Synthesis and evaluation of atorvastatin esters as prodrugs metabolically activated by human carboxylesterases

Article abstract of DOI:10.1016/j.bmcl.2015.12.069

We synthesized 11 kinds of prodrug with an esterified carboxylic acid moiety of atorvastatin in moderate to high yields. We discovered that they underwent metabolic activation specifically by the human carboxylesterase 1 (CES1) isozyme. The results sugges

Full text of DOI:10.1016/j.bmcl.2015.12.069

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of atorvastatin esters as prodrugs
metabolically activated by human carboxylesterases
⇑
Kenta Mizoi, Masato Takahashi, Masami Haba, Masakiyo Hosokawa
Faculty of Pharmacy, Chiba Institute of Science, 15-8, Shiomi-cho, Choshi, Chiba 288-0025, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized 11 kinds of prodrug with an esterified carboxylic acid moiety of atorvastatin in moderate
to high yields. We discovered that they underwent metabolic activation specifically by the human car-
boxylesterase 1 (CES1) isozyme. The results suggested that these ester compounds of atorvastatin have
the potential to act as prodrugs in vivo.
Received 21 October 2015
Revised 28 November 2015
Accepted 19 December 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Carboxylesterase
CES1
Atorvastatin
Prodrug
Metabolism
Carboxylesterases (CESs) are members of an
a
,b-hydrolase-fold
and it is thus one factor decreasing bioavailability (BA). Atorvas-
tatin has the lowest BA among statins.19 If its BA is increased by
improvement of absorbency, it should be possible to reduce its
dose.
family and play important roles in biotransformation of ester- or
amide-type prodrugs. CESs can be classified into five major groups
denominated CES1–CES5, and the majority of CESs belong to the
CES1 or CES2 group in various mammals.1 Drug-metabolizing
enzymes that are present predominantly in the liver are involved
in the biotransformation of both endogenous and exogenous com-
pounds to polar products to facilitate their elimination. These reac-
tions are categorized into phase I and phase II reactions. CESs are
categorized as phase I drug-metabolizing enzymes that can hydro-
lyze a variety of ester-containing drugs and prodrugs, such as anti-
tumor drugs (CPT-11 and Capecitabin),2–6 angiotensin-converting
enzyme inhibitors (temocapril, cilazapril, quinapril, and imi-
dapril),7–10 and narcotics (cocaine, heroin, and meperidine).11–13
Atorvastatin (1) has activity for a 3-hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA) reductase inhibitor (statin), and it is
therefore used as a therapeutic agent for dyslipidemia.14 The total
synthesis of atorvastatin, the most frequently used statin in a
clinical setting, has been reported and has been attracting
attention.15–18 Statins have a 3,5-dihydroxycarboxylic acid moiety
in their active center, but that moiety causes a decrease in
lipophilicity and prevents the absorption from the small intestine,
Therefore, we focused on atorvastatin and the syntheses of pro-
drugs that have the potential for improved BA. Here, we report on
two points. First, various compounds were synthesized from ester-
ification of the carboxylic acid moiety of atorvastatin. Second,
these compounds were metabolized to atorvastatin by CESs.
Primarily, we report on esterification of the carboxylic acid moi-
ety of atorvastatin. We synthesized compounds (2a–f) using the
Fischer esterification reaction.20,21 As shown in Scheme 1, only
straight-chain aliphatic primary alcohols were introduced into
atorvastatin. When other kinds of alcohol were used, lactone (2l)
was obtained as the major product because intramolecular con-
densation reaction with the 5-position of the hydroxyl group pref-
erentially occurred. Therefore, we synthesized acetal-protected
atorvastatin (4) from 2a in two steps in order to suppress the gen-
eration of 2l. In addition, a variety of alcohols and 4 were con-
densed by using EDC as a condensing agent.20,22 Subsequently,
the compounds (2g–k) were obtained by deprotecting the acetal
group. The results are presented in Scheme 2. From the above
results, 11 kinds of ester compounds (2a–k) were collected.
As mentioned in the introduction, CESs have the ability to
hydrolyze the ester structure. Thus, as the second point, it was
examined whether atorvastatin esters (2a–k) and lactone (2l) are
subjected to hydrolysis by CESs. In this study, it was investigated
Abbreviations: DMP, 2,2-dimethoxypropane; TsOH, p-toluenesulfonic acid; THF,
tetrahydrofuran; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; DMAP,
4-dimethylaminopyridine.
⇑
Corresponding author. Tel.: +81 479 30 4683; fax: +81 479 30 4683.
E-mail address: masakiyo@cis.ac.jp (M. Hosokawa).
http://dx.doi.org/10.1016/j.bmcl.2015.12.069
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mizoi, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.069

2
K. Mizoi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
results are supported by the hydrolysis mechanism of CESs.1
Hydrolysis reaction of CESs are involving the serine ester interme-
diate. This intermediate can be attacked by the phenoxide or alkox-
ide ion, instead of water, and then it proceed to the inverse
reaction. Since phenoxide ion has a lower nucleophilic activity
than alkoxide ion, the inverse reaction tends to not occur. There-
fore, esters (2i–k) have a high hydrolytic activity despite having
sterically–bulky aryl group in HLM and HIM. On the other hand,
lactone (2l) was hardly hydrolyzed in either HLM or HIM.
In contrast, the hydrolytic activity of these esters (2a, 2c)
toward HIM was increased in relation to chain length. We have
already reported that human CES1A was highly expressed in the
liver and that human CES2A was highly expressed in the small
intestine.1 The substrate specificities of CES1 and CES2 are signifi-
cantly different. CES1 isozymes mainly hydrolyze a substrate with
a small alcohol group and large acyl group, whereas CES2 isozymes
hydrolyze a substrate with a small acyl group and large alcohol
group.1,23 Our results for linear aliphatic esters are in agreement
with the theory of substrate specificity of CESs. From the above
results, since these ester compounds (2a–k) have hydrolytic activ-
ity in HLM, they may undergo metabolic activation in the CES1
isozyme.
Scheme 1. Esterification of atorvastatin. (a) Isolated yield. (b) Reagents and
conditions: (A) R1OH, H2SO4 (cat), rt.
Furthermore, the results presented in Table 1 show that ator-
vastatin ethylester (2b) has the highest hydrolyzing activity ratio
of HLM/HIM. Hence, this compound has the best utility as a pro-
drug among these esters (2a–l) because this prodrug is required
to be metabolized in the human liver after it has been absorbed
in the human small intestine without being metabolized. We
obtained new findings regarding the structure activity relationship
for CESs, due to differences in the size and electron density of the
ester moiety for atorvastatin.
Scheme 2. Synthesis of atorvastatin ester via an acetal—protected intermediate. (a)
Isolated yield. (b) Reagents and conditions: (B) DMP, TsOH (cat), CH2Cl2, reflux. (C)
1 M NaOH aq, THF, rt. (D) (1) R1OH, EDC, DMAP, CH2Cl2, rt. (2) 1 M HCl aq, MeOH, rt.
However, there was no evidence that the ester compounds were
activated by CESs because these metabolic reactions were per-
formed in microsomes that contained various enzymes. Finally,
we compared the hydrolyzing activities of several CESs including
human CES1b, human CES1c, human CES2, and human arylac-
etamide deacetylase (AADAC). Esters 2a, 2f, and 2j, which showed
the highest activity among the aliphatic esters (2a–e, 2h, 2l), the
allylic or benzylic esters (2f, 2g), and the aromatic esters (2i–k),
respectively, were used as a substrate. As shown in Figure 2, the
metabolic activity of these esters (2a, 2f, 2j) was specific to CES1.
This result corresponds to previously reported observations.1,23 In
addition, it has been reported that AADAC is expressed in the
human liver and gastrointestinal tissues. The characteristic of
AADAC is that it recognizes the substrates including an N-ary-
lamide and/or ester with a small acyl group and large alcohol
group.23,24 Accordingly, it was appropriate result that these esters
(2a, 2f, 2j) having a small alcohol group had low hydrolytic activity
in the presence of AADAC. From these results, it is suggested that
the hydrolysis experiment of esters (2a, 2f, 2j) in HLM were selec-
tively hydrolyzed by CES1.
Figure 1. Enzyme activity assays of microsomes. Values are means S.D. (n = 3).
whether human liver microsomes (HLM) and/or human small
intestine microsomes (HIM), tissues that express major CES iso-
zymes, have hydrolytic activity. The results are summarized in Fig-
ure 1. The hydrolytic activity of primary linear aliphatic esters (2a–
e) was reduced in accordance with a linear increase in HLM. It was
presumed that this result was caused by steric repulsion of access
to the active pocket or nucleophilic attack of water. Especially,
despite a difference of one carbon atom, hydrolytic activity was
significantly different compare with 2a and 2b. This result show
that even the tiny difference of alcohol group in ester structure
was recognized by CES1. This consideration is also supported by
the fact that hydrolytic activity of 2g was lower than that of 2f
for inversely correlated bulkiness despite the fact that there were
nearly equal electronic properties of 2f and 2g. The hydrolytic
activity of a secondary aliphatic ester (2h) was decreased in rela-
tion to bulkiness. Thus, we assume that these results, the biological
hydrolysis reaction and the chemical hydrolysis reaction of esters,
are correlated. We consider that high hydrolytic activity of the aro-
matic esters (2i–k) is natural because the phenoxide ion has higher
ability than the alkoxide ion for elimination. Furthermore, these
In conclusion, CESs have an important role in the metabolic
activation of prodrugs. There is the possibility that the ester com-
Table 1
Hydrolyzing activity ratio of HLM/HIMa
Ratio
Ratio
(HLM/HIM)
Compound R1=
(HLM/HIM) Compound R1=
2a
2b
2c
2d
2e
2f
–CH3
30.5
40.1
5.7
5.3
4.4
2g
2h
2i
2j
2k
2l
–Bn
–Cy
–Ph
–p-F-Ph
–p-OCH3-Ph 3.4
Lactone 1.7
9.1
4.9
3.8
1.9
–Et
–nBu
–Oc
–De
–Allyl
5.1
a
The data were calculated from Figure 1.
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K. Mizoi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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References and notes
1. Hosokawa, M. Molecules 2008, 13, 412.
2. Sanghani, S. P.; Quinney, S. K.; Fredenburg, T. B.; Sun, Z.; Davis, W. I.; Murry, D.
J.; Cummings, O. W.; Seitz, D. E.; Bosron, W. F. Clin. Cancer Res. 2003, 9, 4983.
3. Humerickhouse, R.; Lohrbach, K.; Li, L.; Bosron, W. F.; Dolan, M. E. Cancer Res.
2000, 60, 1189.
4. Kojima, A.; Hackett, N. R.; Ohwada, A.; Crystal, R. G. J. Clin. Invest. 1998, 101,
1789.
5. Satoh, T.; Hosokawa, M.; Atsumi, R.; Suzuki, W.; Hakusui, H.; Nagai, E. Biol.
Pharm. Bull. 1994, 17, 662.
6. Tabata, T.; Katoh, M.; Tokudome, S.; Nakajima, M.; Yokoi, T. Drug Metab. Dispos.
2004, 32, 1103.
7. Geshi, E.; Kimura, T.; Yoshimura, M.; Suzuki, H.; Koba, S.; Sakai, T.; Saito, T.;
Koga, A.; Muramatsu, M.; Katagiri, T. Hypertens. Res. 2005, 28, 719.
8. Furihata, T.; Hosokawa, M.; Koyano, N.; Nakamura, T.; Satoh, T.; Chiba, K. Drug
Metab. Dispos. 2004, 32, 1170.
9. Mori, M.; Hosokawa, M.; Ogasawara, Y.; Tsukada, E.; Chiba, K. FEBS Lett. 1999,
458, 17.
10. Takai, S.; Matsuda, A.; Usami, Y.; Adachi, T.; Sugiyama, T.; Katagiri, Y.;
Tatematsu, M.; Hirano, K. Biol. Pharm. Bull. 1997, 20, 869.
11. Brzezinski, M. R.; Spink, B. J.; Dean, R. A.; Berkman, C. E.; Cashman, J. R.; Bosron,
W. F. Drug Metab. Dispos. 1997, 25, 1089.
12. Kamendulis, L. M.; Brzezinski, M. R.; Pindel, E. V.; Bosron, W. F.; Dean, R. A. J.
Pharmacol. Exp. Ther. 1996, 279, 713.
13. Zhang, J.; Burnell, J. C.; Dumaual, N.; Bosron, W. F. J. Pharmacol. Exp. Ther. 1999,
290, 314.
Figure 2. Enzyme activity assays of CESs. Values are means S.D. (n = 3).
pound of atorvastatin can be used as a prodrug. This possibility is
supported by results showing that these compounds were specifi-
cally metabolized by CES1, which is mainly expressed in the
human liver. Thus, atorvastatin esters can behave as prodrugs that
have been hydrolyzed not by the human small intestine but by the
human liver. The results of further studies should contribute to the
design of ester prodrugs in various substrates that are highly
metabolized in the human liver.
14. Endo, A. J. Lipid Res. 1992, 33, 1569.
15. Chen, X.; Xiong, F.; Chen, W.; He, Q.; Chen, F. J. Org. Chem. 2014, 79, 2723.
16. Kawato, Y.; Chaudhary, S.; Kumagai, N.; Shibasaki, M. Chem. Eur. J. 2013, 19,
3802.
17. Andrushko, N.; Andrushko, V.; Tararov, V.; Korostylev, A.; König, G.; Börner, A.
Chirality 2010, 22, 534.
18. George, S.; Sudalai, A. Tetrahedron Lett. 2007, 48, 8544.
19. Christians, U.; Jacobsen, W.; Floren, L. C. Pharmacol. Ther. 1998, 80, 1.
20. Smith, M. B.; March, J. March’s Advanced Organic Chemistry, 6th ed.; Wiley: New
York, 2007; pp 1414–1419.
21. McMurry, J. Organic Chemistry, 6th ed.; Thomson: Belmont, CA, 2004; pp 781–
782.
22. Sheehan, J. C.; Cruickshank, P. A.; Boshart, G. L. J. Org. Chem. 1961, 26, 2525.
23. Fukami, T.; Yokoi, T. Drug Metab. Pharmacokinet. 2012, 27, 466.
24. Fukami, T.; Kariya, M.; Kurokawa, T.; Iida, A.; Nakajima, M. Eur. J. Pharm. Sci.
2015, 78, 47.
Acknowledgment
We thank the Human and Animal Bridging (HAB) Research
Organization (Japan) for providing human livers.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.
069.
Please cite this article in press as: Mizoi, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.069