Synthesis and antifungal activity of novel sclerotiorin analogues

Article abstract of DOI:10.1021/jf300610j

Sclerotiorin 1, first isolated from Penicillium sclerotiorum, has weak antifungal activity and belongs to the azaphilone-type family of natural products. Several series of sclerotiorin analogues were designed and synthesized with the aim of discovering novel fungicides with improved activity. The syntheses involved two key steps, cycloisomerization and then oxidation, and used a simple and efficient Sonogashira cross-coupling reaction to construct the required functionalized precursor. With sclerotiorin as a control, the activities of the newly synthesized analogues were evaluated against seven fungal pathogens, and several promising candidates (compounds 3a₁, 3d₂, 3e₂, 3f₂ and 3k₂) with greater activity and simpler structures than sclerotiorin were discovered. In addition, preliminary structure-activity relationships were studied, which revealed that not only the chlorine or bromine substituent at the 5-position of the nucleus but also the phenyl group at the 3-position and the substituent pattern on it contributed crucially to the observed antifungal activity. Analogues with a methyl substituent at the 1-position have reduced levels of activity, while those with a free hydroxyl group in place of acetoxy at the quaternary center of the bicyclic ring system retain activity.

Full text of DOI:10.1021/jf300610j

Article
pubs.acs.org/JAFC
Synthesis and Antifungal Activity of Novel Sclerotiorin Analogues
Long Lin,†,‡ Nick Mulholland,‡,§ Qiong-You Wu,†,‡ David Beattie,§ Shao-Wei Huang,† Dianne Irwin,§
John Clough,§ Yu-Cheng Gu,*,§ and Guang-Fu Yang*,†
†Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University,
Wuhan 430079, P. R. China
§Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, U.K.
ABSTRACT: Sclerotiorin 1, first isolated from Penicillium sclerotiorum, has weak antifungal activity and belongs to the
azaphilone-type family of natural products. Several series of sclerotiorin analogues were designed and synthesized with the aim of
discovering novel fungicides with improved activity. The syntheses involved two key steps, cycloisomerization and then
oxidation, and used a simple and efficient Sonogashira cross-coupling reaction to construct the required functionalized precursor.
With sclerotiorin as a control, the activities of the newly synthesized analogues were evaluated against seven fungal pathogens,
and several promising candidates (compounds 3a1, 3d2, 3e2, 3f2 and 3k2) with greater activity and simpler structures than
sclerotiorin were discovered. In addition, preliminary structure−activity relationships were studied, which revealed that not only
the chlorine or bromine substituent at the 5-position of the nucleus but also the phenyl group at the 3-position and the
substituent pattern on it contributed crucially to the observed antifungal activity. Analogues with a methyl substituent at the
1-position have reduced levels of activity, while those with a free hydroxyl group in place of acetoxy at the quaternary center of
the bicyclic ring system retain activity.
KEYWORDS: sclerotiorin, azaphilone, fungicides, natural product
earlier approaches, Porco’s protocol achieved the target
azaphilones in higher yield and in a shorter reaction time. In
particular, this approach takes advantage of readily available
alkynes to construct the o-alkynylbenzaldehydes and results in
the formation of azaphilones with diverse side chains at the
3-position which, we realized, would allow us to investigate the
effect of modifications at this position on the activity of sclero-
tiorin analogues. Till now, little attention has been paid to the
structural optimization of the antifungal activity of the azaphi-
lones. Therefore, we envisioned that sclerotiorin, which had
preciously shown weak antifungal activity in Syngenta’s screens,
might be a lead for the discovery of novel agricultural fungicides.
Structurally, sclerotiorin features a nine-carbon aliphatic
diene side chain and a bicyclic core in which aromaticity is in-
terrupted by a chiral quaternary center. To simplify the struc-
ture of sclerotiorin, we first synthesized compounds 2, 3, and 4
(Figure 1) by replacement of the long aliphatic side chain with
an aromatic ring. This modification may also be helpful in
improving the photostability of sclerotiorin. In the meantime,
compound 5 was designed to determine the importance of
conjugation in the sclerotiorin scaffold. Thus, a methylene
bridge is inserted in compound 5, to break the conjugation
between the bicyclic core and the side chain. In addition, com-
pounds 6 were designed to get further insight into the structure−
activity relationships. For all of these designed compounds, the
bicyclic core of sclerotiorin was retained to mimic the natural
parent structure. In all of the analogues described in this paper, the
1. INTRODUCTION
Natural products are well-known as one of the key sources for
lead discovery in agrochemical development. Natural products
with novel scaffolds always afford an opportunity to discover
novel antifungal agents that operate by modes of action differ-
ent from those that are already known. Consequently, the result-
ing new agrochemicals have the opportunity to overcome
resistance to chemicals currently in use. Notable examples of
the successful utilization of natural products are strobilurin-type
fungicides,1 analogues of the naturally occurring β-methox-
yacrylate strobilurin A, which are inhibitors of the cytochrome
bc1 complex.2
Sclerotiorin 1, first isolated from Penicillium sclerotiorum,3
belongs to the azaphilone-type family of natural products which
share two common characteristics, a highly oxygenated bicyclic
core and a quaternary center which breaks the aromaticity of
the ring system. To date, over 170 different azaphilones have
been identified.4 They have been reported to exhibit a wide
range of biological activities, such as inhibition of monoamine
oxidase,5 inhibition of the formation of the P53-MDM2 complex,6
inhibition of the gp120-CD4 binding reaction,7 and inhibition
of fatty acid synthase,8 as well as having antifungal activity.9,10
Because of their broad spectrum biological properties, a
number of studies aimed at the synthesis of racemic azaphilones
have been reported.11−14 In general, pyronoquinones11 and
pyrylium salts12,13 have been used as precursors. Whalley and
his co-workers reported the synthesis of the bicyclic core of
sclerotioirin by cyclization and then oxidation of a suitable
o-formyl-benzyl ketone.12,13 Recently, Porco’s group described an
efficient gold-catalyzed cycloisomerization of o-alkynylbenzal-
dehydes into 2-benzopyrylium salts and subsequent IBX oxida-
tion to form the azaphilone ring system.15 In comparison to the
Received: November 1, 2011
Revised: March 19, 2012
Accepted: March 22, 2012
Published: March 22, 2012
© 2012 American Chemical Society
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Figure 1. Sclerotiorin and the designed analogues.
Data for 2a. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.75 (d,
J = 6.6 Hz 2H), 7.54−7.50 (m, 3H), 6.78 (s, 1H), 5.70 (s, 1H), 3.98
(br, 1H), 1.60 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 196.1, 195.5,
157.3, 152.6, 143.8, 131.6, 130.0, 129.1, 125.6, 115.8, 106.8, 106.2,
83.5, 28.5. HRMS (MALDI): calcd for C16H12O4 [M + Na]+
291.0633, found 291.0638.
absolute stereochemistry of the chiral center of the bicyclic
system was ignored, and racemic compounds were prepared.
Herein, we report the synthesis and characterization of these
sclerotiorin analogues as well as an assessment of their
antifungal activities.
Data for 2b. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.70 (d,
J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.67 (s, 1H), 5.66 (s, 1H),
3.97 (br, 1H), 3.89 (s, 3H), 1.59 (s, 3H). HRMS (MALDI): calcd for
C17H14O5 [M + Na]+ 321.0739, found 321.0721.
MATERIALS AND METHODS
■
All chemical reagents were commercially available and treated with
standard methods before use. Solvents were dried and redistilled
Data for 2c. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.64 (d,
J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 5.68 (s, 1H),
3.97 (br, 1H), 2.43 (s, 3H), 1.60 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 196.1, 195.6, 157.6, 152.6, 144.2, 142.4, 129.8, 127.3, 125.5,
115.8, 106.0, 105.8, 83.5, 28.6, 21.5. HRMS (MALDI): calcd for
C17H14O4 [M + Na]+ 305.0790, found 305.0786.
1
before use. H NMR spectra were recorded in CDCl3 on a Varian
Mercury 600 spectrometer, and chemical shifts (δ) are given in
ppm relative to tetramethylsilane. 13C NMR spectra were recorded
in CDCl3 on a Varian Mercury 600 (150 MHz) spectrometer, and
chemical shifts (δ) are given in ppm relative to the center line of a
triplet at 77.0 ppm of chloroform-d. The following abbreviations are
used to designate mutiplicities: s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad. HRMS were obtained on a Waters MALDI
SYNAPT G2 HDMS equipped with an electrospray source (Milford,
MA, USA).
General Procedure for the Preparation of 2-Alknylbenzal-
dehydes 9a−9s. An oven-dried two-necked flask was evacuated and
backfilled with argon (the cycle was performed twice) and then
charged under a positive pressure of argon with 2-bromobenzaldehyde
(500 mg, 2.16 mmol), PdCl2(PPh3)2 (75.8 mg, 0.108 mmol, 5 mol %),
and CuI (20.5 mg, 0.108 mmol, 5 mol %), followed by anhydrous
DMF (20 mL) and Et3N (654.5 mg, 6.48 mmol). The resulting
suspension was stirred for 10 min, then the alkyne (2.37 mmol) was
injected, and the reaction mixture was stirred at 60 °C for the indicated
period of time. The resulting suspension was allowed to cool to room
temperature and was then diluted with water (50 mL), neutralized
with 0.1 N HCl, and extracted with ethyl acetate (3 × 50 mL). The
combined organic extracts were washed with water (3 × 100 mL) and
brine (3 × 100 mL), dried over Na2SO4, and concentrated, and the
residue was purified by flash chromatography on silica gel to provide
the desired product.
General Procedure for the Preparation of Azaphilones 2a−2s.
To the alkynylbenzaldehyde 9 (0.5 mmol) and AgNO3 (4.25 mg,
0.025 mmol) were added 2.0 mL of dichloroethane and 200 μL of
trifluoroacetic acid, and the mixture was stirred at rt until TLC
monitoring indicated the disappearance of the material 9 (5 min). To
the resulting mixture were added o-iodoxybenzoic acid (IBX) (155 mg,
0.55 mmol) and tetrabutylammonium iodide (9.25 mg, 0.025 mmol),
and the reaction mixture was stirred at rt for a further 1 h, then then
quenched with saturated Na2S2O3, and extracted three times with
ethyl acetate. The organic extracts were combined, washed with
brine, dried over Na2SO4, and concentrated. Purification by flash
chromatography on silica gel (petroleum ether:ethyl acetate = 3:1)
afforded compounds 2a−2s.
1
Data for 2d. H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 7.79−
7.75 (m, 2H), 7.22−7.17 (m, 2H), 6.75 (s, 1H), 5.70 (s, 1H), 1.60 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 196.0, 195.4, 165.8, 163.3,
156.3, 152.4, 143.6, 127.8, 127.7, 126.3, 116.4, 116.2, 115.7, 106.5,
106.3, 83.5, 28.4. HRMS (MALDI): calcd for C16H11FO4 [M + Na]+
309.0539, found 309.0545.
Data for 2e. 1H NMR (600 MHz, CDCl3): δ 8.06 (s, 1H), 7.91 (d,
J = 8.4 Hz, 2H), 7.76 (d, J = 9.0 Hz, 2H), 6.94 (s, 1H), 5.75 (s, 1H),
1.59 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 196.1, 195.5, 155.0,
152.1, 142.5, 133.2, 132.5, 132.3, 125.7, 125.6, 124.1, 122.3, 115.6,
108.2, 107.3, 83.4, 27.8. HRMS (MALDI): calcd for C17H11F3O4
[M + Na]+ 359.0507, found 359.0510.
Data for 2f. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.68 (d,
J = 8.8 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 5.69 (s, 1H),
3.97 (br, 1H), 1.60 (s, 3H), 1.37 (s, 9H). 13C NMR (100 MHz,
CDCl3): δ 196.1, 195.6, 157.6, 155.5, 152.7, 144.1, 127.2, 126.1, 125.4,
115.8, 106.1, 105.8, 83.5, 35.0, 31.0, 28.6. HRMS (MALDI): calcd for
C20H20O4 [M + Na]+ 347.1259, found 347.1265.
1
Data for 2g. H NMR (600 MHz, CDCl3): δ 8.36 (d, J = 8.4 Hz,
2H), 8.04 (s, 1H), 7.94 (d, J = 9.0 Hz, 2H), 6.92 (s, 1H), 5.78 (s, 1H),
1.61 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 196.8, 196.4, 153.1,
152.9, 148.4, 141.8, 136.1, 126.6, 124.2, 115.4, 110.1, 108.5, 83.0, 26.4.
HRMS (MALDI): calcd for C16H11NO6 [M + Na]+ 336.0484, found
336.0455.
Data for 2h. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.40 (t,
J = 7.8 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.24 (s, 1H), 7.06 (d, J = 8.4
Hz, 1H), 6.76 (s, 1H), 5.69 (s, 1H), 3.88 (s, 3H), 1.60 (s, 3H). 13C
NMR (150 MHz, CDCl3): δ 196.1, 195.5, 159.9, 157.0, 152.6, 143.7,
131.3, 130.1, 117.9, 117.1, 115.7, 111.0, 107.0, 106.2, 83.5, 55.4, 28.4.
HRMS (MALDI): calcd for C17H14O5 [M + Na]+ 321.0739, found
321.0701.
1
Data for 2i. H NMR (600 MHz, DMSO-d6): δ 8.25 (s, 1H),
7.68 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H),
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7.38 (d, J = 7.8 Hz, 1H), 7.30 (s, 1H), 5.58 (s, 1H), 2.39 (s, 3H),
1.36 (s, 3H). HRMS (MALDI): calcd for C17H14O4 [M + Na]+
305.0790, found 305.0776.
stirred at rt until TLC analysis indicated that the starting material had
disappeared. The resulting mixture was washed with water and brine
and dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure, and the residue was purified by flash chromatog-
raphy on silica gel (petroleum ether:ethyl acetate = 5:1) to give the
product 3.
1
Data for 2j. H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 7.55−
7.53 (m, 1H), 7.51−7.43 (m, 2H), 7.25−7.21 (m, 1H), 6.78 (s, 1H),
5.72 (s, 1H), 3.95 (br, 1H), 1.60 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 196.1, 195.3, 164.1, 161.7, 155.8, 152.4, 143.2, 132.2, 132.1,
130.9, 130.8, 121.2, 118.6, 118.4, 115.8, 112.8, 112.5, 107.6, 106.9,
83.6, 28.4. HRMS (MALDI): calcd for C16H11FO4 [M + Na]+
309.0539, found 309.0491.
Data for 3a1. 1H NMR (600 MHz, CDCl3): δ 8.08 (s, 1H), 7.83−
7.82 (m, 2H), 7.58−7.53 (m, 3H), 7.22 (s, 1H), 3.96 (br, 1H), 1.63 (s,
3H). HRMS (MALDI): calcd for C16H11ClO4 [M + Na]+ 325.0244,
found 325.0240.
Data for 2k. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.73 (s,
1H), 7.63 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 7.8
Hz, 1H), 6.78 (s, 1H), 5.72 (s, 1H), 3.93 (br, 1H), 1.60 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 196.1, 195.3, 155.6, 152.4, 143.1, 135.3,
131.8, 131.5, 130.4, 125.6, 123.6, 115.8, 107.6, 106.9, 83.6, 28.4.
HRMS (MALDI): calcd for C16H11ClO4 [M + Na]+ 325.0244, found
325.0223.
1
Data for 3a2. H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.83
(d, J = 7.2 Hz, 2H), 7.59−7.53 (m, 3H), 7.26 (s, 1H), 3.96 (br, 1H),
1.63 (s, 3H). HRMS (MALDI): calcd for C16H11BrO4 [M + Na]+
368.9738, found 368.9748.
Data for 3a3. 1H NMR (400 MHz, CDCl3): δ 7.95 (s, 1H), 7.85−
7.83 (m, 2H), 7.59−7.54 (m, 3H), 7.27 (s, 1H), 3.96 (br, 1H), 1.61 (s,
3H). HRMS (MALDI): calcd for C16H11IO4 [M + Na]+ 416.9600,
found 416.9604.
Data for 2l. 1H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.69 (d,
J = 8.4 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.16 (s, 1H), 7.07 (t, J =
7.8 Hz, 1H), 7.04 (d, J = 7.8 Hz, 1H), 5.65 (s, 1H), 3.96 (s, 3H), 1.60
(s, 3H). 13C NMR (150 MHz, CDCl3): δ 196.2, 195.8, 157.5, 154.4,
152.8, 144.5, 132.5, 128.4, 120.8, 118.9, 115.3, 111.6, 105.8, 83.4, 55.7,
55.6, 28.5. HRMS (MALDI): calcd for C17H14O5 [M + Na]+ 321.0739,
found 321.0752.
1
Data for 3b2. H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.79
(d, J = 7.8 Hz, 2H), 7.16 (s, 1H), 7.03 (d, J = 7.8 Hz, 2H), 3.97 (br,
1H), 3.91 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for
C17H13BrO5 [M + Na]+ 398.9844, found 398.9808.
Data for 3c1. 1H NMR (600 MHz, CDCl3): δ 8.06 (s, 1H), 7.72 (d,
J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.20 (s, 1H), 3.95 (br, 1H),
2.45 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for C17H13ClNO4
[M + Na]+ 339.0400, found 339.0405.
Data for 2m. 1H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.43−
7.41 (m, 2H), 7.31−7.30 (m, 2H), 6.43 (s, 1H), 5.65 (s, 1H), 3.96 (br,
1H), 2.44 (s, 3H), 1.62 (s, 3H). 13C NMR (150 MHz, CDCl3): δ
196.1, 195.6, 159.5, 152.7, 143.7, 136.6, 131.2, 130.9, 130.8, 128.9,
126.2, 115.6, 111.0, 105.9, 83.5, 28.3, 20.4. HRMS (MALDI): calcd for
C17H14O4 [M + Na]+ 305.0790, found 305.0774.
Data for 3c2. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.73 (d,
J = 8.4 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 7.22 (s, 1H), 3.95 (br, 1H),
2.45 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for C17H13BrO4
[M + H]+ 361.0076, found 361.0027.
1
Data for 2n. H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.76−
Data for 3c3. 1H NMR (600 MHz, CDCl3): δ 7.93 (s, 1H), 7.73 (d,
J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 7.22 (s, 1H), 3.97 (br, 1H), 2.45
(s, 3H), 1.61 (s, 3H). HRMS (MALDI): calcd for C17H13IO4 [M + Na]+
430.9756, found 430.9765.
7.72 (m, 1H), 7.52−7.49 (m, 1H), 7.31−7.28 (m, 1H), 7.25−7.22 (m,
1H), 6.97 (s, 1H), 5.71 (s, 1H), 3.96 (br, 1H), 1.61 (s, 3H). 13C NMR
(150 MHz, CDCl3): δ 196.2, 195.4, 161.0, 159.3, 152.5, 152.4, 152.2,
143.4, 132.9, 128.1, 124.8, 118.6, 118.5, 116.9, 116.7, 115.5, 111.8,
111.7, 106.9, 83.6, 30.8, 28.4. HRMS (MALDI): calcd for C16H11FO4
[M + Na]+ 309.0539, found 309.0511.
Data for 3d1. 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.84−7.82
(m, 2H), 7.24−7.21 (m, 2H), 7.16 (s, 1H), 3.91 (br, 1H), 1.62 (s, 3H).
HRMS (MALDI): calcd for C16H10ClFO4 [M + Na]+ 343.0149, found
343.0126.
Data for 2o. 1H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.55 (d,
J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.40
(t, J = 7.2 Hz, 1H), 6.70 (s, 1H), 5.68 (s, 1H), 3.96 (br, 1H), 1.62 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 196.2, 195.5, 155.7, 152.9,
143.2, 132.5, 132.0, 130.8, 130.2, 129.9, 127.2, 115.5, 112.5, 106.6,
83.6, 28.3. HRMS (MALDI): calcd for C16H11ClO4 [M + Na]+
325.0244, found 325.0224.
Data for 3d2. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.86−
7.83 (m, 2H), 7.25−7.20 (m, 3H), 1.62 (s, 3H). HRMS (MALDI):
calcd for C16H10BrFO4 [M + Na]+ 386.9644, found 386.9635.
Data for 3d3. 1H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H), 7.87−
7.83 (m, 2H), 7.26−7.20 (m, 3H), 4.04 (br, 1H), 1.61 (s, 3H). HRMS
(MALDI): calcd for C16H10FIO4 [M + Na]+ 434.9506, found
434.9510.
Data for 2p. 1H NMR (600 MHz, CDCl3): δ 7.97 (s, 1H), 7.83 (d,
J = 7.2 Hz, 1H), 7.71−7.67 (m, 2H), 7.59−7.57 (m, 1H), 6.47 (s, 1H),
5.66 (s, 1H), 3.96 (br, 1H), 1.62 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 196.3, 195.4, 156.9, 152.6, 143.0, 132.2, 131.2, 130.8, 129.6,
128.9, 128.6, 127.1, 124.5, 121.8, 115.6, 111.7, 106.8, 83.7, 28.3.
HRMS (MALDI): calcd for C17H11F3O4 [M + Na]+ 359.0507, found
359.0490.
Data for 3e1. 1H NMR (600 MHz, CDCl3): δ 8.08 (s, 1H), 7.96 (d,
J = 7.8 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.29 (s, 1H), 3.89 (br, 1H),
1.64 (s, 3H). HRMS (MALDI): calcd for C17H10ClF3O4 [M + Na]+
393.0117, found 393.0107.
Data for 3e2. 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.96 (d,
J = 7.8 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 3.96 (br, 1H),
1.63 (s, 3H). HRMS (MALDI): calcd for C17H10BrF3O4 [M + Na]+
436.9612, found 436.9595.
Data for 2q. 1H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.84 (s,
1H), 7.59−7.57 (m, 2H), 6.78 (s, 1H), 5.72 (s, 1H), 1.59 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 196.1, 195.2, 154.8, 152.2, 142.9, 136.0,
133.8, 131.2, 130.0, 127.3, 124.5, 115.8, 107.8, 107.2, 83.7, 28.4.
HRMS (MALDI): calcd for C16H10Cl2O4 [M + Na]+ 358.9854, found
358.9827.
1
Data for 3e3. H NMR (400 MHz, CDCl3): δ 7.95 (m, 3H), 7.80
(d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 3.94 (br, 1H), 1.62 (s, 3H). HRMS
(MALDI): calcd for C17H10IF3O4 [M + Na]+ 484.9474, found
484.9477.
1
Data for 2r. H NMR (400 MHz, CDCl3): δ 8.01 (s, 1H), 7.59−
Data for 3f1. 1H NMR (600 MHz, CDCl3): δ 8.07 (s, 1H), 7.76 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.18 (s, 1H), 3.97 (br, 1H),
1.63 (s, 3H), 1.37 (s, 9H). HRMS (MALDI): calcd for C20H19ClO4
[M + Na]+ 381.0870, found 381.0875.
7.52 (m, 2H), 6.71 (s, 1H), 5.72 (s, 1H), 3.91 (br, 1H), 1.59 (s, 3H).
HRMS (MALDI): calcd for C16H10F2O4 [M + Na]+ 327.0445, found
327.0427.
1
Data for 2s. H NMR (600 MHz, CDCl3): δ 7.86 (s, 1H), 7.40−
Data for 3f2. 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.76 (d,
J = 7.8 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 3.97 (br, 1H),
1.62 (s, 3H), 1.37 (s, 9H). HRMS (MALDI): calcd for C20H19BrO4
[M + Na]+ 425.0364, found 425.0367.
7.37 (m, 2H), 7.35−7.33 (m, 1H), 7.26−7.25 (m, 2H), 6.05 (s, 1H),
5.50 (s, 1H), 3.89 (s, 1H), 3.73 (s, 2H), 1.54 (s, 3H). 13C NMR (150
MHz CDCl3): δ 196.1, 195.5, 161.3, 152.8, 143.7, 133.9, 129.0, 128.9,
127.6, 115.6, 109.1, 105.6, 83.4, 39.2, 28.3. HRMS (MALDI): calcd for
C17H14O5 [M + Na]+ 321.0739, found 321.0721.
Data for 3f3. 1H NMR (600 MHz, CDCl3): δ 7.94 (1H, s), 7.77 (d,
J = 7.8 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 3.97 (br, 1H),
1.61 (s, 3H), 1.37 (s, 9H). HRMS (MALDI): calcd for C20H19IO4
[M + Na]+ 473.0226, found 473.0223.
General Procedure for the Preparation of Halogenated
Azaphilones 3. To a stirred solution of compound 2 (0.05 mmol) in
CH2Cl2 was added NXS (0.055 mmol). The resulting mixture was
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1
Data for 3g2. H NMR (600 MHz, CDCl3): δ 8.39 (d, J = 8.4 Hz,
Data for 3n2. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.79−
7.77 (m, 1H), 7.55−7.54 (m, 1H), 7.47 (s, 1H), 7.33−7.31 (m, 1H),
7.28−7.25 (m, 1H), 3.92 (br, 1H), 1.63 (s, 3H). HRMS (MALDI):
calcd for C16H10BrFO4 [M + Na]+ 386.9644, found 386.9616.
2H), 8.04 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.38 (s, 1H), 3.89 (br, 1H),
1.64 (s, 3H). HRMS (MALDI): calcd for C16H10BrNO6 [M + Na]+
413.9589, found 413.9579.
Data for 3h1. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.46−
7.41 (m, 2H), 7.31 (s, 1H), 7.24 (s, 1H), 7.11 (t, J = 7.2 Hz, 1H), 3.90
(s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for C17H13ClO5
[M + Na]+ 355.0349, found 355.0330.
Data for 3o1. H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.60
1
(d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H),
7.43 (t, J = 7.2 Hz, 1H), 7.19 (s, 1H), 3.90 (br, 1H), 1.65 (s, 3H).
HRMS (MALDI): calcd for C16H10Cl2O4 [M + Na]+ 358.9854, found
358.9852.
Data for 3h2. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.48−
7.39 (m, 2H), 7.31 (s, 1H), 7.24 (s, 1H), 7.10 (d, J = 7.6 Hz, 1H), 3.93
(br, 1H), 3.90 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for
C17H13BrO5 [M + Na]+ 398.9844, found 398.9856.
1
Data for 3o2. H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.61
(d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H),
7.43 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 3.91 (br, 1H), 1.65 (s, 3H).
HRMS (MALDI): calcd for C16H10BrClO4 [M + Na]+ 402.9349,
found 402.9348.
1
Data for 3i1. H NMR (600 MHz, CDCl3): δ 8.07 (s, 1H), 7.63−
7.62 (m, 2H), 7.41 (t, J = 7.8 Hz, 1H), 7.38 (d, J = 6.6 Hz, 1H), 7.20
(s, 1H), 3.92 (br, 1H), 2.46 (s, 3H), 1.63 (s, 3H). HRMS (MALDI):
calcd for C17H13ClO4 [M + Na]+ 339.0400, found 339.0410.
1
Data for 3p1. H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.85
(d, J = 7.8 Hz, 1H), 7.73−7.71 (m, 2H), 7.64−7.63 (m, 1H), 6.92 (s,
1H), 3.87 (br, 1H), 1.65 (s, 3H). HRMS (MALDI): calcd for
C17H10ClF3O4 [M + Na]+ 393.0117, found 393.0110.
1
Data for 3i2. H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.64−
7.62 (m, 2H), 7.43−7.38 (m, 2H), 7.24 (s, 1H), 3.95 (br, 1H), 2.46 (s,
3H), 1.62 (s, 3H). HRMS (MALDI): calcd for C17H13BrO4 [M + Na]+
382.9895, found 382.9886.
Data for 3p2. 1H NMR (600 MHz, CDCl3): δ 7.99 (s, 1H), 7.86−
7.85 (m, 1H), 7.74−7.70 (m, 2H), 7.65−7.64 (m, 1H), 6.96 (s, 1H),
3.90 (br, 1H), 1.65 (s, 3H). HRMS (MALDI): calcd for C17H10BrF3O4
[M + Na]+ 436.9612, found 436.9620.
1
Data for 3i3. H NMR (600 MHz, CDCl3): δ 7.94 (s, 1H), 7.64−
7.62 (m, 2H), 7.44−7.38 (m, 2H), 7.24 (s, 1H), 4.47 (s, 3H), 1.61 (s,
3H). HRMS (MALDI): calcd for C17H13IO4 [M + Na]+ 430.9756,
found 430.9742.
Data for 3q1. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.90 (s,
1H), 7.65−7.62 (m, 2H), 7.19 (s, 1H), 3.92 (br, 1H), 1.64 (s, 3H).
HRMS (MALDI): calcd for C16H9Cl3O4 [M + Na]+ 392.9464, found
392.9453.
1
Data for 3j1. H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 1H),
7.84−7.80 (m, 2H), 7.65−7.58 (m, 1H), 7.49−7.41 (m, 1H), 7.35
(s, 1H), 1.39 (s, 3H). HRMS (MALDI): calcd for C16H10ClFO4
[M + H]+ 321.0330, found 321.0319.
Data for 3q2. 1H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.91 (s,
1H), 7.66−7.61 (m, 2H), 7.23 (s, 1H), 3.92 (br, 1H), 1.62 (s, 3H);
Data for 3j2. 1H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.63 (d,
J = 8.4 Hz, 1H), 7.53−7.51 (m, 2H), 7.28−7.26 (m, 2H), 3.94 (br, 1H),
1.63 (s, 3H). HRMS (MALDI): calcd for C16H10BrFO4 [M + Na]+
386.9644, found 386.9655.
1
Data for 3r1. H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.67−
7.60 (m, 2H), 7.35−7.33 (m, 1H), 7.15 (s, 1H), 3.88 (br, 1H), 1.62 (s,
3H). HRMS (MALDI): calcd for C16H9ClF2O4 [M + Na]+ 361.0055,
found 361.0027.
Data for 3j3. 1H NMR (600 MHz, CDCl3): δ 7.93 (s, 1H), 7.64 (d,
J = 7.2 Hz, 1H), 7.53−7.51 (m, 2H), 7.29−7.26 (m, 2H), 3.95 (br,
1H), 1.61 (s, 3H). HRMS (MALDI): calcd for C16H10IFO4 [M + Na]+
434.9506, found 434.9435.
1
Data for 3r2. H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.68−
7.61 (m, 2H), 7.37−7.32 (m, 1H), 7.19 (s, 1H), 3.84 (br, 1H), 1.62 (s,
3H). HRMS (MALDI): calcd for C16H9BrF2O4 [M + Na]+ 404.9550,
found 404.9554.
1
Data for 3k1. H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.80
General Procedure for the Preparation of Acetylated
Azaphilones 4. To a stirred solution of azaphilone 3 (0.035 mmol)
and acetic anhydride (7.14 mg, 0.070 mmol) in 1.5 mL of CH2Cl2 were
added triethylamine (7.07 mg, 0.070 mmol) and 4-(dimethylamino)-
pyridine (4.27 mg, 0.035 mmol). The resulting mixture was stirred
at rt for 30 min and concentrated in vacuo. Purification on silica gel
(hexane:EtOAc = 10:1 to 5:1) provided 4.
(s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.48 (t, J =
7.8 Hz, 1H), 7.21 (s, 1H), 3.91 (br, 1H), 1.63 (s, 3H). HRMS
(MALDI): calcd for C16H10Cl2O4 [M + Na]+ 358.9854, found 358.9854.
1
Data for 3k2. H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.80
(s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.48 (t, J =
7.8 Hz, 1H), 7.25 (s, 1H), 3.93 (br, 1H), 1.63 (s, 3H). HRMS
(MALDI): calcd for C16H10BrClO4 [M + Na]+ 402.9349, found 402.9354.
Data for 3k3. 1H NMR (600 MHz, CDCl3): δ 7.92 (s, 1H), 7.80 (s,
1H), 7.72 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.49 (t, J = 7.8
Hz, 1H), 7.25 (s, 1H), 3.95 (br, 1H), 1.61 (s, 3H). HRMS (MALDI):
calcd for C16H10IClO4 [M + Na]+ 450.9210, found 450.9217.
Data for 3l1. 1H NMR (600 MHz, CDCl3): δ 8.07 (s, 1H), 7.75 (d,
J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8
Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H), 3.91 (br, 1H), 1.63 (s,
3H). HRMS (MALDI): calcd for C17H13ClO5 [M + Na]+ 355.0349,
found 355.0325.
Data for 4a1. 1H NMR (600 MHz, CDCl3): δ 8.07 (s, 1H), 8.82−
8.81 (m, 2H), 7.56−7.53 (m, 3H), 7.24 (s, 1H), 2.20 (s, 3H), 1.60 (s,
3H). HRMS (MALDI): calcd for C18H13ClO5 [M + Na]+ 367.0349,
found 367.0353.
Data for 4a2. 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.82−
7.81 (m, 2H), 7.58−7.52 (m, 3H), 7.29 (s, 1H), 2.19 (s, 3H), 1.60 (s,
3H). HRMS (MALDI): calcd for C18H13BrO5 [M + Na]+ 410.9844,
found 410.9850.
1
Data for 4b2. H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.77
(d, J = 8.4 Hz, 2H), 7.18 (s, 1H), 7.02 (d, J = 8.4 Hz, 2H), 3.90 (s,
3H), 2.19 (s, 3H), 1.59 (s, 3H). HRMS (MALDI): calcd for
C19H15BrO6 [M + Na]+ 440.9950, found 440.9965.
1
Data for 3l2. H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.76−
7.75 (m, 2H), 7.52 (t, J = 7.8 Hz, 1H), 7.11−7.06 (m, 2H), 4.00 (s,
3H), 1.63 (s, 3H). HRMS (MALDI): calcd for C17H13BrO5 [M + Na]+
398.9844, found 398.9840.
Data for 4c1. 1H NMR (600 MHz, CDCl3): δ 8.06 (s, 1H), 7.71 (d,
J = 8.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.20 (s, 1H), 2.44 (s, 3H),
2.19 (s, 3H), 1.60 (s, 3H). HRMS (MALDI): calcd for C19H15ClO5
[M + Na]+ 381.0506, found 381.0491.
1
Data for 3m1. H NMR (600 MHz, CDCl3): δ 8.06 (s, 1H), 7.50
(d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.35−7.32 (m, 2H), 6.91
(s, 1H), 3.90 (br, 1H), 2.48 (s, 3H), 1.65 (s, 3H). HRMS (MALDI):
calcd for C17H13ClO4 [M + Na]+ 339.0400, found 339.0383.
Data for 4c2. 1H NMR (600 MHz, CDCl3): δ 8.04 (1H, s), 7.71 (d,
J = 7.8 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.24 (s, 1H), 2.44 (s, 3H),
2.19 (s, 3H), 1.59 (s, 3H). HRMS (MALDI): calcd for C19H15BrO5
[M + Na]+ 425.0001, found 425.0005.
1
Data for 3m2. H NMR (600 MHz, CDCl3): δ 8.03 (s, 1H), 7.51
(d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.34−7.33 (m, 2H), 6.96
(s, 1H), 3.94 (br, 1H), 2.48 (s, 3H), 1.65 (s, 3H). HRMS (MALDI):
calcd for C17H13BrO4 [M + Na]+ 382.9895, found 382.9874.
Data for 3n1. 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.79−
7.76 (m, 1H), 7.55−7.54 (m, 1H), 7.42 (s, 1H), 7.33−7.31 (m, 1H),
7.27−7.24 (m, 1H), 3.93 (br, 1H), 1.63 (s, 3H). HRMS (MALDI):
calcd for C16H10ClFO4 [M + Na]+ 343.0149, found 343.0106.
Data for 4c3. 1H NMR (600 MHz, CDCl3): δ 7.94 (s, 1H), 7.71 (d,
J = 8.4 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.26 (s, 1H), 2.45 (s, 3H),
2.19 (s, 3H), 1.58 (s, 3H). HRMS (MALDI): calcd for C19H15IO5
[M + Na]+ 472.9862, found 472.9846.
Data for 4d2. 1H NMR (400 MHz, CDCl3): δ 8.02 (s, 1H), 7.84−
7.81 (m, 2H), 7.24−7.20 (m, 3H), 2.19 (s, 3H), 1.59 (s, 3H). HRMS
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(MALDI): calcd for C18H12FBrO5 [M + Na]+ 428.9750, found
428.9756.
Data for 4m2. H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.49
(d, J = 7.2 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.33−7.32 (m, 2H), 6.98
(s, 1H), 2.48 (s, 3H), 2.20 (s, 3H), 1.62 (s, 3H). HRMS (MALDI):
calcd for C19H15BrO5 [M + Na]+ 425.0001, found 425.0004.
Data for 4n1. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.77−
7.74 (m, 1H), 7.53−7.52 (m, 1H), 7.42 (s, 1H), 7.32−7.29 (m, 1H),
7.25−7.23 (m, 1H), 2.19 (s, 3H), 1.60 (s, 3H). HRMS (MALDI):
calcd for C18H12ClFO5 [M + Na]+ 385.0255, found 385.0229.
Data for 4n2. 1H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.77−
7.74 (m, 1H), 7.55−7.52 (m, 1H), 7.48 (s, 1H), 7.32−7.29 (m, 1H),
7.25−7.23 (m, 1H), 2.19 (s, 3H), 1.60 (s, 3H). HRMS (MALDI):
calcd for C18H12BrFO5 [M + Na]+ 428.9750, found 428.9757.
Data for 4d3. 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.84−
7.80 (m, 2H), 7.25−7.20 (m, 3H), 2.18 (s, 3H), 1.58 (s, 3H). HRMS
(MALDI): calcd for C18H12FIO5 [M + Na]+ 476.9611, found
476.9618.
Data for 4e2. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.94 (d,
J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 2.19 (s, 3H),
1.60 (s, 3H). HRMS (MALDI): calcd for C19H12BrF3O5 [M + Na]+
478.9718, found 478.9721.
1
Data for 4e3. H NMR (600 MHz, CDCl3): δ 7.94 (m, 3H), 7.79
(m, 2H), 7.36 (s, 1H), 2.19 (s, 3H), 1.58 (s, 3H). HRMS (MALDI):
calcd for C19H12IF3O5 [M + Na]+ 526.9579, found 526.9606.
Data for 4f1. 1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.20 (s, 1H), 2.19 (s, 3H),
1.60 (s, 3H), 1.36 (s, 9H). HRMS (MALDI): calcd for C22H21ClO5
[M + Na]+ 423.0975, found 423.1004.
1
Data for 4o1. H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.57
(d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H),
7.42 (t, J = 7.8 Hz, 1H), 7.18 (s, 1H), 2.20 (s, 3H), 1.62 (s, 3H).
HRMS (MALDI): calcd for C18H12Cl2O5 [M + Na]+ 400.9959, found
400.9963.
Data for 4f2. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.53 (d, J = 9.2 Hz, 2H), 7.25 (s, 1H), 2.19 (s, 3H),
1.60 (s, 3H), 1.35 (s, 9H). HRMS (MALDI): calcd for C22H21BrO5
[M + Na]+ 467.0470, found 467.0451.
1
Data for 4o2. H NMR (600 MHz, CDCl3): δ 8.01 (s, 1H), 7.58
(d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H),
7.42 (t, J = 7.2 Hz, 1H), 7.24 (s, 1H), 2.20 (s, 3H), 1.61 (s, 3H).
HRMS (MALDI): calcd for C18H12BrClO5 [M + Na]+ 444.9454,
found 444.9465.
Data for 4f3. 1H NMR (400 MHz, CDCl3): δ 7.94 (1H, s), 7.75 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.26 (s, 1H), 2.18 (s, 3H), 1.58
(s, 3H), 1.37 (s, 9H). HRMS (MALDI): calcd for C22H21IO5 [M + Na]+
515.0331, found 515.0330.
1
Data for 4p1. H NMR (600 MHz, CDCl3): δ 8.00 (s, 1H), 7.84
(d, J = 6.6 Hz, 1H), 7.72−7.70 (m, 2H), 7.63 (d, J = 6.6 Hz, 1H), 6.94
(s, 1H), 2.20 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for
C19H12ClF3O5 [M + Na]+ 435.0223, found 435.0222.
Data for 4h2. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.44−
7.41 (m, 2H), 7.30−7.26 (m, 2H), 7.10−7.09 (d, J = 7.2 Hz, 1H), 3.89
(s, 3H), 2.19 (s, 3H), 1.60 (s, 3H). HRMS (MALDI): calcd for
C19H15BrO6 [M + Na]+ 440.9950, found 440.9958.
1
Data for 4p2. H NMR (600 MHz, CDCl3): δ 7.98 (s, 1H), 7.85
(d, J = 7.2 Hz, 1H), 7.72−7.70 (m, 2H), 7.63 (d, J = 6.6 Hz, 1H), 6.99
(s, 1H), 2.20 (s, 3H), 1.62 (s, 3H). HRMS (MALDI): calcd for
C19H12BrF3O5 [M + Na]+ 478.9718, found 478.9710.
1
Data for 4i1. H NMR (400 MHz, CDCl3): δ 8.06 (s, 1H), 7.62−
Data for 4q2. 1H NMR (400 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.26
(s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 2.13 (s,
3H), 1.52 (s, 3H). HRMS (MALDI): calcd for C18H11BrCl2O5 [M +
Na]+ 478.9065, found 478.9060.
7.60 (m, 2H), 7.42−7.35 (m, 2H), 7.22 (s, 1H), 2.45 (s, 3H), 2.19 (s,
3H), 1.60 (s, 3H). HRMS (MALDI): calcd for C19H15ClO5 [M + Na]+
381.0506, found 381.0498.
1
Data for 4i2. H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.62−
1
7.60 (m, 2H), 7.41−7.38 (m, 2H), 7.27 (s, 1H), 2.46 (s, 3H), 2.19 (s,
3H), 1.60 (s, 3H). HRMS (MALDI): calcd for C19H15BrO5 [M + Na]+
425.0001, found 425.0026.
Data for 4r1. H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.65−
7.62 (m, 1H), 7.60−7.58 (m, 1H), 7.35−7.31 (m, 1H), 7.17 (s, 1H),
2.19 (s, 3H), 1.59 (s, 3H). HRMS (MALDI): calcd for C18H11ClF2O5
[M + Na]+ 403.0161, found 403.0156.
1
Data for 4i3. H NMR (600 MHz, DMSO-d6): δ 8.34 (s, 1H),
1
7.72−7.70 (m, 2H), 7.51−7.45 (m, 2H), 7.24 (s, 1H), 2.42 (s, 3H),
2.12 (s, 3H), 1.50 (s, 3H). HRMS (MALDI): calcd for C19H15IO5
[M + Na]+ 472.9862, found 472.9854.
Data for 4r2. H NMR (600 MHz, CDCl3): δ 8.00 (s, 1H), 7.65−
7.59 (m, 2H), 7.35−7.31 (m, 1H), 7.22 (s, 1H), 2.19 (s, 3H), 1.59 (s,
3H). HRMS (MALDI): calcd for C18H11BrF2O5 [M + Na]+ 446.9656,
found 446.9647.
Data for 4j1. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.61 (d,
J = 8.0 Hz, 1H), 7.53−7.48 (m, 2H), 7.28−7.23 (m, 2H), 2.19 (s, 3H),
1.60 (s, 3H). HRMS (MALDI): calcd for C18H12ClFO5 [M + Na]+
385.0255, found 385.0256.
Preparation of 1-(2,4-Dimethoxy-3-methylphenyl)ethanone
(17). To a solution of 2,6-dimethoxytoluene (3.04 g, 20 mmol) in
dichloromethane (15 mL) was added AlCl3 (3.2 g, 24 mmol) at 0 °C.
The mixture was stirred for 0.5 h, and a solution of acetyl chloride (1.4
mL) in dichloromethane (15 mL) was added. The progress of the
reaction was monitored by TLC and, when it was complete, the
reaction mixture was poured into ice water and neutralized to pH 7
with a 3% solution of NaOH. The separated organic phase was washed
with water and brine and dried over Na2SO4. The solvent was removed
under reduced pressure, and the residue was purified by flash
chromatography on silica gel (petroleum ether:ethyl acetate = 25:1) to
give pale 17 as a yellow oil (2.29 g, 59%). 1H NMR (600 MHz,
CDCl3): δ 7.62 (d, J = 9 Hz, 2H), 6.68 (d, J = 9 Hz, 2H), 3.88 (s, 3H),
3.76 (s, 3H), 2.63 (s, 3H), 2.17 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 198.8, 162.0, 159.2, 128.8, 125.4, 120.1, 105.8, 61.7, 55.6,
30.1, 8.7. ESI-MS 194.0 (M+).
Preparation of 1-(2,4-Dimethoxy-3-methyl-5-nitrophenyl)-
ethanone (18). A solution of 17 (4.85 g, 25 mmol) in 15 mL of acetic
anhydride was added slowly to a mixture of Cu(NO3)2·3H2O (3.62 g,
15 mmol) in 15 mL of acetic anhydride at 0 °C. The mixture was
stirred vigorously, and the reaction was monitored by TLC. When
it was complete, the mixture was poured into ice water, stirred for
30 min, and then extracted three times with ethyl acetate. The combined
ethyl acetate extracts were washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue was
purified by flash chromatography on silica gel (petroleum ether:ethyl
1
Data for 4j2. H NMR (600 MHz, DMSO-d6): δ 8.46 (s, 1H),
7.84−7.81 (m, 2H), 7.67−7.63 (m, 1H), 7.51−7.48 (m, 1H), 7.39 (s,
1H), 2.13 (s, 3H), 1.52 (s, 3H). HRMS (MALDI): calcd for
C18H12BrFO5 [M + Na]+ 428.9750, found 428.9773.
Data for 4j3. 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H), 7.62 (d,
J = 8.0 Hz, 1H), 7.53−7.49 (m, 2H), 7.29−7.26 (m, 2H), 2.19 (s, 3H),
1.58 (s, 3H). HRMS (MALDI): calcd for C18H12IFO5 [M + Na]+
476.9611, found 476.9622.
1
Data for 4k2. H NMR (400 MHz, CDCl3): δ 8.02 (s, 1H), 7.78
(s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.52−7.47 (m, 2H), 7.27 (s, 1H),
2.19 (s, 3H), 1.59 (s, 3H). HRMS (MALDI): calcd for C18H12BrClO5
[M + Na]+ 444.9454, found 444.9462.
Data for 4l1. 1H NMR (600 MHz, CDCl3): δ 8.06 (s, 1H), 7.72 (d,
J = 7.8 Hz, 1H), 7.67 (s, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.09 (t, J = 7.8
Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 2.19 (s, 3H), 1.60 (s,
3H). HRMS (MALDI): calcd for C19H15ClO6 [M + Na]+ 397.0455,
found 397.0459.
Data for 4l2. 1H NMR (600 MHz, CDCl3): δ 8.04 (s, 1H), 7.75 (s,
1H), 7.72 (t, J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.10−7.05 (m,
2H), 3.99 (s, 3H), 2.19 (s, 3H), 1.60 (s, 3H). HRMS (MALDI): calcd
for C19H15BrO6 [M + Na]+ 440.9950, found 440.9951.
1
Data for 4m1. H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.48
(d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.33−7.32 (m, 2H), 6.93
(s, 1H), 2.47 (s, 3H), 2.20 (s, 3H), 1.62 (s, 3H). HRMS (MALDI):
calcd for C19H15ClO5 [M + Na]+ 381.0506, found 381.0468.
1
acetate = 8:1) to provide 18 as a pale yellow solid (3.53 g, 59%). H
NMR (400 MHz, CDCl3): δ 8.09 (s, 1H), 3.94 (s, 3H), 3.84 (s, 3H),
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2.65 (s, 3H), 2.31 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 197.3,
162.2, 155.8, 139.9, 128.8, 128.6, 124.7, 62.2, 62.0, 30.2, 9.9. ESI-MS
239.1 (M+).
160.5, 131.1, 129.3, 128.9, 122.2, 122.0, 113.8,113.2, 112.5, 95.3, 89.8,
31.5, 8.0. HRMS (MALDI): calcd for C17H14O3 [M + H]+ 267.1021,
found 267.0989.
Preparation of 1-(3-Halogen-4,6-dihydroxy-5-methyl-2-(2-
phenylethynyl)phenyl)ethanone (23). To a solution of compound
22 (106 mg, 0.4 mmol) in 4 mL of THF was added concentrated HX (10
equiv of concentrated HCl and HBr for 23a and 23b, respectively), and
the mixture was stirred for 3 min. IBX (0.37 g, 1.20 mmol) and TBAI (7
mg) were added successively. Progress of the reaction was monitored by
TLC, and, after 30 min, the reaction mixture was poured into water and
neutralized with saturated aqueous Na2CO3. The resulting mixture was
extracted three times with ethyl acetate. The combined organic extracts
were washed with brine and dried over anhydrous Na2SO4. The solvent
was evaporated under reduced pressure, and the residue was purified by
flash chromatography on silica gel (petroleum ether:ethyl acetate = 12:1),
affording yellow product 23.
Preparation of 1-(3-Amino-2-bromo-4,6-dimethoxy-5-
methylphenyl)ethanone (19). To a solution of 18 (2.39 g, 10
mmol) in 18 mL of anhydrous THF was added 10% Pd/C (0.25 g),
and the resulting mixture was hydrogenated under H2 at room
temperature. The progress of the reaction was monitored by TLC,
and, when it was complete, the mixture was filtered through Celite and
washed with ethyl acetate. The filtrate was concentrated under reduced
pressure to give the crude aniline, which was employed in the next step
without further purification. The crude aniline was dissolved in 18 mL
of acetic acid, and a solution of bromine (0.57 mL, 11 mmol) in 6 mL
of acetic acid was slowly added at 10 °C. Monitoring the reaction by
TLC indicated the disappearance of the aniline, at which point the
reaction mixture was quenched by the addition of aqueous Na2S2O3.
The mixture was extracted three times with ethyl acetate and the
combined organic extracts were washed successively with saturated
aqueous NaHCO3, water, and brine, then dried over anhydrous Na2SO4,
and concentrated. The residue was purified by flash chromatography on
silica gel (petroleum ether:ethyl acetate = 4:1) to give 19 as a pale yellow
1
Data for 23a. H NMR (600 MHz, CDCl3): δ 13.74 (s, 1H),
7.57−7.56 (m, 2H), 7.418−7.40 (m, 3H), 6.26 (s, 1H), 3.00 (s, 3H),
2.22 (s, 3H). HRMS(MALDI): calcd for C17H13ClO3 [M + H]+
301.0631, found 301.0581.
1
Data for 23b. H NMR (600 MHz, CDCl3): δ 13.72 (s, 1H),
1
7.57−7.56 (m, 2H), 7.418−7.40 (m, 3H), 6.24 (s, 1H), 2.99 (s, 3H),
2.22 (s, 3H). HRMS (MALDI): calcd for C17H13BrO3 [M + H]+
345.0126, found 345.0085.
product (2.22 g, 77%). H NMR (600 MHz, CDCl3): δ 4.13 (br, 2H),
3.75 (s, 3H), 3.64 (s, 3H), 2.52 (s, 3H), 2.18 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 202.6, 146.5, 146.1, 135.6, 133.7, 124.6, 100.3, 62.6, 59.3, 31.7,
9.3. ESI-MS 287.0 (M+).
Preparation of Azaphilone 6a and 6b. A mixture of 23a or 23b
(0.24 mmol), Au(OAc)3 (4.5 mg, 0.012 mmol), and trifluoroacetic acid
(1.5 mL) in 1.5 mL of 1,2-dichloroethane was stirred for 20 min at room
temperature. Then IBX (75 mg) and TBAI (4.5 mg) were added. Progress
of the reaction was monitored by TLC, and, when it was complete, the
mixture was quenched by addition of aqueous Na2S2O3. The reactant was
extracted three times with ethyl acetate. The combined organic extracts
were washed with water and brine and dried with anhydrous Na2SO4. The
solvent was evaporated under reduced pressure, and the residue was
purified by flash chromatography on silica gel (petroleum ether:ethyl
acetate = 5:1) to give 6a or 6b as a yellow solid (32 mg, 36%).
Preparation of 1-(6-Bromo-2,4-dimethoxy-3-methylphenyl)-
ethanone (20). To a solution of compound 19 (2.02 g, 7.0 mmol) in
30 mL of THF and 20 mL of distilled water was slowly added 5 mL of
concentrated HCl, and the reaction mixture was cooled to −5 °C.
Sodium nitrite (0.53 g, 7.68 mmol) in 2 mL of H2O was added
dropwise, and the mixture was stirred for 30 min at −5 °C before the
addition of urea (0.1 g, 1.67 mmol). Aqueous hypophosphorous acid
(50% w/w) (16 mL) was then added over 1 h, and the resulting
mixture was allowed to warm to 0 °C, stirred for an additional 6 h, and
then extracted three times with ethyl acetate. The combined extracts
were washed with brine, dried over anhydrous Na2SO4, and evaporated
under reduced pressure to give a residue, which was purified by flash
chromatography (petroleum ether:ethyl acetate = 15:1) to give 20 as
yellow oil (1.61 g, 84%). 1H NMR (600 MHz, CDCl3): δ 6.81 (s, 1H),
3.83 (s, 3H), 3.69 (s, 3H), 2.52 (s, 3H), 2.08 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 202.4, 159.4, 156.0, 130.6, 119.8, 114.3, 110.6, 62.4, 55.9, 31.8,
1
Data for 6a. H NMR (600 MHz, CDCl3): δ 7.83 (d, J = 7.2 Hz,
2H), 7.57−7.52 (m, 3H), 7.26 (s, 1H), 2.70 (s, 3H), 1.61 (s, 3H). 13C
NMR (150 MHz CDCl3): δ 195.8, 189.4, 165.4, 157.8, 141.4, 132.0,
130.0, 129.2, 125.9, 111.7, 108.7, 103.6, 84.3, 28.5, 19.3. HRMS
(MALDI): calcd for C17H13ClO4 [M + Na]+ 339.0400, found 339.0448.
1
Data for 6b: H NMR (600 MHz, CDCl3): δ 7.84 (d, J = 7.2 Hz,
+
8.8. ESI-MS 272.0 (M − H) , 274.0 (M + H)+.
2H), 7.52−7.57 (m, 3H), 7.32 (s, 1H), 4.00 (br, 1H), 2.70 (s, 3H),
1.62 (s, 3H). 13C NMR (150 MHz CDCl3): δ 195.9, 189.7, 165.4,
158.1, 143.6, 132.0, 129.9, 129.2, 125.9, 112.5, 106.3, 100.5, 84.3, 28.5,
19.2. HRMS (MALDI): calcd for C17H13BrO4 [M + Na]+ 382.9895,
found 382.9861.
Biological Testing. All testing was undertaken on 96-well micro-
titer plates. Pathogen species, rates, and number of replicates are given
in Table 1 for the assays conducted on leaf pieces, and Table 2 for the
assays conducted in artificial media.
Preparation of 1-(6-Bromo-2,4-dihydroxy-3-methylphenyl)-
ethanone (21). To the compound 20 (0.273 g, 1.0 mmol) in 5 mL of
freshly distilled toluene was added AlCl3 (1.3 g, 1.0 mmol). The
mixture was heated under reflux for 1 h (TLC monitoring) and then
poured into ice water and extracted three times with ethyl acetate. The
combined organic extracts were washed with water and brine and dried
with anhydrous Na2SO4. The solvent was concentrated, and the
residue was purified by flash chromatography (petroleum ether:ethyl
1
acetate = 8:1) to give 21 as a yellow solid (0.18 g, 74%). H NMR
(400 MHz, CDCl3): δ 13.49 (s, 1H), 6.76 (s, 1H), 5.43 (s, 1H), 2.86
(s, 3H), 2.08 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 202.5,
158.5, 157.1, 120.2, 116.2, 112.0, 111.4, 32.1, 8.2. ESI-MS 244.0 (M − H) +,
276.0 (M + H)+.
Table 1. Details of the Primary-Level Leaf-Piece Assays
a
b
pathogen
host
rates
replicates
Phytophthora infestans
Septoria tritici
tomato
wheat
200, 60
100
2
3
2
Preparation of 1-(2,4-dihydroxy-3-methyl-6-(2-
phenylethynyl)phenyl)ethanone (22). To a mixture of compound
21 (98 mg, 0.40 mmol), Pd(PPh3)2Cl2 (14 mg, 0.02 mmol), and CuI
(2 mg, mmol) in degassed DMF (3 mL) and under an inert atmo-
sphere were added successively phenylacetylene (49.1 mg, 0.48 mmol)
and Et3N (3 mL). The resulting mixture was heated and stirred at
50 °C for about 5 h. After cooling to room temperature, the mixture
was poured into water, neutralized with 1% aqueous HCl, and then
extracted three times with ethyl acetate. The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4, and then
concentrated under reduced pressure. The residue was purified directly on
a column of silica gel (petroleum ether:ethyl acetate = 8:1), affording 22
as a yellow solid (90.4 mg, 85%). 1H NMR (600 MHz, CDCl3): δ 13.79
(s, 1H), 10.76 (s, 1H), 7.58 (br, 2H), 7.47 (br, 3H), 6.75 (s, 1H), 2.89 (s,
3H), 2.00 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 203.6, 163.3,
Uromyces viciae-fabae
field bean
100
a
Rates are given as parts per million a.i. in the formulated sample
b
applied to the leaf piece. Number of replicates plates per rate.
For the leaf-piece assays, 200−300 μL of water agar was dispensed
into each well of the assay plates. Leaf pieces of the appropriate host
species (6 mm diameter for tomato and bean, 6 mm length for wheat)
were cut either automatically or by hand and transferred onto the
surface of the agar. Dried sample of the active ingredient was formu-
lated to the necessary rate using a robotic liquid-handling system, and
10 μL was dispensed onto the surface of each leaf piece.
Spore suspensions of the pathogen species were made up to the
necessary rate (approximately 150,000 sporangia/mL for Phytophthora
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In this synthetic procedure, 2-bromobenzaldehyde 10 is a key
intermediate for the Sonogashira coupling reaction. A suitable
preparation of this intermediate is outlined in Scheme 1. In this
route, the cheap and commercially available 2-methylresorcinol
was selected as the starting material. The preparation of the
dimethoxybenzaldehyde 13 was achieved by methylation of the
resorcinol 11 and then formylation with POCl3 in DMF under
standard Vilsmeier reaction conditions. Nitration of com-
pound 13 with Cu(NO3)2 afforded 14 in which the aldehyde
was protected in situ as geminal diacetate. The nitro group of
14 was hydrogenated, and the resulting aniline was treated
with bromine to give the fully substituted benzene 15, which
was deaminated by reduction of the corresponding diazonium
salt with H3PO2, under which conditions deprotection of
the geminal diacetate took place to generate 16. The two
methoxyl groups of compound 16 were deprotected using
BBr3 in dichloromethane to afford the desired 2-bromo-
benzaldehyde smoothly and in excellent yield. The inter-
mediate 10 could be obtained on a multigram scale using this
synthetic route.
With the key intermediate 10 in hand, we then investigated
its coupling reactions with phenylacetylenes bearing diverse
aromatic substituents. We initially attempted to optimize the
Sonogashira coupling reaction between 2-bromobenzaldehyde
and phenylacetylene by screening the catalysts [Pd(PPh3)4,
Pd(PhCN)2Cl2, Pd(PPh3)2Cl2], bases (K2CO3, triethylamine,
etc.), and solvents (CH3CN, THF, DMF, toluene, etc.). Optimal
conditions were obtained with the cocatalyst system of
Pd(PPh3)2Cl2/CuI in the presence of triethylamine in DMF
at 60 °C. A variety of aromatic terminal acetylenes were
employed in this palladium/copper cocatalyzed process, and
good to excellent yields were achieved in most cases (Table 4,
entries 1−18). It is worth noting that the presence of either
electron-donating or electron-withdrawing groups on the
phenylacetylene did not significantly affect the yield of the
Sonogashira coupling. In addition, no noteworthy steric effects
were observed (Table 4, entry 6). However, the benzyl-acetylene
1-(prop-2-ynyl)benzene showed lower reactivity in this coupling
reaction, and a much lower yield was observed (entry 19). In this
case, microwave irradiation was employed to improve the yield.
The optimum reaction conditions were found to be 5% PdCl2-
(PPh3)2, 5% CuI, and Et3N (3 equiv) in a 4:1 mixture of dioxane
and water at 80 °C under microwave irradiation for 15 min, and
the isolated yield improved significantly to 75%.
Table 2. Details of the Primary-Level Assays Conducted in
Artificial Media
a
b
pathogen
Pythium dissimile
rates
replicates
20, 2
20, 2
20, 2
20, 2
2
2
2
2
Alternaria solani
Botryotinia fuckeliana (Botrytis cinerea)
Gibberella zeae (Fusarium graminearum)
a
Rates are given as parts per million a.i. in the final artificial media
b
mixture. Number of replicates plates per rate.
infestans, 1,000,000 conidia/mL for Septoria tritici, and 0.3 mg of
spores/mL for Uromyces viciae-fabae), and applied to the treated leaf
pieces using a hand-held spray gun. Lids were placed on the plates, and
they were stored in appropriate controlled environment conditions for
between 5 and 14 days, depending on the test species.
For the artificial medium assays, stock cultures of the target species
were grown in appropriate conditions on artificial media in 90 mm
Petri dishes. For all pathogens apart from Pythium dissimile, spore
suspensions were prepared in water from these stock plates and made
into a 3% nutrient agar (final spore concentrations of 10,000 sp/mL
for Gibberella zeae, 15,000 sp/mL for Botryotinia fuckeliana, and 20,000
sp/mL for Alternaria solani). For Pythium dissimile, a water suspension
of fragments of surface mycelia was made from the stock cultures and
made into a 3% agar as for the other species, adjusted to a final optical
density reading of 0.5 at 425 nm.
The chemicals to be tested were formulated and transferred to the
assay plates using the liquid handling robot as for the leaf-piece assays,
with adjustments in concentration for the high and low rate replicates.
The wells of these plates were then loaded with 90 μL of the spore/
agar suspensions of the appropriate species using an automated liquid
handling system, to give final rates of target compound in the media of
20 ppm (high rate) or 2 ppm (low rate). The plates were then stored
in controlled environments set to appropriate conditions, until
assessment between 5 and 14 days later, depending on the species.
Assessment for each species was carried out by eye, with each
individual well scored on a three-band pattern with additional result
types for assay failure and phytotoxic activity on the leaf piece (Table 3).
Table 3. Assessment Criteria for the Biological Assays
scoring band
well condition
0
0 to 49% control of disease or pathogen
50 to 80% control of disease or pathogen
81 to 100% control of disease or pathogen
not captured (too phytotoxic)
55
99
NCH
NC
not captured (test failure)
The subsequent cycloisomerization with a Lewis acid (AgNO3)
at room temperature using a mixture of 1,2-dichloroethane and
trifluoroacetic acid as solvent afforded the pyrylium salts, which
were oxidized without isolation with o-iodoxybenzoic acid (IBX)
to afford the azaphilones 2 in satisfactory yield. This one-pot
procedure greatly simplifies the synthesis. The intermediate
o-alkynylbenzaldehydes 9 are all tolerated in this two-step one-
pot transformation, and were converted smoothly into the
corresponding azaphilones. Remarkably, electron-donating or
electron-withdrawing aromatic substituents on the o-alkynyl-
benzaldehydes, irrespective of their position on the benzene
The resulting data were collated for each compound, and averages across
replicates were used to make a judgment of the overall activity level of
the compound.
RESULTS AND DISCUSSION
■
Chemistry. According to the retrosynthetic analysis as
shown in Figure 2, we planned to prepare the core structures
2−5 by the oxidation of the appropriate benzopyrylium salts 8,
which may be derived from the alkynylbenzaldehyde 9, which
should be readily accessible using Sonogashira coupling.
Figure 2. Retrosynthetic analysis of the designed azaphilone analogues.
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a
Scheme 1
a
Reagents and conditions: (a) (CH3)2SO4, K2CO3, 87%; (b) POCl3, DMF, 88%; (c) Cu(NO3)2·3H2O, Ac2O, 81%; (d) (i) Pd/C, H2; (ii) Br2, 88%
in two steps; (e) NaNO2, H3PO2, 83%; (f) BBr3, CH2Cl2, −78 °C, 90%.
Table 4. Synthesis of 2 and 3 via the Sonogashira reaction and Subsequent Cycloisomerization
a
yield (%)
no.
R
9
2
1
C6H5−
9a (78)
9b (70)
9c (83)
9d (83)
9e (76)
9f (77)
9g (55)
9h (69)
9i (90)
9j (73)
9k (63)
9l (92)
9m (92)
9n (75)
9o (84)
9p (90)
9q (87)
9r (86)
2a (63)
2b (55)
2c (54)
2d (55)
2e (72)
2f (65)
2g (55)
2h (60)
2i (65)
2j (61)
2k (65)
2l (52)
2m (66)
2n (67)
2o (81)
2p (78)
2q (66)
2r (42)
2
4-CH3O−C6H4−
4-CH3−C6H4−
4-F−C6H4−
4-CF3−C6H4−
4-(CH3)3C−C6H4−
4-NO2−C6H4−
3-CH3O−C6H4−
3-CH3−C6H4−
3-F−C6H4−
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3-Cl−C6H4−
2-CH3O−C6H4−
2-CH3−C6H4−
2-F−C6H4−
2-Cl−C6H4−
2-CF3−C6H4−
3,4-di-Cl−C6H3−
3,4-di-F−C6H3−
C6H5CH2−
b
c
9s (48, 75 )
2s (28, 48 )
a
b
c
Isolated yield. The reaction was run in a dioxane−water mixture (4:1) at 80°C under microwave irradiation. The reaction temperature was
adjusted to 0 °C.
ring, did not affect the yields of azaphilones. In the case of entry
19, only 28% isolated yield was achieved. However, when the
temperature of cycloisomerization was reduced to 0 °C, the
yield improved to 48%.
Selected azaphilones 2 were treated with N-chlorosuccini-
mide (NCS), N-bromosuccinimide (NBS) or N-iodosuccini-
mide (NIS) in dichloromethane to produce derivatives which
were halogenated at the 5-position, and these were then
converted into the corresponding acetates by treatment with
acetic anhydride in the presence of triethylamine and DMAP, as
shown in Table 5. Compounds 5a and 5b were prepared in the
same way, by halogenation of the compound 2s with NCS or
NBS, respectively.
It was prepared by the synthetic route illustrated in Scheme 2,
which parallels the route shown in Scheme 1. Regioselective
Friedel−Crafts acylation of the dimethylated compound 12
provided the intermediate 17, which underwent nitration with
Cu(NO3)2 in acetic acid to afford compound 18. The fully
substituted benzene 19 was then obtained by successive
reduction and bromination of compound 18. Compound 19
was then transformed into the key intermediate 21 by deamina-
tion and demethylation. It is worth noting that, compared with
the BBr3 method, the demethylation of 20 with AlCl3 possesses
many advantages, such as the low cost of the reagent, the mild
reaction conditionsm, and the convenient workup procedure.
Sonogashira coupling of 21 with phenylacetylene afforded com-
pound 22. Unfortunately, when we used the conditions pre-
viously explored to carry out the subsequent cycloisomerization
Compound 6 has a chlorine or bromine atom at the 5-
position and an additional methyl group at the 1-position.
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Table 5. Synthesis of Compounds 3 and 4 via Halogenation and Acetylation
yield (%)
yield (%)
no.
X
R
3
4
no.
X
R
3
4
1
Cl
Br
I
C6H5−
C6H5−
C6H5−
4-CH3O−C6H4−
4-CH3−C6H4−
4-CH3−C6H4−
4-CH3−C6H4−
4-F−C6H4−
3a1 (60)
3a2 (70)
3a3 (58)
3b2 (55)
3c1 (52)
3c2 (49)
3c3 (60)
3d1 (50)
3d2 (80)
3d3 (80)
3e1 (78)
3e2 (65)
3e3 (59)
3f1 (74)
3f2 (40)
3f3 (74)
3g2 (63)
3h1 (40)
3h2 (50)
3i1 (56)
3i2 (53)
4a1 (50)
4a2 (56)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
I
3-CH3−C6H4−
3-F−C6H4−
3-F−C6H4−
3-F−C6H4−
3-Cl−C6H4−
3i3 (50)
3j1 (63)
3j2 (66)
3j3 (54)
3k1 (59
4i3 (42)
2
Cl
Br
I
4j1 (40)
4j2 (45)
4j3 (22)
3
4
Br
Cl
Br
I
4b2 (50)
4c1 (67)
4c2 (43)
4c3 (54)
4d1 (45)
4d2 (49)
4d3 (52)
5
Cl
Br
I
6
3-Cl−C6H4−
3-Cl−C6H4−
3k2 (71)
3k3 (59)
3l1 (38)
3l2 (66)
3m1 (63)
3m2 (70)
3n1 (52)
3n2 (29)
3o1 (40)
3o2 (67)
3p1 (50)
3p2 (65)
3q1 (69)
3q2 (56)
3r1 (67)
3r2 (63)
4k2 (36)
4k3 (17)
4l1 (47)
4l2 (67)
7
8
Cl
Br
I
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
2-CH3O−C6H4−
2-CH3O−C6H4−
2-CH3−C6H4−
2-CH3−C6H4−
2-F−C6H4−
2-F−C6H4−
2-Cl−C6H4−
2-Cl−C6H4−
2-CF3−C6H4−
2-CF3−C6H4−
3,4-di-Cl−C6H3−
3,4-di-Cl−C6H3−
3,4-di-F−C6H3−
3,4-di-F−C6H3−
9
4-F−C6H4−
4-F−C6H4−
10
11
12
13
14
15
16
17
18
19
20
21
Cl
Br
I
4-CF3−C6H4−
4-CF3−C6H4−
4-CF3−C6H4−
4-(CH3)3C−C6H4−
4-(CH3)3C−C6H4−
4-(CH3)3C−C6H4−
4-NO2−C6H4−
3-CH3O−C6H4−
3-CH3O−C6H4−
3-CH3−C6H4−
3-CH3−C6H4−
4m2 (44)
4e2 (74)
4e3 (21)
4f1 (55)
4f2 (38)
4f3 (58)
4g2 (42)
4h1 (27)
4h2 (31)
4i1 (55)
4i2 (53)
4n2 (31)
4o1 (49)
4o2 (58)
Cl
Br
I
Br
Cl
Br
Cl
Br
4p2 (83)
4q2 30
4r1 (38)
4r2 (42)
a
Scheme 2
a
Reagents and conditions: (a) Me2SO4, K2CO3, 87%; (b) CH3COCl, AlCl3, 59%; (c) Cu(NO3)2, Ac2O, 59%; (d) Pd/C, H2; (e) Br2, 77% over two
steps; (f) HCl, NaNO2, H3PO2, 84%; (g) AlCl3, toluene, refluxing, 74%; (h) phenylacetylene, Pd(PPh3)2Cl2, CuI, Et3N, DMF, 85%; (i) for 31a, 10
equiv of HCl, 3 equiv of IBX, 23%; (j) for 31b, 10 equiv of HBr, 3 equiv of IBX, 75%; (k) Au(OAc)3, DCE/TFA (10:1); (l) IBX, TBAB, 15% or
36% for X = Cl or Br in two steps, respectively.
and oxidation, we failed to obtain compound 6a. However,
compounds 6a and 6b were obtained successfully when we
used compound 23 obtained by halogenation of compound 22
to perform the cycloisomerization reaction. This result showed
that the halogen atom at the 3-position is favorable to this
transformation.
Antifungal Activity. In general, antifungal activity from
these compounds was limited to those assays conducted in
artificial media. Activity on leaf-piece assays (particularly the
Septoria tritici assay) was rare throughout. In general, compounds
which displayed activity tended to lack potency, with activity on
lower rates infrequent.
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a
Table 6. Antifungal Activity of Selected Compounds
hydroxyl analogues
acetyl analogues
Pd
b
Pi
St
Uvf
100
Pd
As
Bf
Gz
Pi
St
Uvf
100
As
Bf
Gz
c
compd 200/60
100
20/2
20/2
20/2
20/2
compd
200/60
100
20/2
20/2
20/2
20/2
3a1
3a2
3a3
3b2
3c1
3c2
3c3
3d1
3d2
3d3
3e1
3e2
3e3
3f1
3f2
3f3
3g2
3h1
3h2
3i1
0/0
0
0
99/99
99/77
0/0
99/27
99/0
0/0
27/0
0/0
99/0
99/0
0/0
4a1
4a2
0/−
0/−
99
49
0
0
99/77
99/99
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0
0
0/0
0
0
0/0
0/0
0
27
55
99
0
0/0
0/0
0/0
0/0
4b2
4c1
4c2
4c3
0/0
0/0
27/0
0/0
18
0
NC
55
0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/77
0/0
0
99/49
49/49
0/0
99/0
0/0
99/0
27/27
0/0
99/0
0/0
0/0
51
0
18
0
55/0
99/0
0/0
0/0
0/0
49
49/27
49/0
0/0
0
55
0
99/0
99/0
0/0
0/0
0/0
0/0
0
99/0
0/0
77/0
0/0
77/0
0/0
4d2
4d3
27/0
27/0
0
0
55
99
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0
0
49/0
27/0
49/0
0/0
0
77
NCH
0
0/0
0/0
27/0
99/27
0/0
0/0
18
0
99/49
0/0
99/0
0/0
99/0
0/0
4e2
4e3
4f1
4f2
4f3
0/0
0/0
0/0
0/0
0/0
0
0
0
0
0
77
99
77
99
99
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0
NCH
55
27
27
0
0/0
0/0
0/49
99/0
0/0
0/0
99/49
0/0
36
0
99/0
0/0
99/0
0/0
99/0
0/0
77/27
27/49
49/55
0/49
0/49
0/0
0
0/0
0/0
0/0
0/0
0
99/0
99/55
99/0
99/0
99/0
99/49
0/0
0/0
0/0
99/0
99/0
99/0
99/0
77/0
77/0
0/0
0
0
55/0
77/0
77/0
99/0
99/0
0/0
77/0
77/0
77/0
55/0
55/0
0/0
4h2
4i1
4i2
4j1
4j2
0/0
0
0
0
0
0
0
27/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
18
69
0
55
55
0
77/0
77/0
0/0
18
0
3i2
3j1
0
3j2
0/0
0
0
49/0
0
3j3
0/0
0
0
3k1
3k2
3k3
3l1
27/49
0/0
0
49
27
27
0
0/49
99/49
0/0
0/0
0/0
0/0
33
0
99/0
0/0
99/0
0/0
99/0
0/0
4k2
0/0
0
55
0/0
0/0
0/0
0/0
0/0
49/0
0/0
0
0/0
0/0
0/0
0/0
4l1
0/49
0/0
0/0
0/0
0/0
0/27
0/0
0/0
0
0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
27/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
55/0
0/0
0/0
0/0
0/0
0/0
0/0
3l2
0
0
77/27
99/99
99/27
99/49
0/0
0/0
27/0
55/0
55/0
55/0
0/0
0/0
4l2
0
27
27
0
99/0
27/0
27/0
49/0
0/0
3m1
3m2
3n1
3n2
3o1
3o2
3p1
3p2
3q1
3q2
3r1
3r2
1
49/49
0/55
49/49
0/0
0
0
77/0
0/0
99/0
99/0
99/0
0/0
4m1
4m2
4n1
4n2
4o1
4o2
0
18
0
0
0
0
77/0
55/0
55/0
55/0
0/0
0
49
0
0
0
0
27/77
27/0
0/0
0
0
99/0
99/49
99/77
99/55
0/0
55/0
99/0
0/0
99/0
99/0
27/0
55/0
27/0
27/0
0/0
0
55
27
55/0
49/0
18
0
55
0
18
0/27
0/0
0
0
0/0
27/0
55/0
27/27
0/0
0
0
49/0
0/0
0/0
0
0
0/0
4q2
4r1
4r2
0/0
0/0
0/0
0
0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
49/0
77/49
0/0
0
0
99/0
99/0
99/0
99/49
27/0
27/0
0/0
0/0
18
18
55
0
27/0
0/0
18
0
0
0/0
0/0
0/0
NCH
0
0/0
0/0
0/0
2a
0/0
0
0/0
0/0
0/0
5a
0/0
0
0
0/0
0/0
99/0
99/0
99/0
99/0
5b
0/0
18
0
55
27
0
0/0
0/0
6a
0/0
0/0
0/0
6b
0/0
0
27/0
0/0
0/0
a
Equivalent hydroxyl and acetyl analogues are presented side by side for clear comparison. Values are mean assessment scores across replicates, as
defined in Tables 2 and 3. Higher values indicate greater activity; NC indicates a test failure; NCH that no assessment was possible due to phytotoxic
b
effects on the leaf pieces and “−” that the compound was not tested at that rate. Pi = Phytophthora infestans (tested on tomato leaf pieces), St =
Septoria tritici (tested on wheat leaf pieces), Uvf = Uromyces viciae-fabae (tested on bean leaf pieces); Pd = Pythium dissimile, As = Alternaria solani,
c
Bf = Botryotinia fuckeliana, Gz = Gibberella zeae (all tested on artificial media). Tested rates in ppm.
It is possible to draw some conclusions about the structure−
activity relationship of the azaphilone derivatives. Initial testing
of the series 2a−r produced activity that was typically absent or
limited to a single species on artificial media (compound 2a is
included in Table 6 as an example). Substituting a halogen at
the 5-position (3a−r) improved the spectrum of activity in
some cases. It was also clear that the free hydroxyl group plays
an important role in activity, as compounds in which it is
derivatized as the acetate (4a−r) were all found to be less active
than their equivalent unprotected analogues.
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However, there was some variation in the data with regard to
the relative effects of different halogens tried at the 5-position.
While it is clear that substituting with iodine in this position
(3[n]3) results in poor activity, the effect of substitution with
chlorine (3[n]1) or bromine (3[n]2) is dependent on the
substitution on the side phenyl ring. For example, substituting a
methyl group in the para position produces a broader spectrum
of activity in the chloro-substituted analogue (3c1 > 3c2),
whereas a CF3 in the para position results in broader spectrum
in the bromo-substituted analogue (3e2 > 3e1). Similarly, where
the phenyl-ring substitution is a fluorine atom, placing it in the
para position gave better activity in the bromo-substituted
compound (3d2 > 3d1), the meta position resulted in roughly
equal activity in the two analogues (3j1 = 3j2), while if the fluorine
is placed in the ortho position the chloro-substituted analogue is
clearly more active (3n1 > 3n2). It also appears that polysubsti-
tution on the phenyl ring is detrimental to the antifungal activity
(3q, 3r) regardless of the substitution at the 5-position.
Compounds with para substitutions of strongly electron-
withdrawing (NO2, compound 3g2) or electron-donating (OCH3,
compound 3b2) groups were found to have a poor spectrum of
antifungal activity. However, these were only tested as bromo-
substituted examples and, given the variation in spectrum pro-
duced by other combinations of substituents, firm conclusions
cannot be drawn about the significance of these data. Methoxy
groups were tested in other positions in combination with both
halogen variants (3h, 3l), but only the meta position and
bromo-substituted combination (3h2) showed broad-spectrum
activity.
ortho position but this may change with different substituents
in the 5-position.
In conclusion, we have succeeded in simultaneously simpli-
fying the structure of sclerotiorin and improving activity in the
new analogues. However, the antifungal activity of these newly
synthesized compounds is confined to the liquid culture assays
and there is no significant activity in the leaf disk assays. The
reason for this limitation is not clear at the present stage, and
research to explain this difference is underway.
AUTHOR INFORMATION
Corresponding Author
*E-mail: gfyang@mail.ccnu.edu.cn (G.-F.Y.); yucheng.gu@
syngenta.com (Y.-C.G.).
■
Author Contributions
‡Co-first authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Biology Team at Syngenta for their kind help in
screening the compounds for biological activity.
■
REFERENCES
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