Synthesis and photophysical properties of deuteration of pirfenidone

Article abstract of DOI:10.1016/j.saa.2018.06.016

In order to improve the metabolism of pirfenidone (5-methyl-1-phenylpyridin-2-one, PFD), the methyl-deuterated version of pirfenidone via the substitution of hydrogen (H) at C-5 by its isotope deuterium (D, 5D-PFD) was synthesized and its photophysical properties were investigated. The negative solvatochrom was observed in absorption and fluorescence spectra with increasing solvent polarity, which implied that intermolecular charge transfer (ICT) involved n → π* transition for both of PFD and 5D-PFD. The ground state and excited state dipole moment was calculated as 5.30 D and 3.30 D for PFD, and 3.70 D and 2.18 D for 5D-PFD, respectively, which suggested the more polar nature of PFD in the ground state than that of excited state compared with 5D-PFD. Density functional theory (DFT) results demonstrated a significant propensity of ICT from the electron-donor, methyl and carbonyl group to the amine group as an electron donor. The binding of metal ions with PFD or 5D-PFD induced a red-shift of π → π* transition and blue-shift of n → π* transition, respectively, indicating that the pyridone ring showed more stability upon binding of unoccupied orbital of metal ions with lone-pair electron of oxygen atom and thus prompted the electronic distribution on phenyl unit. Upon addition of metal ions, the aromatic region presented the characteristic upfield shifts, and the resonance contributed by 3-H showed a significant downfield chemical shift/deshielding effect, indicating the deduced resonance of 3-H and the improved electron distribution of phenyl unit. The binding and docking of human serum albumin showed that the affinity of 5D-PFD with HSA was lower than that of PFD, and also 5D-PFD might prefer to present free forms in the blood with better efficacy comparing with PFD. The pharmacokinetic of half-time (T_1/2) for oral and i.v. administration of 5D-PFD was found around 19 and 30 min, higher than that of i.v. administration of PFD, 8.6 min, reported by Giri et al. The results of this work suggest that the deuteration enhances the metabolism of PFD significantly with little change of physical-chemical property.

Full text of DOI:10.1016/j.saa.2018.06.016

Accepted Manuscript
Synthesis and photophysical properties of deuteration of
pirfenidone
Qiuju Yin, Yujie Chen, Meng Zhou, Xiangsheng Jiang, Junjun
Wu, Yang Sun
PII:
DOI:
Reference:
S1386-1425(18)30550-X
doi:10.1016/j.saa.2018.06.016
SAA 16175
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 23 April 2018
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Accepted
date:
29 May 2018
4 June 2018
Please cite this article as: Qiuju Yin, Yujie Chen, Meng Zhou, Xiangsheng Jiang, Junjun
Wu, Yang Sun , Synthesis and photophysical properties of deuteration of pirfenidone. Saa
(2017), doi:10.1016/j.saa.2018.06.016
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ACCEPTED MANUSCRIPT
Synthesis and photophysical properties of deuteration of pirfenidone
Qiuju Yina1, Yujie Chena1, Meng Zhoua1, Xiangsheng Jianga, Junjun Wua, Yang
Suna,b*
a
School of Food Science and Technology·School of Chemical Engineering, Hubei
University of Arts and Science, No. 296 Longzhong Road, Xiangyang, Hubei 441053,
China
b School of Life Sciences, Tsinghua University, Beijing 100084, China
Corresponding author email: sunyang@if.usp.br (Y. Sun),
1 These authors are co-first author of this work
1

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Abstract
In order to improve the metabolism of pirfenidone (5-methyl-1-phenylpyridin-2-one,
PFD), the methyl-deuterated version of pirfenidone via the substitution of hydrogen
(H) at C-5 by its isotope deuterium (D, 5D-PFD) was synthesized and its
photophysical properties were investigated. The negative solvatochrom was observed
in absorption and fluorescence spectra with increasing solvent polarity, which implied
that intermolecular charge transfer (ICT) involved n→π* transition for both of PFD
and 5D-PFD. The ground state and excited state dipole moment was calculated as
5.30 D and 3.30 D for PFD, and 3.70 D and 2.18 D for 5D-PFD, respectively, which
suggested the more polar nature of PFD in the ground state than that of excited state
compared with 5D-PFD. Density functional theory (DFT) results demonstrated a
significant propensity of ICT from the electron-donor, methyl and carbonyl group to
the amine group as an electron donor. The binding of metal ions with PFD or 5D-PFD
induced a red-shift of π→π* transition and blue-shift of n→π* transition, respectively,
indicating that the pyridone ring showed more stability upon binding of unoccupied
orbital of metal ions with lone-pair electron of oxygen atom and thus prompted the
electronic distribution on phenyl unit. Upon addition of metal ions, the aromatic
region presented the characteristic upfield shifts, and the resonance contributed by
3-H showed a significant downfield chemical shift/deshielding effect, indicating the
deduced resonance of 3-H and the improved electron distribution of phenyl unit. The
binding and docking of human serum albumin showed that the affinity of 5D-PFD
with HSA was lower than that of PFD, and also 5D-PFD might prefer to present free
forms in the blood with better efficacy comparing with PFD. The pharmacokinetic of
half-time (T1/2) for oral and i.v. administration of 5D-PFD was found around 19 and
30 min, higher than that of i.v. administration of PFD, 8.6 min, reported by Giri et al.
[1]. The results of this work suggest that the deuteration enhances the metabolism of
PFD significantly with little change of physical-chemical property.
Keywords: Pirfenidone; isotope deuterium; negative solvatochrom; density functional
theory; metal ions; human serum albumin.
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1. Introduction
Pirfenidone (PFD) is a widespread, universal antifibrotic pyridine ketone compound,
chemical name is 5-methyl-1-phenyl-2-[1H]-pyridone, which possesses antifibrotic
effects by regulating the key fibrotic cytokines and growth factors, inhibiting several
inflammatory mediators, attenuating the fibroblast proliferation, differentiating related
collagen synthesis, and restoring the immune response balance [2]. These beneficial
effects of PFD inhibits progression of fibrosis in vivo in a variety of animal models of
lung [3], kidney [4], hepatic [5] and cardiac fibrosis [6]. And PFD is used clinically
for treating idiopathic pulmonary fibrosis [7]. However, PFD-induced phototoxicity
occurred after oral administration as an adverse event in previous clinical trials [7]
and animal model [8, 9]. According to Seto et al., a high dose of PFD (160 mg/kg)
might cause phototoxic responses through the generation of ROS in the skin, and PFD
is photoreactive, and the generation of ROS from UV-exposed PFD might lead to a
photo-irritation risk, but not to photo-genotoxic potential [10].
The alteration of the oxidative metabolism of drug candidates to improve their
pharmacokinetic or reduce the toxicological properties is important aspect of the drug
discovery process. Since Belleau et al. [11] among the first reported the
pharmacodynamic effect of deuteration with αα-dideuterated p-tyramine, there has
been a burgeoning interest, particularly from intellectual property aspects, for
enhancing the pharmacokinetic, pharmacodynamic, and reducing the toxicological
properties of drug [12-14]. Deuteration of drugs to enhance their pharmacokinetic,
pharmacodynamic, or toxicological properties has gained momentum as judged by a
search of the SciFinder database with the search term “deuterated drugs”[15].
Although the deuteration would be benefit for clinical use of medicine, the
photophysical and photochemical properties of deuterated compounds have not been
fully elucidated, and also the mechanisms of deuterated compounds reducing
phototoxicity are not clear, since the molecular structure mainly determines the
activity of the compound [16]. Apart from substitution changes, solvent effect plays
an important part in chemical and physical properties in solution [17]. The
solvent-induced changes in the electronic transition of solutes, namely
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solvatochromism, are helpful for understanding the solvent effects [18, 19].
So far, few studies have been reported the photophysical and photochemical
properties of PFD and its derivatives. Therefore, in this work, the 5D-PFD was
synthetized by replacing CH-5 as CD-5 in order to improve the metabolism of PFD,
and its structure was characterized. Falb et al. have reported a Suzuki–Miyaura
methylation of pyridines leading to the PFD, which focus on a green methylation route
to avoid the environmental and economic impact of employing alkyllithium at cryogenic
temperatures, simplifying the synthesis process and reducing the cost for synthesis
[16]. In this work, besides the deuterated methyl of pyridone, the fluorine is
introduced into phenyl ring in order to impede the oxidation rate of phenyl group and
further improve the metabolism of PFD. Moreover, we propose a green reaction
avoiding the use of the benzene solvent, prompting the environmental protection. The
solvent effect of PFD and 5D-PFD was studied by absorption and fluorescence
spectra, and the solvatochromic parameters were calculated according to the spectral
data. Moreover, the density functional theory (DFT) provided the ground state
properties of PFD and 5D-PFD, such as geometrical parameters, dipole moment and
charge transfer. Beside these, the attempts were made to assess if there was any
deuteration-induced difference in human serum albumin (HSA) binding with PFD and
5D-PFD, including binding site, binding energy and type of interaction force. These
results would have a great significance in pharmacology and clinical medicine as well
as methodology.
2. Experimental
2.1. Materials
5-Bromo-2-methylpyridine, n-Buli, MTBE (methyl tert-butyl ether), iodomethane-D3,
were purchased from Aldrich. Stock solution of PFD and 5D-PFD (0.1 mM) was
prepared by ethanol and kept in darkness. All the solvents were of analytic grade.
2.2. Methods
2.3.1. Synthesis of 5D-PFD
5-Bromo-2-methylpyridine (3.67 g) was dissolved in 10 m MTBE under a N2
atmosphere with a temperature of -78°C. Then, 8.8 mL (2.5 M) n-Buli was added
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dropwise over a period of 1.5 h. Thereafter, the resulting mixture was added by CD3I
dropwise between -60°C to -80 °C over 1.5 hours and then the mixture was warmed
up to room temperature. After the completion of the reaction (monitored by TLC), the
mixture was extracted by 6N HCl (30mL × 2) and the water phase was collected and
then refluxed to react over 48 h (monitored by LC-MS). The reaction mixture was
cooled to room temperature and gave a pH around 12, after that, the solution of above
mixture was stirred at reflux for 24 h. LC-MS detected 65% reacted, another 24 h,
LC-MS detected over 95% 5-bromo-2-methylpyridine consumed. After cooling to
room temperature, the reaction mixture was filtered to isolate the resulting precipitate,
which was then washed sequentially with toluene (40 mL × 2) and 40 mL water.
Drying the organic phase and the solvent was evaporated under reduced pressure to
give the residue, which was then purified through flash chromatography (DCM:
MeOH=20: 1) to give a white solid, yield 71.3%, purity ≥ 98%. The synthetic route
o
was listed in Fig. 1. m.p. 292–295 C.; IR (cm-1): Need to add IR data and nuclear
magnetic data3189 (νNH2); 2831 (νCH); 1734 (νC=O);; 1245 (νC–O); 1131 (νC–O–
C). 1H NMR (500 MHz, DMSO-d6) δ: 8.43 (d, J = 6.4 Hz, 1H), 8.10-7.93 (d, J = 5.1
Hz, 2H), 7.57 (t, J =7.3 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 4.11 (s, 2H), 3.75-3.86 (t, J
= 9.2 Hz, 4H), 3.17 (s, 4H), 2.65-2.74 (t, J = 4H), 2.35 (s, 6H).
2.3.2. Molecular modeling and theoretical calculation
The crystal structure of HSA with resolution of 2.25 Å was obtained from the Protein
Data Bank (PDB code: 2BXH). The docking scheme was summarized as follows.
Firstly, the preprocessing of HSA was carried out by using the programs of Discovery
Studio 2.5 (Accelrys, Inc., USA), which included removing water, adding hydrogen
atoms and assigning CHARMM like force field. PFD and 5D-PFD were built and
geometry optimized using SYBYL with convergent thresholds of 0.02Å. The
molecular docking program GOLD3.2 was used for molecular docking studies and the
binding site was defined as a sphere containing the residues that stay within 10 Å
from ligand in this crystal structure. One hundred binding conformations were
preserved for each ligand, and the docking software automatically determines the
docking space based on the algorithm and automatically specifies the atomic type of
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the ligand atom, and the remaining parameters are set as the default values. The
optimized conformations were selected and the binding energy of ligands with two
binding sites using the ACFIS online server (ACFIS: a web server for fragment-based
drug discovery).
DFT calculations in the present study were performed by using the Amsterdam
Density Functional package (ADF) 2009.01 program [20]. Geometry optimization in
the ground state was carried out using B3LYP density functional calculations[21],
with the DZP basis sets (all-electron double zeta plus polarization function) [22].
2.3. Apparatus
EQUINOX55 Fourier transformed infrared spectrometry (Bruker Company,
Germany). AVANCF600 MHz digital superconducting NMR Fourier (Bruker
Company, Germany). X-5 microscopic melting point apparatus (Tech Instrument Co.,
Ltd. Beijing, China). The absorption spectra were recorded by UV–vis ratio recording
spectrophotometer (Hitachi model U-2501). The fluorescence measurements were
carried out on an F-2700 fluorescence spectrometer (Hitachi, Japan) equipped with a
150W Xenon lamp. A 1.0 cm quartz cell was used for measurements. Elemental
analysis data were obtained on a Perkin–Elmer 240c instrument.
2.2. Chromatographic Equipment and Conditions
The UHPLC analysis was performed with a Waters Acquity™ UPLC system (USA).
Chromatographic separation was carried out at 40°C on an ACQUITY UPLC BEH
C18 column 1.7 µm (2.1 × 50 mm,). The mobile phase consisted of 0.1% formic acid
water solution (A) and 0.1% formic acid ACN solution (B). The optimized UHPLC
elution conditions were: 0 min, 70% A-30% B, 0.4-0.8 min, 5% A-95% B; 0.81-1.2
min, 70% A-30% B. The autosampler was maintained at 4°C. The sample volume
injected was 2.5 µL, the flow rate was 0.55 mL min-1, and the detector wavelength
was set at 310 nm. Data were acquired and processed with an Empower 2.0
chromatography data system. For UHPLC determination, calibration standards of
5D-PFD at concentration levels of 0.1, 0.2, 0.5, 1.0, 5.0, 8.0, 10.0 μM were prepare by
spiking the appropriate amounts of the standard solutions into blank plasma obtained
from healthy rats. The quality control (QC) samples were separately prepared in a
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similar manner as those used for the calibration curves. The blood samples
(approximately 200 μL) were collected from the ophthalmic vein in heparinized tubes
at control and 1, 5, 10, 15, 20, 30, 40, 60, 150 and 300 min after oral or i.v.
administration. Samples were immediately centrifuged at 6000 g for 10 min and the
plasma was frozen at -25°C and stored until analysis according to our previously
described method [23]. Pharmacokinetics analysis was carried out by a
non-compartmental method with the aid of DAS 2.0 software.
3. Results and discussion
3.1. Solvent effect on spectra of PFD and 5D-PFD
The optimized ground-state geometries of PFD and 5D-PFD were confirmed to be
minimum-energy conformations using ADF program at B3LYP–DZP level, as shown
in Fig.2, the pyridine ring substituents are coplanar and the phenyl group is found to
be almost perpendicular with pyridone ring. This perpendicular structure would be
unfavorable because it hinders charge transfer from the donor, pyridone group, to
acceptor unit, phenyl ring. As expected, there is little conformation difference
between PFD and 5D-PFD. The weak π-conjugation effect probably contributes to the
non-stability of PFD, and the ROS generation from PFD exposes to UV radiation,
which may potentially cause photochemical reactions and lead phototoxic skin
responses [10]. An important experimental technique for probing solute-solvent
interactions is the solvent dependence of the absorption and fluorescence spectra, in
which the different electronic properties (multipole moments, polarizabilities, etc.) of
the ground and excited state lead to a shift in the electronic excitation energy or
spectra, and this shift is a direct measure of the specific interactions of solute-solvent
[24].
The absorption and fluorescence spectra of PFD and 5D-PFD in different solvents
were shown in Fig. 2, and the corresponding values of wavenumber of maximum
absorbance and emission were listed in Table1. As seen from Figs. 2A and 2C, the
absorption maximum of PFD in water and hexane is 311 nm and 334 nm, and the
absorption maximum of 5D-PFD in water and hexane is 314 nm and 327 nm,
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respectively. Meantime, the fluorescence spectra in different solvents (Figs. 2B and
2D) show that as the solvent polarity increases (hexane to water), the emission of PFD
shifts from 409 to 391 nm (hypsochromic shift) and emission of 5D-PFD in water and
hexane is 404 nm and 390 nm, respectively. The hypsochromic shift happens when
the dipole moment of the compound decreases during the electronic transition i.e., the
dipole moment of ground state S0 is higher compared to that in the excited state S1
(μe>μg), in which the S0 state is energetically stabilized relative to the relaxed S1 state
and the S0 state is formed in solvent cage of already partly oriented solvent molecules,
consequently, thus a significant hypsochromic shift of the spectra is observed.
Moreover, the Stokes shift of PDF in hexane is 57 nm, whereas the Stokes shift in
water increases to 98 nm, and the Stokes shift of 5D-PDF in hexane is 63 nm, while
the Stokes shift in water is 90 nm, respectively. The increased Stokes shift in polar
protic solvents suggests that PFD and 5D-PFD may present the feature of the
intramolecular charge transfer (ICT), and the weak intermolecular hydrogen bonding
of carbonyl group with protic solvents may induce electron distributions. One group
of chromophores containing carbonyl group have characteristically weak n→π*
absorption bands [25]. For an n→π* electronic excitation, when one strong localized
electron in an n orbital goes to a more delocalized π* orbital, leads a meaningful
reduction of the dipole moment, and a labile lone-pair in the system provides
electronic donor properties. Therefore, it fosters hydrogen bonding with protic
solvents, which induces an energy stabilization of the lone-pair, and be larger in the
ground state than in the n→π* excited state, thereby blue-shift of transition is
observed [26]. In the n→π* transition in pyridone, an ICT occurs from the carbonyl
oxygen lone-pair into the antibonding π* orbital, localizing above and beneath the
plane spanned by the carbonyl group. This excitation leads to a reduction of the dipole
moment of PFD and 5D-PFD and consequently, a different solvation of the electronic
ground and excited states occurs. Since the dipole moment has a decreased magnitude
in the n→π* S1 state as compared to the S0 state, PFD or 5D-PFD is more favorably
stabilized in the S1 state through intermolecular hydrogen bonding interactions of
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oxygen atom with protic solvent. Solvatochromic shifts of dependent the electronic
absorption spectra of molecules, solvent polarity scales have been established
including the popular ETN scale based on Reichardt’s betaine dye [27, 28]. In order to
investigate the main type of interaction of PDF or 5D-PFD with solvents, the multiple
regression approaches based on solvatochromic equations were used and the Onsager
model was taken into consideration:
Lippert-Mataga equation [29]:
v vf  mLippertMataga LippertMataga
F
(,n)constant
a
(1)
(2)
Bakhshiev’s equation[30]:
a
f
Bakhshiev Bakhshiev
Kawski-Chamma-Viallet’s equation[31, 32]:
(va vf )/2 mKawskiChammaViallet KawskiChammaVialle(t,n)constant
F
(3)
(4)
(5)
McRae’s equation [33]:
va m F ()constant
McRae McRae
Suppan’s equation [34]:
va m F ()constant
Suppan
Suppan
where va and vf is the wavenumber of the absorption and emission maxima
respectively, the symbol h and c are Planck's constant and the velocity of light in
vacuum, respectively. FLippert–Mataga, FBakhshiev, FKawski–Chamma–Viallet, FMcRae and FSuppan
are solvent polarity functions and given as:
 1 n2 1
F (,n) 

LM
2 1 2n2 1
(6)
(7)
(8)
2
2


2n 1  1 n 1
FB(,n) 




n2 2  2 n2 2

2
2





2n 1  1 n 1
3

n4 1





FKCV (,n) 


2 
2
2
2

n2 2

 2
2
n 2

n 2






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2
 2

 1

FM () 
(9)
2

 1

F () 
S
2 1
(10)
4



3 n 1

g(n) 
2
2



2

n 2


(11)
(12)
(,n)FB(,n)2g(n)
g(n) is the solvent polarity parameter, and the symbol ε and n is dielectric constant
and refractive index of the solvent, respectively. The fitting of linear curve of va - vf vs
FLippert–Mataga, va - vf vs FBakhshiev, (va + vf)/2 vs FKawski–Chamma–Viallet, va vs FMcRae and va
vs FSuppan gives the slope of mL–P, mB, mK–C–V, mB and mS, respectively, and is given as:
2
3
0
m
 2(
g
 e（L  M )) / hca
(13)
LM
2
3
0
mB  2(  e(B)) / hca
g
(14)
(15)
2
3
0
m
 2(g  e2(K  C  V ))/ hca
K C V

g
(
g
 e(M ))
(16)
(17)
m 
3
M
hca
0
g
(
g
 e(S))
m 
3
0
S
hca
where μg is the ground state dipole moment, μe(L-M), μe(K-C-V), μe(M) and μe(S) is
the excited state dipole moment from the correlation of Lippert–Mataga, Bakhshiev,
Kawski–Chamma–Viallet, McRae and Suppan, respectively. The parameter mL-M, mB,
mK-C-V, mM and mS can be calculated from Eqs. (1)-(5). The symbol ‘a’ is the Onsager
radius of the solute molecule, with the values evaluated by using atomic increment
method [35]. If it can be supposed that ground and excited state dipole moment are
parallel, then:

3 1/2
mKCV mB hca
0




g 
2
2mB


(18)
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
3 1/2
mKCV mB hca
0




e 
2
2mB


(19)
mKCV mB
mKCV mB

e g

(mK-C-V >mB)
(20)
ETN
The normalized value of
, based on the empirical polarity scale, is used for
estimating dipole moment difference (Δμ) from solvatochromic shift [36, 37],
correlating the effect of polarity of solvent and also the effect of protic hydrogen
bonding on the spectral shift [38],

3


2


B
aB
a0
N

 
 



va vf 11307.6
ET constant









(21)
where ΔμB=9 D and aB=6.2 Å are the dipole moment difference of excitation and
Onsager radius of reference betaine dye, respectively. The symbol a0 is the Onsager
radius for the solute molecule, which indicated as:
1/ 3

3M
4N


a 
0


(22)
where N is the Avogadro’s number, M is the solute’s molecular weight and δ is the
solid-state density of solute. For PFD and 5D-PFD, δ is 1.146 g/cm3 and 1.198 g/cm3
with M of 185.22 g/mol and 205.22 g/mol and thus, a0 of PFD and 5D-PFD is 4.01 Å
and 4.08 Å.
The Δμ can be evaluated from the slope of Stokes shift vs ETN plot and is given by
the equation can be determined as [39, 40]:
3




2

B
a
B 
a






m 11307.6
ETN










(23)
ETN is defined using water and tetramethylsilane (TMS) as extreme reference solvents
with an equation:
ET (solvent)ET (TMS) ET (solvent)30.7
ETN =

ET (water)ET (TMS)
32.4
(24)
ET (solven)t hcNv 2.8591103va
a
where va (cm-1) is the absorption maxima of the standard betaine dye in solvent.
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Therefore, the difference of dipole moment is obtained from:
mEN
1.707a03
T
  g  e 
(25)
N
N
where, mET is the slope of the linear curve of Stokes shift vs ET , obtained from Eq.
(21).
The plots of Stokes shift (νa-νf and νa+νf) versus solvatochromic parameters (f(ε, n)
and f(ε, n) + 2g(n)) and ETN were listed in Fig. 3, and the results of dipole moment of
PFD and 5D-PFD were listed in table 3. One can see that the μg and μe of PFD is 5.30
D and 3.30 D, and the μg and μe of 5D-PFD is 3.70 D and 2.18 D, which is agreement
the results of energy transition mentioned above. The dipole moments of PFD in S0
and S1 are higher than that of 5D-PFD, suggesting the more polar nature of PFD in the
ground state than that of excited state compared with 5D-PFD. The time-dependent
density functional theory (TD-DFT) [41] was used to verify the results of dipole
moment of compounds, and the values of μg and μe for PFD is 7.41 D and 5.44 D, and
values of μg and μe for 5D-PFD is 4.77 D and 3.10 D. The values calculated by
TD-DFT are larger than that of calculated by solvatochromic method, since the
calculation of TD-DFT is processed in gas phase, and also the practical function and
solvent effect is not considered. This result may imply that the ground state energy
distribution is not affected to a greater extent possibly due to the more polar nature of
the drug in the ground state than in excited state. Similar negative solvatochromism
behavior has been reported in some articles [41, 42].
The absorption spectra of organic molecules are induced by electron transfers from
ground state to excited state after absorbs energy, and the maximum absorption
wavelength electronic transition is from HOMO (highest occupied molecular orbital)
to LUMO (lowest unoccupied molecular orbital). In order to obtain more insight into
the electronic distribution of PFD and 5D-PFD, the density functional theory (DFT)
calculations has been performed. As seen from Fig. 5, the HOMO or LUMO of PFD
and 5D-PFD corresponding to the n→π* transition mentioned above shows similarity,
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the charge distributes around methyl and carbonyl group acting as an electron donor
for HOMO, and the charge transfer to phenyl group and mainly locates at amine unit
contributing to the electron withdrawing effect of amine in the case of LUMO. These
indicate that the deuteration shows few effects on the charge transition property, and
the significant ICT characteristic of electron immigration from methyl group to
phenyl group is found for both of PFD and 5D-PFD.
Effect of metal ions on the absorption and NMR spectra
As seen from Figs. 6A and 6B, the absorption of PFD or 5D-PFD presents two bands,
the shorter wavelength at range of 200-250 nm originated from the π→π* transition of
phenyl group, the longer wavelength at range of 300-360 nm contributed by n→π*
transition (HOMO−LUMO) as mentioned above. With addition of metal ions (Ag+,
Al3+, Cu2+, Fe3+, K+, Mg2+, Pb2+ and Zn2+), PFD and 5D-PFD show similar spectral
change, the slight blue-shift of 325 nm absorption band, but significant red-shift of
225 nm 225 nm band, suggesting that the pyridone ring shows more stability upon
binding of unoccupied orbital of metal ions with lone-pair electron of oxygen atom.
This prompts the electronic distribution on phenyl group and thus decreases the
stability of phenyl group. The effects of metal ions (Ag+, Al3+, Cu2+, Fe3+, K+, Mg2+,
Pb2+ and Zn2+) on the 1H NMR spectra of PFD and 5D-PFD were investigated and the
results were shown in Figs. 6C-6F, and the Fe3+ ion was excluded since its
paramagnetic effect. The resonance from 7.55 ppm to 7.30 ppm is contributed by
protons of phenyl group, and this region shows the characteristic upfield shifts with
addition of metal ions, suggesting that the binding of metal ions improves the π-π*
transition thus prompts the electron density of phenyl group. And the resonance from
6.42 ppm to 6.42 ppm contributed by 3-H shows a strong downfield chemical
shifts/deshielding effect upon metal binding, which indicates that the binding of
unoccupied orbital of metal ions with lone-pair electron of oxygen atom presents the
electron withdrawing effect, and thus deduces the energy of 3-H, which is agreement
with the results of absorption spectra.
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Analysis of binding ability of human serum albumin and the pharmacokinetic
Human serum albumin (HSA) is an important protein in plasma, it can not only
transport fatty acids, pigments, steroid hormones, metal ions and other important
physiological active substances, but also carry many drug molecules. HSA also plays
a decisive role in some pharmacological properties of drugs (practical aspects of the
ligand-binding and enzymatic properties of HSA). HSA has seven ligand binding
pockets, while the Drug site 1 and Drug site 2 is the major drug binding pocket.
The effects of PFD and 5D-PFD on the fluorescence spectra of HSA have been
investigated in order to elucidate the binding mechanism. As shown in Figs. 7A and
7B, the fluorescence intensity of HSA decreases regularly with little shit of emission
upon increasing concentrations of PFD or 5D-PFD, and the maximum emission of
both PFD and 5D-PFD are around 397 nm with the excitation wavelength of 280 nm.
The fluorescence quenching is usually described by the Stern–Volmer equation [19]:
F0/F=1+Ksv[L]=1+Kqτ0[L]
(27)
where F0 and F is the fluorescence intensity in the absence and presence of ligand
(PFD or 5D-PFD), Ksv is the Stern–Volmer quenching constant and [L] is the ligand
concentration, Kq is the biomacromolecule quenching rate constant and τ0 is the
average lifetime biomacromolecule, usually as 10−8 s [8]. The binding constant (Ka)
was calculated following as:
log(F0−F)/F=log Ka + n log[L]
(28)
where, n is the number of binding sites and F0, F and [L] have the same meaning as in
Eq. (27).
As seen from Figs. 7C and 7D, the value of Ka for PFD-HSA and 5D-PFD-HSA is
0.67 μM-1 and 0.41 μM-1, and corresponding value of n is 1.75 and 1.20, respectively.
This indicates that the affinity of 5D-PFD with HSA is lower than that of PFD, and
14

ACCEPTED MANUSCRIPT
also 5D-PFD may prefer to present free forms in the blood with better efficacy
comparing with PFD, despite the slight difference of binding parameters between two
ligands with HSA.
In order to investigate the binding mechanism of PFD or 5D-PFD with HSA, the
molecular docking using GOLD program has been employed by situating PFD or
5D-PFD within sub-domain IIA hydrophobic cavity of HSA, site I and site II,
respectively. As shown in Figs. 8A and 8B, for both of PFD and 5D-PFD, the phenyl
group is found to be almost coplanar with pyridone ring in the site I, and the oxygen
atom of carbonyl group forms the significant hydrogen bonding with G196 and
pyridone ring contacts with Trp214 and Lys199 by Van de Waal and hydrophobic
interaction. The phenyl group is adjacent to K199 with considerable π-π interaction,
and the hydrophobic interactions of H242, A291, V238 and Y150 with PFD also
promote the matching of ligand with active cavity. On the other hand, as seen from
Fig. 8C, two aromatic rings of PFD are close to be perpendicular inside of site II,
carbonyl group forms two hydrogen bonds with R410 meantime, and the van de waals
and hydrophobic interaction play key role in connecting of pyridone ring with Y441
and L457. The phenyl group inserts the hydrophobic cavity formed by L407, L430,
L453, L387, V433 and F403, which is contributed mainly by hydrophobic interaction.
Moreover, as shown in Figs. 8B and 8D, the 5D-PFD presents little difference of
binding model with HSA comparing with native one. The result of docking suggests
that PFD and 5D-PFD show the similar binding model and mechanism. However,
GOLD program can’t provide the energy information about binding, thus, the
MM_PBSA method of AMBER8 program to calculate the binding energy of PFD and
5D-PFD with HSA [43]. As seen from Table 4, the △GPB of site II is higher than that
of site I and the value of △GPB of 5D-PFD is slight lower than that of PFD, indicating
that ligands could form hydrophobic and van de waals interaction inside of site II and
dominate the binding, and also the infinity of 5D-PFD with HSA is lower than that of
PFD, which is agreement with the spectral results.
15

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Moreover, the pharmacokinetics of 5D-PFD has been evaluated after oral (20 mg kg-1)
and i.v. (5 mg kg-1) administration into normal rats by the method of the previous
report [44], and the time course plasma levels is listed in Fig. 9 and pharmacokinetic
parameters are summarized in Table 5. Plasma levels of 5D-PFD present a peak
around 25 min for oral administration and plasma levels of 5D-PFD fall significantly
in a biexponential fashion for i.v. administration (Fig. 9). The T1/2 value oral and i.v.
administration of 5D-PFD is around 19 and 30 min, higher than the T1/2 value of i.v.
administration of PFD, 8.6 min, reported by Giri et al. [1], which suggests that the
deuteration enhances the pharmacokinetic of PFD significantly with little change of
physical-chemical properties. And the enhanced metabolism and pharmacokinetic of
5D-PFD in rat clearly underscores the need for appropriate kinetic studies in humans.
16

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4. Conclusion
PFD has shown anti-inflammatory and anti-fibrotic properties and suppressed the
progression of fibrotic and inflammatory, however, the rapid metabolism and
clearance restrains the further clinical application of PFD. Thus, in this work, in order
to improve the metabolism of PFD, 5D-PFD has been synthesized by substituted
hydrogen at C-5 with its isotope deuterium (D), and the photophysical properties have
been investigated. For both of PFD and 5D-PFD, the ICT involves n→π* transition
from the electron-donor methyl and carbonyl group to the amine group acting as an
electron withdrawing effect. The diploe moment calculation results suggest that PFD
shows a more polarity in the ground state than excited state compared with 5D-PFD.
The binding of metal ions with PFD or 5D-PFD improves the stability of pyridone
ring due to the binding of unoccupied orbital of metal ions with lone-pair electron of
oxygen atom. The binding and docking of HSA results demonstrate that the affinity of
5D-PFD with HSA is lower than that of PFD, and also 5D-PFD may prefer to present
free forms in the blood with better efficacy comparing with PFD, despite the little
different binding energy and site of two ligands with HSA. The pharmacokinetic of
T1/2 values by oral and i.v. administration of 5D-PFD is found around 19 and 30 min,
which is almost 4 times than that of value reported by Giri et al. [1]. The experimental
and theoretical results offer a rationale for understanding the photophysical properties
of PFD and its deuterium derivates, 5D-PFD, which would be available for wider
clinical application of PFD as active drug.
Acknowledgement
This work is supported by the Natural Science Foundation of Hubei Province
(2016CFB317) and the Doctoral Initiate Foundation of Hubei University of Arts and
Science.
17

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Table 1. Absorption (νa) and fluorescence (νf) spectral wavelengths for PFD and 5D-PFD (described in cm-1)
PFD
5D-PFD
Solvents
νa
νf
νa-νf
(νa+νf)
(νa+νf)/2
νa
νf
νa-νf
(νa+νf)
(νa+νf)/2
water
32154.34 25575.45 6578.89 57729.79 28864.89 31847.13 25641.03 6206.11 57488.16 28744.08
25000 6152.65 56152.65 28076.32
28093.65 31112.76 24981.54 6131.22 56094.3 28047.15
31847.13 25445.29 6401.84 57292.42 28646.21 31645.57 25380.71 6264.86 57026.28 28513.14
31545.74 25000 6545.74 56545.74 28272.87 31438.92 25316.46 6229.29 56862.2 28431.1
Acetonitrile 31055.90 25188.92 5866.98 56244.82 28122.41 31152.65
DMF
31043.76 25143.54 5900.22 56187.3
Methanol
Ethanol
Acetone
n-Butanol
31003.42 24901.31 6102.11 55904.73 27952.365 31086.14 24832.52 6253.62 55918.66 27959.33
31347.96 24937.66 6410.3 56285.62 28142.81 31347.96 25125.63 6222.33 56473.59 28236.8
Ethyl acetate 30674.85 24813.98 5860.87 55488.83 27744.415 31055.9 24937.66 6118.24 55993.56 27996.78
Chloroform 30487.81 24691.36 5796.45 55179.17 27589.585 30864.2 24875.62 5988.58 55739.82 27869.91
Toluene
Benzene
30211.48 24509.81 5701.67 54721.29 27360.645 30674.85 24813.9 5860.95 55488.74 27744.37
30193.72 24494.23 5699.49 54687.95 27343.975 30637.19 24772.36 5864.83 55409.55 27704.78
Cyclohexane 30112.13 24462.69 5649.44 54574.82 27287.41 30602.24 24761.73 5840.51 55363.97 27681.99
n-Hexane
29940.12 24449.88 5490.24
54390
27195
30581.04 24752.48 5828.56 55333.52 27666.76
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Table 2 Solvatochromic parameters
ETN
Solvents
ε
N
α
β
(, n)a
g(n)
FL-M(, n)
FB(, n)b
FK-C-V(, n)
FM()
FS()
water
Acetonitrile
DMF
80.40
38.01
36.71
33.71
24.55
20.56
17.40
6.02
1.331
1.344
1.431
1.328
1.361
1.359
1.399
1.372
1.446
1.497
1.559
1.427
1.375
1.17
0.19
0.00
0.98
0.86
0.08
0.84
0.00
0.20
0.00
0.00
0
0.47
0.40
0.69
0.66
0.75
0.48
0.84
0.45
0.10
0.11
0.10
0
1.3653
1.3328
1.4201
1.3047
1.3045
1.2780
1.2917
0.9955
0.9753
0.6999
0.7163
0.5756
0.5076
0.2255
0.2343
0.2924
0.2235
0.2457
0.2444
0.2711
0.2531
0.3022
0.3354
0.3750
0.2897
0.2551
0.3209
0.3057
0.2742
0.3094
0.2889
0.2840
0.2633
0.1998
0.1482
0.0132
-0.0148
-0.0019
-0.0014
0.9143
0.8643
0.8354
0.8578
0.8131
0.7893
0.7494
0.4893
0.3709
0.0290
-0.0337
-0.0038
-0.0026
0.6826
0.6664
0.7101
0.6523
0.6523
0.6390
0.6459
0.4977
0.4877
0.3499
0.3582
0.2878
0.2538
1.9272
1.8500
1.8450
1.8320
1.7740
1.7340
1.6907
1.2519
1.1189
0.6301
0.5948
0.5075
0.4536
0.9815
0.9610
0.9597
0.9562
0.9401
0.9288
0.9162
0.7699
0.7175
0.4792
0.4585
0.4048
0.3697
1.000
0.460
0.386
0.762
0.654
0.355
0.241
0.228
0.259
0.099
0.111
0.164
0.009
Methanol
Ethanol
Acetone
n-Butanol
Ethyl acetate
Chloroform
Toluene
4.81
2.38
Benzene
2.27
Cyclohexane
2.02
n-Hexane
1.88
0
0
a (, n)= f (, n)+2g(n)
b FB(, n)=f (, n)
19

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Table 3 Calculated parameters according spectral data of PFD and 5D-PFD in solvents.
Onsager
b
d
e
f
g
h
Compound Equation Intercept
Slope
R
μg
μec
μe(L-M)
μe(B)
μe(K-C-V)
μe(M)
μe(S)
μe/μj
radius (Å)
L-M1
B2
K-C-V3
M4
S5
L-M1
B2
5674.43
5605.27
1920.21
840.11
0.87
0.86
0.81
0.79
0.81
0.90
0.89
0.82
0.87
0.88
PFD
26386.73
29473.04
28952.13
5884.14
2845.68
1073.17
2564.31
967.15
4.01
5.65
3.70
3.30
4.19
4.69
3.02
5.37
5.23
3.33
4.64
2.81
0.58
5855.82
425.01
5D-PFD
K-C-V3
M4
27179.04
30264.44
28952.13
1648.10
622.65
4.08
2.18
2.67
3.68
0.59
S5
1492.18
a) 1D = 3.33564 × 10-30 C. m. = 10-18 esu cm. Equation: 1 Lippert–Mataga, 2 Bakhshiev, 3 Kawski–Chamma–Viallet, 4 McRae and 5 Suppan.
b) Ground state dipole moment calculated according to Bilot–Kawski, Eq. (18).
c) Excited state dipole moment calculated according to Bilot–Kawski, Eq. (19).
d) Calculated according to Lippert–Mataga correlation, Eq. (13).
e) Calculated according to Bakhshiev correlation, Eq. (14).
f) Calculated according to Kawski–Chamma–Viallet correlation, Eq. (15).
g) Calculated according to McRae correlation, Eq. (16).
h) Calculated according to Suppan correlation, Eq. (17).
i) The angle between the ground state and excited state dipole moment calculated from Eq. (20).
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Table 4 Binding energy of PFD and 5D-PFD with active site I and II of HSA
PFD-site I
-39.87
-28.02
-67.89
-4.01
5D-PFD-site I
-38.53
-28.50
-67.03
-4.09
PFD-site II
-27.58
-26.65
-54.23
-3.77
5D-PFD-site II
-25.15
ΔEele
ΔEvdw
ΔEgas
ΔGnp
-27.58
-52.73
-3.86
ΔGpol
ΔGsolv
ΔGPB
66.50
65.53
43.63
42.60
62.49
61.44
39.85
38.74
-5.40
-5.59
-14.38
-13.98
Table 5 The pharmacokinetic parameters (mean ± S.D.) of 5D-PFD in rats following oral and i.v. administration at dose of 20 mg/kg and 5 mg/kg
(n = 6), respectively.
T1/2
Tmax
Cmax
AUC0→∞
Vz/F
(mL/kg)
-
CLz/F
(mL/min /kg)
-
MRT0-∞
(min)
Formulation
(mim)
(min)
(μg/mL)
7.27±0.51
(min μg/mL)
473.32±61.01
Oral 5D-PFD
29.83±8.01
18.89±1.52
16.67±2.89
1.00±0.01
46.15±3.00
24.87±1.17
i.v. 5D-PFD
10.69±0.93 193.89±17.79
703.83±16.31
25.94±2.48
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Figure 1. Scheme of synthetic route of 5D-PFD
Figure 2. Optimum configuration B3LYP/DZP of PFD (A) and 5D-PFD (B).
Figure 3 Absorption and fluorescence spectra of PFD (A, B) and 5D-PFD (C, D) in
different solvents, respectively.
Figure 4. A-C: plots of νa-νf vs. f(ε, n), νa+νf vs. f(ε, n) + 2g(n) and νa-νf vs. ETN for
PFD in different solvents. D-F: plots of νa-νf vs. f(ε, n), νa+νf vs. f(ε, n) + 2g(n) and
νa-νf vs. ETN for 5D-PFD in different solvents.
Figure 5. Frontier orbital of PFD and 5D-PFD calculated by the ADF program.
Figure 6. Effect of metal ions on the absorption spectra of PFD (A) and 5D-PFD (B).
NMR titration of metal ions into PFD (C, D) and 5D-PFD (E, F), respectively.
Figure 7. (A, B): Fluorescence spectra of HSA with 1-8 μM PFD and 5D-PFD,
respectively. (λex = 280 nm). Stern–Volmer (C) and modified Stern–Volmer (D) plots
for the PFD–HSA and 5D-PFD–HSA system, respectively.
Figure 8. Binding of PFD (A) and 5D-PFD (B) in the active site I of HSA. Binding of
PFD (C) and 5D-PFD (D) in the active site II of HSA. Red dotted lines represented
the hydrogen bonding between ligands and HSA.
Figure 9. Mean plasma concentration-time curve of 5D-PFD following oral and i.v.
administration at dose of 20 mg/kg and 5 mg/kg in rats.
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Synthesis and photophysical properties of deuteration of pirfenidone
Qiuju Yina1, Yujie Chena1, Meng Zhoua, Xiangsheng Jianga, Junjun Wua, Yang Suna,b*
a
School of Chemical Engineering, Hubei University of Arts and Science, No. 296
Longzhong Road, Xiangyang, Hubei 441053, China
b School of Life Sciences, Tsinghua University, Beijing 100084, China
Corresponding author email: sunyang@if.usp.br (Y. Sun),
1 These authors are co-first author of this work
Highlights
 The deuterium of pirfenidone (5-methyl-1-phenylpyridin-2-one, PFD) by the
substitution of hydrogen (H) at C-5 with isotope deuterium (D, 5D-PFD) has
been synthesized
 The negative solvatochrom was observed in absorption and fluorescence
spectra with increasing solvent polarity, which implied that intermolecular
charge transfer involved n→π* transition for both of PFD and 5D-PFD
 A significant propensity for intramolecular charge transfer from the
electron-donor methyl and carbonyl group to the amine group as an electron
donor has been observed.
 The pyridone ring showed more stability upon binding of unoccupied orbital
of metal ions with lone-pair electron of oxygen atom and thus prompted the
electronic distribution on phenyl unit.
 The human serum binding and docking results demonstrated that the affinity
of 5D-PFD with HSA was lower than that of PFD, and also 5D-PFD may
prefer to present free forms in the blood with better efficacy comparing with
PFD

The pharmacokinetic of T1/2 values by oral and i.v. administration of 5D-PFD
was found around 19 and 30 min.
26

Graphics Abstract

Figure 1

Figure 2