S. Kang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2364–2367
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acid amide (4), and 1-methyl-2-(2 -carboxyethyl) maleic acid
degradation process, protonation of the nitrogen at an acidic pH
by a general acid catalyst can facilitate the degradation. Moreover,
the rate and equilibrium constants of the cyclization reaction are
affected profoundly by the substituent of the cis-double bond.
Bulky alkyl substituents can squeeze the angle between the car-
boxylate and amide groups for more effective internal attack than
hydrogen substituents in maleic acid amide. As a result, mono- and
dialkylmaleic acid amide showed significantly higher rate of cycli-
zation than maleic acid amide, with dialkylmaleic acid amide
showing the highest rate.
amide (5)—was compared using 1H NMR and HPLC at various pH
conditions and 37 °C. The results can provide essential information
for the future development of pH-sensitive biomaterials with tai-
lored kinetics of the release of drugs, change of charge density,
and degradation of scaffold.
Maleic acid amide or maleamic acid derivatives have been syn-
thesized from the corresponding anhydride and n-butylamine as a
model drug under a basic condition using pyridine as a proton
scavenger (Fig. 1a). Amides 1, 2, and 3 were prepared using com-
mercially available anhydrides, maleic anhydride, citraconic anhy-
dride, and cis-aconitic anhydride, whereas amides 4 and 5 were
prepared using freshly synthesized anhydrides, 6 and 7. All five
maleic acid amide derivatives exhibit pH-dependent charge con-
version or control of charge density, because the positively charged
amine group is exposed by the degradation of the maleic acid
amide derivatives that initially have negatively charged carboxyl-
On the basis of the information about degradability, we
compared the pH-dependent stability of five maleic acid amide
derivatives at 37 °C. Except for a few extreme pH conditions in
physiological fluids, we measured the degradation rate of each
1
maleic acid amide at pH 7.4 and 5.5 by H NMR. The stability at
a more acidic extreme pH value (pH 3.0) was also compared for
facilitating the degradation. All NMR samples were incubated in
potassium phosphate buffers in D O. The extent of degradation of
2
1
9
ate groups. Moreover, amides 3, 4, and 5 can be used as pH-
sensitive linkers since they contain two carboxylate groups, the
b-carboxylate group for pH degradability and the other group for
linking with a drug carrier or a polymeric scaffold.20
maleic acid amide derivatives could be measured by comparing
the rate of integration of the methylene protons next to the
nitrogen in the intact amide (peak a) and that in the released
0
Amide 3 can be obtained by the reaction between n-butylamine
and cis-aconitic anhydride; however significant side reactions oc-
curred during amidation to produce allyl-shifted isomers or decar-
boxylated itaconic acid amide.21 The side products showed no
pH-sensitive degradability, and careful control of reactions or puri-
fication conditions was required for a higher yield of the desired
pH-sensitive cis-aconitic acid amide. To overcome these draw-
backs, amide 5 was used instead of amide 3 as a pH-sensitive linker
in several previous reports because amide 5 could inhibit the allyl
shift and decarboxylation.22 The corresponding anhydride 7 was
synthesized by the Horner–Wadsworth–Emmons reaction
between triehtyl-2-phophonopropioninate and dimethyl-2-
n-butylamine after degradation (peak a ) (Fig. 2).
Figure 3 shows the pH-dependent stability based on the 1
H
NMR spectra. Amide 1 with no alkyl substituent and amides 2, 3,
and 4 with one alkyl substituent exhibited chemical stability at
pH 7.4 for 24 h. Only amide 5 with two dimethyl substituents
exhibited significant degradation at pH 7.4. Amide 5 showed about
40% of degradation as soon as the pH value was adjusted to 7.4.
Stability of the maleic acid amide derivatives was reduced with de-
creases in pH value. Although amide 1 showed moderate stability
at pH 5.5, amides 2, 3, and 4 showed over 50% degradation after
8 h of incubation. Amide 5, of course, showed almost complete
degradation at this time point at pH 5.5. At pH 3.0, even maleic acid
amide 1 was degraded at a significant rate and degraded almost
completely after 24 h. All other amides showed over 90% degrada-
tion after incubation for 8 h at pH 3.0.
2
3
oxoglutarate (Fig. 1b). A Z-alkene bond was formed by the
reaction between the phosphonate and the carbonyl groups. Fur-
thermore, we also synthesized a new maleic acid amide derivative
(
4), by removing a 1-methyl group in amide 5 for the precise con-
trol of pH-dependent degradability. The corresponding anhydride
was similarly synthesized to anhydride 7, but using triethyl-2-
The degradation kinetics of maleic acid amide derivatives can
be compared in more detail by HPLC (Fig. 4). The detailed method
is described in Supporting information. All maleic acid amide
derivatives showed hyperbolic degradation profiles. Amide 1 with
two hydrogen substituents showed negligible degradation at pH
7.4, slow degradation at pH 5.5 with a half-life of around 16 h,
and accelerated degradation at pH 3.0 with a half-life of around
7 h (Fig. 4a). Amide 2 with one methyl and one hydrogen substitu-
ent showed a similar stability at pH 7.4, but much faster degrada-
tion at pH 5.5 with a half-life of around 1.5 h (Fig. 4b). At pH 3.0,
over 80% of amide 2 was degraded within 0.5 h. A subtle change
from a hydrogen to a methyl group decreased its stability
drastically at an acidic pH. Amide 3 with one carboxyethyl and
6
phophonoacetate instead of triehtyl-2-phophonopropionate.
Kirby’s group has compared the kinetics of degradation of
maleic acid amide derivatives based on the spectrophotometric
analysis and proposed a possible mechanism of the degradation
in their previous reports.1
2,13
The b-carboxylate group of maleic
acid amide derivatives can easily attack the carbonyl group of
the amide due to the cis-geometric configuration of the carboxylate
and amide groups to form a tetrahedral intermediate with a
five-membered ring (Fig. S1). As the release of the amine from
the tetrahedral structure is the rate-determining step of the
0
0
Figure 1. Synthetic scheme of (a) maleic acid amide derivatives, and (b) 2-(2 -carboxyethyl) maleic anhydride and 1-methyl-2-(2 -carboxyethyl) maleic anhydride.