),
11.23 (1H, s, 7-OH); 13C NMR (75 MHz, CDCl3) δ 56.9 (9-OCH3),
100.1 (C12), 100.5 (C9), 102.8 (C10), 118.0 (C2), 120.0 (C4), 120.5
3H, 2-CH3), 7.24 (d, 1H, J = 8.4 Hz, H4), 7.27 (dd, 1H, J = 8.4, 1.2
Hz, H3), 7.56 (ddd, 1H, J = 7.8, 7.2, 1.5 Hz, H8), 7.80 (ddd, 1H, J =
7.8, 7.2, 1.5 Hz, H9), 7.82 (d, J = 1.2 Hz, H1), 8.10 (ddd, 1H, J = 7.8,
1.5, 0.6 Hz, H7), 8.39 (ddd, 1H, J = 7.8, 1.5, 0.6 Hz, H10); 13C NMR
(75 MHz, CDCl3) δ 21.3 (2-CH3), 117.7 (C4), 117.9 (C11), 121.5
(C12), 121.8 (C10), 123.0 (C8), 129.0 (C3), 130.8 (C7), 131.6 (C1),
134.3 (C13), 134.9 (C9), 135.0 (C2), 149.6 (C14), 161.6 (C6);
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(C3), 127.3 (C1), 134.2 (C13), 135.9 (C11), 149.8 (C14), 163.8
(C7), 164.2 (C9), 167.2 (C6); HRMS (ESI TOF-MS) calcd for
C14H9BrNaO4 342.9576; found 342.9579.
(C2), 159.7 (d, J = 243.3 Hz, C9), 161.0 (C5); HRMS (ESI TOF-MS)
calcd for C12H6FNNaO2 238.0275; found 238.0289.
+
+
9-Methyl-5H-chromene[4,3-b]pyridin-5-one (7r). White solid; mp
182.1−184.3 °C; IR: 3066, 3004, 2926, 2864, 1754, 1720, 1641, 1599,
1588, 1542, 1399, 1264, 1196, 1138, 1113, 1045, 823, 795, 730, 621,
3-Fluoro-8,9-dihydroxy-6H-benzo[c]chromen-6-one (7l). Color-
less solid; mp 283.8−284.4 °C; IR (νmax) 3485, 3138, 3071, 2362,
1705, 1607, 1535, 1518, 1456, 1384, 1363, 1327, 1269, 1185, 1147,
1112, 1063, 980, 856, 803, 766 cm−1; 1H NMR (300 MHz, CDCl3) δ
7.18 (1H, td, J = 8.8, 8.8, 2.6 Hz, H2), 7.27 (1H, dd, J = 9.6, 2.6 Hz,
H4), 7.52 (1H, s, H10), 7.54 (1H, s, H7), 8.07 (1H, dd, J = 8.8, 6.2
Hz, H1), 10.27 (1H, s, 8-OH), 10.51 (1H, s, 9-OH); 13C NMR (75
MHz, CDCl3) δ 105.0 (d, J = 24.6 Hz, C4), 108.5 (C7), 112.3 (C12),
112.7 (d, J = 24.2 Hz, C2), 114.9 (C10), 115.6 (C13), 125.2 (d, J = 9.6
Hz, C1), 128.4 (C11), 147.9 (C8), 151.6 (d, J = 12.6 Hz, C14), 154.2
(C9), 160.4 (C6), 162.7 (d, J = 244.8 Hz, C3); HRMS (ESI TOF-MS)
1
606, 581 cm−1; H NMR (300 MHz, CDCl3) δ 2.47 (s, 3H, 9-CH3),
7.27 (d, 1H, J = 8.4 Hz, H7), 7.38 (ddd, 1H, J = 8.4, 2.1, 0.6 Hz, H8),
7.51 (dd, 1H, J = 8.0, 4.7 Hz, H3), 8.35−8.37 (m, 1H, H10), 8.61 (dd,
1H, J = 8.0, 1.8 Hz, H4), 9.00 (dd, 1H, J = 4.7, 1.8 Hz, H2); 13C NMR
(75 MHz, CDCl3) δ 21.2 (9-CH3), 117.2 (C7), 117.7 (C11), 119.1
(C14), 123.9 (C3), 124.7 (C10), 133.4 (C8), 135.0 (C9), 138.5 (C4),
150.9 (C13), 152.2 (C12), 155.8 (C2), 161.5 (C5); HRMS (ESI
+
TOF-MS) calcd for C13H9NNaO2 234.0525; found 234.0534.
7-Bromo-9-fluoro-5H-chromene[4,3-b]pyridin-5-one (7s). White
solid; mp 213.5−214.2 °C; IR (νmax) 3456, 3082, 2064, 2962, 1748,
1588, 1561, 1493, 1463, 1403, 1262, 1235, 1208, 1163, 1078, 865, 810,
786, 687, 607 cm−1; 1H NMR (300 MHz, CDCl3) δ 7.57 (dd, 1H, J =
7.5, 3.0 Hz, H8), 7.60 (dd, 1H, J = 8.0, 4.8 Hz, H3), 8.27 (dd, 1H, J =
8.4, 3.0 Hz, H10), 8.65 (dd, 1H, J = 8.0, 1.8 Hz, H4), 9.05 (dd, 1H, J =
4.8, 1.8 Hz, H2); 13C NMR (75 MHz, CDCl3) δ 110.3 (d, J = 25.1 Hz,
C10), 111.5 (d, J = 9.8 Hz, C7), 117.6 (C14), 121.9 (d, J = 8.0 Hz,
C11), 123.5 (d, J = 26.9 Hz, C8), 125.1 (C3), 138.6 (C4), 138.7
(C13), 150.8 (C12), 159.0 (d, J = 246.7 Hz, C9), 160.0 (C5); HRMS
+
calcd for C13H7FNaO4 269.0221; found 269.0202.
2-Fluoro-8,9-dimethoxy-6H-benzo[c]chromen-6-one (7m). White
solid; mp 191.3−192.2 °C; IR (νmax) 3075, 3000, 2947, 2848, 1706,
1598, 1501, 1426, 1411, 1371, 1299, 1280, 1176, 1160, 1073, 1025,
875, 805, 792 cm−1; 1H NMR (300 MHz, CDCl3) 3.89 (3H, s,
OCH3), 4.00 (3H, s, OCH3), 7.31 (1H, td, J = 9.0, 2.7 Hz, H3), 7.37
(1H, dd, J = 9.0, 5.1 Hz, H4), 7.53 (1H, s, H10), 7.75 (1H, s, H7),
8.27 (1H, d, J = 9.9 Hz, H1); 13C NMR (75 MHz, CDCl3) δ 56.7, (8-
OCH3), 57.3 (9-OCH3), 105.7 (C10), 110.2 (d, J = 25.3 Hz, C3),
111.0 (C7), 114.3 (C12), 117.3 (d, J = 24.5 Hz, C1), 119.4 (d, J = 8.9
Hz, C4), 120.0 (d, J = 8.7 Hz, C13), 129.5 (C11), 147.4 (C14), 151.3
(C8), 156.1 (C9), 157.9 (C6), 160.7 (d, J = 60.4 Hz, C2); HRMS
+
(ESI TOF-MS) calcd for C12H5FBrNNaO2 315.9378; found
315.9397.
2-Fluoro-8,9-dihydroxy-6H-benzo[c]chromen-6-one (7t). White
solid; mp 234.1−236.0 °C; IR (νmax) 3075, 3000, 2947, 2848, 1706,
1598, 1501, 1426, 1411, 1371, 1299, 1280, 1176, 1160, 1073, 1025,
+
(ESI TOF-MS) calcd for C15H11FNaO4 297.0534; found 297.0528.
9-Fluoro-5H-chromene[4,3-c]pyridin-5-one (7n). White solid; mp
1
186.1−187.9 °C; IR (νmax) 3077, 2953, 2921, 2852, 1720, 1603, 1590,
875, 805, 792 cm−1; H NMR (300 MHz, CDCl3) 7.28 (td, 1H, J =
1
1490, 1407, 1270, 1244, 1232, 1183, 1072, 1024, 876, 818 cm−1; H
8.6, 8.6, 2.7 Hz, H3), 7.38 (dd, 1H, J = 8.6, 4.8 Hz, H4), 7.56 (s, 1H,
H10), 7.61 (s, 1H, H7), 7.92 (dd, J = 9.9, 2.7 Hz, H1), 10.24 (s, 2H, 8-
OHand 9-OH); 13C NMR (75 MHz, CDCl3) δ 105.7 (C10), 110.2 (d,
J = 25.3 Hz, C3), 111.0 (C7), 114.3 (C12), 117.3 (d, J = 24.5 Hz, C1),
119.4 (d, J = 8.9 Hz, C4), 120.0 (d, J = 8.7 Hz, C13), 129.5 (C11),
147.4 (C14), 151.3 (C8), 156.1 (C9), 157.9 (C6), 160.7 (d, J = 60.4
NMR (300 MHz, CDCl3) δ 7.28 (ddd, 1H, J = 9.1, 8.8, 2.9 Hz, H8),
7.40 (dd, 1H, J = 9.1, 4.7 Hz, H7), 7.85 (dd, 1H, J = 8.7, 2.9 Hz, H10),
8.19 (d, 1H, J = 5.0 Hz, H4), 8.92 (s, 1H, H3), 9.49 (s, 1H, H1); 13C
NMR (75 MHz, CDCl3) δ 108.8 (d, J = 25.0 Hz, C10), 117.1 (d, J =
8.3 Hz, C11), 119.1 (d, J = 24.3 Hz, C8), 119.9 (d, J = 8.5 Hz, C7),
122.4 (C4), 127.2 (C13), 145.7 (C3), 148.2 (C12), 150.4 (C1), 159.0
(C5), 159.6 (d, J = 240.0 Hz, C9); HRMS (ESI TOF-MS) calcd for
+
Hz, C2); HRMS (ESI TOF-MS) calcd for C13H7FNaO4 269.0221;
found 269.0222.
+
C12H6FNNaO2 238.0275; found 238.0278.
Hill Reaction Measurement. The basal rate of electron transport
was measured on photosynthetically active thylakoid membranes
isolated from market spinach (Spinacia oleracea L.) leaves by following
ferricyanide reduction.41,42 Plant material was suspended in 5 mL g−1
of ice-cold 20 mM Tricine-NaOH buffer, pH 8.0, containing 10 mM
NaCl, 5 mM MgCl2, and 0.4 M sucrose, and homogenized for 30 s in a
blender at maximal speed. The homogenate was filtered through
surgical gauze, and the filtrate was centrifuged at 4 °C for 1 min at
500g; the supernatant was further centrifuged for 10 min at 1500g.
Pelleted chloroplasts were osmotically swollen by resuspension in
sucrose-lacking buffer. The suspension was immediately diluted 1:1
with sucrose-containing buffer, kept on ice in the dark, and used within
a few hours from preparation. Following proper dilution with 80% (v/
v) acetone and absorbance measurement at 645 and 663 nm, the
chlorophyll content of each preparation was calculated on the basis of
the Arnon’s formula. Aliquots of membrane preparations correspond-
ing to 15 μg of chlorophyll were incubated at 24 °C in 1-mL cuvettes
containing 20 mM Tricine-NaOH buffer, pH 8.0, 10 mM NaCl, 5 mM
MgCl2, 0.2 M sucrose, and 1 mM K3Fe(CN)6. The assay was initiated
by exposure to saturating light (800 μmol m−2 s−1), and the rate of
ferricyanide reduction was measured at 1 min intervals for up to 20
min against an exact blank at 420 nm. Activity was calculated over the
linear portion of the curve from an extinction coefficient of 1000 M−1
cm−1. Phosphorylating conditions were obtained by adding 2 mM
K2HPO4 and 0.5 mM ADP to the reaction mixture. The addition of 2
mM NH4Cl allowed measurement of uncoupled activity.
9-Chloro-5H-chromene[4,3-c]pyridin-5-one (7o). White solid; mp
193.9−194.4 °C; IR (νmax) 3108, 3071, 2919, 2849, 1723, 1583, 1484,
1
1404, 1295, 1239, 1209, 1073, 1013, 809 cm−1; H NMR (300 MHz,
CDCl3), δ 7.35 (d, 1H, J = 8.8 Hz, H7), 7.50 (dd, 1H, J = 8.8, 2.4 Hz,
H8), 8.14 (d, 1H, J = 2.4 Hz, H10), 8.17 (d, 1H, J = 5.1 Hz, H4), 8.91
(d, 1H, J = 5.1 Hz, H3), 9.51 (s, 1H, H1); 13C NMR (75 MHz,
CDCl3), δ 117.1 (C9), 119.4 (C4), 122.1 (C10), 122.2 (C7), 126.9
(C13), 127.4 (C11), 130.8 (C14), 131.4 (C8), 145.3 (C3), 149.9
(C12), 150.2 (C1), 159.1 (C5); HRMS (ESI TOF-MS) calcd for
+
C12H6ClNNaO2 253.9979; found 253.9985.
7-Bromo-9-fluoro-5H-chromene[4,3-c]pyridin-5-one (7p). White
solid; mp 233.2−234.8 °C; IR (νmax) 2921, 2850, 1757, 1581, 1567,
1470, 1425, 1419, 1299, 1269, 1221, 1173, 1103, 1075, 1050, 1031,
1
886, 859, 817, 787, 722, 703, 607, 589 cm−1; H NMR (300 MHz,
CDCl3) δ 7.92 (dd, 1H, J = 7.8, 3.0 Hz, H8), 8.07 (dd, 1H, J = 5.1, 0.9
Hz, H3), 8.49 (dd, 1H, J = 9.3, 3.0 Hz, H10), 8.91 (d, 1H, J = 5.1 Hz,
H4), 9.79 (bs, 1H, H1); 13C NMR (75 MHz, CDCl3) δ 110.1 (d, J =
17.7 Hz, C10), 111.6 (d, J = 10.8 Hz, C7), 119.2 (d, J = 9.8 Hz, C11),
121.9 (C4), 122.4 (d, J = 27.0 Hz, C8), 127.8 (C13), 127.9 (C14),
147.4 (C3), 151.2 (C1), 158.8 (d, J = 242.8 Hz, C9), 159.2 (C5);
+
HRMS (ESI TOF-MS) calcd for C12H5BrFNNaO2 315.9380; found
315.9387.
9-Fluoro-5H-chromene[4,3-b]pyridin-5-one (7q). White solid; mp
184.4−185.5 °C; IR (νmax) 3083, 3044, 1759, 1742, 1585, 1570, 1500,
1470, 1452, 1403, 1305, 1271, 1261, 1176, 1127, 1098, 1081, 1041,
1
882, 817, 788, 722, 607, 594 cm−1; H NMR (300 MHz, CDCl3) δ
Alternariol monomethyl ether from Alternaria alternata (A3171)
was purchased from Sigma-Aldrich (St. Louis, MO), and was dissolved
and diluted in DMSO. Alternariol analogues were also dissolved in
DMSO so as to obtain 20 mM solutions that were then water-diluted,
as appropriate. Their effect upon the photosynthetic electron transport
was evaluated in parallel assays in which the compounds were added to
the reaction mixture to a final concentration of 1, 2, 5, 10, 20, 50, or
7.29 (ddd, 1H, J = 8.7, 8.3, 3.0 Hz, H8), 7.38 (dd, 1H, J = 8.3, 4.5 Hz,
H7), 7.56 (dd, 1H, J = 8.0, 4.5 Hz, H3), 8.25 (dd, 1H, J = 8.7, 3.0 Hz,
H10), 8.63 (dd, 1H, J = 8.0, 1.7 Hz, H4), 9.03 (dd, 1H, J = 4.5, 1.7 Hz,
H2); 13C NMR (75 MHz, CDCl3) δ 105.0 (C14), 110.8 (d, J = 25.1
Hz, C10), 117.7 (C11), 119.1 (d, J = 8.0 Hz, C7), 119.8 (d, J = 25.1
Hz, C8), 124.6 (C3), 138.5 (C4), 148.9 (C13), 151.4 (C2), 156.0
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100 μM. In the case of alternariol monomethyl ether (2), controls
were added with the corresponding volume of DMSO. For each
concentration, the assay was carried out in triplicate. The
concentrations causing 50% inhibition (IC50) were estimated utilizing
the linear regression equation of activity values, expressed as
percentage of untreated controls, plotted against the logarithm of
inhibitor concentration. At least three points in the rectilinear part of
the resulting sigmoid curve were considered. Confidence limits of IC50
values were computed according to Snedecor and Cochran.44
Cyanobacterial Cultures. Synechococcus elongatus, strain PCC
6301, was grown at 24 1 °C under 14 h days (150 μmol m−2 s−1
PAR) and 10 h nights as described.45 Growth was followed by
measuring chlorophyll concentration.46 Following logarithmic trans-
formation of data, growth constants and generation times were
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ASSOCIATED CONTENT
* Supporting Information
Spectroscopic data for alternariol analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Authors
*L.C.A.B. Phone: +55 31 34093396. Fax: +55 31 38993065. E-
mail: lcab@ufmg.br.
*G.F. Phone: +39 0532 455311. Fax: +39 0532 249761. E-mail:
flg@unife.it.
■
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Notes
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(29) Vishnumurthy, K.; Makriyannis, A. J. Comb. Chem. 2010, 12,
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to the following Brazilian agencies: CNPq
(Conselho Nacional de Desenvolvimento Cientifico e
■
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(32) Teixeira, R. R.; Barbosa, L. C. A.; Maltha, C. R. A.; Rocha, M. E.;
Bezerra, D. P.; Costa-Lotufo, L. V.; Pessoa, C.; Moraes, M. O.
Molecules 2007, 12, 1101−1116.
Tecnolog
́
ico) for financial support and research fellowships
o de Amparo a
(A.J.D., L.C.A.B.) and FAPEMIG (Fundaca
̧
̀
̃
́
Pesquisa do Estado de Minas Geraıs) for financial support and
a student fellowship (G.G.C. and C.S.). Support from the
University of Ferrara (Fondo di Ateneo per la Ricerca 2011 and
2012) is also acknowledged.
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R.; Valente, V. M. M.; Ferreira, J. R. O.; Costa-Lotufo, L. V.; Moraes,
M. O.; Pessoa, C. Molecules 2010, 15, 5629−5643.
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