Synthesis and biological activity of both enantiomers of kujigamberol isolated from 85-million-years-old Kuji amber

Article abstract of DOI:10.1016/j.bmcl.2012.05.022

The full-structure of a norlabdane terpenoid, kujigamberol (1) was determined by total synthesis. Key features of the total synthesis are (1) installation of isopentyl group through an o-lithiation of benzamide, (2) construction of tetralone by the RCM reaction, and (3) optical resolution of (±)-1 using chromatographical separation of the corresponding camphanates. X-ray crystallographical analysis of p-bromobenzoate obtained from the more polar camphanate that was identical with a natural derivative, revealed natural kujigamberol to have an S-configuration. Both the natural enantiomer and its (R)-antipode showed the same inhibitory activity toward the mutant yeast and HL-60 cells, while simple analogs without alkyl groups at the C-8 and 9 positions of (±)-1 had no such activity.

Full text of DOI:10.1016/j.bmcl.2012.05.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4259–4262
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of both enantiomers of kujigamberol isolated
from 85-million-years-old Kuji amber
Yue Qi Ye a, Hiroyuki Koshino a, Daisuke Hashizume a, Yuki Minamikawa b, Ken-ichi Kimura b,
Shunya Takahashi a,
⇑
a RIKEN ASI, Wako-shi, Saitama 351-0198, Japan
b Graduate School of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The full-structure of a norlabdane terpenoid, kujigamberol (1) was determined by total synthesis. Key
features of the total synthesis are (1) installation of isopentyl group through an o-lithiation of benzamide,
(2) construction of tetralone by the RCM reaction, and (3) optical resolution of ( )-1 using chromato-
graphical separation of the corresponding camphanates. X-ray crystallographical analysis of p-bro-
mobenzoate obtained from the more polar camphanate that was identical with a natural derivative,
revealed natural kujigamberol to have an S-configuration. Both the natural enantiomer and its (R)-anti-
pode showed the same inhibitory activity toward the mutant yeast and HL-60 cells, while simple analogs
without alkyl groups at the C-8 and 9 positions of ( )-1 had no such activity
Received 30 March 2012
Revised 7 May 2012
Accepted 8 May 2012
Available online 15 May 2012
Keywords:
Kuji amber
Kujigamberol
Norlabdane terpenoid
RCM reaction
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibitory activity toward HL-60 cells
Ambers are fossilized tree resins which are formed under favor-
able circumstances in soils and sediments, where the hardened
resins can be preserved for up to hundreds of millions of years.
Several kinds of origins and/or geological ages are reported in am-
bers such as Dominican (Dominican Republic), Mexican (Mexico),
Baltic (Poland), Cedar Lake (Canada), New Jersey (USA), Spain
(Spain), Lebanese (Lebanon) and Dolomites (Italy).1,2 Among them,
one of the oldest types is Kuji (Iwate Prefecture in Japan) amber
whose geological age is dated at 85 million years. Recently, several
papers have appeared dealing with chemical composition of am-
ber.3,4 However, there is no precedent for the study of compounds
from any amber focused on their biological activity and activity
guided fractionation. In our screening program for a new Ca2+-sig-
nal transduction inhibitor by using the mutant yeast (Saccharomy-
Described herein is the total synthesis of kujigamberol and its
antipode, thus establishing the absolute configuration of the natu-
ral product. Their inhibitory activities are also discussed.
In the synthetic study of kujigamberol, introduction of isopentyl
group into the aromatic ring at the later stage of total synthesis
was estimated to be difficult. Hence, the synthetic strategy is de-
signed as shown in Scheme 1. Both enantiomers would be obtained
by an optical resolution of ( )-1. The quaternary carbon center of 1
would be constructed from a ketone at the C-4 position of tetralone
2. C1–C2 bond7 cleavage in 2 and changing the oxidation level can
revert it back to c-lactone 3. This could be synthesized from 4 via a
chain elongation including Stille coupling.8 These retrosynthetic
analyses allowed us to select benzamide 59 as the starting material.
Synthesis of ( )-1 began with installation of isopentyl group on
an aromatic ring in 5 (Scheme 2). Since o-lithiation10 of 5 followed
by alkylation with an alkyl halide such as prenyl bromide failed,
the aryl lithium was trapped by DMF. The aldehyde 6 thus ob-
tained was treated with the phosphorane prepared from isobutyl-
ces cerevisiae, zds1
D erg3D pdr1D pdr3D
),5 we found the MeOH
extracts of Kuji amber to be positive for the assay. Extensive stud-
ies using spectroscopic methods revealed that the active principle
was a new norlabdanoid named kujigamberol (Fig. 1).6 However,
the absolute configuration has not been determined. Its occurrence
in nature was also limited because of the rarity of the origin. To
determine the absolute configuration of kujigamberol and evaluate
the biological activity exactly, development of a method for prep-
aration of both enantiomers of kujigamberol was required.
phosphonium bromide and n-BuLi in THF to give
7 after
hydrogenation reaction. Demethylation of 7 provided 8, which
was transformed into the corresponding triflate 4. Stille coupling11
of 4 with tributyl(vinyl)tin was effected by treatment with dichlor-
obis(triphenylphosphine)palladium in the presence of lithium
chloride and triphenylphosphine to give 9 in high yield. Hydride
reduction of 9 did not proceed cleanly or gave the 1,6-reduction
product predominantly. Direct hydrolysis of the amide carbonyl
under acidic or basic conditions was also found to be difficult. After
⇑
Corresponding author. Fax: +81 48 462 4627.
E-mail address: shunyat@riken.jp (S. Takahashi).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.022

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Y. Q. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4259–4262
Figure 1. Planar structure of kujigamberol.
Scheme 1. Synthetic plan of kujigamberol.
all, this transformation was attained by the neighboring group par-
ticipation of an internal hydroxyl group as follows. Thus, 9 was ini-
tially oxidized by Lemieux–Johnson oxidation to give aldehyde 10.
Reaction of 10 with allyltributyltin12 provided the corresponding
alcohol, which was successively treated with p-TsOH to give rise
to lactonization, giving c
-lactone 313 in high yield.
For construction of the tetralone ring, 3 was reduced with DI-
BALH and the resulting hemiacetal reacted with methylenetri-
phenylphosphorane under forcing conditions, affording 11
(Scheme 3). Ring-closing metathesis of this with Grubbs cat. 2nd
generation14 proceeded nicely to give 12 in good yield. Hydrogena-
tion of 12 followed by PCC oxidation provided tetralone 2.13 One
carbon homologation15 from 2 was performed by the action of
methoxymethylene triphenylphosphorane to give the correspond-
ing enol ether. Hydrolysis of the product under acidic conditions
resulted in a low yield of a tetralin carbaldehyde derivative, while
on treatment with NBS in aq THF, hydroxy aldehyde 13 was ob-
tained in high yield. Since hydrogenolysis of 13 led to a consider-
able amount of 2, 13 was subjected to dehydration reaction with
Scheme 2. Synthesis of c-lactone 3.
sponding p-bromobenzoate 20, establishing that the natural
product has an S-configuration. Similarly, unnatural form, (+)-(R)-
1 was prepared.
Next we evaluated the biological activities22,23 of the two enan-
tiomers. Synthetic (S)-1 showed a clear growth restored zone
against the mutant yeast at 0.5–0.0156
against human promyelocytic leukemia cell line HL60
(IC50 = 24.0 M). These results were not distinguished from those
lg/spot and cytotoxicity
l
of the natural product. Interestingly, the R-enantiomer and 16 also
showed the same inhibitory activities. Encouraged by the above re-
sults, we examined the biological activities of simple analogs 21–
23 without alkyl groups at the C-8 and 9 positions of 16, 17, or
( )-1, respectively (Fig. 2).24 All analogs, however, had no inhibi-
tory activity. The results suggest that the C4 stereochemistry does
not affect the inhibitory activity and that alkyl groups at the C-8
and/or 9 positions rather than substituents around the C-4 are
important for inhibition potential.
In summary, the absolute configuration of kujigamberol was
determined to be S by the total synthesis. Key features of our total
synthesis are (1) installation of the isopentyl group on the aromatic
ring, (2) construction of tetralone 2 by the RCM reaction, and (3)
optical resolution of ( )-1 using (1S)-(ꢀ)-camphanic chloride. Both
natural enantiomer and its antipode showed the same inhibitory
activity toward the mutant yeast and HL60 cells.
p-TsOH to give
a,b-unsaturated aldehyde 14. This compound
was, after hydrogenation, oxidized to provide the corresponding
methyl ester 15. The lithium enolate generated from the ester by
the action of LDA reacted with iodomethane, and the resulting
compound18 was reduced with LAH, affording ( )-1.19 The 1H and
13C NMR spectra of the synthetic sample were identical with those
of the natural product.
For optical resolution of ( )-1, the racemic alcohol was acylated
with (1S)-(ꢀ)-camphanic chloride to give a diastereomeric mixture
of the corresponding camphanates 18 and 19.13 Each compound
was separated by chromatography on silica gel with n-hexane–
ethyl acetate. More polar isomer 19 was identical in all respects
with an authentic sample derived from the natural product.20
Hydrolysis of 19 gave (ꢀ)-1.13 Its absolute configuration was deter-
mined to be S by X-ray crystallographical analysis21 of the corre-

Y. Q. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4259–4262
4261
Scheme 3. Total synthesis of kujigamberol 1.
9. Hoffmann, B.; Lackner, H. Liebigs Ann. 1995, 87.
10. Snieckus, V. Chem. Rev. 1990, 90, 879.
11. Saá, J. M.; Martorell, G.; García-Raso, A. J. Org. Chem. 1992, 57, 678.
12. Treatment of 10 with allylmagnesium bromide furnished the corresponding
homoallyic alcohol in a low yield.
13. Spectral data of representative compounds: Compound (S)-1: ½a D26
ꢀ11.2 (c =
ꢁ
0.23, methanol) {lit.6
½
a 2D9
ꢁ
ꢀ8.5 (c = 1.0, methanol)}; 1H NMR (600 MHz, CDCl3):
Figure 2. Simple analogs of kujigamberol.
d 7.07 (1H, d, J = 8.2 Hz), 7.00 (1H, br d, J = 8.2 Hz), 3.78 (1H, d, J = 11.0 Hz), 3.51
(1H, d, J = 11.0 Hz), 2.76 (1H, ddd, J = 16.5, 5.9, 5.9 Hz), 2.68 (1H, ddd, J = 16.5,
8.2, 5.5 Hz), 2.57 (2H, t, J = 8.8 Hz), 2.28 (3H, s), 1.97 (1H, ddd, J = 13.0, 10.0,
3.1 Hz), 1.88 (1H, m), 1.79 (1H, m), 1.68 (1H, m), 1.52 (1H, m), 1.36–1.23 (2H,
m), 1.24 (3H, s), 0.98 (6H, d, J = 6.9 Hz); 13C NMR (150 MHz, CDCl3): d 139.8,
138.8, 136.1, 133.6, 128.0, 123.9, 71.9, 39.5, 37.9, 32.9, 29.0, 27.4, 27.2, 26.9,
22.45, 22.44, 19.6, 19.5; HRMS (ESI+) calcd for C18H28ONa [M+Na]+ 283.2038,
found 283.2044.
Acknowledgments
We are grateful to Drs. T. Nakamura and Y. Hongo (mass spec-
tral measurements) in RIKEN. This work was supported in part by
the Chemical Genomics Project (RIKEN) and a Grant-in-Aid for Sci-
entific Research from the Ministry of Education, Science, Sports,
and Culture of Japan.
Compound 2: 1H NMR (500 MHz, CDCl3): d 7.84 (1H, d, J = 8.0 Hz), 7.11 (1H, d,
J = 8.0 Hz), 2.93 (2H, t J = 6.1 Hz), 2.65–2.59 (4H, m), 2.36 (3H, s), 2.14–2.11 (2H,
m), 1.70 (1H, m), 1.35–1.30 (2H, m), 0.99 (6H, d, J = 6.9 Hz); 13C NMR (125 MHz,
CDCl3): d 198.7, 142.1, 139.3, 131.2, 128.6, 124.8, 38.5, 38.0, 28.7, 27.1, 26.2,
22.9, 22.4, 20.4; HRMS (EI+) calcd for C16H22O [M]+ 230.1671, found 230.1677.
Compound 3: 1H NMR (500 MHz, CDCl3): d 7.39 (1H, d, J = 7.8 Hz), 7.13 (1H, d,
J = 7.8 Hz), 5.76 (1H, m), 5.36 (1H, t, J = 5.9 Hz), 5.17 (1H, ddd, J = 17.1, 2.9,
1.5 Hz), 5.13 (1H, ddd, J = 10.3, 2.9, 1.2 Hz), 3.13–3.09 (2H, m), 2.69 (1H, dt,
J = 14.5, 6.6 Hz), 2.59 (1H, dt, J = 14.5, 6.6 Hz), 2.38 (3H, s), 1.74 (1H, m), 1.40–
1.37 (2H, m), 0.99 (6H, d, J = 6.6 Hz); 13C NMR (125 MHz, CDCl3): d 170.3, 147.9,
143.4, 137.3, 135.8, 131.6, 123.2, 119.3, 118.7, 78.5, 39.1, 39.0, 28.7, 25.3,
22.43, 22.40, 18.5; HRMS (EI+) calcd for C17H22O2 [M]+ 258.1620, found
258.1615.
References and notes
1. Ragazzi, E.; Roghi, G.; Giaretta, A.; Gianolla, P. Thermochim. Acta 2003, 404, 43.
2. Tonidandel, L.; Ragazzi, E.; Roghi, G.; Traldi, P. Rapid Commun. Mass Spectrom.
2008, 22, 630.
3. (a) Mills, J. S.; White, R.; Gough, L. J. Chem. Geol. 1984, 47, 15; (b) Lambert, J. B.;
Santiago-Blay, J. A.; Anderson, K. B. Angew. Chem., Int. Ed. 2008, 47, 9608; (c)
Jossang, J.; Bel-Kassaoui, H.; Jossang, A.; Seuleiman, M.; Nel, A. J. Org. Chem.
2008, 73, 412; (d) Bray, P. S.; Anderson, K. B. Science 2009, 326, 132.
4. For investigation of Kuji amber by modern analytical techniques such as
Brillouin light scattering (BLS), see Yoshihara, A.; Maeda, T.; Imai, Y. Vib.
Spectrosc. 2009, 50, 250.
5. (a) Mizunuma, M.; Hirata, D.; Miyahara, K.; Tsuchiya, E.; Miyakawa, T. Nature
1998, 392, 303; (b) Shitamukai, A.; Mizunuma, M.; Hirata, D.; Takahashi, H.;
Miyakawa, T. Biosci., Biotechnol., Biochem. 2000, 64, 1942; (c) Chanklan, R.;
Aihara, E.; Koga, S.; Takahashi, H.; Mizunuma, M.; Miyakawa, T. Biosci.,
Biotechnol., Biochem. 2008, 72, 132.
6. Kimura, K.; Minamikawa, Y.; Ogasawara, Y.; Yoshida, J.; Saitoh, K.; Shinden, H.;
Ye, Y-Q.; Takahashi, S.; Miyakawa, T.; Koshino, H. Fitoterapia, 2012, in press.
doi; 10.1016/j.fitote.2012.03.024.
Compound 18: ½a 2D4
ꢁ
+14.5 (c = 1.02, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.06
(2H, br d, J = 7.7 Hz), 6.97 (2H, br d, J = 7.7 Hz), 4.47 (1H, d, J = 11.0 Hz), 4.10
(1H, d, J = 11.0 Hz), 2.75 (1H, dt, J = 17.0, 6.0 Hz), 2.68 (1H, ddd, J = 17.0, 6.9, 6.0
Hz), 2.55 (2H, m), 2.28 (1H, m), 2.26 (3H, s), 1.94 (1H, m), 1.91–1.85 (2H, m),
1.80 (1H, m), 1.69–1.58 (3H, m), 1.57 (3H, s), 1.33 (3H, s), 1.30–1.25 (3H, m),
1.08 (3H, s), 0.98 (6H, d, J = 6.9 Hz). 0.94 (3H, s), 0.77 (3H, s); 13C NMR
(150 MHz, CDCl3): d 178.2, 167.3, 139.6, 138.0, 135.3, 133.9, 127.9, 124.3, 91.3,
73.0, 54.8, 53.9, 37.9, 37.7, 33.4, 30.5, 28.94, 28.88, 27.3, 27.0, 26.9, 22.4, 19.6,
19.3, 16.7, 16.5, 9.7; HRMS (ESI+) calcd for C28H40O4Na [M+Na]+ 463.2824,
found 463.2837.
Compound 19: ½a 2D4
ꢁ
ꢀ30.3 (c = 1.13, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.06
(2H, br d, J = 7.8 Hz), 6.97 (2H, br d, J = 7.8 Hz), 4.44 (1H, d, J = 11.0 Hz), 4.12
(1H, d, J = 11.0 Hz), 2.77 (1H, dt, J = 17.0, 6.0 Hz), 2.68 (1H, ddd, J = 17.0, 7.3,
6.0 Hz), 2.55 (2H, m), 2.32 (1H, m), 2.26 (3H, s), 1.98 (1H, m), 1.93–1.75 (3H,
m), 1.70–1.55 (3H, m), 1.32 (3H, s), 1.30–1.25 (3H, m), 1.07 (3H, s), 0.98 (6H, d,
J = 6.4 Hz). 0.81 (3H, s), 0.80 (3H, s); 13CNMR (150 MHz, CDCl3): d 178.1, 167.3,
139.6, 138.0, 135.2, 133.9, 128.0, 124.2, 91.3, 73.1, 54.7, 53.9, 37.9, 37.7, 33.4,
7. The carbon numbering system was conveniently according one employed in
the labdanoide diterpene as shown in Scheme 1.
8. (a) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508; (b) Stille, J. K. Pure Appl.
Chem. 1985, 57, 1771; (c) Scott, W. J.; McMurry, J. E. Acc. Chem. Res. 1988, 21, 47.
and references cited therein.

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Y. Q. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4259–4262
30.6, 30.3, 29.0, 28.9, 27.3, 27.1, 26.9, 22.4, 19.6, 19.3, 16.5, 16.4, 9.7; HRMS
21. The carbon numbering system is different from that employed in Scheme 1.
The detailed crystallographic data for 20 have been deposited with the
Cambridge Crystallographic Data Centre: Deposition code CCDC 852847. A
copy of the data can be obtained free of charge via http://www.ccdc.cam.ac.uk/
products/csd/request.
(ESI+) calcd for C28H40O4Na [M+Na]+ 463.2824, found 463.2818.
14. Handbook of Metathesis; Grubbs, R. H., Ed.; Wiley-VCH, 2003; Vols. 1–3,.
15. Reaction of this enolizable ketone with other reagents such as vinyl lithium,
TosMIC,16 and BnOCH2Cl–SmI2 failed.
17
16. Oldenziel, O. H.; van Leusen, D.; van Leusen, A. M. J. Org. Chem. 1977, 42, 3114.
17. (a) White, J. D.; Somers, T. C. J. Am. Chem. Soc. 1987, 109, 4424; (b) Imamoto, T.;
Takeyama, T.; Yokoyama, M. Tetrahedron Lett. 1984, 25, 3225.
18. This compound was shown to be an inseparable mixture of 17 and recovered
15 by 1H NMR analysis. From a practical point of view, separation of the
mixture after the following reduction was found to be more efficient.
19. Along with 1, alcohol 16 derived from unreacted 15 was also isolated in 11%
yield.
22. Ogasawara, Y.; Yoshida, J.; Shiono, Y.; Miyakawa, T.; Kimura, K. J. Antibiot. 2008,
61, 496.
23. Aburai, N.; Yoshida, M.; Ohnishi, M.; Kimura, K. Phytomedicine 2010, 17, 782.
24. Although these compounds were known compounds,25 we prepared similarly
them from a-tetralone according to the method described in Scheme 3.
25. (a) Maiti, S. B.; Chaudhuri, S. R. R.; Chatterjee, A. Synthesis 1987, 806; (b) Wilt, J.
W., ; Pawlikowski, W. W., Jr.; Wieczorek, J. J. J. Org. Chem. 1972, 37, 820; (c)
Newman, M. S.; O’Leary, T. J. J. Am. Chem. Soc. 1946, 68, 258.
20. It was revealed that the natural product contained no antipode by TLC and 1H
NMR analyses (500 MHz) of the crude products after esterification with (1S)-
(ꢀ)-camphanic chloride.