Efficient syntheses of a series of trehalose dimycolate (TDM)/trehalose dicorynomycolate (TDCM) analogues and their interleukin-6 level enhancement activity in mice sera

Article abstract of DOI:10.1021/jo062018j

We found an IL-6 level-enhancing compound during our synthetic study of trehalose-6,6′-dimycolate (1, TDM, formerly called cord factor) analogues. TDM is a glycolipid distributed in the cell wall of Mycobacterium tuberculosis and shows significant antitum

Full text of DOI:10.1021/jo062018j

Efficient Syntheses of a Series of Trehalose Dimycolate (TDM)/
Trehalose Dicorynomycolate (TDCM) Analogues and Their
Interleukin-6 Level Enhancement Activity in Mice Sera
Mugio Nishizawa,*,† Hirofumi Yamamoto,† Hiroshi Imagawa,†
Ve´ronique Barbier-Chassefie`re,‡ Emmanuel Petit,‡ Ichiro Azuma,¶ and Dulce Papy-Garcia‡
Faculty of Pharmaceutical Sciences, Tokushima Bunri UniVersity, Yamashiro-cho,
Tokushima 770-8514, Japan, Laboratory CRRET CNRS UMR 7149, UniVersite de Paris 12 Val de Marne,
AVenne du General de Gaulle 94010 Creteil cedex, France, and Hokkaido Pharmaceutical UniVersity,
Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan
mugi@ph.bunri-u.ac.jp
ReceiVed September 29, 2006
We found an IL-6 level-enhancing compound during our synthetic study of trehalose-6,6′-dimycolate (1,
TDM, formerly called cord factor) analogues. TDM is a glycolipid distributed in the cell wall of
Mycobacterium tuberculosis and shows significant antitumor activity based on an immunoadjuvant activity.
However, due to its significant toxicity, TDM is not yet applicable for practical use. In 1993, Datta and
Takayama reported the purification of trehalose-6,6′-dicorynomycolate (2c, TDCM) from Corynebacterium
spp. We have previously reported the synthesis of four diastereomeric TDCMs and showed that the
synthetic (2R,3R,2′R,3′R)-TDCM (2c, hereafter abbreviated RRRR-TDCM-C14) is identical to natural
TDCM; we also demonstrated that 2c and SSSS-TDCM-C14 (3c) showed significant antitumor activity
as well as inhibitory activity in experimental lung metastasis based on the immunoadjuvant activity.
Furthermore, we found that the significant lethal toxicity in mice by TDM (1) was no longer observed
with the shorter-chain analogues of TDCMs. Therefore, we have elucidated that the 2,3-antistereochemistry
(RR or SS) of the fatty acid residue is promising for biological activities. The chain length of the fatty
acid residue should also be important for the biological activity, and thus, we designed a general synthetic
procedure for trehalose diesters with 2,3-antistereochemistry and a series of chain lengths by using Noyori’s
asymmetric reduction of â,â-ketoesters followed by antiselective alkylation according to Frater to give
â,â-hydroxy alcohols as the key steps. Thus, we prepared trehalose diesters (TDCM) 2a-d, 3a-d, and
4a-d as well as monoesters (TMCM) 5a-d and 6a-d. Immunological activities of TDCMs and TMCMs
were evaluated by determining IL-6 level enhancement in mouse serum, and we found that RRRR-TDCM-
C14 (2c) and RRSS-TDCM-C14 (4c) showed significant IL-6 level enhancement activities.
Introduction
autoimmune diseases and inflammations.3,4 Although conflicting
evidence has reported its involvement in tumor cell growth, the
available information about its source and its pathophysiological
regulation in cancer cells is limited. While IL-6 has been shown
Interleukin-6 (IL-6)1,2 is a multifunctional cytokine exhibiting
important roles in host defense, acute phase reaction, immune
responses, nerve cell function, and hematopoiesis. Elevated
serum IL-6 levels have been observed in a number of pathologi-
cal conditions, including bacterial and viral infections, trauma,
(1) Hirano, T.; Yasukawa, K.; Harada, H.; Taga, T.; Watanabe, Y.;
Matsuda, T.; Kashiwamura, S.; Nakajima, K.; Koyama, K.; Iwamatsu, A.;
Tsunasawa, S.; Sakiyama, F.; Matsui, H.; Takahara, Y.; Taniguchi, T.;
Kitamoto, T. Nature 1986, 324, 73-76.
(2) Hirano, T. International ReView of Immunology; 1998; Vol. 16, pp
249-284.
† Tokushima Bunri University, Yamashiro-cho.
‡ Universite de Paris 12 Val de Marne.
¶ Hokkaido Pharmaceutical University.
10.1021/jo062018j CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/08/2007
J. Org. Chem. 2007, 72, 1627-1633
1627

Nishizawa et al.
to inhibit the proliferation of some human cancer cell lines and
to reduce tumorigenicity in syngeneic animals, it has also been
shown to behave as an autocrine growth factor in cases such as
multiple myeloma and human prostate cancer.5 In some au-
toimmune inflammatory diseases, such as rheumatoid arthritis
and Crohn’s disease, regulating the function of cytokines may
facilitate the restoration of the disequilibrium between pro-
inflammatory cytokines and anti-inflammatory cytokines or
cytokine inhibitors.6,7 We have found an IL-6 level-enhancing
compound during our synthetic study of trehalose-6,6′-dimy-
colate (1, TDM, formerly called cord factor) analogues. TDM
is a glycolipid distributed in the cell wall of Mycobacterium
tuberculosis8,9 and shows significant antitumor activity based
on an immunoadjuvant activity.10,11 However, due to its
significant toxicity, TDM is not yet applicable for practical use.
In 1963, Ioneda reported the existence of smaller glycolipids
on the cell surface of related Corynebacterium diphtheriae;
however, it was shown to be a mixture of three components.12
In 1993, Datta and Takayama reported the purification of
trehalose-6,6′-dicorynomycolate (2c, TDCM) from Corynebac-
terium spp.13 Synthesis of the fatty acid residues of TDCM have
been reported, and the stereochemistries were determined to be
2R,3R.14 We have reported the synthesis of four diastereomeric
TDCMs based on a strategy of the Katsuki-Sharpless epoxi-
dation15 and then on Noyori’s BINAP-Ru-catalyzed asymmetric
reduction of a racemic â-ketolactone.16 Therefore, we showed
that the synthetic (2R,3R,2′R,3′R)-TDCM (2c, hereafter ab-
breviated RRRR-TDCM-C14) is identical to natural TDCM and
also demonstrated that 2c and SSSS-TDCM-C14 (3c) showed
significant antitumor activity as well as inhibitory activity in
experimental lung metastasis based on the immunoadjuvant
activity.17 Furthermore, we found that the significant lethal
toxicity in mice by TDM (1) was no longer observed with the
shorter-chain analogues of TDCMs.17 Therefore, we have
elucidated that the 2,3-antistereochemistry (RR or SS) of the
fatty acid residue is promising for biological activities. The chain
length of the fatty acid residue should also be important for the
biological activity, and thus, we designed a general synthetic
procedure for trehalose diesters with 2,3-antistereochemistry and
a series of chain lengths by using Noyori’s asymmetric reduction
of â-ketoesters18 followed by antiselective alkylation according
to Frater19 to give R-alkyl-â-hydroxyesters as the key steps.
Thus, we prepared trehalose diesters (TDCM) 2a-d, 3a-d, and
4a-d as well as monoesters (TMCM) 5a-d and 6a-d.
Immunological activities of TDCMs and TMCMs were evalu-
ated by determining the IL-6 level enhancement in mouse serum,
and we found that RRRR-TDCM-C14 (2c) and RRSS-TDCM-
C14 (4c) showed significant enhancement activities.
Results and Discussion
Improved synthesis of natural RRRR-TDCM-C14 (2c) was
achieved as follows. A dianion derived from the reaction of
tert-butyl acetoacetate (7) with NaH and then n-BuLi was treated
with C14H29I in THF to afford 8c in 78% yield. Exposure to 70
kgf/cm2 of hydrogen in the presence of RuCl2[(R)-BINAP] (0.1
mol %) in MeOH afforded (R)-alcohol 9c in 94% yield. The
R-stereochemistry of 9c was confirmed by Kusumi’s method,20
and the optical purity was estimated to be higher than 98% ee
based upon the 19F NMR of the MTPA derivative.21The dianion
derived from reaction of â,â-hydroxyester 9c with LDA was
again treated with C14H29I in the presence of HMPA at -48
°C to provide (RR)-10c in 55% yield. Diastereoselectivity of
the latter alkylation leading to 10c was confirmed to be higher
than 99% de based on 1H NMR as well as HPLC of the derived
methyl ester as compared with that of the authentic 2S,3R-
diastereomer.14,15 Our original reductive etherification of the
silylether of 10c at room temperature gave (RR)-benzyl ether
carboxylic acid 11c directly in 96% yield.22,23 Benzyl-protected
(3) (a) Ito, H. Curr. Drug Targets: Inflammation Allergy 2003, 2, 125-
130. (b) Matsumoto, T. Rinaho Byori 1999, 47, 494-500.
(4) (a) Lowe, G. D. Ann. Periodontol. 2000, 6, 1-8. (b) Volante, E.;
Moretti, S.; Pisani, F.; Bevilacqua, G. J. Matern. Fetal Neonat. Med. 2004,
16, 13-16.
(5) Riedel, F. Anticancer Res. 2005, 25, 2761-2764.
(6) Nishino, N. Autoimmunity 2005, 38, 359-367.
(7) Spellman, K.; Burns, J.; Nichols, D.; Winters, N.; Ottersberg, S.;
Tenborg, M. Altern. Med. ReV. 2006, 11, 128-150.
(8) (a) Bloch, H. J. Exp. Med. 1950, 91, 197-217. (b) Noll, H.; Bloch,
H.; Asselineau, J.; Lederer, E. Biochim. Biophys. Acta 1956, 20, 299-399.
(9) Ryll, R.; Kumazawa, Y.; Yano, I. Microbiol. Immunol. 2001, 45,
801-811.
(10) Azuma, I.; Yamamura, Y. J. Biochem. 1962, 52, 82-91.
(11) Numata, F.; Nishimura, K.; Ishida, H.; Ukei, S.; Tone, Y.; Ichihara,
C.; Saiki, I.; Sekikawa, I.; Azuma, I. Chem. Pharm. Bull. 1985, 33, 4544-
4555.
(12) (a) Ioneda, T.; Lenz, M.; Pudles, J. Biochem. Biophys. Res. Commun.
1963, 13, 110-114. (b) Senn, M.; Ioneda, T.; Pudles, J.; Lederer, E. Eur.
J. Biochem. 1967, 1, 353-356. (c) Puzo, G.; Tissie, G.; Aurelle, H.; Lacave,
C.; Prome, J. C. Eur. J. Biochem. 1979, 98, 99-105.
(18) Noyori, R.; Ohkuma, T.; Kitamura, M.; Takaya, H.; Sayo, N.;
Kumobayashi, H.; Akutagawa, S. J. Am. Chem. Soc. 1987, 109, 5856-
5858.
(19) (a) Frater, G. HelV. Chim. Acta 1979, 62, 2825-2828. (b) Wallace,
G. A.; Heathcock, C. H. J. Org. Chem. 2001, 66, 450-454.
(20) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4096.
(21) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512-519.
(22) Hatakeyama, S.; Mori, H.; Kitano, K.; Yamada, H.; Nishizawa, M.
Tetrahedron Lett. 1994, 35, 4367-4370. When the reaction was carried
out at 0 °C or at room temperature, cleavage of the tert-butyl ester group
also took place. See also ref 24.
(23) TMSOTf-catalyzed cleavage of prenyl ester was developed recently
in our group, and the same procedure was applied for the cleavage of tert-
butyl ester. Nishizawa, M.; Yamamoto, H.; Seo, K.; Imagawa, H.; Sugihara,
T. Org. Lett. 2002, 4, 1947-1949.
(13) Datta, A. K.; Takayama, K. Carbohydr. Res. 1993, 245, 151-158.
(14) (a) Tocanne, J.-F.; Asselineau, C. Bull. Soc. Chim. Fr. 1968, 4519-
4525. (b) Kitano, Y.; Kobayashi, Y.; Sato, F. J. Chem. Soc., Chem. Commun.
1985, 498-499. (c) Utaka, M.; Higashi, H.; Takeda, A. J. Chem. Soc.,
Chem. Commun. 1987, 1368-1369.
(15) Nishizawa, M.; Minagawa, R.; Garc´ıa, D. M.; Hatakeyama, S.;
Yamada, H. Tetrahedron Lett. 1994, 35, 5891-5894.
(16) Nishizawa, M.; Garc´ıa, D. M.; Minagawa, R.; Noguchi, Y.;
Imagawa, H.; Yamada, H.; Watanabe, R.; Yoo, Y. C.; Azuma, I. Synlett
1996, 452-454.
(17) Watanabe, R.; Yoo, Y. C.; Hata, K.; Motobe, M.; Koike, Y.;
Nishizawa, M.; Garc´ıa, D. M.; Noguchi, Y.; Imagawa, H.; Yamada, H.;
Azuma, I. Vaccine 1999, 17, 1484-1492.
1628 J. Org. Chem., Vol. 72, No. 5, 2007

TDM/TDCM Analogues and Their IL-6 LeVel Enhancement
(SS)-carboxylic acid 14c was also prepared by the same
procedure, except it utilized (S)-BINAP-Ru catalyst for the
asymmetric reduction of ketoester 8c to 12c and followed the
antiselective alkylation to 13c and reductive etherification/ester
cleavage to 14c.16,17 Condensation of trehalose derivative 1524
and carboxylic acid 11c (1.5 equiv), carried out using EDCI
and DMAP in CH2Cl2 at room temperature, afforded diester
16c in 50% yield along with monoester 19c in 42% yield.25
Reaction of monoester 19c with (SS)-acid 14c (1.2 equiv) under
the same conditions gave (RRSS)-diester 18c in 88% yield.
Esterification of 15 with (SS)-carboxylic acid 14c (1.5 equiv)
using EDCI generated (SSSS)-diester 17c in 48% yield along
with (SS)-monoester 20c in 41% yield. Catalytic hydrogenation
of esters 16c, 17c, 18c, 19c, and 20c using Pd(OH)2 as the
catalyst in methanol-chloroform (1:1) afforded RRRR-TDCM-
C14 (2c), SSSS-TDCM-C14 (3c), RRSS-TDCM-C14 (4c), RR-
TMCM-C14 (5c), and SS-TMCM-C14 (6c), respectively, in
91-97% yields.
solidification of the material at low temperature. Thus, we
switched to the ozonolysis-Wittig strategy for long-chain alkyl
derivatives and examined Noyori’s asymmetric reduction of
allylation product 21. However, the double bond was also
reduced to give the saturated hydroxyester. Prenylation product
22 was efficiently converted to hydroxyester 23 via Noyori
reduction and antiselective allylation; however, a cyclization
product 24 was obtained under reductive etherification condi-
tions even at low temperature, probably via an oxoniumion
intermediate 25. Finally, we decided to employ bis-homoprenyl
iodide 26 for the alkylation of 7.
Thus, the tert-butyl acetoacetate (7) was converted to 27 by
the reaction of its dianion with iodide 26 in 74% yield. Noyori
reduction of 27 using the (R)-BINAP-Ru catalyst afforded (R)-
alcohol 28 in 81% yield. None of the saturated product was
detected at all. Dianion generated from 28 with LDA was treated
with n-C20H41I to give anti-product 29 in 55% yield. Reductive
etherification of the TMS ether of 29 at -78 °C afforded benzyl
ether tert-butyl ester 30 in 96% yield.22,23 Ozonolysis of 30 and
the subsequent Wittig reaction afforded alkene 32. The E/Z ratio
was not determined. Treatment of 32 with 5 mol % of TMSOTf
at room temperature afforded benzyl-protected unsaturated
carboxylic acid (RR)-33 in 61% yield in three steps.22,23 The
corresponding (SS)-carboxylic acid 39 was prepared by the same
procedure except using (S)-BINAP-Ru catalyst for the Noyori
reduction of 27 to give 34 and successive reactions via 35, 36,
37, and 38. Condensation of trehalose derivative 15 and (RR)-
carboxylic acid 33 (1.5 equiv) was carried out using EDCI and
DMAP in CH2Cl2 at room temperature, affording (RRRR)-diester
40 in 44% yield along with (RR)-monoester 43 in 41% yield.
Reaction of (RR)-monoester 43 with (SS)-acid 39 (1.2 equiv)
under the same conditions gave (RRSS)-diester 42 in 88% yield.
Esterification of 15 with SS-carboxylic acid 39 (1.5 equiv) with
EDCI generated (SSSS)-diester 41 in 39% yield along with (SS)-
monoester 44 in 43% yield. Catalytic hydrogenation of esters
40, 41, 42, 43, and 44 using Pd(OH)2 as the catalyst in ethanol-
ethyl acetate afforded RRRR-TDCM-C20 (2d), SSSS-TDCM-
C20 (3d), RRSS-TDCM-C20 (4d), RR-TMCM-C20 (5d), and
SS-TMCM-C20 (6d), respectively, in 79-92% yield.
(RR)-Carboxylic acids 11a and 11b as well as (SS)-carboxylic
acids 14a and 14b were also prepared by the same procedure
from ketoester 7. Carboxylic acids 11a, 11b, 14a, and 14b were
condensed with 15 by the Keck procedure to give 16a, 16b,
17a, 17b, 18a, and 18b,25 and following catalytic hydrogenation
afforded the corresponding TDCM derivatives RRRR-TDCM-
C6 (2a), RRRR-TDCM-C10 (2b), SSSS-TDCM-C6 (3a), SSSS-
TDCM-C10 (3b), RRSS-TDCM-C6 (4a), and RRSS-TDCM-
With 20 different TDCM analogues in hand, we investigated
the immune activation of this class of compounds by evaluating
the enhancement of IL-6 levels in mice serum. This assay
employs a quantitative sandwich enzyme immunoassay tech-
nique.27 A monoclonal antibody specific for mouse IL-6 was
precoated onto a microplate. Standards, controls, and samples
were pipetted into the well, and any mouse IL-6 present was
C
10 (4b). TMCM derivatives RR-TMCM-C6 (5a), RR-TMCM-
C10 (5b), SS-TMCM-C6 (6a), and SS-TMCM-C10 (6b) were
also prepared via monoesters 19a, 19b, 20a, and 20b.
When we examined the Frater alkylation of the Noyori
reduction product 9d, which has a very long carbon chain, the
alkylation product was not obtained at all probably due to
(26) (a) Rondinone, C. M. Endocrine 2006, 29, 81-90. (b) Hu, Y.; Sun,
H.; Drake, J.; Kittrell, F.; Abba, M. C.; Deng, L.; Gaddis, S.; Sahin, A.;
Baggerly, K.; Medina, D.; Aldaz, C. M. Cancer Res. 2004, 64, 7748-
7755.
(24) Imagawa, H.; Tsuchihashi, T.; Singh, R. K.; Yamamoto, H.;
Sugihara, T.; Nishizawa, M. Org. Lett. 2003, 5, 153-155.
(25) Bodem, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394-2395.
(27) Vormittag, R.; Hsieh, K.; Kaider, A.; Minar, E.; Bialonczyk, C.;
Hirschl, M.; Mannhalter, C.; Pabinger, I. Thromb Haemostasis 2006, 95,
802-806.
J. Org. Chem, Vol. 72, No. 5, 2007 1629

Nishizawa et al.
As seen in Table 1, RRRR-TDCM-C14 (2c) induced the
highest IL-6 plasma levels, 456.3 ( 160.0 pg/mL, 1 day after
its administration; these IL-6 levels were 67-fold higher than
those of the control animals. RRSS-TDCM-C14 (4c) also
showed significant IL-6 enhancement activity, 195.7 ( 67.1
pg/mL, 1 day after its administration; this corresponds to a 29-
fold higher response than that in the control animals. Significant
enhancement by 4c was also observed at day 3. Compounds
not listed in Table 1 did not show any IL-6 level enhancement
activity. Except for C20 analogues 5d and 6d, the monoester
derivatives were inactive. These results indicate that the TDCM
diester analogues effectively and potently enhance IL-6 levels
in the plasma of treated animals. The 2,3-antistereochemistry
RR on the TDCM derivatives together with a precise C14 length
on the fatty acid residue are structural features necessary for
inducing an effective and significant increase of the plasma level
of cytokines. Although these results were obtained in healthy
animals, the effect of compound 2c is consistent with previously
proposed immunoadjuvant-based antitumor and antimetastasic
activities.17 Moreover, these results afford further evidence for
the positive role of IL-6 against tumor development, which has
been a point of controversy.26 Thus, use of TDCMs may advance
the knowledge of IL-6 regulation in cancer cells. Based on its
nontoxicity, RRRR-TDCM-C14 (2c) certainly constitutes a new
potential therapeutic alternative in the immunological treatment
of cancer or of other pathological conditions.
We are currently working on the activity of our synthetic
TDCM as well as TMCM analogues to other cytokines such as
TNF-R and IL-8. We also would like to examine the structure-
toxicity relationship of these series of compounds. These results
will be published elsewhere.
Experimental Section
tert-Butyl-3-oxooctadecanoate (8c). To a stirred suspension of
NaH (847 mg, 35.3 mmol) in THF (100 mL) was dropwise added
tert-butyl acetoacetate 7 (4.65 g, 29.4 mmol) at 0 °C and stirred
for 10 min. Subsequently, n-BuLi (1.6 M hexane solution, 20.2
mL, 32.3 mmol) was added, and the mixture was stirred for an
additional 30 min at the same temperature. To the resulting mixture
was added 1-iodotetradecane (14.3 g, 44.1 mmol), and the mixture
was stirred for 30 min at 0 °C. The reaction was quenched by the
addition of 1 N HCl solution, and the organic materials were
extracted with ether. The dried and concentrated material was
subjected to column chromatography on silica gel using hexane
and ethyl acetate (20:1) as an eluent to give tert-butyl-3-oxoocta-
decanoate (8c) (8.13 g, 78%) as a colorless syrup. 8c: FT-IR (neat)
bound by the immobilized antibody. After washing to remove
any unbound substances, an enzyme-linked polyclonal antibody
specific for mouse IL-6 was added to the well. Following a
wash to remove any unbound antibody-enzyme reagent, a
substrate solution was added to the well. The enzyme reaction
yields a blue product that turns yellow when the reaction is
stopped. The intensity of the color is proportional to the amount
of mouse IL-6 bound in the initial step, and the sample values
were determined by comparison with a standard curve.
TABLE 1. IL-6 Induction on Mice Sera (pg/mL, Mean ( SD)e
compound
controla
RR-TMCM-C6 (5a)
SS-TMCM-C6 (6a)
RRRR-TDCM-C10 (2b)
SSSS-TDCM-C10 (3b)
RRSS-TDCM-C10 (4b)
RRRR-TDCM-C14 (2c)
SSSS-TDCM-C14 (3c)
RRSS-TDCM-C14 (4c)
RRRR-TDCM-C20 (2d)
SSSS-TDCM-C20 (3d)
RRSS-TDCM-C20 (4d)
RR-TMCM-C20 (5d)
SS-TMCM-C20 (6d)
day 1
day 2
day 3
day 4
day 5
6.8 ( 0.8
8.3 ( 1.3
5.1 ( 1.0
12.0 ( 2.1c
10.5 ( 2.9c
13.6 ( 3.8
13.2 ( 2.1
13.6 ( 2.9
26.2 ( 12.7c
28.8 ( 5.4d
15.0 ( 1.6
7.7 ( 2.9
4.0 ( 1.4
6.9 ( 0.8
3.5 ( 0.5
7.5 ( 1.6
4.7 ( 0.8
7.5 ( 1.7
4.9 ( 1.1
10.0 ( 1.6b
9.6 ( 3.5
8.1 ( 1.6
37.8 ( 13.7c
87.2 ( 23.6d
8.9 ( 1.8
40.7 ( 21.0c
13.6 ( 4.7
91.2 ( 26.8d
26.9 ( 9.4c
87.0 ( 25.5d
64.8 ( 21.5d
4.2 ( 1.8
40.3 ( 25.2b
19.2 ( 3.9
26.4 ( 8.8
71.6 ( 9.4d
75.2 ( 13.0d
141.7 ( 7.0d
14.2 ( 1.5
3.6 ( 0.8
8.6 ( 2.6
15.9 ( 2.6
24.3 ( 4.4
25.8 ( 4.9c
28.6 ( 8.1c
24.6 ( 2.9
6.6 ( 1.8
456.3 ( 160.0d
144.3 ( 9.4d
195.7 ( 67.7d
17.3 ( 0.6c
6.8 ( 2.3
23.4 ( 8.0c
41.0 ( 16.4
41.8 ( 18.9c
16.9 ( 4.9c
2.8 ( 1.3
3.0 ( 1.0
33.5 ( 8.5
11.0 ( 3.2
7.6 ( 1.6
16.8 ( 4.2
4.0 ( 3.0
24.8 ( 5.0
19.2 ( 8.1
16.3 ( 3.0c
15.4 ( 2.5
13.2 ( 3.6
13.3 ( 2.7b
14.6 ( 5.6c
a Control corresponds to mice injected with vehicle and maintained under the same condition as the treated mice. b p < 0.05. c p < 0.01. d p < 0.005.
e Endogenous level of IL-6: 5-10 pg/mL.
1630 J. Org. Chem., Vol. 72, No. 5, 2007

TDM/TDCM Analogues and Their IL-6 LeVel Enhancement
1
2935, 2858, 1740 cm-1; H NMR (300 MHz in CDCl3) δ 0.88
) 11.4 Hz), 4.60 (1H, d, J ) 11.4 Hz), 7.24-7.36 (5H, m); 13C
NMR (75 MHz in CDCl3) δ 14.1, 22.7, 24.7, 27.6, 29.4, 29.6 (2C),
29.7 (many), 31.0, 31.9, 49.7, 72.0, 79.9, 127.5, 127.8, 128.2, 138.1,
180.6; CIMS m/z (%) 588 (30, M+ + H+), 569 (25), 481 (100),
434 (40); HRMS (CI+) m/z calcd for C39H71O3 (M+ + H+),
587.5403; found, 587.5397.
(3H, t, J ) 6.9 Hz), 1.25 (22H, m), 1.47 (9H, s), 1.58 (4H, m),
2.51 (2H, t, J ) 7.5 Hz), 3.33 (2H, s); 13C NMR (75 MHz in CDCl3)
δ 14.1, 22.6, 23.4, 27.9, 29.0, 29.3, 29.4 (many), 29.6 (2C), 31.9,
42.9, 50.6, 81.7, 166.5, 203.4; CIMS m/z (%) 355 (2, M+ + H+),
339 (20), 299 (100); HRMS (CI+) m/z calcd for C22H43O3 (M+
+
1), 355.3212; found, 355.3215.
6,6′-Bis-O-[(2R,3R)-3-benzyloxy-2-tetradecyloctadecanoyl]-
2,3,4,2′,3′,4′-hexabenzyl-R,R’-trehalose (16c) and 6-O-[(2R,3R)-
3-Benzyloxy-2-tetradecyloctadecanoyl]-2,3,4,2′,3′,4′-hexabenzy]-
R,R′-trehalose (19c). A mixture of carboxylic acid 11c (291 mg,
49.6 µmol), trehalose derivative 15 (292 mg, 33.0 µmol), EDCI
(190 mg, 99.0 µmol), DMAP (20.2 mg, 17.0 µmol), and 4 Å
molecular sieves powder in CH2Cl2 (4 mL) was stirred for 7 h at
room temperature. The filtrate was concentrated, and the resulting
material was subjected to column chromatography on silica gel
using hexane and ethyl acetate (20:1 to 5:1) as the eluent to give
6,6′-bis-O-[(2R,3R)-3-benzyloxy-2-tetradecyloctadecanoyl]-2,3,
4,2′,3′,4′-hexabenzyl-R,R′-trehalose (16c) (333 mg, 50%) and 6-O-
[(2R,3R)-3-benzyloxy-2-tetradecyloctadecanoyl]-2,3,4,2′,3′,4′-hexa-
benzy]-R,R′-trehalose (19c) (201 mg, 42%) as colorless syrups.
16c: [R]21D +43.5 (c 1.0, CHCl3); FT-IR (neat) 3090, 3063, 3029,
(R)-tert-Butyl-3-hydroxyoctadecanoate (9c). â-Ketoester 8c
(1.78 g, 5.02 mmol) and dichloro[(R)-(+)-2,2′-bis(diphenylphos-
phino)-1,1′-binaphthyl]ruthenium(II) (hereafter abbreviated to (R)-
BINAP-RuCl2) (3.97 mg, 0.50 µmol) were dissolved in MeOH
(3 mL) and deoxygenated by the freeze/melt method under an argon
atmosphere. The mixture was stirred for 72 h under H2 (70 kgf/
cm2) using a TAIATSU SUS316 microautoclave. The concentrated
mixture was subjected to column chromatography on silica gel using
hexane and ethyl acetate (25:2) as an eluent to give (R)-tert-butyl-
3-hydroxyoctadecanoate (9c) (1.68 g, 94%) as a colorless syrup.
9c: [R]19D -15.2 (c 1.1, CHCl3); FT-IR (neat) 3452, 2923, 2851,
1718 cm-1; H NMR (300 MHz in CDCl3) δ 0.88 (3H, t, J ) 6.9
1
Hz), 1.25 (24H, m), 1.42 (4H, m), 1.47 (9H, s), 2.31 (1H, dd, J )
16.2 and 8.7 Hz), 2.43 (1H, dd, J ) 16.2 and 3.3 Hz), 3.12 (OH,
d, J ) 3.6 Hz), 3.95 (1H, m); 13C NMR (75 MHz in CDCl3) δ
14.1, 22.6, 25.4, 28.0, 29.3, 29.5, 29.6 (many), 31.9, 36.4, 42.3,
68.0, 81.0, 172.5; CIMS m/z (%) 357 (1, M+ + H+), 343 (5), 301
(100), 283 (55); HRMS (CI+) m/z calcd for C22H45O3 (M+ + H+),
357.3369; found, 357.3383.
1
2944, 2868, 1742 cm-1; H NMR (300 MHz in CDCl3) δ 0.88
(12H, t, J ) 6.9 Hz), 1.12-1.61 (108H, m), 2.66 (2H, ddd, J )
10.2, 6.6, and 3.2 Hz), 3.49 (2H, dd, J ) 9.6 and 3.6 Hz), 3.56
(2H, t, J ) 9.3 Hz), 3.62 (2H, m), 4.01 (2H, t, J ) 9.6 Hz), 4.09
(2H, dd, J ) 12.3 and 3.0 Hz), 4.17 (4H, m), 4.45 (4H, s), 4.51
(2H, d, J ) 10.5 Hz), 4.61 (2H, d, J ) 12.3 Hz), 4.67 (2H, d, J )
12.3 Hz), 4.83 (2H, d, J ) 10.5 Hz), 4.84 (2H, d, J ) 10.8 Hz),
4.98 (2H, d, J ) 10.8 Hz), 5.10 (2H, d, J ) 3.6 Hz), 7.16-7.36
(40H, m); 13C NMR (50 MHz in CDCl3) δ 14.1, 22.7, 24.9, 27.7,
27.8, 29.4 (2C), 29.7 (many), 31.1, 31.9, 49.8, 62.2, 69.1, 71.9,
72.9, 75.2, 75.6, 77.7, 79.6, 80.2, 81.5, 93.9, 127.4, 127.5, 127.7,
127.8, 127.9 (2C), 128.2, 128.3, 128.4 (2C), 137.8, 137.9, 138.6
(2R,3R)-tert-Butyl-3-hydroxy-2-tetradecyloctadecanoate (10c).
To a stirred solution of diisopropylamine (668 mg, 6.60 mmol) in
THF (5 mL) was added MeLi (2.2 M ether solution, 2.95 mL, 6.50
mmol) at -78 °C and stirred at 0 °C for 30 min. Then the mixture
was cooled to -48 °C, and a solution of 9c (713 mg, 2.00 mmol)
in THF (2 mL) was added. After stirring for 30 min at the same
temperature, HMPA (2 mL) and a solution of 1-iododecane (973
mg, 3.00 mmol) in THF (2 mL) were added, and the mixture was
stirred for 6 h at -48 °C. To this was added saturated NH4Cl, which
was then extracted with ether. The dried and concentrated extract
was subjected to column chromatography on silica gel using hexane
and ethyl acetate (20:1) as an eluent to give (2R,3R)-tert-butyl-3-
hydroxy-2-tetradecyloctadecanoate (10c) (607 mg, 55%) as a
(2C), 174.3; HRMS (TOF) m/z calcd for C132H194O15Na (M+
+
Na+), 2042.4315; found, 2042.4277. 19c: [R]19 +75.6 (c 1.0,
D
CHCl3); FT-IR (neat) 3501, 3088, 3063, 3031, 2925, 2853, 1947,
1
1870, 1807, 1736 cm-1; H NMR (300 MHz in CDCl3) δ 0.88
(6H, t, J ) 7.2 Hz), 1.12-1.64 (54H, m), 2.65 (1H, ddd, J ) 10.2,
6.9, and 3.3 Hz), 3.55 (6H, m), 3.61 (1H, m), 4.04 (3H, m), 4.14
(1H, dd, J ) 12.9 and 3.6 Hz), 4.21 (2H, m), 4.46 (2H, s), 4.53
(1H, d, J ) 10.5 Hz), 4.65 (4H, m), 4.71 (1H, d, J ) 12.0 Hz),
4.85 (2H, d, J ) 11.7 Hz), 4.86 (2H, d, J ) 10.8 Hz), 4.98 (1H, br
d, J ) 10.8 Hz), 5.08 (1H, d, J ) 3.6 Hz), 5.14 (1H, d, J ) 3.6
Hz), 7.17-7.38 (35H, m); 13C NMR (50 MHz in CDCl3) δ 14.1,
22.9, 27.7, 27.9, 29.4, 29.5, 29.7 (many), 31.1, 31.9, 49.8, 61.5,
62.2, 69.1, 71.2, 71.9, 73.0, 73.1, 75.0, 75.2, 75.6 (2C), 77.4, 77.8,
79.5, 79.7, 80.3, 81.5, 93.7, 93.9, 127.4, 127.6, 127.7, 127.9 (3C),
128.1, 128.2, 128.4 (2C), 137.8, 138.0, 138.2, 138.7 (2C), 138.8,
174.4; HRMS (TOF) m/z calcd for C93H126O13Na (M+ + Na+),
1473.9096; found, 1473.9141.
colorless syrup. 10c: [R]20 +5.3 (c 1.0, CHCl3); FT-IR (neat)
D
3493, 2925, 2853, 1730, 1708 cm-1; 1H NMR (300 MHz in CDCl3)
δ 0.88 (6H, t, J ) 6.9 Hz), 1.25 (52H, m), 1.47 (9H, s), 1.62 (2H,
m), 2.29 (1H, dt, J ) 9.0 and 5.1 Hz), 2.65 (OH, d, J ) 8.4 Hz),
3.59 (1H, m); 13C NMR (75 MHz in CDCl3) δ 14.1, 22.7, 25.8,
27.3, 28.1, 29.4, 29.5 (2C), 29.6, 29.7 (many), 29.8, 31.9, 35.8,
51.2, 72.5, 81.0, 175.3; CIMS m/z (%) 554 (5, M+ + H+), 498
(70), 367 (100), 285 (55), 256 (60); HRMS (CI+) m/z calcd for
C36H73O3 (M+ + H+), 553.5560; found, 553.5531.
(2R,3R)-3-Benzyloxy-2-tetradecyloctadecanoic Acid (11c). To
a solution of 10c (553 mg, 1.00 mmol) in CH2Cl2 (3 mL) were
added Et3N (304 mg, 3.00 mmol) and TMSCl (162 mg, 1.50 mmol),
and the mixture was stirred for 30 min at room temperature. After
addition of brine, the organic materials were extracted with CH2-
Cl2. The dried and concentrated extract was further dried by
azeotropic reflux with CH2Cl2 using a Soxhlet-type apparatus
passing through a 4 Å molecular sieves column, and it cooled to
-78 °C. To the mixture were successively added benzaldehyde
(159 mg, 1.50 mmol), triethylsilane (174 mg, 1.50 mmol), and
trimethylsilyltriflate (111 mg, 50.0 mmol). After stirring for 15 min
at -78 °C and then at room temperature for 2 h, the reaction was
quenched by the addition of saturated NaHCO3, and the organic
materials were extracted with ether. The dried and concentrated
extract was subjected to column chromatography on silica gel using
hexane and ethyl acetate (10:1) to give (2R,3R)-3-benzyloxy-2-
tetradecyloctadecanoic acid (11c) (562 mg, 96%) as a colorless
syrup. 11c: [R]19D +1.0 (c 1.0, CHCl3); FT-IR (neat) 3087, 3065,
6-O-[(2R,3R)-3-Benzyloxy-2-tetradecyloctadecanoyl]-6′-O-
[(2S,3S)-3-benzyloxy-2-tetradecyloctadecanoyl]-2,3,4,2′,3′,4′-
hexabenzyl-R,R′-trehalose (18c). A mixture of carboxylic acid 14c
(48.0 mg, 8.20 µmol), monoester 19c (99.0 mg, 6.80 µmol), EDCI
(19.6 mg, 10.2 µmol), DMAP (4.15 mg, 3.40 µmol), and 4 Å
molecular sieves powder in CH2Cl2 (1 mL) was stirred for 7 h at
room temperature. After filtration and concentration, the residue
was subjected to column chromatography on silica gel using hexane
and ethyl acetate (20:1) as an eluent to give 6-O-[(2R,3R)-3-
benzyloxy-2-tetradecyloctadecanoyl]-6′-O-[(2S,3S)-3-benzyloxy-2-
tetradecyloctadecanoyl]-2,3,4,2′,3′,4′-hexabenzyl-R,R′-trehalose (18c)
(121 mg, 88%) as a colorless syrup. 18c: [R]19 +49.0 (c 1.1,
D
CHCl3); FT-IR (neat) 3088, 3064, 3031, 2929, 2855, 1946, 1870,
1
1805, 1739 cm-1; H NMR (600 MHz in CDCl3) δ 0.88 (12H, t,
J ) 7.8 Hz), 1.12-62 (108H, m), 2.65 (1H, ddd, J ) 10.8, 7.2,
and 3.6 Hz), 2.69 (1H, ddd, J ) 10.8, 7.2, and 3.6 Hz), 3.48 (1H,
dd, J ) 9.6 and 3.6 Hz), 3.51 (1H, dd, J ) 9.6 and 3.6 Hz), 3.55
(1H, t, J ) 9.6 Hz), 3.56 (1H, t, J ) 9.6 Hz), 3.62 (2H, m), 4.00
(1H, t, J ) 9.6 Hz), 4.01 (1H, t, J ) 9.6 Hz), 4.08 (2H, m), 4.18
3031, 2931, 2854, 1707 cm-1; H NMR (300 MHz in CDCl3) δ
0.88 (6H, t, J ) 7.2 Hz), 1.25 (50H, m), 1.60 (4H, m), 2.65 (1H,
dt, J ) 10.2 and 5.7 Hz), 3.64 (1H, q, J ) 6.0 Hz), 4.52 (1H, d, J
1
J. Org. Chem, Vol. 72, No. 5, 2007 1631

Nishizawa et al.
(4H, m), 4.44 (1H, d, J ) 11.4 Hz), 4.45 (2H, s), 4.47 (1H, d, J )
11.4 Hz), 4.51 (1H, d, J ) 10.2 Hz), 4.61 (1H, d, J ) 11.4 Hz),
4.64 (1H, d, J ) 12.0 Hz), 4.66 (1H, t, J ) 12.0 Hz), 4.68 (1H, t,
J ) 12.0 Hz), 4.74 (1H, d, J ) 10.8 Hz), 4.83 (4H, m), 4.97 (1H,
d, J ) 11.4 Hz), 4.98 (1H, d, J ) 10.8 Hz), 5.08 (1H, d, J ) 3.6
Hz), 5.11 (1H, d, J ) 3.6 Hz), 7.13-7.36 (40H, m); 13C NMR
(150 ΜΗz in CDCl3) δ 14.1, 22.7, 24.9 (2C), 27.5, 27.7, 27.8 (2C),
29.4, 29.5 (2C), 29.6 (many), 29.7 (2C), 29.8 (2C), 31.0, 31.1, 31.9,
49.6, 49.8, 62.1, 62.2, 69.1, 71.8, 71.9, 72.9, 73.0, 75.2, 75.6, 75.7,
77.7, 79.5, 79.7, 80.0, 80.3, 81.5, 94.0, 127.4 (2C), 127.6, 127.7,
127.8, 127.9 (2C), 128.0, 128.2, 128.4 (4C), 137.8, 138.0 (2C),
138.6, 138.7, 174.1, 174.4; HRMS (TOF) m/z calcd for C132H194O15-
Na (M+ + Na), 2042.4315; found, 2042.4372.
6,6′-Bis-O-[(2R,3R)-3-hydroxy-2-tetradecyloctadecanoyl]-R,R′-
trehalose (2c). A mixture of diester 16c (233 mg, 11.5 µmol) and
Pd(OH)2 on C (16.1 mg, 2.30 µmol) in CHCl3 and MeOH (1:1) (4
mL) was stirred at room temperature for 7 h under H2 (1 atm).
After filtration, the concentrated residue was subjected to column
chromatography on silica gel using CH2Cl2/MeOH (10:1) as the
eluent to give 6,6′-bis-O-[(2R,3R)-3-hydroxy-2-tetradecyloctade-
canoyl]-R,R′-trehalose (2c) (141 mg, 94%) as a colorless syrup.
2c: [R]24D +69.1 (c 1.0, CHCl3); FT-IR (neat) 3385, 2928, 2855,
1730 cm-1; 1H NMR (600 MHz in C5D5N) δ 0.87 (12H, t, J ) 7.1
Hz), 1.28-1.88 (106H, m), 1.98 (2H, m), 2.91 (2H, m), 4.16 (2H,
t, J ) 9.5 Hz), 4.21 (2H, m), 4.29 (2H, dd, J ) 9.5 and 3.7 Hz),
4.69 (2H, t, J ) 9.5 Hz), 4.86 (2H, dd, J ) 11.7 and 5.7 Hz), 5.15
(4H, m), 5.86 (2H, d, J ) 3.7 Hz); 13C NMR (50 MHz in C5D5N)
δ 14.4, 23.0, 26.4, 28.2, 29.5, 29.7 (many), 30.4, 32.2, 35.5, 54.0,
64.4, 71.6, 72.4, 72.6, 73.4, 74.8, 96.1, 175.1; FAB-MS m/z (%)
1322 (5, M+ + Na+), 413 (100), 329 (80), 307 (100); HRMS
(FAB+) m/z calcd for C76H146O15Na (M+ + Na+), 1322.0550; found,
1322.0530.
tert-Butyl-9-methyl-3-oxo-8-decenoate (27). To a stirred sus-
pension of NaH (1.68 g, 70.2 mmol) in THF (250 mL) was
dropwise added tert-butyl acetoacetate (7) (9.25 g, 58.5 mmol) at
0 °C, and the mixture was stirred for 10 min. After addition of
n-BuLi (1.6 M hexane solution, 40.2 mL, 64.4 mmol), the mixture
was stirred for an additional 30 min at the same temperature. To
the resulting mixture was added 6-iodo-2-methyl-2-hexene (26)
(19.6 g, 87.8 mmol), and it was stirred for 30 min at 0 °C. After
addition of a saturated NH4Cl solution, the organic materials were
extracted with ether, and the dried and concentrated material was
subjected to column chromatography on silica gel using hexane
and ethyl acetate (20:1) as an eluent to give tert-butyl-9-methyl-
3-oxo-8-decenoate (27) (11.0 g, 74%) as a colorless syrup. 27: FT-
IR (neat) 2978, 2931, 2858, 1739, 1717 cm-1; 1H NMR (300 MHz
in CDCl3) δ 1.35 (2H, m), 1.47 (9H, s), 1.59 (3H, br s), 1.60 (2H,
m), 1.68 (3H, br s), 1.98 (2H, q, J ) 7.5 Hz), 2.52 (2H, t, J ) 7.2
Hz), 3.33 (2H, s), 5.09 (1H, m); 13C NMR (75 MHz in CDCl3) δ
17.7, 23.1, 25.7, 27.7, 28.0, 28.3, 29.3, 42.9, 50.7, 81.8, 124.1,
131.7, 166.5; CIMS m/z (%) 255 (1, M+ + H+), 199 (40), 181
(100); HRMS (CI+) m/z calcd for C15H27O3 (M+ + H+), 255.1960;
found, 255.1967.
M+ + H+), 201 (100), 185 (12); HRMS (CI+) m/z calcd for
C15H29O3 (M+ + H+), 257.2116; found, 257.2113.
(2R,3R)-tert-Butyl-2-icosy-3-hydroxy-9-methyl-8-decenoate (29).
To a stirred solution of diisopropylamine (5.21 g, 51.5 mmol) in
THF (21 mL) was added MeLi (2.2 M ether solution, 22.6 mL,
49.9 mmol) at -78 °C, and the mixture was stirred at 0 °C for 30
min. The mixture was cooled to -48 °C, and a solution of 28 (4.00
g, 15.6 mmol) in THF (10 mL) was added. After stirring for 30
min at the same temperature, HMPA (18 mL) and a solution of
1-iodoicosane (9.56 g, 23.4 mmol) in THF (14 mL) were added,
and the reaction mixture was stirred for 6 h at -48 °C. After
addition of saturated NH4Cl, the organic materials were extracted
with ether. The dried and concentrated extract was subjected to
column chromatography on silica gel using hexane and ethyl acetate
(25:1) as an eluent to give (2R,3R)-tert-butyl-2-icosy-3-hydroxy-
9-methyl-8-decenoate (29) (4.60 g, 55%) as a colorless syrup. 29:
[R]20D +7.1 (c 1.3, CHCl3); FT-IR (neat) 3509, 2934, 2856, 1728,
1
1707 cm-1; H NMR (300 MHz in CDCl3) δ 0.88 (3H, t, J ) 6.9
Hz), 1.10-1.41 (44H, m), 1.47 (9H, s), 1.59 (3H, br s), 1.68 (3H,
d, J ) 0.9 Hz), 1.98 (2H, m), 2.30 (1H, dt, J ) 8.7 and 5.4 Hz),
2.65 (OH, d, J ) 8.7 Hz), 3.60 (1H, m), 5.11 (1H, m); 13C NMR
(75 MHz in CDCl3) δ 14.1, 17.6, 22.7, 25.5, 25.7, 27.3, 27.9, 28.1,
29.3, 29.4, 29.5, 29.7 (many), 29.8, 31.9, 35.7, 51.3, 72.4, 80.9,
124.6, 131.2, 175.2; CIMS m/z (%) 537 (1, M+ + H+), 506 (10),
463 (100); HRMS (CI+) m/z calcd for C35H69O3 (M+ + H+),
537.5240; found, 537.5247.
(2R,3R)-tert-Butyl-3-benzyloxy-2-icosy-9-methyl-8-deceno-
ate (30). To a solution of 29 (2.60 g, 4.84 mmol) in CH2Cl2 (25
mL) were added Et3N (1.47 g, 14.5 mmol) and then TMSCl (788
mg, 7.26 mmol), and the mixture was stirred for 30 min at room
temperature. After addition of brine, the organic materials were
extracted with CH2Cl2. The dried and concentrated extract was
further dried by azeotropic reflux with CH2Cl2 using a Soxray-
type apparatus passing through a 4 Å molecular sieves column,
and it was cooled to -78 °C. To the mixture were successively
added benzaldehyde (770 mg, 7.26 mmol), triethylsilane (844 mg,
7.26 mol), and trimethylsilyltriflate (807 mg, 3.63 mmol). After
stirring for 10 min at -78 °C, the reaction was quenched by the
addition of saturated NaHCO3, and the organic materials were
extracted with ether. The dried and concentrated material was
subjected to column chromatography on silica gel using hexane
and ethyl acetate (10:1) as an eluent to give (2R,3R)-tert-butyl-3-
benzyloxy-2-icosy-9-methyl-8-decenoate (30) (2.91 g, 96%) as a
colorless syrup. 30: [R]20D +3.5 (c 2.0, CHCl3); FT-IR (neat) 3089,
1
3064, 3032, 2930, 2854, 1947, 1871, 1806, 1726 cm-1; H NMR
(300 MHz in CDCl3) δ 0.88 (3H, t, J ) 6.9 Hz), 1.18-1.59 (44H,
m), 1.43 (9H, s), 1.59 (3H, br s), 1.68 (3H, d, J ) 1.2 Hz), 1.96
(2H, m), 2.55 (1H, ddd, J ) 11.1, 7.5, and 3.6 Hz), 3.63 (1H, dt,
J ) 6.9 and 3.6 Hz), 4.50 (2H, s), 5.09 (1H, m), 7.27-7.38 (5H,
m); 13C NMR (75 MHz in CDCl3) δ 14.1, 17.7, 22.7, 24.3, 25.7,
27.5, 27.8, 27.9, 28.1, 29.4, 29.5 (2C), 29.6 (many), 29.7, 30.0,
30.7, 31.9, 50.3, 71.8, 80.0, 80.4, 124.7, 127.4, 127.8, 128.1, 131.2,
138.7, 174.0; CIMS m/z (%) 627 (3, M+ + H+), 596 (7), 571 (100);
HRMS (CI+) m/z calcd for C42H75O3 (M+ + H+), 627.5716; found,
627.5721.
(R)-tert-Butyl-3-hydroxy-9-methyl-8-decenoate (28). â-Ke-
toester 27 (5.00 g, 19.7 mmol) and (R)-BINAP-RuCl2 (15.5 mg,
1.95 µmol) were dissolved in MeOH (3 mL) and deoxygenated by
the freeze/melt method under an argon atmosphere. The mixture
was stirred for 36 h under H2 (50 kgf/cm2) using a TAIATSU
SUS316 microautoclave. The concentrated mixture was subjected
to column chromatography on silica gel using hexane and ethyl
acetate (25:2) as an eluent to give (R)-tert-butyl-3-hydroxy-9-
methyl-8-decenoate (28) (4.10 g, 81%) as a colorless syrup. 28:
[R]20D -17.6 (c 1.4, CHCl3); FT-IR (neat) 3454, 2978, 2931, 2857,
(2R,3R)-tert-Butyl-2-(1-benzyloxy-6-oxohexyl)docosanoate (31).
Ozone gas was bubbled through a solution of 30 (1.30 g, 2.07
mmol) in CHCl3 (60 mL) for 30 min at -48 °C. Excess ozone was
evacuated by bubbling argon, and to this was added Me2S (1,29 g,
20.7 mmol) at -48 °C. After warming to room temperature,
saturated NaHCO3 was added, and organic materials were extracted
with CH2Cl2. The dried and concentrated material was subjected
to column chromatography on silica gel using hexane and ethyl
acetate (10:1) as an eluent to give (2R,3R)-tert-butyl-2-(1-benzy-
loxy-6-oxohexyl)docosanoate (31) (871 mg, 70%) as a colorless
syrup. 31: [R]19D +6.7 (c 1.9, CHCl3); FT-IR (neat) 3089, 3064,
1
1731 cm-1; H NMR (300 MHz in CDCl3) δ 1.34 (6H, m), 1.47
(9H, s), 1.60 (3H, br s), 1.68 (3H, d, J ) 1.2 Hz), 1.98 (2H, m),
2.31 (1H, dd, J ) 16.5 and 9.0 Hz), 2.43 (1H, dd, J ) 16.5 and
3.0 Hz), 3.07 (OH, d, J ) 3.9 Hz), 3.94 (1H, m), 5.10 (1H, m);
13C NMR (75 MHz in CDCl3) δ 17.5, 25.0, 25.6, 27.8, 28.0, 29.7,
36.4, 42.3, 68.0, 81.0, 124.5, 131.2, 172.4; CIMS m/z (%) 257 (20,
1
3032, 2927, 2853, 1728 cm-1; H NMR (300 MHz in CDCl3) δ
0.88 (3H, t, J ) 7.2 Hz), 1.18-1.43 (40H, m), 1.43 (9H, s), 1.56
(4H, m), 2.40 (2H, dt, J ) 7.2 and 1.8 Hz), 2.55 (1H, ddd, J )
1632 J. Org. Chem., Vol. 72, No. 5, 2007

TDM/TDCM Analogues and Their IL-6 LeVel Enhancement
10.8, 7.2, and 3.6 Hz), 3.63 (1H, dt, J ) 6.9 and 3.6 Hz), 4.47
(1H, d, J ) 11.1 Hz), 4.53 (1H, d, J ) 11.1 Hz), 7.28-7.34 (5H,
m), 9.74 (1H, t, J ) 1.8 Hz); 13C NMR (75 MHz in CDCl3) δ
14.0, 22.1, 22.6, 24.2, 27.5, 27.6, 28.0, 29.3, 29.4 (2C), 29.5, 29.6
(many), 30.4, 31.8, 43.7, 50.0, 71.8, 80.0, 80.1, 127.4, 127.7, 128.1,
138.5, 173.7, 202.1; CIMS m/z (%) 601 (2, M+ + H+), 584 (5),
6,6′-Bis-O-[(2R,3R)-2-icosyl-3-hydroxytetracosanoyl]-R,R′-tre-
halose (2d). A mixture of diester 40 (199 mg, 8.49 µmol) and Pd-
(OH)2 on C (11.9 mg, 1.67 µmol) in CHCl3 and MeOH (3:2) (8
mL) was stirred at room temperature for 7 h under H2 (1 atm).
Filtrated and concentrated residue was subjected to column chro-
matography on silica gel using CH2Cl2 and MeOH (10:1) as the
eluent to give 6,6′-bis-O-[(2R,3R)-2-icosyl-3-hydroxytetracosanoyl]-
527 (70), 437 (100); HRMS (CI+) m/z calcd for C39H69O4 (M+
+
H+), 601.5196; found, 601.5195.
R,R′-trehalose (2d) (128 mg, 92%) as a colorless syrup. 2d: [R]20
D
+58.5 (c 0.3, CHCl3); FT-IR (neat) 3396, 2919, 2849, 1722 cm-1
;
(2R,3R)-tert-Butyl-3-benzyloxy-2-icosyl-8-tetracosenoate (32).
To a solution of hexadecanytriphenylphosphonium iodide (1.07 g,
1.74 mmol) in THF (7 mL) was added n-BuLi (1.6 M hexane
solution, 1.00 mL, 1.60 mmol) at -78 °C, and the mixture was
stirred at room temperature for 30 min and cooled to 0 °C. To this
was added a solution of 31 (871 mg, 1.45 mmol) in THF (5 mL),
and the mixture was stirred for an additional 30 min. After addition
of saturated NH4Cl, the organic materials were extracted with ether.
The dried and concentrated material was subjected to column
chromatography on silica gel using hexane and ethyl acetate (30:
1) as the eluent to give (2R,3R)-tert-butyl-3-benzyloxy-2-icosyl-
8-tetracosenoate (32) (1.15 g, 98%) as a colorless syrup. 32: FT-
IR (neat) 3088, 3064, 3030, 3004, 2935, 2857, 1943, 1866, 1801,
1H NMR (300 MHz in C5D5N) δ 0.88 (12H, t, J ) 7.2 Hz), 1.14-
2.06 (156H, m), 2.91 (2H, ddd, J ) 10.8, 6.6, and 4.2 Hz), 4.15
(2H, t, J ) 9.6 Hz), 4.21 (2H, m), 4.28 (2H, dd, J ) 9.3 and 3.9
Hz), 4.69 (2H, t, J ) 9.3 Hz), 4.85 (2H, dd, J ) 11.7 and 5.7 Hz),
5.14 (4H, m) 5.85 (2H, d, J ) 3.6 Hz); 13C NMR (75 MHz in
C5D5N) δ 14.2, 22.9, 26.3, 28.1, 29.4, 29.6, 29.8, 29.9, 30.0 (many),
32.1, 35.4, 53.8, 64.3, 71.5, 72.2, 72.5, 73.3, 74.6, 95.9, 175.1;
HRMS (TOF) m/z calcd for C100H194O15Na (M+ + Na+), 1658.4315;
found, 1658.4276.
Determination of the IL-6 Level on Mice Sera.27 Briefly, a
monoclonal antibody specific for mouse IL-6 was precoated onto
a microplate. Recombinant IL-6 standards (ranging from 0 to 500
pg/mL), sera from TDCM-treated mice (five animals for each time),
and sera from control mice (five animals for each time), corre-
sponding to mice injected with the vehicle and maintained under
the same condition as the treated mice, were pipetted into wells,
and sample IL-6 was bound by the immobilized antibody. After
washing to remove any unbound substances, a horseradish peroxi-
dase-linked polyclonal antibody specific for mouse IL-6 was added
to the well. Following a wash to remove any unbound antibody, a
substrate solution composed of hydrogen peroxide and tetrameth-
ylbenzidine was added to the well. The enzyme reaction yielded a
blue product that turned yellow when the reaction was stopped.
The intensity of the color was proportional to the amount of mouse
IL-6 bound in the initial step. The sample values were determined
by comparison with a standard curve prepared with recombinant
IL-6 (linearity assessed from 0 to 500 pg/mL; R2 ) 0.9991). For
statistical evaluation, sample values were compared to the same
day control values.
1
1730 cm-1; H NMR (300 MHz in CDCl3) δ 0.88 (6H, t, J ) 6.9
Hz), 1.18-1.62 (79H, m), 2.01 (4H, m), 2.55 (1H, ddd, J ) 11.1,
7.5, and 3.6 Hz), 3.63 (1H, dt, J ) 7.2 and 3.6 Hz), 4.51 (2H, s),
5.35 (2H, m), 7.21-7.36 (5H, m); 13C NMR (75 MHz in CDCl3)
δ 14.1, 22.7, 24.3, 27.2, 27.3, 27.5, 27.8, 28.1, 29.4, 29.5 (2C),
29.6, 29.7 (many), 29.8, 29.9, 30.7, 31.9, 50.3, 71.9, 80.1, 80.4,
127.3, 127.7, 128.1, 129.5, 130.1, 138.7, 173.9; FAB-MS m/z (%)
832 (0.1, M+ + H+), 107 (10), 91 (100); HRMS (FAB+) m/z calcd
for C55H100O3Na (M+ + Na+), 831.7570; found, 831.7601.
(2R,3R)-3-Benzyloxy-2-icosyl-8-tetracosenoic Acid (33). To a
solution of 32 (1.00 g, 1.24 mmol) in CH2Cl2 (4 mL) was added
trimethylsilyltriflate (55.0 mg, 24.3 µmol) at room temperature, and
the mixture was stirred for 2 h at the same temperature. After
addition of saturated NaHCO3, organic materials were extracted
with CH2Cl2. The dried and concentrated material was subjected
to column chromatography on silica gel using hexane and ethyl
acetate (10:1) as an eluent to give (2R,3R)-3-benzyloxy-2-icosyl-
8-tetracosenoic acid (33) (831 mg, 89%) as an amorphous solid.
33: FT-IR (neat) 3087, 3064, 3030, 3004, 2928, 2853, 1944, 1866,
1
1806, 1704 cm-1; H NMR (300 MHz in CDCl3) δ 0.88 (6H, t, J
Acknowledgment. This research was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and a
MEXT.HAITEKU, 2002-2007.
) 6.9 Hz), 1.14-1.72 (70H, m), 2.01 (4H, m), 2.65 (1H, dt, J )
10.8 and 5.1 Hz), 3.64 (1H, q, J ) 5.1 Hz), 4.52 (1H, d, J ) 11.4
Hz), 4.59 (1H, d, J ) 11.4 Hz), 5.37 (2H, m), 7.20-7.35 (5H, m);
13C NMR (75 MHz in CDCl3) δ 14.1, 22.7, 24.5, 27.1, 27.3, 27.7,
29.3, 29.4 (2C), 29.6 (2C), 29.7 (2C), 29.8 (many), 30.9, 31.0, 31.9,
32.4, 32.6, 49.7, 72.1, 79.9, 127.6, 127.8, 128.3, 129.4, 130.2, 138.1,
180.1; FAB-MS m/z (%) 776 (50, M+ + Na+), 176 (28), 91 (100);
HRMS (FAB+) m/z calcd for C51H92O3Na (M+ + Na+), 775.6944;
found, 775.6923.
Supporting Information Available: 1H and 13C NMR spectra
of all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO062018J
J. Org. Chem, Vol. 72, No. 5, 2007 1633