Bitterless guaifenesin prodrugs—design, synthesis, characterization, in vitro kinetics, and bitterness studies

Article abstract of DOI:10.1111/cbdd.13409

A respected number of drugs suffer from bitter taste which results in patient incompliance. With the aim of solving the bitterness of guaifenesin, dimethyl maleate, maleate, glutarate, succinate, and dimethyl succinate prodrugs were designed and synthesized. Molecular orbital methods were utilized for the design of the ester prodrugs. The density functional theory (DFT) calculations revealed that the hydrolysis efficiency of the synthesized prodrugs is significantly sensitive to the pattern of substitution on C=C bond and distance between the nucleophile and the electrophile. The hydrolysis of the prodrugs was largely affected by the pH of the medium. The experimental t_1/2 for the hydrolysis of guaifenesin dimaleate ester prodrugs in 1N HCl was the least and for guaifenesin dimethyl succinate was the highest. Functional heterologous expression of TAS2R14, a broadly tuned bitter taste receptor responding to guaifenesin, and experiments using these prodrugs revealed that, while some of the prodrugs still activated the receptor similarly or even stronger than the parent substance, succinate derivatization resulted in the complete loss of receptor responses. The predicted binding modes of guaifenesin and its prodrugs to the TAS2R14 homology model suggest that the decreased activity of the succinate derivatives may be caused by a clash with Phe247.

Full text of DOI:10.1111/cbdd.13409

DR MAIK BEHRENS (Orcid ID : 0000-0003-2082-8860)
PROFESSOR RAFIK KARAMAN (Orcid ID : 0000-0001-5526-4490)
Article type : Research Article
Article type: Research Article
Bitterless Guaifenesin Prodrugs – Design, Synthesis, Characterization, In
Vitro Kinetics and Bitterness Studies
1
1
2
1
Amin Thawabteh , Filomena Lelario , Laura Scrano , Sabino A. Bufo
3
3,4
5
5
Stefanie Nowak , Maik Behrens , Antonella Di Pizio , Masha Y Niv
1
,6
and Rafik Karaman*
1
Department of Sciences, University of Basilicata, 85100,Potenza, Italy.
2
Department of European Cultures (DICEM), University of Basilicata, Potenza 85100, Italy
3
German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany
4
Leibniz-Institute for Food Systems Biology at the Technical University of Munich, 85354
Freising, Germany
5
The Institute of Biochemistry, Food and Nutrition, The Robert H Smith Faculty of
Agriculture, Food and Environment, The Hebrew University and The Fritz Haber Center for
Molecular Dynamics, The Hebrew University
Rehovot, IL
6
Pharmaceutical Sciences Department, Faculty of Pharmacy, Al-Quds University, Jerusalem,
Palestine.
*Corresponding author:
Rafik Karaman
Department of Sciences, University of Basilicata, 85100,Potenza, Italy
Pharmaceutical Sciences Department, Faculty of Pharmacy, Al-Quds University, Jerusalem,
Palestine.
Tel.: 00972-59-8755052
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13409
This article is protected by copyright. All rights reserved.

Email: dr_karaman@yahoo.com
Keywords: Bitter taste, prodrugs, guaifenesin, DFT calculations, acid-catalyzed ester
hydrolysis, molecular modeling.
Running title: Bitterless Guaifenesin Prodrugs.
Abstract:
A respected number of drugs suffer from bitter taste which results in patient incompliance.
With the aim of solving the bitterness of guaifenesin, dimethyl maleate, maleate, glutarate,
succinate and dimethyl succinate prodrugs were designed and synthesized. Molecular orbital
methods were utilized for the design of the ester prodrugs.
The density functional theory (DFT) calculations revealed that the hydrolysis efficiency of
the synthesized prodrugs is significantly sensitive to the pattern of substitution on C=C bond
and distance between the nucleophile and the electrophile. The hydrolysis of the prodrugs
was largely affected by the pH of the medium. The experimental t1/2for the hydrolysis of
guaifenesin dimaleate ester prodrugs in 1N HCl was the least and for guaifenesin dimethyl
succinate was the highest.
Functional heterologous expression of TAS2R14, a broadly tuned bitter taste receptor
responding to guaifenesin, and experiments using these prodrugs revealed that, while some of
the prodrugs still activated the receptor similarly or even stronger than the parent substance,
succinate derivatization resulted in the complete loss of receptor responses. The predicted
binding modes of guaifenesin and its prodrugs to the TAS2R14 homology model suggest that
the decreased activity of the succinate derivatives may be caused by a clash with Phe247.
1. Introduction:
A significant number of prescribed and OTC (over-the-counter) drugs are bitter. The
bitter and unpleasant taste of drugs is a major obstacle in formulating pediatrics and geriatrics
drugs and is considered as a great challenge to the health community [1,2].The most
commonly used bitter tasting drugs include paracetamol, ampicillin, azithromycin,
diphenhydramine, erythromycin, ibuprofen, penicillin, pseudoephedrine and guaifenesin [3-
5].Nowadays, the medicinal industry has recognized the importance of taste masking,
however, the development of an appropriate formulation is relatively expensive and time
consuming [6, 7].
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A vast number of techniques have been invoked for concealing the unpleasant taste of
drugs [8-10], including polymer coating, complex formation with β-cyclodextrin, ion
exchange resins and solubility reducing methods. On the other hand, traditional taste masking
techniques such as the use of sweeteners and flavoring agents, such as anise oil, cardamom,
lemon and orange, or artificial flavors, alone resulted in inadequate masking of the taste of
extremely bitter drugs. It is worth noting that excipients such as sweetening flavors, menthol
and chloroform to anaesthetize taste receptors, have failed to mask 70% of the tested drugs.
Micro-encapsulation technique is important in masking bitter tasting medicines through
coating of the drug particles by a suitable polymer; the drug has no contact with the taste
buds in the mouth when dosage forms are given orally to patients [11-17].For example, the
unpleasant taste of diclofenac sodium was masked using a micro-encapsulation method
without affecting the active ingredient’s release rate. In addition, the results demonstrated that
the extent of taste masking was influenced by the presence of additives such as cellulose and
lactose within the core, as well as the plasticizer and the core size was found to have an effect
on the parent drug’s release rate [15]. However, effective methods of avoiding unpleasant
perception for adults are problematic for pediatric formulations.
Guaifenesin, the glyceryl ether of guaiacol, is one of the most commonly used OTC drugs. It
is a component of numerous cough and cold preparations available world-wide, termed as an
expectorant. This medication is used to loosen mucus and phlegm and eventually clear the
symptoms of congestion resulting from a cold or allergy [18-23]. Guaifenesin (Figure 1) was
discovered in the 1500’s, but its strong bitter taste made it unacceptable for pediatric and
geriatric patients [20].
Since most of the above mentioned physical approaches were found to be limited, different
strategies should be developed in order to overcome the unpleasant taste of guaifenesin.
Bitterness detection in the oral cavity of humans is facilitated by a family of ~25 bitter taste
receptors (TAS2Rs) belonging to the G protein-coupled receptor (GPCR) super-family [24-
2
6]. Despite low sequence similarity, TAS2Rs are classified as class A GPCRs [27]. The
human sense of bitter taste is commonly categorized as a warning sense against the ingestion
of toxic food components, although, in particular during later phases in life, moderate
bitterness is well tolerated and even appreciated in the context of some food items and
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beverages [28]. On the tongue, subsets of TAS2R genes are co-expressed in bitter taste
receptor cells that represent a subpopulation of type II cells within taste buds [29,30].The
functional screening of human TAS2Rs has so far resulted in the identification of bitter
agonists for 21 of the 25 receptors Moreover, it became evident that TAS2Rs recognize a
highly variable number of structurally diverse bitter compounds ranging from single or few
compounds to dozens [31-34] . These differences in tuning breadths suggest that receptors
may contribute disproportionally to human bitter sensing. Among the 3 most broadly tuned
receptors, TAS2R10 [35], TAS2R14 [36], and TAS2R46 [37], the TAS2R14 exhibits to date
the largest panel of identified bitter agonists, which frequently represent drugs with profound
pharmacological activities [38].
The recent novel prodrug chemical approach invoked by Karaman’s group involves
the design of prodrugs for masking the bitter taste of pharmaceuticals based on
intramolecular processes. In this approach no enzyme is needed to catalyze the reconversion
of a prodrug to its corresponding parent drug. The rate of the drug release is controlled by the
nature of the promoiety bound to the drug. The role of the latter is to block those functional
groups located on the parent drug and responsible for the interactions between the drug and
its bitter taste receptor/s [8, 9].This approach succeeded to design paracetamol prodrugs that
mask the bitterness of the parent drug [39].Similarly, guaifenesin prodrugs may reduce or
eliminate the parent drug’s bitterness by altering its ability to interact with the cognate bitter
taste receptor/s [39-42].
In this study, guaifenesin prodrugs consisting of mono- and di-esters were designed and
synthesized aiming to provide non-bitter entities which upon exposure to the stomach
medium undergo cleavage to provide the parent drug, guaifenesin and a non-toxic linker.
2. Material and methods:
2.1 Computational analyses
QM studies - The Becke three-parameter, hybrid functional combined with the Lee,
Yang, and Parr correlation functional, denoted B3LYP, were employed in the calculations
using density functional theory (DFT). All calculations were carried out based on the
restricted Hartree-Fock method using the quantum chemical package Gaussian-2009 [43].
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The starting geometries of all calculated molecules were obtained using the Argus Lab
program [44] and were initially optimized at AM1 and HF/6-31G (d,p) level of theory,
followed by optimization by DFT at the B3LYP/6-31G(d,p) level. The search for the global
minimum structure in the guaifenesin derivatives studied was accomplished by 36 rotations
around the ester O-C(O) bond in increments of 10°, and calculation of the energies of the
resulting conformers. An energy minimum (a stable compound or a reactive intermediate) has
no negative vibrational force constant. Transition states were located first by the normal
reaction coordinate method [45] where the enthalpy changes were 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 [46]. The “reaction coordinate method” was used to
calculate the activation energy in the designed mono- and di-ester prodrugs. In this method,
one bond length is constrained for the appropriate degree of freedom while all other variables
are freely optimized [47]. The activation energy values for the prodrug interconversion to
guaifenesin were calculated from the difference in energies of the global minimum structures
(
GM) and the derived transition states. Verification of the desired reactants and products was
accomplished using the “intrinsic coordinate method” [48]. The transition state structures
were verified by their only one negative frequency. Full optimization of the transition states
was accomplished after removing any constrains imposed while executing the energy profile.
The activation energies obtained from DFT at B3LYP/6-31G (d,p) level of theory for
GDEProD1-3and GMEProD1-3( Figure 1) were calculated with and without the inclusion
of solvent (water and ether). The calculations with the incorporation of a solvent were
performed using the integral equation formalism model of the Polarizable Continuum Model
(PCM) [49, 50].
2.2 Chemistry
The chemicals used in the present study were procured from Sigma Aldrich (USA).
-1
IR spectra were obtained from a KBr matrix (4000–400 cm ) using a Thermo Scientific
Nicolet iS10 FT-IR Spectrometer. The LC-MS system used was LC system coupled to a
hybrid quadrupole ion trap (LTQ) - Fourier transforms ion cyclotron resonance (FT-ICR)
mass spectrometer (Thermo fisher scientific Bremen, Germany). 1H nuclear magnetic
resonance (NMR) experiments were performed with I 500 MHz NMR Varian Unity INOVA
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spectrometer (University of Basilicata). Samples were injected into Agilent technologies
200 series (Avondale, PA, USA) HPLC-PDA. The optimal HPLC British pharmacopoeia
1
validated method (with some modification in the percentage of the mobile phase component)
used for the analysis of guaifenesin [51]; Eclipse XDB-C18 (3 μm particle size, 4.6 mm ×
1
50 mm) column (Phenomenex-USA), a mixture of water: acetonitrile (water pH adjusted to
.5 using diluted phosphoric acid) (55:45 v/v) as a mobile phase, a flow rate of 0.5
5
mL/minute and a diode array 1200 HPLC system consisting of an HP-G1311A Quat pump,
HP G1311A UV detection at a wavelength of 276 nm in University of Basilicata.
2.2.1Synthesis of the Prodrugs
2
.2.1.1 Guaifenesin mono-ester prodrugs,GMEProD1-3(Scheme S1):
In a 250 mL round-bottom flask guaifenesin (5 mmol) was dissolved in
tetrahydrofuran (THF) (50 mL), 0.125 g (5 mmol) of sodium hydride (60% suspended
powder) was added, the resulting solution was stirred for 30 minutes then a THF solution of 5
mmol maleic anhydride, succinic anhydride or glutaric anhydride, was slowly added to the
reaction mixture which then was stirred at room temperature for overnight. The reaction was
monitored by Thin Layer Chromatography (TLC) which was performed on regular basis to
check the reactions completion. 1N HCl (50 mL) was added while the round-bottom flask
was placed in an ice bath. The aqueous layer was extracted with ether (90 mL) three times
and the combined ether layer was dried over MgSO anhydrous, filtered and evaporated to
4
dryness, then the product was washed with hexane three times and dried.
2
.2.1.2 Guaifenesin di-ester prodrugs, GDEProD1-3 (Scheme S2):
In a 250 mL RBF guaifenesin (5 mmol) was dissolved in tetrahydrofuran (THF) (50
mL), 0.250 g (10 mmol) of sodium hydride (60% suspended powder) was added, the
resulting solution was stirred for 30 minutes then a THF solution of 10 mmol 2,3-dimethyl
maleic anhydride, maleic anhydride or 2,2-dimethyl succinic anhydride, was slowly added to
the reaction mixture which then was stirred at room temperature for overnight. The reaction
was monitored by Thin Layer Chromatography (TLC) which was performed on regular basis
to check the reactions completion. 1N HCl (50 mL) was added while the round-bottom flask
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was placed in an ice bath. The aqueous layer was extracted with ether (90 mL) three times
and the combined ether layer was dried over MgSO anhydrous, filtered and evaporated to
dryness, then the product was washed with hexane three times and dried (Figure 1).
4
2.2.2Characterization by H-NMR, FTIR and LC-MS:
1
Guaifenesin Drug: H NMR (CDCl , δ ppm) δ 7.01 – 6.69 (m, 4H), 4.19 – 3.85 (m, 5H), 3.84
3
–1
-1
(
6 5
s, 3H), 3.81 (s, 1H), 3.27 (s, 1H). FT-IR (KBr/νmax cm ): (O-H) 3251cm , (C=C of C H )
-1
13
1
596 cm . C NMR (CDCl
3
, δ ppm) δ 149.50, 148.10, 121.59, 121.20, 114.30, 112.00,
71.30, 70.30, 63.80, 56.10 (Figure S1).
1
GMEProD1: Yield: 61.5%, Solid (decomposes before melting), H NMR (CDCl
3
, δ ppm): δ
6.88-7.15 (m, 4H), 6.31-6.14 (m, 2H), 4.27 (m, 1H), 4.03-4.23 (m, 2H), 3.98-4.10 (m, 2H),
–1
-1
-1
-
3
.86 (s, 3H). FT-IR (KBr/νmax cm ): (O-H) 3063 cm , (C=O) 1738 cm , (CH=CH) 1634 cm
1
.
m/z: 295.08(100%), 295.11(25%), 295.14(12%) (M-1) (Figure S1).
1
GMEProD2: Yield: 58%, Oily, H-NMR (CDCl
3
, δ ppm) δ 6.88-7.12 (m, 4H), 4.48 (m, 1H),
–1
4
.09-4.31 (m, 2H), 4.03-4.24 (m, 2H), 3.84 (s, 3H), 2.97 (m, 4H). FT-IR: (KBr/νmax cm ):
-1
-1
(
O-H) 3017 cm , (C=O) 1738cm , and m/z: 297.09 (100%), 298.10(13.9%) (M-1) (Figure
S1).
1
GMEProD3: Yield: 58%, Oily, H NMR (CDCl
3
, δ ppm) δ 6.87-7.09 (m, 4H), 4.50 (m,1H),
4.16-4.33 (m, 2H), 4.05-4.24 (m, 2H), 3.70 (s, 3H), 2.39-2.45 (m, 4H), 1.91-1.97 (m, 2H).
–1
-1
-1
FT-IR: (KBr/νmax cm ): (O-H) 3066 cm , (C=O) 1736 cm , and m/z 311.113 (100%),
312.116(13.0%) (M-1) (Figure S1).
1
GDEProD1: Yield: 73%, Oily, H NMR (CDCl
3
, δ ppm) δ 11.10 (s, 2H), 7.07 – 6.60 (m, 4H),
5.28 (p, J = 5.0 Hz, 1H), 4.48 (d, J = 5.0, 1.4 Hz, 2H), 4.21 (d, J = 5.0, 1.4 Hz, 2H), 3.84 (s,
–1
-1
-1
3
H), 2.02 – 1.69 (m, 12H). FT-IR: (KBr/νmax cm ): (O-H) 3220 cm , (C=O) 1720 cm ,
1
3
(
(
(
3
C=C) 1637. C NMR (CDCl , δ ppm) δ 170.77 – 170.59 (m, 2C), 169.28 (2C), 149.50
1C), 148.10 (1C), 138.82 (1C), 138.63 (1C), 138.34 (d, J = 3.0 Hz, 2C), 121.59 (1C), 121.20
1C), 114.30 (1C), 112.00 (1C), 74.61 (1C), 69.16 (1C), 62.39 (1C), 56.10 (1C), 15.36 –
1
4.04 (m, 4C), and m/z 449.15 (100%), 450.16 (25.3%), 451.12 (5.1%) (M-1) (Figure S1).
1
GDEProD2: Yield: 80%, Oily, H NMR (CDCl , δ ppm) δ 13.06 (s, 2H), 7.10 – 6.56 (m, 4H),
3
6.33 – 5.38 (m, 4H), 5.38 – 5.06 (m, 1H), 4.34 (d, J = 5.0, 1.4 Hz, 4H), 3.84 (s, 3H). FT-IR:
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–
1
-1
-1
-1 13
(
KBr/νmax cm ): (O-H) 3224 cm , (C=O) 1734 cm , (C=C) 1644 cm . C NMR (CDCl
ppm) δ 167.03 (1C), 166.55 – 166.40 (m, 3C), 149.50 (1C), 148.10 (1C), 136.01 – 135.41 (m,
C), 132.46 (1C), 131.01 (1C), 121.59 (1C), 121.20 (1C), 114.30 (1C), 112.00 (1C), 73.06
3
, δ
2
(1C), 69.16 (1C), 62.39 (1C), 56.10 (1C)and m/z 393.09 (100%), 394.07 (20.4%), 395.10
(2.2%) (M-1) (Figure S1).
1
GDEProD3: Yield: 90%, Oily, H NMR (CDCl , δ ppm) δ 12.00 (s, 2H), 7.00 – 6.70 (m, 4H),
3
5.28 (t, J = 5.0 Hz, 1H), 4.26 (d, J = 15.0, 5.0 Hz, 4H), 3.84 (s, 3H), 2.72 (d, J = 3.7, 1.9 Hz,
–1
-1
-1 13
4
H), 1.16 (s, 12H). FT-IR: (KBr/νmax cm ): (O-H) 3212 cm , (C=O) 1732 cm . C NMR
(
(
(
(
CDCl , δ ppm) δ 178.00 – 177.72 (m, 2C), 170.93 (1C), 170.42 (1C), 149.50 (1C), 148.10
3
1C), 121.59 (1C), 121.20 (1C), 114.30 (1C), 112.00 (1C), 71.65 (1C), 69.16 (1C), 62.14
1C), 56.10 (1C), 46.23 (1C), 45.61 (1C), 43.58 (2C), 25.14– 24.92 (m, 4C),and m/z 453.18
100%), 454.17 (25.3%), 455.19 (5.1%) (M-1) (Figure S1).
2
.2.3Kinetic Methods
After the preparation of the calibration curves for guaifenesin and all the prodrugs, a sample
from each prodrug was prepared in 1N HCl, buffer pH 3.3, buffer pH 5.5 or buffer pH 7.4,
and all samples were injected into HPLC-PDA using the method mentioned above.
2
.2.3.1Hydrolysis of guaifenesin prodrugs:
0
Guaifenesin prodrugs hydrolysis rates were studied at 37 C in buffer solutions at different
pHs (1N HCl (pH 0.2), 0.1N HCl (pH 1.2), pH 3.3, pH 5.5 and pH 7.4). Samples from the
reaction mixtures were analyzed directly by HPLC; the decreased area percentage of the
prodrug peaks and the increased area of guaifenesin peak were monitored during the reaction
progress, then area under the peaks of guaifenesin and its prodrugs vs. time were plotted.
2.3 Functional calcium imaging experiments
Functional heterologous expression of TAS2R14 was done as previously reported [52].
Briefly, TAS2R14 cDNA flanked by a 5’ sst3-tag and a 3’ hsv-tag in the vector pcDNA5FRT
36] was transiently transfected into HEK 293T-Gα16gust44 cells. After ~24 h cells were
[
loaded with Fluo-4 AM in the presence of 2.5 mM probenecid, then washed several times
tetra
with C1-buffer and placed in a fluorometric imaging plate reader (FLIPR , Molecular
Devices). Test stimuli were automatically applied and changes in fluorescence were
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monitored. Cells transfected with an empty expression vector were used as a negative control
and directly compared to receptor transfected cells. Two independent experiments performed
in triplicates were used for the calculations.
2.4 Molecular modeling
TAS2R14 receptor model generated in Karaman et al. and refined in Di Pizio et al [52.]
was used to investigate the binding modes of guaifenesin, by using the Induced-Fit docking
protocol (Glide version 7.5; Prime version 4.8, Schrödinger, LLC, New York, NY, 2017)
[
53,54]. Side chains of residues within 5.0 Å of the ligand were refined. The docking was
performed with the Standard Precision (SP) mode of Glide. The binding poses of the
prodrugs were obtained aligning the ligand structures to the guaifenesin in its docking pose
with Phase Shape Screening (Phase, version 5.1, Schrödinger, LLC, New York, NY, 2017).
Therefore binding energies of the ligands with receptor were estimated by employing the
Multi Ligand Bimolecular Association with Energetics (MBAE) [55], using EMBRACE
minimization of Macro Model (Schrödinger, LLC, New York, NY, 2017). All complexes
have been minimized to a derivative convergence of 0.05 kJ/mol Å using the Polak-Ribiere
Conjugate Gradient (PRCG) minimization algorithm, the OPLS3 force field. A shell of 5 Å
around the ligand was set to be free to move, another shell of 3 Å minimized applying a force
constant of 200 kJ/mol Å2.
Embrace minimization was performed by opting energy difference mode. The calculation
was performed first on the receptor, then on the ligand, and finally on the complex. The
energy difference is then calculated using the equation:
∆
E (ligand binding energy) =Ecomplex – Eligand – Eprotein
Since all compounds were synthesized and tested in racemic mixture, we have submitted all
stereoisomers to the simulations and used the stereoisomers the showed better performance
for the analysis.
Transmembrane (TM) residues are identified by a superscript number system according to the
Ballesteros-Weinstein numbering method [56].
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3. Result and discussion:
We have successfully obtained six guaifenesin prodrugs with five different linkers,
GDEProD1, GDEProD2, GDEProD3,GMEProD1, GMEProD2, GMEProD3 (Schemes
S1and S2). These prodrugs were characterized by FT-IR, 1H-NMR and LC-MS techniques as
shown in Figures S1 (A,B and C).
The kinetics of the acid-catalyzed hydrolysis for guaifenesin prodrugs were carried out in
aqueous buffers in a manner similar to that done by Bruice’s group on methyl phenyl acetals
of formaldehyde [57]. The kinetic study aimed to investigate whether guaifenesin prodrugs
are hydrolyzed to release the parent drug, guaifenesin in aqueous medium and to what extent.
Acid-catalyzed hydrolysis of the synthesized guaifenesin prodrugs was studied in four
different aqueous media: HCl and buffers pH 3, pH 5 and pH 7.4 and the hydrolysis reaction
was monitored by HPLC (Scheme S3). The appearance of the parent drug’s peak and
disappearance of the prodrug’s peak were monitored to determine the rates of the
interconversion of each of the six prodrugs and the results are summarized in Figures S2
(A,B and C).
For guaifenesin prodrugs, at constant temperature (37°C) and 1N HCl (pH 0.2), the reaction
displayed strict first-order kinetics for GDEProD1, GDEProD3 and GMEProD1 as the kobs
was fairly constant and a straight line was obtained by plotting ln concentration of the
guaifenesin prodrugs versus time in hours, and zero order for the other prodrugs according to
the best linearity of concentration versus time plotting as shown in Tables S1 andS2. The rate
constant (kobs) for guaifenesin prodrugs in 1N HCl was calculated from the linear regression
equation correlating the concentration of the prodrug versus time according to the integral
rate law equation of the zero order law (eq. 1), and the linear regression equation correlating
the concentration of the prodrug versus time according to the integral rate law equation of the
first order (eq. 2) (Tables S1 andS2). The rate constant (k ) obtained from the kinetics for
obs
-3
-3
-
GDEProD1, GDEProD3 and GMEProD1 was found to be 5.2 x 10 , 0.02 x 10 , 1.6 x 10
3
-1
-1
-3
-3
-3
-1
-1
mol x L x h , and 12.2 x 10 , 11.75 x 10 , 11.14 x 10 mol x L x h for GDEProD2,
GMEProD2, and GMEProD3, respectively. The complete hydrolysis of the guaifenesin
prodrugs to their parent drug, guaifenesin in 1N HCl required 1.67, 5.33, 11.00, 12.00, 13.5,
and 96 hours, respectively. Whereas, in 0.1 N HCl the hydrolysis rate constant of
-3
-1
-1
GEDProD1 was 14.01 x 10 mol x L x h as zero order reaction, and required 6 hours for
complete hydrolysis to guaifenesin, as shown in Figure S2 (D).
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Using the equations derived from the integral zero and first law (eqs. 3 and 4) and the
experimental rate conversion value we have calculated the t1/2 values (the time needed for the
conversion of 50% of the reactants to products) for the conversion of GDEProD1-3 and
GMEProD1-3 to their parent drug. The t½ values for GDEProD1-3 and GMEProD1-3
were 0.3, 4.81, 73.13, 1.45, 7.06 and 7.16 h in 1N HCl, and 3.81 h for GDEProD1 in 0.1N
HCl, respectively.
ꢀ
ꢁ] = ꢀꢁ°] − ꢂ ∗ ꢃ … … ꢄꢄꢄꢄꢅꢆ. ꢇ
ꢈꢉꢀꢁ] = ꢈꢉꢀꢁ°] − ꢂꢃ … … . ꢅꢆ. ꢊ
ꢈꢉꢊ
ꢃꢄꢋꢌꢍꢈꢎꢏ = ꢀꢁ°]/ꢊꢂ] … … ꢅꢆ. ꢐ
ꢃꢋꢌꢍꢈꢎꢏ =
… … ꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢄꢅꢆ. ꢑ
ꢂ
0
Where [A]=[A ] / 2, [A
concentration.
t
] is the concentration during time and [A] is the initial
3.1 Computational analysis
Continuing the strategy for exploring enzyme models in the design of novel prodrugs,
Bruice s enzyme model (hydrolysis of di-carboxylic semi-esters) was employed in the design
̓
of guaifenesin prodrugs (Schemes S1and S2) which have the potential to be more
bioavailable and to elicit less bitter sensation. Furthermore, it is planned that their intra-
conversion rate to guaifenesin will be programmed according to the nature of the prodrug
linker [57,58].
The calculations at B3LYP/6-31G (d,p) level for the ring-closing reactions of di-
carboxylic semi-esters, GMEProD1-3 and GDEProD1-3 (Schemes S1 andS2), were
directed toward elucidation of the transition and ground state structures (reactants,
intermediates and products). Calculations for all ground states, intermediates, transition states
and products were run in water (dielectric constant of 78.39) and in 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.
The orientation of the carboxylate anion to the ester carboxyl moiety is very important and
affects the mode and rate of the ring-closing reaction. The distance (dGM) between the
nucleophile (O1) and the electrophile (C6) is shorter in the di-carboxylic semi-ester exists in
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the syn (condensed) conformation than the anti (extended) conformation (Scheme S3). This
is the reason that the cyclization in syn conformation is more efficient than in the anti
(extended) conformation.
In addition, the activation energy of the prodrug to drug intraconversion is largely affected by
the strain energy of the prodrug conformation. If the ground state is strained, the free energy
of the reactant will be higher than the less strained conformation, so it is predicted that the
activation energy of the strained reactant conformers will be less than that of the less-strained
conformers. We were concerned with the identification of the most stable conformation
(Global Minimum, GM) for each of dicaboxylic semi-esters GMEProD1-3 and GDEProD1-
3.
The DFT calculations of the starting geometries in GMEProD1-3 and GDEProD1-3
demonstrated that the global minimum structures (GM) for all prodrugs exist in the syn
(condensed) conformation, as shown in Figure S3 (A).
3
.1.1Optimized Geometries for the Entities Involved in the Ring-Closing Reactions
of GMEProD1-3 and GDEProD1-3
.1.1.1Reactants (GM)
3
The global minimum structures for GMEProD1-3 and GDEProD1-3 are illustrated
in Figure S3 (A). The dGM values for GDEProD1-3 were 3.147Å, 3.254 Å and 4.115 Å,
respectively, and for GMEProD2-3 were 3.468 Å 3.497 Å, respectively, where the GM for
GDEProD1 was with the shortest distance and for the GM of GDEProD3 with the longest
distances.
3
.1.1.2Transition State Geometries (TS)
The calculated DFT transition state structures for the cyclization reactions of
GMEProD1-3 and GDEProD1-3 are shown in Figure S3 (B). Examination of the optimized
TS structures indicates that all of them resemble that of the corresponding tetrahedral
intermediates. Furthermore, the calculated O-C distances, O1-C6, O8-C6 and O1-C2
(
SchemeS4) are significantly different. The distance range for O1-C6 in the TS of
GMEProD1-3 and TS of GDEProD1-3 was 1.378 Å – 1.449 Å, for O8-C6 was 1.792 Å –
.803 Å and for O1-C2 was 1.371 Å - 1.413 Å.
1
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3.1.2Reaction Mechanism Investigation
The DFT calculations at B3LYP 6-31G (d,p) level were performed for calculating the
kinetic parameters for all entities involved in the reaction shown in Scheme S5. The
mechanism involves two steps; the first is the approach of the anionic carboxylate oxygen
(O1) toward the carboxylic carbon (C6) to give a tetrahedral intermediate and the second is
the dissociation of the tetrahedral intermediate to yield a cyclic anhydride and guaifenesin-
enolate anion. The ‘reaction coordinate’ calculations for both steps revealed the following:
(a) no transition state structures were found for the approach processes in GMEProD1-3 and
GDEProD1-3. (b) The ‘reaction coordinates’ and frequency calculations for the intermediate
dissociation route in GMEProD1-3 and GDEProD1-3 demonstrated the presence of a
transition state. Further, monitoring the dissociation processes revealed that upon increasing
the distance between O8 and C6, opening of the cyclic ring was observed in GMEProD1-3
and GDEProD1-3. However, the cycle opening magnitude (the distances O1-C6, O8-C6 and
O1-C2) was found to be dependent on the transition state nature.
Using the calculated B3LYP 6-31G (d,p) values for the enthalpy (H) and entropy (S)
of the global minimum (GM) and the transition state (TS) structures for the ring-closing
reactions of di-carboxylic esters GMEProD1-3 and GDEProD1-3, (Table S3), the activation
energy values for the rate-limiting step (∆G) were calculated in the gas phase as well as in a
dielectric constant of 78.39 (water). The calculated activation energy values in both media,
∆
GGP and ∆GH2O, respectively, are summarized in Table S4. Inspection of the ∆G values
shown in Table S4 demonstrated that the cyclization rate is largely affected by the nature
(structural features) of the reactant.
To examine whether the discrepancy in the rates for the reactions of GMEProD2-3
and GDEProD1-3 stems from proximity orientation or is due to steric effects (strain energy)
we calculated using molecular modeling (MM2) method the strain energy values (E ) for the
s
reactants and intermediates in systems GMEProD2-3 and GDEProD1-3. The calculated Es
values for the reactions of GMEProD2-3 and GDEProD1-3 were examined for the
correlation with the experimental t1/2 values and the correlation results are depicted in
equations 5 and 6 and illustrated graphically in Figure S3(C, I). Eq. 5 and Figure S3 (C, I)
demonstrate a good correlation between the experimental t1/2 and the MM2-calculated strain
s
energy values (E ) with a correlation coefficient (R) of 0.94.
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It should be noted, that attempts to correlate the distance O1-C6 (dGM) with t1/2 gave random
correlation. For example, the calculated O1-C6 distances for GDEProD1 and
GMEProD2were similar (3.147 Å- 3.254 Å), whereas the calculated Es and t1/2 experimental
values differ significantly (t1/2; 0.3 h and 1.45h, Es: 22.2 and 10.61, respectively). These
results suggest that the driving force for the enhancement in the ring-closing reaction is
driven by strain effects in contrast to that suggested by Bruice et al. [57-58]. In order to
further support this conclusion, the B3LYP 6-31G (d,p) for GMEProD2-3 and GDEProD1-
3
activation energy values in water (∆G H2O) were examined for correlations with both t1/2
and Es and the results are shown in equations 7-8 and represented graphically in Figure.
S3(C, II,III).
Again, the correlation results of the calculated ∆GH2O with the experimental half-lives (t1/2
)
revealed to the same conclusions; the driving force for acceleration is due to strain effects and
not to proximity orientation.
Es = 0.6797 t1/2 + 16.271 (R=0.9)…………..(eq. 5)
∆
∆
GH2O (B3LYP/6-31G) = 1.4042 t1/2 + 3.2675 (R=0.9)…………….(eq. 6)
s
G (B3LYP/6-31G) = 1.3469 E + 19.351 (R=0.9)……………(eq. 7)
t 0 t 0
ln [A ] = ln [A ]-K.t, ([A ] = [A ]/2 (for t1/2 calculation)………………(eq. 8)
The half-lives, t for Processes GDEProD1-3 and GMEProD1-3
1
/2
The experimental half-lives for the intramolecular ring-closing reactions of GDEProD1-3
and GMEProD1-3 were obtained from the integral first order law (eq. 8).
Since an excellent correlation was obtained between the activation free energy values
(
∆GH2O) for GDEProD1-3 and GMEProD1-3 and the strain energy values (eq. 7), the
calculated values of E for GDEProD1-3 and GMEProD1-3 were used in eq. 5 to calculate
the t1/2 in equation 7 as shown in eq 7, half-life= [(∆G H2O (B3LYP/6-31G))/1.3469 –
6.271]/0.6797. The calculated half-lives for GDEProD1-3 and GMEProD1-3 obtained
s
1
were: 5.69, 19.10, 3.78, 10.826, 26.513 and 29.610 h, respectively. Examination of the
calculated half-lives demonstrates that GDEProD1 and GMEProD1 are the most efficient
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processes among all the systems investigated, whereas the least efficient are GMEProD2 and
GMEProD3.
Using the integral rate law first order equation (eq. 8) the calculated rate conversion for
GDEProD1 andGDEProD3 and GMEProD1-3 obtained were: 0.1217, 0.0640, 0.0362,
-1
0
.0261, and 0.0234 h , respectively.
For obtaining credibility to our calculation results, we introduce our computation rational for
calculating the (∆G) values for GDEProD1-3 and GMEProD1-3 based on the DFT
calculated enthalpy of activation (∆H) and entropy of activation for GDEProD1-3 and
ꢒ
GMEProD1-3, experimental free activation energies (ExpΔ ) were calculated using equation
9, which describes the ∆G parameter as a function of the rate conversion values.
∆
G = -RT lnK ……..(Eq. 9)
-3
Where R is the Ideal Gas constant (1.9872036x10 kcal/mol K) and T is the temperature in
Kelvin.
The ExpΔ
ꢒ
for GDEProD1-3 were 3.114, 4.345, 6.406 kcal/mol x Kelvin, and for
GMEProD1-3 were, 3.811, 4.500 and 4.524 kcal/mol x Kelvin, respectively.
In addition, for further support to the credibility of our DFT calculations, the calculated free
activation energies in water (∆G) were correlated with the experimental free activation
energies (Exp∆G). Strong correlation was obtained with R value of 0.92 (Figure S3 (D)).
Figure S3(D)indicates that although the calculated and experimental ∆G values are
comparable, their absolute values slightly differ. This might be due to the fact that the
experimental ∆G values for GDEProD1-3 and GMEProD1-3 were measured in the presence
of aqueous acid, whereas the DFT calculations were run in plain water. The dielectric
constant value for a mixture of acid/water is expected to be different from pure water (78.39)
and hence the discrepancy in the calculated and experimental ∆G values.
3.2 TAS2R14 activation
To investigate if the derivatization of guaifenesin may enable the creation of
TAS2R14 non-agonists which can be bioactivated, we challenged HEK 293T-Gα16gust44
cells transiently transfected with TAS2R14 cDNA with different concentrations of
derivatives and the parental substance guaifenesin and monitored calcium responses
(FigureS4)Whereas guaifenesin, guaifenesin maleate, guaifenesin glutarate, and guaifenesin
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dimaleate elicited cellular responses, the two succinate derivatives guaifenesin succinate and
guaifenesin disuccinate failed to activate TAS2R14 expressing cells at all concentrations.
Hence, derivatization of guaifenesin with succinate would prevent bitterness mediated by
TAS2R14, whereas other modifications would not eradicate TAS2R14 activation or even, in
case of guaifenesin glutarate, result in increased responses. As the masking of the guaifenesin
moiety should not be too different between e.g. the agonist guaifenesin dimaleate and the
non-agonist guaifenesin disuccinate, the succinate side-chains may adopt a conformation that
prevents receptor activation.
3.3 Structural analysis
We have investigated the conformation assumed by guaifenesin in the receptor’s
binding pocket [27], by Induced Fit Docking simulations [53,54]. Guaifenesin establishes π-π
interactions with Phe2476.55, hydrophobic interactions with Phe1865.46, and H-bonds with
Asn933.36 and Ser2657.38 side chains of TAS2R14 (FigureS5). We have aligned prodrugs
to guaifenesin in the predicted binding conformation in order to maintain the binding pose
and the main interactions, i.e. π-π interactions with Phe2476.55 and H-bond with Asn933.36.
This allowed us to understand how the chemical modifications of synthesized prodrugs affect
TAS2R14 activity in comparison to the bitter guaifenesin. Guaifenesin glutarate
(GMEProD3) can establish π-π interactions with Phe1865.46 and points the glutarate ester
towards TMs 6 and 7, establishing H-bonds with Ser2657.38 and Ser2466.54. Moreover, the
ligand conformation is stabilized by an intramolecular H-bond between the carboxyl and the
hydroxyl groups (FigureS5). Guaifenesin maleate (GMEProD1) binds to TAS2R14 in a very
similar mode establishing the same interactions observed for the glutarate derivative
(FigureS5). The additional maleate moiety of GDEProD2 points to the extracellular region
and forms H-bonds with Asn157, Ser167 and Ser169 of the ECL2 (FigureS5). This breaks
the intramolecular H-bond and causes a slight modification of the conformation of the
carboxyl group that interacts with Ser2657.38 and Ser2466.54, and a different orientation of
the aromatic ring that cannot establish the π-π interactions with Phe1865.46, as observed for
GMEProD3 and GMEProD1. Guaifenesin succinate and disuccinate show binding modes
similar to guifenesin maleate and dimaleate but the saturated esters increase the volume of the
molecules that clash with Phe2476.55, whose position is restrained because of the π-π
interactions with the aromatic ring (FigureS5).
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4. Summary and Conclusion
Taken together we have shown that prodrugs of guaifenesin can be rationally designed. Using
multiple derivatization strategies, a set of prodrugs has been established showing sufficient
stability at neutral pH-values and rapid release under low pH-conditions as observed during
stomach passage. Using bitter taste receptor assays, we confirmed that the most promiscuous
human bitter taste receptor responsible for the bitter perception of many drugs, the TAS2R14,
is not activated by some of the prodrugs. We conclude that our approach could be used to
design better drugs showing reduced bitterness in particular for pediatric formulations.
5. Acknowledgements
Authors are thankful to Università degli Studi Della Basilicata and Al-Quds University for
supporting the present study.
6. Conflict of Interest
The authors declare no conflicts of interest.
7. Figure 1 legend:
Figure 1:.Chemical structures of: 1. guaifenesin, 2. GMEProD1, 3. GMEProD2, 4.
GMEProD3, 5. GDEProD1, 6. GDEProD2, and 7. GDEProD3.
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