Design, synthesis and in vitro kinetic study of tranexamic acid prodrugs for the treatment of bleeding conditions

Article abstract of DOI:10.1007/s10822-013-9666-2

Based on density functional theory (DFT) calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives four tranexamic acid prodrugs were designed. The DFT results on the acid catalyzed hydrolysis revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. When the amine leaving group was a primary amine or tranexamic acid moiety, the tetrahedral intermediate collapse was the rate-limiting step, whereas in the cases by which the amine leaving group was aciclovir or cefuroxime the rate-limiting step was the tetrahedral intermediate formation. The linear correlation between the calculated DFT and experimental rates for N-methylmaleamic acids 1-7 provided a credible basis for designing tranexamic acid prodrugs that have the potential to release the parent drug in a sustained release fashion. For example, based on the calculated B3LYP/6-31G(d,p) rates the predicted t_1/2 (a time needed for 50 % of the prodrug to be converted into drug) values for tranexamic acid prodrugs ProD 1-ProD 4 at pH 2 were 556 h [50.5 h as calculated by B3LYP/311+G(d,p)] and 6.2 h as calculated by GGA: MPW1K), 253 h, 70 s and 1.7 h, respectively. Kinetic study on the interconversion of the newly synthesized tranexamic acid prodrug ProD 1 revealed that the t_1/2 for its conversion to the parent drug was largely affected by the pH of the medium. The experimental t_1/2 values in 1 N HCl, buffer pH 2 and buffer pH 5 were 54 min, 23.9 and 270 h, respectively. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10822-013-9666-2

J Comput Aided Mol Des (2013) 27:615–635
DOI 10.1007/s10822-013-9666-2
Design, synthesis and in vitro kinetic study of tranexamic acid
prodrugs for the treatment of bleeding conditions
•
Rafik Karaman Hiba Ghareeb Khuloud Kamal Dajani
•
•
•
Laura Scrano Hussein Hallak Saleh Abu-Lafi
•
•
•
Gennaro Mecca Sabino A. Bufo
Received: 28 February 2013 / Accepted: 6 July 2013 / Published online: 24 July 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Based on density functional theory (DFT) cal-
culations for the acid-catalyzed hydrolysis of several
maleamic acid amide derivatives four tranexamic acid
prodrugs were designed. The DFT results on the acid cat-
alyzed hydrolysis revealed that the reaction rate-limiting
step is determined on the nature of the amine leaving
group. When the amine leaving group was a primary amine
or tranexamic acid moiety, the tetrahedral intermediate
collapse was the rate-limiting step, whereas in the cases by
which the amine leaving group was aciclovir or cefuroxime
the rate-limiting step was the tetrahedral intermediate for-
mation. The linear correlation between the calculated DFT
and experimental rates for N-methylmaleamic acids 1–7
provided a credible basis for designing tranexamic acid
prodrugs that have the potential to release the parent drug
in a sustained release fashion. For example, based on the
calculated B3LYP/6-31G(d,p) rates the predicted t1/2 (a
time needed for 50 % of the prodrug to be converted into
drug) values for tranexamic acid prodrugs ProD 1–ProD 4
at pH 2 were 556 h [50.5 h as calculated by B3LYP/
311?G(d,p)] and 6.2 h as calculated by GGA: MPW1K),
253 h, 70 s and 1.7 h, respectively. Kinetic study on the
interconversion of the newly synthesized tranexamic acid
prodrug ProD 1 revealed that the t1/2 for its conversion to
the parent drug was largely affected by the pH of the
medium. The experimental t1/2 values in 1 N HCl, buffer
pH 2 and buffer pH 5 were 54 min, 23.9 and 270 h,
respectively.
Keywords Tranexamic acid ꢀ Prodrugs ꢀ Menstrual
bleeding ꢀ Fibrinolysis ꢀ Proton transfer ꢀ Traumatic
haemorrhage ꢀ Hemophilia
Introduction
Tranexamic acid is a synthetic lysine amino acid deriva-
tive. It was originally developed to prevent and reduce
excessive hemorrhage in hemophilia patients and reduce
the need for replacement therapy during and following
tooth extraction. It is often prescribed for excessive
bleeding. The mechanism by which tranexamic acid exerts
its antifibrinolytic activity is by competitively inhibits the
activation of plasminogen to plasmin, a molecule respon-
sible for the degradation of fibrin. Tranexamic acid has
roughly 8 times the antifibrinolytic activity of an older
analogue, e-aminocaproic acid [1]. Over the past few years,
the use of tranexamic acid has been expanding beyond the
small number of hemophilia patients; it is an important
agent in decreasing mortality rate due to bleeding in trauma
patients; this can be seen from CRASH-2 study which
concludes that all cause mortality, relative risk and relative
death due to bleeding were reduced with tranexamic acid
Electronic supplementary material The online version of this
article (doi:10.1007/s10822-013-9666-2) contains supplementary
material, which is available to authorized users.
R. Karaman (&) ꢀ H. Ghareeb ꢀ H. Hallak ꢀ S. Abu-Lafi
Bioorganic Chemistry Department, Faculty of Pharmacy,
Al-Quds University, P. O. Box 20002, Jerusalem, Israel
e-mail: dr_karaman@yahoo.com
R. Karaman ꢀ L. Scrano ꢀ S. A. Bufo
Department of Sciences, University of Basilicata,
Via dell’Ateneo Lucano 10, 85100 Potenza, Italy
K. K. Dajani
Faculty of Public Health Sciences, Al-Quds University,
Box 20002, Jerusalem, Israel
G. Mecca
Exo Research Organization, Potenza, Italy
123

616
J Comput Aided Mol Des (2013) 27:615–635
group more than placebo group [2]. In addition, tranexamic
acid was found to reduce blood loss during and after sur-
gery in women who undergo lower segment cesarean
section [3]. Finally, tranexamic acid was shown to be
pharmacologically active in reducing the need for intra-
operative blood transfusion during heart surgery, hip and
knee replacement surgery and liver transplant surgery [4].
Tranexamic acid pharmacokinetic profile indicates that
after an intravenous dose of 1 g the plasma concentration
time curve shows a terminal elimination half-life of about
2 h [1]. Furthermore, tranexamic acid in CRASH-2 study
was administered using a loading dose of 1 g by intrave-
nous infusion over 10 min followed by 1 g infusion over
8 h. Although an 8 h IV infusion may be an easy option in
a hospital setting, such option may not be available in
under-developed countries [2].
complications. Thus, tranexamic acid can be used safely
and effectively to reduce bleeding resulting from CS
[9–11].
After the withdrawing of aprotinin from worldwide
market in November 2009, tranexamic acid is the only
marketed antifibrinolytic agent available in the market [12].
Further, it was found that tranexamic acid is also
effective in inhibiting the activity of urokinase in urine [13]
and it is safe and effective for treating severe hematourea
in patient with chronic renal impairment that poorly
respond to conventional therapy [14].
Recent studies showed that tranexamic acid inhibits the
ultraviolet radiation induced pigmentation activity, thus it
can be used as bleaching agents [15]. Oral tranexamic acid
dosage form was found to be effective and safe in treating
melasma, a hypermelanosis disease that occurs in Asian
women [16].
Recently, a new oral formulation of tranexamic acid was
shown to be safe and effective for treatment of heavy
menstrual bleeding [5]. Oral administration of tranexamic
acid results in a 45 % oral bioavailability. The total oral
dose recommended in women with heavy menstrual
bleeding was two 650 mg tablets 3 times daily for 5 days.
Accumulation following multiple dosing was reported to
be minimal [6].
Since tranexamic acid is an amino acid derivative and
undergoes ionization in physiologic environments its oral
bioavailability is expected to be low due to inefficient
absorption through membranes. Note the log P (partition
coefficient) for tranexamic acid is -1.6 [17]. Hence, there
is a necessity to design and synthesis a relatively more
lipophylic tranexamic acid prodrugs that can provide the
parent drug in a sustained release manner which might
result in better clinical outcome, more convenient dosing
regimens and potentially less side effects than the original
medication.
Post-partum hemorrhage is a leading cause of maternal
mortality, accounting for about 100,000 maternal deaths
every year. Medications used to control postpartum hem-
orrhage (PPH) are in the category of uterotonic drugs.
These drugs stimulate contraction of the uterine muscle,
helping to control PPH. The two medications most com-
monly used for treatment include oxytocin or misoprostol.
In addition, patients are commonly given an IV blood
transfusion in cases of severe hemorrhage [7, 8]. In third
world countries, availability of blood and fluid replacement
may be an issue. One approach to decrease the risk of
maternal hemorrhage may be to improve the availability of
blood and fluid replacement. An alternative approach is to
decrease the likelihood of maternal hemorrhage. Further-
more, all the treatment options mentioned above are
intended for intravenous administration; this may not be a
viable option in under-developed countries. Therefore, a
cheaper oral alternative may be better suited for such
circumstances.
In general, such tranexamic acid derivatives need to be
pharmacologically inactive chemicals that could be used to
alter the physicochemical properties of tranexamic acid, in
a temporary manner. To enhance the oral pharmacokinetic
properties of tranexamic acid and increase its usefulness,
lipophylic linkers need to covalently bound to the parent
drug and such linkers need to e able to be converted in vivo
to the active drug molecule, enzymatically or nonenzy-
matically, to exert a therapeutic effect. Ideally, the pro-
drugs should be converted to the original drug as soon as
the goal is achieved, followed by the subsequent rapid
elimination of the released linker group. For example,
tranexamic acid is given by continuous IV infusion
resulting in peak plasma concentration following admin-
istration. If an IV slow release prodrug can be prepared,
then tranexamic acid Cmax related side effects may be
avoided and longer duration of exposure may be achieved
resulting in potentially better maintenance paradigm.
In addition, improvement of tranexamic acid pharma-
cokinetic properties and hence its effectiveness may
increase the absorption of the drug via a variety of
administration routes, especially the oral and SC injection
routes of administration.
Several preliminary studies were conducted to evaluate
the effect of tranexamic acid in PPH. Recent analysis of the
available data indicates that two studies provided the most
reliable data. Both studies explored the efficacy and safety
of tranexamic acid at caesarian section (CS). Tranexamic
acid was administered via slow IV infusion (over 5 min) at
a dose of 1 g, 10 min before incision. Both studies con-
cluded that tranexamic acid statistically reduces the extent
of bleeding from placental delivery to 2 h postpartum and
its use was not associated with any side effects or
Modern computational methods can be used for the
design of innovative prodrugs for medicines that contain
123

J Comput Aided Mol Des (2013) 27:615–635
617
hydroxyl or amine groups. For example, mechanisms of
some enzyme models that have been used to gain a better
understanding of enzyme catalysis have been recently
investigated and utilized for the design of novel prodrug
linkers [18–36]. Using computational methods such as
density functional theory (DFT), molecular mechanics and
ab initio, various enzyme models were investigated for
assigning the factors affecting the rate-determining step
and playing dominant roles in governing the reaction rate.
Among these enzyme models are: (a) proton transfer
between two oxygens in Kirby’s acetals [37–43] and proton
transfer between ammonium nitrogen and oxygen in Kir-
by’s enzyme model [37–43]; (b) intramolecular acid-cata-
lyzed hydrolysis in some of Kirby’s N-alkylmaleamic acid
derivatives [37–43]; (c) proton transfer between carboxylic
oxygen and alcoholic oxygen in rigid systems as investi-
gated by Menger [44–47]; (d) the acid-catalyzed lacton-
ization of hydroxy-acids as studied by Milstein and Cohen
[48–50] and Menger [44–47]; and (e) the SN2-based ring
closing reactions as studied by Brown, Bruice, and
Mandolini [51–54].
useful in the optimization of the clinical application of a
drug. Numerous prodrugs have been designed and devel-
oped to overcome pharmaceutical and pharmacokinetic
barriers in clinical drug application, such as low oral drug
absorption, lack of site specificity, chemical instability,
toxicity, and poor patient acceptance (bad taste, odor, pain
at injection site, etc.). However, till now no experimental
study has been reported for prodrug systems consisting of a
covalently linked drug to a host (prodrug) that is capable of
releasing the parental drug via a controlled cleavage
reaction.
It is worthy to note that our previous studies [18–36] on
enzyme models emphasize the necessity to explore the
reaction mechanism and to assign the driving force
affecting the reaction rate in order to design an efficient
chemical device (pro-drug). The designed prodrugs should
have the potential to undergo cleavage reactions in physi-
ological environments at rates that are completely depen-
dent on the structural features of the host (inactive linker).
By doing so, a variety of linkers could be used to obtain
different prodrugs that release the parental drug at different
rates depending mainly on the nature or the structural
features of the linker.
These studies have revealed to the followings: (1) rates
acceleration in intramolecular processes is a result of both
entropy and enthalpy effects. In intramolecular ring-closing
reactions where enthalpic effects were predominant, steric
effects were the determining factor for the acceleration,
whereas proximity orientation was the determining factor
in proton-transfer reactions. (2) The distance between the
two reacting centers is the main factor in determining
whether the reaction type is intermolecular or intramolec-
Continuing our study on how to utilize enzyme models
as potential carriers for drugs containing amine and
hydroxyl groups [55–60], we sought to study the proton
transfer reactions in the acid-catalyzed hydrolysis of N-
alkylmaleamic acids 1–7 (Kirby’s enzyme model, Fig. 1)
reported by Kirby and Lancaster [61]. Based on the cal-
culation results of this system, we propose four tranexamic
acid prodrugs, tranexamic prodrugs ProD 1–ProD 4
(Fig. 2).
˚
ular. When the distance exceeded 3 A, an intermolecular
engagement was preferred because of the engagement with
a water molecule (solvent). When the distance between the
˚
As shown in Fig. 2, tranexamic acid prodrugs, ProD 1–
ProD 4 have a carboxylic group (hydrophilic moiety) and a
lipophilic moiety (the rest of the prodrug), where the com-
bination of both moieties secures a relatively moderate HLB.
In most of the physiologic environments (pH 1–8.0)
tranexamic acid will exist primary in the ionized forms
(Eq. 1) while its prodrugs, ProD 1–ProD 4, will equili-
brate between the ionic and the free acid forms (Eq. 2)
especially in the physiological pH environment of 5.5–6.8
(intestine). Thus, it is expected that prodrugs ProD 1–
ProD 4 may have a better bioavailability and pharmaco-
kinetic profile than that of the parent drug due to neutral-
izing the ionization of the amine group which results in the
oral absorption improvement. In addition, these prodrugs
may be used in different dosage forms (i.e. enteric coated
tablets, topical use and etc.) because of their potential
solubility in organic and aqueous media due to the ability
of the carboxylic group to be converted to the corre-
sponding carboxylate anion in a physiological pH of
around 6.0.
electrophile and nucleophile was \3 A, an intramolecular
reaction was dominant. (3) The efficiency of proton
transfer between two oxygens and between nitrogen and
oxygen in Kirby’s enzyme models is attributed to a rela-
tively strong hydrogen bonding in the products and the
transition states leading to them [18–36].
It was concluded from the studies on intramolecularity
that there is a need to further investigate the reaction
mechanism for assigning the factors determining the
reaction rate. This would allow for better design of an
efficient chemical device that can be used as a prodrug
linker and have the potential to chemically and not enzy-
matically liberate the active drug in a programmable or
controlled manner. Among the various approaches to
minimize the undesirable drug properties while retaining
the desirable therapeutic activity, the chemical approach
using drug derivatization offers perhaps the highest flexi-
bility and has been demonstrated as an important means of
improving drug efficacy. The prodrug approach can be
123

618
J Comput Aided Mol Des (2013) 27:615–635
Fig. 1 Acid-catalyzed
hydrolysis in Kirby’s N-
methylmaleamic acids 1–7
O
O
O
R1
R2
R1
R2
R1
NHMe
OH
OH
OH
H2O
H2O
O
NH2Me
R2
O
O
O
1; R1=R2=H
2; R1=R2=Me
3 ;R1=H; R2=Me
4; R1,R2 CycIopent-l-ene-1,2-diyl
5; R1, R2 Cyclohex-l-ene-1,2-diyl
6; R1=H; R2=Et
7; R1=H; R2=n-Propyl
Fig. 2 Acid-catalyzed
hydrolysis in tranexamic acid
prodrugs, ProD 1–ProD 4
O
O
O
O
O
COOH
R1
R2
N
R1
R1
R2
H
OH
OH
H2O
O
H2O
OH
Tranexamic Acid
R2
O
ProD1; R1=R2=H
ProD2; R1=R2=Me
ProD3; R1=H; R2=Me
O
O
Me
COOH
Me
Me
O
O
N
H
Me
Me
Me
Me
H2O
H2O
OH
OH
OH
O
Tranexamic Acid
Me
Me
Me
Me
Me
O
O
ProD 4
OH
H
COOH
H2N
COO
H2N
O
ð1Þ
NH
O
NH
NH
O
HOOC
HO
OOC
O
HO
HOOC
O
O
O
ð2Þ
O
NH
OOC
O
O
It should be emphasized that at pH 5.5–6.5 (SC, skin,
mouth cavity and intestine physiologic environments)
the carboxylic group of the prodrugs will equilibrate with
the corresponding carboxylate form (Eq. 2). Subsequently,
the free acid form will undergo proton transfer reaction
(rate limiting step) to yield the antifibrinolytic drug, tran-
examic acid, and the inactive linker as a by-product
(Fig. 2).
123

J Comput Aided Mol Des (2013) 27:615–635
619
It is worth noting that our proposal is to exploit tran-
examic acid prodrugs ProD 1–ProD 4 for oral use via
enteric coated tablets. At this physiologic environment
these prodrugs will exist in the acidic and ionic forms
where the equilibrium constant for the exchange between
the two forms is dependent on the pKa of the given prodrug
(Eq. 2). The experimentally determined pKa for tranexamic
acid ProD 1–ProD 4 linkers are in the range of 4–6.
Therefore, it is expected that the pKas of the corresponding
prodrugs will have similar pKa range as for the carboxylic
linkers. Since the pH for the small intestine lies in the range
of 5.5–6.8, the calculated unionized (acidic)/ionized ratio
will be in the range of 10–50 %. Although the percentage
of the acidic form is not significantly high, we expect these
prodrugs to undergo an efficient proton transfer (rate lim-
iting step) to yield the antifibrinolytic drug, tranexamic
acid especially ProD 4 that have a pKa close to 6.5. In the
blood circulation at pH 7.4, the calculated acidic form is
around 1 % and it is expected that the efficiency for
delivering the parent drug will be relatively low. Improving
the efficiency could be achieved by using carboxylic
linkers having pKa close to that of the blood circulation
(pH 7.4).
water molecule and with two water molecules at the HF/6-
31G level of theory, followed by optimization at the
B3LYP/6-31G(d,p),
B3LYP/6-311?G(d,p),
MPW1k
[mpwpw91/6-31?G(d,p)] or MP2/6-31G(d,p) levels. Total
geometry optimizations included all internal rotations.
Second derivatives were estimated for all 3N-6 geometrical
parameters during optimization. The search for the global
minimum structure in each of 1–7 and tranexamic acid
ProD 1–ProD 4 was accomplished by 360° rotation of the
carboxylic group about the C6–C7 bond (i.e. variation of
the dihedral angle O1/C7/C6/C5, Fig. 3), and 360° rotation
of the carbonyl amide group about the C4–C5 bond (i.e.
variation of the dihedral angle O3/C4/C5/C6) in increments
of 10° and calculation of the conformational energies (see
Fig. 3). For systems 1–7, and tranexamic acid prodrugs
ProD 1–ProD 4 two types of conformations in particular
were considered: one in which the amide carbonyl is per-
pendicular to the carboxyl carbonyl group and another in
which it is planar. An energy minimum (a stable com-
pound or a reactive intermediate) has no negative vibra-
tional force constant. A transition state is a saddle point
which has only one negative vibrational force constant
[67]. Transition states were located first by the normal
reaction coordinate method [68] where the enthalpy chan-
ges was monitored by stepwise changing the interatomic
distance between two specific atoms. The geometry at the
highest point on the energy profile was re-optimized by
using the energy gradient method at the B3LYP/6-31G(d,p)
level of theory [65]. The ‘‘reaction coordinate method’’
[68] was used to calculate the activation energy in
all maleamic (4-amino-4-oxo-2-butenoic) acid amides
(Figs. 1–2). In this method, one bond length is constrained
for the appropriate degree of freedom while all other
variables are freely optimized. The activation energy val-
ues for the approach processes in 1–7 and tranexamic acid
prodrugs ProD 1–ProD 4 (the approach of O1 towards C4,
Fig. 3) were calculated from the difference in energies of
the global minimum structures (GM) and the derived
transition states (tetrahedral intermediate formation). In the
same manner, the activation energies of the dissociation
processes in 1–7 and tranexamic acid prodrugs ProD
1–ProD 4 (the breakdown of C4–N9 bond, Fig. 3) were
calculated from the difference in energies of the global
minimum structures (GM) and the corresponding transition
states (tetrahedral intermediate breakdown). Full optimi-
zation of the transition states was accomplished after
removing any constrains imposed while executing the
energy profile. The activation energies obtained from the
DFT at B3LYP/6-31G(d,p) level of theory for all mole-
cules were calculated, in the presence of one water mole-
cule, with and without the inclusion of water (dielectric
constant of water, 78.39). The calculations with the
incorporation of a water molecule were performed using
In this manuscript, we describe our theoretical and
experimental study which includes: (1) DFT quantum
molecular orbital investigations of ground and transition
states structures, vibrational frequencies, and reaction tra-
jectories for the intramolecular proton transfer in seven
N-methylmaleamic acids, 1–7, and four proposed tranex-
amic acid prodrugs ProD 1–ProD 4 (Figs. 1, 2). It is
expected that the study on proton transfers in these systems
will provide a good basis for the prediction of the phar-
macokinetic behavior of the prodrugs of the type ProD 1–
ProD 4 and (2) synthesis, characterization and kinetic
study of the interconversion of tranexamic acid ProD 1 in
different media; 1 N HCl. Buffer pH 2, buffer pH 5 and
buffer pH 7.4.
Calculations methods
The Becke three-parameter, hybrid functional combined
with the Lee, Yang, and Parr correlation functional,
denoted B3LYP, were employed in the calculations using
DFT. The DFT calculations at B3LYP/6-31G(d,p) and
B3LYP/311?G(d,p) levels, MP2 calculations and the
density functional from Truhlar group (hybrid GGA:
MPW1k) [62–64] were carried out using the quantum
chemical package Gaussian-2009 [65]. Calculations were
carried out based on the restricted Hartree–Fock method
[65]. The starting geometries of all calculated molecules
were obtained using the Argus Lab program [66] and were
initially optimized without the presence of water, with one
123

620
J Comput Aided Mol Des (2013) 27:615–635
Fig. 3 Chemical representation
of the perpendicular and planar
conformations for the global
minimum structures (GM) of
1–7 and tranexamic acid
2
H
R3
1
6
2
9
O
H
1
3
O
HN
O
prodrugs ProD 1–ProD 4
R3
4
4
8
O
9
O
O
3
8
7
N
7
H
5
6
5
R2
R2
R1
R1
Planar
Perpendicular
the integral equation formalism model of the Polariz-
able Continuum Model (PCM) [69–72]. In this model the
cavity is created via a series of overlapping spheres. The
radii type employed was the United Atom Topological
Model on radii optimized for the PBE0/6-31G(d) level of
theory.
buffers. The Sep-Pack C18 6 cc (1 g) cartridges were
purchased form Waters (Milford, MA, USA). 1H-NMR
experiments were performed with a Bruker AvanceII 400
spectrometer equipped with a 5 mm BBO probe. All
infrared spectra (FTIR) were obtained from a KBr matrix
(4,000–400 cm-1) using a PerkinElmer Precisely, Spec-
trum 100, FT-IR spectrometer.
Experimental
Preparation of tranexamic acid ProD 1 (Scheme 1)
General
In
a 250 mL round-bottom flask, tranexamic acid
Inorganic salts were of analytical grade and were used
without further purification. Organic buffer components
were distilled or recrystallized. Distilled water was redis-
tilled twice before use from all-glass apparatus. Maleic
anhydride, anhydrous sodium dihydrogen phosphate,
sodium lauryl sulfate, triethyl amine and tranexamic acid
were commercially obtained from sigma Aldrich. HPLC
grade solvents of methanol, acetonitrile and water were
purchased from Sigma Aldrich. High purity chloroform,
THF and diethyl ether ([99 %) were purchased from
Biolab (Israel). The LC/ESI–MS/MS system used was
Agilent 1200 series liquid chromatography coupled with a
6520 accurate mass quadruple-time of flight mass spec-
trometer (Q-TOF LC/MS). The analysis was performed in
the positive electrospray ionization mode. The capillary
voltage was 4.0 kV, the scanned mass range was
200–540 m/z (MS).
(10 mmol) was dissolved in THF (100 mL), a solution of
0.5 g NaOH in dry THF was added (50 mL), the resulting
solution was stirred for 30 min, then maleic anhydride
(10 mmol) was slowly added to the reaction mixture and
stirred at room temperature for 10 h, then the reaction
mixture was refluxed for 2 h and cooled to room temper-
ature. The white precipitate formed was collected and dried
(yield 87 %). M.P. 201 °C (not corrected). 1H-NMR d
(ppm) CD3OD– 1.03–1.06 (q, 2H, J = 3.2 Hz, CH–CH2–
CH2), 1.36–1.40 (q, 2H, J = 3.6 Hz, CH–CH2–CH2),
1.54–1.56 (m, 1H, CH2–CH–CH2–CH2), 1.84–1.89 (m, 2H,
CH2–CH2–CH), 1.99–2.02 (m, 2H, CH2–CH2–CH),
2.03–2.22 (m, 1H, CH2–CH–CH2–CH2), 3.16–3.17 (d, 2H,
J = 6.8 Hz, CH2–N), 6.23–6.25 (d, 1H, J = 12.8 Hz,
HC = CH), 6.27–6.44 (d, 1H, J = 12.8 Hz, HC = CH). IR
(KBr/mmax cm-1) 1,712 (C = O), 1,643 (C = O), 1,587
(C = C), 1,420, 1,280, 1,200, 1,132, 1,058. m/z 256.1179
(M ? 1)? (for further details see Supplementary data).
The high pressure liquid chromatography (HPLC) sys-
tem consisted of an Alliance 2695 module equipped with
2996 Photodiode array detector from Waters (Germany).
Data acquisition and control were carried out using
Empower 2TM software (Waters, Germany). Analytes were
separated on a 4.6 mm 9 150 mm XBridgeÒ C18 column
Kinetic methods
Buffer preparation 6.8 g potassium dihydrogen phos-
phate were dissolve in 900 mL water for HPLC, the pH of
buffer 2 was adjusted by diluted o-phosphoric acid and
water was added to a final volume of 1,000 mL (0.05 M).
The same procedure was done for the preparation of buf-
fers pH 5 and 7.4, however, the required pH was adjusted
using 1 N NaOH.
(5 lm particle size) used in conjunction with
a
4.6 9 20 mm, XBridgeÒ C18 guard column. Microfilters
of 0.45 lm porosity were normally used (AcrodiscÒ GHP,
Waters). pH meter model HM-30G: TOA electronicsTM
was used in this study to measure the pH value for the
123

J Comput Aided Mol Des (2013) 27:615–635
621
THF
COO Na
COOH
H2N
NaOH
H2N
Under N2
Tranexamic Acid
O
O
H
O
H
H
H
COOH
N
THF
H
OH
O
O
Tranexamic Acid ProD 1
Maleic Anhydride
Scheme 1 Synthesis of tranexamic acid ProD 1 from its parent drug, tranexamic acid
Interconversion of 500 ppm tranexamic acid ProD 1
solution, in 1 N HCl, buffer pH 2, buffer pH 5 or buffer pH
7.4, to its parent drug, tranexamic acid, was followed by
HPLC monitoring at a wavelength of 220 nm. Conversion
reactions were run mostly at 37.0 °C.
The progression of reaction was followed by monitoring
the disappearance of the prodrug peak and the appearance
of tranexamic acid and maleic anhydride versus time.
Results and discussion
Calibration curve for tranexamic acid and tranexamic acid
prodrug ProD 1 To construct a calibration curve for
tranexamic acid ProD 1 and the parent drug, tranexamic
acid, several concentrations (0.1, 1.0, 2.0, 5.0, 10.0, 20.0, 50,
100 and 200.0 ppm) were prepared. 20 lL of each solution
was injected into the HPLC and the peak for the pharma-
ceutical was recorded using the following HPLC conditions:
4.6 mm 9 250 mm, 5 lm, XBridgeÒ C18 column using
mobile phase containing 11 g anhydrous sodium dihydrogen
phosphate, 1.4 g Sodium Lauryl Sulfate, 5 mL triethyl
amine dissolved in 600 mL water and 400 mL methanol (pH
2.5 using diluted phosphoric acid), a flow rate of 0.9 mL/min
and UV detection at a wavelength of 220 nm.
Kirby et al. studied the efficiency of intramolecular catal-
ysis of amide hydrolysis by the carboxy-group of a number
of substituted N-methylmaleamic acids, 1–7 (Fig. 1) and
found that the reaction is remarkably sensitive to the pat-
tern of substitution on the carbon–carbon double bond. In
addition, their study revealed that the rates of hydrolysis of
the studied dialkyl-N-methylmaleamic acids range over
more than ten powers of ten, and the ‘‘effective concen-
tration’’ of the carboxy-group of the most reactive amide,
dimethyl-N-n-propylmaleamic acid, is greater than 1010 M.
This acid amide was found to be converted into the more
stable dimethylmaleic anhydride with a half-life of less
than 1 s at 39 °C below pH 3 [61].
Furthermore, Kirby and Lancaster’s [61] study demon-
strated that the amide bond cleavage is due to intramo-
lecular nucleophilic catalysis by the adjacent carboxylic
acid group and the dissociation of the tetrahedral inter-
mediate is the rate-limiting step. Later on Kluger and Chin
[73] researched the intramolecular hydrolysis mechanism
of a series of N-alkylmaleamic acids derived from aliphatic
amines having a wide range of basicity. Their study
revealed that the identity of the rate-limiting step is a
function of both the basicity of the leaving group and the
acidity of the solution.
Peak area versus concentration of the pharmaceutical
(ppm) was then plotted, and R2 value of the plot was
recorded.
Preparation of standard and sample solution 500 ppm of
standard tranexamic acid was prepared by dissolving
50 mg of drug in 100 mL of 1 N HCl, buffer pH 2, buffer
pH 5 or buffer pH 7.4, then each sample was injected into
HPLC to detect the retention time of tranexamic acid.
500 ppm of standard maleic anhydride was prepared by
dissolving 50 mg of drug in 100 mL of 1 N HCl, buffer pH
2, buffer pH 5 or buffer pH 7.4, then each sample was
injected into HPLC to detect the retention time of maleic
anhydride.
To utilize N-alkylmaleamic acids, 1–7, as prodrug
linkers for atenolol, acyclovir, cefuroxime and other drugs
having poor bioavailability or/and undesirable (bitter) taste,
we have unraveled the mechanism for their acid-catalyzed
hydrolysis using DFT and molecular mechanics methods.
500 ppm of tranexamic acid ProD 1 was prepared by
dissolving 50 mg of the prodrug in 100 mL of 1 N HCl,
buffer pH 2, buffer pH 5 or buffer pH 7.4 then each sample
was injected into HPLC to detect the retention time.
123

622
J Comput Aided Mol Des (2013) 27:615–635
Our DFT calculation results were found to be in accor-
dance with the reports by Kirby and Lancaster [61] and
Kluger and Chin [73]. Our findings demonstrated that the
N-alkylmaleamic acids hydrolysis occurs by one mecha-
nism where the rate-limiting step was found to be largely
dependent on the nature of the amine leaving group and the
medium solvent. Two different rate-limiting steps were
proposed: one involves the formation of a tetrahedral
intermediate [74], and the second involves a tetrahedral
intermediate dissociation (Fig. 4) [61, 73]. The gas phase
DFT calculations showed that the rate-limiting step for the
hydrolysis of all maleamic acid amides studied regardless
of the nature of the amine leaving group is a tetrahedral
formation. On the other hand, when the DFT calculations
were done in a dielectric constant of 78.39 (water) the rate-
limiting step for the hydrolysis of acid amides having
primary amine as a leaving group was the dissociation of
the tetrahedral intermediate, whereas that having acyclovir
or cefuroxime moieties the rate-limiting step was the tet-
rahedral intermediate formation.
reaction entities were done, in the presence of one mole-
cule of water, in a dielectric constant of 78.39 (water) and
the gas phase. It is expected that the stability of the ground
and transition states will be different in solvent having low
dielectric constant, such as the gas phase and solvent with
high dielectric constant, such as water.
General consideration
The ground state energy of the N-alkylmaleamic acid
moiety is vastly affected by the orientation of both the
carboxylic acid and amide groups especially when there is
a possibility for engagement in intramolecular hydrogen
bonding. Therefore, we were concerned with the identifi-
cation of the most stable conformer (global minimum) for
each of Kirby’s N-alkylmaleamic acids 1–7 and tranexamic
acid prodrugs ProD 1–ProD 4. The global minimum
search was achieved by 360° rotation of the carboxylic
group about the C6–C7 bond (i.e. variation of the dihedral
angle O1C7C6C5, Fig. 3), and 360° rotation of the car-
bonyl amide group about the C4–C5 bond (i.e. variation of
the dihedral angle O3C4C5C6) in increments of 10° and
calculation of the conformational energies (see Fig. 3).
In the DFT calculations for 1–7 and tranexamic acid
ProD 1–ProD 4, two types of conformers in particular
In similar manner to that done for systems 1–7, the DFT
calculations for the acid-catalyzed hydrolysis of tranex-
amic acid prodrugs ProD 1–ProD 4 were directed toward
elucidation of the transition and ground state structures
(reactants, intermediates and products). Calculations for all
R
R
R
HN
HN
HN
OH
R1
R1
R2
R1
R2
O
OH
O
Approach
Proton Transfer
H2O/H+
H
O
O
R2
O
O
INT
O
OR
GM
TSf
R
HN
H
O
O
H
H
R
H
R
NH
NH
O
O
O
O
OH
OH
O
R1
R2
R1
R2
R1
R2
Dissociation
O
H
RNH2
O
Protonated INT
O
Protonated TSd
R-NH- =
Methylamine,
Tranexamic acid,
Atenolol,
or
Cefuroxime
Aciclovir
Fig. 4 Mechanistic pathway for the acid-catalyzed hydrolysis of 1–7 and tranexamic acid ProD 1–ProD 4. GM, INT, TSf and TSd are global
minimum structure, tetrahedral intermediate 1, transition state for the formation step and transition state for the dissociation step, respectively
123

J Comput Aided Mol Des (2013) 27:615–635
623
Fig. 5 a DFT optimized
structures for the global
minimum (GM) in tranexamic
acid ProD 1–ProD 4. b DFT
optimized structures for the
tetrahedral intermediate (INT)
in tranexamic acid ProD 1–
ProD 4. c DFT optimized
structures for the tetrahedral
intermediate dissociation step
(TSd) in tranexamic acid ProD
1–ProD 4. d DFT optimized
structures for the tetrahedral
intermediate formation step
(TSf) in tranexamic acid ProD
1–ProD 4
123

624
J Comput Aided Mol Des (2013) 27:615–635
Fig. 5 continued
were considered: one in which the amide carbonyl is per-
pendicular to the carboxyl group and another in which it is
planar. It was found that the global minimum structures for
1–7 and tranexamic acid ProD 1–ProD 4 all exist in the
perpendicular conformation (see Figs. 3, 5a).
water. The calculations were done by placing water
molecule which is hydrogen bonded with the O8 (Fig. 3)
of the carboxylic group such that the bond angle
O8ꢀꢀꢀHꢀꢀꢀO is in the range of 170–180. The calculations
with two water molecules were executed by placing one
water molecule that is hydrogen bonded with O8 of the
carboxylic group and the other with O3 (Fig. 3) of the
carbonyl amide group. The calculation results reveal that
the presence of water has no significant effect on either
the enthalpy or activation energies. For example, the gas
phase calculated DFT enthalpic energy for system 2
without water, with one molecule of water and with two
molecules of water were 12.96, 13.93 and 13.07 kcal/mol,
respectively, whereas the calculated values in dielectric
constant of 78.39 (water) were 16.93, 17.56 and
17.5 kcal/mol, respectively.
Since the acid-catalyzed hydrolysis of N-alkylmaleamic
acids involves two steps: tetrahedral intermediate forma-
tion and dissociation, it was our concern to examine if
water molecules have any effect on the barriers involved
in the reaction. Interaction of a water molecule with the
carboxylic group might have an effect on its nucleophil-
icy. On the other hand, water molecule when interacts
with the amide carbonyl group the ability of the N-alkyl
amine as a leaving group might be altered. Therefore, the
starting geometries for the reactants were calculated with
one and two water molecules and without the presence of
123

J Comput Aided Mol Des (2013) 27:615–635
625
Fig. 5 continued
It should be emphasized that the orientation of the water
molecules was positioned in a similar manner in all sys-
tems studied (1–7 and ProD 1–ProD 4).
calculated DFT angles a and b values in the global mini-
mum structures (ProD 1GM–ProD 3GM) were in the
range of 125.2°–133.4° and 123.2°–130.1°, respectively.
On the other hand the angle a and b values in tranexamic
acid ProD 4GM were 109.6° and 108.0°, respectively.
Optimized structures for the entities involved in the acid-
catalyzed hydrolysis of tranexamic acid prodrugs ProD
1–ProD 4
Tetrahedral intermediate geometries (INT) The optimized
structures for the tetrahedral intermediates, ProD 1INT–
4INT, are shown in Fig. 5b. Inspection of the optimized
structures demonstrates that angles a and b values in the
intermediates are much smaller than the values of the corre-
sponding angles in the reactants. The a range values was
103.1°–107.8° whereas that for the b was 103.5°–110.4°.
Furthermore, the angles around the tetrahedral carbon were
found to be similar to that of regular tetrahedral intermediates.
Global minimum structures (GM) The global minimum
structures for ProD 1GM–ProD 4GM are illustrated in
Fig. 5a. Careful inspection of the calculated structures in
Fig. 5a reveals that all the global minima of the reactants,
except ProD 4GM, exist in conformation by which their
carboxyl group is engaging intramolecularly in a hydrogen
bonding net to form a seven-membered ring. Further, the
123

626
J Comput Aided Mol Des (2013) 27:615–635
The ranges for O1–C7 and O1–C4 bond distances were
˚
1.354–1.365 and 1.438–1.465 A, respectively.
ated ProD 1TSd–ProD 4TSd and Fig. 5a–d illustrate their
DFT optimized structures, respectively.
Using the calculated B3LYP/6-31G(d,p) enthalpy and
entropy values for the GM and transition states involved in
the acid-catalyzed hydrolysis of tranexamic acid prodrugs
ProD 1–ProD 4 the barriers (DGꢀ) for all steps described
in Fig. 4 were calculated in the gas phase as well as in
water (dielectric constant of 78.39). The calculated DFT
enthalpy and activation energy values for the barriers are
listed in Table 2. Careful examination of the DGꢀ (DGfꢀ,
activation energy for the tetrahedral intermediate formation
and DGꢀd, activation energy of the tetrahedral intermediate
dissociation) values in Table 2 demonstrates that while the
rate-limiting step for all prodrugs as calculated in the gas
phase is the tetrahedral intermediate formation the picture
is somewhat different when the calculations were done in
water. The DFT calculations in water indicate that the rate-
limiting step in the acid-catalyzed hydrolysis of tranexamic
acid ProD 4 was the tetrahedral intermediate formation
while that for ProD 1–ProD 3 was the tetrahedral inter-
mediate collapse.
Transition state structures for the acid-catalyzed hydrolysis
of tranexamic acid prodrugs ProD 1–ProD 4
The calculated DFT optimized structures for the transition
states of the tetrahedral intermediate dissociation (rate-
limiting step) in ProD 1–ProD 4 (ProD 1TSd–ProD 4TSd)
and tetrahedral formation (rate-limiting step) in ProD
1–ProD 4 (ProD 1TSf–ProD 4TSf) are shown in Fig. 5c,
d, respectively. Inspection of the DFT optimized transition
state structures for ProD 1TSd–ProD 3TSd revealed that
the geometries of the transition states resemble that of the
corresponding tetrahedral intermediates. The angle a and b
values are quite identical in both the tetrahedral interme-
diates and their corresponding transition states. The range
of the a values were found in the range 106.7°–107.9° and
that of b was in the range 108.5°–111.2°.
On the other hand, the rate-limiting step in the acid-
catalyzed hydrolysis of tranexamic acid ProD 4 was the
tetrahedral intermediate formation and not its collapse. The
transition state optimized geometries for the formation step
in tranexamic acid ProD 1–ProD 4 (ProD 1TSf–ProD
4TSf) are depicted in Fig. 5d. As shown in Fig. 5d, the
calculated C–O distance (the distance between the nucle-
ophile O– and the electrophile C) in ProD 1TSf–ProD
For an evaluation of the factors playing dominate role in
determining the acid-catalyzed hydrolysis rate in tranex-
amic acid prodrugs ProD 1–ProD 4 we have made a
comparison of the calculated DFT properties for tranex-
amic acid prodrugs ProD 1–ProD 4 with previously cal-
culated properties for the acid-catalyzed hydrolysis of 1–7
(Fig. 1), atenolol Prodrugs ProD 1–ProD 2 (Fig. 6) acy-
clovir prodrugs ProD 1–ProD 4 (Fig. 6) and cefuroxime
ProD 1–ProD 4 (Fig. 6).
˚
4TSf was 2.30 A and the angle a and b were quite similar
to that of the corresponding global minimum structures and
were 111.1°–118.5° and 112.6°–122.4°, respectively.
Itshouldbeemphasizedthatthe calculationsfortheentities
involved in the acid-catalyzed hydrolysis for the systems
studied in the presence of a water molecule and without water
were comparable. When the calculated DFT activation energy
values (Tables S1 and S2) in the presence and absence of a
water molecule were examined for correlation strong corre-
lation with a correlation coefficient r = 0.98 was obtained
(DGꢀ (GP) = 0.8625 DGꢀ (H2O) ? 6.97).
Table 2 lists the activation energy barrier values for the
intermediate formation (DGꢀf ) and dissociation (DGbꢀ) for
1–7, atenolol Prodrugs ProD 1–ProD 2, acyclovir prodrugs
ProD 1–ProD 4 and cefuroxime ProD 1–ProD 4.
Inspection of the energy values listed in Table 2 revealed
that the rate-limiting step (higher barrier) in the gas phase
for all systems studied is the tetrahedral intermediate for-
mation (the energy barrier for the tetrahedral intermediate
formation is about 0.5–20 kcal/mol higher than that for its
breakdown). On the other hand, the picture is quite dif-
ferent when the calculations were done in dielectric con-
stant of 78.39 (water medium). While for systems 1–7 and
atenolol prodrugs ProD 1–ProD 2 the rate-limiting step
was the breakdown of the tetrahedral intermediate (energy
difference between the breakdown and formation barriers
is 3–9 kcal/mol) in the reactions of cefuroxime prodrugs
ProD 1–ProD 4 and acyclovir prodrugs ProD 1–ProD 4
the rate-limiting step was found to be the formation of the
tetrahedral intermediate (energy difference between the
formation and break-down barriers is 14–30 kcal/mol).
Since the stability of the ground and transition states is
largely dependent on the reaction solvent and based on
Mechanistic investigation The DFT at B3LYP/6-
31G(d,p) level of kinetic and thermodynamic properties for
tranexamic acid prodrugs ProD 1–ProD 4 (Fig. 2) were
calculated using the quantum chemical package Gaussian-
2009 [62]. The enthalpy and entropy energy values for all
structures involved in the acid-catalyzed hydrolysis, global
minimum (GM), transition states (TS), intermediates (INT)
and products (P), were calculated, with the presence of a
water molecule, in the gas phase and water (dielectric
constant 78.39). Table 1 lists the enthalpy and entropy
energy values for ProD 1GM–ProD 4GM, ProD 1TSf–
ProD 4TSf, ProD 1INT–ProD 4INT, protonated ProD
1INT–ProD 4INT, ProD 1TSd–ProD 4TSd and proton-
123

J Comput Aided Mol Des (2013) 27:615–635
627
Table 1 DFT [B3LYP/6-31G(d,p)] calculated properties for the acid catalyzed hydrolysis of tranexamic acid ProD 1–ProD 4
Compound
Enthalpy, H (gas phase) (Ha)
Entropy, S (gas phase) (cal/mol-K)
Frequency(cm-1
)
ProD 1GMa
ProD 1TSaf
ProD 1TETINTa
ProD 1TETINT (protonated)
ProD 1TSad
-974.8819081
-974.8326548
-974.8471076
-898.7944564
-974.8065994
-898.7843298
-1,014.2048171
-1,014.1594626
-1,014.1723376
-938.2658956
-1,014.1327548
-938.1152433
-1,053.5140615
-1,053.4868204
-1,053.4978521
-977.4522428
-1,053.4581183
-977.4423073
-1,133.3773287
-1,133.3096986
-1,133.3474671
-1,057.2912653
-1,133.3678845
-1,057.2889832
163.12
148.66
150.98
137.23
149.45
135.69
171.51
156.97
157.60
146.56
157.67
145.14
179.62
171.73
163.08
154.44
164.32
154.45
177.52
171.68
175.66
154.43
173.21
161.75
–
145.69i
–
152.8i
84.33
–
ProD 1TSd (protonated)
ProD 2GMa
ProD 2TSaf
ProD 2TETINTa
ProD 2TETINT (protonated)
ProD 2TSad
115.95i
–
119.96i
78.44
–
ProD 2TSd (protonated)
ProD 3GMa
ProD 3TSaf
ProD 3TETINTa
ProD 3TETINT (protonated)
ProD 3TSad
102.39i
–
172.63i
167.48
–
ProD 3TSd (protonated)
ProD 4GMa
ProD 4TSaf
ProD 4TETINTa
ProD 4TETINT (protonated)
ProD 4TSd
145.69i
–
41.02i
79.44i
ProD 4TSd (protonated)
GM, TETINT and TS are global minimum, tetrahedral intermediate and transition state structures, respectively. f and d refer to tetrahedral intermediate formation
and dissociation, respectively
a
Calculated in the presence of a water molecule
Kirby’s findings and our previous DFT calculations on this
type of systems [24, 61], it is expected that the tetrahedral
intermediate formation and dissociation barriers will be
quite different when calculated in the gas phase or water. In
fact, examination of the barriers values for the tetrahedral
intermediate formation process (DGꢀf ) and tetrahedral
to shift the equilibrium to the reactants by destabilizing TSf
and thus making the tetrahedral intermediate formation as
the rate-limiting step whereas solvents with high dielectric
constants such as water (78.39) interact via dipole–dipole
interactions with the ionic transition state to shift the
equilibrium to the right side (see Eq. 3).
NH2
NH
O
NH
OH
NH
HOOC
OH
HOOC
HO
HOOC
O
HOOC
O
O
O
ð3Þ
O
O
O
Intermediate
dissociation
Intermediate
formation
O
intermediate breakdown process (DGꢀd) tabulated in
Table 2 clearly indicates that solvents with low dielectric
constant such as the gas phase (dielectric constant =1) tend
The dipole–dipole (hydrogen bonding) interactions stabi-
lize the transition state for the tetrahedral intermediate
formation (TSf) which results in lowering the approach of
123

628
J Comput Aided Mol Des (2013) 27:615–635
123

J Comput Aided Mol Des (2013) 27:615–635
629
O
N
H
N
H
H
N
CH3
N
N
O
O
O
O
HN
O
HO
OH
H3C
H3C
HO
OH
N
H
CH3
CH3
CH3
CH3
O
OH
O
O
O
O
O
HO
H2N
Aciclovir ProD 1
H2N
Atenolol ProD 2
Atenolol ProD 1
O
O
N
O
N
N
O
HN
N
HN
O
O
O
O
HN
H
H
H
F3C
F3C
N
N
O
N
N
N
H
N
N
H
N
H
O
O
OH
OH
H3C
OH
OH
OH
O
HO
O
Aciclovir ProD 3
Aciclovir ProD 4
Aciclovir ProD 2
O
O
OH
OH
O
O
OH
O
O
NH
NH
O
H
H
O
N
H3C
S
NH
N
O
H
S
O
O
O
N
OH
S
H
O
OH
H
H3C
O
H3C
O
NH
O
NH
OH
O
H
O
O
NH
O
O
O
N
N
O
O
O
N
O
Cefuroxime ProD 2
Cefuroxime ProD 3
Cefuroxime ProD 1
O
OH
O
O
NH
N
F3C
F3C
S
O
O
OH
H
O
NH
O
O
N
O
Cefuroxime ProD 4
Fig. 6 Chemical structures for atenolol ProD 1–ProD 2, acyclovir ProD 1–ProD 4 and cefuroxime ProD 1–ProD 4
the nucleophile to the electrophile barrier; thus the reaction
rate will be dependent on the tetrahedral intermediate
collapse step (the step via TSd is the rate-limiting).
correlated with the calculated DFT activation energy val-
ues, (DGꢀd) (Table 2). Good correlation was obtained
between the Es (INT) in 1–7 and tranexamic acid ProD 1–
ProD 3 and the activation energies for the tetrahedral
intermediate breakdown (DGꢀd) with a correlation coeffi-
cient values r = 0.86 (Fig. 7a). Attempts to correlate the
strain energies for the global minimum structures (Es for
GM) with either the experimental log krel or DGꢀd resulted
in a random correlation with r less than 0.40.
For assigning the factor determining the rate-limiting
step, the strain energy values for the reactants (GM), and
intermediates (INT2) in 1–7 and tranexamic acid prodrugs
ProD 1–ProD 4 were calculated using Allinger’s MM2
method [75]. The MM2 calculated strain energies (Es)
values are depicted in Table 2. The Es values were
123

630
J Comput Aided Mol Des (2013) 27:615–635
Fig. 7 a Plot of activation
energy for tetrahedral
intermediate dissociation DGdꢀ
versus strain energy of the
tetrahedral intermediate (EsINT
)
in systems 1–7 and tranexamic
acid ProD 1–ProD 3. b Plot of
acid-catalyzed hydrolysis
experimental rate (log krel
versus intermediate strain
)
energy (EsINT) for processes
1–7. c Plot of the DFT
calculated effective molarity
(EMcalc) versus the
experimental effective molarity
(EMexp) for systems 1–7
Fig. 8 Schematic
representation of the
intermolecular process Inter
O
O
H
H
NHCH3
CH3COOH
OH
NH2CH3
H3C
H3C
Inter
In addition, strong correlation was found between the
experimental relative rate (log krel) and Es (INT) in 1–7
with a correlation coefficient r of 0.95 (Fig. 7b).
process. It is generally accepted that the measure for intra-
molecular efficiency is the effective molarity (EM) which is
defined as a ratio of the intramolecular rate (kintra) and its
corresponding intermolecular (kinter) where both processes
are driven by identical mechanisms. The major factors
affecting the EM value are ring size, solvent and reaction
type. Values in the order of 109–1013 M have been measured
for the EM in intramolecular processes occurring through
nucleophilic addition. Whereas for proton transfer processes
EM values of less than 10 M were reported [43] until
recently where values of 1010 was reported by Kirby on the
hydrolysis of some enzyme models [37–41, 43, 76–80].
For obtaining credibility to our calculation results we
introduce our computation rational for calculating the EM
values for processes 1–7 and tranexamic acid ProD
1–ProD 4 based on the DFT calculated activation energies
(DGꢀ) of 1–7 and tranexamic acid ProD 1–ProD 4, and the
corresponding intermolecular process Inter (Fig. 8).
The intermolecular process Inter (Fig. 8) was calculated
to be used in the calculation of the effective molarity
Figure 7a, b and Table 2 demonstrate that the reaction
rate for systems 1–7 and tranexamic acid ProD 1–ProD 3
is dependent on the tetrahedral intermediate breakdown
and its value is largely affected by the strain energy of the
tetrahedral intermediate formed. Systems with less-strained
intermediates such as 2 and tranexamic acid ProD 3
undergo hydrolysis with higher rates than that having more
strained intermediates such as 4 and tranexamic acid ProD
1. This might be attributed to the fact that the transition
state structures in those systems resemble that of the cor-
responding intermediates.
The effective molarities (EM) for the reactions of 1–7
and tranexamic acid ProD 1–ProD 4
The effective molarity parameter is considered an excellent
tool to describe the efficiency of a certain intramolecular
123

J Comput Aided Mol Des (2013) 27:615–635
631
values (EM) for the corresponding intramolecular pro-
cesses 1–7 and tranexamic acid ProD 1–ProD 4.
For obtaining the EM values for the reactions of 1–7 and
tranexamic acid ProD 1–ProD 4 we have calculated the
kinetic and thermodynamic parameters for their corre-
sponding intermolecular process, Inter (Fig. 8).
log EMcalc ¼ 0:809 log EMexp þ 4:75
ð9Þ
In order to confirm that the DFT calculations at B3LYP/
6-31G(d,p) are not dependent on the specific theoretical
method used processes 1–3 were calculated at B3LYP/
311?G(d,p), hybrid GGA: MPW1k and MP2/6-31G(d,p)
levels. The comparisons between the activation energy
values calculated by the different methods are listed in
Table 3. Careful examination of Table 3 reveals that the
calculated values at B3LYP/6-31G(d,p) and B3LYP/
311?G(d,p) were very close, whereas, those obtained by
either MPW1k [mpwpw91/6-31?G(d,p)] and MP2/6-
31G(d,p) were about 2–3 kcal/mol smaller.
Using Eqs. 4–7, we have derived Eq. 8 which describes
the EM term as a function of the difference in the activa-
tion energies of the intra- and the corresponding inter-
molecular processes. The values calculated using Eq. 8 for
processes 1–7 and tranexamic acid ProD 1–ProD 4 in
water are listed in Table 2.
EM ¼ kintra=kinter
ð4Þ
ð5Þ
ð6Þ
ð7Þ
Using the calculated energies by the different methods
we have calculated the EM values for tranexamic acid
ProD 1 and systems 1–3. The calculated EM values for
tranexamic acid ProD 1 at B3LYP/6-31G, B3LYP/
311?G(d,p) and hybrid GGA: MPW1K [mpwpw91/6-
31?G(d,p)] were 9.53, 10.58 and 11.48, respectively.
Using the t1/2 value for process 2 (t1/2 = 1 s) [61] and the
calculated EM values using the three different methods we
have calculated the t1/2 values for tranexamic acid ProD 1
which were 556 h (as calculated by B3LYP/6-31G(d,p),
50.5 h [as calculated by B3LYP/311?G(d,p)] and 6.2 h [as
calculated by mpwpw91/6-31?G(d,p)].
z
DGinter ¼ ꢁRT ln kinter
z
DGintra ¼ ꢁRT ln kintra
z
z
DGintra ꢁ DGinter ¼ ꢁRT ln kintra=kinter
z
z
ꢁDGintraÞ=RT
EM ¼ eꢁðDG
ð8Þ
inter
where T is 298 °K and R is the gas constant.
The calculated log EM values listed in Table 2 were
examined for correlation with the corresponding log EM
experimental values [24, 61]. Good correlation was
obtained with r value of 0.93 (Fig. 7c). Inspection of the
log EM values listed in Table 2 and Fig. 7c reveals that 2,
5 and tranexamic acid ProD 3 were the most efficient
processes among all processes where by which the tetra-
hedral intermediate dissociation is the rate-limiting step,
whereas processes 1 and 4 were the least. The discrepancy
in rates between 2 and 5 on one hand and 1 and 4 on the
other hand is attributed to strain effects.
Hydrolysis studies
The kinetics of the acid-catalyzed hydrolysis studies were
carried out in aqueous buffer in the same manner as that
done by Kirby on Kirby’s enzyme model 1–7. This is in
order to explore whether the prodrug hydrolyzes in aqueous
medium and to what extent or not, suggesting the fate of
the prodrug in the system. Acid-catalyzed hydrolysis
kinetics of the synthesized tranexamic acid ProD 1 was
studied in four different aqueous media: 1 N HCl, buffer
pH 2, buffer pH 5 and buffer pH 7.4. Under the
Although the calculated and experimental EM values are
comparable there is a discrepancy in their absolute values.
This is due to the fact that the experimental measurement
of the EM values in 1–7 was conducted in the presence of
aqueous acid whereas the DFT calculations were done in
water. The dielectric constant value for a mixture of acid/
water is expected to be different from that of water (78.39)
and hence the discrepancy in the calculated and experi-
mental EM values [24, 69–72].
Table 3 Comparison between calculated energies for 1–3 using
B3LYP/6-31G(d,p), B3LYP/6-311?G(d,p), MPW1K and MP2
methods
System
B3L
DHꢀd
B3L311
DHꢀd
MPW1k
DHꢀd
MP2
DHꢀd
1
27.31
13.93
24.41
28.19
27.42
14.02
23.85
26.76
25.07
11.96
22.61
25.52
25.75
10.41
21.51
–
Calculation of the t1/2 values for the cleavage reactions
2
of tranexamic acid prodrugs ProD 1–ProD 4
3
ProD 1
Using Eq. 9 obtained from the correlation of log EMcalc
versus log EMexp (Fig. 7d) and the t1/2 value for process 2
(t1/2 = 1 s) [61], we have calculated the t1/2 values for
tranexamic acid ProD 1–ProD 4. The predicted t1/2 at pH 2
for ProD 1–ProD 4 as calculated by B3LYP/6-31G(d,p)
method are 556, 253 h, 70 s and 1.7 h, respectively.
B3L, B3L311, MPW1k [mpwpw91/6-31?G(d,p)] and MP2 refer to
B3LYP/6-31G (d,p), B3LYP/6-311?G(d,p), MPW1K/6-31G(d,p)
and MP2/6-31G(d,p) methods, respectively. DHꢀ is the calculated
enthalpy activation energy (kcal/mol). d refers to tetrahedral inter-
mediate dissociation (see Fig. 4). All values were calculated with a
molecule of water in the gas phase
123

632
J Comput Aided Mol Des (2013) 27:615–635
2 and 5 (Fig. 9). At 1 N HCl the prodrug was hydrolyzed to
release the parent drug in less than 1 h. On the other hand,
at pH 7.4, the prodrug was entirely stable and no release of
the parent drug was observed. Since the pKa of tranexamic
acid ProD 1 is in the range of 3–4, it is expected at pH 5
the anionic form of the prodrug will be dominant and the
percentage of the free acidic form that undergoes the acid-
catalyzed hydrolysis will be relatively low. At 1 N HCl and
pH 2 most of the prodrug will exist as the free acid form
and at pH 7.4 most of the prodrug will be in the anionic
form. Thus, the difference in rates at the different pH
buffers.
Table 4 The observed k value and t1/2 of tranexamic acid prodrug
(ProD 1) In 1 N HCl and at pH 2, 5 and 7.4
Medium
kobs (h-1
)
t (h)
‘
1 N HCl
5.13 9 10-3
3.92 9 10-5
3.92 9 10-6
No reaction
0.9
Buffer pH 2
Buffer pH 5
Buffer pH 7.4
23.9
270
No reaction
experimental conditions the target compounds hydrolyzed
to release the parent drug (Fig. 6) as evident by HPLC
analysis. At constant pH and temperature the reaction
displayed strict first order kinetics as the kobs was fairly
constant and a straight plot was obtained on plotting log
concentration of residual prodrug verves time. The rate
constant (kobs) and the corresponding half-lives (t1/2) for
tranexamic acid prodrug ProD 1 in the different media
were calculated from the linear regression equation corre-
lating the log concentration of the residual prodrug verses
time. The kinetic data are listed in Table 4. The 1 N HCl,
pH 2 and pH 5 were selected to examine the interconver-
sion of the tranexamic acid prodrug in pH as of stomach,
because the mean fasting stomach pH of adult is approxi-
mately 1–2 and increases up to 5 following ingestion of
food. In addition, buffer pH 5 mimics the beginning small
intestine pathway. Finally, pH 7.4 was selected to examine
the interconversion of the tested prodrug in blood circula-
tion system. Acid-catalyzed hydrolysis of the tranexamic
acid ProD 1 was found to be higher in 1 N HCl than at pH
Conclusions and future directions
The DFT calculations of the acid-catalyzed hydrolysis in
Kirby’s acid amides 1–7 and tranexamic acid ProD
1–ProD revealed that the reaction rate-limiting step
depends on the reaction medium. In aqueous medium the
rate-limiting step is a dissociation of the tetrahedral inter-
mediate whereas in the gas phase the formation of the
tetrahedral intermediate is the rate-limiting step. In addi-
tion, the calculations demonstrated that the efficiency of
the processes studied is largely sensitive to the pattern of
substitution on the carbon–carbon double bond and the
nature of the amide N-alkyl group. The reaction rate was
found to be linearly correlated with the strain energy of the
intermediate (Es INT). In addition, a linear correlation
Fig. 9 First order hydrolysis
plot of tranexamic acid prodrugs
1 in a 1 N HCl, b buffer of pH 2
and c buffer pH 5
123

J Comput Aided Mol Des (2013) 27:615–635
633
to Nardene Karaman, Angi Karaman, Donia Karaman, and Rowan
Karaman for technical assistance.
between the calculated and experimental EM values rein-
force the credibility of using DFT methods in predicting
energies as well as rates for reactions of the type described
herein [18–36].
References
Using the correlation equation obtained from the plot of
the calculated and experimental EM values we have cal-
culated the t1/2 of four different tranexamic acid prodrugs
(ProD 1–ProD 4).
1. CyklokapronÒ tranexamic acid injection prescribing information:
http://www.pfizer.com/files/products/uspi_cyklokapron.pdf
2. CRASH-2 trial collaborators (2010) Effects of tranexamic acid on
death, vascular occlusive events, and blood transfusion in trauma
patients with significant haemorrhage (CRASH-2): a randomised,
placebo-controlled trial. Lancet 6736(10):60835
3. Gohel M, Patel P, Gupta A, Desai P (2007) Efficacy of tranex-
amic acid in decreasing blood loss during and after cesarean
section: a randomized case controlled prospective study. J Obstet
Gynaecol India 57(3):227–230
Comparison between the calculated t1/2 values (556 h)
for tranexamic acid ProD 1 to the experimental value
(23.9 h) indicates that while the value obtained by B3LYP/
6-31G(d,p) is overestimated (about 23 times larger than the
experimental) the values obtained by B3LYP/6-
311?G(d,p) and mpwpw91/6-31?G(d,p) were much more
closer [50.5 h as calculated by B3LYP/311?G(d,p)] and
[6.2 h as calculated by mpwpw91/6-31?G(d,p)].
4. Giancarlo L, Francesco B, Angela L, Pierluigi P, Gina R (2011)
Recommendations for the transfusion management of patients in
the peri-operative period. II. The intra-operative period. Blood
Transfus 9:189–217
The discrepancy between the calculated B3LYP/6-
31G(d,p) and the experimental values might be attributed
to (1) B3LYP/6-31G(d,p) is a DFT method without dis-
persion corrections and (2) PCM solvation model (calcu-
lations in presence of solvent) is not capable for handling
calculations in acidic aqueous solvent (medium) since the
dielectric constant for pH 2 aqueous solutions is not
known. In the study calculations the value of 78.39
(dielectric constant for pure water) was used instead.
The t1/2 experimental value at pH 5 was 270 h and at pH
7.4 no interconversion was observed. The lack of the
reaction at the latter pH might be due to the fact that at this
pH tranexamic acid ProD 1 exists solely in the ionized
form (pKa about 4). As mentioned before the free acid form
is a mandatory requirement for the reaction to proceed.
On the other hand, tranexamic acid ProD 4 has a higher
pKa than tranexamic acid ProD 1 (about 6 vs. 4). There-
fore, it is expected that the interconversion rate of tranex-
amic acid ProD 4 to its parent drug, tranexamic acid, at all
pHs studied will be higher (log EM for ProD 4 is 14.33 vs.
9.53 for ProD 1).
5. Lukes AS, Kouides PA, Moore KA (2011) Tranexamic acid: a
novel oral formulation for the treatment of heavy menstrual
bleeding. Womens Health (Lond Engl) 7(2):151–158
6. Lukes AS, Moore KA, Muse KN, Gersten JK, Hecht BR, Edlund
M, Richter HE, Eder SE, Attia GR, Patrick DL, Rubin A,
Shangold GA (2010) Tranexamic acid treatment for heavy
menstrual bleeding: a randomized controlled trial. Obstet Gyne-
col 116(4):865–875
7. Ducloy-Bouthors AS, Jude B, Duhamel A, Broisin F, Huissoud
C, Keita-Meyer H, Mandelbrot L, Tillouche N, Fontaine S, Le
Goueff F, Depret-Mosser S, Vallet B, EXADELI Study Group,
Susen S (2011) High-dose tranexamic acid reduces blood loss in
postpartum haemorrhage. Crit Care 15(2):R117. doi:10.1186/
cc10143 (Epub 2011 Apr 15)
8. Ayedi M, Jarraya A, Smaoui M, Zouari J, Smaoui L, Kolsi K
(2011) Effect of tranexamic acid on post partum hemorrhage by
uterine atony: a preliminary result of a randomized, placebo-
controlled trial: 11AP4-7. Eur J Anaesthesiol 28:165
˘
9. Gungorduk K, Yıldırım G, Asıcıoglu O, Gungorduk OC, Sudol-
mus S, Ark C (2011) Efficacy of intravenous tranexamic acid in
reducing blood loss after elective cesarean section: a prospective,
randomized, double-blind, placebo-controlled study. Am J Per-
inatol 28(3):233–240
10. Sekhavat L, Tabatabaii A, Dalili M, Farajkhoda T, Tafti AD
(2009) Efficacy of tranexamic acid in reducing blood loss after
cesarean section. J Matern Fetal Neonatal Med 22(1):72–75
11. Sentu¨rk MB, Cakmak Y, Yildiz G, Yildiz P (2012) Tranexamic
acid for cesarean section: a double-blind, placebo-controlled,
randomized clinical trial. Arch Gynecol Obstet. Nov 11 [Epub
ahead of print]
12. Cardone D, Klein AA (2009) Perioperative blood conservation.
Eur J Anaesthesiol 26:722–729
13. Nilsson IM (1980) Clinical pharmacology of aminocaproic and
tranexamic acids. J Clin Pathol 33(14):41–47
14. Turki A, Michael W (2011) Tranexamic acid treatment of life-
threatening hematuria in polycystic kidney disease (case report).
Int J Nephrol 2011:203579. doi:10.4061/2011/203579
15. Maedaa K, Naganuma M (1998) Topical trans-4-aminomethyl
cyclohexane carboxylic acid prevents ultraviolet radiation-
induced pigmentation. J Photochem Photobiol B 47:136–141
16. Sufan W, Hangyan S, Hua W, Sheng Y, Jincai G, Yi S, Lei P
(2012) Treatment of melasma with oral administration of tran-
examic acid. Aesthet Plast Surg 36:964–970
Future strategy to achieve desirable tranexamic acid
prodrugs capable of releasing tranexamic acid in a con-
trolled manner and enhancing the parent drug bioavail-
ability is: (2) synthesis of tranexamic acid ProD 4; (2)
kinetic studies (in vitro) of ProD 4 will be performed in at
pH 6.5 (intestine) and pH 7.4 (blood circulation system) (3)
in vivo pharmacokinetic studies will be done in order to
determine the bioavailability and the duration of action of
the tested prodrug. Furthermore, based on the in vivo
pharmacokinetics characteristics of tranexamic acid ProD
4 new prodrugs may be design and synthesized.
Acknowledgments The author would like to acknowledge funding
by the German Research Foundation (DFG, ME 1024/8-1) and Exo
Research Organization, Potenza, Italy. Special thanks are also given
123

634
J Comput Aided Mol Des (2013) 27:615–635
17. Pilbrant A, Schannong M, Vessman J (1981) Pharmacokinetics
and bioavailability of tranexamic acid. Eur J Clin Pharmacol
20(1):65–72
40. Kirby AJ, Williams NH (1991) Efficient intramolecular general
acid catalysis of vinyl ether hydrolysis by the neighbouring car-
boxylic acid group. J Chem Soc Chem Commun 1643–1644
41. Kirby AJ (1996) Enzyme mechanisms, models, and mimics.
Angew Chem Int Ed Engl 35:706–724
18. Karaman
R (2008) Analysis of Menger’s spatiotemporal
hypothesis. Tetrahedron Lett 49:5998–6002
19. Karaman R (2009) Cleavage of Menger’s aliphatic amide: a
model for peptidase enzyme solely explained by proximity ori-
entation in intramolecular proton transfer. J Mol Struct (Theo-
chem) 910:27–33
20. KaramanR(2010)Theefficiencyof protontransferinKirby’senzyme
model, a computational approach. Tetrahedron Lett 51:2130–2135
21. Karaman R, Pascal RA (2010) Computational analysis of intra-
molecularity in proton transfer reactions. Org Biomol Chem 8:
5174–5178
22. Karaman R (2010) A general equation correlating intramolecular
rates with ‘attack’ parameters: distance and angle. Tetrahedron
Lett 51:5185–5190
23. Karaman R (2011) Analyzing the efficiency of proton transfer to
carbon in Kirby’s enzyme model—a computational approach.
Tetrahedron Lett 52:699–704
24. Karaman R (2011) Analyzing the efficiency in intramolecular
amide hydrolysis of Kirby’s N-alkylmaleamic acids—a compu-
tational approach. Comput Theor Chem 974:133–142
25. Karaman R (2009) A new mathematical equation relating acti-
vation energy to bond angle and distance: a key for understanding
the role of acceleration in lactonization of the trimethyl lock
system. Bioorg Chem 37:11–25
42. Fife TH, Przystas TJ (1979) Intramolecular general acid catalysis
in the hydrolysis of acetals with aliphatic alcohol leaving groups.
J Am Chem Soc 101:1202–1210
43. Kirby AJ (2005) Effective molarities for intramolecular reactions.
J Phys Org Chem 18:101–278
44. Menger FM, Ladika M (1988) Fast hydrolysis of an aliphatic
amide at neutral pH and ambient temperature. A peptidase model.
J Am Chem Soc 110:6794–6796
45. Menger FM (1985) On the source of intramolecular and enzy-
matic reactivity. Acc Chem Res 18:128–134
46. Menger FM, Chow JF, Kaiserman H, Vasquez PC (1983)
Directionality of proton transfer in solution. Three systems of
known angularity. J Am Chem Soc 105:4996–5002
47. Menger FM, Galloway AL, Musaev DG (2003) Relationship
between rate and distance. Chem Commun 2370–2371
48. Milstein S, Cohen LA (1970) Concurrent general-acid and gen-
eral-base catalysis of esterification.
92:4377–4382
J
Am Chem Soc
49. Milstein S, Cohen LA (1970) Rate acceleration by stereopopu-
lation control: models for enzyme action. Proc Natl Acad Sci
USA 67:1143–1147
50. Milstein S, Cohen LA (1972) Stereopopulation control I. Rate
enhancement in the lactonizations of o-hydroxyhydrocinnamic
acids. J Am Chem Soc 94:9158–9165
26. Karaman R (2009) Revaluation of Bruice’s proximity orientation.
Tetrahedron Lett 50:452–458
27. Karaman R (2009) Accelerations in the lactonization of trimethyl
lock systems are due to proximity orientation and not to strain
effects. Res Lett Org Chem. doi:10.1155/2009/240253
28. Karaman R (2009) The gem-disubstituent effect—a computa-
tional study that exposes the relevance of existing theoretical
models. Tetrahedron Lett 50:6083–6087
29. Karaman R (2009) Analyzing Kirby’s amine olefin—a model for
amino-acid ammonia lyases. Tetrahedron Lett 50:7304–7309
30. Karaman R (2009) The effective molarity (EM) puzzle in proton
transfer reactions. Bioorg Chem 37:106–110
31. Karaman R (2010) Effects of substitution on the effective
molarity (EM) for five membered ring-closure reactions—a
computational approach. J Mol Struct (Theochem) 939:69–74
32. Karaman R (2010) The effective molarity (EM) puzzle in intra-
molecular ring-closing reactions. J Mol Struct (Theochem)
940:70–75
33. Menger FM, Karaman R (2010) A singularity model for chemical
reactivity. Eur J Chem 16:1420–1427
34. Karaman R (2010) The effective molarity (EM)—a computa-
tional approach. Bioorg Chem 38:165–172
35. Karaman R (2010) Proximity vs. strain in ring-closing reactions
of bifunctional chain molecules—a computational approach.
J Mol Phys 108:1723–1730
36. Karaman R (2011) The role of proximity orientation in intra-
molecular proton transfer reactions. J Comput Theor Chem
966:311–321
37. Barber SE, Dean KES, Kirby AJ (1999) A mechanism for effi-
cient proton-transfer catalysis. Intramolecular general acid
catalysis of the hydrolysis of 1-arylethyl ethers of salicylic acid.
Can J Chem 77:792–801
38. Kirby AJ, de Silva MF, Lima D, Roussev CD, Nome F (2006)
Efficient intramolecular general acid catalysis of nucleophilic
attack on a phosphodiester. J Am Chem Soc 128:16944–16952
39. Kirby AJ, Williams NH (1994) Efficient intramolecular general
acid catalysis of enol ether hydrolysis. Hydrogen-bonding sta-
bilization of the transition state for proton transfer to carbon.
J Chem Soc Perkin Trans 2:643–648
51. Brown RF, van Gulick NM (1956) The geminal alkyl effect on
the rates of ring closure of bromobutylamines. J Org Chem
21:1046–1049
52. Bruice TC, Pandit UK (1960) The effect of geminal substitution
ring size and rotamer distribution on the intra molecular nucle-
ophilic catalysis of the hydrolysis of monophenyl esters of dibasic
acids and the solvolysis of the intermediate anhydrides. J Am
Chem Soc 82:5858–5865
53. Bruice TC, Pandit UK (1960) Intramolecular models depicting
the kinetic importance of ‘‘Fit’’ in enzymatic catalysis. Proc Natl
Acad Sci USA 46:402–404
54. Galli C, Mandolini L (2000) The role of ring strain on the ease of
ring closure of bifunctional chain molecules. Eur J Org Chem
2000:3117–3125
55. Karaman R (2010) Prodrugs of aza nucleosides based on proton
transfer reactions. J Comput Aided Mol Des 24:961–970
56. Karaman R (2011) Computational aided design for dopamine
prodrugs based on novel chemical approach. Chem Biol Drug
Des 78:853–863
57. Karaman R, Dajani KK, Qtait A, Khamis M (2012) Prodrugs of
acyclovir—a computational approach. Chem Biol Drug Des
79:819–834
58. Karaman R, Hallak H (2010) Computer-assisted design of pro-
drugs for antimalarial atovaquone. Chem Biol Drug Des
76:350–360
59. Karaman R, Dajani KK, Hallak H (2012) Computer-assisted
design for atenolol prodrugs for the use in aqueous formulations.
J Mol Model 18:1523–1540
60. Hejaz H, Karaman R, Khamis M (2012) Computer-assisted
design for paracetamol masking bitter taste prodrugs. J Mol
Model 18:103–114
61. Kirby AJ, Lancaster PW (1972) Structure and efficiency in
intramolecular and enzymatic catalysis. Catalysis of amide
hydrolysis by the carboxy-group of substituted maleamic acids.
J Chem Soc Perkin Trans 2:1206–1214
62. Zhao Y, Truhlar DG (2008) Density functionals with broad
applicability in chemistry. Acc Chem Res 41(2):157–167
123

J Comput Aided Mol Des (2013) 27:615–635
635
`
63. Zhao Y, Truhlar DG (2008) Exploring the limit of accuracy of the
global hybrid meta density functional for main-group thermo-
chemistry, kinetics, and noncovalent interactions.’’. J Chem
Theory Comput 4(11):1849–1868
64. Zheng J, Zhao Y, Truhlar DG (2009) The DBH24/08 database
and its use to assess electronic structure model chemistries for
chemical reaction barrier heights. J Chem Theory Comput
5(4):808–821
65. Gaussian 09, Revision A.1, Frisch MJ, Trucks GW, Schlegel HB,
Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V,
Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X,
Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL,
Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M,
Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery
Jr JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E,
Kudin K. N, Staroverov V. N, Kobayashi R, Normand J, Ragh-
avachari K, Rendell A, Burant J. C, Iyengar S. S, Tomasi J, Cossi
M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V,
Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL,
Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannen-
71. Mennucci B, Cances MT, Tomasi J (1997) Evaluation of solvent
effects in isotropic and anisotropic dielectrics and in ionic solu-
tions with a unified integral equation method: theoretical bases,
computational implementation, and numerical applications.
J Phys Chem B 101:10506–10517
`
72. Tomasi J, Mennucci B, Cances MT (1997) The IEF version of the
PCM solvation method: an overview of a new method addressed
to study molecular solutes at the QM ab initio level. J Mol Struct
(Theochem) 464:211–226
73. Kluger R, Chin J (1982) Carboxylic acid participation in amide
hydrolysis. Evidence that separation of a nonbonded complex can
be rate determining. J Am Chem Soc 104:2891–2897
74. Katagi T (1990) AM1 study of acid-catalyzed hydrolysis of
maleamic (4-amino-4-oxo-2-butenoic) acids. J Comput Chem
11(9):1094–1100
75. Burker U, Allinger NL (1982) Molecular mechanics. American
Chemical Society, Washington, DC
76. Kirby AJ, Parkinson A (1994) Most efficient intramolecular
general acid catalysis of acetal hydrolysis by the carboxyl group.
J Chem Soc Chem Commun 707–708
77. Brown CJ, Kirby AJ (1997) Efficiency of proton transfer catal-
ysis. Intramolecular general acid catalysis of the hydrolysis of
dialkyl acetals of benzaldehyde. J Chem Soc Perkin Trans
2:1081–1094
78. Craze G-A, Kirby AJ (1974) The role of the carboxy-group in
intramolecular catalysis of acetal hydrolysis. The hydrolysis of
substituted 2- methoxymethoxybenzoic acids. J Chem Soc Perkin
Trans 2:61–66
¨
berg JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz
JV, Cioslowski J, Fox DJ (2009) Gaussian, Inc., Wallingford CT
66. Casewit CJ, Colwell KS, Rappe’ AK (1992) Application of a
universal force field to main group compounds. J Am Chem Soc
114:10046–10053
67. Murrell JN, Laidler KJ (1968) Symmetries of activated com-
plexes. Trans Faraday Soc 64:371–377
68. Muller K (1980) Reaction paths on multidimensional energy
hypersurfaces. Angew Chem Int Ed Engl 19:1–13
79. Hartwell E, Hodgson DRW, Kirby AJ (2000) Exploring the limits
of efficiency of proton-transfer catalysis in models and enzymes.
J Am Chem Soc 122:9326–9327
`
69. Cances MT, Mennucci B, Tomasi J (1997) A new integral
equation formalism for the polarizable continuum model: theo-
retical background and applications to isotropic and anisotropic
dielectrics. J Chem Phys 107:3032–3041
80. Kirby AJ (1997) Efficiency of proton transfer catalysis in models
and enzymes. Acc Chem Res 30:290–296
70. Mennucci B, Tomasi J (1997) Coninuum solvation models: a new
approach to the problem of solute’s charge distribution and cavity
boundaries. J Chem Phys 106:5151
123