Molecules 2008, 13, 519-547 [600720]

Molecules 2008, 13, 519-547
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules

Review

Prodrugs for Amines

Ana L. Simplício 1,2,*, John M. Clancy 3 and John F. Gilmer 3

1 Instituto de Tecnologia Química e Biológica, Univ ersidade Nova de Lisboa, Av. da República –
EAN, 2780-157 Oeiras, Portugal
2 IBET, Apartado 12, 2781-901 Oeiras, Portugal
3 School of Pharmacy, Trinity College, Dublin 2, Ireland; E-mails: [anonimizat]; [anonimizat]

* Author to whom correspondence should be addressed; E-mail: [anonimizat]

Received: 14 December 2007; in revised form: 25 February 2008 / Accepted: 25 February 2008 /
Published: 3 March 2008

Abstract: The purpose of this work is to review the published strategies for the production
of prodrugs of amines. The review is divided in two main groups of approaches: those that
rely on enzymatic activation and those that take advantage of physiological chemical
conditions for release of the drugs. A compila tion of the most important approaches is
presented in the form of a table, where the main advantages and disadvantages of each
strategy are also referred.

Keywords: Prodrugs, amines, enzymatic activation, chemical activation

Introduction

The prodrug strategy may be defined as the tem porary derivatisation of a functional group of a
drug in order to improve its pharmaceutical utility. Sometimes the functional group is merely a handle
for the introduction of a moiety th at confers on the new entity some desirable characteristic; more
frequently, the group is intimately connected with the pharmaceutical deficiency and its masking
directly addresses the deficiency. Of the commonl y occurring drug functional groups, perhaps greatest
effort has been directed at temporarily masking the amino group. The most easily identified liability of
candidate amino drugs is their tendency to ionise under physiological conditions, leading to poor

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520
membrane penetration by passive diffusion. The impact of this is amplified for the large number of
amino drugs that are required to penetrate th e blood brain barrier in order to reach their
pharmacological targets. A second issue that can aff ect the development of amino drugs is instability.
An example of this is the tendency of primar y amines to undergo first-pass metabolism due to N –
acetylation and oxidation by monoaminooxidase (MAO) [1]. The same applies to peptides [2]
containing basic amino acid side-chains. Low wa ter solubility, poor stability and low permeability
through biological membranes often hinder the clini cal development of biologically active peptides
[3]. The major problem in designing amine prodrugs is the general robustness of amine derivatives
particularly those, such as amides, in which the capacity to ionize has been obviated. On the other
hand, the very robustness of amino derivatives means th at subtle drug targeting effects can be achieved
if an appropriate local vector can be identified and accommodated in the design process. A number of prodrugs of cytotoxic agents fit this description. In structuring the review we have in the first instance
sought to classify prodrugs according to whethe r in vivo activation is enzymatic or pH/redox
dependent. Within this major division the various t ypes are identified by the nature of the derivative
functional group. The approach is admittedly somewh at arbitrary as prodrug systems rarely undergo
activation exclusively by one route. Also, a deriva tive type that undergoes unmasking enzymatically in
one prodrug, may be removed primarily chemically in another because of overall structural differences. Finally, although peptides can be deri vatised on other functionalities, we have included
only those approaches that involve the amino group alone or conjointly with other functional groups.
Table 1 presents a summary of the most importa nt amine prodrug designs along with their dominant
activation mechanism and the advantages/disadva ntages of each approach. The table should be
consulted where difficulties arise in identifying the chemical structure of a group from its name.

Prodrugs that rely (mostly) on enzymatic activation

N-Alkylation
Secondary and tertiary alkyl amines are reported to undergo dealkylation mediated by MAO-B to
an amine and the corresponding aldehyde or ketone [4]. This has been investigated as a prodrug
approach for the CNS active agent 2-phenylethylamine (PEA). In comparison with the free drug, N,N –
dipropargyl-2-phenylethylamine and N-propargyl-2-phenylethylamine produced increased levels of
PEA in the brain of rats [5,6]. N-(2-cyanoethyl)-2-phenylethylamine [7] and N-(3-chloropropyl)-2-
phenylethylamine ( 1) [8] also caused sustained elevations of PEA in rat brain.

H
N Cl
1

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521
Table 1 . Compilation of a series of published prodrug approaches to amine drugs.
Prodrug typemechanism of
transformation advantages disadvantages
lipid solubility
enzymatic, may be improved Needs to be activated by an
amides and carbamates pH activated slow release electron withrawing substitute
enzymatic followed improved only applicable
N-acyloxyakylderivatives by spontaneous lipoph ilicity to tertiary amines
N-acyloxyalkoxy enzymatic followed produces neutral usually not suitable
carbonyl derivatives by spontaneous compounds for primary amines
lowers pKA easily hydrolysed
β-aminoketones pH activated increases lipoph ilicity in aqueous solution
(oxodioxolenyl)methyl base catalysis also applicable
derivatives and/or enzymatic to primary amines
lowers pKa formation of formaldehyde
N-Mannich bases base catalysis up to 3 units low stab ility
easily hydrolysed
imines (Schiff bases) pH activated lowers pKa in aqueous solution
enamines and lowers pKa
enaminones chemical improved lipoph ilicity not stable e nough at low pH
possibility of only applicable
azo compounds azo-reductases targeting to aromatic amines
possible to
lactonization enzymatic followed manipulate phys/chem poor aqueous solub ility
systems by spontaneous characteristics in most cases
enzymatic and improved only applicable
THTT chemical lipoph ilicity to primary amines
chemical or possibility of
redox sytems enzymatic activation targeting oxidation in solid state
chemical and improved need association with
PEG enzymatic activation solubility other systemsNH C
RO
NH C
ORO
N
RR1
NH C
CR
R2R1NH C
OO
CH
OR
O
R1
NNNH C
HCR
C
R1ONH CH 2
NR
OR1
R3
R4 R5NHR
O
OO
R4
N
SNR
SR
RN O2OHO NHRO O
R1N+CH OR
C
R1O
HN
C
OOOOO
NO
RO N

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522
N-Acylation: amides and simple carbamates

Bioreversible masking of the hydroxyl group with an ester functionality is a practical prodrug
approach as ester compounds are in general readily hydrolysed by the rich variety of hydrolase enzymes present in the human body. Acylation of amin es is ostensibly less promising because of the
chemical and enzymatic stability of amides and car bamates: in this context, mammalian amidases
appear to be less promiscuous than esterases. Indeed amides also undergo hydrolysis by esterases in
vitro [9], but the rates are usually too slow and insu fficiently competitive to be used for amine release
in vivo .
Nevertheless, several successful N-acyl prodrugs are in clinical use or in development. In some
cases, activation relies on specific substrate-peptidase relationships while in others, slow prodrug hydrolysis by non-specific enzymes is not undesirabl e. A well-known example of the former is the
exploitation of renal γ-glutamyl transpeptidase as a vector for amino drug release from γ-glutamic acid
conjugates [10]. Dopamine has been a candidate for this and numerous other prodrug approaches
because it is inactivated by sulfotransferase, MAO and COMT in the intestin al wall and liver [11]
following oral administration [12]. The dopamine double prodrug, γ-glutamyl-
L-dopa (gludopa),
achieves kidney dopamine levels about five-fold highe r than those obtained with an equimolar quantity
of the single prodrug L-dopa [13]. Gludopa itself however, suffers from poor oral bioavailability [14].
Docarpamine, [ N-(N-acetyl- L-methionyl)- O,O-bis(ethoxycarbonyl)dopamine), 2], a pseudopeptide
prodrug of dopamine, is administered orally in the treatment of renal and cardiovascular pathologies
[15,16].
C2H5OCO O
C2H5OCO OH
N
OHN O
S
2

Docarpamine is well absorbed from the oral route, has weak intrinsic vasodilatory effects [17], has
no effect in the CNS even at high doses [11], and is easily converted to dopamine in vivo [15]. Non-
peptide N-acyl derivatives of dopamine have also been evaluated [18]. Several other amine conjugate
with amino acids via amide bonds have been inves tigated with significant improvement in solubility,
for example, dapsone [19,20].
More recently, amino acids have been linked to anti-tumour amines to produce water-soluble
amide prodrugs that release, in vivo , the parent amine [21]. Glycine and valine amides of
monoaminooxidase A (MAO-A) inhibitors were also prepared as an attempt to deliver the active
compounds to the brain before inhibiting MAO-A in the intestinal mucosa which leads to the
potentiation of tyramine induced hypertension [22].
Derivatization with amino acids has also been used to target intestinal transporters such as PEPT1.
One such case is the vasoconstrictor midodrine, which releases the active form 1-(2',5'-dimethoxy-
phenyl)-2-aminoethanol) following cleavage of the glycine residue [23].

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523
One synthetic amide prodrug type system that has been extensively studied are the N-acyl
derivatives of allopurinol, which are more lipophilic than the parent drug, while at the same time being
in some cases more water soluble [24]. The acetyl , veleroyl and nicotinoyl amides of stobadine ( 3) are
highly lipophilic and penetrate the BBB [25].
N
HN
3

Simple N-acyl prodrugs in which slow cleavage is desirable are the [(2-sulfo)-9-
fluorenylmethoxycarbonyl]-3 derivatives of gl ucose lowering drugs like insulin [26] ( 4, R=H) and
exendin-4 [27]. These prodrugs have the advantage of delivering the drugs slowly to the systemic
circulation reducing the risk of hypoglycaemia.
R
HN
GlyA1AsnA21OH
RHNPheB1
NHLysB29ThrB30OHHO3S
4OO
R
R=

Another system that involves carbamate and urea prodrugs has been developed for antibody-
directed enzyme prodrug therapy [ADEPT]. In th is system, the carbamate and the urea groups are
substrates for the enzyme tyrosinase present in melanomas, where it triggers the release of the drug
[28]. New cephalosporins have also been subjected to acyl derivatisation in order to increase solubility [29]. The prototypical non-sedating antihistamine loratadine ( 5) is an ethyl carbamate, which
undergoes a CP450–mediated conversion in vivo to the active desloratadine ( 6) [30]. It is interesting to
note that loratadine was not designed in response to any apparent pharmaceutical liability in desloratadine, and it was certainly not clear at the outset that one acted as a precursor for the other [31].
N
N
ClOON
HN
Cl5 6

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524
The anti-cancer drug capecitabine is a cascade-type prodrug that produces 5-fluorouracil (5-FU) in
the liver and in response to enzymes overexpressed in cancer cells. The first step in the cascade is
hydrolysis of a pentyl carbamate mediated by liv er carboxylesterase (CES-1). Significant species
differences were observed when a panel of homologous carbamates was subjected to hydrolysis by
human, monkey and mouse carboxylesterases and the disc overy scientists point out that the correct
analogue would not have been selected for development without the availability of human intestinal
and liver carboxylesterases [32]. Capecitabine has lo wer gastrointestinal toxicity than 5FU, better
tumour targeting and is widely used now clin ically [33]. Pentyl PABC-Doxaz (PPD) is a doxaz
carbamate prodrug that contains a pentylcarbamate of an anilide, though in this case hydrolysis is
mainly mediated by human intestinal carboxyleste rase (CES-2) [34]. Neith er of these pentyl
carbamates is an amine prodrug in the truest sens e, since in neither case is the pharmacologically
active moiety an amine. However they illustrate that carbamates of anilides may be processed by
intestinal or liver carboxylases, predominantly CES-2 or CES-1 respectively.

N-Acyloxyalkylation, N-hydroxyalkyl ation and N-(phosphoryloxy)alkylation

N-Acyloxyalkyl derivatives of primary and seconda ry amines are not usually suitable as prodrugs
due to their high lability in aqueous solution. However, with tertiary or N -heterocyclic amines it is
possible to produce stable quaternary ammonium salts that are nonetheless susceptible to enzymatic
hydrolysis by esterases and subsequent spontaneous decomposition. An example of this type is the
tetradecyloxymethyl quaternary salt of pilocarpine (7) [35].
OOC2H5
N+
NO (CH 2)12CH3O
7

N-acyloxyalkyl derivatives 8 of the topical antiproliferative dr ug theophylline have been evaluated.
No increase in skin permeability was apparent with alkyl chains of up to five carbons; however, the
intermediate 7-hydroxymethyltheophylline, possesses twice the permeability of the drug itself [36].
N
NNNO
OO
R
O
8

Some quaternary derivatives of tertiary amines have been mentioned as potential prodrugs since
these compounds degrade at physiological pH releasing the parent amine [10] in a similar fashion to
the acyloxyalkyl derivatives. Another a pproach involves a salt form of an N-phosphoryloxymethyl
prodrug, from which the parent drug is released by a first step enzyme-catalysed rate-determining
dephosphorylation, followed by spontane ous chemical breakdown of the N-hydroxymethyl
intermediate [37]. This approach which has been applied to loxapine, improves the aqueous solubility

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525
and stability of the drug [38] and in vivo tests suggest that there is quantitative reversion of the prodrug
to the parent drug [39]. The phosphoryloxymethyl spacer approach has also been successfully
employed in fosphenytoin (Scheme 1), a prodrug of the anti-epileptic phenytoin [40].

Scheme 1 . Tripartite prodrug system for tertiary amines (illustrated for fosphenytoin).

OPO-
O-O
phosphatases spontaneous
NHN
OOOH
NHN
OO
NHH
N
OO

(Acyloxy)alkyl and (phosphoryloxy)alkyl carbamates

N-Acyloxyalkoxycarbonyl derivatives or (acyloxy)alkyl carbamates (R 4= alkyl or aryl), have
received attention as possible prodrug types for am ines [41-44]. The compounds possess an esterase
sensitive terminal group, whose hydrolysis triggers a spontaneous decomposition of the intermediate
(hydroxyalkoxy)carbonyl derivative liberating the parent amine (Scheme 2). This approach neatly
addresses the relative enzymatic stability of simple N-acyl groups.

Scheme 2 . Hydrolysis of (acyloxy)alkyl carbamate and amine prodrugs.
R1
N OO
OR3
R4O
R1
N OO
OHR3
R1
N OHO
R1
N
HR2 R2
R2R2enzymatic

The system has limited applicability to primary amines whose N-acyloxyalkoxycarbonyl adducts
can undergo intramolecular acyl transfer, leading to the formation of stable N-acyl compounds
(Scheme 3) [45].

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526
Scheme 3 . Intramolecular acyl transfer in N-acyloxyalkoxycarbonyl derivatives of primary amines.
O O
NHO
R2R3O
R1R1H
NR 3
OR2 CHO CO2 ++

(Acyloxy)alkyl carbamates derivatives of hydrophilic beta-blockers exhibited several fold increase
in rat skin and rabbit cornea permeation in comparison with the original drugs [46]. Other examples of
(acyloxy)alkyl carbamates are the taste-masking prodrugs 9 of the bitter anti-bacterial norfloxacin ( 10)
[47] and XP13512, a prodrug of gabapentin rec ognized by MCT1 and the sodium-dependent
multivitamin transporter (SMVT) [ 48]. XP13512 was efficiently absorbed and rapidly converted to
gabapentin after oral dosing. In monkeys, the or al bioavailability of gabapentin from XP13512 was
about 84% compared to 25% after similar oral administration of gabapentin [49].

N
OCOOH FNHN
N
OCOOH FNNO
OR O
O
9 10

The use of (acyloxy)methyl esters as bridging groups (Scheme 2, R 2=H) is generally a topic of
controversy due to the generation of formaldehyde during breakdown. For this reason, (acyloxy)ethyl
esters (Scheme 2, R 2=CH 3) are usually preferred. On the ot her hand, (acyloxy)ethyl derivatives
introduce a chiral centre into the system; if the drug already has a chiral centre, diastereomers are formed, which can display very different hydrolytic rates [50].
(Alkoxycarbonyloxy)methyl carbamates have also been prepared (Scheme 2, R
4= alkoxy or
aryloxy) [43], as well as (phosphoryloxy)methyl carbamates 11 which would, in vivo , be cleaved by
alkaline phosphatases. In vitro tests with the phosphate esters showed that following the initial
enzymatic triggering, a spontaneous cascade leads to the release of the amine [51].
R1
N
R2O O
OPO
O-O-11

Azo compounds

Numerous azo compounds have been investigated as site-specific prodrugs that exploit the facile
reduction of the azo linkage by azo-reductase enzyme s. A remarkable story of an early amine azo
prodrug is that of Prontosil ( 12), which was used for the treatme nt of streptococcal infections.
Prontosil is a prodrug of sulphanilamide ( 13) and it was developed by Gerhard Domagk in 1932,

Molecules 2008, 13

527
probably for commercial reasons rather than pha rmacokinetic ones, as the active compound was not
patentable. A group at the Pasteur Institute later specu lated that the azo link might not be necessary for
therapeutic efficacy, and that the active principal might be formed by reduction of the azo bond. They
later proved that the antibacterial activity resided in the sufanilamide portion of the molecule [52].
S
OH2NO
NNH2N
NH2
S
OH2NO
NH212
13
A well-known azo-drug class that exploits azoreduc tases for site-specific drug release are the 5-
aminosalicylic acid prodrugs (e.g. olsalazine, 14). These pass unaffected through the intestine where
they are poorly absorbed [10, 45], but are reduced by azoreductases associated with the high levels of
colonic bacteria.
NHOOC
HO NCOOH
OH
14
Other clinically used prodrugs in this class in clude balsalazide and ipsalazide, in which the 5-
aminosalicylic acid moiety is conjugated to 4-aminobenzoyl- β-alanine and 4-aminobenzoylglycine,
respectively [53]. It should be noted that the azo-approach is generally restricted to primary aromatic
amines, azo derivatives of aliphatic amines being unstable. More recently, conventional N -acyl type
conjugates of 5-aminosalicylic acid with various amino acid derivatives were evaluated for colon
specific delivery. 5-Aminosalicylic acid- L-aspartic acid was effectively delivered to the large intestine,
releasing about half of the administered dose of 5-aminosalicylic acid [54].
Redox systems

The design of prodrug systems poi sed to undergo redox reaction receives growing attention. The
principal merit of this type of drug-release triggeri ng mechanism is its potential to achieve site-specific
delivery. For example, several reductive systems which are selectively activated in hypoxic conditions have been developed and applied in cancer therapy. The systems involve amines latent as nitro groups
which, after conversion, form adducts with DNA. The reduction pathway involves several radical
intermediates as well as nitroso, hydroxylamine and amine intermediates (Scheme 4).

Molecules 2008, 13

528
Scheme 4 . Generalised reduction pathway for nitro-heterocycles [55].

RN O 2
O2 O2
RN H 2RN O 2
2H+
H+RN HRNO
H+
-OH-H+RN O H
R NHOHe-e-e-
e-
e-e-

This type of drug delivery system has been eval uated for the 2-nitroimidazoles, mitomycin C,
tirapazamine [55] and indolequinone s [56], amongst others. The reduction can be facilitated by the
action of endogenous or specially delivered enzyme s [ADEPT or GDEPT]. A similar approach has
been applied to aromatic mustards 15 to produce prodrugs to target hypoxic tumours. Although the
direct use of nitroaromatic mustards 16 could be envisaged as a hypoxic activated system, its
application is limited because both the nitro and the alkylating groups are attached to the same
aromatic rings and have opposing electronic requirements. This means that the prodrug has a very low
reduction potential with consequent low hypoxic sel ectivity. Substitution on the aromatic ring with
electron-withdrawing groups greatly reduced the cyto toxicity of the drug [57]. Nevertheless a water-
soluble phosphate ester of a 3,5-dinitrobenzamide-2-n itrogen mustard is currently under clinical trials.
The prodrug is converted in vivo to the correspondi ng alocohol and afterwards activated by reduction
to the corresponding 5-hydroxylamine and 5-amine [58].
ClNN H 2Cl
R
ClNN O 2Cl
R15 16

However, the design of a reductively activated system where the reduction potential can be
manipulated independently (R 1) allows the production of prodr ugs of the more cytotoxic non-
substituted mustards. Moreover, the selection of th e linker group X (Scheme 5) allows some control
over the rates of cyclization [57].
A series of N-dinitrophenylamino acid amides that also release primary amines via nitroreduction
and intramolecular cyclization has been studied. This system does not seem to be efficiently activated
by nitroreductases but the reduction can be radiation- induced, which is a possible approach in cancer
therapy [59].

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529
Scheme 5 . Mechanism of activation of 2-aminoaryl derivatives.
ClNN
HCl
XOO2N
R1
ClNN
HCl
XOH2N
R1
ClN N
HCl
XHOH2+
N
ClNN H 2Cl
XO
NH
+R R
R
R

Another system that can be activated by reduc tion is the 4-azidobenzyloxycarbonyl (Scheme 6). In
this system the drug is linked through a carbamate to an aromatic azide that is converted by reduction
to an amine: a cascade reaction eliminates the carba mic acid, which is readily converted to the amine
drug [60].

Scheme 6 . Activation of the 4-azidobenzyloxycarbonyl prodrug system.
-O N
HONO2H2NO N
HONO2
+H2N-CO 2
H2O
H2NNO2N3O NHONO
2
H2NOH
+ +

N-oxides have been suggested as bior eductive prodrugs for tertiary amines. N-oxidation masks the
cationic charge of the amine reducing their DNA binding affinity and toxicity. The prodrugs are
activated by metabolic reduction under hypoxic conditions [61].
The dihydropyridinepyridinium salt system [62,63] (S cheme 7) is an example of a site-specific
prodrug, developed for brain penetration of amines (but that is also applicable to alcohols and
carboxylic acids), which employs an oxidative pathway for prodrug localisation. Application of this
system to amines was illustrated for desipramine, amongst others. In this case, although there was no

Molecules 2008, 13

530
evidence of a more efficient delivery, there was a pr olonged presence of the drug in the rat brain at a
constant level [64].

Scheme 7 . Redox carrier system to the brain.

NDrugO
NDrugO
NHOO
+enzymatic
oxidationenzymatic
hydrolysisDrug

The system has also been applied to dopamine [65,66]. A dimeric form of this progroup has also
been used [17]. The system has also been modified to include an activated carbamate ester 17.
.HO
HOH
N
17O
NO

The relatively lipophilic prodrug penetrates th e BBB and the salt obtained after oxidation is
trapped in the brain, where it slowly releases th e drug. Any oxidation in periphery results in rapid
elimination of the intermediate due to its high polarity. This way, the drug can be preferentially
accumulated in the brain. The approach has wide applicability to other functional groups such as
alcohols and carboxylic acids [67,68]. One significant lim itation to the design is the facile oxidation of
the dihydropyridine function, which makes the developm ent of a stable formulation difficult [13]. The
application of this method to several different drugs has been reviewed [69]. An analogous thiazolium
system [70] and carrier groups that involve alkoxycarbonyl methyl derivatives of 7,4-dihydropyridine-
3,5-dicarboxilate [71] have been proposed to overcome the stability problems of the dihydropyridine type prodrugs.

PEG and other macromolecular systems

Double prodrug systems that consist of poly(ethylene glycol) (PEG) linked through a spacer to the
amine drug ( 18) have been explored for drug solubilizati on and for extending the plasma half-life of
the drug. In one approach, ester, carbonate, carbama te or amide bonds are introduced as spacers and
triggers for enzymatic activation and release of th e PEG group. After that, the drug, latentiated in the
form of a carbamate or an ester, is released by a spontaneous 1,4- or 1,6-benzyl elimination [72,73].

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531
XO
R3
PEGR2
R1O
ONH
X=O or NH18R

PEG can also be used in conjunction with the "trimethyl lock" system to produce prodrugs for
amines with improved solubility and potentially capable of targeting specific tumors. In this system,
which has been applied to Daunorubicin, PEG is c onnected through a spacer to the phenol group of the
open lactone. Manipulation of the spacer or the substitu ents in the aromatic ring allows for tuning of
release rates [73-76]. Analogous conjugates have been prepared for Doxorubicin with different results
from the ones obtained for Daunorubicin [77], re vealing that individual compounds should be
evaluated with different linkers in order to determine the most effective combination.
Low molecular weight proteins (LMWP) have also been used to prepare systems for kidney
targeting that can be cleaved by aminopeptidases or lysosomal lysates. The LMWP can also be linked to the drug through an acid sensitive spacer. Using
β-naphthylamine ( β-naph) as a model compound, it
was found that the Leu- β-naph and the Gly-Phen- β-naph conjugates were stable in buffer solution, but
released the amine completely in cortex homogena tes and lysosomal lysates solutions. However the
results were not as promising with adriamycin, triametrene and sulfamethoxasole as model drugs [78].
Dextran conjugates have also been prepared using aminocarboxilic acids as spacers [79]. The
concept has been applied for slow release of Mitomycin C; different release rates were observed for
different linkers.

Prodrugs that rely mostly on chemical activation

β-aminoketones

A recently reported design i nvolves the preparation of β-aminoketones, which are usually stable in
acidic conditions but cleave into the parent amine and an α,β-unsaturated ketone at neutral to basic pH
[80,81]. Elimination from β-aminoketones by retro-Michael reaction is well known, but hadn’t been
used previously in prodrug systems for amines. Placem ent of the amine in a benzylic position seems to
have a positive effect on the rate of elimination in comparison with aliphatic systems. This is probably
due to the extended conjugation of the double bond upon elimination of the amine.
Half-lives at pH=7.4 range from less than one minute to several hours depending on the amine and
on the progroup. The indanone prodrug of atenolol 19 has a half-life of 1.3 minutes in pH=7.4 buffer
and 2.3 minutes in plasma.

Molecules 2008, 13

532
O
N
HO
O
NH2O19

Schiff bases

Imines (Schiff bases), are usually too easily hydrolysed to be useful as amine prodrugs.
Nevertheless, in some particular cases they may be surprisingly stable and useful as they impart increased lipophilicity to the parent amine and depress the pKa values. The anticonvulsant progabide under the trade name Gabrene
© (20) was prepared as a prodrug form of γ-aminobutyric acid (GABA)
since it crosses the BBB, while the free dr ug doesn't. The prodrug is converted to γ-aminobutiramide
and GABA (Scheme 8), which are then trapped if produced in the brain. However, it might not be
considered a true prodrug as it possesses intrinsic pharmacological activity [10].

Scheme 8 . Metabolism of progabide.

OH
FN
NH2O
ClH2N
NH2O
H2N
OHO
20

Chemically activated azomethine prodrugs 21 [82,83] of the reference histamine H3 receptor
agonist R-α-methylhistamine have been evaluated, as well as enzymatically [84] activated prodrugs
(amide, esters and carbamates).
N
HNN
H3CHHO
21

The new compounds, being more lipophilic, have improved oral absorption and better BBB
penetration than the original drug.

Molecules 2008, 13

533
S and N-Mannich bases

N-Mannich bases are synthesised through the Mannich reaction, which involves a NH-acidic
compound, an aldehyde (usually formaldehyde) and an amine in ethanol (Scheme 9).
Scheme 9. Synthesis of N-Mannich bases.
RN H 2O
+H
HO
HNR1
R2+
RN
HO
N+H2O

This system can be used to produce prodrugs that are more soluble than the parent [85], not only
for amines but also for amides, as in the case of rolitetracycline [45]. N-Mannich bases are useful when
an increase in the lipophilicity of amines is desirable. N-Mannich base formation also suppresses the
pKa (a difference of up to 4 units) with respect to the amine, which means that an important proportion
remains unionized at the pH of the intestine [1]. However, the range of biologically acceptable amide
type transport groups affording an appropriate cleavage rate is limited [86].
Cleavage of the prodrug in this case is stri ctly pH dependent: it has been found that N -Mannich
bases of salicylamide and different aliphatic amin es and amino acids show a bell shaped pH/rate
profile with a high breakdown rate at pH 7.4. In the case of salicylamide, the hydroxyl group is
thought to be responsible for the high reactivit y, possibly by intramolecular catalysis, when the
compound is in the neutral or zwitterionic forms. At high pH, when the compound is in the anionic
form, the reactivity decreases markedly [87] . However, derivatisation of this group by
acyloxymethylation, provides new possibilities of controlling in vivo cleavage as well as improved in
vitro stability (Scheme 10) [88].

Scheme 10. Esterase sensitive N -Mannich bases of salicylamide as prodrugs for amines.

N
HO
NR1
R2
O
O
O R3N
HO
NR1
R2
O
OH
N
HO
NR1
R2
OH
NH2O
HNR1
R2
OHH2CO ++enzymaticspontaneous
spontaneous

Molecules 2008, 13

534
It was established that the acidity of the pare nt amide and steric effects within the amine
component correlate with reactivity [85,89]. For amines with similar steric properties, a decreasing
basicity is associated with decreasing reactivity [85]. For example, th e rate of breakdown of N-
Mannich bases of aromatic amines with succina mide increases markedly with increasing amine
basicity [1]. For amines with similar pKas, some correlation was found between reaction rates and the
difference in pKa between the amine and the corres ponding Mannich base (for the same amide) [90].
A drawback of the N-Mannich bases is their limited in vitro stability, raising some
stability/formulation problems [45]. The unavoidable release of formaldehyde during decomposition is another factor that has to be taken into consideration due to its toxicity [85].
An example of an S-Mannich base is dipyrone (metamizole) the methanesulfonic acid of the
analgesic 4-(methylamino)antipyrine, which is highl y water soluble and therefore more suitable for
parenteral administration. When administered ora lly, dipyrone appears to unde rgo fast hydrolysis in
the stomach followed by intestinal absorption of the active form [91].

(Oxodioxolenyl)methyl carbamates and other selfimmolative systems

(Oxodioxolenyl)methyl carbamates were prepared as an attempt to avoid the drawbacks of
(acyloxy)ethyl and (acyloxy)methyl esters. The prodr ugs break by base catalysis according to Scheme
11, but the rate of hydrolysis in plasma solutions is higher than in pH 7.4 buffer solutions [43,50].

Scheme 11. (Oxodioxolenyl)methyl carbamate prodrugs of amines: mechanism of base-
catalysed cleavage.
OO
R3O
ONO R1
R2HO- O
OR1
HN
R2+ CO 2 +spontaneous
and/or enzymatic

In these systems, the cleavage of the dioxole none ring by the amino group attack on the reactive
vinylene carbonate function is precluded, which makes th is approach potentially applicable to primary
amines. Aryl R 3 substituents generally have a destabilising influence, reducing the half-life of the
prodrugs [50]. The system has been applied to pseudomycins and some of the prodrugs exhibited
comparable in vivo efficacy to that achieved by the parent compounds, with reduced side effects [92].
In a similar approach a selfimmolative linker wa s used to attach tryptophan to a bisphosphonate
component through a carbonate-labile linker, 4-hydroxy-3,5-dimethoxybenzyl alcohol, which was
further attached through a stable carbamate li nkage to the amine group of tryptophan [93]. The
carbonate linkage hydrolysed with a half-life of 90 h, but it can be modulated through the nature of the
substituent on the aromatic ring of the self-immolative linker.
Another example using oxodioxolenyl group is that of Prulifloxacin (Scheme 12) which is a
fluoroquinolone antibacterial agent with a broa d spectrum of activity against Gram-positive
and -negative bacteria [94] currently under clinical phase 3 trials. The prodrug uses the (5-methyl-2-
oxo-1,3-dioxolyl)methyl promoiety linked to the secondary amine group of the active form by N-alkylation. Amine release is triggered by esterase attack on a distal oxodioxolenyl group, but formally

Molecules 2008, 13

535
speaking, the connection to the amine group being masked is an alkyl group . Prulifloxacin is available
for oral use, and after absorption is metabolized to the active form ulifloxacin by esterases, mainly
paraoxonase [95].

Scheme 12. Enzymatic hydrolysis of Prulifloxacin [95].

N
SNO
OHO
F
NO
OO
N
SNO
OHO
F
NO
OOHN
SNO
OHO
F
HN
esterasespontaneous

Enamines and enaminones

Enamines [96], (α,β -unsaturated amines), just like imines, are generally unstable particularly at low
pH, which make them unsuitable for the preparati on of prodrugs for oral delivery. Nevertheless, an
enamine prodrug of ampicillin was found to prom ote the rectal absorption of the drug [97].
Enaminones, which are enamines of β-dicarbonyl compounds are more st able, probably due to keto-
enol and imine-enamine tautomeric equilibria (Sch eme 13) [91], and were thought to have potential
use as prodrugs [10].

Scheme 13. Stabilisation of enaminones.

The hydrolysis of enaminones derived from some amino acids and antibiotics is rapid, releasing
the amine and a diketone (Scheme 14). OH NR3
R2 R1OH NR3
R2 R1ONR3
R2 R1

Molecules 2008, 13

536
Scheme 14. Hydrolysis of enaminones.
R3 R4O NR2 R1
R3 R4O
N
HR2 R1O
+

The prodrugs are more lipophilic than the corresponding drugs, which usually results in improved
absorption [10]. The system seems to be relatively insensitive to the type of amine used, but very
sensitive to minor changes in the structure of the 1,3-dicarbonyl compound used to produce the
prodrug. Closed structures like the compounds derive d from cyclohexane-1,3-dione show considerably
lower rates of hydrolysis. This is probably due to thei r rigid geometry and the inherent stability of this
system. The maximum rate of hydrolysis occurs in the pH range 2-5 [98].
Based on chemical stability considerations, enaminones do not seem promising as prodrugs.
However it has been speculated that enaminones obtai ned from ketoesters and lactones may be better
candidates as they may be subjected to enzyme-catalysed degradation [99].

"Trimethyl lock" and coumarin systems

Phenolic amides derived from lactones can be used as amine prodrug systems as they release the
amine and a lactone at physiological pH [100] (Schem e 15). In this system, referred to generally as a
"trimethyl lock", the side chain is folded back to bring the amide carbonyl group into proximity with
the nucleophilic phenolic oxygen. This conformation may account for the facile cyclisation that occurs
independently of the drug attached to the side ch ain. However the half-lives of these systems are
usually less than 1 min, which is too short for useful application [101].

Scheme 15. "Trimethyl lock" prodrug system for amino drugs.
OH
R2R1N
HO
R
O
R2R1 RNH 2O
+spontaneous

The design has been has been modified to pr oduce compounds that are esterase [102] or redox
[103] sensitive (Figure 1). These derivatisations i nvolve the protection of the nucleophilic hydroxyl in
a bioreversible manner. The rate-limiting step then becomes the enzymatic exposure of the phenolic
group. A further variation to this system involved the introduction of phosphate esters as the phenolic
masking group [104].

Molecules 2008, 13

537
Figure 1 . Tripartite "trimethyl lock" systems.
O NO O
R1R
O NO
PO
HOR O NO
R
OH
O
Esterase sensitive Alkaline phosphatase sensitive redox sensitiveHHH

A conceptually similar system exploits the facile cyclization of coumarinic acid and its derivatives
[105] (Scheme 16). The presence of the phenolic hydroxyl group and the cis-geometry of the double
bond allows lactonization at rates comparable to t hose of the "trimethyl lock" system. The phenol
group is protected by an ester or a phosphate group that serves as an esterase or phosphatase sensitive
biological triggering mechanism.

Scheme 16. Coumarin-based esterase sensitive system for amino drugs.
R1
R2 R3NHR
O
OO
R4
R1
R2 R3NHR
O
OH
R1
R2 R3O
O
+ NH 2Resterase spontaneous

The lactonization rate is higher for primary amines than for secondary amines; it also depends on
steric features of the amine to be released [106]. For secondary amines with higher pKas, the system is
sometimes undesirably slow. A further refinenment to the design is the use of ring mesomeric effects
to tune drug release rates: increases of up to 16-fold can be achieved by placing electron releasing
groups on the aromatic ring [107,108].
More recently, an attempt was made to prepare a tripartite prodrug (double prodrug) that uses the
coumarin system as a spacer between the drug (linke d to the side chain) and a carrier group, a peptide
or an amino acid, connected to the hydroxyl group of the coumarin. The advantage of this system
would be the possibility of targeting drug specific proteases for the cleavage that would release the
carrier, which would be followed by spontaneous lactonization, releasing the drug. Poor aqueous
solubility has, however, limited the expl oitation of this system [109].

THTT

A tetrahydrothiadiazine-2-thione (THTT, Figure 2) was proposed as a prodrug system for primary
amines [110], amino acids [111] a nd peptide drugs [112]. In this system the nitrogen atom from the
drug is included in a six membered ring, which is more lipophilic than the original drug. The prodrugs

Molecules 2008, 13

538
are enzyme and chemically sensitive at physiol ogical pH, but are stable under acidic conditions.
Despite the apparent promise of this system, it does not seem to have been subjected to further
development.

Figure 2 . THTT prodrug system for amines and amino acids.
N
SNR
SR

Cyclic derivatives of polyfunctional drugs containing the amino group

Making prodrugs of compounds with multiple functi onal groups may be a challenge, particularly
in the case of peptides, where the amine group poses a problem of its own due to the lack of suitable
biologically reversible masking groups. The "trimet hyl lock" and coumarin systems mentioned before
as prodrug systems for amines, have also been test ed as prodrugs for peptides (linked to the progroup
through an amide and an ester link) with promising results (Figure 3) [113-116].

Figure 3 . Coumarin system applied to peptides.
ONH
OOPeptide
Peptide = -D-Leu-Phen Gly- D-Ala- Tyr-

The concept has been applied to peptides such as DADLE, an opioid peptide. The C and N
terminal ends of the linear peptide are masked by forming an ester and an amide bond with the phenol
hydroxyl and side chain carboxyl groups, respectively, of the linker [107]
Oxazolidines 22, which are cyclic condensation products of β-aminoalcohols and aldehydes or
ketones, are a possible means of formation of pe ptide prodrugs [86]. These compounds are less basic
and more lipophilic than the corresponding β-aminoalcohols and hydrolyse completely in aqueous
solution [117]. Thiazolidines can be used for β-aminothiols.

NOR1
R2
22N
OHNR1
R2
23

Molecules 2008, 13

539
4-Imidazolidinones 23 [118-120] have been proposed for the α-aminoamide moiety, in particular
as prodrugs of Leu-enkephalin and prilocaine. The derivatives of Leu-enkephalin afford protection
against aminopeptidase-N and angiotensin conver ting enzyme (ACE) and are cleaved slowly in
buffered solutions at pH=7.4, with half-lives of some hundred minutes [120]. The hydrolysis of some
prilocaine derivatives, at basic pHs, proceeds to an equilibrium due to reversible kinetics [119].
Lactams and pyrrolines have been shown to reve rt to the corresponding amino acids by enzymatic
action. Moreover, they pass the BBB while the opened structures don't. One example is the cyclic derivative (24) of GABA depicted in Scheme 17.

Scheme 17. Conversion of 2-pyrrolidinone to GABA.
. NHO
H2NCOOHenzymatic
24
Conclusions

Three major themes emerge in amine prodrug chemis try: (i) suppression of ionization in order to
promote passive diffusion; (ii) increasing the meta bolic stability especially of primary amines and
peptides; (iii) tissue targeting, particularly tumor tissue targeting. Other objectives include increasing
the water solubility of the amine. Overall then, amino drugs may benefit significantly from prodrug
design, but designing appropriate prodrugs for amin es has been challenging. This challenge has
provoked a markedly disparate variety of responses from pharmaceutical researchers. We considered it
timely to gather these into one revi ew article as a sort of catalogue that developers might find useful to
consult during the development of new amino drugs or improvement of existing ones. Predicting the
suitability of any single approach to a new situati on, however, is still problematic. It seems prudent to
investigate a number of approaches in parallel with appraisal in a panel of the most relevant human biological matrices, for example, intestinal and liver microsome preparations as well as plasma.

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