Synthesis of highly water-soluble fibrate derivatives via BGLation

Article abstract of DOI:10.1016/j.bmcl.2012.08.057

Three water-soluble fibrates (fenofibrate, bezafibrate and chlofibrate) conjugated with a symmetrically branched glyceryl trimer (BGL003) were synthesized, and an evaluation of the fenofibrate-BGL003 conjugate as a candidate for anti-hyperlipemia drug was carried out using rats. The water-solubility of the fenofibrate-BGL003 conjugate was several thousand times greater than that of the original fenofibrate. The lipid-lowering effects of the fenofibrate-BGL003 conjugate were as strong as those of the same grams of fenofibrate. The actual active species of fenofibrate, fenofibric acid, was detected in rats' blood, but neither the fenofibrate-BGL003 conjugate nor fenofibrate was detected, probably due to enzymatic hydrolysis of the ester bond. The plasma concentration of fenofibric acid derived from the fenofibrate-BGL003 conjugate was five times higher than that derived from fenofibrate 4 h after administration.

Full text of DOI:10.1016/j.bmcl.2012.08.057

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6425–6428
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of highly water-soluble fibrate derivatives via BGLation
Hisao Nemoto a, , Masaki Kamiya a, Aki Nakamoto a, Tsuyoshi Matsushita a, Kosuke Matsumura a,
⇑
Hatsuhiko Hattori a, Tomoyuki Kawamura a, Chiaki Taoka b, Shinji Abe c, Keisuke Ishizawa b, Licht
Miyamoto a,b, Koichiro Tsuchiya b
a Department of Pharmaceutical Chemistry, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
b Department of Medical Pharmacology, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
c Practice Room for Clinical Pharmacy, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2012
Revised 18 July 2012
Accepted 16 August 2012
Available online 23 August 2012
Three water-soluble fibrates (fenofibrate, bezafibrate and chlofibrate) conjugated with a symmetrically
branched glyceryl trimer (BGL003) were synthesized, and an evaluation of the fenofibrate–BGL003 con-
jugate as a candidate for anti-hyperlipemia drug was carried out using rats. The water-solubility of the
fenofibrate–BGL003 conjugate was several thousand times greater than that of the original fenofibrate.
The lipid-lowering effects of the fenofibrate–BGL003 conjugate were as strong as those of the same grams
of fenofibrate. The actual active species of fenofibrate, fenofibric acid, was detected in rats’ blood, but nei-
ther the fenofibrate–BGL003 conjugate nor fenofibrate was detected, probably due to enzymatic hydro-
lysis of the ester bond. The plasma concentration of fenofibric acid derived from the fenofibrate–BGL003
conjugate was five times higher than that derived from fenofibrate 4 h after administration.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Anti-hyperpemia
Water-solubility
Fibrates
Permiability
Fibrates1 such as fenofibrate (1a), bezafibrate (1b), and chlofi-
brate (1c) have occupied a large market share among anti-hyperli-
pemia drugs in recent years (Fig. 1).
These fibrates must be taken postprandially because they are
extremely water-insoluble, and their permeability via the alimen-
tary canal increases after a meal. Postprandial administration also
helps to achieve a reproducibly stable blood drug concentration. A
standard dosage of the traditional form of 1a (LipantilÒ) in Japan
was 200–300 mg per day.2 However, recent formulation technol-
ogy consisting of fine pulverization (producing a drug of brand
name is LipidilÒ)3 allowed reduction of the dosage by two-thirds
compared to the traditional formulation.
Figure 1. Typical commercially available fibrates.
Such improvements in formulation are meritorious. However, it
may be proved, paradoxically, that the extreme water-insolubility of
1a inhibits its absorption via the alimentary canal. It is contrastive
to the common-sense approach that increasing water-solubility
inhibitsadrug’sthepermeabilitythroughcellmembrane. Itispossible
that such a common sense may be acceptable when the lipophilicity
and hydrophilicity of the substrates are balanced at a certain range.
It is likely that permeation of 1a through the cell membrane is
greatly inhibited because, in the aqueous conditions around the ali-
mentary canal, a significant amount of 1a coheres into large lumps
(Fig. 2). Thus, we hypothesized that a water-soluble derivative of
⇑
Corresponding author.
E-mail address: nem@ph.tokushima-u.ac.jp (H. Nemoto).
Figure 2. Hypothesis of permeability of water-soluble and poorly water-soluble
compounds in alimentary canal.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.057

6426
H. Nemoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6425–6428
OR
OR
OR
OR
acetonides in 4a–4c was performed in anhydrous methanol in
the presence of Amberlyst-15 at room temperature to afford 5a,
5b, and 5c in 87%, 76%, and 89% yields, respectively.
Terminus
O
O
Apex
OR
OR
OR
OR
O
O
Although we did not quantitatively measure the water-solubil-
ity of the three BGLated fibrates, each 5a–5c (5 mg, 0.0085–
0.0114 mmol) was completely dissolved in 5 mL of deionized
water at 25 °C to confirm their water-solubility above 1 g/L
(approximately >2 mmol/L). It is noteworthy that the molecular
weights of 5a–5c are less than twice the molecular weights of ori-
ginal compounds 1a–1c in spite of the high performance of water-
solubilization by BGL003 (Table 1).
Next, we focused on fenofibrate (1a) and continued the research
on BGLation. For the in vivo experiments using rats, a large quan-
tity of 5a was synthesized starting from acid chloride 69 without
the use of EDC, as shown in Scheme 3.
HO
Poorly
water-soluble
residue
–R
OH
OH
OH
OH
O
O
X
OH
O
OH
OH
OH
O
BGLated derivative
Scheme 1. BGLation using BGL003.
Condensation of 6 and 3 was carried out in the presence of
DMAP in dichloromethane, followed by deprotection of acetonides
to afford 5a in 87% yield (Scheme 3). The water-solubility of 5a was
consistently obtained over 1–4 g/L, which is several thousand
times higher than the water-solubility of 1a.10 In contrast, the par-
tition coefficient (water/1-octanol) was 1:55. Accordingly, 5a is
sufficiently lipophilic for the required purpose in spite of its high
water-solubility. This result was encouraging because it meant
the cell membrane permeability of 5a might be much higher than
we expected.
For comparison, the mono-ol 7 was also prepared from 6 via con-
densation with ethylene glycol in 62% yield. The diol 8 was prepared
from 6 via condensation with cis-2-phenyl-1,3-dioxan-5-ol,11 fol-
lowed by acidic deprotection of the benzylidene group to give the
product in 83% overall yield. Incidentally, reductive deprotection
using a palladium catalyst under a hydrogen atmosphere gave the
over-reduced 9 as the main product. In contrast, under acidic depro-
tection conditions, 1–5% of 10 was contaminated although 8 was ob-
tained as a major product. This isomerization is classified as a
general acyl migration between the acylated glycerides 11 and 12,
which has been reported in a number of papers (Scheme 4).12
This migration between 11 and 12 is unavoidable other than by
using biological technology such as enzymatic protection–depro-
tection procedures. Thus, 8 was not purified and used for the sub-
sequent assay in vivo. In contrast, we never observed isomerization
between 13 and 14, including in the case of 5a–5c. Thus, chemical
purity of 13, which is mono-acylated at the apex of BGL, can gen-
erally be guaranteed.
Next, we conducted biological evaluation of 5a, 7, 8 and 1a in
rats using 1.5% carboxymethylcellulose sodium salt (CMC) as a
vehicle. 100 mg/kg of 5a, 7, 8 or 1a (100 mg/mL) was orally admin-
istered once a day for 8 days to male SD rats at the age of 7 weeks.
Plasma triglyceride concentrations after treatments with 5a, 7 and
8 were found to have decreased to the same extent as after treat-
ment with 1a (Fig. 3) (⁄p <0.05; by ANOVA and Dunnett’s test).
Accordingly, in spite of their chemical modifications, reducing ef-
fect of the triglyceride in plasma in vivo of 5a, 7 and 8 was pre-
served.13 The rats’ body weights were unchanged by the
treatment, and we did not notice any aberrant behavior by the rats
during the study. Thus, it is suggested that these compounds exhi-
bit adequate tolerability in vivo.
The carboxylic acid form of fenofibrate, fenofibric acid (2a),
which is produced by hydrolysis, has been shown to be the actual
active species in vivo.14,15 Since all three chemically modified
fenofibrates lowered plasma triglyceride concentrations, it is sug-
gested that the ester bond in all of these compounds are signifi-
cantly cleaved in vivo.
1a might permeate the cell membrane more efficiently than 1a
itself.4
As shown in Scheme 1, the method we developed, which in-
volves covalent bond formation at the apex of symmetrically
branched oligoglycerols (BGL),5 and sometimes followed by depro-
tection of R groups at the terminal hydroxides, is a suitable for con-
verting such poorly water-soluble medicinal compounds into
water-soluble derivatives as a single isomer,6 because BGL has nei-
ther an asymmetric center nor a molecular weight distribution.
In contrast, another oligo- or polyglycerols7 have a number of
uncontrolled asymmetric centers with a molecular weight distri-
bution due to preparation methods involving oligomerization or
polymerization to afford no single isomer. Although polyethylene-
glycohol (PEG)4 has no asymmetric center (but does have a molec-
ular weight distribution), a large degree of polymerization of
ethyleneglycol unit (of the order of several dozens or more than
hundreds) is required for water-solubilization because the func-
tional groups with water-affinity are mainly ether groups. In con-
trast, BGL has a number of very polar primary hydroxyl groups.
Therefore, the net weight of medicinally active residues occupying
the entire molecule is small, due to the large PEG region, which re-
sults in increases in both the price of drug and the absolute dose.
In this Letter, we report the synthesis of three water-soluble fi-
brate derivatives, 5a–5c, by BGLation (Scheme 2), and evaluation of
BGLated fenofibrate (5a) using 7 weeks old Sprague–Dawley (SD)
male rats. Condensation of fenofibric acid (2a) and diacetonide-
protected BGL003 (3)8 was carried out with ethyl 3-(N,N-dimethyl-
amino)propyl carbodiimide hydrochloride (EDC) in the presence of
4-(N,N-dimethylamino)pyridine (DMAP) in dichloromethane at
room temperature for 2 h to afford ester 4a in 96% yield. Using
the same conditions, 2b and 2c were also transformed to 4b and
4c with yields of 98% and 87%, respectively. Removal of the two
R
2a: R = 4-Cl–C6H4–CO-–
OH
2b: R = 4-Cl–C6H4–CONHCH2CH2–
2c: R = Cl–
O
2a–2c
O
O
O
O
O
O
O
EDC•HCl, DMAP
CH2Cl2, rt
HO
1.5–2 h
EDC = ethyl-3-(N,N-dimethyl)propyl carbodiimide
DMAP = 4-(N,N'-dimethylamino)pyridine
3
O
O
O
O
OH
OH
OH
OH
O
O
R
O
R
Amberlyst-15
O
O
O
4a–4c
MeOH
O
O
rt, 24–36 h
O
Next, we determined the plasma concentrations of 2a after a
single oral administration of 1a or 5a. We collected plasma from
male SD rats (7 wk) 4 h after administration of a stoichiometrically
equivalent amount (0.277 mmol/kg) of 1a (100 mg/kg) or 5a
O
5a–5c
Scheme 2. Synthesis of water-soluble fibrates.

H. Nemoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6425–6428
6427
Table 1
Ratios of molecular weights (BGLated/original)
Compound
Molecular weight of original 1 Mw1
Molecular weight after BGLation Mw5
Mw5/Mw1
a series
b series
c series
360.83
361.82
242.70
540.99
584.06
436.88
1.50
1.61
1.80
Figure 4. Comparison of plasma fenofibric acid concentration 4 h after single oral
administration of fenofibrate (1a) or fenofibrate–BGL003 conjugate (5a).
Scheme 3. Synthesis of 5a and comparator compounds 7 and 8 on a large scale.
Scheme 5. Hydrolysis of fenofibrate–BGL003 conjugate to fenofibric acid and
BGL003 (15).
Finally, we conducted a biological examination of BGL003 (15)
itself, because the hydrolysis of 5a produces not only 2a but also
15 (Scheme 5). To clarify whether the presence of 15 influences
the results of biological examinations, repetitive oral administra-
tion of 15 (58 mg/kg/day) was carried out for 6 days, and no ad-
verse effects were found, including changes in body weight.16
In conclusion, we designed and successfully synthesized novel
water-soluble derivatives of fibrates, which have potential as po-
tent anti-hyperlipemia drugs, via BGLation. The lipid-lowering ef-
fects of the BGLated derivatives were as significant as those of
the same amount of the original fenofibrate in healthy rats. Fur-
thermore, administration of the fenofibrate–BGL003 conjugate re-
Scheme 4. Comparison of acyl migration.
sulted in
a plasma concentration of the active metabolite,
fenofibric acid, that was five times higher than that caused by
fenofibrate. This is the first demonstration of BGLation of a com-
mercially available popular medicinal molecule for the purpose
both synthesis and preliminary biological examinations.
Acknowledgments
Figure 3. Concentration of triglycerides in plasma after eight-day administration in
normal rats.
This work was partly supported by the Japan Science and Tech-
nology Agency (JST), Adaptable and Seamless Technology Transfer
Program through Target-driven R&D in 2009–2012, and the Re-
search for Promoting Technological Seeds in 2006–2007 and in
2007–2008.
(150 mg/kg), since the molecular weight of 5a is 1.50 times greater
than that of 1a, as shown in Table 1. The compounds in the plasma
were extracted with ether and chromatographically separated by
HPLC with absorbance monitoring at 254 nm. As previously re-
ported,14 2a, which is derived from 5a or 1a by hydrolysis, was
clearly observed but neither 5a nor 1a was present in the plasma
4 h after administration (Fig. 4). It is noteworthy that the peak area
of 2a from 5a was five times greater than that derived from 1a.
Supplementary data
Supplementary data (the preparation of 5a–5c, 7 and 8 and the
investigation of synthetic intermediates by IR, 1H NMR, 13C NMR

6428
H. Nemoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6425–6428
M.; Kawamura, T.; Matsushita, T.; Matsumura, K.; Itou, T.; Hattori, H.; Tamaki,
M.; Ishizawa, K.; Miyamoto, L.; Abe, S.; Tsuchiya, K. Bioorg. Med. Chem., in press.
7. Applications and preparations of oligoglycerols: (a) Jayaraman, M.; Fréchet, J.
M. J. J. Am. Chem. Soc. 1998, 120, 12996; (b) Grayson, S. M.; Fréchet, J. M. J. Chem.
Rev. 2001, 101, 3819; (c) Haag, R.; Sunder, A.; Stumbé, J.-F. J. Am. Chem. Soc.
2000, 122, 2954; (d) Caldero’n, M.; Quadir, M. A.; Sharma, S. K.; Haag, R. Adv.
Mater. 2010, 22, 190; (e) Calderón, M.; Quadir, M. A.; Strumia, M.; Haag, R.
Biochimie 2010, 92, 1242; (f) Allcock, H. R.; Ravikiran, R.; O’Connor, S. J. M.
Macromolecules 1997, 30, 3184; (g) Cassel, S.; Debaig, C.; Benvegnu, T.;
Chaimbault, P.; Lafosse, M.; Plusequellec, D.; Rollin, P. Eur. J. Org. Chem. 2001,
875.
8. Preparation of 3: Nemoto, H.; Kamiya, M.; Nakamoto, A.; Katagiri, A.;
Yoshitomi, K.; Kawamura, T.; Hattori, H. Chem. Lett. 2010, 39, 856.
9. Preparation of 6: Lafon, L. United States Patent 4146,728.
10. A detailed study of the water-solubility of 5a and 1a is underway, and will be
published in the near future.
11. Preparation of cis-2-phenyl-1,3-dioxan-5-ol: (a) Carlsen, P. H. J.; Sørbye, K.;
Ulven, T.; Aasbø, K. Acta Chem. Scand. 1996, 50, 185; (b) Crich, D.; Beckwith, A.
L. J.; Chen, C.; Yao, Q.; Davison, I. G. E.; Longmore, R. W.; de Anaya, P. C.;
Quintero-Cortes, L.; Sandoval-Ramirez, J. J. Am. Chem. Soc. 1995, 117, 8757.
12. (a) Isai, S. V.; Usol’tsev, A. A.; Stiba, E. N. Chem. Nat. Compd. 2003, 39, 325; (b)
Kodali, D. R.; Tercyak, A.; Fahey, D. A. Chem. Phys. Lipids 1990, 52, 163; (c)
Crossley, A.; Freeman, I. P.; Hudson, B. J. F.; Pierce, J. E. J. Chem. Soc. 1959, 760;
(d) Lee, G.-C.; Wang, D.-L.; Ho, Y.-F.; Shaw, J.-F. J. Am. Oil Chem. Soc. 2004, 81,
533.
13. Although another attempts were carried out using the lower amount of
compounds, no significant difference between 1a and 5a was observed. In
some cases, the results were further confusable because the difference
between the control and fenofibrate itself (1a) was not observed. Then, we
explored further experimental from a different point of view, and found the
result shown in Figure 4. However, the result shown in Figure 3 is also
important because it shows the suitability of the method for the chemical
modification (= BGLation).
and high-resolution mass spectroscopy (HRMS)) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2012.08.057.
References and notes
1. (a) Monk, J. P.; Todd, P. A. Drugs 1987, 33, 539; (b) Miller, D. B.; Spence, J. D. Clin.
Pharmacokinet. 1998, 34, 155; (c) Chong, P. H.; Bachenheimer, B. S. Drugs 2000,
60, 55; (d) Staels, B.; Dallongeville, J.; Auwerx, J.; Schoojhans, K.; Leitersdorf, E.;
Fruchart, J. C. Circulation 1998, 98, 2088; (e) Goldenberg, I.; Benderly, M.;
Goldbourt, U. Vasc. Health Risk Manag. 2008, 4, 131.
2. http://www.aska-pharma.co.jp/iryouiyaku/seihin/if/if_lipidil.pdf.
3. (a) Guichard, J. P.; Sauron, R. L. P. Curr. Ther. Res. 1993, 54, 610; (b) Shepherd, J.
Atherosclerosis 1994, 110, S55; (c) Munoz, A.; Guichard, J. P.; Reginault, Ph.
Atherosclerosis 1994, 110, S45.
4. Although study of physical mixture of fenofibrate and PEG was reported in the
following paper, the chemical modification to the water-soluble derivatives of
fenofibrate has not been reported. (a) Palmieri, G. F.; Antonini, I.; Martelli, S.
STP Pharma. Sci. 1996, 6, 188; (b) Jamzad, S.; Fassihi, R. AAPS PharmSci. Technol.
2006, 7, E1.
5. Preparation of BGL family: (a) Nemoto, H.; Wilson, J. G.; Nakamura, H.;
Yamamoto, Y. J. Org. Chem. 1992, 57, 435. , and J. Org. Chem., 1995, 60, 1478.
[Erratum]; (b) Nemoto, H.; Araki, T.; Kamiya, M.; Kawamura, T.; Hino, T. A. Eur.
J. Org. Chem. 2007, 3003; (c) Nemoto, H.; Kamiya, M.; Minami, Y.; Araki, T.;
Kawamura, T. Synlett 2007, 2091; (d) Nemoto, H.; Atsushi, I.; Araki, T.; Katagiri,
A.; Kamiya, M.; Matsushita, T.; Hattori, H.; Mimura, Y.; Tomoda, Y.; Yamasaki,
M. Bioorg. Med. Chem. Lett. 2011, 4724.
6. Studies for BGLated medicinal molecules. (a) Nemoto, H.; Iwamoto, S.;
Nakamura, H.; Yamamoto, Y. Chem. Lett. 1993, 465; (b) Nemoto, H.; Cai, J.;
Iwamoto, S.; Yamamoto, Y. J. Med. Chem. 1995, 38, 1673; (c) Yamamoto, Y.; Cai,
J.; Nakamura, H.; Sadayori, N.; Asao, N.; Nemoto, H. J. Org. Chem. 1995, 60, 3352;
(d) Takagaki, M.; Ono, K.; Oda, Y.; Kikuchi, H.; Nemoto, H.; Iwamoto, S.; Cai, J.;
Yamamoto, Y. Cancer Res. 1996, 56, 2017; (e) Nemoto, H.; Kikuishi, J.; Yanagida,
S.; Kawano, T.; Yamada, M.; Harashima, H.; Kiwada, H.; Shibuya, M. Bioorg. Med.
Chem. Lett. 1999, 9, 205; (f) Yamaguchi, H.; Suzawa, T.; Araki, T.; Kamiya, M.;
Nemoto, H.; Yamasaki, M. Biochim. Biophys. Acta 2008, 1780, 680; (g) Ishihara,
A.; Yamauchi, M.; Kusano, H.; Mimura, M.; Nakakura, M.; Kamiya, M.; Katagiri,
A.; Kawano, M.; Nemoto, H.; Suzawa, T.; Yamasaki, M. Int. J. Pharm. 2010, 391,
237; (h) Ishihara, A.; Yamauchi, M.; Tsuchiya, T.; Mimura, M.; Tomoda, Y.;
Katagiri, A.; Kamiya, M.; Nemoto, H.; Suzawa, T.; Yamasaki, M.; J. Biom. Sci.,
Polymer Ed., in press.; (i) Nemoto, H.; Katagiri, A.; Kamiya, M.; Matsushita, T.;
Hattori, H.; Matsumura, K.; Itou, T.; Kawamura, T.; Kita, T.; Nishida, H.; Arakaki,
N. Bioorg. Med. Chem. Lett. 2012, 22, 5051; (j) Nemoto, H.; Katagiri, A.; Kamiya,
14. Hanafy, A.; Spahn-Langguth, H.; Vergnault, G.; Grenier, P.; Grozdanis, M. T.;
Lenhardt, T.; Langguth, P. Adv. Drug Deliv. Rev. 2007, 59, 419.
15. The ester 5a was stable for both the conditions: 2 h at 37 °C in 1 N HClaq and
2 h at 37 °C in fresh rat serum. Therefore, hydrolysis of 5a may have occurred
between alimentary canal and the liver, but not in the stomach or in the blood.
16. A detailed examination of the toxicity of 15 (a)Miyamoto, L.; Watanabe, M.;
Kono, M.; Matsushita, T.; Hattori, H.; Ishizawa, K.; Nemoto, H.; Tsuchiya, K. J.
Toxicol. Sci. in press. (b) Miyamoto, L.; Watanabe, M.; Tomida, Y.; Kono, M.;
Matsushita, T.; Hattori, H.; Ishizawa, K.; Nemoto, H.; Tsuchiya, K. submitted to
a pharmacological journal. will be submitted to a pharmacological journal.