Synthesis and SAR of 4-methyl-5-pentylbenzene-1,3-diol (MPBD), produced by Dictyostelium discoideum

Article abstract of DOI:10.1016/j.bmcl.2016.01.067

4-Methyl-5-pentylbenzene-1,3-diol (MPBD) is a secondary metabolite of SteelyA polyketide synthase, which controls cell aggregation and spore maturation of Dictyostelium discoideum. In this study, chemical synthesis of MPBD and its derivatives was achieved. Structure-activity relationship (SAR) studies for antimicrobial activities against Escherichia coli and Bacillus subtilis were also conducted.

Full text of DOI:10.1016/j.bmcl.2016.01.067

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1428–1433
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of 4-methyl-5-pentylbenzene-1,3-diol (MPBD),
produced by Dictyostelium discoideum
Chihiro Murata, Tetsuhiro Ogura, Shuhei Narita, Anna P. Kondo, Natsumi Iwasaki, Tamao Saito,
⇑
Toyonobu Usuki
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Methyl-5-pentylbenzene-1,3-diol (MPBD) is a secondary metabolite of SteelyA polyketide synthase,
which controls cell aggregation and spore maturation of Dictyostelium discoideum. In this study, chemical
synthesis of MPBD and its derivatives was achieved. Structure–activity relationship (SAR) studies for
antimicrobial activities against Escherichia coli and Bacillus subtilis were also conducted.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 30 December 2015
Revised 18 January 2016
Accepted 22 January 2016
Available online 22 January 2016
Keywords:
4-Methyl-5-pentylbenzene-1,3-diol
Dictyostelium discoideum
SAR study
Antimicrobial activity
The cellular slime mold Dictyostelium discoideum is an excellent
model organism for the study of cell and developmental systems
because it shows a unique two-stage life cycle.1 Ordinarily,
D. discoideum inhabit in the soil and predaceous bacteria as a
unicellular amoeba. However, when starved, they aggregate and
prompt a multicellular mound. The aggregation forms fruiting bod-
ies consisting of spores and stalk at the end of its differentiation.
In recent years, D. discoideum has attracted increasing attention
as the organisms which have the ability of the production as
secondary metabolites for leading novel medical drugs. The
D. discoideum genome contains more than 40 polyketide synthase
genes, indicating that it has huge potential for polyketide produc-
tion.2,3 Various biologically active compounds from D. discoideum
are thus reported.4–7 For example, differentiation-inducing
factor-1 (DIF-1) and its analogs produced by SteelyB are signal
molecules that induce stalk cell formation.8–10 They exhibit various
bioactivities such as anti-tumor activity,11 sugar metabolism
promoting activity,12 interleukin-2 production inhibitor,13 and
amyloid-b production inhibitor.14
the first total synthesis of 1 was reported,17 development of a strat-
egy that allowed for the easier synthesis of MPBD 1 derivatives for
structure–activity relationship (SAR) studies was required. Herein,
we report the synthesis of MPBD 1 and its derivatives, and their
antimicrobial activities by means of SAR.
In our overall retrosynthesis of MPBD 1 and its derivatives, the
target molecule 1 could be prepared via the Wittig reaction and
halogen alkylation of compound 2 (Scheme 1).19 Intermediate 2
could be obtained from commercially available 1,3-dihydroxyben-
zoic acid 3.
The total synthesis of MPBD 1 is shown in Scheme 2. First, 3 was
methylated by treatment with dimethyl sulfate ((MeO)2SO2) and
potassium carbonate (K2CO3) in acetone under reflux conditions
to give 4 in 92% yield. Reduction of the methyl ester group on 4
using lithium aluminum hydride (LiAlH4) afforded benzyl alcohol
5 in 98% yield. Regioselective iodination of 5 using N-iodosuccin-
imide (NIS) afforded the desired product 6 in 94% yield,20 which
was subjected to Swern oxidation to obtain the common interme-
diate 2 in 96% yield. Wittig reaction between aldehyde 2 and
nBuPPh3Br3 gave olefin 7 (E/Z = 1.3/1) in 87% yield. Lithiation of 7
using n-butyllithium (nBuLi), followed by treatment with
iodomethane (MeI), gave 8 in 97% yield. Olefin 8 was then hydro-
genated using hydrogen gas with Pd/C to give 9 in 88% yield.
Removal of the methyl protecting groups of 9 using BBr3 afforded
the desired MPBD 1 in 93% yield. Thus, the total synthesis of 1
was accomplished in eight steps, in 56% overall yield.
4-Methyl-5-pentylbenzene-1,3-diol (MPBD 1, Figure 1), which
is produced by SteelyA polyketide synthase,15 is also a signal mole-
cule that regulates cell aggregation in early stage development16
and induces spore cell formation in the later stages.17 However,
the only known biological activities of MPBD 1 are chemotaxis reg-
ulation, spore maturation activity, and anti-tumor activity.18 While
As MPBD derivatives for SAR study, 4-ethyl-5-pentylbenzene-
1,3-diol (EPBD) and 4-n-propyl-5-pentylbenzene-1,3-diol (PPBD)
⇑
Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361.
E-mail address: t-usuki@sophia.ac.jp (T. Usuki).
http://dx.doi.org/10.1016/j.bmcl.2016.01.067
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

C. Murata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1428–1433
1429
Figure 1. Structure of MPBD 1. The carbon numbering of 1 was adapted from Ref.
17.
were designed and synthesized from compound 2 (Scheme 3). The
lithiation–alkylation strategy using iodoethane or 1-iodopropane
toward intermediate 7 did not afford the target products (data
not shown), probably due to the steric repulsion resulting from
the aryl anion in the SN2 reaction. Ethyl and n-propyl substitutes
were hence introduced by Negishi cross-coupling reactions of
iodoethane and 1-iodopropane with zinc, respectively.21 The reac-
tions were carried out using 20 mol % pyridine-enhanced precata-
lyst preparation stabilization and initiation (PEPPSI)-IPr to give 10
and 11 in 77% and 69% yields, respectively. Wittig reaction of 10
and 11 with nBuPPh3Br afforded olefins 12 (E/Z = 1/1.4) and 13
(E/Z = 1/1.3) in 73% yield. Interestingly, the E/Z ratios of 12 and
13 were the inverse of that observed in the MPBD synthesis, prob-
ably due to the change of steric environment. Thus, the desired
products EPBD 14 and PPBD 15 were obtained via hydrogenation
and deprotection in 84% and 82% yields, respectively, in two steps.
We also designed and synthesized a compound without a
hydroxyl group at C3 for the SAR study (Scheme 4). Iodination of
Scheme 3. Synthesis of EPBD 14 and PPBD 15. Reagents and conditions: (a) Zn,
TMSCl, iodoethane or 1-iodopropane, then, PEPPSI-IPr, DMF, rt, 2 h, 77% (10), 69%
(11); (b) nBuLi, nBuPPh3Br, THF, rt, 2 h, 73% (12), 73% (13); (c) H2, Pd/C, THF, rt, 12 h;
(d) BBr3, CH2Cl2, ꢀ78 °C to rt, 21 h, 84%; (14), 82% (15) (two steps).
the starting material 4-amino-1-hydroxybensoic acid 16 was car-
ried out by the Sandmeyer reaction to yield 17 in quantitatively.22
Di-methylation of 17 with MeI gave 18 in 97% yield.23 Negishi
cross-coupling reaction of 18 with iodomethane and zinc was then
carried out using PEPPSI-IPr to give 19 in 99% yield. After reduction
of 19 using LiAlH4, followed by Swern oxidation, Witting reaction
of aldehyde 21 with nBuPPh3Br was carried out to obtain olefin
22 (E/Z = 1/1.6) in 78% yield. Reduction of the olefin and removal
Scheme 1. Retrosynthetic analysis of MPBD (1).
Scheme 2. Total synthesis of MPBD 1. Reagents and conditions: (a) (MeO)2SO2, K2CO3, acetone, reflux, 4 h, 92%; (b) LiAlH4, THF, 0 °C to rt, 2 h, 98%; (c) NIS, DMF, 40 °C, 4 h,
94%; (d) DMSO, (COCl)2, Et3N, CH2Cl2, ꢀ78 °C to rt, 3 h, 96%; (e) nBuLi, nBuPPh3Br, THF, 0 °C to rt, 30 min, 87%; (f) nBuLi, MeI, Et2O, ꢀ78 °C to rt, 12 h, 97%; (g) H2, Pd/C, EtOAc,
rt, 24 h, 88%; (h) BBr3, CH2Cl2, ꢀ78 °C to rt, 9 h, 93%.

1430
C. Murata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1428–1433
Scheme 4. Synthesis of MPP 23. Reagents and conditions: (a) (i) NaNO2, HCl, H2O, 0 °C, 30 min, (ii) KI, H2O, 0 °C to 90 °C, 50 min, quant; (b) MeI, K2CO2, acetone, DMF, rt, 4 h,
97%; (c) Zn, TMSCl, MeI, then, PEPPSI-IPr, DMF, rt, 2 h, 99%; (d) LiAlH4, THF, 0 °C to rt, 1 h, 99%; (e) DMSO, (COCl)2, Et3N, CH2Cl2, ꢀ78 °C to rt, 2 h, 89%; (f) nBuLi, nBuPPh3Br,
THF, 0 °C to rt, 30 min, 78%; (g) H2, Pd/C, EtOAc, rt, 24 h; (h) BBr3, CH2Cl2, ꢀ78 °C to rt, 7 h, 76% (two steps).
MPP 23) were also synthesized24 and subjected to SAR study. The
Table 1
Antimicrobial assay
antimicrobial activities of these compounds against E. coli and B.
subtilis were evaluated by disk diffusion assay.25 These obtained
results would provide some insight for the bioorganic studies of
compounds produced by D. discoideum.26
a
Compounds
E. colib (mm)
B. subtilisb (mm)
MPBD (1)
5
—
4–6
4–5
1–2
7–10
1–4
—
1–2
4–6
1
DiMe-MPBD (9)
EPBD (14)
PPBD (15)
MPP (23)
Acknowledgments
Ampicillin
7–12
This work was supported by a Sophia University Collaborative
Research Grant (to T.S. and T.U.).
a
Concentrations of 1, 9, 14, 15, 23: 100 mM; concentration of ampicillin: 50 mM.
Sizes (radii) of inhibition ring.
b
Supplementary data
of the methyl group afforded the desired 4-methyl-5-pentylphenol
(MPP, 23) in 76% yield in two steps.
Supplementary data (NMR spectra and disk diffusion assay)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.01.067.
The disk diffusion assays for Escherichia coli and Bacillus subtilis
were performed to evaluate their antimicrobial activity for the five
synthesized compounds (1, 9, 14, 15, and 23), which were purified
by reversed-phase high performance liquid chromatography (RP-
HPLC), as well as ampicillin (Table 1). MPBD 1, EPBD 14, and PPBD
15 showed antimicrobial activity against E. coli; compounds 1 and
15 showed antimicrobial activity against B. subtilis, with PPBD 15
showing potent B. subtilis activity. However, diMe-MPBD 9 and
MPP 23 did not show antimicrobial activity. This is attributed to
the methyl protecting groups at C1 and C3 on diMe-MPBD 9 and
the lack of a C3-hydroxyl group on MPP 23, respectively, did not
showed antimicrobial activities.
Based on the results of the SAR study, the following conclusions
could be drawn: (1) free hydroxyl groups at C1 and C3 are neces-
sary for antimicrobial activity; (2) existence of a hydroxyl group
at C3 is crucial for antimicrobial activity; (3) the length of the car-
bon chain at C4 of MPBD 1 does not affect the activities against
E. coli; (4) the n-propyl group at C4 is important for activity against
B. subtilis.
References and notes
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C. Murata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1428–1433
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washed with saturated Na2S2O3 solution, brine, dried over Na2SO4, and
concentrated in vacuo. Purification by silica gel column chromatography
(hexane/EtOAc = 5:1) afforded 6 as a colorless solid (1.65 g, 5.62 mmol, 94%); Rf
0.66 (hexane/EtOAc = 1:1); mp 97–98 °C; 1H NMR (300 MHz, CDCl3) d 6.73 (1H,
d, J = 2.7 Hz, H6), 6.39 (1H, t, J = 2.7 Hz, H2), 4.69 (2H, s, H10), 3.87 (3H, s, OMe),
3.83 (3H, s, OMe); 13C NMR (75 MHz, CDCl3) d 161.5, 158.7, 144.9, 105.3, 98.2,
69.7, 56.6, 55.7; EI-MS (m/z) calcd for C9H11IO4 [M]+ 293.98, found 294.05.
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36.0 mmol) in CH2Cl2 (10 mL) cool to ꢀ78 °C was dropwise added oxalyl
chloride (0.618 mL, 7.21 mmol). The resulting mixture was stirred at ꢀ78 °C for
20 min. To the mixture was cannulated a solution of (4-iodo-1,3-
dimethoxyphenyl)methanol (6, 1.06 g, 3.60 mmol) in CH2Cl2 (7.5 mL). After
stirring at ꢀ78 °C for 1 h, Et3N (2.52 mL, 18.0 mmol) was added. The reaction
mixture was stirred at ꢀ78 °C for 1 h and warmed to room temperature and
stirred for 1 h. The reaction was then quenched with 1 N HCl and extracted
with CH2Cl2. The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/EtOAc = 5:1) afforded 3 as a yellow solid (1.01 g,
3.48 mmol, 96%); Rf 0.60 (hexane/EtOAc = 3:1); mp 100–102 °C; 1H NMR
(300 MHz, CDCl3) d 10.19 (1H, s, H10), 7.08 (1H, d, J = 2.8 Hz, H6), 6.67 (1H, d,
J = 2.7 Hz, H2), 3.91 (3H, s, OMe), 3.86 (3H, s, OMe); 13C NMR (75 MHz, CDCl3) d
196.4, 161.4, 159.1, 136.6, 105.0, 104.7, 84.3, 56.9, 55.9; EI-MS (m/z) calcd for
C9H9IO3 [M]+ 291.96, found 291.85.
18. Kikuchi, H.; Oshima, Y.; Ichimura, A.; Gokan, N.; Hasegawa, A.; Hosaka, K.;
Kubohara, Y. Life Sci. 2006, 80, 160.
4-Iodo-1,3-dimethoxy-5-pent-1-enylbenzene (7). To
a
suspension of n-
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butylphosphonium bromide (281 mg, 0.703 mmol) in THF (2 mL) was
dropwise added nBuLi (2.64 M in hexane, 0.244 mL, 0.645 mmol). The
suspension was stirred at 0 °C for 15 min. To the orange suspension at 0 °C
was then added a solution of 4-iodo-1,3-dimethoxybenzaldehyde (2, 171 mg,
0.586 mmol) in THF (1.5 mL). After stirring at room temperature for 30 min,
the reaction mixture was quenched with saturated NH4Cl solution, and
extracted with EtOAc. The combined organic layers were then washed with
brine, dried over Na2SO4, and concentrated in vacuo. The crude product was
filtered through a short silica gel column (hexane/EtOAc = 3:1) to remove
phosphine oxide. Purification by silica gel column chromatography (hexane/
24. General procedures. All non-aqueous reactions were conducted under an
atmosphere of nitrogen with magnetic stirring using freshly distilled
solvents unless otherwise indicated. Dichloromethane (CH2Cl2), diethyl ether
(Et2O), N,N-dimethylformamide (DMF) and tetrahydrofuran (THF) were
distilled and stored over activated molecular sieves. All reagents were
obtained from commercial suppliers and used without further purification
unless otherwise stated. Analytical thin layer chromatography (TLC) was
performed on Silica gel 60 F254 plates produced by Merck KGaA (Darmstadt,
Germany). HPLC grade methanol (MeOH), HPLC grade distilled water (H2O)
was purchased from Kanto Chemicals, Co. Ltd (Tokyo, Japan). Column
chromatography was performed with acidic Silica gel 60 (spherical, 40–
EtOAc = 10:1) afforded
7 as a colorless oil (169 mg, 0.509 mmol, 87%, E/
Z = 1.3:1); Rf 0.63 (hexane/EtOAc = 3:1); IR (neat, cmꢀ1) 2957, 1579, 1459,
1414, 1325, 1205, 1159, 1080, 1009, 963, 831, 733; 1H NMR (300 MHz, CDCl3) d
6.72–6.30 (3H, m, H2/6/10), 6.18–6.11 (1H, m, H20-E), 5.78–5.64 (1H, m, H20-Z),
3.90–3.75 (6H, m, OMe), 2.30–2.02 (2H, m, H30), 1.62–1.36 (2H, m, H40), 1.04–
0.82 (3H, m, H50); 13C NMR (75 MHz, CDCl3) d 161.00, 160.56, 158.9, 143.4,
143.1, 134.42, 134.38, 133.48, 133.44, 107.1, 103.4, 97.6, 97.2, 56.62, 56.56,
55.6, 35.1, 30.6, 22.9, 22.5, 14.0, 13.9; EI-HRMS (m/z) calcd for C13H17IO2 [M]+
332.0273, found 332.0270.
1,3-Dimethoxy-4-methyl-5-pent-1-enylbenzene (8). To a solution of 4-iodo-1,3-
dimethoxy-5-pent-1-enylbenzene (7, 81.0 mg, 0.2438 mmol) in Et2O (1.5 mL)
50 l
m) produced by Kanto Chemical, Co., Ltd (Tokyo, Japan). 1H and 13C NMR
spectra were recorded on a JEOL JNM-EXC 300 spectrometer or on a JEOL-ECA
500 spectrometer. 1H NMR data are reported as follows: chemical shift (d,
ppm), integration, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet), coupling constants (J) in Hz, assignments. 13C NMR data are
reported in terms of chemical shift (d, ppm). EI-MS spectra are reported on a
Shimadzu GCMS QP-5050 instrument or JEOL JMS-700 instrument. FAB-MS
spectra were also observed on the JEOL JMS-700. HPLC purifications were
performed with SHIMADZU LC-6AD, degasser unit DGU-20A3R, refractive index
detector RID-10A, UV–Vis SPD-20A, valve unit FCV-12AH, and fraction
collector FRC-10A.
Methyl 1,3-dimethoxybenzoate (4). To a suspension of 1,3-dihydroxybenzoic
acid (3, 3.00 g, 19.5 mmol) and K2CO3 (10.8 g, 77.9 mmol) in acetone (30 mL)
was added (MeO)2SO2 (6.46 mL, 68.1 mmol). After stirring at reflux for 4 h, the
reaction mixture was cooled to room temperature and filtered. After
concentration to remove acetone, EtOAc and saturated NaHCO3 solution
were added and stirred for 5 min, and then extracted with EtOAc. The
combined organic layers were washed with 5% HCl, saturated NaHCO3
solution, brine, dried over Na2SO4, and concentrated in vacuo. Purification by
cooled to ꢀ78 °C was added nBuLi (2.64 M in hexane, 185
After stirring at ꢀ78 °C for 1 h, to the solution at ꢀ78 °C was added MeI
(46.0 L, 0.731 mmol). The solution was then allowed to warm up to room
lL, 0.488 mmol).
l
temperature. After stirring for 12 h, water was added and extracted with
EtOAc. The combined organic layers were dried over Na2SO4 and concentrated
in vacuo. Purification by silica gel column chromatography (hexane/
EtOAc = 10:1) afforded 8 as a colorless oil (51.9 mg, 0.236 mmol, 92%); Rf
0.71 (hexane/EtOAc = 3:1); IR (neat, cmꢀ1) 2953, 1595, 1461, 1320, 1201, 1150,
1104, 1056, 964, 833; 1H NMR (300 MHz, CDCl3) d 6.65–6.20 (3H, m, H2/6/10),
6.15–6.00 (1H, m, H20-E), 5.78–5.60 (1H, m, H20-Z), 3.85–3.70 (6H, m, OMe),
2.30–2.00 (5H, m, H7/30), 1.60–1.27 (2H, m, H40), 1.02–0.80 (3H, m, H50); 13C
NMR (125 MHz, CDCl3) d 158.6, 158.3, 138.6, 133.0, 132.8, 128.6, 128.3, 101.9,
97.3, 96.9, 55.7, 55.6, 55.42, 55.40, 35.5, 30.7, 23.0, 22.7, 13.94, 13.85, 11.0; EI-
HRMS (m/z) calcd for C10H12O4 [M]+ 220.1463, found 220.1455.
1,3-Dimethoxy-4-methyl-5-pentylbenzene (9). A mixture of 1,3-dimethoxy-4-
methyl-5-pent-1-enylbenzene (8, 48.5 mg, 0.220 mmol) and 10% Pd/C (5.0 mg)
in EtOAc (1 mL) was stirred under an atmosphere of H2 at room temperature
for 24 h. The reaction mixture was then filtered through a pad of Celite and
wash with EtOAc. Concentration and purification by silica gel column
chromatography (hexane/EtOAc = 20:1) afforded 9 as a colorless oil (43.3 mg,
0.195 mmol, 88%); Rf 0.57 (hexane/EtOAc = 10:1); IR (neat, cmꢀ1) 2932, 1601,
1462, 1314, 1200, 1151, 1060, 831; 1H NMR (300 MHz, CDCl3) d 6.37–6.28 (2H,
m, H2/6), 3.84–3.76 (6H, m, OMe), 2.58–2.55 (2H, t, J = 7.5 Hz, H1), 2.09 (3H, s,
H7), 1.64–1.48 (2H, m, H20), 1.44–1.20 (4H, m, H30/40), 1.00–0.80 (3H, m, H50);
13C NMR (75 MHz, CDCl3) d 158.6, 158.2, 143.1, 106.6, 105.6, 95.9, 55.6, 55.4,
silica gel column chromatography (hexane/EtOAc = 10:1) afforded
4 as a
colorless solid (3.50 g, 17.8 mmol, 92%); Rf 0.31 (hexane/EtOAc = 10:1); mp 42–
44 °C; 1H NMR (300 MHz, CDCl3) d 7.19 (2H, d, J = 2.4 Hz, H4/6), 6.65 (1H, t,
J = 2.4 Hz, H2), 3.83 (6H, s, OMe); 13C NMR (75 MHz, CDCl3) d 167.0, 160.7,
132.1, 107.2, 105.9, 55.7, 52.4; EI-MS (m/z) calcd for C10H12O4 [M]+ 196.07,
found 196.15.
(1,3-Dimethoxy-phenyl)-methanol (5). To
a suspention of LiAlH4 (867 mg,
23.2 mmol) in THF (15 mL) cooled to 0 °C was added methyl 1,3-
dimethoxybenzoate (4, 3.04 g, 15.5 mmol) in THF (17 mL). After stirring at
room temperature for 2 h, the reaction mixture was cooled to 0 °C and water
was carefully added. After 1 N HCl was added and stirred, the mixture was
filtered through a pad of Celite. The filtrate was then extracted with EtOAc. The
combined organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. Purification by silica gel column chromatography
(hexane/EtOAc = 3:1) afforded 5 as a colorless solid (2.54 g, 15.1 mmol, 98%); Rf
0.60 (hexane/EtOAc = 1:1); mp 48–50 °C; 1H NMR (300 MHz, CDCl3) d 6.53 (2H,
d, J = 2.1 Hz, H4/6), 6.39 (1H, t, J = 2.2 Hz, H2), 4.64 (2H, s, H10), 3.80 (6H, s,
OMe); 13C NMR (75 MHz, CDCl3) d 161.1, 143.5, 104.7, 99.7, 65.4, 55.4; EI-MS
(m/z) calcd for C9H12O3[M]+ 168.08, found 168.15.
34.2, 32.0, 30.4, 22.7, 14.2, 10.8; EI-HRMS (m/z) calcd for C
10H12O4 [M]+
222.1620, found 222.1614.
4-Methyl-5-pentylbenzene-1,3-diol (MPBD, 1). To a solution of 1,3-dimethoxy-4-
methyl-5-pentylbenzene (9, 40.2 mg, 18.1 mmol) in CH2Cl2 (1.5 mL) cooled to
ꢀ78 °C was added BBr3 (1.0 M in CH2Cl2, 0.904 mL, 0.904 mmol). The reaction
temperature was gradually raised to room temperature. After stirring for 9 h,
the reaction mixture was quenched with saturated NaHCO3 solution and then
extracted with EtOAc. The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. Purification by silica gel column chromatography
(hexane/EtOAc = 10:1) afforded 1 as a yellow oil (3.50 mg, 17.84 mmol, 92%);
Rf 0.31 (hexane/EtOAc = 10:1); IR (neat, cmꢀ1) 3701, 3357, 2929, 2864, 2359,
1707, 1605, 1466, 1278, 1142, 1058, 1000, 840, 727; 1H NMR (300 MHz, CDCl3)
d 6.25 (1H, t, J = 2.8 Hz, H6), 6.22–6.10 (1H, m, H2), 2.52 (2H, t, J = 7.9 Hz, H10),
2.09 (3H, s, H7), 1.68–1.46 (2H, m, H20), 1.42–1.20 (4H, m, H30/40), 1.00–0.80
(3H, m, H50); 13C NMR (75 MHz, CDCl3) d 154.5, 153.4, 144.2, 114.6, 108.8,
100.6, 33.9, 31.9, 30.1, 22.7, 14.2, 10.7; EI-HRMS (m/z) calcd for C10H12O4 [M]+
(4-Iodo-1,3-dimethoxy-phenyl)methanol (6). To a solution of (1,3-dimethoxy-
phenyl)-methanol (5, 1.00 g, 5.95 mmol) in DMF (12 mL) cooled to 0 °C was
added NIS (1.61 g, 7.14 mmol). After stirring at 40 °C for 3 h, Et2O and water
were added, and then extracted with EtOAc. The combined organic layers were

1432
C. Murata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1428–1433
194.1307, found 194.1305.
4-Ethyl-1,3-dimethoxy-benzaldehyde (10). Zinc dust (134 mg, 2.05 mmol) was
placed in a nitrogen-purged 1.5 ml microtube. DMF (150 L) and trimethylsilyl
chloride (TMSCl, 60 L, 0.47 mmol) were added, and the resulting mixture was
stirred vigorously for 15 min at room temperature. After stirring, and the
solution was removed by micro syringe. The remaining solid was dried using a
hot air gun at reduced pressure. The activated zinc was cooled to room
(6H, m, OMe), 2.70–2.42 (2H, m, H7), 2.30–2.00 (2H, m, H30), 1.60–1.35 (2H, m,
H40), 1.05–0.80 (6H, m, H8/50); 13C NMR (75 MHz, CDCl3) d 158.7, 158.5, 158.4,
157.8, 138.4, 133.1, 132.8, 128.5, 128.1, 122.3, 121.6, 105.8, 101.8, 97.4, 97.1,
55.7, 55.5, 55.4, 35.5, 30.7, 28.3, 27.4, 23.5, 23.1, 23.0, 22.7, 14.44, 14.40, 14.0,
13.8; EI-HRMS (m/z) calcd for C16H24O2 [M]+ 248.1776, found 248.1757.
l
l
4-Ethyl-5-pentylbenzene-1,3-diol (EPBD, 14).
A
mixture of 4-ethyl-1,3-
mol) and 10% Pd/C
dimethoxy-5-pent-1-enyl-benzene (12, 17.2 mg, 73.4
l
temperature, and DMF (200
l
L) and iodoethane (27
l
L, 0.342 mmol, 2.0 equiv)
(1.7 mg) in THF (0.5 mL) was stirred under an atmosphere of H2 at room
temperature for 12 h. The reaction mixture was then filtered through a pad of
Celite and wash with EtOAc. Concentration in vacuo gave a colorless oil
(19.5 mg).
were added to the activated zinc. The reaction mixture was stirred at room
temperature for 1 h, after which time stirring was stopped and the zinc duct
was allowed to settle using a centrifuge separator. The solution was removed
from the activated zinc via micro syringe with 200
l
L DMF and added to a
The crude oil was then diluted with CH2Cl2 (1 mL). To the solution cooled to
ꢀ78 °C was added BBr3 (1.0 M in CH2Cl2, 0.413 mL, 0.413 mmol). The reaction
temperature was gradually raised to room temperature. After stirring for 21 h,
the reaction mixture was quenched with saturated NaHCO3 solution and then
extracted with EtOAc. The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. Purification by silica gel column chromatography
flask, containing PEPPSI-IPr (23.3 mg, 34.2 mol, 20 mol %) and the 4-iodo-1,3-
l
dimethoxybenzaldehyde (2, 50.0 mg, 0.171 mmol, 1.0 equiv). Stirring was
continued at room temperature for 2 h, the reaction mixture was quenched
with a saturated NH4Cl solution. The aqueous layer was then extracted with
EtOAc. The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo to give the crude product. Purification by
silica gel column chromatography (hexane/EtOAc = 10:1) afforded 10 as a
colorless oil (25.5 mg, 0.131 mmol, 77%); Rf 0.66 (hexane/EtOAc = 10:1); IR
(neat, cmꢀ1) 2967, 2363, 1689, 1604, 1463, 1400, 1348, 1297, 1197, 1150,
1051, 928, 878, 629; 1H NMR (300 MHz, CDCl3) d 10.34 (1H, s, H10), 6.97 (1H, d,
J = 2.7 Hz, H6), 6.67 (1H, d, J = 2.4 Hz, H2), 3.84 (6H, s, OMe), 3.00 (2H, q,
J = 7.6 Hz, H7), 1.17 (3H, t, J = 7.6 Hz, H8); 13C NMR (75 MHz, CDCl3) d 191.7,
158.9, 158.8, 134.6, 130.1, 104.8, 102.6, 55.9, 55.6, 17.1, 16.2; EI-HRMS (m/z)
calcd for C11H14O3 [M]+ 194.0943, found 194.0945.
(hexane/EtOAc = 3:1) afforded 14 as a colorless oil (12.8 mg, 61.5 lmol, 84%);
Rf 0.20 (hexane/EtOAc = 3:1); IR (neat, cmꢀ1); 1H NMR (300 MHz, CDCl3) d 6.25
(1H, d, J = 2.4 Hz, H6), 6.17 (1H, d, J = 2.4 Hz, H2), 2.66–2.42 (4H, m, H7/10),
1.66–1.46 (2H, m, H20), 1.43–1.28 (2H, m, H30), 1.12 (3H, t, J = 7.6 Hz, H8), 0.95–
0.84 (3H, m, H50); 13C NMR (75 MHz, CDCl3) d 154.5, 153.8, 143.5, 120.8, 108.8,
100.7, 33.0, 32.0, 31.2, 22.7, 18.7, 14.7, 14.2; EI-HRMS (m/z) calcd for C13H20O2
[M]+ 208.1463, found 208.1443.
5-Pentyl-4-propylbenzene-1,3-diol (PPBD, 15). A mixture of 1,3-dimethoxy-5-
pent-1-enyl-4-propyl-benzene (13, 18.6 mg, 74.9 lmol) and 10% Pd/C (1.9 mg)
1,3-Dimethoxy-4-propylbenzaldehyde (11). Zinc dust (134 mg, 2.05 mmol) was
placed in a nitrogen-purged 1.5 ml microtube. DMF (150 lL) and TMSCl (60 lL,
0.47 mmol) were added, and the resulting mixture was stirred vigorously for
15 min at room temperature. After stirring, and the solution was removed by
micro syringe. The remaining solid was dried using a hot air gun at reduced
pressure. The activated zinc was cooled to room temperature, and DMF
in THF (0.5 mL) was then stirred under an atmosphere of H2 at room
temperature for 12 h. The reaction mixture was filtered through a pad of
Celite and wash with EtOAc. Concentration in vacuo gave colorless oil
(19.8 mg).
The crude oil was then diluted with CH2Cl2 (1 mL). To the solution cooled to
ꢀ78 °C was added BBr3 (1.0 M in CH2Cl2, 0.413 mL, 0.413 mmol). The reaction
temperature was gradually raised to room temperature. After stirring for 21 h,
the reaction mixture was quenched with saturated NaHCO3 solution and then
extracted with EtOAc. The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. Purification by silica gel column chromatography
(200 lL) and 1-iodopropane (33 lL, 0.342 mmol, 2.0 equiv) were added to the
activated zinc. The reaction mixture was stirred at room temperature for 1 h,
after which time stirring was stopped and the zinc duct was allowed to settle
using a centrifuge separator. The solution was removed from the activated zinc
via micro syringe with 200
l
L DMF and added to a flask, containing PEPPSI-IPr
(hexane/EtOAc = 3:1) afforded 15 as a colorless oil (13.6 mg, 61.2 lmol, 82%);
(23.3 mg, 34.2 mol, 20 mol %) and the 4-iodo-1,3-dimethoxybenzaldehyde (2,
l
Rf 0.20 (hexane/EtOAc = 3:1); IR (neat, cmꢀ1); 1H NMR (300 MHz, CDCl3) d 6.25
(1H, d, J = 2.8 Hz, H6), 6.17 (1H, d, J = 2.4 Hz, H2), 2.60–2.40 (4H, m, H7/10),
1.62–1.42 (4H, m, H8/20), 1.42–1.30 (2H, m, H30), 0.99 (3H, t, J = 7.4 Hz, H9),
0.91 (3H, t, J = 7.2 Hz, H50); 13C NMR (75 MHz, CDCl3) d 154.7, 153.9, 143.8,
119.4, 108.7, 100.6, 33.1, 32.1, 31.1, 27.7, 23.5, 22.7, 14.5, 14.2; EI-HRMS (m/z)
calcd for C14H22O2 [M]+ 222.1620, found 222.1608.
50.0 mg, 0.171 mmol, 1.0 equiv). Stirring was continued at room temperature
for 2 h, the reaction mixture was quenched with a saturated NH4Cl solution.
The aqueous layer was then extracted with EtOAc. The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated in vacuo
to give the crude product. Purification by silica gel column chromatography
(hexane/EtOAc = 10:1) afforded 11 as a colorless oil (24.5 mg, 0.118 mmol,
69%); Rf 0.66 (hexane/EtOAc = 3:1); IR (neat, cmꢀ1) 2960, 2363, 1690, 1604,
1465, 1400, 1295, 1206, 1151, 1055, 937, 849; 1H NMR (300 MHz, CDCl3) d
10.34 (1H, s, H10), 6.98 (1H, d, J = 2.4 Hz, H6), 6.67 (1H, d, J = 2.4 Hz, H2), 3.84
(3H, s, OMe), 3.83 (3H, s, OMe), 2.95 (2H, t, J = 7.6 Hz, H7), 1.65–1.45 (2H, m,
H8), 0.96 (3H, t, J = 7.2 Hz, H9); 13C NMR (75 MHz, CDCl3) d 191.7, 159.1, 158.8,
135.0, 128.6, 104.8, 102.3, 55.9, 55.6, 25.4, 25.1, 14.1; EI-HRMS (m/z) calcd for
1-Hydroxy-4-iodobenzoic acid (17). A solution of NaNO2 (1.1 g, 15.67 mmol) in
water (28 mL) was added slowly to a cold (0 °C) stirred solution of 4-amino-1-
hydroxybenzoic acid (16) (2.0 g, 13.06 mmol), concentrated HCl (10 mL). After
addition was completed, the solution was stirred for additional 30 min at 0 °C.
A solution of KI (3.25 g, 19.59 mmol) in water (5 mL) was added slowly. The
resulting mixture was stirred for 20 min at 0 °C and then heated 90 °C for
30 min. The miture was cooled to room temperature, extracted with EtOAc and
washed with Na2S2O3. The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. A brown solid was given and used without further
purification (3.48 g, 13.18 mmol, quant); Rf 0.60 (MeOH/CH2Cl2 = 2:1); 1H NMR
(300 MHz, CD3OD) d 7.74 (1H, d, J = 8.6 Hz, H3), 7.23 (1H, d, J = 8.9, H6), 6.68
(1H, dd, J = 9.0, 3.3 Hz, H2); 13C NMR (75 MHz, CD3OD) d 170.3, 158.9, 143.1,
138.4, 121.4, 118.9, 80.9, 80.8; FAB-HRMS (m/z) calcd for C7H5IO3 [M]+
262.9180, found 263.9283.
C
12H16O3 [M]+ 208.1099, found 208.1096.
4-Ethyl-1,3-dimethoxy-5-pent-1-enylbenzene (12). To
butylphosphonium bromide (57.0 mg, 0.143 mmol) in THF (0.8 mL) was
added dropwise nBuLi (2.64 M in hexane, 50.0 L, 0.131 mmol). The
suspension was then stirred at 0 °C for 15 min. To the orange suspension at
0 °C was added solution of 4-ethyl-1,3-dimethoxy-benzaldehyde (10,
a
suspension of n-
l
a
23.1 mg, 0.119 mmol) in THF (0.7 mL). After stirring at room temperature for
30 min, the reaction mixture was quenched with saturated NH4Cl solution, and
extracted with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The crude product was filtered
through a short silica gel column (hexane/EtOAc = 3:1) to remove phosphine
oxide. Purification by silica gel column chromatography (hexane/EtOAc = 10:1)
Methyl 4-iodo-1-methoxybenzoate (18). 1-Hydroxy-4-iodo-benzoic acid (17,
29.7 mg, 0.113 mmol) was dissolved in acetone (67
lL) and DMF (0.68 mL).
K2CO3 (119 mg, 0.859 mmol) and MeI (54 L, 0.859 mmol) were then added
l
and the mixture was stirred at room temperature for 4 h. The mixture was then
concentrated under reduced pressure to remove the excessive MeI. The residue
was extracted with CH3Cl and water. The combined organic layers were dried
over Na2SO4, and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/EtOAc = 15:1) afforded 18 as a yellow oil (32.5 mg,
0.113 mmol, 97%); Rf 0.70 (hexane/EtOAc = 2:1) 1H NMR (300 MHz, CDCl3) d
7.83 (1H, d, J = 7.8 Hz, H3), 7.34 (1H, d, J = 2.9 Hz, H6), 6.75 (1H, dd, J = 8.8,
3.1 Hz, H2), 3.93 (3H, s, OMe), 3.82 (3H, s, CO2Me), 2.52 (3H, s, H7); 13C NMR
(75 MHz, CDCl3) d 166.6, 159.4, 141.8, 135.7, 119.3, 116.4, 82.4, 55.5, 52.5;
FAB-HRMS (m/z) calcd for C9H9IO3 [M]+ 291.9584, found 291.9596.
afforded 12 as a colorless oil (20.4 mg, 87.1 lmol, 73%, E/Z = 1:1.4); Rf 0.57
(hexane/EtOAc = 10:1); IR (neat, cmꢀ1) 2960, 2363, 1836, 1596, 1462, 1303,
1200, 1148, 1052, 965, 835; 1H NMR (300 MHz, CDCl3) d 6.65–6.30 (3H, m, H2/
6/10), 6.15–6.00 (1H, m, H20-E), 5.75–5.60 (1H, m, H20-Z), 3.88–3.65 (6H, m,
OMe), 2.75–2.45 (2H, m, H7), 2.55–2.00 (2H, m, H30), 1.60–1.30 (2H, m, H40),
1.10–0.75 (6H, m, H50); 13C NMR (75 MHz, CDCl3) d 158.43, 158.39, 158.34,
157.8, 138.1, 133.2, 133.0, 128.2, 127.9, 123.7, 123.0, 105.8, 101.9, 97.5, 97.1,
55.7, 55.6, 55.4, 35.5, 30.7, 23.1, 22.8, 19.5, 18.6, 14.7, 14.2, 14.0, 13.9; EI-HRMS
(m/z) calcd for C15H22O2 [M]+ 234.1620, found 234.1611.
Methyl 1-methoxy-4-methylbenzoate (19). Zinc dust (389.5 mg, 5.96 mmol) was
1,3-Dimethoxy-5-pent-1-enyl-4-propylbenzene (13). To
butylphosphonium bromide (50.4 mg, 0.126 mmol) in THF (0.5 mL) was
added dropwise nBuLi (2.64 M in hexane, 44.0 L, 0.116 mmol). The
suspension was then stirred at 0 °C for 15 min. To the orange suspension at
0 °C was added solution of 1,3-dimethoxy-4-propyl-benzaldehyde (11,
21.9 mg, 0.116 mmol) in THF (0.5 mL). After stirring at room temperature for
30 min, the reaction mixture was quenched with saturated NH4Cl solution, and
extracted with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The crude product was filtered
through a short silica gel column (hexane/EtOAc = 3:1) to remove phosphine
oxide. Purification by silica gel column chromatography (hexane/EtOAc = 10:1)
a
suspension of n-
divided in two nitrogen-purged 1.5 ml microtube. DMF (225
lL) and TMSCl
(85 L, 0.69 mmol) were added respectively, and the resulting mixtures were
l
l
stirred vigorously for 15 min at room temperature. After stirring, and the
solutions were removed by micro syringe. The remaining solid was dried using
a hot air gun at reduced pressure. The activated zinc was cooled to room
a
temperature, and DMF (275 lL) and MeI (35 lL, 0.50 mmol, 2.0 equiv) were
added to the activated zinc respectively. The reaction mixture was stirred at
room temperature for 1 h, after which time stirring was stopped and the zinc
duct was allowed to settle using a centrifuge separator. The solution was
removed from the activated zinc via micro syringe with 270
lL DMF and added
to a flask, containing PEPPSI-IPr (67.5 mg, 99.3 mol, 20 mol %) and the methyl
l
afforded 13 as a colorless oil (19.1 mg, 76.9
lmol, 73%, E/Z = 1:1.3); Rf 0.57
4-iodo-1-methoxy-benzoate (18, 150.0 mg, 0.514 mmol, 1.0 equiv). Stirring
was continued at room temperature for 2 h, the reaction mixture was
(hexane/EtOAc = 10:1); IR (neat, cmꢀ1) 3734, 2957, 2363, 1836, 1595, 1463,
1311, 1202, 1147, 1056, 960, 833, 735; 1H NMR (300 MHz, CDCl3) d 6.70–6.25
(3H, m, H2/6/10), 6.22–5.86 (1H, m, H20-E), 5.84–5.50 (1H, m, H20-Z), 4.00–3.60
quenched with
a saturated NH4Cl solution. The aqueous layer was then
extracted with EtOAc/hexane (1:1). The combined organic layers were washed

C. Murata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1428–1433
1433
with brine, dried over Na2SO4, and concentrated in vacuo to give the crude
product. Purification by silica gel column chromatography (hexane/
EtOAc = 30:1) afforded 19 as a colorless oil (91.9 mg, 0.51 mmol, 99%); Rf
0.53 (hexane/EtOAc = 5:1); 1H NMR (300 MHz, CDCl3) d 7.44 (1H, d, J = 2.6 Hz,
H6), 7.14 (1H, d, J = 8.3 Hz, H3), 6.96 (1H, dd, J = 8.8, 2.6 Hz, H2), 3.89 (3H, s,
OMe), 3.77 (3H, s, CO2Me), 2.52 (3H, s, H7); EI-MS (m/z) calcd for C10H12O3 [M]+
180.08, found 180.05.
(300 MHz, CDCl3) d 7.09–6.67 (3H, m, H2/3/6), 6.53 (1H, d, J = 15.0 Hz, H10-
E), 6.40 (1H, d, J = 12.6 Hz, H10-Z), 6.13–6.04 (1H, m, H20-E), 5.75–5.66 (1H, m,
H20-Z), 3.79 (3H, s, OMe), 2.26 (3H, s, H7), 2.25–2.11 (3H, m, H30), 1.54–11.39
(2H, m, H40), 0.98–0.87 (3H, m, H50); 13C NMR (75 MHz, CDCl3) d 156.9, 137.6,
132.6, 132.2, 130.7, 130.2, 127.7, 114.4, 112.0, 111.6,110.4, 55.0, 54.98, 30.15,
22.7, 22.3, 18.63, 18.61, 13.5, 13.5, 13.4; EI-MS (m/z) calcd for C13H18O [M]+
190.14, found 190.10.
(1-Methoxy-4-methyl-phenyl)-methanol (20). To a suspention of LiAlH4 (2.4 M
in THF, 0.173 mL, 0.42 mmol) in THF (0.9 mL) cooled to 0 °C was added methyl
1-methoxy-4-methyl-benzoate (19, 51.9 mg, 0.288 mmol) in THF (0.9 mL).
After stirring at room temperature for 2 h, the reaction mixture was cooled to
0 °C and water was carefully added. After 1 N HCl was added and stirred, the
mixture was filtered through a pad of Celite. The filtrate was extracted with
EtOAc. The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/EtOAc = 4:1) afforded 20 as a colorless oil (43.6 mg,
0.286 mmol, 99%); Rf 0.27 (hexane/EtOAc = 3:1); 1H NMR (300 MHz, CDCl3) d
7.08 (1H, d, J = 8.5 Hz, H3), 6.97 (1H, d, J = 2.9 Hz, H6), 6.75 (1H, dd, J = 7.9,
2.3 Hz, H2), 4.67 (2H, s, H10), 3.80 (3H, s, OMe), 2.07 (3H, s, H7). 13C NMR
(75 MHz, CDCl3) d 158.6, 140.4, 131.7, 128.2, 113.5, 113.3, 64.0, 55.9, 18.2; EI-
MS (m/z) calcd for C9H12O2 [M]+ 152.08, found 152.05.
4-Methyl-5-pentyl-phenol (MPP, 23). A mixture of 1-methoxy-4-methyl-5-pent-
1-enyl-benzene (22, 19.2 mg, 0.101 mol) and 10% Pd/C (2.5 mg) in EtOAc
(0.5 mL) was stirred under an atmosphere of H2 at room temperature for 24 h.
The reaction mixture was then filtered through a pad of Celite and wash with
EtOAc. Concentration in vacuo gave colorless oil (16.0 mg).
The crude oil was then diluted with CH2Cl2 (0.5 mL). To the solution cooled to
ꢀ78 °C was added BBr3 (1.0 M in CH2Cl2, 0.191 mL, 0.191 mmol). The reaction
temperature was gradually raised to room temperature. After stirring for 7 h,
the reaction mixture was quenched with saturated NaHCO3 solution and then
extracted with EtOAc. The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. Purification by silica gel column chromatography
(hexane/EtOAc = 5:1) afforded 23 as a yellow oil (12.4 mg, 69.6 lmol, 76%); Rf
0.13 (hexane/EtOAc = 10:1); 1H NMR (300 MHz, CDCl3) d 7.04 (1H, d, J = 8.5 Hz,
H6), 6.71 (1H, d, J = 2.7 Hz, H3), 6.65 (1H, dd, J = 7.9, 2.7 Hz, H2), 3.78 (3H, s,
OMe), 2.55 (1H, t, J = 8.6 Hz, H10), 2.23 (3H, s, H7), 1.59–1.52 (2H, m, H20), 1.40–
1.30 (4H, m, H30/40), 0.93–0.88 (3H, m, H50); EI-MS (m/z) calcd for C2H18O2 [M]+
178.14, found 178.10.
1-Methoxy-4-methyl-benzaldehyde (21). To
2.86 mmol) in CH2Cl2 (0.50 mL) cool to ꢀ78 °C was dropwise added oxalyl
chloride (49
L, 0.573 mmol). The resulting mixture was stirred at ꢀ78 °C for
a solution of DMSO (204 lL,
l
20 min. To the mixture was cannulated a solution of (1-methoxy-2-methyl-
phenyl)-methanol (20, 43.6 mg, 0.287 mmol) in CH2Cl2 (0.35 mL). After stirring
at ꢀ78 °C for 1 h, Et3N (0.20 mL, 1.43 mmol) was added. The reaction mixture
was then stirred at ꢀ78 °C for 1 h and warmed to room temperature and
stirred for 2 h. The reaction was quenched with 1 N HCl and extracted with
CH2Cl2. The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/EtOAc = 8:1) afforded 21 as a colorless oil (38.4 mg,
0.256 mmol, 89%); Rf 0.60 (hexane/EtOAc = 3:1); 1H NMR (300 MHz, CDCl3) d
10.28 (1H, s, H10), 7.33 (1H, d, J = 2.7 Hz, H6), 7.17 (1H, d, J = 8.7 Hz, H3), 7.05
(1H, dd, J = 8.7, 2.7 Hz, H2), 3.84 (3H, s, OMe), 2.62 (3H, s, H7).
Purification of 1, 9, 14, 15, and 23 by HPLC. MPBD (1), EPBD (14), and PPBD (15)
were purified by HPLC under the condition (column: Cosmosil 5C18-AR-II
10 ꢁ 250 mm; solvent: MeOH/H2O = 8:2; flow rate: 3.0 mL/min; detection:
254 nm; temperature: 30 °C; tR = 15.8, 17.7, and 21.8 min, for 1, 14, and 15,
respectively). diMe-MPBD (9) was purified by HPLC under the condition
(column: Cosmosil 5C18-AR-II 10 ꢁ 250 mm; solvent: MeOH/H2O = 95:5; flow
rate: 3.0 mL/min; detection: 254 nm; temperature: 30 °C; tR = 19.7 min). MPP
(23) was purified by HPLC under the condition (column: Cosmosil 5C18-AR-II
10 ꢁ 250 mm; solvent: MeOH/H2O = 9:1; flow rate: 3.0 mL/min; detection:
254 nm; temperature: 30 °C; tR = 14.0 min).
25. Disk diffusion assay. Simplified disk diffusion assay was used to examine the
antibacterial activities. Bacterial cultures were grown in Lysogeny broth (LB)
1-Methoxy-4-methyl-5-pent-1-enylbenzene (22). To
butylphosphonium bromide (118 mg, 0.296 mmol) in THF (1.0 mL) was
added dropwise nBuLi (1.60 M in hexane, 169 L, 0.271 mmol). The
a
suspension of n-
and spread on LB or LP (Lactose peptone) agar plates using
a sterilized
l
spreader. The plates were then dried for 10 min before placing filter paper
disks with 6 mm diameter (purchased from Advantec). Five disks were placed
suspension was then stirred at 0 °C for 15 min. To the orange suspension at
0 °C was added a solution of 1-methoxy-4-methyl-benzaldehyde (21, 37.0 mg,
0.246 mmol) in THF (1.0 mL). After stirring at room temperature for 30 min,
the reaction mixture was quenched with saturated NH4Cl solution, and
extracted with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column
chromatography (hexane/EtOAc = 3:1) afforded 22 as a yellow oil (36.6 mg,
0.192 mmol, 78%, E/Z = 1:1.6); Rf 0.87 (hexane/EtOAc = 3:1); 1H NMR
with at least 3 cm apart from each other in one plate. Each disk contained 4
ll
of 100 mM MPBD 1 and its derivatives. For a positive control, 4 l of 50 mM
l
ampicillin was used. The plates were incubated for 16–24 h at 37 °C. The zone
of clearance around the disk was confirmed and photographed. Escherichia coli
and Bacillus subtilis were used for Gram negative and Gram positive standards.
26. Other SAR studies of 1 including the inducement of spore cell formation will be
reported separately.