Undesired versus designed enzymatic cleavage of linkers for liver targeting

Article abstract of DOI:10.1016/j.bmcl.2013.12.126

A design for the selective release of drug molecules in the liver was tested, involving the attachment of a representative active agent by an ester linkage to various 2-substituted 5-aminovaleric acid carbamates. The anticipated pathway of carboxylesterase-1-mediated carbamate cleavage followed by lactamization and drug release was frustrated by unexpectedly high sensitivity of the ester linkage toward hydrolysis by carboxylesterase-2 and other microsomal components.

Full text of DOI:10.1016/j.bmcl.2013.12.126

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Undesired versus designed enzymatic cleavage of linkers for liver
targeting
Srinivas R. Chirapu a, Jonathan N. Bauman c, Heather Eng c, Theunis C. Goosen c, Timothy J. Strelevitz c,
Subhash C. Sinha b, Robert L. Dow d, , M. G. Finn a,
⇑
⇑
,
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
b Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
c Pfizer Global Research & Development, Eastern Point Road, Groton, CT 06340, USA
d Pfizer Global Research & Development, 620 Memorial Drive, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A design for the selective release of drug molecules in the liver was tested, involving the attachment of a
representative active agent by an ester linkage to various 2-substituted 5-aminovaleric acid carbamates.
The anticipated pathway of carboxylesterase-1-mediated carbamate cleavage followed by lactamization
and drug release was frustrated by unexpectedly high sensitivity of the ester linkage toward hydrolysis
by carboxylesterase-2 and other microsomal components.
Received 22 July 2013
Revised 29 December 2013
Accepted 31 December 2013
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Cleavable linkers
Drug targeting
Liver targeting
Carboxylesterase
The development of antibodies and other targeting molecules
for the delivery of therapeutic agents1 has spurred an accompany-
ing interest in linkages that can release the cargo at its destina-
tion.2 Cleavable linkers also enable many other applications in
solid-phase synthesis,3 materials science,4 and other fields. In the
biomedical context, the use of proteases, esterases, or other endog-
enous enzymes to release materials in specific environments or cell
types represents an elegant and widely-practiced strategy.5,6
For the selective release of drug molecules in the liver, the
carboxylesterases are a natural choice, since these enzymes are
abundant in that organ and contribute to both the metabolism of
biologically active compounds7,8 and the activation of a variety of
prodrugs.9–11 Carboxylesterase-1 (CE-1) is predominately ex-
pressed in human hepatocytes and recognizes substrates contain-
ing small (C1–C5) alcohols, but is quite promiscuous with regards
to the acyl moiety of the ester.7,12,13 The other major isoform,
carboxylesterase-2 (CE-2), is predominately expressed in the
intestine and exhibits the opposite substrate recognition pattern
to CE-1. Many examples exist of prodrugs that respond to one or
both of these enzymes,7,10,14–18 but the need for release of
unmodified drug has often led to the installation of tethers such
as p-aminomethylphenol which fragments to quinone methide-
type species. We had hoped with the approach detailed below to
take advantage of differences in substrate recognition to initiate
a tissue-specific carboxylester-initiated reaction cascade in the li-
ver without releasing such electrophilic (and therefore potentially
toxic) agents.
Since CE-1 cleavage of O-alkylcarbamate functionalities is
known,19,20 a methyl carbamate initiation element was incorpo-
rated in the general structure 1 (Fig. 1). Enzymatic processing of
the structure in the liver would lead to six-membered ring closure
of the lactam, releasing the drug. The feasibility of this approach
was recently assessed by measurement of rates for five-membered
ring closure with release of a phenolic leaving group; here, c-lac-
tam formation from the unprotected amine was rapid at 37 °C
(t1/2 <1 min) and relatively insensitive to steric hindrance at the
position
a
to the ester carbonyl.21 Internal lactamization to liberate
a drug entity has also been recently reported based on the in situ
formation of anilines from diazo intermediates in the colon.22 Even
for a weakly nucleophilic aniline, cyclization occurred with a half-
life of 57 min at 37 °C.
The synthesis of the requisite compounds beginning with val-
erolactam methyl ether is shown in Scheme 1. Four different sub-
stituents
a to the ester group were installed by alkylation of the
⇑
Corresponding authors.
E-mail addresses: robert.l.dow@pfizer.com (R.L. Dow), mgfinn@gatech.edu (M.G.
derived lithium aza-enolate, and three disubstituted variants were
also prepared. To model later attachment of cell-targeting
moieties, benzyl azide was added by Cu-catalyzed azide–alkyne
Finn).
Present address: School of Chemistry
Technology, Atlanta, GA 30332, USA.
& Biochemistry, Georgia Institute of
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.126
Please cite this article in press as: Chirapu, S. R.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2013.12.126

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S. R. Chirapu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Design of sequential enzymatic carbamate cleavage and d-lactamization steps for release of drug conjugates targeted to the liver.
Scheme 1. Reagents and conditions: (a) (i) t-BuLi, À78 °C, THF, warm to 0 °C, 15 min; (ii) R1X 60–80%. (b) (For R2 = Me, Bn) (i) t-BuLi, KOtBu, À78 °C, THF, warm to 0 °C,
15 min; (ii) R2X 60–80%. (c) 0.1 M HCl, CHCl3, rt, overnight, 78%. (d) Methyl chloroformate, iPr2NEt, 0 °C–rt, 2 h, 85%. (e) LiOH, THF/MeOH/H2O (3:1:1), rt, 6 h, 75–85%; or 1 N
NaOH, THF/H2O (1:1), reflux, 6 h, 75%. (f) BnN3, CuSO4, Na ascorbate, DMF/H2O (3:1), rt, 1 h, 90%. (g) EDCI, DMAP, DMF, rt, 6 h, 90%.
cycloaddition as well.23 Mild acidic hydrolysis followed by carba-
mate formation and ester hydrolysis gave the free acids 4a–h in
good yields.
distinguished from direct ester hydrolysis by the simultaneous
appearance of the GR modulator (6 or 7) and lactam 11.
As summarized in Table 1, the compounds were found to be sta-
ble toward hydrolysis in buffer, but were rapidly metabolized by
microsomal preparations, with decomposition rates varying over
a range of approximately 10-fold for the series. All of the com-
pounds were metabolized more quickly by intestinal microsomes
than by liver microsomes, suggesting turnover predominantly
mediated through CE-2 rather than CE-1. To investigate this
hypothesis, compounds 8f, 8g, 9c, and 9g (Fig. 2) were incubated
with purified recombinant CE-1 and CE-2 enzymes, and were
found to be completely resistant to the former but sensitive to
the latter (moderate to extensive hydrolysis within 60 min at
37 °C; see Supplementary information). These two sets of data
are consistent, since CE-2 is present in both HLM and HIM,
although other esterases could also be participating.
Chromatographic analysis of these metabolic reactions (HLM,
HIM, or recombinant CE-1 and CE-2) failed to find measurable
quantities of the d-lactams 11 (synthesized independently to
provide authentic samples) expected from preferential carbamate
cleavage and cyclization. These results show that both the
aromatic and aliphatic ester linkages in these molecules are much
more sensitive to general esterase activity than we expected. We
attempted to alleviate this problem by installing steric hindrance
As candidate cargo molecules, we chose the glucocorticoid
receptor (GR) modulators 6 and 7 (Scheme 1),24 which are struc-
tural variants of a series originally developed at Abbott laborato-
ries.25–27 Glucocorticoid receptors are expressed in almost every
cell in the body and regulate a myriad of functions. In the liver
the endogenous GR ligand cortisol leads to increased hepatic glu-
cose production via the upregulation of key gluconeogenic en-
zymes. Thus, targeting GR modulators to the liver is desired for
the treatment of such disorders as diabetes, without the undesired
side effects of systemic GR antagonism in such tissues as bone or
the hypothalamic–pituitary–adrenal axis. The poor aqueous solu-
bility of 6 makes it an excellent candidate for attachment to solu-
bilizing and cell targeting groups by a cleavable linker. Aromatic
ester adducts of 6, and aliphatic ester analogues using a hydroxy-
ethyl spacer (7), were prepared by carbodiimide coupling, giving
structures 8 and 9, respectively (Scheme 1).
The suitability of these molecules for liver-specific cleavage was
assessed by measuring their stabilities in the presence of human
liver microsome (HLM) or human intestinal microsome (HIM)
preparations, surrogates for CE-1 or CE-2 activity, respectively.
The immediate product from methylcarbamate cleavage (10) was
not expected to be observed, but carbamate cleavage could be
a
to the ester carbonyl, testing the idea that intermolecular
Please cite this article in press as: Chirapu, S. R.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2013.12.126

S. R. Chirapu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Metabolic stability in human liver microsomes (HLM) and human intestinal microsomes (HIM), in the absence of added NADPH
a
Half life (t1/2, min)
CLint,in
(mL minÀ1 mgÀ1
)
vitro
Compound
R1, R2
HLM
HIM
HLM
HIM
8ab
8cb
8eb
8gc
8bb
8db
8fb
H, Me
H, propargyl
Me, Me
H, CH2-trz-Bn
H, i-Bu
H, Bn
Me, Bn
H, propargyl
H, CH2-trz-Bn
1.3
2.5
5.7
6.7
17
19
73
1.8
5.3
0.53
0.84
1.4
1.9
6.3
9.9
32
0.73
3.8
1.0
2.6
1.7
1.0
0.48
0.22
0.14
0.043
1.9
0.55
0.24
0.14
0.082
0.073
0.019
0.77
0.17
9cb
9gc
0.24
a
b
c
The expected d-lactam was not detected in any case. All compounds were stable (no hydrolysis observed after 3 h in the presence of 1% bovine serum albumin in buffer.
Microsomal incubations contained 0.5 mg/mL protein.
Microsomal incubations contained 0.76 mg/mL protein.
Figure 2. Potential cleavage products from test compounds 8 and 9.
(enzyme catalyzed) hydrolysis would be much more sensitive to
this parameter than intramolecular cyclization to form the lactam.
Indeed, d-lactamization rates were found to increase slightly with
increasing substitution in a related case.21 While a modest effect
was observed (e.g. 8a vs 8b), it was not sufficient to allow CE-1
mediated carbamate cleavage to dominate metabolic clearance.
Still, we believe that carbamate derivatives employed in this way
have some promise in tissue-specific drug release.
These results comprise the first comparison of esterase-medi-
ated cleavage of hindered esters and terminal carbamates, and
highlight the need for careful biochemical evaluation of release
mechanisms in investigations of prodrug or carrier-drug potency.
Observation of cytotoxicity in vitro20 is necessary but not sufficient
to have confidence that complex esterase-containing mixtures are
acting as expected in cascade methods of drug release.
Reagents and characterization: Pooled mixed gender (N = 50)
human liver microsomes and pooled mixed gender (N = 6) intesti-
nal microsomes (Cat. no. 452210, lot no. 41279) were purchased
from BD Biosciences (Woburn, MA). Cypex recombinant CE-1
(Cat. no. CYP152, lot no. INT042E4A) and CE-2 (Cat. no. CYP153, lot
no. 153001) bactosomes and control Escherichia coli cytosol (Cat.
no. CYP099, lot no. INT016E18B) were purchased from Xenotech
(Lenexa, KS). Bovine serum albumin (BSA) was purchased from Sig-
ma–Aldrich. Solvents used for analysis were of analytical or HPLC
grade (Fisher Scientific). All synthesized compounds were charac-
terized by thin-layer chromatography (single spot) and electro-
spray ionization mass spectrometry (strong (M+H)+ or (M+Na)+
parent ions).
incubations were 0.025% and 0.98%, respectively. Microsomes were
thawed on ice and diluted to a final protein concentration of 0.5 or
0.76 mg/mL in 100 mM potassium phosphate buffer (pH 7.4).
Microsomes at the final dilution were pre-warmed to 37 °C and
maintained at that temperature for 5 min before adding substrate.
Periodically (0–60 min), aliquots (50
l
L) of the incubation mixture
were added to acetonitrile (200 L) containing 0.2
l
l
g mLÀ1 terfena-
dine (internal standard). Samples were centrifuged at 2300g for
10 min. Supernatants were mixed with an equal volume of water
containing 0.2% formic acid and then analyzed for the disappear-
ance of 8 or 9 by liquid chromatography tandem mass spectrometry
(LC–MS/MS). To determine stability in the absence of microsomes,
incubations were conducted in 1% (10 mg/mL) BSA dissolved in
100 mM potassium phosphate buffer (pH 7.4), following the same
procedure outlined above. In vitro t1/2 and CLint,in vitro were calcu-
lated using Microsoft Excel. To estimate CLint,in vitro, the in vitro
t1/2 of
8 and 9 were scaled using the following equation:
CLint,in vitro = [0.693Á(mL incubation)]/[(t1/2)Á(microsomal protein
concentration in incubation)].
Metabolite identification in microsomes and recombinant enzymes:
Stock solutions of 8f, 8g, 9c, and 9g were prepared in DMSO at
10 mM and diluted to 1 mM in acetonitrile. Each compound (final
concentration 10 lM) was incubated with human liver micro-
somes, human intestinal microsomes, recombinant CE-1 bacto-
somes, recombinant CE-2 bactosomes, or control E. coli cytosol
(n = 1) at 37 °C (pH 7.4), in the manner described above. One hour
after substrate addition, each incubation mixture (1 mL) was trans-
ferred to a vial containing acetonitrile (5 mL). To generate the initial
Intrinsic clearance (CLint,in vitro) determination in microsomes: Stock
solutions of 8 or 9 were prepared in dimethyl sulfoxide (DMSO) at
4 mM and diluted to 0.1 mM in acetonitrile. Compounds 8 or 9 (final
(t0) samples, 495
added to a vial containing 5 mL acetonitrile, followed by addition
of 5 L of substrate stock solution. Samples were vortexed then
lL of microsomes, bactosomes, or control was
l
concentration, 1
l
M) were incubated with human liver or intestinal
centrifuged at 2300g for 10 min. The supernatants were dried under
a steady nitrogen stream. The residue was reconstituted with mo-
bile phase and analyzed for metabolite formation by LC–MS/MS.
microsomes (n = 2) at 37 °C (pH 7.4). Total incubation volume was
0.5 mL and the final DMSO and acetonitrile concentrations in the
Please cite this article in press as: Chirapu, S. R.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2013.12.126

4
S. R. Chirapu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
LC–MS/MS conditions: The concentrations of 8 or 9 were deter-
Acknowledgements
mined on a Sciex API4000 Qtrap LC–MS/MS triple quadrupole mass
spectrometer fitted with a Turbo ion-spray (TIS) interface operated
in the positive-ion mode. A Shimadzu LC-20AD HPLC system with
We gratefully acknowledge support from Pfizer Global Research
& Development, and the NIH (P50 GM103368-01).
a CTC Leap autosampler was programmed to inject 10
lL of sample
on a Supelco Discovery 3
lm C18 50 Â 2.1 mm column. Analytes
Supplementary data
were eluted with a binary gradient mixture consisting of 0.1%
(v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid
in acetonitrile (solvent B) at a flow rate of 0.6 mL minÀ1 and
monitored using the multiple reaction monitoring (MRM) mode
for the mass-to-charge (m/z) transitions: 8a 660.3 ? 370.2, 8b
702.4 ? 412.4, 8c 684.4 ? 394.2, 8d 736.4 ? 446.3, 8e 674.3 ?
384.4, 8f 750.5 ? 460.2, 8g 817.5 ? 90.5, 9c 728.5 ? 394.3, 9g
861.6 ?90.5, terfenadine 472.5 ? 57.2. The concentrations of 8
and 9 in the samples were determined by interpolation from
standard curves with a dynamic range from 0.5–2000 nM (8e, 8b
and 8c), 1–2000 nM (8a, 8f and 9c), 5–2000 nM (8d and 9g), or
20–2000 nM (8h) using synthetic standards, analyzed with Analyst
1.4 software (Applied Biosystems).
General esterification procedure (8a–g, 9c, 9g), and representative
examples: A solution of the carboxylic acid (5a–g, 2.01 mmol) in
CH2Cl2 (20 mL) was treated with 6 (2.45 mmol, 1.22 equiv), EDCI
(3.02 mmol, 1.5 equiv), and DMAP (0.193 mmol, 0.09 equiv). The
mixture was stirred for 1 h, and the precipitate was filtered and
rinsed with CH2Cl2. The combined organic layers were dried over
MgSO4, filtered, concentrated under vacuum, and the crude prod-
uct was purified by column chromatography.
4-(4-((Benzyl(2-methyl-3-(methylsulfonamido)phenyl)amino)
methyl)phenoxy)phenyl 5-((methoxycarbonyl)amino)-2-methylpent-
anoate (8a): 1H NMR (600 MHz, CDCl3) d 7.35–6.84 (m, 16H),
4.05 (d, J = 19.2 Hz, 4H), 3.74–3.58 (m, 3H), 2.99 (m, 5H), 2.40 (s,
3H), 1.37–1.10 (m, 7H). ESI-MS m/z: 660.2 (M+H+).
2-(4-(4((Benzyl(2methyl3(methylsulfonamido)phenyl)amino)
methyl)phenoxy)phenoxy)ethyl 2-isobutyl-5-((methoxycarbonyl)amino)
pentanoate (8b): 1H NMR (600 MHz, CDCl3) d 7.27–6.87 (m, 16H),
4.06 (d, J = 18.6 Hz, 4H), 3.66 (d, J = 6.2 Hz, 3H), 2.95 (s, 3H),
2.71 (t, J = 6.8, 2H), 2.34 (s, 3H), 1.58 (m, 4H), 1.25 (s, 4H),
0.93–0.85 (m, 4H). ESI-MS m/z: 702.3 (M+H+).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.
126.
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4-(4-((Benzyl(2-methyl-3-(methylsulfonamido)phenyl)amino)
methyl)phenoxy)phenyl 5-((methoxycarbonyl)amino)-2,2-dimethyl-
pentanoate (8e): 1H NMR (500 MHz, CDCl3) d 7.41–6.82 (m,
16H), 4.06 (d, J = 15.5 Hz, 4H), 3.69 (d, J = 6.3 Hz, 3H), 2.98 (m,
5H), 2.38 (s, 3H), 1.41–1.18 (m, 10H). ESI-MS m/z: 674.3 (M+H+).
4-(4-((Benzyl(2-methyl-3-(methylsulfonamido)phenyl)amino)
methyl) phenoxy)phenyl 2-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-
((methoxycarbonyl)amino)pentanoate (8g): 1H NMR (600 MHz,
CDCl3) d 7.41–7.01 (m, 17H), 6.88–6.81 (m, 5H), 5.55–5.45
(m, 2H), 4.02 (d, J = 17.5 Hz, 4H), 3.64 (d, J = 2.4 Hz, 3H), 3.56
(s, 2H), 3.48 (s, 2H), 3.19 (m, 3H), 2.95 (m, 5H), 2.36 (s, 3H),
1.81–1.03 (m, 4H). ESI-MS m/z: 817.3 (M+H+).
Please cite this article in press as: Chirapu, S. R.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2013.12.126