Design and total synthesis of (-)-codonopsinine, (-)-codonopsine and codonopsinine analogues by O-(2-oxopyrrolidin-5-yl)trichloroacetimidate as amidoalkylating agent with improved antimicrobial activity via solid lipid nanoparticle formulations

Article abstract of DOI:10.1016/j.bmc.2019.02.021

A general strategy towards total synthesis of (-)-codonopsinine, (-)-codonopsine and codonopsinine analogues has been developed from (D)-tartaric acid via the intermediate (3S,4R)-1-methyl-2-oxo-5-(2,2,2-trichloroacetamido)pyrrolidinediacetate (7). α-amidoalkylation studies of 7 with electron rich benzene derivative 8a-g as C-nucleophiles afforded (aryl derivatives) 9a-g. The target compounds 1, 2 and 13c-g were readily obtained from 10a-g via Grignard addition to the homochiral lactam which was produced by deoxygenation using Lewis-acid followed by deacetylation. The synthesized compounds were loaded onto solid lipid nanoparticle formulations (SLNs) prepared by hot emulsification-ultrasonication technique using Compritol as solid lipid and Pluronic f68 as surfactant. SLNs were fully evaluated and the permeation of synthesized compound from SLNs was assayed against non-formulated compounds through dialysis membranes using Franz cell. The data indicated good physical characteristics of the prepared SLNs, sustaining of release profiles and significant improvement of permeation ability when compared to the non-formulated compounds. The antibacterial and antifungal activities of 1, 2 and 13c-g were determined by disc diffusion and microbroth dilution method to determine the minimum inhibitory concentrations (MIC) against seven microorganisms (Staphyloccus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii and Candida albicans). The most active compounds against the Gram positive S. aureus were 1, 13C, 13d, and 13g. Also, 13c, 13d, and 13e had antibacterial activity but not 13f against some Gram negative organisms (E. coli, and P. mirabilis). MIC concentrations against P. aeruginosa, and K. pneumoniae were ≥512 μg/ml, while that against A. baumannii was ≥128 μg/ml except for nanoformulae of 13e and 13f that were 16 and 64 μg/ml, respectively. No antifungal activity against Candida albicans was recorded for all compounds and their nanoformulae (MIC > 1024 μg/ml). SLNs were found to decrease the MIC values for some of the compounds with no effect on the antifungal activity. In conclusion, we demonstrated a novel, straight-forward and economical procedure for the total synthesis of (-)-codonopsinine 1, (-)-codonopsine 2 and codonopsinine analogues 13c-g from simple and commercially available starting materials; D-tartaric acid; with antimicrobial activities against Gram positive and Gram-negative organisms that were improved by SLNs formulations.

Full text of DOI:10.1016/j.bmc.2019.02.021

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and total synthesis of (-)-codonopsinine, (-)-codonopsine and
codonopsinine analogues by O-(2-oxopyrrolidin-5-yl)trichloroacetimidate as
amidoalkylating agent with improved antimicrobial activity via solid lipid
nanoparticle formulations
⁎
Ahmed O.H. El-Nezhawya,b, , Majed Alrobaianc, Ahmed Khamesc,d, Mohamed F. El-Badawye,f,
Sayed F. Abdelwahabe,g
a Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif 21974, Saudi Arabia
b Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki 12622, Cairo, Egypt
c Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Taif University, Taif 21974, Saudi Arabia
d Department of Pharmaceutics and Industrial Pharmacy, Beni-Suef University, Beni-Suef 62521, Egypt
e Division of Pharmaceutical Microbiology, Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Taif University, Taif 21974, Saudi Arabia
f Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology, Al-Motamayez District, P.O. Box 77, 6th of October City
12568, Egypt
g Department of Microbiology and Immunology, Faculty of Medicine, Minia University, Minia 61511, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A general strategy towards total synthesis of (-)-codonopsinine, (-)-codonopsine and codonopsinine analogues
has been developed from (D)-tartaric acid via the intermediate (3S,4R)-1-methyl-2-oxo-5-(2,2,2-tri-
chloroacetamido)pyrrolidinediacetate (7). α-amidoalkylation studies of 7 with electron rich benzene derivative
8a-g as C-nucleophiles afforded (aryl derivatives) 9a-g. The target compounds 1, 2 and 13c-g were readily
obtained from 10a-g via Grignard addition to the homochiral lactam which was produced by deoxygenation
using Lewis-acid followed by deacetylation. The synthesized compounds were loaded onto solid lipid nano-
particle formulations (SLNs) prepared by hot emulsification-ultrasonication technique using Compritol as solid
lipid and Pluronic f68 as surfactant. SLNs were fully evaluated and the permeation of synthesized compound
from SLNs was assayed against non-formulated compounds through dialysis membranes using Franz cell. The
data indicated good physical characteristics of the prepared SLNs, sustaining of release profiles and significant
improvement of permeation ability when compared to the non-formulated compounds. The antibacterial and
antifungal activities of 1, 2 and 13c-g were determined by disc diffusion and microbroth dilution method to
determine the minimum inhibitory concentrations (MIC) against seven microorganisms (Staphyloccus aureus,
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii and
Candida albicans). The most active compounds against the Gram positive S. aureus were 1, 13C, 13d, and 13g.
Also, 13c, 13d, and 13e had antibacterial activity but not 13f against some Gram negative organisms (E. coli,
and P. mirabilis). MIC concentrations against P. aeruginosa, and K. pneumoniae were ≥512 μg/ml, while that
against A. baumannii was ≥128 μg/ml except for nanoformulae of 13e and 13f that were 16 and 64 μg/ml,
respectively. No antifungal activity against Candida albicans was recorded for all compounds and their nano-
formulae (MIC > 1024 μg/ml). SLNs were found to decrease the MIC values for some of the compounds with no
effect on the antifungal activity. In conclusion, we demonstrated a novel, straight-forward and economical
procedure for the total synthesis of (-)-codonopsinine 1, (-)-codonopsine 2 and codonopsinine analogues 13c-g
from simple and commercially available starting materials; D-tartaric acid; with antimicrobial activities against
Gram positive and Gram-negative organisms that were improved by SLNs formulations.
(-)-Codonopsinine
(-)-Codonopsine
Codonopsinine analogues
(D)-tartaric acid
α-Amidoalkylation
Trichloroacetimidate
Antimicrobial activity
Solid lipid nanoparticles
⁎ Corresponding author at: Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Saudi Arabia.
E-mail address: ah.omer@tu.edu.sa (A.O.H. El-Nezhawy).
https://doi.org/10.1016/j.bmc.2019.02.021
Received 10 December 2018; Received in revised form 28 January 2019; Accepted 12 February 2019
0968-0896/PublishedbyElsevierLtd.
Pleasecitethisarticleas:AhmedO.H.El-Nezhawy,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.02.021

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Figure 3. Retrosynthesis of (-)-codonopsinine 1, (-)-codonopsine 2 and codo-
nopsinine analogues 13a-g.
Figure 1. Structure of (-)-codonopsinine 1 and (-)-codonopsine 2.
1. Introduction
(acetyloxy)-1-methyl-5-trichloroacetimidoyloxy-2-oxopyrrolidin-3-yl
and can provide the target compounds (1, 2 and 13c-g) Din-taarntaerffiiccaiecnidt
acetate intermediate (7) can be readily available from
Design and total synthesis of natural products have played im-
portant roles in many fields especially organic chemistry and medicinal
chemistry. However, five membered-ring alkaloids as natural poly-
hydroxylated pyrrolidines (or iminosugars) and their derivatives ac-
quired significant values in the last decade due to their promising
pharmacological activities as antimicrobial, antiviral, antidiabetic and
α-glucosidase inhibition.1–10 Many polyhydroxylated pyrrolidine alka-
loids exhibit potent and selective pharmacologically active compounds
such as steviamine, swainsonine, casuarine uniflorine, and hyacintha-
cine.11–16 Polyhydroxylated pyrrolidine alkaloids, which carry methyl
group on position 5 and aromatic substituent on the 2 position of the
pyrrolidine ring, are unusual and important class that is found in
nature.17 (-)-Codonopsinine 1 and (-)-codonopsine 2 (Fig. 1) separated
from Codonopsis clematidea in 1969 by Russian scientists18,19 are two
manner.
Nanotechnology was recently applied either for developing new
therapeutic pathways and/or optimizing the traditionally available
ones.37 Solid lipid nanoparticles (SLNs) are modified colloidal carrier
systems based on physiologically compatible solid lipids dispersed
within an aqueous surfactant solution to obtain nano-particulate sys-
tems with a diameter in the range of 100–150 nm. They have high drug
loading capability, extended stability, resist drug leak out of the lipid
phase and high physiological tolerability. They can control and sustain
the entrapped drug release as well as targeting to a specified tissue.
They can, also, be prepared on a large scale. The lipophilic nature and
small particle size of these systems allow efficient penetration through
physiological membranes and increase cellular drug concentration with
subsequent significant improvement of therapeutic action.38
types of complex pyrrolidine alkaloids. (-)-Codonopsinine
1 and
(-)-codonopsine 2 display antibiotic activity as well as hypotensive
activity without effects on the central nervous system.20,21 Structural
characterization of (-)-codonopsinine 1 and (-)-codonopsine 218,22
showed a novel class of simple pyrrolidine alkaloids that possess
1,2,3,4,5 penta-substituted pyrrolidine structures with four adjacent
stereo-genic centers, that are located in all trans positions. The absolute
configuration of the (-)-codonopsinine antibiotic 1 was established as
(2R,3R,4R,5R).21,23 This was additionally verified by X-ray crystal-
lography of (-)-codonopsine 2 separated by chromatography.24 Due to
the high biological activity and unique structural features, these pyr-
rolidine alkaloids 1 and 2 have gained substantial interest by several
organic chemists. Numerous total syntheses of codonopsinine 1 were
described: most of these methods are enantio-specific, starting from
There is an enduring need for the discovery of new antibiotics. As a
continuation of efforts of drug discovery39–44, we report herein an ef-
fective methodology for constructing (-)-codonopsinine 1, (-)-codo-
nopsine 2 and codonopsinine analogues in enantiomerically pure form
from cheap D-tartaric acid via a simple synthetic route with high yields.
These compounds had antimicrobial activities against Gram positive
and Gram negative organisms that was improved by SLN formulation.
2. Results and discussion
2.1. Chemistry
In this study, a novel methodology for synthesis of (-)-codonopsi-
nine 1, (-)-codonopsine 2 and codonopsinine analogues 13c-g was
utilized to obtain these target compounds from simple and commer-
cially available starting material; D-tartaric acid (3), which was trans-
formed into the anhydride 4 by reflux with acetyl chloride for 30 h then
the anhydride 4 was refluxed with methylamine in acetonitrile to give
imide 5.25,36 The reduction of 5 with sodium borohydride in methanol
afforded (3R,4R)-2-hydroxy-1-methyl-5-oxopyrrolidine-3,4-diyl diace-
tate (6). The reaction of 5-hydroxy pyrrolidinone derivative 6 with
trichloroacetonitrile in the presence of 1,8-diazabicyclo[5.4.0]-undec-
7-ene (DBU) as catalytic base furnished (3R,4R)-1-methyl-2-oxo-5-
(2,2,2-trichloroacetamido)pyrrolidine-3,4-diyl diacetate (7) in high
yield (Scheme 1).
either D-tartaric acid25
,
L-tartaric acid26 D-ribose27
,
D-alanine,22,24 L-
pyroglutamic acid,28 (S)-3-chloropropan-1,2-diol,29 via gold-mediated
tandem-catalyzed pyrrole synthesis,30 or by ring-closing iodoamination
of homoallylic amines.31
Synthesis of substituted pyrrolidine has been studied via creation of
quaternary stereocenters by the addition of allyltributylin or methyl
silyloxyfuran to chiral N-acyliminium ions.32,33 Glycosidic bond for-
mation is usually carried out by the transformation of O,O- and N,O-
hemiacetals into trichloroacetimidoyl derivatives and their acid-cata-
lyzed activation (Fig. 2).34,35
Total synthesis of natural (-)-lentiginosine via O-(2-oxopyrrolidin-5-
36
yl)trichloro acetimidate as amidoalkylating agent promote us to use
this concept in the synthesis of (-)-codonopsinine 1, (-)-codonopsine 2
and codonopsinine analogues 13c-g in an enantiomerically pure form
from D-tartaric acid. The retrosynthesis (Fig. 3) shows that (3R,4R)-4-
Only one diastereoisomer of 7 was recovered with enantiomeric
excess (100% ee) as confirmed by NMR. This was based on the J4,5
coupling constant of 2.4 Hz might be the (5R)-isomer.
Trichloroacetimidate 7 was used for the α-amidoalkylation experiments
with different C-nucleophile. Reaction of (3R,4R)-1-methyl-2-oxo-5-
(2,2,2-trichloroacetamido)pyrrolidine-3,4-diyl diacetate (7) with elec-
tron-rich anisole, 1,2-dimethoxybenzene, 1-fluoro-3-methoxybenzene,
(methoxymethyl)benzene, benzene, toluene or 1-fluoro-3,4-dimethoxy-
2-methylbenzene (8a-g) as the C-nucleophile in the presence of tri-
methylsilyl trifluoromethane sulfonate (TMSOTf) as catalyst led to
compounds 9a-g in good yields (scheme 2). Only one stereoisomer of
each of 9a-g was produced in high yield (100% ee) as confirmed by
NMR. This was based on the J4,5-coupling constant of ca. 5 Hz the (R)-
configuration was assigned to C-5. Nucleophilic addition of methyl-
magnesium bromide to 2-pyrrolidinone 9a-g at −78 ˚C afforded the
labile quaternary α-hydroxy pyrrolidine 10a-g, which equilibrated to
Figure 2. Alkylation of nucleophiles by acid-catalysis with O-alkyl tri-
chloroacetimidate derivatives.
2

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of trichloroacetimidate 7. Reagents and conditions: (a) acetyl chloride, reflux 30 h (b) CH3NH2, THF, AcCl, reflux, 5: 88% (c) NaBH4, MeOH,
−7 °C, 12 min, 6: 89% (d) CCl3CN, DBU, 0 °C, 10 min. 7: 72%.
the open keto form 11a-g. The mixture was submitted to reductive
deoxygenation45 with Et3SiH in the presence of BF3.OEt2, cleanly
leading to 12a-g as a single stereoisomer (100% ee) in good yield.
Deacetylation of the pyrrolidine derivative 12a-g in the presence of
NaOMe in methanol gave the corresponding (-)-codonopsinine 1,
(-)-codonopsine 2 or codonopsinine analogues 13c-g in high yield
(Scheme 2). The NMR, IR and mass spectra and microanalysis of the
synthezsised (-)-codonopsinine 1 were in agreement with the published
data of natural (-)-codonopsinine.24
physical stability of the prepared SLNs due to the induced particle ag-
glomeration.46 These repulsive forces are described in terms of elec-
trical surface properties and expressed by Zeta potential (Z.P.). SLNs
with Z.P. of
30 mV are usually physically stable formula.47 The
value of zeta potential of the prepared SLN formulations are usually
controlled by surfactant addition during formulation that, also, lowers
the interfacial tension at the lipid/water interface with subsequent
stabilizing effect on the prepared formulae.38 The data presented in
Table 1 indicate that the recorded Z.P. values for all prepared formulae
were acceptable (−21.5 to −34.7 mV) with small differences mainly
due to fixation of preparation conditions (homogenization and sonica-
tion speed and time). Increasing particle size results in higher Z.P. va-
lues as a result of increasing the charge density with increasing surface
area of the particle.
In summary, easily obtained trichloroacetimidates 7 are outstanding
precursors for α-amidoalkylation reactions with electron rich C-nu-
cleophiles. This concept can be employed to the synthesis of substituted
pyrrolidine alkaloids, as exhibited in the synthesis of (-)-codonopsinine
1, (-)-codonopsine 2 or codonopsinine analogues 13c-g.
2.2. Characterization of the prepared SLN formulations
2.2.4. Release studies
In-vitro drug release data from the prepared SLN formulations were
studied using dialysis bag diffusion technique in comparison to non-
formulated compound release profiles. The release data are shown in
Fig. 4. The release profiles of non-formulated compounds showed
faster, complete dissolution within 2 to 3 h depending on the compound
solubility in dissolution medium, where compounds 13e and 13f
showed faster, and complete drug dissolution within two hours in
comparison to 95.2%, 96.7%, 91.8% and 87.8% for compounds 1, 13c,
13d and 2, respectively, within the same time period. Compound 13g
showed a slower dissolution profile; where only 78.2% of the com-
pound was dissolved after two hours. These data are in accordance with
the solubility characteristics of the synthesized compounds at the tested
experimental conditions. SLN formulations showed slow, and extended
release profiles of the synthesized compounds over a 12 h period, where
the percentage released reached 93.3%, 93.2%, 90.2%, 92.1%, 91.2%,
90.1%, and 92.2% from SLN formula (F)1 to F7, respectively (Fig. 4).
The release profile from the prepared SLN formulations followed bi-
phasic pattern with an initial burst high release during the first three
hours, followed by a slower and sustained release within the remaining
12 h. This release pattern could be correlated to the matrix nature of the
prepared SLNs, where the initial burst release results from the dis-
solution of the drug adsorbed on the particle surface and solubilized
within particle outer most layers.48,49 By time, the release rate slowed
down depending on dissolution medium diffusion rate through deeper
lipid layers and/or biodegradation and surface erosion of the matrix
resulting in a slower and more extended dissolution process.38
2.2.1. Entrapment efficacy
Table 1 shows the characterization results for the prepared SLN
formulations. All prepared formulations showed good percentage of
entrapment efficacy for all tested compounds with small difference
ranging from 70.8% (F7) to 86.9% (F8). It could be noted that the
percentage of entrapment efficacy was dependent on the synthesized
compound’s solubility and molecular weight, where the higher the li-
pophilicity of the tested compound, the higher was its solubility within
the applied lipid core and hence more amount was entrapped within the
SLN formula.38
2.2.2. Particle size determination and size distribution
Data in Table 1 shows that all prepared SLN formulae had accep-
table particle size ranging from 75 to 126 nm with satisfying narrow
particle size distribution as indicated by the small Polydispersity Index
(PDI) values (0.21
0.015 to 0.37
0.10). Particle size of SLN for-
mulations is a complicated process that is affected by many factors
including lipid type, concentration, emulsifying properties, surfactant
type (HLB), crystallization conditions including homogenization/soni-
cation time and speed.38 Despite fixed lipid concentration and constant
preparation conditions that resulted in proximity of recorded particle
sizes for different formulation, it could be noted that drug content of the
different formulations had positive effect on particle size and this could
be explained by the increase of viscosity of formula mixture with in-
creasing drug content.
2.2.3. Zeta potential measurement
2.2.5. In-vitro permeation studies
During the preparation of SLN formulae, high shearing conditions
are usually applied to affect particle size reduction that results in higher
surface free energy values at the lipid/water interface and affects the
The lipophilic characteristics and nano-size of SLNs increase its
permeation through membranes especially those of lipid nature and this
causes significant accumulation of the loaded drug within the targeted
3

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 2. Synthesis of target compounds. Reagents and conditions: (a) CH2Cl2, TMSOTf, r.t., 30–120 min. 9a: 68%, 9b: 75%, 9c: 55%, 9d: 69%, 9e: 74%, 9f: 69%,
9 g: 57% (b) MeMgBr, ether, −78–0 °C (c) Et3SiH, BF3OEt2, DCM, −78 °C, 12a: 62%, 12b: 68%, 12c: 66%, 12d: 69%, 12e: 75%, 12f: 75%, 12 g: 73% (d) NaOMe,
MeOH, 0 °C, 1: 90%, 2: 92%, 13c: 87%, 13d: 93%, 13e: 88%, 13f: 90%, 13 g: 93%
tissue. The in vitro permeation of the prepared SLN formulations
through simulated cellular (cellophane) membrane was studied by
measuring the percentage of drug accumulation in the acceptor
chamber of Franz cell. The data shown in Fig. 5 showed the cumulative
percentage of permeated compound against time (hours) for 6 h. The
synthesized compounds showed complete permeation from the pre-
pared SLN formulations within 6 h, while the percentages of com-
pounds permeated were 60.7%, 67.2%, 59.1%, 62.7%, 49.8%, 56.3%
and 72.9% for non-formulated compounds 1, 2, 13c, 13d, 13e, 13f and
13g, respectively. Further investigation showed that the synthesized
compound lipophilicity had a positive effect on the permeation results,
where 13g and 2 showed higher permeation results when compared to
the other synthesized compounds. These data confirm the ability of the
prepared SLN formulations to increase the permeation through mem-
branes and improve cellular uptake of the loaded compound with an
Table 1
Characterization of the prepared SLN formulations of the synthesized com-
pounds.
Formula
Original
E.E.*(%)
P.S.(nm)
Z.P.(mV) PDI
Code
compound
F1
F2
F3
F4
F5
F6
F7
1
78.09
83.96
77.98
81.14
70.83
74.61
86.94
2.85 88
2.63
− 24.1
0.33
0.27
0.21
0.31
0.32
0.22
0.37
0.053
0.071
0.015
0.019
0.075
0.054
0.10
2
1.95 118
1.53 94
3.15 109
4.01 77
3.86 75
3.87 126
3.13 29.8 -
13c
13d
13e
13f
13 g
2.19
23.8 -
2.73 25.7 -
1.17
21.5 -
21.3 -
1.81
3.93 34.7 -
* EE, entrapment efficiency; P.S., particle size; Z.P. Zeta potential; DPI,
Polydispersity Index
4

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
100
80
60
40
20
0
F4
F1
F3
F2
F5
F6
13d
1
13c
2
13e
13f
0
2
4
6
8
10
12
Time (hr)
Figure 4. Release profiles for formulated and non-formulated synthesized
compounds in phosphate buffer (pH 6.8).
100
80
Figure 6. An example of antimicrobial susceptibility testing by disc diffusion
method against S. aureus for the synthesized compounds.
to the parent compounds. On the other hand, the MIC value for A.
baumannii for the same compound decreased from > 1024 µg/ml to
256 µg/ml. A similar decrease in MIC was seen for compounds 13e and
13g against P. mirabilis and A. baumannii and for 13e against P. mir-
abilis, P. aeruginosa and A. baumannii. Regarding compound 13g, the
present study revealed that the nano-formulation of such compound
was found to decrease the MIC values for E. coli, P. mirabilis and A.
baumannii isolates from 64, 1024 and 512 µg/ml to 16, 128 and 256 µg/
ml, respectively (Table 2). With respect to compound 13d, nano-for-
mulation was found to decrease the MIC value for P. mirabilis from 16 to
8 µg/ml. On the other hand, the nano-formulation for that compound
didn’t affect the MIC values for the other tested isolates except for K.
pneumoniae, for which the MIC was increased from 256 to 512 μg/ml.
These data show that the 13e nano-formulation exhibited a great effect
on the MIC value for A. baumannii in which the MIC decreased from 128
to 16 µg/ml.
60
40
F3
13d
1
F2
F5
13c
F4
F1
2
13e
20
0
0
1
2
3
4
5
6
Time (hr)
Figure 5. Permeation profiles for formulated and non-formulated synthesized
compounds after 6 h.
expected increase in the antibacterial action.
For compound 13f, it was found that nano-formulation for such
compound was able to decrease the MIC value for A. baumannii and S.
aureus from 256 and > 1024 µg/ml to 64 and 1024 µg/ml, respectively
(Table 2). Finally, nano-formulation of compounds 1 and 13f was found
to decrease the MIC values for K. pneumonaie from > 1024 and
1024 µg/ml to 1024 and 512 µg/ml, respectively. On the other hand,
the nano-formulation for all tested codonopsinine derivatives didn’t
affect or improve the antifungal activity against C. albicans indicating
lack of antifungal activity of such compounds. Interestingly, the MIC of
13d, and 13g against K. pneumoniae was increased. The same applies for
2, 13c, and 13f against E. coli (Table 2).
2.3. Antimicrobial activity testing:
2.3.1. Antimicrobial susceptibility screening by modified Kirby-Bauer disc
diffusion method:
As a preliminary screening step, we tested the synthesized com-
pounds against seven microorganisms including Gram positive, and
Gram negative organisms and yeast by disc diffusion method as de-
scribed in the Experimental section. The data showed that compounds 1
and 13c were the most effective codonopsinine derivatives against S.
aureus (a Gram positive organism), where the inhibition zone diameters
for these compounds were 32 and 30 mm, respectively. Also, com-
pounds 2, 13d and 13g showed obvious activity against S. aureus as
shown in Figure 6 (and also in Table 2 as shown below). Apart from S.
aureus, the current disc diffusion data showed that compound 13e was
the most effective codonopsinine derivative against Gram negative
isolates, where the inhibition zone diameters for E. coli, K. pneumoniae,
P. mirabilis, P. aeruginosa and A. baumannii were 40, 22, 34, 18 and
32 mm, respectively (data not shown). On the other hand, none of the
codonopsinine derivatives were found to have antifungal activity
against Candida albicans (not shown).
In summary, the data presented above suggest that 1, 13d and 13g
were the most potent derivatives against the Gram-positive bacterium;
S. aureus. On the other hand, 13c, 13d, and 13e are the most potent
antibacterial compounds against Gram negative bacteria particularly E.
coli and P. mirabilis. These compounds had MIC values ≤ 16 μg/ml ei-
ther in the pure and/or the nano-formulae. In addition, nano-formula-
tions improved the antibacterial activity of the synthesized compounds
against some microorganisms while it had a deteriorating effect on
others. As known, the genetic makeup of the bacterium greatly impacts
its susceptibility to different antimicrobial agents. This could have an
impact on our results. As indicated above, nano-formulations improved
the activity of certain codonopsinine derivatives while it did not affect
or deteriorated the activity of others. We speculate that the increased
activity by nano-formulation can be attributed to increased uptake of
the formulated compounds by facilitating the entry of the compounds to
the bacterial cells or up regulation of membrane transporters of such
compounds. Also, formulation could enhance the binding of such
2.3.2. Determination of minimum inhibitory concentration of the
synthesized compounds and their nanoformulae by microbroth dilution
method:
The minimum inhibitory concentration (MIC) of the tested deriva-
tives and their nanoformulae are presented in Table 2. As shown, the
MIC values for the nano-formula of compound 1 against E. coli and P.
mirabilis decreased from 128 µg/ml to 64 µg/ml, each, when compared
5

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 2
MIC values for non-nano-formulated and nano-formulated codonopsinine derivatives against Gram positive, Gram negative microorganisms and yeast. The MIC of
the test compounds against seven microorganisms were determined as described in the Experimental section and the MIC values for the non-nano-formulated and
nano-formulated compounds are shown in μg/ml.
CPD
Gram Positive
Gram Negative
Yeast
S. aureus
E. coli
K. pneumoniae
P. mirabilis
P. aeruginosa
A. baumannii
C. Albicans
ATCC 29213
ATCC 25922
ATCC 700603
ATCC 14153
ATCC 27853
Clinical Isolate
10231
Non-Nano
Nano
Non-Nano
Nano
Non-Nano
Nano
Non-Nano
Nano
Non-Nano
Nano
Non-Nano
Nano
Non-Nano
Nano
1
≤0.5
32
≤0.5
32
128
64
4
64
256
16
8
˃1024
˃1024
˃1024
256
1024
˃1024
1024
512
128
64
1024
˃1024
˃1024
˃1024
˃1024
512
˃1024
˃1024
512
256
˃1024
512
˃1024
16
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
˃1024
2
˃1024
˃1024
16
˃1024
˃1024
8
˃1024
˃1024
˃1024
1024
13c
13d
13e
13f
13g
≤0.5
2
≤0.5
2
8
˃1024
128
1024
˃1024
4
1024
1024
4
16
128
64
16
512
16
1024
1024
512
˃1024
512
4
2
1024
1024
512
128
1024
1024
256
64
˃1024
1024
˃1024
512
256
compounds to their target sites. On the other hand, the deteriorating
effect of nano-formulation on the MIC of some of the codonopsinine
derivatives may be attributed to down regulation of certain transport
proteins at the cell envelope of different bacteria or the up regulation of
certain efflux pumps that decrease the intracellular effective con-
centration of the compounds. To this end, the differential impact of
nano-formulations on the MIC may be attributed to the interaction of
these compounds in their native or formulated conditions with the
bacterial envelope and the permeation, retention and/or efflux of these
compounds by the different bacterial strains.
perform mass spectra. Analytical analysis of carbon, hydrogen and ni-
trogen were carried at the Microanalytical Center of Cairo University,
Egypt.
4.1.1. (3R,4R)-1-methyl-2,5-dioxopyrrolidine-3,4-diyl diacetate (5)
acid (10 g, 66.7 mmol) and acetyl chloride (50 ml,
0.7 Dm-toalr)tamriicxture was stirred under reflux for 25 h till the reaction
mixture became homogeneous. Distillation was used to remove excess
acetyl chloride at 760 mmHg, and the remaining amounts were re-
moved by vacuum. The crude product was dissolved in tetrahydrofuran
(100 ml) and methylamine (10 ml, 324 mmol) was added gradually.
After stirring for two hours, the solution was concentrated in vacuo and
the residue was kept under reflux with acetyl chloride (50 ml, 0.7 mol)
for another 4 h. After the reaction mixture concentration in vacuo, the
residue was purified using column chromatography (petroleum ether-
ethyl acetate, 1:1) to give 5 (yield 87%) as a pale yellow oil. [α]D = −
51.5 (c = 4, MeOH). IR (neat) ν 2950, 1740, 1690, 1610 cm−1. 1H NMR
(400 MHz, CDCl3) δ 2.05 (s, 6H, CH3CO), 2.93 (s, 3H, NCH3), 5.40 (s,
2H, CHOAc). 13C NMR (100 MHz, CDCl3) δ 19.9, 24.9, 72.4, 169.4,
169.7. MS (EI): m/z (%) = 229 (12) [M+], 187 (68), 169 (1 0 0), 145
(60), 127 (65), 116 (26), 101 (92); Analytical Calculation for
C9H11NO6:C, 47.16; H, 4.83; N, 6.11. Found: C, 47.20; H, 4.90; N, 6.12.
Finally, one the drawbacks of this study is that we did not examine
the antibacterial activity of (+)-codonopsinine or its analogues and
compare it with the (-)-codonopsinine and its analogues. This is cur-
rently under investigation.
3. Conclusion
In conclusion, we have demonstrated a novel, simple and efficient
procedure for the total synthesis of (-)-codonopsinine 1, (-)-codo-
nopsine 2 and codonopsinine analogues 13c-g from simple and com-
mercially available starting materials (D-tartaric acid) via the reaction
of intermediate trichloroacetamidate 7 with electron rich compounds as
C-nucleophiles. Loading of the synthesized compounds on SLNs showed
sustaining of release for 12 h and also significant improvement of per-
meation ability when compared to non-formulated compounds.
Importantly, the synthesized compounds had antimicrobial activities
against Gram positive and Gram-negative organisms that were im-
proved by SLNs formulation.
4.1.2. (3R,4R)-2-hydroxy-1-methyl-5-oxopyrrolidine-3,4-diyl
diacetate
(6)
To a solution of 5 (4.57 mmol) in 125 ml of methanol was cooled to
−10 °C in an ice-salt bath, (875 mg, 23.1 mmol) of sodium borohydride
were added in one portion. The reaction mixture was stirred for 15 min
and partitioned between 150 ml of CH2Cl2, 150 ml of saturated aqueous
Na2CO3 and 100 ml of water. The aqueous phase was separated and
extracted with three 100 ml fractions of dichloromethane. The organic
phase was dehydrated using MgSO4 and concentrated in vacuo to give 6
as a white solid (88%) mp 75–76 °C. The product was purified using
silica gel column chromatography (petroleum ether-ethyl acetate, 1:1).
[α]D = -24.9 (c = 1.8, MeOH). IR (neat) ν 3379, 2950, 1740, 1690,
4. Experimental
4.1. Chemistry
All chemicals were obtained from Fluka and Sigma-Aldrich
(Germany) and were used as is. All reactions were carried out under
inert gas and dry solvents. Also, all reactions were checked by thin layer
chromatography (TLC) using Merck’s silica gel-coated plastic sheets (60
F254; E. Merck, layer thickness 0.2 mm) under UV light. Detection was
accomplished by treatment with either a solution of 20 g of ammonium
molybdate and 0.4 g of cerium (IV) sulfate in 400 ml of 10% H2SO4 or
with 15% H2SO4 and heating at 150 °C. Melting points were measured
on a Gallenkamp melting point apparatus and were uncorrected.
Optical rotations were measured by a Perkin-Elmer 241 MC polarimeter
in a 1-dm cell. Infra-red spectra were determined on a JASCO FT/IR-
4600 and reported as cm−1. Bruker Unit 400 (400 MHz for 1H NMR and
100 MHz for 13C NMR) instrument was used with CDCl3 as a solvent
and tetramethylsilane as an internal standard. Thermo Finnigan LCQ
Advantage spectrometer in ESI mode, spray voltage 4.8 kV was used to
1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.06 (s, 3H, CH3CO), 2.07 (s,
3H, CH3CO), 2.84 (s, 3H, NCH3), 4.91 (br, s 1H, OH, exchangeable with
D2O), 5.02 (d, J = 2.8 Hz, 1H CHOAc), 5.05–5.16 (m, 2H, CHOAc and
NCHO). 13C NMR (100 MHz, CDCl3) δ 20.3, 20.5, 26.5, 73.8, 78.7, 84.9,
167.0, 170.1, 170.2. MS (EI): m/z (%) = 230 (10) [M+], 170 (60), 128
(1 0 0), 111 (40), 101 (70), 70 (30), 59 (80); Analytical Calculation for
C9H13NO6: C, 46.75; H, 5.67; N, 6.05. Found: C, 46.55; H, 5.57; N, 6.01.
4.1.3. (3S,4R)-1-methyl-2-oxo-5-(2,2,2-trichloroacetamido)pyrrolidine-
3,4-diyl diacetate (7)
A solution of 6 (2.5 mmol) in dry dichloromethane (20 ml) and
trichloroacetonitrile (25 mmol) was treated with DBU (70 μl) at
−10–0 °C and the reaction mixture was stirred for 12 min. The solvent
6

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
was evaporated and column chromatography was used to purify the
product using triethylamine (TEA) in toluene 5% to give 7; which is
pale yellow oil (75%). [α]D = + 50.5 (c = 1.9, MeOH). IR (neat) ν
3350, 2950, 1740, 1690, 1610 cm−1. 1H NMR (400 MHz, CDCl3) δ 2.02
(s, 3H, CH3CO), 2.04 (s, 3H, CH3CO), 2.90 (s, 3H, NCH3), 5.32 (m, 2H,
3-H, 4-H), 6.20 (d, J = 2.5 Hz, 1H, 5-H), 8.64 (br, s, 1H, NH, ex-
changeable with D2O). 13C NMR (100 MHz, CDCl3) δ 20.4, 20.5, 26.9,
74.1, 78.6, 85.1, 162.2, 167.9, 170.1, 170.3.
4.86 (s, 2H, phCH2O), 4.67 (d, = J 5.0 Hz, 1H, 5-H), 5.19 (dd, = J 5.0
and 4.9 Hz, 1H, 4-H), 5.49 (d, J = 5.1 Hz, 1H, 3-H), 6.90–7.16 (m, 4H,
ph-H). 13C NMR (100 MHz, CDCl3) δ 20.4, 21.3, 30.9, 56.3, 58.9, 70.2,
74.8, 85.5, 125.9, 126.4 130.9, 131.1, 138.8, 140.8, 168.7, 169.8,
171.6. MS: (EI) m/z (%) = 335 (30) [M+], 219 (65), 204 (1 0 0);
Analytical Calculation for C17H21NO6: C, 60.89; H, 6.31; N, 4.18.
Found: C, 60.96; H, 6.49; N, 4.23.
4.1.4.5. (3S,4R,5R)-1-methyl-2-oxo-5-phenylpyrrolidine-3,4-diyl diacetate
(9e). The colorless oil (74%) was purified using silica gel column
chromatography (petroleum ether-ethyl acetate, 10:1). [α]D = –22.26
4.1.4. General procedure for the synthesis of (9a–g)
A solution of trichloroacetimidate 7 (1.4 mmol) and the nucleophile
anisole, 1,2-dimethoxybenzene, 1-fluoro-3-methoxybenzene, (methox-
ymethyl)benzene, benzene, toluene or 1-fluoro-3,4-dimethoxy-2-me-
thylbenzene (8a-g) (1.4 mmol) in dry CH2Cl2 (20 ml) were treated with
TMSOTf (0.16 ml) and then stirred for 20–100 min. Addition of solid
sodium bicarbonate quenched the reaction, which was diluted with
CH2Cl2. The reaction mixture was filtered and concentrated to give 9a-
g.
(c = 1.9, MeOH). IR (neat) ν 2940, 1742, 1694, 1610 cm−1
.
1H NMR
(400 MHz, CDCl3) δ 2.10 (s, 3H, CH3CO), 2.11 (s, 3H, CH3CO), 3.35 (s,
3H, NCH3), 4.62 (d, J 5.0 Hz, 1H, 5-H), 5.29 (dd, J = 5.0 and 5.2 Hz,
1H, 4-H), 5.48 (d, J = 5.0 Hz, 1H, 3-H), 7.26–7.54 (m, 5H, ph-H). 13C
NMR (100 MHz, CDCl3) δ 20.3, 21.2, 31.3, 56.9, 75.8, 86.9, 125.9,
126.4, 129.5, 130.1, 130.9, 131.1, 168.5, 169.0, 172.1. MS (EI): m/z
(%) = 291 (40) [M+], 173 (1 0 0), 160 (75); Analytical Calculation for
C15H17NO5: C, 61.85; H, 5.88; N, 4.81. Found: C, 61.95; H, 5.92; N,
4.1.4.1. (2R,3R,4S)-2-(4-methoxyphenyl)-1-methyl-5-oxopyrrolidine-3,4-
diyl diacetate (9a). The colorless oil (68%) was purified using silica gel
column chromatography (petroleum ether-ethyl acetate, 1:1). [α]D = -
4.99.
4.1.4.6. (3S,4R,5R)-1-methyl-2-oxo-5-(p-tolyl)pyrrolidine-3,4-diyl
diacetate (9f). The colorless oil (69%) was purified using silica gel
column chromatography (petroleum ether-ethyl acetate, 10:1).
21.68 (c = 1.9, MeOH). IR (neat) ν 2940, 1742, 1694, 1610 cm−1
.
1H
NMR (400 MHz, CDCl3) δ 2.03 (s, 3H, CH3CO), 2.11 (s, 3H, CH3CO),
2.62 (s, 3H, NCH3), 3.80 (s, 3H, OCH3), 4.70 (d, J = 5.0 Hz, 1H, 5-H),
5.42 (dd, J = 5.1 and 4.9 Hz, 1H, 4-H), 5.50 (d, J = 5.1 Hz, 1H, 3-H),
6.95–7.31 (m, 4H, Ph-H). 13C NMR (100 MHz, CDCl3) δ 20.6, 27.9,
55.3, 62.2, 74.6, 76.5, 111.0, 114.4, 120.8, 123.6, 128.8, 130.2, 157.6,
167.8, 169.6, 169.8. MS (EI): m/z (%) = 320 (30) [M+], 260 (60), 218
(1 0 0), 201 (30), 149 (50). Analytical Calculation for C16H19NO6: C,
59.81; H, 5.96; N, 4.36. Found: C, 59.89; H, 6.06; N, 4.43.
[α]D = -30.20 (c = 1.9, MeOH). IR (neat)
ν 2950, 1745, 1690,
1614 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.12 (s, 3H, CH3CO), 2.13
(s, 3H, CH3CO), 2.45 (s, 3H, CH3), 3.38 (s, 3H, NCH3), 4.60 (d,
J = 5.1 Hz, 1H, 5-H), 5.35 (dd, J = 5.1 and 5.1 Hz, 1H, 4-H), 5.44 (d,
J = 5.1 Hz, 1H, 3-H), 7.26 (d, J = 12.2 Hz, 2H, ph-H), 7.34 (d,
J = 11.9 Hz, 2H, ph-H). 13C NMR (100 MHz, CDCl3) δ 20.3, 20.6,
21.2, 30.8, 76.5, 85.8, 125.9, 126.1, 129.9, 130.1, 130.9, 131.1, 168.2,
169.1, 172.6. MS (EI): m/z (%) = 305 (20) [M+], 189 (1 0 0), 174 (90);
Analytical Calculation for C16H19NO5: C, 62.94; H, 6.27; N, 4.59.
Found: C, 62.85; H, 6.62; N, 4.68.
4.1.4.2. (2R,3R,4S)-2-(3,4-dimethoxyphenyl)-1-methyl-5-oxopyrrolidine-
3,4-diyl diacetate (9b). Th colorless oil (75%) was purified using silica
gel column chromatography (petroleum ether-ethyl acetate, 1:1).
[α]D = –32.46 (c = 1.9, MeOH). IR (neat)
ν
2950, 1740, 1690,
4.1.4.7. (2R,3R,4S)-2-(2-fluoro-4,5-dimethoxy-3-methylphenyl)-1-
methyl-5-oxopyrrolidine-3,4-diyl diacetate (9 g). The colorless oil (57%)
was purified using silica gel column chromatography (petroleum ether-
ethyl acetate, 10:1). [α]D = –32.18 (c = 1.9, MeOH). IR (neat) ν 2940,
1620 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.12 (s, 3H, CH3CO), 2.13
(s, 3H, CH3CO), 2.65 (s, 3H, NCH3), 3.81 (s, 6H, OCH3), 4.73 (d,
J = 5.1 Hz, 1H, 5-H), 5.38 (dd, J = 5.1 and 4.9 Hz, 1H, 4-H), 5.53 (d,
J = 5.0 Hz, 1H, 3-H), 6.91 (s, 1H, ph-H), 7.08–7.20 (m, 2H, Ph-H). 13C
NMR (100 MHz, CDCl3) δ 20.3, 21.0, 28.9, 56.7, 62.2, 70.6, 75.5,
112.0, 116.9, 121.6, 124.7, 128.9, 130.8, 156.9, 168.3, 169.9, 170.2.
MS (EI): m/z (%) = 351 (45) [M+], 235 (35), 220 (1 0 0); Analytical
Calculation for C17H21NO7: C, 58.11; H, 6.02; N, 3.99. Found: C, 58.29;
H, 6.09; N, 4.13.
1740, 1690, 1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.10 (s, 3H,
CH3CO), 2.12 (s, 3H, CH3CO), 2.45 (s, 3H, CH3), 3.35 (s, 3H, NCH3),
3.85 (s, OCH3), 3.87 (s, OCH3), 4.63 (d, J = 5.2 Hz, 1H, 5-H), 5.44 (dd,
J = 5.1 and 5.2 Hz, 1H, 4-H), 5.44 (d, J = 5.0 Hz, 1H, 3-H), 7.18 (s, 1H,
ph-H). 13C NMR (100 MHz, CDCl3) δ 20.7, 20.9, 21.2, 30.8, 49.2, 56.2,
60.1, 76.9, 86.8, 116.6, 117.9, 129.7, 145.4, 150.9, 160.1, 168.7,
169.7, 171.9. MS (EI): m/z (%) = 383 (30) [M+], 265 (1 0 0), 252 (60);
Analytical Calculation for C18H22FNO7: C, 56.39; H, 5.78; F, 4.96; N,
3.65. Found: C, 56.49; H, 5.82; F, 5.03; N, 3.78.
4.1.4.3. (2R,3R,4S)-2-(2-fluoro-4-methoxyphenyl)-1-methyl-5-
oxopyrrolidine-3,4-diyl diacetate (9c). The colorless oil (55%) was
purified using silica gel column chromatography (petroleum ether-
ethyl acetate, 5:1). [α]D = –22.26 (c = 1.9, MeOH). IR (neat) ν 2946,
4.1.5. General procedure for the synthesis of (10a–g)
1745, 1684, 1615 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.14 (s, 3H,
Methyl magnesium bromide (0.28 ml, 0.84 mmol, 1.2 equiv. as a
3 M solution in diethyl ether) was slowly added to a cooled (-78 °C)
solution of 9a-g (0.6 mmol) in diethyl ether (10 ml). Stirring continued
for 2 h at −78 °C for 2 h. The reaction mixture was quenched with
saturated NaHCO3 (4 ml), washed with water (2 × 4 ml) and brine
(3 ml), dried over MgSO4, filtered and concentrated in vacuo, then used
as crude in the next step.
CH3CO), 2.15 (s, 3H, CH3CO), 3.30 (s, 3H, NCH3), 3.89 (s, OCH3), 4.53
(d, J = 5.1 Hz, 1H, 5-H), 5.20 (dd, J = 5.1 and 4.9 Hz, 1H, 4-H), 5.43
(d, J = 5.1 Hz, 1H, 3-H), 7.02–7.07 (m, 2H, Ph-H), 7.18 (s, 1H, Ph-H).
13C NMR (100 MHz, CDCl3) δ 20.1, 20.3, 30.9, 54.6, 56.0, 70.6, 79.5,
124.9, 130.9, 138.8, 140.8, 157.1, 160.6, 168.3, 169.9, 170.2. MS (EI):
m/z (%) = 339 (50) [M+], 221 (35), 208 (1 0 0); Analytical Calculation
for C16H18FNO6: C, 56.63; H, 5.35; F, 5.60; N, 4.13. Found: C, 56.69; H,
5.41; F, 5.52; N, 4.23.
4.1.6. General procedure for the synthesis of (12a–g)
To a solution of hydroxy lactam 10a-g (0.35 mmol) in CH2Cl2
(5 ml), under an argon atmosphere at −78 °C, Et3SiH (0.15 ml,
1.05 mmol) was added followed by BF3.OEt2 (0.08 ml, 0.070 mmol)
dropwise. Stirring continued for 20–30 min at 0 °C. The reaction was
quenched with H2O (10 ml) and extracted with dichloromethane
(3 × 20 ml). The organic phase was washed with brine (5 ml), dried
over MgSO4, and the solvent was removed under reduced pressure to
4.1.4.4. 4.1.4.4.(2R,3R,4S)-2-(4-(methoxymethyl)phenyl)-1-methyl-5-
oxopyrrolidine-3,4-diyl diacetate (9d). The colorless oil (69%) was
purified using silica gel column chromatography (petroleum ether-
ethyl acetate, 1:1). [α]D = –32.16 (c = 1.9, MeOH). IR (neat) ν 2943,
1742, 1690, 1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 2.11 (s, 3H,
CH3CO), 2.12 (s, 3H, CH3CO), 3.35 (s, 3H, NCH3), 3.48 (s, 3H, OCH3),
7

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
give 12a-g.
(400 MHz, CDCl3) δ 1.11 (d, J = 6.4 Hz, 3H, CH3), 2.10 (s, 3H, CH3CO),
2.11 (s, 3H, CH3CO), 2.18 (s, 3H, NCH3), 3.55–3.59 (m, 1H, 5- H), 4.15
(d, J = 6.4 Hz, 1H, 2-H), 4.47 (dd, J = 4.4, 4.0 Hz, 1H, 4-H), 5.16 (dd,
J = 6.4 and 4.4 Hz, 1H 3-H), 7.26–7.34 (m, 5H, Ph-H). 13C NMR
(100 MHz, CDCl3) δ 11.0, 21.2, 21.3, 38.3, 57.4, 66.4, 76.8, 77.1,
124.3, 126.4, 126.8, 128.2, 138.9, 140.6, 171.3, 171.5. MS (EI): m/z
(%) = 291 (30) [M+], 173 (40), 160 (1 0 0); Analytical Calculation for
4.1.6.1. (2R,3R,4R,5R)-2-(4-methoxyphenyl)-1,5-dimethylpyrrolidine-
3,4-diyl diacetate (12a). The colorless oil (319 mg, 62%) was purified
using silica gel column chromatography (petroleum ether-ethyl acetate,
1:1). [α]D = -31.60 (c = 2.0, MeOH), IR (neat)
ν 2945, 1745,
1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 1.23 (d, J = 6.5 Hz, 3H,
CH3), 2.11 (s, 3H, CH3CO), 2.13 (s, 3H, CH3CO), 2.28 (s, 3H, NCH3),
3.59 (s, 3H, OCH3), 3.69–3.73 (m, 1H, 5- H), 4.13 (d, J = 6.5 Hz, 1H, 2-
H), 4.45 (dd, J = 4.4, 4.0 Hz, 1H, 4-H), 5.04 (dd, J = 6.4 and 4.4 Hz,
1H 3-H), 6.90 (d, J = 8.9 Hz, 2H, ph-H), 7.26 (d, J = 8.9 Hz, 2H, Ph-H).
13C NMR (100 MHz, CDCl3) δ 12.1, 20.9, 21.1, 38.1, 56.2, 56.8, 66.1,
75.1, 76.7, 114.2, 128.9, 129.2, 158.4, 170.1, 170.3. MS (EI): m/z
(%) = 321 (40) [M+], 203 (1 0 0), 190 (80); Analytical Calculation for
C16H21NO4: C, 65.96; H, 7.27; N, 4.81. Found: C, 65.99; H 7.28; N,
4.86.
4.1.6.6. (2R,3R,4R,5R)-1,2-dimethyl-5-(p-tolyl)pyrrolidine-3,4-diyl
diacetate (12f). The colorless oil (75%) was purified using silica gel
column chromatography (petroleum ether-ethyl acetate, 4:1). [α]D = -
26.20 (c = 2.0, MeOH), IR (neat) ν 2940, 1743, 1600 cm−1
.
1H NMR
C
17H23NO5: C, 63.54; H, 7.21; N, 4.36. Found: C, 63.60; H, 7.32; N,
(400 MHz, CDCl3) δ 1.13 (d, J = 6.5 Hz, 3H, CH3), 2.11 (s, 3H, CH3CO),
2.13 (s, 3H, CH3CO), 2.22 (s, 3H, NCH3), 2.35 (s, 3H, CH3), 3.65–3.69
(m, 1H, 5- H), 4.14 (d, J = 6.5 Hz, 1H, 2-H), 4.48 (dd, J = 4.2, 3.98 Hz,
1H, 4-H), 5.20 (dd, J = 6.5 and 4.2 Hz, 1H 3-H), 7.16 (d, J = 8.8 Hz,
2H, ph-H), 7.18 (d, J = 8.8 Hz, 2H, Ph-H). 13C NMR (100 MHz, CDCl3) δ
11.5, 21.1, 21.3, 22.8, 39.3, 58.4, 65.4, 75.7, 76.8, 126.1, 126.4, 128.8,
129.2, 133.9, 135.5, 170.7, 170.9. MS (EI): m/z (%) = 305 (30) [M+],
187 (40), 174 (1 0 0); Analytical Calculation for C17H23NO4: C, 66.86;
H, 7.59; N, 4.59. Found: C, 66.90; H 7.66; N, 4.61.
4.38.
4.1.6.2. (2R,3R,4R,5R)-2-(3,4-dimethoxyphenyl)-1,5-
dimethylpyrrolidine-3,4-diyl diacetate (12b). The colorless oil (68%) was
purified using silica gel column chromatography (petroleum ether-ethyl
acetate, 1:1). [α]D = –22.31 (c = 2.0, MeOH), IR (neat) ν 2950, 1740,
1600 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.20 (d, J = 6.8 Hz, 3H, CH3),
2.10 (s, 3H, CH3CO), 2.12 (s, 3H, CH3CO), 2.20 (s, 3H, NCH3), 3.85 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 3.66–3.70 (m, 1H, 5- H), 4.22 (d,
J = 6.9 Hz, 1H, 2-H), 4.40 (dd, J = 4.6, 4.2 Hz, 1H, 4-H), 5.14 (dd,
J = 6.9 and 4.6 Hz, 1H 3-H), 6.75–6.82 (m, 2H, ph-H), 6.95 (s, 1H, Ph-
H). 13C NMR (100 MHz, CDCl3) δ 11.6, 21.1, 21.3, 38.8, 55.3, 56.7,
65.1, 76.3, 76.9, 113.7, 129.9, 130.8, 159.2, 170.4, 170.9. MS (EI): m/z
(%) = 351 (30) [M+], 235 (70), 220 (1 0 0); Analytical Calculation for
4.1.6.7. (2R,3R,4R,5R)-2-(2-fluoro-4,5-dimethoxy-3-methylphenyl)-1,5-
dimethylpyrrolidine-3,4-diyl diacetate (12 g). The colorless oil (73%) was
purified using silica gel column chromatography (petroleum ether-ethyl
acetate, 2:1). [α]D = –32.38 (c = 2.0, MeOH), IR (neat) ν 2950, 1742,
1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 1.12 (d, J = 6.5 Hz, 3H CH3),
C
18H25NO6: C, 61.52; H, 7.17; N, 3.99. Found: C, 61.66; H, 7.18; N,
2.11 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.24 (s, 3H, NCH3), 2.34 (s, 3H, CH3),
3.67–3.71 (m, 1H, 5- H), 3.83 (s, OCH3), 3.85 (s, OCH3), 4.29 (d,
J = 6.5 Hz, 1H, 2-H), 4.47 (dd, J = 4.4, 4.0 Hz, 1H, 4-H), 5.24 (dd,
J = 6.5 and 4.4 Hz, 1H 3-H), 7.09 (s, 1H, Ph-H). 13C NMR (100 MHz,
CDCl3) δ 20.5, 20.7, 21.1, 29.8, 49.8, 56.3, 60.8, 75.6, 85.4, 117.6,
118.5, 129.8, 144.8, 151.7, 160.0, 170.7, 171.9. MS (EI): m/z
(%) = 383 (40) [M+], 265 (50), 252 (1 0 0); Analytical Calculation
for C19H26FNO6: C, 59.52; H, 6.84; F, 4.96; N, 3.65. Found: C, 59.75; H,
6.98; F, 5.03; N, 3.75.
3.88.
4.1.6.3. 4.1.6.3.(2R,3R,4R,5R)-2-(2-fluoro-4-methoxyphenyl)-1,5-
dimethylpyrrolidine-3,4-diyldiacetate (12c). The colorless oil (66%) was
purified using silica gel column chromatography (petroleum ether-ethyl
acetate, 2:1). [α]D = –32.21 (c = 2.0, MeOH), IR (neat) ν 2955, 1744,
1610 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.12 (d, J = 6.6 Hz, 3H, CH3),
2.11 (s, 3H, CH3CO), 2.13 (s, 3H, CH3CO), 2.26 (s, 3H, NCH3),
3.61–3.63 (m, 1H, 5- H), 3.87 (s, 3H, OCH3), 4.18 (d, J = 6.7 Hz, 1H,
2-H), 4.47 (dd, J = 4.6, 4.0 Hz, 1H, 4-H), 5.09 (dd, J = 6.7 and 4.0 Hz,
1H 3-H), 6.92–6.98 (m, 2H, ph-H), 7.15 (s, 1H, Ph-H). 13C NMR
(100 MHz, CDCl3) δ 20.0, 20.2, 31.3, 55.0, 56.0, 71.2, 80.3, 125.5,
130.1, 139.2, 141.3, 157.3, 160.9, 169.9, 170.1. MS (EI): m/z
(%) = 339 (40) [M+], 221 (1 0 0), 208 (70); Analytical Calculation
for C17H22FNO5: C, 60.17; H, 6.53; F, 5.60; N, 4.13. Found: C, 60.25; H,
6.58; F, 5.71; N, 4.18.
4.1.7. General procedure for the synthesis of (-)-codonopsinine (1),
(-)-codonopsine (2) and codonopsinine analogues (13c–g)
A stirred solution of the compound 12a-g (2.4 mmol) in dry me-
thanol (15–20 ml) was cooled to 0 °C and then treated with a freshly
prepared solution of 0.1 M sodium methoxide (3–5 ml). Stirring then
continued at room temperature for 2–3 h and the solution was stirred
with Amberlite IR-120 (H+) until the solution became neutral. The
resin was filtered off, and the solution was concentrated under reduced
pressure to give 1, 2 and 13-c-g.
4.1.6.4. 4.1.6.4.(2R,3R,4R,5R)-2-(4-(methoxymethyl)phenyl)-1,5-
dimethylpyrrolidine-3,4-diyl diacetate (12d). The colorless oil (69%) was
purified using silica gel column chromatography (petroleum ether-ethyl
acetate, 2:1). [α]D = -26.50 (c = 2.0, MeOH), IR (neat) ν 2943, 1742,
1600 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.18 (d, J = 6.7 Hz, 3H, CH3),
2.09 (s, 3H, CH3CO), 2.12 (s, 3H, CH3CO), 2.20 (s, 3H, NCH3), 3.43 (s,
3H, OCH3), 3.59–3.63 (m, 1H, 5- H), 4.10 (d, J = 6.7 Hz, 1H, 2-H), 4.40
(dd, J = 4.6, 3.98 Hz, 1H, 4-H), 4.87 (s, 2H, OCH2), 5.10 (dd, J = 6.7
and 4.6 Hz, 1H 3-H), 7.06 (d, J = 8.6 Hz, 2H, ph-H), 7.10 (d,
J = 8.6 Hz, 2H, Ph-H). 13C NMR (100 MHz, CDCl3) δ 11.2, 21.1, 21.3,
39.4, 56.9, 58.8, 65.1, 75.9, 76.9, 77.8, 126.2, 126.9, 128.7, 129.1,
134.1, 138.9, 170.7, 170.9. MS (EI): m/z (%) = 335 (20) [M+], 188
(1 0 0), 175 (60); Analytical Calculation for C18H25NO5: C, 64.46; H,
7.51; N, 4.18. Found: C, 64.59; H, 7.62; N, 4.28.
4.1.7.1. (2R,3R,4R,5R)-2-(4-methoxyphenyl)-1,5-dimethylpyrrolidine-
3,4-diol [(-)-codonopsinine] (1)21,25,50. The white needles (90%) mp
168–170 °C22,35 was purified using silica gel column chromatography
(ethyl acetate–methanol, 2:1). [α]D = − 10.5 (c = 1.1, MeOH); IR
(neat) ν 3365, 2950, 1610 cm−1
;
1H NMR (400 MHz, CDCl3) δ 1.34 (d,
J = 6.8 Hz, 3H, CCH3), 2.24 (s, 3H, NCH3), 3.64 (s, 3H, OCH3),
3.67–3.72 (m, 1H, 5- H), 4.06 (d, J = 6.8 Hz, 1H, 2-H), 4.39 (dd,
J = 4.4 and 3.8 Hz, 1H, 4-H), 4.65 (dd, J = 6.8 and 4.4 Hz, 1H, 3-H),
5.26 (br s, OH) 6.95 (d, J = 8.7 Hz, 2H, ph-H), 7.32 (d, J = 8.7 Hz, 2H,
ph-H). 13C NMR (100 MHz, CDCl3) δ 14.3, 35.2, 55.5, 65.7, 74.6, 85.6,
88.0, 114.7, 130.2, 135.4, 160.1. MS (EI): m/z (%) = 237 (20) [M+],
205 (50), 190 (1 0 0); Analytical Calculation for C13H19NO3: C, 65.80;
H, 8.07; N, 5.90. Found: C, 65.85; H, 8.08; N, 5.95.
4.1.6.5. (2R,3R,4R,5R)-1,2-dimethyl-5-phenylpyrrolidine-3,4-diyl
diacetate (12e). The colorless oil (79%) was purified using silica gel
column chromatography (petroleum ether-ethyl acetate, 1:1). [α]D = -
4.1.7.2. (2R,3R,4R,5R)-2-(3,4-dimethoxyphenyl)-1,5-
dimethylpyrrolidine-3,4-diol [(-)-codonopsine] (2)24. The white needles
(92%) mp 149–150 °C was purified using silica gel column
46.10 (c = 2.0, MeOH), IR (neat) ν 2940, 1742, 1610 cm−1
.
1H NMR
8

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
chromatography (ethyl acetate–methanol, 2:1). [α]D = − 16 (c = 1.2,
MeOH), IR (neat) ν 3360, 2955, 1610 cm−1 1H NMR (400 MHz, CDCl3)
δ 1.29 (d, J = 6.7 Hz, 3H, CH3), 2.20 (s, 3H, NCH3), 3.64–3.37 (m, 1H,
5- H), 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.19 (d, J = 6.7 Hz, 1H,
2-H), 4.43 (dd, J = 4.7, 4.4 Hz, 1H, 4-H), 5.14 (dd, J = 6.7 and 4.4 Hz,
1H 3-H), 5.30 (br s, OH), 6.80–6.83 (m, 2H, ph-H), 7.01 (s, 1H, Ph-H).
13C NMR (100 MHz, CDCl3) δ 13.0, 37.4, 56.6, 59.2, 67.2, 77.1, 78.9,
111.7, 125.1, 130.4, 152.1, 155.2. MS (EI): m/z (%) = 267 (40) [M+],
235 (60), 220 (1 0 0); Analytical Calculation for C14H21NO4: C, 62.90;
H, 7.92; N, 5.24. Found: C, 62.96; H, 7.98; N, 5.28.
4.1.7.7. (2R,3R,4R,5R)-2-(2-fluoro-4,5-dimethoxy-3-methylphenyl)-1,5-
dimethylpyrrolidine-3,4-diol (13 g). The colorless oil (93%) was purified
using silica gel column chromatography (petroleum ether-ethyl acetate,
3:1). [α]D = -12.60 (c = 1.0, MeOH), IR (neat)
ν 3360, 2950,
1600 cm−1
.
1H NMR (400 MHz, CDCl3) δ 1.14 (d, J = 6.5 Hz, 3H,
CH3), 2.26 (s, 3H, NCH3), 2.35 (s, 3H, CH3), 3.65–3.69 (m, 1H, 5- H),
3.81 (s, OCH3), 3.83 (s, OCH3), 4.29 (d, J = 6.5 Hz, 1H, 2-H), 4.45 (dd,
J = 4.4, 4.0 Hz, 1H, 4-H), 5.23 (dd, J = 6.5 and 4.4 Hz, 1H 3-H), 5.34
(br s, OH), 7.06 (s, 1H, Ph-H). 13C NMR (100 MHz, CDCl3) δ 11.3, 18.5,
39.0, 56.4, 59.5, 60.8, 63.4, 75.0, 82.4, 100.8, 111.6, 144.8, 150.7,
160.8. MS (EI): m/z (%) = 299 (60) [M+], 265 (70), 252 (1 0 0);
Analytical Calculation for C15H22FNO4: C, 60.19; H, 7.41; F, 6.35; N,
4.68. Found: C, 60.30; H, 7.48; F, 6.42; N, 4.73.
4.1.7.3. (2R,3R,4R,5R)-2-(2-fluoro-4-methoxyphenyl)-1,5-
dimethylpyrrolidine-3,4-diol (13c). The colorless oil (87%) was purified
using silica gel column chromatography (petroleum ether-ethyl acetate,
4.2. Preparation of solid lipid nanoparticles
2:1). [α]D = –22.20 (c = 1.0, MeOH), IR (neat)
ν 3365, 2950,
1610 cm−1 1H NMR (400 MHz, CDCl3) δ 1.20 (d, J = 6.5 Hz, 3H,
CH3), 2.23 (s, 3H, NCH3), 3.54–3.57 (m, 1H, 5- H), 3.79 (s, OCH3), 4.20
(d, J = 6.5 Hz, 1H, 2-H), 4.43 (dd, J = 4.4, 4.0 Hz, 1H, 4-H), 5.11 (dd,
J = 6.5 and 4.0 Hz, 1H 3-H), 5.25 (br s, OH), 7.04 (m, 2H, ph-H), 7.20
(s, 1H, Ph-H). 13C NMR (100 MHz, CDCl3) δ 11.7, 39.1, 59.0, 61.7, 75.8,
80.8, 103.9, 109.8, 134.2, 136.6, 158.6, 161.6. MS (EI): m/z (%) = 255
(50) [M+], 221 (1 0 0), 208 (80); Analytical Calculation for
Solid lipid nanoparticles (SLNs) were prepared using Compritol 888
(Saint-Priest Cedex, France) as a solid lipid and Pluronic f68 (Merck
KGaA, Darmstadt, Germany) as a surfactant using hot emulsification-
ultrasonication technique.51 For that, the specified weights of tested
compounds 1, 2 and 13c-g were dispersed in Compritol (5%) by heating
at 75 °C (above lipid melting point). The aqueous phase was prepared
by dissolving Pluronic f68 in double distilled water to make a 3% so-
lution. The melted lipid phase was dispersed in the aqueous surfactant
solution at the same temperature and gently mixed to prepare a pre-
emulsion. The obtained pre-emulsion was, then, homogenized at
10,000 rpm (at 75 °C) for 15 min (Kinematica Polytron PT-MR 3100 D
Homogenizer, Switzerland) followed by sonication (Rivotek,probe so-
nicator, Mumbai, India) for 10 min to reduce the globule size of the
prepared hot oil in water (o/w) emulsion. The mixture was cooled by
sudden immersion in an ice bath to stimulate lipid crystallization and
the formation of SLNs. The applied percentage of lipid and surfactant
were selected based on preliminary data (not shown) and proposed to
give the optimum SLNs characteristics.
C
13H18FNO3: C, 61.16; H, 7.11; F, 7.44; N, 5.49. Found: C, 61.17; H,
7.13; F, 7.50; N, 5.63.
4.1.7.4. (2R,3R,4R,5R)-2-(4-(methoxymethyl)phenyl)-1,5-
dimethylpyrrolidine-3,4-diol (13d). The colorless oil (93%) was purified
using silica gel column chromatography (petroleum ether-ethyl acetate,
3:1). [α]D = -16.50 (c = 1.0, MeOH), IR (neat)
ν 3360, 2955,
1610 cm−1
.
1H NMR (400 MHz, CDCl3) δ 1.24 (d, J = 6.5 Hz, 3H,
CH3), 2.28 (s, 3H, NCH3), 3.45 (s, 3H, OCH3), 3.69–3.73 (m, 1H, 5- H),
4.12 (d, J = 6.5 Hz, 1H, 2-H), 4.44 (dd, J = 4.4, 4.0 Hz, 1H, 4-H), 4.79
(s, 2H, OCH2–), 5.13 (dd, J = 6.5 and 4.4 Hz, 1H 3-H), 5.32 (br s, OH),
7.10 (d, J = 8.6 Hz, 2H, ph-H), 7.15 (d, J = 8.6 Hz, 2H, Ph-H). 13C NMR
(100 MHz, CDCl3) δ 11.4, 39.7, 58.9, 59.8, 65.7, 74.9, 76.8, 81.7,
126.3, 126.8, 129.1, 129.7, 134.5, 135.7MS (EI): m/z (%) = 351 (40)
[M+], 219 (1 0 0), 204 (70); Analytical Calculation for C14H21NO3: C,
66.91; H, 8.42; N, 5.57. Found: C, 66.99; H, 8.51; N, 5.67.
4.2.1. Characterization of the prepared SLNs
The prepared solid lipid nanoparticles were evaluated by mea-
suring:
4.2.1.1. Particle size (P.S.) and size distribution. Photon Correlation
Spectroscopy (PCS) using a Zetasizer 4 (Nano ZS, Zen 3600, Malvern
Instruments Ltd., UK) was applied to measure the average diameter
(nm) and Polydispersity Index (PDI) of the prepared SLN formulae at
25 °C without dilution. Each value is the average of three
measurements.
4.1.7.5. (2R,3R,4R,5R)-1,2-dimethyl-5-phenylpyrrolidine-3,4-diol
(13e). The colorless oil (88%) was purified using silica gel column
chromatography (petroleum ether-ethyl acetate, 1:1). [α]D = -26.20
(c = 1.0, MeOH), IR (neat)
ν
3360, 2945, 1600 cm−1 1H NMR
(400 MHz, CDCl3) δ 1.21 (d, J = 6.4 Hz, 3H, CH3), 2.28 (s, 3H,
NCH3), 3.50–3.54 (m, 1H, 5- H), 4.17 (d, J = 6.4 Hz, 1H, 2-H), 4.48
(dd, J = 4.4, 4.0 Hz, 1H, 4-H), 5.14 (dd, J = 6.4 and 4.4 Hz, 1H 3-H),
5.32 (br s, OH), 7.25–7.36 (m, 5H, ph-H). 13C NMR (100 MHz, CDCl3) δ
11.4, 39.4, 59.4, 68.5, 76.5, 80.1, 124.1, 125.4, 126.1, 128.5, 129.2,
138.6. MS (EI): m/z (%) = 207 (40) [M+], 173 (50), 160 (1 0 0);
Analytical Calculation for C12H17NO2: C, 69.54; H, 8.27; N, 6.76.
Found: C, 69.70; H 8.37; N, 6.81.
4.2.1.2. Zeta potential measurement (Z.P.). For determination of
electrical surface properties of the prepared SLN formulations, Z.P.
was quantified using a Zetasizer 4 (Nano ZS, Zen 3600, Malvern
Instruments Ltd., UK), at 25 °C after suitable dilution with distilled
water to an adequate intensity at pH 6.5–7.5. The value was measured
in triplicate.
4.1.7.6. (2R,3R,4R,5R)-1,2-dimethyl-5-(p-tolyl)pyrrolidine-3,4-diol
(13f). The colorless oil (90%) which was isolated by column
chromatography on silica gel (petroleum ether/ethyl acetate, 2:1).
[α]D = -36.10 (c = 1.0, MeOH), IR (neat) ν 3365, 2950, 1610 cm−1
1H NMR (400 MHz, CDCl3) δ 1.14 (d, J = 6.6 Hz, 3H, CH3), 2.23 (s, 3H,
NCH3), 2.36 (s, 3H, CH3), 3.64–3.67 (m, 1H, 5- H), 4.16 (d, J = 6.6 Hz,
1H, 2-H), 4.48 (dd, J = 4.2, 3.98 Hz, 1H, 4-H), 5.201(dd, J = 6.5 and
4.2 Hz, 1H 3-H), 5.32 (br s, OH), 7.18 (d, J = 8.7 Hz, 2H, ph-H), 7.20
(d, J = 8.7 Hz, 2H, Ph-H). 13C NMR (100 MHz, CDCl3) δ 11.7, 21.5,
39.8, 59.7, 66.8, 81.2, 126.3, 126.5, 128.9, 129.1, 133.7, 136.3. MS
(EI): m/z (%) = 221 (40) [M+], 187 (60), 174 (1 0 0); Analytical
Calculation for C13H19NO2: C, 70.56; H, 8.65; N, 6.33. Found: C,
70.80; H, 8.66; N, 6.42.
4.2.1.3. Determination of entrapment efficiency (E.E.). Undiluted sample
(5 ml) of the prepared SLNs was centrifuged at 5000 rpm for an hour for
complete precipitation of the solid lipid layer. The aqueous layer was,
then, collected and the absorbance of the tested compound was
spectrophotometrically
measured
at
the
determined
λmax
(274–283 nm on Shimadzu UV/Vis double beam spectrophotometer)
after filtration on a 0.45 μm membrane filter. The free compound
concentration in the dispersion medium was calculated using an
equation obtained from a previously constructed standard calibration
curve. The entrapment efficiency (E.E.) percentage of the prepared
SLNs was calculated as follows:
E.E. (%) = (DI
DF/DF)100
9

A.O.H. El-Nezhawy, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
where DI = Initial compound amount added during preparation,
DF = Amount of free compound in the aqueous phase
nutrient broth (DSNB) containing the tested isolates and mixed well, by
pipetting 3–6 times. The plates were incubated at 37 °C for 18 h.55
4.2.1.4. Determination of release rate from the prepared SLN
formulations. Dialysis bag diffusion technique38 was applied to
investigate the release rate profiles of the tested compounds from the
prepared SLN formulations in comparison to non-formulated
compounds using the USP XXIV dissolution apparatus II (UDT-804
paddle, Logan, Utah, USA). Formulae and compound suspensions (3 ml)
were packed into dialysis bags (12 kDa MWCO) and immersed in
500 ml of a phosphate buffer dissolution medium (with a pH of 6.8)
4.3.3. Inoculum preparation of the test organisms (direct colony suspension
method)
Direct colony suspension method was performed by suspending a
few colonies of the tested isolates in sterile normal saline. The turbidity
of the prepared suspension was adjusted using 0.5 McFarland standard
and this is equivalent to 1–2 × 108 CFU/ml, which is further diluted to
obtain a final inoculum concentration of 5 × 105 CFU in each well of
the microtiter plates. This was done by adding 35 µl from 0.5
McFarland adjusted inoculum to 35 ml of DSNB. The inoculated broth
was used directly within 15 min of preparation.55
without enzymes and kept at 37
0.5 °C with constant stirring speed
(50 rpm). At pre-specified time intervals, 5 ml samples were withdrawn
with replacement for 12 h and the cumulative percentage release rate of
the tested compound was calculated as previously described in E.E.
determination. The mean of six determinations was considered.
Acknowledgment
This study was funded by the Deanship of Scientific Research, Taif
University, KSA (Research Project No. 1-438-5843).
Disclosure statement.
4.2.1.5. In-vitro permeation studies. In-vitro permeation of the prepared
SLN formulations in comparison to non-formulated compound aqueous
dispersion was studied on a dialysis membrane using glass modified
Franz diffusion cell (PermeGear, Inc., Hellertown, PA, USA). The
receptor cell was filled with fresh phosphate buffer (pH = 6.8) kept
All authors declare that they do not have any conflict of interest.
References
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0.5 °C with constant stirring using a magnetic stirrer. One ml
of each SLN formula and the compound suspension were separately
packed into the donor chamber placed in contact to the dialysis
membrane and the opening of donor cell was sealed. At
predetermined time intervals, samples were withdrawn from the
receptor chamber with replacement for six hours. The sample
absorbance was spectrophotometrically measured and the
concentration was calculated using the equation generated from a
standard calibration curve as described.52
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