Synthesis, pH dependent, plasma and enzymatic stability of bergenin prodrugs for potential use against rheumatoid arthritis

Article abstract of DOI:10.1016/j.bmc.2017.08.011

Bergenin is a unique C-glycoside natural product possessing anti-inflammatory and anti-arthritic activity. It is hydrophilic molecule and stable under acidic conditions however is unstable at neutral-basic pH conditions. The rate of degradation is directly proportional to the increase in pH which might be one of the reasons for its low oral bioavailability. Thus, herein our objective was to improve its stability using prodrug strategy. Various ester and ether prodrugs were synthesized and studied for lipophilicity, chemical stability and enzymatic hydrolysis in plasma/esterase. The stability of synthesized prodrugs was evaluated in buffers at different pH, in biorelevant media such as SGF, SIF, rat plasma and in esterase enzyme. All prodrugs displayed significantly improved lipophilicity compared with bergenin, which was in accordance with the criteria of drug-like compounds. Acetyl ester 4a₂ appeared to be the most promising prodrug as it remained stable at gastric/intestinal pH and was completely transformed to the parent compound bergenin in plasma as desired for an ideal prodrug. The data presented herein, will help in designing stable prodrugs of unstable molecules with desired physicochemical properties in structurally similar chemotypes.

Full text of DOI:10.1016/j.bmc.2017.08.011

Accepted Manuscript
Synthesis, pH dependent, plasma and enzymatic stability of bergenin prodrugs
for potential use against rheumatoid arthritis
Rohit Singh, Vikas Kumar, Sonali S. Bharate, Ram A. Vishwakarma
PII:
S0968-0896(17)31330-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.011
BMC 13914
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 June 2017
3 August 2017
8 August 2017
Please cite this article as: Singh, R., Kumar, V., Bharate, S.S., Vishwakarma, R.A., Synthesis, pH dependent, plasma
and enzymatic stability of bergenin prodrugs for potential use against rheumatoid arthritis, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.011
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Graphical Abstract
Synthesis, pH dependent, plasma and enzymatic
stability of bergenin prodrugs for potential use against
rheumatoid arthritis
Leave this area blank for abstract info.
Rohit Singha,b, Vikas Kumara,b, Sonali S. Bharate*a, and Ram A. Vishwakarma*,b,c
aPreformulation Laboratory, bAcademy of Scientific and Innovative Research (AcSIR), cMedicinal Chemistry
Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
Improved
modulated log P, log D values
stability
and
Stable in buffers at pH 1.2, 4.0,
6.8 and 7.4
Stable in SGF (pH 1.2) and SIF
(pH 6.8)
Hydrophilic
Log P = –0.555
Log D = – 0.947
Enzyme-mediated
hydrolytic
Drug like properties
Log P = 1.15
Log D = 0.5
activation in plasma and in
presenceof esterase

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis, pH dependent, plasma and enzymatic stability of bergenin prodrugs for
potential use against rheumatoid arthritis
Rohit Singha,b, Vikas Kumara,b, Sonali S. Bharate*a, and Ram A. Vishwakarma*,b,c
aPreformulation Laboratory, bAcademy of Scientific and Innovative Research (AcSIR), cMedicinal Chemistry Division, CSIR-Indian Institute of Integrative
Medicine, Canal Road, Jammu-180001, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Bergenin is a unique C-glycoside natural product possessing anti-inflammatory and anti-arthritic
activity. It is hydrophilic molecule and stable under acidic conditions however is unstable at
neutral-basic pH conditions. The rate of degradation is directly proportional to the increase in
pH which might be one of the reasons for its low oral bioavailability. Thus, herein our objective
was to improve its stability using prodrug strategy. Various ester and ether prodrugs were
synthesized and studied for lipophilicity, chemical stability and enzymatic hydrolysis in plasma/
esterase. The stability of synthesized prodrugs was evaluated in buffers at different pH, in
biorelevant media such as SGF, SIF, rat plasma and in esterase enzyme. All prodrugs displayed
significantly improved lipophilicity compared with bergenin, which was in accordance with the
criteria of drug-like compounds. Acetyl ester 4a2 appeared to be the most promising prodrug as
it remained stable at gastric/intestinal pH and was completely transformed to the parent
compound bergenin in plasma as desired for an ideal prodrug. The data presented herein, will
help in designing stable prodrugs of unstable molecules with desired physicochemical properties
in structurally similar chemotypes.
Received in revised form
Accepted
Available online
Keywords:
Bergenin
Stability
Prodrugs
Lipophilicity
Enzymatic hydrolysis
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Omeprazole was not designed as a prodrug. However later it was
found that it acts as a prodrug and is converted to active inhibitor
Prodrugs are chemically oriented drug delivery tools wherein
pharmacologically potent structures can be optimized so as to
of proton pump in the acidic environment per se. It undergoes
chemical activation via molecular rearrangements in acidic
environment to form the active sulfonamide.4 To prevent this
premature activation, delayed release capsules of omeprazole are
available in the market for oral administration.5 The formulation
consists of enteric coated granules of omeprazole (10, 20 or 40
mg). It is formulated as enteric coated granules because it is acid
labile and enteric coating protect it from pre-absorption
degradation in acidic environment of stomach. Absorption begins
overcome
its
physicochemical,
pharmaceutical
and
biopharmaceutical properties to make it more useful. The parent
drug/s are modulated through a temporary chemical change
through the covalent attachment of
a chemical moiety
(promoiety). The prodrug being a completely new chemical, it
possesses a different physicochemical profile which might allow
easier drug delivery. Prodrugs can be used for several reasons viz.
enhancing solubility, permeability or chemical stability of parent
compound. This may result in improving drug targeting, reduced
side effects and reduced too rapid elimination of parent drug.1
These are bioreversible derivatives of drug/s that undergo an
enzymatic and/or chemical transformation in vivo to release the
parent drug, which can exert the desired pharmacological effect.2
when formulation leave the stomach i.e. 1-3 h after dosing
(gastric emptying time).6,
7
Azacitidine (Vidaza®) is
investigational antineoplastic agent and considered as a potential
inhibitor of nucleic acid biosynthesis. For the treatment of
myelodysplastic syndromes, it is the first DNA hypomethylating
agent approved by FDA. Azacitidine is administered by
intravenous and subcutaneous route. It presents several
drawbacks with respect to oral administration, such as sub-
optimal physiochemical properties (hydrophilic nature: water
solubility 89 mg/mL, Log P -3.1) and hydrolytic instability.8 In
this direction, Xu and coworkers prepared amino acid ester
prodrugs of azacitidine to overcome its hydrolytic instability.9
The aqueous solution of azacitidine readily gets hydrolyzed
however its bisulfite prodrug is stable to such degradation at
acidic pH. The prodrug also gets converted to active drug at
physiological pH.10 NSC-743380 (oncrasin-72) is an anticancer
agent, was found to be unstable in injectable formulations.
There are many literature reports wherein prodrugs have been
utilized for improving stability of parent compound. Omeprazole,
a proton pump inhibitor, is marketed as Prilosec® which has
shown to reduce gastric acid secretion in animals and humans.3
_____________
*Corresponding authors. Tel.: +91-191-2585006; Fax: +91-191-2586333
E-mail addresses: sonalibharate@gmail.com (SSB); ram@iiim.ac.in
(RAV)

Oncrasin-72 tends to form dimers in aqueous solutions resulting
in its loss of activity, especially at low-pH conditions. Wu et al.
reported oncrasin-266, a stable prodrug of oncrasin-72 with
cyclohexylacetic acid as a promoiety.11 The shelf-life of
cefamandole, a second generation broad spectrum cephalosporin
antibiotic, was improved by formation of nafate ester.
Cycloserine (an antibiotics used to treat tuberculosis) under
acidic conditions hydrolyses to hydroxylamine and D-serine.
Merck chemist Norm Jensen discovered stable prodrug of
cycloserine, pentizidone.12 To improve metabolic stability of
compounds, researchers have also attempted ether prodrugs.13, 14
There are many reports of ether prodrugs which were prepared in
order to improve lipophilicity which will eventually improve
permeability of parent compound.13 Japanese company Yamasa
cooperation prepared ether prodrugs of antiviral drug; BV-araU
with an objective to improve its stability in GI tract upon
ingestion.14 This illustrates utility of prodrugs in improving
stability of parent compound/ drug candidates so as to enhance
drug delivery and thus oral bioavailability.
attempts have never been made to improve the stability of
bergenin by prodrug approach. Herein, we report for the first
time, the design and synthesis of prodrugs of bergenin in order to
improve its stability and drug-like properties (Figure 2a and 2b).
Ester and ether prodrugs of bergenin were synthesized using
different promoieties. All synthesized prodrugs were evaluated
for its stability at different pH conditions in order to identify an
appropriate promoieties which will give stable prodrug/s with
balanced lipophilicity. The stability of synthesized prodrugs was
determined at different pH conditions and in biorelevant media
viz. buffer solutions at pH 1.2, 4.0, PBS (pH 6.8), 7.4, SGF (pH
1.2), SIF (pH 6.8), rat plasma and in esterase enzyme from
porcine liver. The experimental log P (water/octanol) and log D
(PBS pH 7.4/ octanol) were determined by miniaturized shake
flask method.
2. Results and discussion
2.1. Synthesis of ester and ether prodrugs of bergenin
Bergenin (1) is a C-glycoside of 4-O-methylgallic acid which
naturally occurs in several plant genera like Bergenia ciliata and
Bergenia ligulata,15, 16 Bergenia stracheyi (rhizomes),17 Astilbe
chinensis (rhizomes),18 Dryobalanops aromatic (stem bark),19
Ardisia elliptica (stem bark)20 and Mallotus japonicas (stem
bark).21 Bergenin (1) exhibits various pharmacological activities
Bergenin is a unique C-glycoside natural product present in
different species of Bergenia. It is one of a major component of
Bergenia ciliata plant. The repeated silica gel column
chromatography of methanolic extract of this plant resulted in
isolation of bergenin in grams. The isolated bergenin was
characterized by detailed spectral analysis including NMR, MS,
IR and 2D NMR analysis.
like
anti-arthritic,22
anti-inflammatory,15
antitussive,23
25
hypolipidemic,24,
anti-arrhythmic,26 hepatoprotective,21
neuroprotective,27 anti-fungal28 etc. Recently, we reported that
bergenin possess selective inhibition of IL-6 cytokine (inhibition
of IL-6, IC50 = 208 nM; TNF-α, IC50 = 12.5 µM) both in in-vitro
as well as in-vivo assays. It also displayed promising anti-
arthritic activity in collagen-induced arthritis model.15 The
overview of various pharmacological activities of bergenin is
shown in Figure 1.
Designing ester and ether prodrugs is one approach to improve
stability of bergenin having hydroxyl groups and modify/alter its
physicochemical properties such as lipophilicity. It has been
reported that ester prodrugs of a compound have been prepared to
improve its stability and is an effective way to optimize drug-like
properties of any compound. The esterases present in the body
fluids are responsible for reversible conversion of prodrug to the
parent form.33, 34
Bergenia ciliata
Higher alkyl chain fatty acids like hexanoic acid chloride, 2f,
have been utilized to generate lipophilic prodrugs such as
haloperidol decanoate, a prodrug of haloperidol.35 It is also
reported that increase in degree of branching or length of the acyl
side chain, 2b and 2e, will result in lipophilic prodrugs. Total 19
ester and ether prodrugs of bergenin were synthesized as depicted
in Figure 2. All 19 compounds were then studied for Log P and
Log D determination and investigated for their chemical and
enzymatic stability. Bergenin on treatment with various acyl
chlorides and alkyl halides resulted in formation of bergenin-
esters and bergenin-ethers, respectively. All prepared prodrugs
Multiple
“Anti-arthritic”
pharmacological
Selective IL-6 inhibitor
activities
• IL-6: IC50 = 0.208 µM
• TNF-α: IC50 = 12.5 µM
In-vivo anti-arthriticactivity
ꢀanti-arthritic
ꢀanti-inflammatory
ꢀantitussive
ꢀhypolipidemic
ꢀanti-arrhythmic
ꢀhepatoprotective
ꢀneuroprotective
ꢀanti-fungal
O
HO
OH
Cons:
• Highly hydrophilic (Log P < 0)
• High aqueous solubility (>1 mg/ml)
• PoorPK profile (less t1/2)
• Unstable at neutral and alkaline pH
O
HO
HO
O
O
OH
Bergenin (1)
Figure 1. Pharmacological activities and physicochemical properties of
bergenin
1
were characterized by NMR, MS, IR and HRMS studies. In H
Literature reports indicate that bergenin gets degraded in
neutral and alkaline conditions.29 However, the drug was reported
to be stable at acidic conditions at pH 1.0, 3.0 and 5.0. This
indicate that bergenin is susceptible to degradation under neutral-
basic pH conditions and the rate of degradation increases with
increase in pH.30 The degradation mechanism of bergenin
indicates that the lactone bond gets hydrolyzed. The reason for
low oral bioavailability of bergenin could be instability in neutral
and alkaline pH such as intestinal tract.31, 32 The physicochemical
properties of bergenin are also reported by Zhou and coworkers.30
They have reported the aqueous solubility of bergenin at different
pH conditions viz. pH 1.0 (1.29 mg/mL), 3.0 (1.08 mg/mL) and
5.0 (1.22 mg/mL). The log P is -1.06 which clearly indicate the
hydrophilic nature of bergenin. A Research group from China29
has reported novel coated floating tablets of bergenin in
combination with cetirizine dihydrochloride. The advantage of
floating formulation is to prolong retention of bergenin in
stomach thus avoiding its intestinal degradation which will in
turn help to enhance its absorption and efficacy. However
NMR study, the presence of additional groups of acyl moiety
confirmed the formation of acyl prodrugs. These observations
were further confirmed by 13C NMR and MS analysis. The
position of acylation or etherification was confirmed by HMBC
analysis, for which we selected four compounds viz. penta-
substituted ester 4d1, mono-acyl product 4i1, diacyl product 4i2
and di-substituted ether 5b. Based on the observed HMBC
correlations (2J and 3J) with adjacent carbons, the positions of
acyl or alkyl groups was assigned. It was observed that
etherification was primarily favored on aromatic hydroxyls e.g.
all di-subsubtituted ethers were formed on two aromatic OH
groups. However, all di-substituted esters were formed on one
aromatic OH and one –CH2OH of sugar moiety. This has been
depicted in Figure 2 and HMBC data is presented in supporting
information.
2.2. Determination of partition coefficient (log P) and
distribution coefficient (log D)

─0.5 and log
of bergenin prodrugs, compounds except 4a1 (log P,
Assessment of log P and log D is associated with
physicochemical and physiological properties of the compound
such as its membrane permeability.36 Compounds with lower log
P (< 0) are more polar and have poor membrane permeability
however the compounds with higher log P (> 3) are more non-
polar and have poor aqueous solubility. Log P and log D of
synthesized esters (4a-k) and ethers (5a-d) was determined. We
also determined the effect of different substituent (-R groups,
aliphatic and aromatic) on partition coefficient and distribution
coefficient of bergenin ester and ether prodrugs (Table 1). The
log P and log D values followed opposite trend wherein the
D, ─1.0), 4e (log P, 6.3 and log D, 6.4), 4f (log P, 5.9 and log D,
6.0), 4j (log P, 6.0 and log D, 6.1) and 4k (log P, 3.7 and log D,
3.9) possess optimal log P and log D values (0-3).
2.3. In-vitro stability studies
It was observed that bergenin was unstable in PBS pH 6.8
(53% degraded in 24 h) and PBS pH 7.4 (70% degraded in 24 h).
The hydrolytic study of synthesized ester and ether prodrugs of
bergenin (4a-k, 5a-d) initiated by determining compound’s
stability in PBS pH 7.4 buffer. The compounds which were found
to be stable in PBS pH 7.4 were taken forward to determine their
stability in different buffer solutions and in biorelevant media.
These compounds were evaluated for stability towards hydrolysis
in different media: 1) Buffer solutions of different pH viz. 1.2,
4.0 and 6.8 to examine their chemical hydrolysis, 2) SGF (pH
1.2) and SIF (pH 6.8) to evaluate their hydrolysis in biorelevant
media, and 3) Rat plasma and esterase from porcine liver to
understand enzymatic hydrolysis of synthesized prodrugs.
˗k
synthesized prodrugs 4a , 5a-d were hydrophobic (log P and
log D in the range of 0.5 to 6.4) as compared to bergenin (log P,
─0.5 and log D, ─0.9). Aliphatic chain length was directly
proportional to log P and log D value of bergenin prodrugs.
Compounds 4e, 4f and 4j had log P/D value of >5.0 indicating
their hydrophobic nature and make them to deviate from the
criteria for drug-like properties. Based on log P and log D values
O
O
R5O
O
OR4
HO
OH
O
O
a
R1O
R2O
HO
HO
R
Cl
+
O
O
O
O
2a-k
OR3
OH
Bergenin (1)
Bergenin esters
4a-k
4a1: R1, R4
=
4a2: R1, R2, R3, R4, R5
=
4a3: R1, R2, R4, R5
=
4b: R1, R4, R5
R2, R3 = H
=
O
O
O
O
R3 = H
R2, R3, R5 = H
F
F
4d1: R1, R2, R3, R4, R5
=
4d2: R1, R4, R5
=
4c: R1, R4
=
O
O
O
R2, R3 = H
R2, R3, R5 = H
O2N
O2N
NO2
4e: R1, R2, R3, R4, R5
=
4g: R1, R4
=
4f: R1, R2, R3, R4, R5
=
O
O
O
R2, R3, R5 = H
NO2
NO2
4h: R1, R2, R4, R5
=
4i1: R1
=
O
O
R3 = H
4i2: R1, R4 =
O
R2, R3, R4, R5 = H
R2, R3, R5 = H
Cl
4j: R1, R2, R3, R4, R5
=
Cl
O
4k: R1, R4
=
O
Cl
R2, R3, R5 = H
2a
O
O
R5O
OR4
HO
OH
H2
C
O
b
O
R1O
R2O
HO
HO
R
Cl
+
O
O
O
O
3a-d
OR3
OH
Bergenin ethers
Bergenin (1)
5a-d
5a: R4, R5
=
5b: R4, R5
=
R1, R2, R3 = H
R1, R2, R3 = H
: R , R
5d
=
5
5c: R4, R5
=
4
R1, R2, R3 = H
R1, R2, R3 = H
2b
Figure 2. Synthesis of ester (4a-k) and ether (5a-d) prodrugs of bergenin (2a). Reagents and conditions: (a) Pyridine, 0-5 °C,
─80 %. Acid chlorides
60
viz. 2c, 2e, 2f, 2i, 2j and 2k were synthesized using thionyl chloride with 2,6-difluorobenzoic acid, 3-
methylbutanoic acid, hexanoic acid, p-nitrobenzoic acid and octanoic acid respectively. (2b). Reagents and conditions: (b) KOH,
DMF, RT with alkyl halide, 60─80 %.

Table 1
Experimental log P, log D and stability in PBS (pH 7.4) of bergenin and its prodrugsa
Compound
Partition coefficient at 25 °C
Log P (water/ n-octanol)
-0.555 0.003
-0.494 0.008
1.155 0.035
1.181 0.020
2.553 0.175
1.801 0.072
0.771 0.055
0.599 0.252
6.358 0.036
5.967 0.025
2.787 0.104
1.751 0.179
1.563 0.172
2.508 0.146
6.011 0.028
3.693 0.155
1.655 0.019
2.495 0.032
1.633 0.113
1.965 0.184
Distribution coefficient at 25 °C
Log D (PBS pH 7.4/ n-octanol)
-0.947 0.045
-1.062 0.102
0.516 0.048
% remaining after 24 h at 37 °C
PBS (pH 7.4)
1
33.786 3.277
41.123 6.877
93.808 10.227
96.450 2.619
5.021 1.617
4a1
4a2
4a3
4b
4c
0.429 0.055
1.773 0.089
1.861 0.027
56.818 20.375
85.268 11.647
81.364 12.738
4.46 0.830
4d1
4d2
4e
0.162 0.019
0.552 0.008
6.453 0.025
4f
6.064 0.008
3.423 1.455
4g
4h
4i1
4i2
4j
1.798 0.008
32.275 10.328
17.985 3.552
53.427 7.952
73.591 9.881
14.996 4.857
89.181 21.547
37.412 13.289
31.041 11.29
24.662 5.469
91.055 17.344
1.608 0.092
1.535 0.061
2.655 0.326
6.113 0.008
4k
5a
5b
5c
3.888 0.206
1.508 0.029
2.835 0.059
1.524 0.018
5d
1.981 0.014
a, all values are average of three determinations and are represented as mean SD; nd, not determined
2.3.1. Chemical (non-enzymatic) hydrolysis in PBS pH 7.4
However, in PBS pH 6.8 buffer solution, compounds 4a2, 4a3,
4d1, 4d2 and 4k were stable with only 4-8% compound liable to
degradation over a period of 24 h at 37 °C. 4i2 and 5d were
susceptible to degradation with 48.2 and 53.4% remaining after
24 h. The compounds viz. 4a2, 4a3, 4d1 and 4d2 were stable to
chemical hydrolysis at pH 1.2, 4.0 and 6.8 over 24 h at 37 °C.
The % hydrolyzed after incubation of compounds in PBS pH 7.4
buffer for 24 h was determined and the values are shown in Table
2. The obtained results indicated that prodrugs viz. 4a2, 4a3, 4d1,
4d2, 4i2, 4k and 5d were stable to hydrolysis in PBS pH 7.4
buffer. The results are shown in Table 1, where % remaining
after 24 h at 37 °C was found to be 93.8, 96.5, 85.3, 81.4, 73.6,
89.2 and 91.1% for 4a2, 4a3, 4d1, 4d2, 4i2, 4k and 5d,
respectively. Therefore only these compounds were considered
further to determine its stability at different pH and in different
media. However, in case of bergenin, only 30% remained stable
over a period of 24 h at 37 °C.
2.3.3. Stability in biorelevant media, SGF (pH 1.2) and SIF (pH
6.8)
In vitro enzymatic hydrolysis pattern of ester prodrugs was
studied in SGF (pH 1.2) and SIF (pH 6.8) at 37 °C. To mimic in
vivo gastric and intestinal digestion, simulated gastric fluid with
pepsin and simulated intestinal fluid with pancreatin were used
for in vitro studies. The % hydrolysis of the compounds is
depicted in Table 2. It was observed that prodrugs 4a2, 4a3, 4d1,
4d2, 4i2 and 4k were stable to hydrolysis in SGF pH 1.2.
However, 5d was hydrolyzed upto 31.2% after 24 h. When these
stock solutions were incubated in SIF pH 6.8, compound 4a2,
4d1, 4d2, 4k and 5d were stable with >85% remaining after 24 h.
In brief, only compounds 4a2, 4d1, 4d2 and 4k were stable to
chemical hydrolysis and in biorelevent media namely SGF (pH
1.2) and SIF (pH 6.8). These four compounds were further
selected to study their stability in rat plasma.
2.3.2. Chemical (non-enzymatic) hydrolysis in different buffers at
pH 1.2, 4.0 and 6.8
The % hydrolyzed after incubation of compounds (4a2, 4a3,
4d1, 4d2, 4i2, 4k and 5d) for 24 h in buffer solutions at pH 1.2,
4.0 and 6.8 was determined and the values are shown in Table 2.
In HCl buffer at pH 1.2, compounds 4i2, 4k and 5d were
degraded to the extent of 60.61, 23.6 and 49.2% respectively. It
was also observed that 4i2, 4k and 5d were unstable in phosphate
buffer pH 4.0 and degree of hydrolysis was 54.9, 24.1 and 47.6
respectively.
Table 2
Stability of selected bergenin prodrugs: chemical (non-enzymatic) and enzymatic hydrolysis
Compound
% remaining after 24 h at 37 °C
HCl buffer pH 1.2 Phosphate buffer pH 4.0
% remaining after 4 h at 37 °C
PBS pH 6.8
SGF (pH 1.2)
111.045 3.60
99.093 3.56
92.321 19.86
108.698 7.01
105.541 3.94
91.567 7.64
86.456 5.17
68.845 9.23
SIF (pH 6.8)
Rat plasma
82.584 6.27
4.103 0.48
ND
1
99.734 10.21
109.05 2.60
111.635 2.23
99.95 0.83
102.431 4.37
118.98 3.72
122.03 13.44
103.7 7.02
95.041 0.96
45.02 6.27
75.99 7.84
52.414 2.35
53.768 3.46
93.339 1.96
94.584 15.96
93.251 5.18
98.13 3.67
51.775 3.75
97.266 5.78
46.60 12.297
100.96 14.16
100.123 0.34
41.834 4.024
86.083 7.80
93.497 2.42
59.553 6.68
87.484 4.35
90.529 5.04
4a2
4a3
4d1
4d2
4i2
4k
5d
96.771 6.49
92.951 13.34
ND
98.356 0.58
40.39 7.70
76.362 0.37
50.862 7.83
86.5 11.43
ND

2.3.4. Stability in rat plasma and esterase from porcine liver
(PLE)
Aesar), pancreatin, porcine pancreas (Sigma-Aldrich), potassium
chloride (Ranbaxy laboratories Ltd.), and sodium hydroxide
(Qualigens Fine Chemicals) were used for preparation of buffers
of different pH and biorelevant media for the study. HPLC grade
methanol and acetonitrile (SD Fine Chemicals, Mumbai) was
used throughout the study. n-Octanol (Sigma Aldrich) was used
for partition and distribution coefficient experiments.
In vitro enzymatic hydrolysis of 4a2, 4d1, 4d2 and 4k was
studied in rat plasma and in esterase from porcine liver at 37 °C.
Compounds 4d1, 4d2 and 4k were stable to hydrolysis in rat
plasma. The reason might be prodrugs (4d1, 4d2 and 4k) prepared
using substituted benzoyl chlorides as promoieties are less prone
to degradation in comparison to prodrugs synthesized using acyl
chloride (4a2). The hydrolysis rate of 4a2 in rat plasma and
esterase was faster than the corresponding rate in HCl buffer (pH
1.2), phosphate buffer (pH 4.0), PBS (pH 7.4), SGF (pH 1.2) and
SIF (pH 6.8). This indicated that the hydrolysis of 4a2 is
facilitated by enzymes and it was completely converted to parent
compound bergenin in rat plasma and in esterase from porcine
liver within 4 h (Table 2). The time dependent hydrolysis of 4a2
in rat plasma and in esterase enzyme showed that 69% and 85%
of 4a2, respectively, was converted immediately to bergenin upon
mixing.
HPLC (Shimadzu, LC-6AD), reversed-phase C18 column
(NeoSphere, 5 µm, 250 mm × 4.6 mm), reversed-phase C8
column (Discovery HS, 5 µm, 250 mm × 4.6 mm), vortex (IKA
vortex Genius 3), microplate shaker (Grant-bio PMS-1000
Microplate Shaker, Digital), pH meter (Thermo electron co-
operation orion 20A+), microcentrifuge 5430R (Eppendorf),
sonicator and micropipettes were used for the study.
1H, 13C and DEPT NMR spectra were recorded on Brucker-
Avance DPX FT-NMR 500 and 400 MHz instruments. 13C NMR
spectra were recorded at 125 MHz or 100 MHz. ESI-MS spectra
were recorded on Agilent 1100 LC-Q-TOF machine. IR spectra
and melting points were recorded on Perkin-Elmer IR
spectrophotometer and digital melting point apparatus,
respectively.
Briefly, the compound 4a2 was found to have ideal properties
that are required for any prodrug. Bergenin, being highly polar
and unstable at intestinal pH, may be limiting factor for its
absorption. Optimal lipophilicity is required for efficient
absorption because most of the drugs are absorbed by passive
diffusion. Thus, the prodrug 4a2 with improved lipophilicity in
comparison with bergenin, may promote its gastrointestinal
absorption. Furthermore, the compound was very stable to
chemical hydrolysis in buffer solutions of different pH and in
SGF (pH 1.2) and SIF (pH 6.8). However, it was completely
converted to parent compound i.e. bergenin in rat plasma and in
presence of esterase from porcine liver in 15 min.
4.2. Isolation and characterization of bergenin (1)
The air-dried and powdered plant material of Bergenia ciliata
(1.5 kg) was extracted with methanol using maceration method.
Evaporation of the solvent produced 300 g of methanolic extract.
MeOH extract was loaded on the silica gel (# 100-200) column
chromatography and column was eluted with EtOAc: hexane
followed by MeOH: CHCl3, which resulted in isolation of
bergenin (1, 12 g). White crystalline powder; HPLC purity:
1
98.8% (tR = 5.68 min); m.p. 237-240 °C; H NMR (400 MHz,
3. Conclusion
DMSO-d6): δ 9.74 (s, 1H), 8.39 (s, 1H), 6.92 (s, 1H), 5.60 (d, J
=4 Hz, 1H), 5.39 (d, J= 4 Hz, 1H), 4.92 (d, J= 12 Hz, 1H), 3.95
(t, J= 8 Hz, 2H), 3.80 (d, J= 16 Hz, 1H), 3.71 (s, 3H), 3.61-3.48
(m, 4H); 13C NMR (125 MHz, DMSO-d6): δ 165.25, 152.82,
149.92, 142.41, 119.92, 117.79, 111.29, 83.59, 81.62, 75.51,
73.92, 72.72, 62.94, 61.67; IR (CHCl3) νmax 3585, 3370, 2922,
2851, 2346, 1653, 1422, 1154, 1021 cm-1; ESI-MS: m/z 329.00
[M+H]+; HRMS: m/z 329.0865 calcd for C14H16O9+H+
(329.0867).
The stability of bergenin was improved successfully via
prodrug approach. In total, 19 ester and ether prodrugs of
bergenin were synthesized and investigated for their stability at
different pH, log P and log D values. The data obtained in this
study demonstrated that ester prodrugs of bergenin possess better
lipophilicity, in comparison to bergenin which was in accordance
with the criteria that is required in drug discovery. Log P and log
D of all ester and ether prodrugs was found to be in the range of
0-3 which is as per the criteria required for the compounds in
drug discovery. The best identified prodrug 4a2 was stable to
hydrolysis under different pH conditions. This was also substrate
for the enzymes in plasma and other esterases leading to its
conversion to the parent compound, bergenin. The selection of
appropriate ester moiety was essential to control the conversion
of ester to free bergenin in the presence of esterase enzymes.
Preventing hydrolysis of ester prodrug in SGF (pH 1.2) and SIF
(pH 6.8) was necessary to retain the molecule sufficiently
lipophilic and in stable form to achieve intestinal absorption.
Thus, stable and lipophilic prodrugs of compounds containing
hydroxyl group may have potential to improve the transport of
the prodrugs.
4.3. Synthesis and purification of ester (4a˗k) and ether (5a-d)
prodrugs of bergenin
Bergenin (328 mg, 1.0 mM, 1 Eq) was dissolved in pyridine
and the mixture was stirred for 10 min in an ice bath. To this
solution, the respective acid chloride (0.5 Eq/OH) was added
drop-wise under stirring. The reaction mixture was stirred at RT
for 3 h and quenched by addition of cold water. The mixture was
washed 3 times with saturated solution of NaHCO3 followed by
washing 3 times with brine. The resulting mixture was
partitioned with ethyl acetate. The EtOAc layer was concentrated
over vacuo rotavapor and the crude products were purified over
silica gel column chromatography using EtOAc: Hexane as
mobile phase to get ester products 4a-k (Figure 2a).
4. Experimental
Bergenin (328 mg, 1.0 mM, 1 Eq) was dissolved in DMF. The
resulting mixture was stirred for 10 min at RT. To this solution,
the respective alkyl halide (1 Eq) was added drop-wise under
stirring and the reaction mixture was further stirred at RT for 3-4
h. The reaction was quenched by addition of cold water and
washed 3 times with NaHCO3 followed by brine, respectively.
The resulting mixture was partitioned with ethyl acetate and this
4.1. General
Sodium dihydrogen phosphate, potassium phosphate
monobasic, sodium acetate, boric acid, disodium hydrogen
phosphate (Alfa Aesar), sodium chloride (Loba Chemie Pvt.
Ltd.), pepsin, porcine stomach (Sigma-Aldrich), hydrochloric
acid (SD Fine Chemicals), monobasic potassium phosphate (Alfa

layer was concentrated to get desired ether prodrugs 5a-d (Figure
2b).
(m, 1H), 5.11-5.01 (m, 2H), 4.71 (m, 1H), 4.15-4.11 (m, 1H),
3.93(s, 3H), 3.46 (d, J= 12 Hz, 1H); 13C NMR (125 MHz,
CDCl3): δ 167.00, 166.97, 166.36, 163.15, 135.66, 134.91,
134.80, 134.55, 131.87, 131.66, 131.39, 131.23, 131.16, 130.89,
130.37, 130.26, 129.98, 129.82, 129.78, 78.71, 77.96, 74.98,
73.95, 70.03, 63.34, 62.88; IR (CHCl3) νmax 3449, 3065, 2925,
2853, 1738, 1602, 1584, 1486, 1451, 1428, 13670, 1328, 1314,
1274, 1244, 1178, 1109, 1067, 1025, 1003 cm-1. ESI-MS: m/z
663.70 [M+Na]+; HRMS: m/z 641.4215 calcd for C35H28O12+H+
(641.1654).
HPLC purity of compounds was carried out using RP-C18
column (NeoSphere, 250 mm × 4.6 mm, 5 µm). Mobile phase
consisted of ACN: water (15:85 v/v) at flow rate of 1.0 mL/min
(pump, Shimadzu LC-6AD). The column oven (CTO-10ASVP)
temperature was 37 °C and the injection volume (SIL-20A HT
Prominence autosampler) was 15 µL. The analysis was
performed using diode array detector (SPD-M20A, Prominence,
Shimadzu).
4.3.1. (8-Acetoxy-3,4,10-trihydroxy-9-methoxy-6-oxo-
2,3,4,4a,6,10b-hexahydropyrano[3,2-c]isochromen-2-yl)methyl
4.3.5. (8-((2,6-Difluorobenzoyl)oxy)-3,4,10-trihydroxy-9-
methoxy-6-oxo-2,3,4,4a,6,10b-hexahydropyrano[3,2-
acetate (4a1): Pale yellow powder; HPLC purity: 99.6% (tR
=
c]isochromen-2-yl)methyl 2,6-difluorobenzoate (4c): Pale white
powder; HPLC purity: 97.13% (tR = 2.75 min); m.p. 182-183 °C;
1H NMR (400 MHz, CDCl3): δ 8.10 (s, 1H), 7.50-7.46 (m, 1H),
7.37-7.33 (m, 1H), 7.01 (t, J= 8 Hz, 2H), 6.89(t, J= 8 Hz, 2H),
5.08-5.00 (m, 2H), 4.57-4.53 (m, 1H), 4.24-4.10 (m, 4H), 3.94 (s,
3H), 3.83-2.78 (m, 2H); 19F NMR (376 MHz, CDCl3): δ -108.60
(s, 2F), -109.27 (s, 2F); 13C NMR (125 MHz, acetone-d6): δ
161.07 (d, 1JCF = 141 Hz), 161.83, 161.78, 161.43, 159.79 (d, J =
1JCF =123 Hz), 159.74, 159.45, 159.40, 148.86, 144.87, 143.68,
134.82 (d, JCF = 20 Hz, 1C), 134.74, 134.0 (d, JCF = 21.4 Hz),
133.91, 122.77, 118.86, 115.96, 112.61, 112.41, 112.21, 79.55,
79.15, 74.34, 73.18, 70.25, 64.47, 59.91; IR (CHCl3) νmax 3396,
3022, 2925, 2852, 2346, 1737, 1626, 1589, 1504, 1471, 1333,
1290, 1252, 1235, 1096, 1057, 1015 cm-1. ESI-MS: m/z 609.50
[M+H]+; HRMS: m/z 609.0991 calcd for C28H20F4O11+H+
(609.1015).
2.35 min.); m.p. 220-223 °C; 1H NMR (400 MHz, DMSO-d6): δ
7.65 (s, 1H), 5.66 (d, J= 4 Hz, 1H), 5.38 (d, J= 4 Hz, 1H), 4.74
(s, 1H, OH), 4.06 (t, J= 8 Hz, 2H), 3.82 (s, 3H), 3.67-3.64 (m,
2H), 3.44-3.33 (m, 2H), 3.15-3.14 (m, 1H), 2.39 (s, 3H), 2.34 (s,
3H); 13C NMR (125 MHz, DMSO-d6): δ 170.51, 170.03, 164.33,
151.09, 144.96, 142.52, 124.89, 120.85, 119.78, 84.11, 81.58,
75.65, 73.40, 72.02, 63.24, 63.04, 22.39, 22.26; IR (CHCl3) νmax
3385, 2922, 2851, 2346, 1738, 1729, 1608, 1653, 1485, 1458,
1372, 1329, 1196, 1096, 1024 cm-1. ESI-MS: m/z 413.3 [M+H]+,
847.8 [2M+Na]+; HRMS: m/z 413.1081 calcd for C18H20O11+H+
(413.1078).
2
2
4.3.2. 2-(Acetoxymethyl)-9-methoxy-6-oxo-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromene-3,4,8,10-tetrayl
tetraacetate (4a2): Dark yellow amorphous powder; HPLC
purity: 97.35% (tR = 5.97 min); m.p. 206-207 °C; 1H NMR (400
MHz, acetone-d6): δ 7.73 (s, 1H), 5.63 (t, J = 8 Hz, 1H), 5.15 (t,
8 Hz, 2H), 4.56 (t, J = 10 Hz, 1H), 4.37 (m, 1H), 4.22 (m, 1H),
4.10 (m, 1H), 3.92 (s, 3H), 2.38 (s, 6H), 2.08 (s, 3H), 2.06 (s,
6H); 13C NMR (125 MHz, acetone-d6): δ 169.93, 169.44, 169.28,
168.26, 167.58, 161.34, 149.99, 144.09, 141.45, 130.09, 123.50,
119.02, 76.65, 76.30, 72.15, 68.35, 61.97, 61.05, 19.85, 19.83,
19.76; IR (CHCl3) νmax 3422, 2925, 2853, 2346, 2111, 1741,
1651, 1484, 1430, 1370, 1328, 1217, 1098, 1034 cm-1. ESI-MS:
m/z 561.0 [M+Na]+; HRMS: m/z 539.1402 calcd for
C24H26O14+H+ (539.1395).
4.3.6. 2-(((4-(Tert-butyl)benzoyl)oxy)methyl)-9-methoxy-6-oxo-
2,3,4,4a,6,10b-hexahydropyrano[3,2-c]isochromene-3,4,8,10-
tetrayl tetrakis(4-(tert-butyl)benzoate) (4d1): White powder;
HPLC purity: 99.65% (tR = 3.52 min); m.p. 182-183 °C; 1H NMR
(400 MHz, CDCl3): δ 8.15 (d, J = 8 Hz, 2H), 8.05 (d, J = 8 Hz,
2H), 7.91(d, J = 8 Hz, 5H), 7.76 (d, J = 12 Hz, 2H), 7.56 (d, J =
12 Hz, 2H), 7.50 (d, J = 8 Hz, 2H), 7.46 (d, J = 8 Hz, 2H), 7.38
(d, J = 8 Hz, 2H), 7.32 (d, J = 8 Hz, 2H), 6.01 (t, J =12 Hz, 1H),
5.69 (m, 1H), 5.08 (m, 1H), 4.70 (t, J = 8 Hz, 1H), 4.08-3.97 (m,
1H), 3.92 (s, 3H), 3.27 (m, 1H), 1.37-1.26 (m, 45H); 13C NMR
(125 MHz, CDCl3): δ 171.46, 165.49, 165.35, 164.78, 161.78,
158.02, 157.48, 157.03, 156.53, 144.54, 130.33, 130.09, 129.89,
129.72, 129.57, 126.87, 126.51, 12627, 126.05, 125.86, 125.78,
125.56, 125.47, 125.35, 125.32, 125.27, 77.28, 76.71, 73.54,
72.34, 68.24, 61.78, 60.93, 35.27, 35.17, 35.09, 35.04, 35.03,
31.14, 31.07, 31.03, 31.00, 30.94; IR (CHCl3) νmax 2964, 2907,
2870, 2346, 1932, 1738, 1733, 1610, 1573, 1485, 1463, 1428,
1365, 1330, 1314, 1265, 1189, 1111, 1017 cm-1. ESI-MS: m/z
1129.40 [M+H]-; HRMS: m/z 1129.4041 calcd for C69H76O14+H-
(1129.5308).
4.3.3. 2-(Acetoxymethyl)-4-hydroxy-9-methoxy-6-oxo-
2,3,4,4a,6,10b-hexahydropyrano[3,2-c]isochromene-3,8,10-triyl
triacetate (4a3): Pale white gummy solid; HPLC purity: 94.59%
(tR = 6.32 min); m.p. 213-215 °C; H NMR (400 MHz, acetone-
1
d6): δ 7.35 (s, 1H), 5.45 (t, J= 12 Hz, 1H), 4.98-4.87 (m, 2H),
4.31 (t, J= 12 Hz, 1H), 4.20 (dd, J= 8 Hz, 12 Hz, 1H), 4.07 (dd, J
= 4 Hz, 8 Hz, 1H), 3.92-3.89 (m, 1H), 3.77 (s, 3H), 2.20 (s, 3H),
1.93 (s, 3H), 1.91 (s, 3H), 1.89 (s, 3H); 13C NMR (125 MHz,
acetone-d6): δ 171.32, 169.96, 169.31, 167.71, 161.93, 151.00,
145.66, 141.35, 123.49, 119.03, 115.72, 76.87, 76.24, 72.11,
68.51, 62.11, 60.37, 19.88, 19.86, 19.81, 19.78; IR (CHCl3) νmax
3585, 3385, 2922, 2851, 2308, 1742, 1609, 1491, 1463, 1370,
1222, 1095, 1037 cm-1. ESI-MS: m/z 495.1 [M-H]-; HRMS: m/z
497.1300 calcd for C22H24O13+H+ (497.1290).
4.3.7. 2-(((4-(Tert-butyl)-benzoyl)oxy)methyl)-3,4-dihydroxy-9-
methoxy-6-oxo-2,3,4,4a,6,10b-hexahydropyrano[3,2-
c]isochromene-8,10-diyl-bis(4-(tert-butyl)benzoate) (4d2): White
powder; HPLC purity: 99.69% (tR = 3.58 min); m.p. 182-183 °C;
1H NMR (400 MHz, CDCl3): δ 8.16 (d, J= 8 Hz, 2H), 8.03-7.99
(m, 2H), 7.94 (d, J= 8 Hz, 2H), 7.94-7.92 (m, 1H), 7.56 (d, J= 8
Hz, 2H), 7.50 (d, J= 8 Hz, 2H), 7.46 (d, J= 12 Hz, 2H), 5.59 (t,
J= 8 Hz, 1H), 4.97-4.95 (m, 1H), 4.57 (t, J= 12 Hz, 1H), 4.46-
4.40 (m, 1H), 3.97 (s, 3H), 3.85-3.70 (m, 1H), 3.55-3.53 (m, 1H),
3.29-3.14 (m, 1H), 1.38-1.33 (m, 27H); 13C NMR (125 MHz,
CDCl3): δ 166.48, 162.05, 162.00, 158.04, 157.27, 157.23,
157.00, 150.24, 144.43, 141.37, 130.34, 130.19, 130.02, 129.81,
4.3.4. 2-((Benzoyloxy)methyl)-3,4-dihydroxy-9-methoxy-6-oxo-
2,3,4,4a,6,10b-hexahydropyrano[3,2-c]isochromene-8,10-diyl
dibenzoate (4b): yellow powder; HPLC purity: 100.0% (tR = 3.68
1
min); m.p. 180-182 °C; H NMR (400 MHz, CDCl3): δ 8.22 (d,
J= 4 Hz, 2H), 7.96 (d, J= 8 Hz, 2H), 7.82 (d, J= 8 Hz, 1H), 7.70
(t, J= 8 Hz, 1H), 7.57-7.28 (m, 10H), 6.03 (t, J= 8 Hz, 1H), 5.70

129.71, 129.58, 126.73, 126.68, 126.39, 125.99, 125.78, 125.61,
125.43, 125.39, 123.37, 118.69, 79.02, 75.51, 73.31, 72.93,
68.69, 61.76, 61.39, 60.40, 35.28, 35.27, 35.15, 35.13, 35.11,
31.12, 31.08, 31.00; IR (CHCl3) νmax 3455, 2964, 2870, 1743,
1609, 1573, 1485, 1462, 1427, 1410, 1365, 1330, 1314, 1261,
1188, 1110, 1063, 1016 cm-1. ESI-MS: m/z 807.3 [M-H]-;
HRMS: m/z 809.3499 calcd for C47H52O12+H+ (809.3532).
methylbenzoate) (4h): Cream amorphous powder; HPLC purity:
99.90% (tR = 19.48 min); m.p. 148-150 °C; 1H NMR (500 MHz,
acetone-d6): δ 7.90-7.82 (m, 4H), 7.65-7.60 (m, 4H), 7.51-7.49
(m, 2H), 7.43-7.41 (m, 1H), 7.36-7.32 (m, 1H), 7.27-7.08 (m,
5H), 6.01 (t, J= 10 Hz, 1H), 5.67-5.62 (m, 1H), 5.22-5.21 (m,
1H), 4.86-4.82 (m, 1H), 4.29-4.13 (m, 1H), 3.95-3.88 (m, 1H),
3.80 (s, 3H), 3.46-3.34 (m, 1H), 2.29 (s, 3H), 2.21 (s, 3H), 2.16
(s, 3H), 2.12 (s, 3H); 13C NMR (125 MHz, acetone-d6): δ 165.35,
165.22, 164.88, 164.20, 161.31, 138.31, 138.25, 134.13, 134.11,
133.72, 130.58, 130.54, 130.03, 129.97,128.88, 128.41, 128.29,
127.37, 126.78, 126.69, 77.09, 76.00, 72.85, 69.13, 61.81, 61.44,
20.40, 20.37, 20.30, 20.24; IR (CHCl3) νmax 3385, 2922, 2852,
2346, 1738, 1654, 1608, 1590, 1485, 1458, 1373, 1329, 1270,
1188, 1105, 1080, 1016 cm-1. ESI-MS: m/z 801.25 [M+H]+;
HRMS: m/z 801.2561 calcd for C46H40O13+H+ (801.2542).
4.3.8. 9-Methoxy-2-(((3-methylbutanoyl)oxy)methyl)-6-oxo-
2,3,4,4a,6,10b-hexahydropyrano[3,2-c]isochromene-3,4,8,10-
tetrayl tetrakis(3-methylbutanoate) (4e): Creamish powder;
1
HPLC purity: 98.88% (tR = 17.85 min); m.p. 182-183 °C; H
NMR (500 MHz, CDCl3): δ 7.70 (s, 1H), 5.56 (t, J= 10 Hz, 1H),
5.18 (t, J= 10 Hz, 1H), 4.79 (d, J= 15 Hz, 1H), 4.32-4.27 (m,
2H), 4.04 (dd, J= 5 Hz, 15 Hz, 1H), 3.84 (s, 3H), 3.75 (d, J= 5
Hz, 1H), 2.46-2.44 (m, 3H), 2.70-1.98 (m, 12H), 1.04-1.02 (m,
12H), 0.95-0.84 (m, 18H); 13C NMR (125 MHz, CDCl3): δ
172.56, 171.92, 171.45, 170.44, 169.81, 161.57, 150.11, 144.28,
141.57, 129.55, 123.99, 118.79, 77.33, 77.08, 76.92, 7682, 76.78,
72.89, 71.62, 67.60, 61.41, 61.35, 43.06, 43.00, 42.91, 42.67,
31.43, 30.17, 29.68, 25.75, 25.64, 25.52, 25.36, 22.41, 22.35,
22.33, 22.30; IR (CHCl3) νmax 2960, 2873, 1746, 1609, 1484,
1467, 1426, 1370, 1327, 1293, 1246, 1183, 1165, 1093, 1018 cm-
1. ESI-MS: m/z 663.3 [M-isovaleryl]-; HRMS: m/z 665.3162
calcd for C39H56O14-C5H8O+ (665.3168).
4.3.12. (3,4,8,10-Tetrahydroxy-9-methoxy-6-oxo-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromen-2-yl)methyl 4-nitrobenzoate
(4i1): Dark yellow amorphous powder; HPLC purity: 94.71% (tR
= 21.47 min); m.p. 202-205 °C; 1H NMR (400 MHz, acetone-d6):
δ 7.80 (s, 1H), 7.80 (s, 2H), 7.59 (s, 2H), 5.72 (t, J= 12 Hz, 1H),
5.25 (d, J = 12 Hz, 1H), 5.14 (t, J= 12 Hz, 1H), 4.70 (t, J= 8 Hz,
1H), 4.51-4.50 (m, 1H), 4.39-4.26 (m, 2H), 4.03 (s, 3H); 13C
NMR (125 MHz, acetone-d6): δ 170.30, 164.02, 151.34, 144.57,
138.30, 137.90, 137.07, 133.51, 133.07, 129.62, 129.09, 124.74,
77.82, 72.78, 72.57, 69.91, 64.97, 62.99; IR (CHCl3) νmax 3585,
3473, 3081, 2926, 2854, 1748, 1673, 1609, 1578, 1550, 1484,
1453, 1426, 1371, 1325, 1266, 1240, 1193, 1156, 1102, 1039 cm-
1. ESI-MS: m/z 495.19 [M+H]+; HRMS: m/z 495.1271 calcd for
C21H19NO20+ K+ (495.1246).
4.3.9. 2-((Hexanoyloxy)methyl)-9-methoxy-6-oxo-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromene-3,4,8,10-tetrayl
tetrahexanoate (4f): Yellow liquid; HPLC purity: 97.99% (tR =
1
4.84 min); H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 5.53 (t,
J=12 Hz, 1H), 5.20 (t, J= 12 Hz, 1H), 4.84 (d, J= 12 Hz, 1H),
4.37-4.26 (m, 2H), 4.15-4.11 (m, 1H), 3.88 (s, 3H), 3.81-3.79 (m,
1H), 2.62 (t, J= 4 Hz, 4H), 2.37-2.27 (m, 6H), 1.79-1.71 (m,
4H), 1.62-1.58 (m, 6H), 1.40-1.29 (m, 20H), 0.95-0.86 (m, 15H);
13C NMR (125 MHz, CDCl3): δ 173.27, 172.67, 172.22, 171.15,
170.54, 161.64, 150.02, 144.28, 141.52, 129.50, 123.91, 118.71,
76.77, 76.71, 71.86, 71.78, 67.74, 61.42, 33.96, 33.89, 33.87,
33.85, 33.70, 31.17, 31.14, 31.12, 24.47, 24.38, 24.31, 22.35,
22.26, 13.84, 13.81; IR (CHCl3) νmax 2956, 2933, 2863, 2873,
1748, 1755, 1610, 1577, 1485, 1462, 1426, 1378, 1327, 1294,
1273, 1243, 1218, 1166, 1097, 1017 cm-1. ESI-MS: m/z 842.00
[M+Na]+; HRMS: m/z 819.4540 calcd for C44H66O14+H+
(819.4525).
4.3.13.
nitrobenzoyl)oxy)methyl)-6-oxo-2,3,4,4a,6,10b-
3,4,10-Trihydroxy-9-methoxy-2-(((4-
hexahydropyrano[3,2-c]isochromen-8-yl 4-nitrobenzoate (4i2):
Creamish amorphous powder; HPLC purity: 91.16% (tR = 2.76
1
min); m.p. 191-193 °C; H NMR (500 MHz, acetone-d6): δ 8.92
(t, J= 5 Hz, 1H), 8.83 (t, J= 5 Hz, 1H), 8.59-8.58 (m, 2H), 8.52-
8.49 (m, 2H), 7.97 (t, J= 10 Hz, 1H), 7.89 (t, J= 10 Hz, 1H), 7.49
(s, 1H), 5.34 (d, J= 10 Hz, 1H), 5.16 (dd, J= 5 Hz, 20 Hz, 2H),
4.64-4.60 (m, 1H), 4.34-4.26 (m, 2H), 4.04-3.90 (m, 1H), 3.91 (s,
3H); 13C NMR (125 MHz, acetone-d6): δ 164.22, 162.55, 162.13,
148.68, 148.57, 148.48, 144.89, 144.07, 135.77,135.33, 131.66,
130.70, 130.42, 128.29, 127.72, 124.55, 124.01, 122.71, 118.73,
116.34, 79.51, 79.18, 74.41, 73.19, 70.61, 64.64, 60.04; IR
(CHCl3) νmax 3585, 3374, 3092, 2924, 2854, 2346, 1738, 1731,
1653, 1616, 1586, 1533, 1481, 1455, 1351, 1292, 1249, 1114,
1092, 1016 cm-1. ESI-MS: m/z 627.45 [M+H]+; HRMS: m/z
627.1099 calcd for C28H22NO15+H+ (627.1093).
4.3.10. 3,4,10-Trihydroxy-9-methoxy-6-oxo-2-(((2,4,6-
trinitrobenzoyl)oxy)methyl)-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromen-8-yl 2,4,6-trinitrobenzoate
(4g): yellowish powder; HPLC purity: 99.98% (tR = 2.62 min);
1
m.p. 172-173 °C; H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H),
7.52-7.44 (m, 4H), 5.51 (d, J= 12 Hz, 1H), 4.91 (s, 1H), 4.69-
4.66 (m, 1H), 4.02-3.94 (m, 5H), 3.71-3.57 (m, 3H); 13C NMR
(125 MHz, CDCl3): δ 164.29, 162.04, 161.96, 153.17, 153.09,
148.90, 143.32, 143.10, 123.41, 120.66, 117.78, 117.31, 107.70,
107.65, 75.45, 69.62, 68.19, 65.72, 62.24, 61.10, 60.98; IR
(CHCl3) νmax 3384, 3015, 2926, 2851, 2346, 1738, 1653, 1590,
1503, 1486, 1463, 1417, 1334, 1233, 1206, 1170, 1128, 1105,
1014 cm-1. ESI-MS: m/z 806.50 [M]+; HRMS: m/z 806.1210
calcd for C28H18N6O23+ (806.0423).
4.3.14. 9-Methoxy-2-((octanoyloxy)methyl)-6-oxo-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromene-3,4,8,10-tetrayl
tetraoctanoate (4j): Yellow liquid; HPLC purity: 98.18% (tR
=
23.19 min); 1H NMR (400 MHz, acetone-d6): δ 7.23 (s, 1H), 5.66
(t, J=12 Hz, 1H), 5.23-5.14 (m, 2H), 4.57 (t, J= 12 Hz, 1H), 4.37-
4.33 (m, 1H), 4.23-4.19 (m, 1H), 4.12-4.08 (m, 1H), 3.90 (s, 3H),
2.73 (t, J= 8 Hz, 4H), 2.42-2.28 (m, 6H), 1.80-1.72 (m, 4H),
1.67-1.59 (m, 6H), 1.38-1.29 (m, 40H), 0.93-0.87 (m, 15H); 13C
NMR (100 MHz, acetone-d6): δ 173.37, 172.90, 172.60, 171.80,
171.14, 162.12, 150.94, 145.16, 142.62, 130.95, 124.35, 120.00,
77.80, 77.39, 73.20, 72.83, 69.01, 62.55, 61.95, 34.48, 32.45,
32.42, 29.74, 29.71, 29.65, 25.56, 25.49, 25.48, 25.47, 23.28,
23.25, 14.32, 14.30; IR (CHCl3) νmax 3585, 3473, 2956, 2928,
2857, 1748, 1609, 1484, 1462, 1426, 1378, 1326, 1264, 1217,
4.3.11. 4-Hydroxy-9-methoxy-3-((2-methylbenzoyl)oxy)-2-(((3-
methylbenzoyl)oxy)methyl)-6-oxo-2,3,4,4a,6,10b-
hexahydropyrano[3,2-c]isochromene-8,10-diyl
bis(3-

1194, 1161, 1100, 1018 cm-1. ESI-MS: m/z 959.45 [M+H]+;
HRMS: m/z 976.6373 calcd for C54H86O14+NH4+ (976.6390).
1237, 1193, 1177, 1130, 1108, 1031 cm-1. ESI-MS: m/z 469.6
[M+H]+; HRMS: m/z 469.2412 calcd for C24H36O9+H+
(469.2432).
4.3.15.
trichlorobenzoyl)oxy)methyl)-2,3,4,4a,6,10b-
3,4,10-Trihydroxy-9-methoxy-6-oxo-2-(((2,4,6-
4.3.19. 8-(Benzyloxy)-2-((benzyloxy)methyl)-3,4,10-trihydroxy-9-
methoxy-2,3,4,4a-tetrahydropyrano[3,2-c]isochromen-6(10bH)-
one (5d): White amorphous powder; HPLC purity: 99.86% (tR =
2.84 min); m.p. 151-153 °C; 1H NMR (400 MHz,
CD3OD+CDCl3): δ 7.57 (s, 1H), 7.49-7.25 (m, 10H), 5.23 (t, J=
12 Hz, 2H), 5.06 (q, J=12 Hz, 16 Hz, 2H), 4.57 (d, J= 8 Hz, 1H),
3.94 (t, J= 12 Hz, 1H), 3.77 (s, 3H), 3.75-3.65 (m, 3H), 3.52 (t, J
= 12 Hz, 1H), 3.32-3.28 (m, 1H); 13C NMR (125 MHz, CD3OD):
δ 164.39, 152.67, 149.91, 149.06, 137.54, 136.42, 128.26,
128.01, 127.86, 127.76, 127.44, 118.95, 111.55, 80.98, 80.27,
75.67, 74.51, 71.85, 70.77, 69.71, 60.52, 60.36; IR (CHCl3) νmax
3330, 2922, 2851, 2354, 1715, 1654, 1594, 1483, 1447, 1426,
1330, 1246, 1133, 1104, 1030 cm-1. ESI-MS: m/z 509.10
[M+H]+; HRMS: m/z 509.1816 calcd for C26H24O9+H+
(509.1819).
hexahydropyrano[3,2-c]isochromen-8-yl 2,4,6-trichlorobenzoate
(4k): Pale yellow amorphous powder; HPLC purity: 99.74% (tR =
1
6.36 min); m.p. 165-167 °C; H NMR (400 MHz, acetone-d6): δ
8.31 (s, 1H), 7.63 (s, 2H), 7.54 (s, 2H), 5.23 (d, J= 12 Hz, 1H),
5.05-4.95 (m, 2H), 4.51 (q, J= 8 Hz, 16 Hz, 1H), 4.19-4.06 (m,
2H), 3.89-3.87 (m, 1H), 3.84 (s, 3H), 3.62-3.57 (m, 1H); 13C
NMR (100 MHz, acetone-d6): δ 165.06, 163.41, 162.80, 150.32,
146.60, 144.57, 138.36, 137.81, 134.01, 133.66, 133.55, 132.57,
129.98, 129.71, 124.40, 120.29, 117.08, 80.96, 80.41, 75.74,
74.60, 71.54, 66.45, 61.47; IR (CHCl3) νmax 3391, 2926, 2346,
2100, 1736, 1653, 1579, 1550, 1507, 1457, 1370, 1332, 1273,
1247, 1190, 1157, 1106, 1037, 1004 cm-1. MS: m/z 744.04
[M+H]+; HRMS: m/z 759.9294 calcd for C28H18O11Cl6+H+
(759.9290).
4.3.16.
(propoxymethyl)- 2,3,4,4a-tetrahydropyrano[3,2-c]isochromen-
6(10bH)-one (5a): Cream powder; HPLC purity: 99.9% (tR
3,4,10-Trihydroxy-9-methoxy-8-propoxy-2-
4.4. Determination of log P and log D
Log P and log D was determined as per the recently published
protocol.37 For log P and log D experiments, organic phase
consisted of n-octanol saturated with water and PBS (pH 7.4),
respectively. The aqueous phase consisted of water and PBS (pH
7.4) saturated with n-octanol. Briefly, in 1.5 mL eppendorf tube,
200 µL of stock solution of compound (1000 µg/mL) was added
and volume was made up to 1 mL with 300 µL of presaturated n-
octanol and 500 µL of presaturated aqueous phase (water / PBS
pH 7.4). The eppendorf tubes were shaken overnight at 500 rpm
and centrifuged at 16000 RCF (G force) for 20 min so as to
separate aqueous and organic layer. The concentration of
compound in organic and aqueous phase was determined by
HPLC method. The HPLC analysis employed reversed-phase
C18 column (NeoSphere, 5 µm, 250 mm × 4.6 mm, Hexon
Laboratories Pvt. Ltd., India) using photodiode detector
(SPD˗M20A, Prominence, Shimadzu). Isocratic mobile phase
consisting of acetonitrile and water in various proportions was
used for the analysis. The details of HPLC method of all
compounds is given in Table S1. Injection volume was 10.0 µL
(SIL˗20A HT Prominence auto-sampler) (pump, LC-6AD
Shimadzu liquid chromatography). The column oven temperature
=
1
2.73 min); m.p. 162-163 °C; H NMR (500 MHz, CD3OD): δ
7.42 (s, 1H), 4.86 (t, J= 20 Hz, 1H), 4.05-3.90 (m, 6H), 3.92 (s,
3H), 3.85-3.81 (m, 2H), 3.61-3.57 (m, 1H), 3.52-3.49 (m, 1H),
1.90-1.77 (m, 4H), 1.12-1.05 (m, 6H); 13C NMR (125 MHz,
CD3OD): δ 164.61, 153.10, 150.42, 148.69, 126.29, 119.00,
110.18, 81.07, 80.32, 75.77, 74.51, 74.84, 70.33, 69.95, 60.91,
60.22, 23.09, 22.18, 9.53, 9.45; IR (CHCl3) νmax 3355, 2964,
1937, 2878, 1733, 1711, 1593, 1486, 1473, 1453, 1428, 1387,
1358, 1331, 1178, 1129, 1112, 1030 cm-1. ESI-MS: m/z 411.1
[M-H]-; HRMS: m/z 413.1807 calcd for C20H28O9+H+
(413.1806).
4.3.17. 8-Butoxy-2-(butoxymethyl)-3,4,10-trihydroxy-9-
methoxy-2,3,4,4a-tetrahydropyrano[3,2-c]isochromen-6(10bH)-
one (5b): Pale white needle shaped crystals; HPLC purity:
1
99.88% (tR = 3.13 min); m.p. 155-158 °C; H NMR (400 MHz,
CD3OD): δ 7.18 (s, 1H), 7.57 (t, J= 4 Hz, 1H), 3.86-3.70 (m, 6H),
3.67 (s, 3H), 3.36-3.57 (m, 2H), 3.38 (t, J= 12 Hz, 1H), 3.29-3.25
(m, 1H), 1.62-1.49 (m, 4H), 1.36-1.25 (m, 4H), 0.78-0.75 (m,
6H); 13C NMR (125 MHz, CD3OD): δ 164.59, 153.09, 150.41,
148.67, 126.29, 119.00, 110.14, 81.06, 80.29, 74.50, 73.94,
71.83, 69.90, 68.51, 60.88, 60.21, 32.05, 30.97, 18.99, 18.86,
12.95, 12.79; IR (CHCl3) νmax 3384, 2958, 2935, 2873, 2346,
1715, 1595, 1486, 1454, 1427, 1384, 1361, 1332, 1306, 1242,
1190, 1179, 1130, 1108, 1035 cm-1. ESI-MS: m/z 441.5 [M+H]+;
HRMS: m/z 441.2119 calcd for C22H32O9+H+ (441.2119).
˗10ASVP). Each
was kept at 37 ºC (column oven, CTO
determination was performed in triplicate.
4.5. In-vitro stability studies
4.5.1. Chemical (non-enzymatic) hydrolysis in different buffers at
pH 1.2, 4.0, 6.8 and 7.4
To test stability of compound, different buffers were prepared
as per the protocol given in Indian Pharmacopoeia 2007.38, 39 The
buffers consisted of hydrochloric acid buffer pH 1.2, phosphate
buffer pH 4.0 and phosphate buffered saline (pH 6.8 and pH
7.4).34, 40-42 A known concentration of compound in DMSO (120
µg/mL) was incubated in buffers of pH 1.2, 4.0, 6.8 and 7.4 at 37
°C for 24 h. From the incubated solutions, aliquot of 50 µL was
removed at two time points i.e. at zero time and after 24 h. These
aliquots were quenched by adding 200 µL of ice-cold
acetonitrile. The resulting solution was centrifuged at 17000 g for
10 min at 4 °C. The supernatant was analyzed, using HPLC
conditions as mentioned in Table 3 and Table S2, to determine
AUC (area under the curve) of the corresponding prodrug ester
4.3.18. 3,4,10-Trihydroxy-9-methoxy-8-(pentyloxy)-2-
((pentyloxy)methyl)-2,3,4,4a-tetrahydropyrano[3,2-
c]isochromen-6(10bH)-one (5c): yellow powder; HPLC purity:
1
96.48% (tR = 3.84 min); m.p. 170-172 °C; H NMR (400 MHz,
CD3OD): δ 7.28 (s, 1H), 4.72-4.68 (m, 1H), 3.99-3.82 (m , 6H),
3.80 (s, 3H), 3.74-3.70 (m, 2H), 3.51 (t, J= 12 Hz, 1H), 3.41-3.37
(m, 1H), 1.77-1.65 (m, 4H), 1.42-1.28 (m, 8H), 0.88 (t, J= 8 Hz,
6H); 13C NMR (125 MHz, CD3OD): δ 166.61, 153.10, 150.42,
148.69, 126.28, 119.02, 110.17, 81.07, 80.32, 74.50, 74.21,
71.85, 69.89, 68.81, 60.87, 60.21, 29.63, 28.56, 28.02, 27.92,
22.24, 22.09, 13.07, 12.99; IR (CHCl3) νmax 3390, 2956, 2933,
2872, 1728, 1594, 1486, 1469, 1454, 1427, 1381, 1361, 1333,

Table 3
HPLC method of bergenin (1) and selected bergenin prodrugs
Compound Details of method
Retention time (min)
Bergenin
Compound
1
Isocratic, mobile phase 80:20- ACN : 0.1% formic acid, flow rate 1.0 mL/min, RP-C8 column
2.857
3.502
3.474
2.857
-
(Discovery HS, 5 µm, 250 mm × 4.6 mm), Detection wavelength- 280 nm, run time - 10 min
Isocratic, mobile phase 70:30- ACN : 0.1% formic acid, flow rate 0.8 mL/min, RP-C8 column
(Discovery HS, 5 µm, 250 mm × 4.6 mm), Detection wavelength- 254 nm, run time - 10 min
Isocratic, mobile phase 90:10- ACN : water, flow rate 0.8 mL/min, RP-C8 column (Discovery
HS, 5 µm, 250 mm × 4.6 mm), Detection wavelength- 254 nm, run time - 10 min
4a2
4a3
5.292
4.394
4d1
4d2
4i2
4k
5d
4.681
4.645
3.547
6.618
3.410
Isocratic, mobile phase 80:20- ACN : 0.1% formic acid, flow rate 1.0 mL/min, RP-C8 column
(Discovery HS, 5 µm, 250 mm × 4.6 mm), Detection wavelength- 240 nm, run time - 10 min
and its metabolite (parent compound i.e. bergenin), if any. The
experiment was performed in triplicate for each compound.
Supplementary Material
Supplementary data associated with this article can be found, in
the online version.
4.5.2. Stability in biorelevant media, SGF (pH 1.2) and SIF (pH
6.8)
References
Biorelevant media viz. SGF (pH 1.2) and SIF (pH 6.8) were
prepared as per the protocol given in USP 2000 43. The stability
of compound in biorelevant media viz. SGF and SIF was
determined as per the protocol mentioned in section 4.5.1. The
solution of compound was incubated in SGF and SIF at 37 °C
and aliquot was removed at 0 and 24 h. The HPLC analysis was
performed to find out degradation/ hydrolysis of ester prodrug to
bergenin, if any, using HPLC method as described in Table 3.
Each analysis was performed in triplicate.
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