Continuous Flow Synthesis of 16-Dehydropregnenolone Acetate, a Key Synthon for Natural Steroids and Drugs

Article abstract of DOI:10.1021/acs.oprd.8b00038

A telescoped multistep process to provide the continuous delivery of 16-dehydropregnenolone acetate (16-DPA) from diosgenin is described. The method was evaluated through batch screenings that helped to identify critical bottlenecks and flowability, and the best conditions were optimized in flow systems before the individual steps were telescoped together into a single integrated flow process. Further highlights of our approach include the use of efficient in-line extraction operations and reaction monitoring, the avoidance of time-consuming purifications between steps, and improvement of efficiency and safety standards.

Full text of DOI:10.1021/acs.oprd.8b00038

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Continuous Flow Synthesis of 16-Dehydropregnenolone
Acetate (16-DPA), a Key Synthon for Natural Steroids and Drugs
Valentina Mancino, Bruno Cerra, Alessandro Piccinno, and Antimo Gioiello
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00038 • Publication Date (Web): 18 Apr 2018
Downloaded from http://pubs.acs.org on April 18, 2018
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Continuous Flow Synthesis of 16-
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Dehydropregnenolone Acetate (16-DPA), a Key
Synthon for Natural Steroids and Drugs
Valentina Mancino, Bruno Cerra, Alessandro Piccinno, Antimo Gioiello*
Laboratory of Medicinal and Advanced Synthetic Chemistry, Department of Pharmaceutical
Sciences, University of Perugia, Via del Liceo 1, 06122 Perugia (Italy).
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Table of Contents
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KEYWORDS: Steroids, flow chemistry, continuous flow processing, in-line work-up, telescoped
process.
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Abstract
A telescoped multi-step process to provide the continuous delivery of 16-dehydropregnenolone
acetate (16-DPA) from diosgenin is described. The method has been evaluated through batch
screenings helpful to identify critical bottlenecks and flowability, and the best conditions have
been optimized in flow systems before telescoping the individual steps together in a single,
integrated flow process. Further highlights of our approach include the use of efficient in-line
extraction operations and reaction monitoring, the avoidance of time-consuming purifications
between steps as well as improvement of efficiency and safety standards.
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1. Introduction
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The most significant expansions of steroid chemistry came with the discovery of cortisone and
progesterone as important therapeutic agents.1 Indeed, to make them available as marketed drugs,
chemists had to surmount a substantial obstacle, i.e. the synthetic accessibility. This led to the so-
called “golden age of steroids” occurred in the 50’s and beyond, when an unusually large number
of the world-leading chemists was attracted to the field to render steroids available for clinic and
drug production.1 Nowadays, the global market of steroidal drugs and intermediates is estimated
to be over a billion dollars preserving the interest in developing new methods for steroid synthesis.
Novel strategies and improved protocols of well-established methods are therefore highly desired
to meet modern synthesis criteria such as sustainability, safety, and cost. To this aim, a major
impetus came with the adoption of technological solutions as flow processing.2 However, while
many chemical practices have already benefited from flow chemistry, assessment of the literature
indicated steroid manipulation to be poorly explored in flow systems.3 Furthermore, to the best of
our knowledge, only one recent example of two step synthesis of steroids under flow conditions
has been reported so far and relates with the production of ursodeoxycholic acid using immobilized
enzymes.4
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In this work, we were challenged to develop a continuous flow process for the preparation of
16-dehydropregnenolone acetate (16-DPA) (
natural steroids, sex hormones, and drugs (Figure 1).5 It is worth noting that most of steroidal drugs
are prepared from 16-DPA (
) with few of which obtained by total synthesis.6
The overall process we pursued to investigate started from diosgenin (22-iso-5-spirostane-3
ol) ( ), a cheap and readily available plant-derived sapogenin obtained by extraction from
1), a key substrate for the production of several
1
β
-
2
Dioscorea floribunda (Scheme 1).7 The method proceeded through (a) acetylation/acetolysis
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reaction of
2
to form the furostenol derivative namely pseudodiosgenin-3,26-diacetate (3), (b)
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oxidative cleavage of the Δ20-22 double bond to get diosone (
hydrolysis followed by elimination to yield the final product
4) that was readily submitted to (c)
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1
.
Figure 1. 16-Dehydropregnenolone acetate (1), a crucial synthetic precursor of natural steroids
and drugs.
Marker’s degradation is the first known route for making 16-DPA (
(Scheme 1).8 The method consisted in refluxing diosgenin (
) in an autoclave with acetic anhydride
(Ac2O) in xylene for 14 h at 5 bar pressure and the resulting pseudodiosgenin was then oxidized
) in 60% overall yield.
1) from diosgenin (2)
2
3
with CrO3 and hydrolysed under acidic conditions to get 16-DPA (
1
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Nowadays, diverse protocols are available for batch synthesis (Table S4, Supporting
Informations),9 and although major advances have been made, further criteria need to be met for
modern manufacturing process. For instance, the first step of acetolysis generally requires harsh
reaction conditions as elevated temperature and pressure that strongly affect the sustainability of
the process and the quality of the end product. Moreover, the high temperature required to promote
the reaction combined with the non-homogenous batch heating is potential cause of side products
formation, unsatisfactory yields, and the need for tedious purifications. The following oxidation
step also needs improvements to avoid the use of hazardous reagents and expensive (co)oxidant
that raise issues in terms of safety, waste production and costs. To this end, other groups reported
the use of various additives like pyridine or the employment of high boiling solvents.8c-e,g,h A recent
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synthesis of 16-DPA (
amounts of Ac2O (3.5 equiv.) at 250 °C for 10 h to give pseudodiosgenin-3,26-diacetate (
that was then reacted with CrO3 in acetic acid at -10/10 °C under ultrasound irradiation for 1 h.8c
16-DPA was obtained in 57% yield by refluxing diosone ( ) in acetic acid for 2 h. Despite the
1
) was realized by heating diosgenin (
2
) in xylene in presence of smaller
3
) (91%)
4
advantages with respect to previous methods, the use of high temperatures, long reaction time and
the difficult control and tuning of ultrasound reaction conditions, make this strategy not ideal for
commercial production and hence alternative methods are needed.
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Scheme 1. General route for converting diosgenin (2) into 16-dehydropregnenolone acetate (1).
Consequently, we aim to develop an integrated flow process that would overcome such
limitations and adhere to modern synthesis principles. We initially proceeded with the evaluation
of the process through batch screenings helpful to identify critical bottlenecks, flowability issues
and best conditions before attempting to telescope the individual reactions together in a single,
integrated flow process.
2. Results and discussion
2.1 From batch to flow
Step a: acetylation/acetolysis of diosgenin
The acetylation/acetolysis step converting diosgenin (
proceeds according to the mechanism depicted in Scheme 2. We previously argued that the
isomerization of into generally required high temperature and pressure, which may lead to
2) into pseudodiosgenin-3,26-diacetate (3)
2
3
extensive decomposition of the starting material and side products formation.8c Considering this
fact, we had the idea of combining the potential of microwave irradiation with flow technology.10
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Unfortunately, although batch screens in neat conditions gave high yields (Table S1, Supporting
Information), preliminary attempts to translate the protocol under flow conditions (Figure S1,
Supporting Information) were negative because of system overpressure. The employment of
different solvent systems (THF, EtOAc/MEK, THF/MEK) and Lewis acids (0.1 equiv.) were also
unsuccessful (Table S2, Supporting Information). The only exceptions were the reactions
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conducted with AlCl3 and BF3∙Et2O that furnished 3 in 30% and 40% yield, respectively. Overall,
these observations prompted us to investigate a different strategy.
Scheme 2. One-pot acetylation/acetolysis mechanism for the formation of pseudodiosgenin-3,26-
diacetate (3) from diosgenin (2) with (B) or without BF3∙
Et2O (A).8i;11
With the desire of promoting the isomerization of
of explorative reactions by employing the more reactive acetic trifluoroacetic anhydride
(ATFAA)10 as the acetyl source in the presence of a stoichiometric amount of BF3
Et2O that was
2 into 3 at room temperature, we run a series
∙
preferable to AlCl3 as it did not generate insoluble salts. ATFAA was freshly prepared by slow
addition of AcOH (1.2 equiv.) to trifluoroacetic anhydride (TFAA) (1.0 equiv.) maintained at room
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temperature in dark conditions and stirring for 5 min. The reaction of
2
with ATFAA and BF3
by means of
TSA)11 employed as
CH2Cl2 soluble base and acid (Scheme 2b). Interestingly, DBU was significantly more efficient
than TSA (37% yield) affording the desired compound
in 90% yield.a
∙Et2O
in CH2Cl2 at room temperature gave intermediate
5 that was then converted into 3
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1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) or -toluenesulfonic acid (
p
p
p
3
At this stage, the reaction with DBU was repeated in flow mode using different set-ups (Table
1). Thus, reagents were fed by loop injection, using two pumps dedicated to the organic solvent
(CH2Cl2), a 10 mL PTFE coil reactor, a BPR (100 psi), an UV detector and a fraction collector
(Figure 2). After the loop injection of the solution A and B, and the switching of the valves to the
loops, the two solutions were mixed in a T-junction, and pumped through the coil reactor at room
temperature. Initially, the outflowing mixture was directly quenched onto DBU (16.0 equiv.) in
semi-batch modality (Table 1, entries 1-4). Subsequently, applying a different set-up, a CH2Cl2
stream containing DBU (0.96 M, flow rate= 2 mL/min) was inserted at the output line of the reactor
and the outflow was collected using a fraction collector (Figure 2). The conversion and reaction
yield were determined by 1H-NMR analysis of the crude mixtures. This approach was suitable to
screen different system set-ups using the minimum amount of the starting material
of the injection loop system and the connection with a fraction collector allowed to process samples
of in succession using the same solvent and varying the experimental conditions. The in-line UV
detector was instrumental to monitor and detect reactions and related components. Thus, the
2. The presence
2
a Reactions were performed on 100 mg of
2
(0.24 mmol) using freshly prepared ATFAA (11.0
TSA. The
equiv.) and BF3∙Et2O (2.0 equiv.) in dry CH2Cl2 (10 min, r.t.), and then adding DBU or
p
relative yield was determined by 1H-NMR analysis of the crude reaction mixture.
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absence of the UV signals from the collected output indicated the end of the reaction and the
possibility to start the next experiment after line washing with CH2Cl2.
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Figure 2. Flow set-up employed for exploring flow set-up and reaction optimization (Tables 1 and
2).
As a result, a complex reaction mixture was obtained when a 0.15 M solution of diosgenin (
2)
in CH2Cl2 was reacted with ATFAA and BF3∙Et2O (Table 1, entries 1 and 2), while when ATFAA
was flowed as the sole reaction component (pump B) a better reaction outcome was achieved
(Table 1, entries 3 and 4). The best yield was reached using the conditions reported in Table 1,
entry 5: in this case, a solution of DBU in CH2Cl2 was pumped via syringe pump, mixed with the
reaction crude in a T-junction, the outflow was quenched with H2O, and the organic phase was
separated from the aqueous waste to afford
3 in 75% yield. Importantly, we also found that DBU
was not necessary to promote the conversion of
5
into 3 under flow conditions (Table 1, entry 6).
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Table 1. Different set-up for the conversion of diosgenin (2) into pseudodiosgenin-3,26-diacetate
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(3
) under flow conditions.a
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Entry
A
2
B
Conversion yieldb
Yieldb
1
ATFAA, BF3
∙
Et2O
Quantitative
(Complex mixture)
BF3∙Et2O
(0.96 M in CH2Cl2)
2
2
, ATFAA
90%
(Complex mixture)
3
2
, BF3
, BF3
, BF3
, BF3
∙
∙
∙
∙
Et2O
Et2O
Et2O
Et2O
ATFAA
Quantitative
Quantitative
Quantitative
Quantitative
60%
50%
75%
75%
4c
5d
2
2
2
ATFAA
ATFAA
6e
ATFAA
a
Reactions performed on 200 mg of
Figure 2. Reagents and conditions
2
(0.48 mmol) using the flow set-up depicted in
Et2O
consumption by
:
2
0.15 M in dry CH2Cl2, ATFAA (11.0 equiv.), BF3∙
b
(2.0 equiv.), = 10 min, r.t., then DBU (16.0 equiv.). Determined as
τ
2
1H-NMR analysis of the crude reaction mixture. DBU was maintained under magnetic
stirring. d DBU (0.96 M) in CH2Cl2 was pumped via syringe pump (flow rate = 2 mL/min)
and mixed with the reaction mixture in a T-junction. Then the mixture was poured into H2O
and the phases separated. e Reaction performed without DBU.
c
We next decided to test the effect of (higher) concentration and flow rate to enhance productivity,
as well as the use of lower amount of BF3
Since a better solubility of diosgenin ( ) in CH2Cl2 was observed in the presence of BF3
reaction was conducted at 0.23 M of that represented the maximum concentration ensuring
solubility. While these conditions and the halving of the residence time gave positive outcomes
(Table 2, entries 1 and 4), the reduction of BF3 Et2O and ATFAA equiv. was not relevant (Table
2, entries 2 and3). It is worth noting that when was mixed with 1.1 equiv. of BF3 Et2O at the
concentration of 0.23 M, the formation of a suspension was observed. Therefore, the best option
turned out to be treating a solution of (0.23 M) and BF3 Et2O (2.0 equiv.) in CH2Cl2 with freshly
∙
Et2O and ATFAA to reduce cost and waste generation.
2
∙
Et2O, the
2
∙
2
∙
2
∙
prepared ATFAA (11.0 equiv.) at room temperature with a residence time of 10 min.
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Table 2. Optimization of the conditions for the flow synthesis of
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pseudodiosgenin-3,26-diacetate (3) from diosgenin (2
) in flow modality.a
BF3∙Et2O ATFAA Residence
Entry Concentration of 2
Yieldb
equiv.
equiv.
11.0
11.0
5.5
time
1
2
3
4
0.23 M
0.15 M
0.15 M
0.15 M
2.0
10 min
10 min
10 min
5 min
80%
75%
68%
79%
1.1
2.0
2.0
11.0
a
Reactions performed on 200 mg of 2 (0.48 mmol) using the flow set-up
depicted in Figure 2. Reagents and conditions: 2 0.23 or 0.15 M in dry CH2Cl2,
ATFAA (5.5 or 11.0 equiv.), BF3∙Et2O (1.1 or 2.0 equiv.), τ = 5 or 10 min, r.t.
b Determined by 1H-NMR analysis of the crude reaction mixture.
Step b: Oxidative cleavage of pseudodiosgenin-3,26-diacetate to diosone
The necessity of removing drawbacks for the oxidative cleavage of pseudodiosgenin-3,26-
diacetate (3) to form diosone (4) (Scheme 1, step b) prompted us to evaluate different protocols
starting from a preliminary batch screen. In the past, we and others found the use of H2O2 to be
beneficial for eco-friendly flow oxidations.12 In line with these findings, we tested the efficacy of
H2O2 as co-oxidant in the presence of different metal oxides employed as oxidants in
substoichiometric amount (Table 3). The best result was obtained using a catalytic amount of OsO4
(0.01 equiv.) in CH2Cl2/DMF that furnished the desired diosone (4) in 70% yield after 30 min at
room temperature (Table 3, entry 7). While we found a novel procedure amenable for flow
applications, the inherent high toxicity of OsO4 led us to proceed with the investigation of diverse
oxidant systems based on the use of KMnO4 and CrO3.13
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Table 3. Oxidative cleavage of pseudodiosgenin-3,26-diacetate (3) to diosone
(4) with H2O2 and metal oxides in batch conditions at room temperature.a
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Reaction
conditions
Entry Reagents (equiv.)
114
Yieldb
AcOH,
ultrasound, 1 h
H2O2 (30% w/w) (2.0 equiv.)
(Complex mixture)
(Complex mixture)
(Complex mixture)
(Complex mixture)
H2O2 (30% w/w) (4.0 equiv.),
CrO3 (0.1 equiv.)
2
AcOH, 1 h
THF, 24 h
THF, 3 h
H2O2 (30% w/w) (4.3 equiv.)
CrO3 (0.08 equiv.)
3
H2O2 (30% w/w) (4.3 equiv.)
SeO2 (0.08 equiv.)
4
H2O2 (30% w/w) (4.3 equiv.)
SeO2 (0.08 equiv.)
NaIO4 (1.5 equiv.)
5
6
THF, 3 h
THF, 3 h
(Complex mixture)
(Complex mixture)
H2O2 (30% w/w) (4.3 equiv.)
SeO2 (0.08 equiv.),
then NaIO4 (1.5 equiv.)
H2O2 (30% w/w) (4.3 equiv.)
V2O5 (0.08 equiv.)
AcOH (2.0 equiv.)
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8
9
THF, 3 h
THF, 3 h
THF, 3 h
40%
H2O2 (30% w/w) (4.3 equiv.)
VO(acac)2 (0.08 equiv.)
(Complex mixture)
(Complex mixture)
H2O2 (30% w/w) (4.3 equiv.)
VO(acac)2 (0.08 equiv.)
AcOH (2.0 equiv.)
H2O2 (30% w/w) (4.0 equiv.)
OsO4 (0.01 equiv.)
DMF (20.0 equiv.)
1015
CH2Cl2, 30 min
70%
a Reaction performed on 100 mg of 3 (0.20 mmol). Reagents and conditions:
H2O2 (30% w/w), oxidant, solvent. Determined by H-NMR analysis of the
crude reaction mixture.
b
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A recent work proposed the use of KMnO4 (5% mol) in the presence of NaIO4 (5% mol) and
triethyl benzyl ammonium bromide (TEBA) (2.2% mol) to synthesize diosone (4) from 3.8a. With
the perspective to use the crude solution of 3 deriving from the previous step, we run a set of
experiments using KMnO4/NaIO4 as oxidizing system and CH2Cl2 as the organic solvent of choice
(Table S3, Supporting Information). In all cases, complex mixtures were obtained. Remarkably,
when KMnO4 was replaced with CrO3 (1.4 equiv.) in a monophasic solvent system constituted by
CH2Cl2/H2O/AcOH (1:1:0.5, v/v/v), 4 was obtained in 80% yield (Table S3). These last conditions
were translated and further optimized into a flow apparatus using the set-up depicted in Figure 3.
Our scope was to evaluate the efficiency of the oxidative species in different solvent systems either
in the presence or not of a phase transfer agent or a co-solvent (Table 4).
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Figure 3. Flow set-up used for the optimization of the reaction conditions reported in Table 4.
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Table 4. E-ring oxidative cleavage of pseudodiosgenin-3,26-diacetate (3) to diosone (4) under
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flow conditions.a
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Phase transfer/
co-solvent
(Pump B)
-
Flow rate
(mL/min)
Reactor
volume (mL)
Conversion
yieldb
Entry
Yieldb
1
2
0.2
0.4
10
20
-
-
-
-
-
TEAI
(0.02 equiv.)
3
4
5
0.4
0.4
2.0
0.4
20
20
15%
10%
TEAI
(0.10 equiv.)
Quantitative 10%
TEAI
(0.02 equiv.)
20
-
-
TEAI
(0.02 equiv.)
6
7
20c
26%
20%
0.2
0.2
THF (5% v/v)
10c
10c
-
-
-
-
8
MeCN (5% v/v)
a
Reaction performed on 100 mg of 3 (0.20 mmol) using the flow set-up
reported in Figure 3. Reagents and conditions: 3 0.05 M in CH2Cl2, CrO3 (1.4
equiv.) 0.07 M in AcOH/H2O (1:2, v/v), solvent B: H2O, phase transfer agent or
co-solvent, r.t. b Determined as 3 consumption by 1H-NMR analysis of the crude
reaction mixture. c Reactor immersed in ultrasound bath.
Unfortunately, only low yields were obtained applying different residence times, either using or
not tetraethylammonium iodide (TEAI, 0.02 or 0.10 equiv.) (Table 4, entries 1-6). Also the
employment of co-solvents as THF and MeCN was not beneficial for the reaction (Table 4, entries
7 and 8). Therefore, the CrO3-promoted oxidative cleavage was studied in different batch
monophasic systems (Table 5). Thus, a solution of 3 in AcOH was reacted with a AcOH/H2O (9:1,
v/v) solution of CrO3 (1.4 equiv.), yielding 90% of 4 in 5 min (Table 5, entry 1). Remarkably, the
same result was attained when the starting material 3 was dissolved in CH2Cl2 (Table 5, entry 2).
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In an attempt to reduce the equiv. of CrO3, the reaction was repeated with a lower amount of
oxidizing agent (1.1 equiv.) (Table 5, entry 3). Sub-stoichiometric amounts of oxidant (0.1 or 0.5
equiv.) were used in the presence of N-methylmorpholine-N-oxide (NMO, 3.0 equiv.) resulting in
complex reaction mixtures and unreacted 3 (Table 7, entries 4-6).
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Table 5. E-ring oxidative cleavage of pseudodiosgenin-3,26-diacetate (3) to diosone (4) with
stoichiometric CrO3 or catalytic CrO3 and NMO in batch conditions.a
Conversion
Entry Reagents
Solvent and conditions
Yieldb
yieldb
CrO3 (1.4 equiv.)
(in AcOH/H2O 9:1, v/v)
AcOH,
5 min
1
2
3
Quantitative
90%
CrO3 (1.4 equiv.)
(in AcOH/H2O 9:1, v/v)
CH2Cl2,
5 min
Quantitative
85%
90%
80%
CrO3 (1.1 equiv.)
(in AcOH/H2O 9:1, v/v)
CH2Cl2,
5 min
CH2Cl2,
AcOH/H2O (9:1 v/v),
o.n.
CrO3 (0.1 equiv.)
NMO (3 equiv.)
4
20%
(traces)
CH2Cl2,
AcOH/H2O (9:1 v/v),
o.n.
CrO3 (0.5 equiv.)
NMO (3 equiv.)
5
6
40%
-
(traces)
-
NMO (3 equiv.)
CH2Cl2, o.n.
a
Reaction performed on 100 mg of 3 (0.20 mmol). Reagents and conditions: 3 in the
b
reported solvent, CrO3 in AcOH/H2O (9:1, v/v) and/or NMO, r.t. Determined as 3
consumption by 1H-NMR analysis of the crude reaction mixture. o.n.: overnight.
The best outcome (Table 5, entry 2) was then explored for solvent and phase-transfer effect using
the set-up depicted in Figure 3. Initially, the reaction was performed in CH2Cl2 to avoid workup
operations. However, at both high and low flow rates, in the presence of TEAI under ultrasound
irradiation, a poor conversion was detected (Table 6, entries 1-3). This might be ascribable to the
formation of H2O/CrO3 dots or CrO3 precipitation. Accordingly, the monophasic mixture of
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AcOH/H2O (9:1, v/v) allowed to obtain the target product 4 in 75% yield (Table 6, entry 4). Under
these conditions, reduction of CrO3 equiv. (1.1) was negative for the reaction outcome (Table 6,
entry 5).
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Table 6. E-ring oxidative cleavage of pseudodiosgenin-3,26-diacetate (3) to diosone (4) under
flow conditions: effect of solvents and phase transfer agents.a
CrO3
equiv.
Flow rate Phase transfer
(mL/min)
Conversion
yieldb
Entry
1c
Solvent B
CH2Cl2
Yieldb
(traces)
30%
(Pump B)
1.4
1.4
2
-
-
TEAI
(0.02 equiv.)
2d
0.6
0.6
2
CH2Cl2
60%
TEAI
(0.02 equiv.)
3d,e
1.4
1.4
1.1
CH2Cl2
60%
30%
AcOH/H2O
(9:1, v/v)
4
-
-
Quantitative 75%
AcOH/H2O
(9:1, v/v)
5
2
70%
62%
a
Reaction performed on 100 mg of 3 (0.20 mmol) using the flow set-up reported in
Figure 3. Reagents and conditions: 3 0.05 M in CH2Cl2, CrO3 (1.4 equiv.) 0.07 M in
b
AcOH/H2O (9:1, v/v), phase transfer agent, r.t. Determined as 3 consumption by 1H-
c
NMR analysis of the crude reaction mixture. H2O (and CrO3) dots observed; H2O
necessary to completely solubilise CrO3. d CrO3 precipitation in loop and reactor. e The
reaction was conducted irradiating the reactor with ultrasound.
The possibility to perform the final hydrolysis/elimination step by a telescoped approach was
next evaluated. Indeed, taking advantage from the acidic environment deriving from the previous
transformation, the crude reaction mixture from the CrO3-mediated oxidative cleavage of 3 was
readily flowed into a second 10 mL thermostated coil reactor spaced by a residence time reactor,
instrumental to collect the reaction mixture before changing the flow rate for the hydrolytic step
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(Figure 4). The outflowing crude 1 was detected by in-line UV sensor before being collected into
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a fraction collector.
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Figure 4. Flow set-up used for the optimization of the reaction conditions reported in Table 7.
Initially, both steps b and c were performed at 1 mL/min to give 40% conversion, 30% yield of
4 and traces of the target product 2 (Table 7, entry 1). When the solution of 3 was reacted with
CrO3 dissolved in AcOH/H2O, at 2 mL/min flow rate for the oxidative step and 0.2 mL/min for
the hydrolysis step, mixtures composed by starting material 3, 4 and 1 were obtained (Table 7,
entries 2 and 3). Having noted the necessity to pre-solubilize the oxidant in H2O to form the active
oxidizing species (Table 7, entries 4 and 5), a remarkable high yield (>90% from 3 to 1) was
obtained when the reaction was performed at 150 °C and using 10% of H2O in line B.
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Table 7. Study of 16-dehydropregnenolone acetate (1) synthesis from pseudodiosgenin-3,26-
diacetate (3) under flow conditions.a
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Flow rate
Resultsb
(mL/min)
Temperature Temperature
Entry H2O
Reactor 1
Reactor 2
Step b Step c
3
4
30%
30%
37%
42%
40%
(traces)
-
1
1c
2c,d
3c
4e
5e
6e
7e
10%
10%
20%
20%
10%
10%
10%
1
2
2
2
2
2
2
1
5 mL
10 mL
10 mL
10 mL
10 mL
10 mL
10 mL
130 °C
130 °C
130 °C
130 °C
130 °C
145 °C
150 °C
60%
(traces)
50%
51%
58%
60%
90%
95%
0.2
0.2
0.2
0.2
0.2
0.2
18%
12%
-
-
-
-
a Reaction performed on 100 mg of 3 (0.20 mmol) using the flow set-up depicted in Figure 4.
Reagents and conditions: 3 0.05 M in CH2Cl2, CrO3 (1.4 equiv.) 0.07 M in AcOH/H2O (9:1 or
8:2, v/v), r.t., then 130-150 °C. b Relative percentages and yields were determined by 1H-NMR
analysis of the crude reaction mixture. c CrO3 was dissolved in the mixture of AcOH and H2O.
d CrO3 precipitation. e CrO3 was dissolved in H2O, then AcOH was added.
Diverse attempts aimed at reducing the total reaction time over the two steps were also
performed by applying ultrasound or microwave irradiation at reactor 2 or by adding catalytic
amounts of pTSA. Unfortunately, none of them gave better results (Table 8, entries 1-5). Finally,
1.1 equiv. of CrO3 afforded 1 in 80% and 60% yield using conventional and microwave heating,
respectively (Table 8, entries 6 and 7).
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Table 8. Attempts to optimise 16-dehydropregnenolone acetate (1) synthesis from
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pseudodiosgenin-3,26-diacetate (3) in flow conditions.a
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Resultsc
CrO3
equiv.
Flow rate
(mL/min)
Entry
Catalyst
Reactor 2b
3
4
1
Coil,
ultrasound,
54-62 °C
1
1.4
-
0.2
-
75%
(traces)
pTSA
(0.1 equiv.)
Coil,
150 °C
2
3
4
5
6
1.4
1.4
1.4
1.4
1.1
1.1
0.4
0.4
0.4
0.3
0.2
0.2
23%
30%
68%
70%
-
Microwave,
150 °C
-
-
-
-
-
Microwave,
160 °Cd
Microwave,
150 °C
18%
12%
20%
66%
80%
60%
Coil,
150 °C
Microwave,
150 °C
7
10%
a
Reaction performed on 100 mg of 3 (0.20 mmol). Reagents and conditions: 3 0.05
M in CH2Cl2, CrO3 (1.1 or 1.4 equiv.) 0.07 M in AcOH/H2O (9:1, v/v), r.t., then heating.
b 10 mL. c Relative percentages and yields were determined by 1H-NMR analysis of the
crude reaction mixture. d The set temperature was not reached.
The information gathered throughout these feasibility studies allow us to develop a more
comprehensive process understanding and as a result enabled us to define the requirement and
appropriate set-up for a flow processing method.
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2.2 Telescoped flow synthesis of 16-dehydropregnenolone acetate from diosgenin
An integrated, telescoped flow transformation of diosgenin (2) into 16-DPA (1) was designed in
order to reduce the downstream operations towards an easy scalable process. The first task to be
tackled was the removal of acidic traces from the crude pseudodiosgenin-3,26-diacetate (3) to
prevent pump injury caused by the acidic media and salt precipitation. To this end, we decided to
attempt an in-line work-up (Figure 5). Initially, the reaction mixture was treated with a 0.2 M
aqueous solution of NaOH and, after phase separation, the organic layer was pumped into the
system. A pump plunger failure was observed as ascribed to salt precipitation. Nevertheless, the
final product 1 was obtained in 65% yield along with traces of diosgenin (2). Therefore, an aqueous
washing was performed and pseudodiosgenin-3,26-diacetate (3) was obtained in 75% yield after a
simple extraction. The crude was further processed to provide 16-DPA (1) in 60% yield without
any pump failure. Finally, after reacting the mixture of 2 and BF3⋅Et2O with ATFAA at room
temperature, a workup with a 0.5 M aqueous solution of NaHCO3 was performed to obtain 3 in
80% yield.
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The improved flow setup was applied to the telescoped synthesis of 16-DPA (1) from diosgenin
(2) (Figure 5). Therefore, a solution of diosgenin (2) and BF3⋅Et2O (2.0 equiv.) in dry CH2Cl2 was
mixed with freshly prepared ATFAA (11.0 equiv.) in a T-junction and pumped through the coil
reactor maintained at room temperature with a total flow rate of 1.0 mL/min. The stream was then
combined in a T-shaped mixing element with a 0.5 M aqueous solution of NaHCO3, and the
biphasic system was sent to a membrane separator. The organic layer was filtered on a Nylon
syringe filter after the aqueous washing, and used as a stock solution of 3 for the next step.
Therefore, the solution of pseudodiosgenin-3,26-diacetate (3) was mixed with CrO3 in AcOH/H2O
(9:1, v/v) in a T-junction and the solution was pumped in a 10 mL coil reactor at room temperaure
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with at 2 mL/min flow rate. The reaction mixture was collected into a residence time reactor before
entering a 10 mL coil reactor heated at 150 °C. After a residence time of 50 min, the outflow was
combined with a 0.5 M aqueous solution of NaHCO3 and CH2Cl2 (0.5 mL/min for each pump) and
the organic layer was separated by a gravity extraction funnel. Concentration of the organic phase
and automated flash chromatography of the crude mixture led to the target product 1 in 95% yield
over two steps (overall yield: 75%).
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Figure 5. Integrated flow set-up for the investigation of diosgenin (2) degradation to 16-
dehydropregnenolone acetate (1).
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Conclusions
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In summary, the set-up and assessment of the telescoped, continuous flow synthesis of 16-DPA
(1) from diosgenin (2) was accomplished. Besides the known benefits offered by flow processing
over batch synthesis, our method supersedes bottlenecks like the use of harsh reaction conditions
and showed important advantages and improvements with respect to previous syntheses (Table
S4, Supporting Information). These include higher efficiency and yields under milder reaction
conditions, the use of lower amount of reagents, reduced manual handling, simple downstream
procedures, that resulted in high quality material and safety standards. Moreover, the process
exhibited a productivity of 2.1 g/day (for single reactor) and it can be easily monitored by in-line
analysis and assisted by automation software. Preliminary attempts of increasing productivity (the
first step can generate up to 150 g/d of intermediate 3, see Supporting Information) using higher
concentrations, peristaltic pumps, and larger tubing reactors showed that the method is robust and
amenable for future process intensification works and kilo scale preparations. In this frame, future
efforts directed towards the optimization of the oxidative cleavage can be foreseen. Our results
suggest that the implementation of an OsO4-catalized continuous method integrated with an
appropriate reagent loading system and in-line scavenging as thiurea resins would improve the
overall synthetic process limiting toxicity, handling, and disposal issue while enabling catalyst
recovery and reuse.
Supporting Information. Experimental procedures, images of flow equipment used,
spectroscopic characterization and copies of NMR, FT-IR, EICs and HRMS spectra, and HPLC
HPLC chromatograms are provided. The following files are
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Corresponding Author
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*Prof. Antimo Gioiello, e-mail: antimo.gioiello@unipg.it
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Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
We gratefully acknowledge financial support by TES Pharma (Perugia, Italy) and Intercept
Pharmaceuticals (New York, USA).
ABBREVIATIONS
16-DPA, 16-dehydropregnenolone acetate; Ac2O, acetic anhydride, ATFAA, acetic trifluoroacetic
anhydride; DBU, 1,8-diazabicyclo(5.4.0)undec-7-ene; MEK, methyl ethyl ketone; NMO, N-
methylmorpholine-N-oxide; pTSA, p-toluenesulfonic acid; TEAI, tetraethylammonium iodide;
TEBA, triethyl benzyl ammonium bromide; TFAA, trifluoroacetic anhydride.
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(13) OsO4 can be used as an alternative to CrO3 with cautions and under appropriate disposals.
Note that OsO4 has a vapour pressure of 9 hPa (20 °C) and low melting point (ca. 40 °C). It is
classified in high hazardous compounds categories for oral, inhalation and dermal acute toxicity
(Regulation EC No 1272/2008). OsO4 is fatal if swallowed, inhaled and in contact with skin,
causing severe damages to eyes, respiratory tract and skin with a LD50 in rat: 14 mg/kg (CrO3
LD50: 80 mg/Kg).
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