3-Aminoxypropionate-based linker system for cyclization activation in prodrug design

Article abstract of DOI:10.1016/j.bmcl.2008.11.097

A novel linker system based on 3-aminoxypropionate was designed and evaluated for drug release using proteolysis as an activation trigger followed by intramolecular cyclization. The hydroxylamine moiety present in the linker system enabled faster release of the parent drug from the linker-drug conjugate at lower pH as compared to an aliphatic amine moiety. Introduction of two methyl groups strategically at the α position to the carboxylate in the linker further improved the rate of cyclization by nearly 2-fold. The 3-aminoxypropionate linker was successfully applied to a model prodrug for protease activation using α-chymotrypsin as the activating enzyme; the activation of the model prodrug bearing the 3-aminoxypropionate linker was 136 times faster than the corresponding model prodrug bearing an amine linker.

Full text of DOI:10.1016/j.bmcl.2008.11.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 941–944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Aminoxypropionate-based linker system for cyclization activation
in prodrug design
Yiyu Ge a, Xinghua Wu a, Dazhi Zhang a, Longqin Hu a,b,
*
a Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel linker system based on 3-aminoxypropionate was designed and evaluated for drug release using
proteolysis as an activation trigger followed by intramolecular cyclization. The hydroxylamine moiety
present in the linker system enabled faster release of the parent drug from the linker–drug conjugate
at lower pH as compared to an aliphatic amine moiety. Introduction of two methyl groups strategically
Received 11 November 2008
Revised 23 November 2008
Accepted 24 November 2008
Available online 3 December 2008
at the
The 3-aminoxypropionate linker was successfully applied to a model prodrug for protease activation
using -chymotrypsin as the activating enzyme; the activation of the model prodrug bearing the 3-amin-
a position to the carboxylate in the linker further improved the rate of cyclization by nearly 2-fold.
Keywords:
Prodrug
Cyclization activation
Proteolysis
Drug design
a
oxypropionate linker was 136 times faster than the corresponding model prodrug bearing an amine
linker.
Ó 2008 Elsevier Ltd. All rights reserved.
FUDR
Cancer chemotherapy
Targeted activation
Hydroxylamine
Non-selective cytotoxicity to normal cells has been the major
problem of cancer chemotherapy. The prodrug strategy using a tu-
mor- or tissue-specific enzyme as the activating enzyme is one of
the potential solutions that have been investigated.1–4 Ideally,
the prodrug is only activated at the site of tumor tissues through
a specific metabolic pathway, such as bioreduction5 or proteoly-
sis.6 In the case of proteolysis-activated prodrugs, insertion of a lin-
ker between the peptide substrate and the parent drug is often
necessary to avoid steric hindrance caused by the bulky parent
drug molecule and to enable efficient cleavage by the target prote-
ase.7,8 After proteolytic cleavage, the linker–drug conjugate goes
through self-immolation to release the parent drug resulting in
activation. Elimination9 and intramolecular cyclization10,11 are
among the most widely used self-immolation mechanisms in the
design of linker systems for proteolysis-activated prodrugs. In this
letter, we report a new type of linker system for proteolysis-acti-
vated prodrugs, which would go through cyclization, an intramo-
lecular nucleophilic addition elimination sequence, to liberate the
parent drug. The nucleophilicity of the trigger group is one critical
factor affecting the rate of cyclization in addition to the size of the
ring formed. An aliphatic or aromatic amino group is often used as
the nucleophilic trigger in the linker and can be deactivated
through formation of an amide bond with the C-terminal carbox-
ylic acid of a peptide substrate. Upon proteolysis, a facile cycliza-
tion will occur as the free amino group undergoes a nucleophilic
attack on the carbonyl group directly attached to the parent drug
leading to the release of the free parent drug as shown in Scheme
1. The rate of cyclization is affected by the nucleophilicity of the
trigger group, the leaving ability of the parent drug, and the size
of the ring formed. Aliphatic amine has been widely used as a trig-
ger group in such linker designs.12,13 However, the optimum pH for
cyclization in this case is much higher than physiological pH; with
pKa between 9 and 11, an aliphatic amine is mostly present in the
protonated form with much reduced nucleophilicity under physio-
logical pH.14
Herein, we report the development of a linker system that
would cyclize readily under physiological conditions and in the
interstitial tissues of solid tumors. The solid tumor environment
is known to be slightly acidic with a pH of around 6. Hydroxyl-
amine is of great interest to us because the pKa of its conjugate acid
is only 5.9, much lower than that of aliphatic amines. At pH ꢀ6,
about 50% of hydroxylamines will be protonated while 99.9% of ali-
phatic amines will be protonated. This difference prompted us to
introduce the more nucleophilic and less basic hydroxylamine in
our linkers as shown in model compound 1 to facilitate cyclization.
In order to further improve the rate of cyclization of hydroxyl-
amine-containing linkers, two methyl groups were strategically
introduced in the linker as in compound 2 to restrict the conforma-
tion of the linker, placing the trigger hydroxylamino group
* Corresponding author. Tel.: +1 732 445 5291; fax: +1 732 445 6312.
E-mail address: LongHu@rutgers.edu (L. Hu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.097

942
Y. Ge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 941–944
Protease
Cyclization
Parent Drug
+ Parent drug
NH
H2N
HN
Peptide
O
Parent Drug
O
O
Peptide
Scheme 1. Two-step activation of peptide-based prodrugs through the cyclization of an amine-containing linker.
spatially close to the carboxylate for nucleophilic attack. Fluorode-
oxyuridine (FUDR) was used as the parent drug to evaluate our lin-
ker system as in model compounds 1–2 and a Cbz–Phe–linker–
FUDR prodrug 4 for detailed kinetic analysis of cyclization, model
enzyme cleavage, and drug release. The corresponding amine ana-
log 3 and its Cbz–Phe conjugate 5 were used as controls to show
the effect of introducing hydroxylamine functionality on the rate
of cyclization and proteolysis.
glycol gave compound 7. N-Hydroxyl phthalimide was attached
to the hydroxyl group in compound 7 through Mitsunobu reac-
tion15 affording intermediate 8. After hydrolysis of the acetal with
concentrated sulfuric acid, Jones oxidation of the resulting alde-
hyde gave the corresponding carboxylic acid 9. The carboxylic
acid 9 was converted to the corresponding tertiary butyl ester
12 with tert-butyl trichloroacetimidate in the presence of cata-
lytic amount of BF3ÁEt2O. The intermediate 11 without the two
methyl substitutions were obtained by the Michael addition of
N-hydroxylphthalimide to tert-butyl acrylate (10).16 After removal
of the phthalimide protecting group with anhydrous hydrazine,
the free amines were acylated with benzyl chloroformate in the
presence of 2,6-lutidine to give compounds 13 and 14. Coupling
O
F
NH
R
R
X
O
O
R'
N
H
N
O
O
a
of the free amine to N -Cbz-phenylalanine through the mixed
OH
anhydride protocol gave compound 15. The tert-butyl in com-
pounds 13–15 was removed with 50% TFA in CH2Cl2 affording
the corresponding carboxylic acids 17, 18, and 20. Compounds
1 R=H, R'=H, X=O
2
R=CH3, R'=H, X=O
3 R=H, R'=H, X=CH2
4 R=CH3, R'=Cbz-Phe, X=O
5
R=H, R'=Cbz-Phe, X=CH2
19 and 21 were synthesized directly from
c-aminobutyric acid
(16). Following Mitsunobu reaction,17,18 the carboxylic acids
17–21 were selectively coupled to FUDR at the primary hydroxyl
to afford compounds 22–24, and the model prodrugs 4 and 5.
Model compounds 1–3 were obtained as 10 mM solutions in
methanol upon hydrogenolysis of compounds 22–24 in methanol
The target compounds were synthesized from the commer-
cially available isobutyraldehyde (6) as shown in Scheme 2. Aldol
condensation with 30% formaldehyde in a sodium hydroxide
solution followed by protection of the aldehyde with ethylene
O
O
H
O
i
ii
N
O
iii
N O
HO
O
OH
O
O
O
O
O
O
O
O
6
7
8
9
t-Bu
O
iv
v
R
R
R R
10
vi
R'
O
O
N
O
O
t-Bu
N
t-Bu
H
O
O
O
9
13
R=H, R'=Cbz
11
12
R=H
14
R=CH3, R'=Cbz
R=CH3
15 R=CH3, R'=Cbz-Phe
O
F
13-15
vii
NH
R
R
R
R
ix
R'
X
OH
R'
X
O
N
N
H
N
O
viii
O
H
H2N(CH2)3COOH
O
O
16
OH
R=H, R'=Cbz, X=O
22
17
R=H, R'=Cbz, X=O
23 R=CH3, R'=Cbz,X=O
18 R=CH3, R'=Cbz, X=O
19 R=H, R'=Cbz, X=CH2
24
4
R=H, R'=Cbz, X=CH2
R=CH3, R'=Cbz-Phe, X=O
20
R=CH3, R'=Cbz-Phe, X=O
5 R=H, R'=Cbz-Phe, X=CH2
21 R=H, R'=Cbz-Phe, X=CH2
O
F
NH
R
R
x
X
O
22-24
O
H2N
N
O
O
OH
1 R=H, X=O
2 R=CH3, X=O
3
R=H, X=CH2
Scheme 2. Synthesis of the aminoxypropionate–FUDR conjugates. Reagents and conditions: (i) a—formaldehyde (30%), NaOH; b—ethylene glycol, p-TsOHÁH2O, toluene,
reflux; (ii) PhtOH, Ph3P, DIAD, THF; (iii) a—concd H2SO4, acetone/water (1:1); b—K2Cr2O7; (iv) PhtOH, CH3OH; (v) tert-butyl trichloroacetimidate, cat BF3ÁEt2O, cyclohexane/
dichloromethylene (2:1); (vi) a—H2NNH2, dichloromethylene; b—for 13/14: CbzCl, 2,6-lutidine, CH2Cl2; for 15: Cbz-Phe-OH, ClCOOiPr, TEA, 0 °C; (vii) for 17, 18, and 20: 50%
TFA/CH2Cl2; (viii) for 19: NaOH, CbzCl; for 21: Cbz-Phe-OSu, NaOH; (ix) FUDR, Ph3P, DIAD, THF; (x) H2, Pd/C, CH3OH, cat concd HCl.

Y. Ge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 941–944
943
under H2 atmosphere in the presence of 5% Pd/C and a drop of
concentrated HCl.
incubation. Compounds 1 and 3 are structurally similar and should
assume similar conformation. The difference in the rate of cycliza-
tion was presumably due to the difference in nucleophilicity and
degree of protonation at the assay pH between hydroxylamine
and aliphatic amine. Comparing the rate of cyclization between
The kinetics of cyclization for compounds 1, 2, and 3 were eval-
uated in 100 mM phosphate buffer at pH 6.0 and 7.4, 37 °C. Ali-
quots withdrawn at different time intervals were quenched with
0.5 N HCl and quickly frozen in dry ice/isopropanol for HPLC anal-
ysis. The kinetics of enzyme cleavage for model prodrugs 4 and 5
were conducted in 100 mM phosphate buffer at pH 7.4 and 37 °C
compounds 1 and 2 at pH 7.4 indicates that the two
a-methyl
groups are able to restrict the conformation to favor cyclization
as shown in Figure 1. This type of conformational restriction
employing the Thorpe-Ingold effect is well-known in prodrug de-
sign through cyclization activation.11,19 It should be noted that
the rate of cyclization was much slower at pH 6 and the effect of
this enhancement was not observed.
in the presence of a-chymotrypsin. The stability of the model pro-
drug 4 was carried out in 100 mM phosphate buffer at pH 7.4 and
37 °C in the absence of the enzyme. Aliquots withdrawn at differ-
ent time intervals were quenched with acetonitrile and quickly fro-
zen for later HPLC analysis.
a-Chymotrypsin is known to cleave peptides after hydrophobic
The results of kinetic analysis of cyclization are shown in Table
1. When incubated at pH 7.4, 37 °C, compound 2 with two methyl
amino acids like phenylalanine and was used as the model enzyme
in our study to evaluate the kinetics of proteolysis of the model
prodrug 4 in 100 mM phosphate buffer at pH 7.4 and 37 °C
groups at the
a position to the carboxylate gave the fastest cycliza-
tion rate with a half life of 43.3 min compared to that of com-
pounds 1 and 3 (t1/2 = 68.6 min and 24.2 h, respectively). When
incubated at a lower pH of 6.0, 37 °C, the rate of cyclization for
compounds 1 and 2 decreased by 20- and 32-fold, respectively,
while compound 3 was stable and did not cyclize after 48 h of
(Scheme 3). The model prodrug 4 was cleaved by a-chymotrypsin
with t1/2 of 36.5 min (Fig. 2, closed circle) with formation of the
intermediate 2 (Fig. 2, closed square). The model prodrug 4 was
stable under the assay conditions in the absence of a-chymotryp-
sin (open circle) indicating the release of the parent drug FUDR
(Fig. 2, open square) was dependent on enzyme cleavage rather
than chemical hydrolysis. The results also suggest that the pep-
tide–linker–drug conjugate can help avoid steric hindrance that
renders direct peptide–drug conjugates resistant to proteolysis.
The rate of proteolysis for model prodrug 4 was 136 times faster
Table 1
Kinetics of cyclization of model linker–drug conjugates 1–3a
O
than the control prodrug
5
(k1: 1.9 Â 10À2 minÀ1 for
4 vs
F
NH
1.4 Â 10À4 minÀ1 for 5). This indicates that the prodrug activation
through proteolysis is facilitated by replacing the amide with
hydroxamate as in our linker system.
R
R
X
O
HCl
N
O
H2N
O
O
OH
kobs (minÀ1
pH 7.4
Compound
Structure
X
)
t1/2
pH 7.4
100
R
pH 6.0
pH 6.0
1
2
3
H
CH3
H
O
O
CH2
5.05 Â 10À4
1.01 Â 10À2
1.60 Â 10À2
4.78 Â 10À4
22.9 h
23.4 h
—
68.6 min
43.3 min
24.2 h
4.94 Â 10À4
100
80
b
b
—
80
a
The assay was performed at 0.2 mM substrate concentration in 100 mM
phosphate buffer at pH 6.0 or 7.4, 37 °C and the cyclization was followed by
measuring the peak area change of the starting substrate using HPLC as monitored
at 268 nm.
60
40
20
0
60
40
20
0
b
No cyclization was detected after 48 h.
0
1000
2000
time (min)
3000
ONH2
CH3
H
H
H3C
H
H3C
CH3
H
H3C
H
CH3
0
100
200
time (min)
300
400
500
H
CO-FUDR
a
H2NO
ONH2
CO-FUDR
b
CO-FUDR
Figure 2. Shown are the kinetic courses of compound 2 at pH 7.4, 37 °C (.), model
prodrug 4 in the presence of -chymotrypsin at pH 7.4, 37 °C (d), model prodrug 4
in the absence of -chymotrypsin at pH 7.4, 37 °C (s), intermediate 2 derived from
model prodrug 4 in the presence of -chymotrypsin at pH 7.4, 37 °C (j), and FUDR
formed from model prodrug 4 in the presence of -chymotrypsin at pH 7.4, 37 °C
c
antiplanar
anticlinal
a
a
Figure 1. Conformational restriction using the two methyl groups in 3-aminoxy-
propionate linkers as in 2. Shown are the three low energy conformations with
a
a
steric hindrance favoring the anticlinal
b and c conformations, placing the
(h). Shown in the inset are those of compound 3 at pH 7.4, 37 °C (Ç) and model
hydroxylamine spatially close to the carboxylate for nucleophilic attack.
prodrug 5 in the presence of
a
-chymotrypsin at pH 7.4, 37 °C (e).
O
O
O
F
F
F
NH
NH
R
X
R
NH
R
R
R
R
cyclization
α-chymotrypsin
O
X
O
HO
X
O
N
O
Cbz-Phe
N
H
N
O
N
O
H2N
+
O
O
O
NH
O
O
k1
k2
OH
R = CH3, X = O
5 R = H, X = CH2
OH
FUDR
OH
R = CH3, X = O
3 R = H, X = CH2
0.019 min-1
0.0097 min-1
N/A
4
21
R = CH3, X = O
22 R = H, X = CH2
2
0.00014 min-1
Scheme 3. Proteolytic cleavage and cyclization activation of model prodrugs 4 and 5 at pH 7.4, 37 °C.

944
Y. Ge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 941–944
In summary, we developed a novel type of linker system for
References and notes
prodrug design by introducing a hydroxylamine moiety and two
1. Hu, L. Drugs 2004, 7, 736.
methyl groups
a to the carbonyl in the linker. The introduction
2. Chen, Y.; Hu, L. Med. Res. Rev. 2008. doi:10.1002/med.20137.
3. Kratz, F.; Müller, I. A.; Ryppa, C.; Warnecke, A. Chem. Med. Chem. 2008, 3, 20.
4. Denny, W. A. Cancer Invest. 2004, 22, 604.
of hydroxylamine moiety enabled faster release of the parent drug
than the corresponding aliphatic amine under physiological condi-
5. Seddon, B.; Kelland, L. R.; Workman, P. Methods Mol. Med. 2004, 90, 515.
6. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A.; Weber, M. J. Proc. Natl.
Acad. Sci. U.S.A. 1980, 77, 2224.
7. Khomenko, T.; Deng, X. M.; Jadus, M. R.; Szabo, S. Biochem. Biophys. Res.
Commun. 2003, 309, 910.
8. Nicolaou, K. C.; Dai, W. M.; Guy, R. Angew. Chem., Int. Ed. Engl. 1994, 33, 15.
9. Papot, S.; Tranoy, I.; Tillequin, F.; Florent, J. C.; Gesson, J. P. Curr. Med. Chem.
Anti-Cancer Agents 2002, 2, 155.
tions. The two methyl groups
icantly facilitated the release of the parent drug. The 3-
aminoxypropionate linker was successfully applied to a model pro-
drug for protease activation using
ing enzyme. It was demonstrated that proteolytic cleavage of
Cbz–Phe–hydroxamate–linker–drug conjugate by -chymotrypsin
a to the carbonyl in the linker signif-
a-chymotrypsin as the activat-
a
10. Gomes, P.; Vale, N.; Moreira, R. Molecules 2007, 12, 2484.
11. Shan, D.; Nicolaou, M. G.; Borchardt, R. T.; Wang, B. J. Pharm. Sci. 1997, 86, 765.
12. Kline, T.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Senter, P. D. Mol.
Pharm. 2004, 1, 9.
13. Suaifan, G. A. R. Y.; Arafat, T.; Threadgill, M. D. Bioorg. Med. Chem. 2007, 15,
3474.
is over 100 times faster than the corresponding conjugate contain-
ing the original amide linkage, representing an additional advan-
tage of our new aminoxypropionate linker. The novel linker
should be of general applicability in the design of prodrugs for pro-
teolytic activation.
14. Suaifan, G. A. R. Y.; Mahon, M. F.; Arafat, T.; Threadgill, M. D. Tetrahedron 2006,
62, 11245.
15. Mitsunobu, O. Synthesis 1981, 1981, 1.
Acknowledgment
16. Wolfe, S.; Wilson, M.-C.; Cheng, M.-H.; Shustov, G. V.; Akuche, C. I. Can. J. Chem.
2003, 81, 937.
17. Hu, L.; Liu, B.; Hacking, D. R. Bioorg. Med. Chem. Lett. 2000, 10, 797.
18. Liu, B.; Hu, L. Bioorg. Med. Chem. 2003, 11, 3889.
19. Jung, M. E.; Piizzi, G. Chem. Rev. 2005, 105, 1735.
We gratefully acknowledge the financial support of a research
scholar grant from the American Cancer Society.