Convenient synthesis of an isoxazole compound, KRIBB3, as an anticancer agent

Article abstract of DOI:10.1080/00397911.2010.546551

A diaryl isoxazole compound, KRIBB3, which exhibits strong antimigratory and antimitotic activities against cancer cells, was prepared in a practical synthetic way. The synthetic method may provide easy access to KRIBB3 analogs with various substituents a

Full text of DOI:10.1080/00397911.2010.546551

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Convenient Synthesis of an Isoxazole
Compound, KRIBB3, as an Anticancer
Agent
Hyeong Kyu Lee a , Eunju Yun a , Ji Hye Min a , Kab Seog Yoon a
Dong-Ho Choung a & Sangku Lee a
,
a Biotherapeutics Research Center, KRIBB , Daejeon , Republic of
Korea
Accepted author version posted online: 07 Dec 2011.Published
online: 26 Mar 2012.
To cite this article: Hyeong Kyu Lee , Eunju Yun , Ji Hye Min , Kab Seog Yoon , Dong-Ho Choung &
Sangku Lee (2012) Convenient Synthesis of an Isoxazole Compound, KRIBB3, as an Anticancer Agent,
Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic
Chemistry, 42:13, 1890-1894, DOI: 10.1080/00397911.2010.546551
To link to this article: http://dx.doi.org/10.1080/00397911.2010.546551
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Synthetic Communications1, 42: 1890–1894, 2012
Copyright # KRIBB
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.546551
CONVENIENT SYNTHESIS OF AN ISOXAZOLE
COMPOUND, KRIBB3, AS AN ANTICANCER AGENT
Hyeong Kyu Lee, Eunju Yun, Ji Hye Min, Kab Seog Yoon,
Dong-Ho Choung, and Sangku Lee
Biotherapeutics Research Center, KRIBB, Daejeon, Republic of Korea
GRAPHICAL ABSTRACT
Abstract A diaryl isoxazole compound, KRIBB3, which exhibits strong antimigratory and
antimitotic activities against cancer cells, was prepared in a practical synthetic way. The
synthetic method may provide easy access to KRIBB3 analogs with various substituents
at an aryl moiety for structure–activity relationships (SAR), as well as a large quantity
of KRIBB3 for in vivo studies.
Keywords Anticancer; antimigratory; cyclization; isoxazole
INTRODUCTION
In the course of searching for an antimigratory compound using cell-based
screening, we synthesized a series of diaryl isoxazole compounds. One of them,
5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)isoxazole (1), named
KRIBB3, showed strong antimigratory and antimitotic activities against cancer cells
(Figure 1).[1] KRIBB3 showed inhibition of proliferation of HCT-116 colorectal can-
cer cells with GI50 value of 0.1 mM and showed six times stronger inhibitory activity
than nocodazol as a reference. As we have been interested in further biological stu-
dies including in vivo activities, we need a practical synthetic approach to KRIBB3,
thereby also enabling access to KRIBB3 analogs for examining structure–activity
relationships. Herein we describe a practical synthesis of KRIBB3.
The synthesis of 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)
isoxazole (1) was reported by two research groups.[2] They constructed the core dia-
ryl isoxazole ring system via ring opening and cyclization by reaction of isoflavones
with hydroxylamine. Their approaches have a drawback in modifying substituents of
Received October 19, 2010.
Address correspondence to Sangku Lee, Biotherapeutics Research Center, KRIBB, Daejeon
305-806, Republic of Korea. E-mail: sangku@kribb.re.kr
1890

SYNTHESIS OF KRIBB3
1891
Scheme 1. Synthesis of KRIBB3.
Figure 1. Structure of KRIBB3 (1).
an aryl moiety for structure–activity relationships of KRIBB3. The substituent at the
2-position of an aryl moiety of diaryl isoxazole derivatives is fixed with a hydroxyl
group because of use of isoflavones in the reaction routes. However, our synthetic
route to the isoxazole ring involves the reaction of diaryl enaminone with
hydroxylamine, providing a way to prepare diaryl isoxazole derivatives with various
substituents at an aryl moiety.
The synthesis started with formylation of commercially available 4-ethylresor-
cinol (2) using POCl3 and dimethylformamide (DMF),[3] followed by methylation
using methyl iodide and potassium carbonate to yield 5-ethyl-2-hydroxy-4-methox-
ybenzaldehyde, which by treatment with benzyl bromide and potassium carbonate
afforded compound 3 (Scheme 1). Aldehyde 3 was converted to ketone 4 by
addition of 4-methoxybenzylmagnesium chloride and tetrapropylammonium per-
ruthenate (TPAP) oxidation[4] of the resulting alcohol. Enaminoketone 5 was pre-
pared in 70% yield from diaryl ethanone 4 by treatment with dimethylformamide
dimethyl acetal (DMFDMA) in refluxing toluene.[5] Finally, heterocyclization of
compound 5 with hydroxylamine in refluxing methanolic AcOH in the presence
of Na2CO3 afforded diary isoxazole 6,[5b] and subsequent debenzylation provided
KRIBB3 (1). This synthetic pathway afforded a practical route easily amenable
to large scale and produced 10-g quantities of KRIBB3 starting from 25 g of
4-ethylresorcinol (2).
In conclusion, KRIBB3 (1), an antimigratory agent, was prepared by a
convenient synthetic method that would bring a large quantity of 1.

1892
H. K. LEE ET AL.
EXPERIMENTAL
Compound 3
POCl3 (18.5 mL, 0.33 mol) was slowly added to dried dimethylformamide
(DMF) (61 mL, 0.75 mol) at 10 ꢀC. The mixture was stirred for 30 min. Then, a
solution of 4-ethylresorcinol (2) (12.5 g, 0.095 mol) in DMF (40 mL) was added,
and the reaction mixture was stirred at room temperature for 1 h. The reaction mix-
ture was cooled to 0 ꢀC, and 2 M NaOH aqueous solution was added to quench the
reaction. The reaction solution was diluted with ethyl acetate and extracted two
times with 2 M NaOH aqueous solution. The aqueous solution was neutralized by
3 N HCl, extracted four times with ethyl acetate, and washed with brine. The com-
bined organic layer was dried over anhydrous magnesium sulfate, filtered, and con-
centrated under reduced pressure. The concentrated residue was purified by silica-gel
column chromatography (hexane–EtOAc 7:1) to give 10 g of formylated compound.
Methyl iodide (9.4 g, 0.065 mol) was added to a mixture of the formylated compound
(10 g, 0.065 mol) and potassium carbonate (17.5 g, 0.13 mol) in DMF (100 mL), and
the reaction mixture was stirred at room temperature for 5 h. The reaction solution
was filtered to remove inorganic salts, and the filtrate was diluted with water,
extracted three times with ethyl acetate, and washed with brine. The combined
organic layer was dried over anhydrous magnesium sulfate, filtered, and concen-
trated under reduced pressure. The concentrated residue was purified by silica-gel
column chromatography (hexane–EtOAc 20:1) to give 6.6 g of methylated com-
pound. Benzyl bromide (7.2 g, 0.05 mol), was added to a mixture of the methylated
compound (6.6 g, 0.04 mol) and potassium carbonate (15 g, 0.11 mol) in DMF
(65 mL), and the reaction mixture was stirred at room temperature for 1 h. The reac-
tion solution was filtered to remove inorganic salts, and the filtrate was diluted with
water, extracted three times with ethyl acetate, and washed with brine. The combined
organic layer was dried over anhydrous magnesium sulfate, filtered, and concen-
trated under reduced pressure. The concentrated residue was purified by silica-gel
column chromatography (hexane–EtOAc 5:1) to give 9.4 g (35% yield in three steps)
of compound 3: white solid, mp 92-93 ꢀC; IR (film) 2868, 1667, 1602, 1443, 1271,
1
1198, 1112, 1057 cmꢁ1; H NMR (300 MHz, CDCl3) d 10.39 (s, 1H), 7.66 (s, 1H),
7.46-7.34 (m, 5H), 6.45 (s, 1H), 5.18 (s, 2H), 3.85 (s, 3H), 2.56 (q, J ¼ 7.8 Hz, 2H),
1.16 (t, J ¼ 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 188.3, 163.7, 161.7, 136.2,
128.7, 128.3, 128.2, 127.2, 125.8, 118.4, 95.6, 70.8, 55.5, 22.2, 13.8; ESIMS m=z
293.3 (Mþ þ Na).
Compound 4
A solution of 4-methoxybenzylchloride (16 g, 0.11 mol) in THF (50 mL) was
slowly added to a mixture of magnesium turnings (7.5 g, 0.32 mol) in THF
(100 mL) at room temperature. The reaction solution was refluxed with heating
for one hour and then cooled down in a 0 ꢀC water bath. The ashy solution was
extracted by using a syringe, which was used as a Grignard reagent. The Grignard
reagent was added slowly to a solution of the compound 3 (9.25 g, 0.035 mol) in
THF (100 mL) at 0 ꢀC, and the reaction mixture was stirred at room temperature
for 1 h. Saturated ammonium chloride solution was added to the reaction solution,

SYNTHESIS OF KRIBB3
1893
and the mixture was extracted with ethyl acetate three times and washed with brine.
The combined organic layer was dried over anhydrous magnesium sulfate, filtered,
and concentrated under reduced pressure. The concentrated residue was purified
by silica-gel column chromatography (hexane–EtOAc 7:1) to give 13 g of alcoholic
compound. Tetrapropylammonium perruthenate (0.55 g) was added to a mixture
of the alcoholic compound (13 g, 0.042 mol), 4-methylmorpholine N-oxide (5.7 g,
˚
0.05 mol) and anhydrous powdered 4 A molecular sieves (13 g) in dichloromethane
(80 mL). The reaction mixture was stirred for 30 min, passed through a short silica-
gel pad by washing with ethyl acetate, and concentrated in vacuo. The residue was
purified by silica-gel column chromatography (dichloromethane as an eluent) to give
12 g (88% yield in two steps) of compound 4: white solid, mp 67–68 ꢀC; IR (film)
2969, 1661, 1598, 1516, 1249, 1125, 1020 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
7.66 (s, 1H), 7.45–7.36 (m, 5H), 7.11–7.02 (m, 2H), 6.85–6.78 (m, 2H), 6.45 (s,
1H), 5.19 (s, 2H), 4.23 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 2.56 (q, J ¼ 7.8 Hz, 2H),
1.16 (t, J ¼ 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 198.3, 161.8, 158.1, 136.2,
131.4, 130.5, 129.2, 128.7, 128.2, 127.8, 127.6, 125.2, 120.1, 113.6, 95.9, 71.0, 55.3,
55.1, 49.0, 22.3, 13.9; HRMS (FAB) m=z 391.1906 [(M þH)þ, calcd for C25H27O4
391.1909].
Compound 5
Dimethylformamide dimethylacetal (DMFDMA) (6.25 mL, 0.065 mol) was
added to a solution of compound 4 (12 g, 0.03 mol) in toluene (40 mL). The reaction
mixture was refluxed with heating for 16 h at 135 ꢀC. The reaction solution was cooled
to 0 ꢀC, concentrated, and purified by silica-gel column chromatography (hexane–
EtOAc 2:1) to give 9.4 g (70% yield) of compound 5: yellow oil; IR (film) 2960,
1
2233, 1566, 1511, 1384, 1240, 1147 cmꢁ1; H NMR (300 MHz, CDCl3) d 7.44–7.26
(m, 5H), 7.15 (s, 1H), 7.09–7.06 (m, 2H), 7.04 (s, 1H), 6.81–6.78 (m, 2H), 6.40 (s,
1H), 5.04 (s, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 2.63 (s, 6H), 2.56 (q, J ¼ 7.8 Hz, 2H),
1.13 (t, J ¼ 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 194.0, 158.1, 158.0, 154.4,
154.0, 137.6, 132.9, 129.4, 129.0, 128.2, 127.5, 127.0, 125.1, 124.8, 113.3, 112.9,
97.9, 71.4, 55.3, 55.1, 43.2, 22.4, 14.1; ESIMS m=z 446.5 (Mþ þ H).
Compound 6
Sodium carbonate (0.7 g, 12 mmol) and NH2OH HCl (16 g, 23 mmol) were
added to a solution of compound 5 (9.4 g, 21 mmol) in methanol (125 mL). The mix-
ture was adjusted to pH 4-5 using acetic acid and then refluxed with heating for 2 h.
The mixture was concentrated in vacuo, diluted with water, adjusted to pH 8 using
saturated ammonium hydroxide aqueous solution, extracted with dichloromethane
four times, dried over anhydrous sodium sulfate, and concentrated in vacuo. The
concentrated residue was purified by silica-gel column chromatography (hexane–
EtOAc 8:1) to give 8.0 g (90% yield) of isoxazole compound 6: white solid, mp
99–100 ꢀC; IR (film) 2960, 1605, 1507, 1444, 1247, 1195 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d 8.42 (s, 1H), 7.25-7.00 (m, 8H), 6.78 (dd, J ¼ 6.9, 2.4 Hz,
2H), 6.48 (s, 1H), 4.84 (s, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 2.56 (q, J ¼ 7.8 Hz, 2H),
1.14 (t, J ¼ 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 162.3, 159.7, 158.7, 155.5,

1894
H. K. LEE ET AL.
150.5, 136.5, 130.8, 128.2, 127.6, 127.0, 125.3, 123.3, 116.6, 113.8, 109.3, 97.0, 70.7,
55.3, 55.2, 22.3, 14.0; HRMS (FAB) m=z 416.1859 [(M þH)þ, calcd. for C26H26NO4
416.1862].
Compound 1
To the solution of the isoxazole compound 6 (8.0 g, 19 mmol) prepared in ethyl
acetate (75 mL) was added 10% Pd=C (0.4 g). The reaction was performed under
60 psi of hydrogen atmosphere for 14 h. The reaction solution was passed through
a short silica-gel pad by washing with ethyl acetate and concentrated in vacuo.
The residue was purified by silica-gel column chromatography (hexane–EtOAc
3:1) to give 5.3 g (90% yield) of the final compound 1: white solid, mp 149–150 ꢀC;
IR (film) 3162, 1613, 1598, 1515, 1242, 1207, 1142 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 8.35 (s, 1H), 7.28 (dd, J ¼ 6.9, 2.4 Hz, 2H), 7.06 (s, 1H), 6.88 (dd,
J ¼ 6.9, 2.4 Hz, 2H), 6.48 (s, 1H), 5.19 (s, 2H), 3.80 (s, 6H), 2.42 (q, J ¼ 7.8 Hz,
2H), 0.98 (t, J ¼ 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 162.3, 160.3, 159.4,
153.6, 151.5, 129.3, 128.5, 125.2, 121.6, 115.2, 114.4, 105.3, 99.6, 55.3, 55.2, 22.0,
13.7; HRMS (FAB) m=z 326.1389 [(M þH)þ, calcd. for C19H20NO4 326.1392].
ACKNOWLEDGMENT
This research was supported by a grant from the KRIBB Research Initiative
Program.
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