Isolation, synthesis, and cytotoxicity evaluation of two impurities in nomegestrol acetate

Article abstract of DOI:10.1002/ardp.201800295

Nomegestrol acetate (NOMAc) is a synthetic progesterone analog and classified as a fourth-generation progestin. It has been approved in many countries for oral contraception, hormonal replacement therapy (HRT), and treatment of various gynecological disorders. There are several synthetic routes reported for the synthesis of NOMAc and they all share the very similar last three to five steps toward the conversion of 6-methylene to 6-methyl-6,7-unsaturated structure. Therefore the final product from different processing routes may have similar impurity profiles. In the analysis of NOMAc, we identified two impurities, impurity A (listed in EP 8.0) and impurity B (not specified in EP 8.0). Both impurities were further confirmed by synthesis. In addition, both impurities and NOMAc were evaluated for their in vitro cytotoxicities against L02 liver cells, mesenchymal stem cells, MCF-7 breast cancer cells, and C33A cervical cancer cells. These three analogs are not cytotoxic to the four cell lines at low concentrations (<20 μM). NOMAc and impurity A showed cytotoxicity to L02, MCF-7, and C33A cells at high concentrations, while impurity B did not show significant cytotoxicity to any of the cell lines tested.

Full text of DOI:10.1002/ardp.201800295

Received: 11 October 2018
Revised: 29 November 2018
Accepted: 5 December 2018
|
|
DOI: 10.1002/ardp.201800295
FULL PAPER
Isolation, synthesis, and cytotoxicity evaluation of two
impurities in nomegestrol acetate
1
1
1
1
|
|
|
|
Bao-Cheng Xie
Shu-Yong Song
Xiao-Yang Xie
Yu-Tong Sun
2
1
1
|
|
Xiao-Yuan Zhang
Dao-Hua Xu
Yun-Sheng Huang
1
Guangdong Medical University School of
Pharmacy, Dongguan, China
Abstract
2
Center for Research and Development,
Nomegestrol acetate (NOMAc) is a synthetic progesterone analog and classified as a
fourth-generation progestin. It has been approved in many countries for oral
contraception, hormonal replacement therapy (HRT), and treatment of various
gynecological disorders. There are several synthetic routes reported for the synthesis
of NOMAc and they all share the very similar last three to five steps toward the
conversion of 6-methylene to 6-methyl-6,7-unsaturated structure. Therefore the final
product from different processing routes may have similar impurity profiles. In the
analysis of NOMAc, we identified two impurities, impurity A (listed in EP 8.0) and
impurity B (not specified in EP 8.0). Both impurities were further confirmed by
synthesis. In addition, both impurities and NOMAc were evaluated for their in vitro
cytotoxicities against L02 liver cells, mesenchymal stem cells, MCF-7 breast cancer
cells, and C33A cervical cancer cells. These three analogs are not cytotoxic to the four
cell lines at low concentrations (<20 μM). NOMAc and impurity A showed cytotoxicity
to L02, MCF-7, and C33A cells at high concentrations, while impurity B did not show
significant cytotoxicity to any of the cell lines tested.
Lijiang Yinghua Biochemical and
Pharmaceutical Co. Ltd., Lijiang, Yunnan,
China
Correspondence
Dr. Yun-Sheng Huang and Dr. Dao-Hua Xu,
Guangdong Medical University School of
Pharmacy, 1 Xincheng Ave, Songshan Lake
Technology Park, Dongguan 523808, China.
Email: yshuang@gdmu.edu.cn (Y.-S.H.);
daohuaxu@gdmu.edu.cn (D.-H.X.)
Funding information
Natural Science Foundation of Guangdong
Province, Grant number: 2016A030313676;
National Science and Technology Support
Program, Grant number: 2016YFC1000904
K E Y W O R D S
cytotoxicity, impurity, nomegestrol acetate, progestin, steroid, synthesis
®
®
®
®
1
|
INTRODUCTION
Zoley , Lutenyl , Uniplant , Naemis , etc. in many countries as oral
[7–11]
contraceptives (OCs) and hormone replacement therapeutics.
Nomegestrol acetate (NOMAc), or 6-methyl-3,20-dioxo-19-norpre-
gna-4,6-dien-17-yl acetate, is a fourth generation synthetic analog of
progesterone. It has high affinity and selectivity for human progester-
one receptor (PR) and is devoid of any estrogenic, androgenic,
glucocorticoid or mineralocorticoid activity. In addition, it has excellent
anti-gonadotropic activity, good progesterone receptor-mediated
Unlike earlier generations of progestins that were derived from the
structure of testosterone (estranes and gonanes), NOMAc and other
fourth generation of progestins, including dienogest (DNG), trimege-
stone (TMG), and nesterone (NES), were derived from the structure of
[12–14]
pregnane.
These newly developed progestins are also called
pure progestins due to their high affinity for PR and lack of affinities for
[15]
[1–6]
anti-estrogenic activity, and moderate anti-androgenic activity.
other steroidal receptors.
Progestins inhibit ovulation by suppress-
[16]
NOMAc has been approved and marked by commercial names such as
ing the natural production of menstrual-cycle hormones,
and
are often used in combination with an estrogen for oral contracep-
[
17–19]
[20,21]
[22–24]
tion,
HRT,
endometrosis,
and gynecological
|
1 of 10
Bao-Cheng Xie and Shu-Yong Song contributed equally to this work.
Arch Pharm Chem Life Sci. 2018;e1800295.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
https://doi.org/10.1002/ardp.201800295

2
of 10
XIE ET AL.
|
disorders, such as menstrual disturbances, dysmenorrhoea, and
line that is a frequent choice to study hepatocellular toxicity of test
drugs.[38–40] MSCs play important roles for tissue repair and
regeneration and can differentiate into a variety of cell types, including
osteoblasts, adipocytes, chondrocytes, myoblasts, and neurons, thus
[25,26]
premenstrual syndrome.
Studies indicated that the use of earlier
[27,28]
generations of progestins may have some minor side effects,
but
their long-term use have been credited with some highly beneficial
effects in prevention of osteoporosis, breast, ovarian, and endometrial
[41–44]
are often used as cell substrates for cytotoxicity assays.
Breast
[45]
[29–32]
cancers.
NOMAc and other fourth generation progestins are
cancer and cervical cancer are highly associated with estradiol.
expected to have fewer or no undesired side effects and are expected
to offer even more health benefits to long-term users.
NOMAc has demonstrated to reduce the production of estradiol and
[46]
thus has inhibitory activity against the growth of cancer cells.
Three synthetic methods for NOMAc (6) share the last five steps
Therefore, MCF-7 (breast cancer cells) and C-33A (cervical cancer
cells) were also used to assess the anticancer activity of impurity A,
impurity B, and NOMAc.
(
Scheme 1), the differences among them are the methods of obtaining
[33–35]
the precursor 1.
Another synthetic method for NOMAc also has
similar steps in the early stage and is followed by the modification at
[36]
the 17 carbon position.
Therefore, all four reported synthetic
procedures may display some common impurities resulted from the
modification at the 6 and 7 positions of the steroidal structure as
shown in Scheme 1. Pharmaceutical impurities have attracted more
and more attention from not only the scientific community but also the
2
|
RESULTS AND DISCUSSION
There are at least four reported methods for the synthesis of NOMAc;
three of them share the last five steps and the other one also has similar
chemistry in its early stages. Impurity A has been specified as a
common and hard to be removed substance in the final product.
Impurity B has not been identified as an impurity of NOMAc in EP 8.0.
One earlier patent reported its preparation and implicated its
[37]
regulatory agencies due to their potential harmful effects.
Studies
are encouraged to identify more unknown impurities and assess their
safety in normal treatment application.
For the product of NOMAc, a known impurity (impurity A) with the
structure of 6-α-methyl-3,20-dioxo-19-norpregna-4-en-17-yl acetate
has been specified in EP 8.0 with a limit of <0.2%. There are other
impurities not specifically identified in EP 8.0, but there is a combined
limit of <0.3%. In our recent analysis of NOMAc, we identified impurity
A and another impurity (B), a hydroxyl substitution at 6-methyl
position of NOMAc, or 6-hydroxymethyl-3,20-dioxo-19-norpregna-
[47]
pharmaceutical application.
Another publication implicated that
this impurity B could be a possible metabolite (metabolite #3) of
[48]
NOMAc, but there has been no confirmation of impurity B being the
metabolite #3 up to today even though crystal structure of impurity B
[49]
has been published.
However, during our analysis of NOMAc, we
identified impurity B as an impurity in the final product at below 0.1%
level. Therefore, it is not likely a metabolite of NOMAc.
4
,6-dien-17-yl acetate (Figure 1). In addition, we developed processes
to synthesize both impurity A and impurity B and obtained both in
multi-gram quantities (Schemes 2 and 3).
The introduction of impurity A is certainly due to the reduction of
the double bond of the 6-methylene of intermediate 5 or the 6,7-
double bond of NOMAc (Figure 1) during the catalytic double bond
translocation reaction. Whereas, the introduction of impurity B is likely
due to the incomplete reduction of intermediate 3 that would become
A preliminary cytotoxicity screening assay was performed for
impurity A, impurity B, and NOMAc against the survival of L02 cells
and mesenchymal stem cells (MSCs). L02 is a human normal liver cell
SCHEME 1 Common procedures for the synthesis of NOMAc

XIE ET AL.
| 3 of 10
FIGURE 1 Chemical structures of impurity A and impurity B
intermediate 8 as a result of the subsequent acidification (Scheme 4).
Intermediate 8 is then subjected to the catalytic double bond
translocation reaction to afford impurity B. Another possible (but
unlikely) route of introducing impurity B is that intermediate 4 does not
dehydrate during the acidification process and form intermediate 9
which would go through the catalytic double bond translocation
reaction and to give impurity B.
undergo epoxidation at the 6-methylene position with mCPBA to
afford intermediate 7 that was then treated with acid to form impurity
B with high purity after silica gel column chromatography (Scheme 3).
Impurities A, B, and intermediate 7 were characterized by both
1
13
H NMR and C NMR. Though impurity A is a confirmed impurity in
NOMAc, no NMR data for impurity A is available in the literature. It is
1
very difficult to assign H NMR resonance peaks to the corresponding
In order to confirm the proposed chemistry for introduction of
impurity A, we carried out a reaction using intermediate 5 to undergo
catalytic double bond translocation reaction with extended reaction
protons due to overlapped signals between 1.0 and 2.5 ppm. But
1
several resonance signals are clearly identifiable in the H NMR spectra
for both the isolated impurity A and the synthesized impurity A.
[
35]
time.
The reaction mixture did have impurity A, NOMAc, and other
Notably, ∼5.87 ppm for C4-H, ∼2.10 ppm for C-CO-CH
carbon, ∼2.05 ppm for CH -CO-O- at #23 carbon, ∼1.10–1.12 ppm
for 6-α-CH or on #24 carbon, ∼0.70 ppm for −CH on the #18 carbon.
3
at #21
by-products (data not shown). When the NOMAc was used to further
undergo catalytic double bond translocation reaction (palladium on
active carbon with added cyclohexene) at extended reaction time
3
3
3
1
Similar resonance peaks are also identifiable in the H NMR spectra for
(
(
Scheme 2), the impurity A was produced in much higher percentage
∼89%) in the reaction mixture and thus much easier to be purified.
isolated impurity B and the synthesized impurity B, such as, ∼6.36 ppm
for C4-H, ∼6.01 ppm for C7-H, ∼4.32 ppm for −CH
carbon, ∼2.10 ppm for C-CO-CH at #21 carbon, ∼2.06 ppm for CH
CO-O- at #23 carbon, ∼0.72 ppm for -CH on the #18 carbon. For
2
-OH on #24
After multi-rounds of re-crystallization, impurity A was obtained with
multi-gram quantity and in high purity. To confirm the formation of
impurity B via intermediate 8, intermediate 3 was acidified to give 8
that then underwent catalytic double bond translocation reaction
3
3
-
3
intermediate 7, a signal at ∼5.92 ppm is assigned for C4-H; a signal
at ∼3.25 ppm is assigned for 1 proton on the epoxide moiety and
∼2.87 ppm for another proton on the epoxide ring; a signal at
(
palladium on active carbon with added cyclohexene), the reaction
mixture did show an impurity B absorption (data not shown), but also
contained lots of other by-products and was hard to be purified.
Efforts to obtain intermediate 9 via acidification of intermediate 4
were not successful (always resulted in intermediate 5). In order to
obtain impurity B more efficiently, intermediate 5 was subjected to
∼2.13 ppm is assigned for C-CO-CH
3
at #21 carbon; a signal
at ∼2.08 ppm is assigned for CH -CO-O- at #23 carbon. One typical
3
resonance at ∼2.93–2.98 ppm (td) is observable on all three analogs’
1
H NMR spectra, it likely belongs to one of the two protons on #16
carbon.
SCHEME 2 Preparation of impurity A

4
of 10
XIE ET AL.
|
SCHEME 3 Preparation of impurity B
1
3
13
C NMR and C NMR-DEPT135 spectra nicely displayed each
(Table 1). For impurity A, the ∼121.57 ppm certainly belongs to C-4
13
13
13
individual carbon ( C NMR) or proton-attached ( C NMR-DEPT135)
carbon (based on C NMR-DEPT135); the signal at ∼96.71 ppm
1
3
signal. Based on the resonance signal information from both C NMR
spectra, each signal was assigned to the corresponding carbon
certainly belongs to C-17 carbon; the signal at ∼204.12 ppm likely
belongs to C-20 carbon; the signal at ∼200.07 ppm likely belongs to
SCHEME 4 Proposed mechanism of introduction of impurities A and B

XIE ET AL.
| 5 of 10
1
3
TABLE 1 Assignment of C NMR spectra of impurities A, B, intermediate 7, and NOMAc
a
a,b
A
B
7
NOMAc
C-1
35.55
26.12
200.07
121.58
170.09
50.24
40.72
38.01
42.78
40.39
25.78
30.31
47.01
48.90
23.64
31.04
96.71
14.47
37.04
26.85
200.17
121.47
156.57
135.32
139.84
41.00
45.32
40.90
24.93
30.31
47.52
48.53
23.19
30.99
96.45
14.20
36.36
26.76
37.51
26.97
C-2
C-3
C-4
124.37
121.77
C-5
C-6
C-7
38.31
41.42
50.35
37.31
25.71
30.31
139.56
41.00
45.56
41.06
24.92
30.26
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
48.32
23.47
30.91
48.66
23.16
30.98
14.47
26.49
14.19
26.40
204.12
26.37
170.74
21.27
17.76
203.94
26.46
170.69
21.20
62.78
21.26
52.34
21.20
19.32
a
13
Data were from DEPT- C NMR only.
b
Data were extracted from Ref. [35] for comparison purpose.
C-3 carbon; the signal at ∼170.74 ppm likely belongs to C-5 carbon;
the signal at ∼170.09 ppm likely belongs to C-22 carbon. Signals at
C-13 NMR spectra for impurity B and intermediate 7, as summarized in
Table 1.
∼
50.24, 48.90, 42.78, 40.39, 38.01 ppm belong to one of the 5 single
proton attached carbon units, C-6, C-8, C-9, C-10 and C-14. Signals at
40.72, 35.55, 31.04, 30.31, 26.12, 25.78 and 23.64 ppm belong to
The human liver cell line L02 and mesenchymal stem cells were
used to investigate the cytotoxicities of impurity A, impurity B, and
NOMAc. The results demonstrated that NOMAc inhibited the viability
of L02 cells line at >20 µM in both 24 and 48 h incubations, suggesting
that it is probably cytotoxic to liver at high doses (Figure 2A). It did not
inhibit the viability of human MSCs (Figure 2B). Breast and cervical
∼
one of the seven double proton attached carbon units, C-1, C-2, C-7,
C-11, C-12, C-15, and C-16. Finally, signals at ∼26.37, 21.27, 17.76,
and 14.47 ppm belong to C-21, C-23, C-24, and C-18, respectively.
Based on the same criteria, we assigned each of the corresponding
[50,51]
cancer are associated with sex hormones.
NOMAc is a selective

6
of 10
XIE ET AL.
|
FIGURE 2 Viability of cells treated with NOMAc at different concentrations. The L02 (A), MSCs (B), MCF-7 (C), or C33A (D) cells were
incubated with different concentrations of nomegestrol acetate for 24 and 48 h. The cell viability was determined by CKK-8 assay. *p < 0.05,
**p < 0.01 compared with control
progestogen and structurally similar to progesterone with anti-
estrogenic activity by inhibiting the production of estradiol. NOMAc
has been reported to have anti-proliferative effect on human breast
will not have any measurable cytotoxicity against the four cell lines
tested under normal therapeutic dosages.
[32]
and endometrial cancer cells.
Our results also indicated that
NOMAc inhibited the viability of MCF-7 breast cancer cells and C-33A
3
|
CONCLUSIONS
cervical cancer cells at ≥40 and ≥20 µM, respectively, in both 24 and
4
8 h incubations (Figure 2C and D). In the 24 h incubation assay,
Two impurities have been identified during our quality analysis of
NOMAc. Impurity A is a known impurity for NOMAc and is listed in EP
8.0 as a “must be measured” substance that has been known to be
introduced during the 6-methylene double bond catalytic transloca-
tion. Impurity B has not been identified to be an impurity in NOMAc
before and for the first time was identified and confirmed as a process
impurity due to the incomplete reduction of 6-formyl group, which was
subsequently transformed to the 6-hydroxymethyl group during the 6-
methylene double bond translocation process. Analytical methods for
both impurities were developed and could be used for NOMAc quality
control. We also developed synthetic procedures to prepare both
impurities A and B and were able to obtain both compounds in multiple
grams with high purity. At high concentrations, both NOMAc and
impurity A showed very milder cytotoxicities to L02 liver cells, MCF-7
breast cancer cells, and C-33A cervical cancer cells. Impurity B did not
show significant cytotoxicity to L02 liver cells, MCF-7 breast cancer
cells, and C-33A cervical cancer cells. All three analogs did not show
cytotoxicities to mesenchymal stem cells, instead impurities A and B
showed some stimulation of growth to mesenchymal stem cells.
Overall, the dosages used for medical treatment contain far less in vivo
concentrations of these analogs that would trigger any cytotoxicity
against the cell lines tested.
NOMAc has IC50 values greater than 100 μM against all four cell lines,
whereas in the 48 h incubation assay, it has IC50 values greater than
8
0 μM against the same cell lines.
Impurity A demonstrated similar pattern of cytotoxicities to those of
NOMAc against these four cell lines with slightly less cytotoxicity.
Impurity A inhibited the viability of L02 cells line at >20 µM, inhibited the
viability of MCF-7 at >40 µM and C33A at >60 µM (Figure 3A, C, D). It is
interesting to note that impurity A increased the viability of human MSCs
at 20 µM (Figure 3B). Impurity Bdid not show significant cytotoxicityup to
60 µM against the viability of L02, MCF-7, and C33A cell lines (Figure 4A,
C, D). It also increased the viability of human MSCs at ≥10 µM (Figure 4B).
Impurity A showed IC50 values greater than 100 μM against all four cell
lines in the 24 h incubation and greater than 100 μM against L02, MSCs,
and MCF-7, and greater than 80 μM against C33A cells in the 48 h
incubation. Whereas impurity B had IC50 values greater than 100 μM
against all four cell lines in both the 24 and 48 h incubations.
NOMAc is currently marketed with doses of 2.5, 3.75, and 5.0 mg
orally with plasma concentrations ranged from 2.5 to 5 nM, which are
far less than the concentrations that showed cytotoxictity in this study.
Impurities A and B have less than 0.2% in the API and thus have plasma
concentrations in the range of 5–10 pM. Therefore, all these analogs

XIE ET AL.
| 7 of 10
FIGURE 3 Viability of cells treated with impurity A at different concentrations. The L02 (A), MSCs (B), MCF-7 (C), or C33A (D) cells were
incubated with different concentrations of nomegestrol acetate for 24 and 48 h. The cell viability was determined by CKK-8 assay. *p < 0.05,
**p < 0.01 compared with control
4
4
|
EXPERIMENTAL
General
4.2
|
Analysis of NOMAc
Analysis of the purified product NOMAc
.1
|
4.2.1
|
[
35]
Intermediate 5 and NOMAc were obtained from Lijiang Yinghua
BioPharmaceutical Company (Lijiang, Yunnan, China). Reference
impurity A was purchased from Guangzhou Daersike Biotech Co.
The NOMAc was synthesized according to procedure of Lu et al.
and obtained in 99.67% purity at 245 nm and 99.97% purity at 290 nm
(Supporting Information Figures S1 and S2). It should be noticed that
both NOMAc and impurity B do not have good UV absorption at
245 nm, whereas impurity A does not have good absorption at 290 nm.
Thus, they have to be analyzed at different wavelengths of UV light in
order to be measured, that is, impurity A is analyzed at 245 nm,
impurity B is analyzed at 290 nm.
(
Panyu, Guangzhou). All other chemicals were purchased commercially
as reagent grade and used directly without further purification. The
reactions were monitored by analytical thin-layer chromatography
(
TLC) on silica gel F254 glass plates and visualized under UV light (254
and 365 nm). Flash column chromatography was performed on silica
1
gel (200–300 mesh). H NMR spectra were recorded with a Bruker
Avance III 400 MHz NMR spectrometer at room temperature.
Chemical shifts (in ppm) were recorded as parts per million (ppm)
downfield to tetramethylsilane (TMS). The following abbreviations are
used for multiplicity of NMR signals: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; dd, doublet of doublets; td, triplet of doublets; dt,
4
.2.2 Purification of impurity A from the mother
|
liquid of NOMAc
A 5.0 kg of mother liquid in methanol solution from the crystallization
of NOMAc (supplied by Lijiang Yinghua BioPharmaceutical Company)
was repeatedly concentrated to get the majority of NOMAc out to
finally obtain 300 mL enriched solution that was then chromato-
graphed on silica gel column with petroleum ether/ethyl acetate (10:1
to 4:1) gradient elution to obtain the crude impurity A (with NOMAc)
and impurity B crudes, respectively. The crude impurity A was
subjected to numerous chromatography on silica gel column (for a total
1
3
doublet of triplets; dq, doublet of quartets; b, broad; C NMR or
13
DEPT-
C
(distortionless enhancement by polarization transfer
C NMR) spectra were recorded with Bruker Avance III 400 MHz
NMR spectrometer (100 MHz) and calibrated with CDCl
δ = 77.23 ppm). LC-mass spectrum was recorded with a Bruker
1
3
3
(
amaZon SL instrument. High-resolution mass spectra were recorded
with a LTQOrbitrap XL (ThermoFisher) mass spectrometer.
of six rounds) with petroleum ether/ethyl acetate (50:1 to 5:1) gradient
1
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
elution to finally give 87 mg of impurity A. H NMR (CDCl
3
, ppm): 5.86
(s, 1H), 2.92–2.97 (td, 1H), 2.37–2.41 (m, 1H), 2.21–2.30 (m, 3H), 2.10

8
of 10
XIE ET AL.
|
FIGURE 4 Viability of cells treated with impurity B at different concentrations. The L02 (A), MSCs (B), MCF-7 (C), or C33A (D) cells were
incubated with different concentrations of nomegestrol acetate for 24 and 48 h. The cell viability was determined by CKK-8 assay. *p < 0.05,
**p < 0.01 compared with control
(
(
(
s, 3H), 2.05 (s, 3H), 1.87–2.00 (m, 3H), 1.70–1.82 (m, 5H), 1.54–1.59
4.3.2 Analysis of impurity A
|
m, 2H), 1.30–1.33 (m, 2H), 1.10–1.12 (d, 3H), 0.84–1.05 (m, 2H), 0.69
The synthesized impurity A was analyzed at both 245 and 290 nm and
showed it has a very weak absorption at 290 nm (Supporting
Information Figure S3). It showed a purity of 99.58% at 245 nm
with a retention time of 19.367 min. The reference impurity A was
analyzed at the same condition and showed a purity of 99.13% at
13
s, 3H); C NMR (DEPT, CDCl
3
, ppm): δ 121.57, 50.24, 48.90, 42.78,
4
2
1
0.72, 40.39, 38.01, 35.55, 31.04, 30.31, 26.37, 26.12, 25.78, 23.64,
1
13
1.27, 17.76, 14.47. Note: Both the H NMR and the C NMR DEPT-
35 spectra indicated the presence of small amounts of impurities in
the isolated impurity A.
2
45 nm with a retention time of 19.414 min (Supporting Information
Figure S4). When mixed of the synthesized impurity A and the
reference impurity A at 1:1 ratio, the mixture showed a purity of
4
.3
|
Synthesis and analysis of impurity A
Synthesis of impurity A
9
9.42% at 245 nm with a retention time of 19.442 min (Supporting
Information Figure S5).
4
.3.1
|
To a 500 mL round bottle was added ethanol (300 mL), 5% Pd/C (5 g),
cyclohexene (5 mL). The solution was heated to reflux and was added
NOMAc (15 g, 40.5 mmol) and continued to stir under reflux for 2 h.
The reaction was cooled to rt and filtered off the solid, concentrated to
dryness. The crude was recrystallized from acetone three times and
4
.4
|
Synthesis and analysis of impurity B
Purification of impurity B from the mother
4
.4.1
|
liquid of NOMAc
then from ethanol twice to afford impurity A (5.2 g, yield 34%, HPLC
The crude impurity B (from above 4.1.2.) was repeatedly chromato-
1
9
1
3
2
3
1
3
9.58% at 245 nm). Mp 173–174°C; H NMR (CDCl
3
, ppm): 5.88 (s,
graphed on silica gel column (for a total of four rounds) with petroleum
H), 2.92–2.99 (td, 1H), 2.37–2.44 (m, 1H), 2.20–2.33 (m, 3H), 2.11 (s,
H), 2.06 (s, 3H), 1.87–2.01 (m, 3H), 1.67–1.82 (m, 5H), 1.48–1.59 (m,
H), 1.26–1.37 (m, 2H), 1.11–1.13 (d, 3H), 0.91–1.08 (m, 2H), 0.70 (s,
ether/ethyl acetate (20:1 to 4:1) gradient elution to finally give 44 mg
1
impurity B. H NMR (CDCl
3
, ppm): 6.36 (s, 1H), 6.01 (s, 1H), 4.32–4.33
(d, 2H), 2.96–3.03 (td, 1H), 2.54–2.58 (d, 1H), 2.30–2.35 (t, 3H), 2.15–
2.20 (t, 1H), 2.10 (s, 3H), 2.06 (s, 3H), 1.93–1.99 (m, 4H), 1.57–1.61 (m,
13
3
H); C NMR (CDCl , ppm): δ 204.12, 200.07, 170.74, 170.09,
[47]
21.57, 96.71, 50.25, 48.92, 47.01, 42.80, 40.74, 40.41, 38.02, 35.55,
4H), 1.25–1.50 (m, 2H), 1.24–1.27 (m, 2H), 0.72 (s, 3H); Ref.
1
1.05, 30.33, 26.37, 26.13, 25.79, 23.64, 21.26, 17.76, 14.48; ESI-MS:
H NMR: 6.37 (s, 1H), 6.01 (s, 1H), 4.34 (s, 2H), 2.10 (s, 3H), 2.07 (s, 3H),
13
+
m/z 373.2349 [M+1] ; calcd. MW for C23
H
32
O
4
372.2301. Anal. calcd.
3
1.63 (s, 1H), 0.73 (s, 3H); C NMR (DEPT, CDCl , ppm): δ 139.79,
(
%) for C23
H
32
O
4
: C 74.16, H 8.66. Found: C 74.05, H 8.52.
121.43, 62.71, 48.54, 45.33, 41.01, 40.89, 37.39, 30.99, 30.30, 26.85,

XIE ET AL.
| 9 of 10
+
3
2
6.44, 24.93, 23.18, 21.18, 14.19; ESI-MS: m/z 387.3 [M+1] ; calcd.
approximately 5 × 10 cells per well in 96-well plates. NOMAc,
impurity A and impurity B were dissolved in DMSO. The solutions were
then added to the culture medium at the concentrations indicated with
final concentration of DMSO at less than 0.1% for 24 and 48 h. The
cytotoxicity was assessed by the CCK-8. The absorbance at 450 nm
was measured by a microplate reader.
MW for C23 386.21.
30 5
H O
4
.4.2 Synthesis of impurity B
|
To a 200 mL round bottle was added intermediate 5 (5.0 g, 13.5 mmol),
dichloromethane (50 mL), and 3-chloroperbenzoic acid (5.0 g,
2
9.0 mmol) in dichloromethane (20 mL). The solution was stirred at
4
.6
|
Statistical analysis
refluxing for 4 h, cooled to rt and poured onto ice-water, extracted
with dichloromethane, washed with brine, dried over sodium sulfate.
The solvent was removed in vacuo and the residue was purified by a
All data were presented as mean ± SD and statistical analysis was
performed using one-way analysis of variance (one-way ANOVA)
among multiple groups. A p-value less than 0.05 was considered
significant for all analysis.
silica gel column to give 7 (3.5 g, yield 67%, HPLC 98.8% at 254 nm).
1
3
H NMR (CDCl , ppm): 5.92–5.93 (d, 1H), 3.25–3.26 (d, 1H), 2.93–2.99
(
td, 1H), 2.87–2.88 (d, 1H), 2.36–2.46 (m, 3H), 2.13 (s, 3H), 2.08 (s, 3H),
.97–2.04 (m, 2H), 1.87–1.92 (m, 1H), 1.78–1.82 (m, 2H), 1.62–1.65
m, 5H), 1.49–1.50 (dd, 1H), 1.35–1.40 (m, 2H), 1.07–1.13 (m, 1H), 0.74
1
ACKNOWLEDGMENTS
(
(
13
s, 3H); C NMR (DEPT, CDCl
3
, ppm): δ 124.37, 52.34, 50.35, 48.32,
1.42, 38.31, 37.31, 36.36, 30.91, 30.31, 26.76, 25.71, 23.47, 21.26,
4.47. Anal. calcd. (%) for C23 : C 71.48, H 7.82. Found: C 71.44,
This research was supported partially by Natural Science Foundation
of Guangdong Province (Grant No. 2016A030313676) and by the
4
1
30 5
H O
1
3th 5-year National Science and Technology Support Program under
H 7.71.
the Ministry of Science and Technology of the People's Republic of
China (grant No. 2016YFC1000904).
To a round bottle was added the above intermediate 7 (3.0 g,
7
.7 mmol), THF (30 mL), and stirred until all solid dissolved. The
2 4
solution was cooled to 0°C and added 1 N H SO (5 mL) and then
stirred at rt overnight. The solution was poured into water, extracted
with dichloromethane, washed with brine, and dried over sodium
sulfate. The solvent was removed in vacuo and the residue was purified
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
by a silica gel column to give impurity B (1.2 g, yield 40%, HPLC 99.73
1
at 290 nm). Mp 105–107°C; H NMR (CDCl
3
, ppm): 6.38 (s, 1H), 6.03
ORCID
(
s, 1H), 4.35 (s, 2H), 2.98–3.06 (td, 1H), 2.57–2.62 (dt, 1H), 2.31–2.38
m, 3H), 2.17–2.23 (t, 1H), 2.09–2.12 (2s, 6H), 1.91–2.02 (m, 3H), 1.81–
Yun-Sheng Huang
http://orcid.org/0000-0003-3730-9338
(
1
1
2
4
2
.89 (m, 1H), 1.60–1.67 (m, 4H), 1.43–1.52 (m, 1H), 1.31–1.42 (qd, 1H),
1
3
3
.23–1.29 (m, 2H), 0.74 (s, 3H); C NMR (CDCl , ppm): δ 203.94,
00.17, 170.69, 156.57, 139.84, 135.32, 121.47, 96.45, 62.78, 48.53,
REFERENCES
7.52, 45.32, 41.00, 40.09, 37.40, 30.98, 30.31, 26.85, 26.45, 24.93,
[
1] A. O. Mueck, R. Sotruk-Ware, Steroids 2011, 76, 531.
+
3.19, 21.20, 14.20; ESI-MS: m/z 387.2146 [M+1] ; calcd. MW for
[
2] S. Christin-Maitre, E. Laroche, L. Bricaire, Women's Health 2013, 9(1),
13.
C
23
H
30
O
5
386.2093. Anal. calcd. (%) for C23
30 5
H O : C 71.48, H 7.82.
[
3] H. A. van Diepen, Reprod. Biol. Endocrinol. 2012, 10, 85.
4] M. G. F. Gerrits, P. G. Schnabel, T. M. Post, P. A. M. Peeters,
Contraception 2013, 87(2), 193.
Found: C 71.39, H 7.68.
[
[
[
[
[
5] S. Lello, Drugs 2010, 70(5), 541.
4
.4.3 Analysis of impurity B
|
6] X. Ruan, H. Seeger, A. O. Mueck, Maturitas 2012, 71, 345.
7] A. Pintiaux, U. Gaspard, M. Nisolle, Rev. Med. Liege 2012, 67(3), 152.
8] C. Westhoff, A. M. Kaunitz, T. Korver, W. Sommer, L. Bahamondes, P.
Darney, C. Verhoeven, Obstet. Gynecol. 2012, 119(5), 989.
9] U. M. Agren, M. Anttila, K. Maenpaa-Liukko, M. L. Rantala, H.
Tautiainen, W. F. Sommer, E. Mommers, Eur. J. Contracept. Reprod.
Health Care 2011, 16(6), 458.
NOMAc was analyzed on a Shimadzu LC-15C liquid chromatograph
with impurity B showing a retention time around 3.755 min and
NOMAc showing a retention time around 11.044 min (Supporting
Information Figure S6). The synthesized impurity B along was analyzed
in the same condition and showed a retention time around 3.722 min
[
[
10] D. Mansour, C. Verhoeven, W. Sommer, E. Weisberg, S. Taneepa-
nichskul, G. B. Melis, I. Sundstrom-Poromaa, T. Korver, Eur. J.
Contracept. Reprod. Health Care 2011, 16(6), 430.
(
Supporting Information Figure S7). When the synthesized impurity B
was added to the NOMAc, the HPLC results indicated an increased
absorption at 3.751 min (Supporting Information Figure S8).
[
11] S. Christin-Maitre, D. Serfaty, N. Chabbert-buffet, E. Ochsenbein, D.
Chassard, J. L. Thomas, Hum. Reprod. 2011, 26(6), 1338.
12] D. Mansour, Obstet. Gynecol. 2006, 8, 229.
[
4.5
|
Evaluation of cytotoxicity in vitro
[13] S. Ouzounian, L. Verstraete, N. Chabbert-Buffet, Expert Rev. Obstet.
Gynecol. 2008, 3(2), 190.
The cytotoxicity was measured using the Cell Counting Kit-8 (CCK-8,
Sigma). Briefly, the cells were harvested and seeded at a density of
[
14] R. Stitruk-Ware, A. Nath, Contraception 2010, 82, 410.
[15] R. Sitruk-Ware, Maturitas 2008, 61, 151.

10 of 10
XIE ET AL.
|
[
16] D. S. Fleischman, C. D. Navarrete, D. M. T. Fessler, Psychol. Sci. 2010,
[41] Y. Jiang, B. N. Jahagirdar, R. L. Reinhardt, R. E. Schwartz, C. D. Keene,
X. R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du,
S. Aldrich, A. Lisberg, W. C. Low, D. A. Largaespada, C. M. Verfaillie,
Nature 2002, 418(6893), 41.
2
1(5), 750.
[
[
[
17] F. Fruzzetti, J. Bitzer, Contraception 2010, 81, 8.
18] L. P. H. Yang, G. L. Plosker, Drugs 2012, 72(14), 1917.
19] C. Bastianlli, M. Farris, E. Rosato, I. Brosens, G. Benagiano, Expert Rev.
Clin. Pharmacol. 2017, 10(3), 315.
[42] R. Volkman, D. Offen, Stem Cells 2017, 35(8), 1867.
[43] P. Ickrath, M. Wagner, A. Scherzad, T. Gehrke, M. Burghartz, R.
Hagen, K. Radeloff, N. Kleinsasser, S. Hackenberg, Int. J. Environ. Res.
Public Health 2017, 14(12), E1590.
[
[
[
[
[
[
[
[
[
20] H. D. Nelson, L. L. Humphrey, P. Nygren, S. M. Teutsch, J. D. Allan,
JAMA 2002, 288(7), 872.
21] R. Louw-du Toit, M. S. Perkins, J. P. Hapgood, D. Africander, Biochem.
Biophys. Res. Commun. 2017, 491(1), 140.
[44] N. C. R. Correa, C. Kuligovski, A. C. C. Paschoal, A. P. R. Abud, C. L. K.
Rebelatto, L. M. B. Leite, A. C. Senegaglia, B. Dallagiovanna, A. M.
Aguiar, Regul. Toxicol. Pharmacol. 2018, 92, 75.
22] C. Charpin, S. Illouz, J. P. Dales, M. N. Lavaut, C. Allasia, L. Boubli, Bull.
Acad. Natl. Med. 2002, 186(1), 125.
[45] J. Shields-Botella, I. Duc, E. Duranti, F. Puccio, P. Bonnet, R.
Delansorne, J. Paris, J. Steroid Biochem. Mol. Biol. 2003, 87, 111.
[46] J. Botella, E. Duranti, I. Duc, A. M. Cognet, R. Delansorne, J. Paris, J.
Steroid Biochem. Mol. Biol. 1994, 50, 41.
23] S. Illouz, J. P. Dales, K. Sferlazzo, S. Garcia, S. Carpentier-Meunier, L.
Boubli, M. N. Lavaut, C. Charpin, Int. J. Mol. Med. 2003, 12(4), 517.
24] P. Florio, A. Imperatore, P. Litta, M. Franchini, S. Calzolari, S. Angioni,
M. Gabbanini, L. Bruni, F. Petraglia, Steroids 2010, 75, 912.
25] P. Persiani, E. Radici, P. A. Lombardi, P. L. Lombardi, Minerva Ginecol.
[47] R. Bucourt, A. Piasco, C. Tchernatinsky, PCT WO9302095A1, 1993.
[48] Q. Huang, X Chen, Y. Zhu, L. Cao, J. E. Riviere, Eur. J. Drug Metab.
Pharmacokinet 2015, 40(4), 435.
1
997, 49(4), 181.
26] E. Weisberg, K. McGeehan, J. Hangan, S. Fraser, Pilot Feasibility Stud.
017, 3, 18.
[49] C. Combes, A. Ratier, J. Jaud, F. Rodriguez, M. Lanquetin, Int. J. Pharm.
2002, 248, 141.
2
27] M. Farris, C. Bastianelli, E. Rosato, I. Brosens, G. Benagiano, Expert
Rev. Clin. Pharmacol. 2017, 7, 1.
[50] J. R. Pasqualini, Gynecol. Endocrinol. 2007, 23(S1), 32.
[51] C. H. Diep, A. R. Daniel, L. J. Mauro, T. P. Knutson, C. A. Lange, J. Mol.
Endocrinol. 2015, 54(2), R31.
28] A. H. Vlieg, F. M. Helmerhorst, J. P. Vandenbroucke, C. J. M. Doggen,
F. R. Rosendaal, BMJ 2009, 339, b2921.
[
[
[
29] J. R. Pasqualini, Maturitas 2009, 65S, S17.
30] L. W. Rice, Gynecol. Oncol. 2010, 118, 202.
SUPPORTING INFORMATION
31] V. De Leo, M. C. Musacchio, V. Cappelli, P. Piomboni, G. Morgante,
Hum. Reprod. Update 2016, 22 (5), 634.
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
[32] A. Y. Ma, S. W. Xie, J. Y. Zhou, Y. Zhu, Int. J. Mol. Sci. 2017, 18, 1337.
[33] J. M. Gastaud, French Patent FR2271833, 1975.
[34] Z. D. Yang, X. Zhang, T. Li, Y. C. Li, Y. F. Wang, Chem. Res. Chin. Univ.
1
13
HPLC analysis and spectroscopic data ( H and C NMR) for impurities
A and B as well as intermediate 7 are available.
2
009, 25(5), 653.
[35] H. L. Lu, Z. W. Wu, S. Y. Song, X. D. Liao, Y. Zhu, Y. S. Huang, Org.
Process Res. Dev. 2014, 18, 431.
[
[
[
[
36] C. Tchernatinsky, PCT WO8501504, 1985.
37] A. Giordani, W. Kobel, H. U. Gally, Eur. J. Pharm. Sci. 2011, 43, 1.
38] G. Prabha, V. Raj, Mater. Sci. Eng. C. Mater. Biol. Appl. 2017, 79, 410.
39] S. Shi, L. Yao, K. Guo, X. Wang, Q. Wang, W. Li, Mol. Med. Rep. 2018,
How to cite this article: Xie B-C, Song S-Y, Xie X-Y, et al.
Isolation, synthesis, and cytotoxicity evaluation of two
impurities in nomegestrol acetate. Arch Pharm Chem Life Sci.
2018;1–10. https://doi.org/10.1002/ardp.201800295
1
7(1), 743.
40] X. Yao, Q. Bai, D. Yan, G. Li, C. Lu, H. Xu, Toxicol. In Vitro 2015, 29(3),
00.
[
6