Design and synthesis of a vialinin A analog with a potent inhibitory activity of TNF-α production and its transformation into a couple of bioprobes

Article abstract of DOI:10.1016/j.bmcl.2012.02.034

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5′,6′-dimethyl-1,1′:4′1″-terphenyl-2′, 3′,4,4″-tetraol (2) with a comparable inhibitory activity (IC ₅₀ = 0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.

Full text of DOI:10.1016/j.bmcl.2012.02.034

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2385–2387
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of a vialinin A analog with a potent inhibitory activity
of TNF-a production and its transformation into a couple of bioprobes
Yue Qi Ye a,b, Jun-ichi Onose b, Naoki Abe b, , Hiroyuki Koshino a, Shunya Takahashi a,
⇑
⇑
a RIKEN ASI, Wako-shi, Saitama 351-0198, Japan
b Department of Nutritional Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-
a production in rat
Received 27 January 2012
Revised 10 February 2012
Accepted 14 February 2012
Available online 23 February 2012
basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced
analog, 50,60-dimethyl-1,10:40100-terphenyl-20,30,4,400-tetraol (2) with a comparable inhibitory activity
(IC50 = 0.02 nM) to that of 1. The synthesis involved double Suzuki–Miyaura coupling as a key step, and
required only five steps from commercially available 3,4-dimethylphenol. For identification of the target
molecule, fluorescent and biotinylated derivatives of 2 were prepared through a ‘click’ coupling process.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Vialinin A
Inhibitor of TNF-a production
Dimethyl analog of vialinin A
Fluorescent bioprobe
Biotinylated bioprobe
Vialinin A (1) is an oxygenated p-terphenyl bearing phenylacet-
oxy groups which we isolated as a powerful DPPH free radical-
scavenger (EC50 = 14 lM vs 10 lM for BHT) from the dry fruiting
is required. The core part in the probe is generally designed based
on the structure of an active library member which would be
gained through SAR studies of the original natural product, and
has been modified by introduction of a tag such as biotin and a
fluorescent label. Therefore, the first problem of this project was
design of the core. In a previous paper, we reported that the posi-
tional isomers of 1 regarding the phenylacetyl group (ganbajunins
bodies of an edible Chinese mushroom, Thelephora vialis (Fig. 1).1
This compound was also isolated from Thelephora terrestris and
Thelephora aurantiotincta by Asakawa et al.2 and by Norikura et
al.,3 respectively. The latter reported that this natural product
had cytotoxicity against human hepatocellular carcinoma cells
(HepG2) and human colonic carcinoma cells (Caco2), but not to
noncancerous human hepatocytes, and suggested that 1 was an
attractive compound for cancer prevention and/or treatment.3
Recently, we found that 1 strongly inhibited tumor necrosis fac-
D and E) showed no inhibitory activity against TNF-a production in
RBL-2H3.11 A similar result was also obtained from the case of
p-terphenyl series carrying the benzoyl group,12 suggesting the
importance of the catechol moiety in the central benzene ring.
The instability of the acyl groups toward several chemical modifi-
cations, in particular, basic conditions was observed in the course
of total synthesis of 1 and its congeners.12,13 These findings
prompted us to design as a probe core a scalable p-terphenyl 2 in
tor (TNF)-
a
production in rat basophilic leukemia (RBL-2H3) cells:
is a potent mul-
IC50 = 90 pM vs IC50 = 0.25 nM for FK-506.4 TNF-
a
tifunctional cytokine that mediates a variety of biological actions
with a central role in the pathogenesis of inflammatory diseases
such as rheumatoid arthritis (RA).5–8 In RA, TNF-
a causes accumu-
lation of inflammatory cells and self-perpetuation of inflammation,
leading to cartilage and bone destruction.9,10 Thus, inhibitors of
TNF-a production in activated mast cells and basophils are prom-
ising candidates for a new type of anti-allergic agent, for example,
RA treatment. The mode of action of 1 and its inhibition mecha-
nism, however, remain unclear. In order to clarify the target mole-
cule of 1 and its inhibition mechanism, development of a bioprobe
⇑
Corresponding authors. Fax: +81 35 477 2735 (N.A., for biological synthesis);
fax: +81 48 462 4627 (S.T., for synthesis).
E-mail addresses: abe@nodai.ac.jp (N. Abe), shunyat@riken.jp (S. Takahashi).
Figure 1. Structures of vialinin A (1) and its advanced analog 2.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.034

2386
Y. Q. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2385–2387
We next examined the effect of 2 on TNF-
a production from
RBL-2H3 Cells (Fig. 2) according to the method4 previously
described. As expected, analog 2 showed strong inhibitory activity
with IC50 = 0.02 nM. Interestingly, its precursor 7 with protected
catechol moiety also showed
a medium inhibitory activity
(IC50 = 0.32 nM) but the activity level was observed to reach satu-
ration at 1 nM, although the reason for this is not clear. These
results suggest that displacement of the phenylacetoxy function
by the methyl group retained the activity to some extent, which
is quite useful information for further rational design of artificial
analogs of 1. Synthesis and biological activity of similar methylated
analogs of terprenin, an immunosuppressive p-terphenyl, were
disclosed by Kawada et al.20
Encouraged by the above results, we planned to transform 2
into the corresponding probe molecules. Due to preservation of
the catechol moiety and easy modification,21 we thought that the
tag including a functional group should be introduced to the hy-
droxyl group at the terminal benzene ring rather than the central
one (e.g., 16), and tried mono O-protection of 7 such as meth-
oxymethylation and benzylation. Disappointingly, the correspond-
ing phenol 8 or 9 was obtained in a low yield (ꢀ30%).
Scheme 1. Synthesis of advanced analog 2.
With this result, we turned our attention to preparing desym-
metrical p-terphenyl by Suzuki–Miyaura coupling using different
types of boronic acids. According to the method described before,22
one-pot coupling was examined. Contrary to the previous case, the
use of Pd(OAc)2 was found to be ineffective for the coupling. The
best result was obtained by using tetrakis(triphenylphosphine)pal-
ladium (Pd(Ph3P)4) as a catalyst. Thus, 5 was treated with 1.2 equiv
of 6 in the presence of Pd(Ph3P)4 (0.05 equiv) and cesium carbonate
(2.0 equiv) in toluene at 100 °C for 9 h, and after the addition
of 10 (1.5 equiv), Pd(Ph3P)4 (0.05 equiv) and cesium carbonate
(2.0 equiv), the reaction was further continued for 10 h to give
the desired desymmetrical product 11 in ꢀ50% yield. The yield
was not improved even though the conditions (e.g., phosphine,
additive and solvent) were changed. From a practical point of view,
isolation of 9 after the following de-silylation was found to be
more efficient. After methoxymethylation of 9, the benzyl group
which the chemically reactive phenylacetoxyl groups in 1 were re-
placed by chemically stable dimethyl groups. Described herein are
the synthesis of 2, its inhibitory activity, and transformation into a
couple of bioprobes 16 and 17.
Synthesis of 2 involved double Suzuki–Miyaura coupling14 of 5
with boronic acid 6 as a key step (Scheme 1). The known bromide
4
15 prepared from 3,4-dimethylphenol (3) in 48% overall yield (two
steps) was protected as dimethoxymethyl ether to afford 5.
Suzuki–Miyaura coupling of 5 with 6 was performed by the action
of 0.05 equiv of palladium acetate (Pd(OAc)2)16 in the presence of
triphenylphosphine (0.15 equiv) and sodium carbonate (4.0 equiv)
in aq. propanol at 100 °C to give terphenyl 717 in high yield.18
Hydrolysis of 7 provided 219 in good yield. The overall yield of 2
from commercially available 3 attained 33%.
Scheme 2. Synthesis of a couple of bioprobes 16 and 17.

Y. Q. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2385–2387
5. Gordon, J. R.; Burd, P. R.; Galli, S. J. Immunol. Today 1990, 11, 458.
2387
6. Vassalli, P. Annu. Rev. Immunol. 1992, 10, 411.
7. Sieper, J.; Braun, J. Exper. Opin. Emerg. Drugs 2002, 2, 235.
8. Holgate, S. T. Cytokine 2004, 28, 152.
9. Brennan, F. M.; Chantry, D.; Jackson, A.; Maini, R.; Feldmann, M. Lancet 1989,
2(8657), 244.
10. Maini, R.; St Clair, E. W.; Breedveld, F.; Furst, D.; Kalden, J.; Weisman, M.;
Smolen, J.; Emery, P.; Harriman, G.; Feldmann, M.; Lipsky, P. Lancet 1999,
354(9194), 1932.
11. Xie, C.; Koshino, H.; Esumi, Y.; Onose, J.; Yoshikawa, K.; Abe, N. Bioorg. Med.
Chem. Lett. 2006, 16, 5424.
12. Ye, Y.-Q.; Koshino, H.; Onose, J.; Negishi, C.; Yoshikawa, K.; Abe, N.; Takahashi,
S. J. Org. Chem 2009, 74, 4642.
13. Ye, Y.-Q.; Koshino, H.; Onose, J.; Yoshikawa, K.; Abe, N.; Takahashi, S. Org. Lett
2007, 9, 4131.
14. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
15. Horner, L.; Sturm, K. Liebigs Ann. Chem. 1955, 597, 1.
16. Huff, B. E.; Koenig, T. M.; Mitchell, D.; Staszak, M. A. Organic Syntheses;
Wiley&Sons: NewYork, 2004, pp.102.
17. This coupling reaction in alkaline aqueous solutions was accompanied by
hydrolysis of TBS group to afford 7 while the TBS group was retained under
aprotic conditions (e.g., preparation of 11 in Scheme 2).
18. As initial attempts for Suzuki–Miyaura coupling of 5 and 6, Pd(Ph3P)4 was
employed as a catalyst. A considerable amount of the corresponding biphenyl,
however, was always isolated along with the desired p-terphenyl 7. These
results made us to conceive the coupling using different types of boronic acids
as shown in Scheme 2.
Figure 2. TNF-a Production Inhibition of 2 and 7.
19. Spectral data of representative compounds
Compound 2: mp >270 °C (n-hexane-ether-methanol); 1H NMR (400 MHz, d6-
acetone): d 8.35 (2H, s), 7.08 (4H, d, J = 8.8 Hz), 6.91 (4H, d, J = 8.8 Hz), 6.65 (2H,
s), 1.93 (6H, s); 13C NMR (100 MHz, d6-acetone): d 157.21, 141.05, 132.36,
in 12 was removed to afford 8. Upon treatment with 3-bromo-1-
propyne and sodium hydride, 8 gave a key intermediate 13.19
1,3-Dipolar Huisgen cycloaddition23 of 13 with 1424 in the pres-
ence of copper sulfate and sodium ascorbate provided a coupling
product, which was treated with TFA, giving a fluorescent probe
1619 in 48% yield from 13. Similarly, reaction of 13 with 1525 pro-
vided a coupling product in a good yield. The final deprotection un-
der acidic conditions, however, resulted in a complex mixture. On
the other hand, the click reaction after deprotection of 13 pro-
ceeded without trouble to give the desired product 1719 in good
yield. Both compounds showed an inhibitory activity against
129.64, 128.95, 126.50, 115.97, 17.41; HRMS (ESI) calcd for
C20H18O4Na
[M+Na]+ 345.1103, found 345.1104.
Compound 13: 1H NMR (500 MHz, CDCl3): d 7.24 (2H, J = 8.8 Hz), 7.22 (2H, d,
J = 8.8 Hz), 7.10 (2H, d, J = 8.8 Hz), 7.04 (2H, J = 8.8 Hz), 5.22 (2H, s), 4.82 (2H, s),
4.81 (2H, s), 4.74 (2H, br s), 3.51 (3H, s), 2.92 (3H, s), 2.89 (3H, s), 2.54 (1H, t,
J = 2.4 Hz), 2.04 (3H, s), 2.03 (3H, s); 13C NMR (125 MHz, CDCl3): d 156.4, 156.1,
145.3, 136.0, 135.9, 131.9, 131.8, 131.67, 131.65, 131.6, 131.4, 115.8, 114.4,
98.88, 98.86, 94.6, 78.6, 75.4, 56.80, 56.77, 56.0, 55.9, 17.94, 17.92; HRMS (ESI+)
calcd for C29H32O7Na [M+Na]+ 515.2046, found 515.2042.
Compound 16: 1H NMR (500 MHz, CDCl3): d 7.67 (1H, s), 7.40 (1H, d, J = 8.5 Hz),
7.23 (2H, d, J = 8.8 Hz), 7.17 (2H, d, J = 8.5 Hz), 7.10 (2H, d, J = 8.8 Hz), 6.97 (2H,
d, J = 8.5 Hz), 6.87 (1H, dd, J = 8.5, 2.5 Hz), 6.85 (1H, d, J = 2.5 Hz), 6.30 (1H, t,
J = 1.5 Hz), 5.62 (1H, br s), 5.27 (2H, s), 5.26 (2H, br d), 5.01 (1H, s), 4.97 (1H, s),
4.40 (2H, t, J = 7.1 Hz), 3.88 (3H, s), 2.47 (2H, t, J = 7.3 Hz), 2.01–1.96 (2H, m),
1.971 (3H, s), 1.967 (3H, s), 1.78–1.72 (2H, m), 1.46–1.39 (2H, m); 13C NMR
(125 MHz, CDCl3): d 172.4, 162.9, 161.0, 157.6, 155.5, 155.2, 149.3, 144.2,
138.62, 138.56, 131.55, 131.45, 129.2, 128.4, 127.41, 127.34, 126.75, 126.74,
124.4, 122.7, 115.9, 115.1, 112.7, 110.5, 109.8, 101.2, 62.1, 61.1, 55.8, 50.1, 33.6,
29.9, 25.9, 24.1, 17.17, 17.15; HRMS (ESI+) calcd for C40H39O9N3Na [M+Na]+
728.2584, found 728.2596.
TNF-
a production from RBL-2H3 cells at a promising level
(IC50 = 0.55 nM for 16 and 8.15 nM for 17). These bioprobes are ex-
pected to be a tool for identifying a vialinin A target. Now the re-
search is under investigation.
We succeeded in synthesizing an advanced analog 2 with a
comparable inhibitory activity to that of vialinin A (1), and trans-
forming it to a couple of bioprobes 16 and 17. The strategy devel-
oped here provides versatility for preparing a variety of functional
molecules such as a photoactive probe as well as 16 and 17 and
therefore is useful in medicinal chemistry.
Compound 17: ½a 2D3
ꢁ
+16.8 (c = 0.11, MeOH); 1H NMR (500 MHz, d4-methanol):
d 8.08 (1H, s), 7.15 (2H, d, J = 8.8 Hz), 7.08 (2H, d, J = 8.8 Hz), 7.05 (2H, d,
J = 8.6 Hz), 6.87 (2H, d, J = 8.6 Hz), 5.22 (2H, s), 4.46 (1H, dd, J = 7.9, 4.9 Hz), 4.42
(2H, t, J = 7.1 Hz), 4.27 (1H, dd, J = 7.9, 4.4 Hz), 3.18–3.08 (3H, m), 2.89 (1H, dd,
J = 12.7, 4.9 Hz), 2.68 (1H, d, J = 12.7 Hz), 2.18 (2H, t, J = 7.1 Hz), 1.95–1.87 (2H,
m), 1.91 (3H, s), 1.90 (3H, s), 1.75–1.37 (12H, m); 13C NMR (125 MHz, d4-
methanol): d 175.9, 166.1, 158.7, 157.5, 145.2, 141.41, 141.37, 132.7, 132.6,
132.4, 130.23, 130.18, 129.6, 127.4, 127.3, 125.3, 116.2, 115.8, 63.4, 62.5, 61.6,
57.0, 51.3, 41.0, 40.2, 36.8, 31.2, 30.2, 29.8, 29.5, 27.3, 27.1, 26.9, 17.48, 17.46;
HRMS (ESI+) calcd for C39H48O6N6SNa [M+Na]+ 751.3254, found 751.3250.
20. Kawada, K.; Ohtani, M.; Suzuki, R.; Arimura, A. US-Patent, 7,101,915, 2006.
21. We had already gained the results of the inhibitory activity of vialinin A related
compounds including its artificial regioisomers.4,11–13 Furthermore, after
several experiments, we found that the reactivity of two hydroxyl groups on
the central benzene ring of 2 was low and that an efficient alkylation at such
position was difficult. These results made us to choose the position of OH group
for introduction of the tag without SAR studies on dimethyl analogs of 1 other
than 2 and 7.
Acknowledgments
We are grateful to Ms. A. Yoshida (technical assistant), Drs. T.
Nakamura and Y. Hongo (mass spectral measurements) in RIKEN.
This work was supported by the Chemical Genomics Project (RI-
KEN). N. A. acknowledges the Advanced Research Project of Tokyo
University of Agriculture.
References and notes
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Phytochemistry 2005, 66, 1052.
24. Compound 14 was prepared from 6-azidohexanoic acid26 and 4-bromomethyl-
7-methoxycoumarin.
25. Inverarity, I. A.; Viguier, R. F. H.; Cohen, P.; Hulme, A. N. Bioconjugate Chem.
2007, 18, 1593.
3. Norikura, T.; Fujiwara, K.; Narita, T.; Yamaguchi, S.; Morinaga, Y.; Iwai, K.;
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1998, 6, 755.
4. Onose, J.; Xie, C.; Ye, Y.-Q.; Takahashi, S.; Koshino, H.; Yasunaga, K.; Abe, N.;
Yoshikawa, K. Biol. Pharm. Bull. 2008, 31, 831.