Large-Scale Practical Synthesis of Boc-Protected 4-Fluoro-l-Proline

Article abstract of DOI:10.1021/acs.oprd.0c00080

A large-scale synthesis process of N-Boc-4-fluoro-l-proline (1) from N-Boc-4-hydroxy-l-proline methyl ester (2) using nosyl fluoride (13) as a deoxyfluorinating agent has been developed. An eco-friendly and large-scale feasible process using a single solvent was developed to afford moderate yields of products with excellent purity >99% by high-performance liquid chromatography. The key feature of the optimization involving chromatography-free purification and isolation on a kilogram-scale at a pilot plant scale is described.

Full text of DOI:10.1021/acs.oprd.0c00080

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Full Paper
Large-Scale Practical Process of Boc-Protected 4-Fluoro-L- Proline
Yasinalli Tamboli, Bharat B Kashid, Ramprasad Yadav, Mohammad Rafeeq, and Arvind Y Merwade
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00080 • Publication Date (Web): 07 Aug 2020
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Large-Scale Practical Process of Boc-Protected 4-Fluoro-L- Proline
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Yasinalli Tamboli, Bharat Kashid, Ram Prasad Yadav, Mohammad Rafeeq, and Arvind
Y Merwade*
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Chemical Research, Wockhardt Research Centre, D-4, MIDC, Chikalthana,
Aurangabad-431 006, India
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Abstract
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A large-scale synthesis of N-Boc-4-Fluoro-L-Proline (1) from N-Boc-4-hydroxy-
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L-Proline methyl ester (2) using Nosyl fluoride (13) as deoxyfluorinating agent has been
developed. Eco-friendly and large scale feasible process using single solvent was
developed to afford excellent purity >99% by HPLC with moderate yield. Key feature of
the optimization included chromatography free purification and isolation on kilogram
scale at pilot plant scale is described.
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Key words
Deoxyfluorination, Nosyl fluoride, N-Boc-4-Fluoro-L-Proline, N-Boc-cis-4-fluoro-L-
proline methyl ester, chromatography free
Introduction
The fluorine substituted natural amino acids have been used as a powerful tool in
medicinal chemistry. Naturally occurring bioactive Proline and its 4-substituted
derivatives are important amino acids and have been extensively used in the
pharmaceutical industry. 4-Fluoroproline is tool for protein design and engineering.1 N-
Boc-4-Fluoro-L-Proline (1) is useful intermediate in the synthesis of several drug
molecules, such as Dipeptidyl peptidase IV inhibitors2a-d, Hepatitis C virus (HCV)
inhibitors3, HIV-1 protease inhibitors4a,b, Anti-alzheimer agents5, Anti-tumor agents6,
GABA uptake inhibitors7, Thrombin inhibitors8, Peptide deformylase (PDF) inhibitors9,
TNF-α production inhibitors10, Pan-aurora kinase inhibitors11, Inhibitors of ROS1 and
NTRK kinase12, GPR119 receptor agonists13, VLA-4 inhibitors14a,b, Fibroblast activation
arotein (FAP) inhibitors.15 Unfortunately methods reported in literature for preparation of
N-Boc-4-Fluoro-L-Proline (1) are extremely expensive and very difficult to synthesize on
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large scale. Short, chromatography-free and scale up suitable process is not reported.
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Herein, we report an efficient and scalable process of N-Boc-4-Fluoro-L-Proline (1).
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CN
O
N
H2N
N
O
F
N
NH
CN
F
N
OH
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N
N
Boc
O
F
F
1
Melogliptin
F
Denagliptin
Results and Discussions
There are a number of drawbacks in the conventional synthesis of an optically
active N-Boc-4-Fluoro-L-Proline (1). (Diethylamino)sulfur trifluoride {DAST}2a,e is
most popular fluorinating agent utilized in small scale deoxyfluorination of N-Boc-4-
hydroxy-L-Proline methyl ester (2). However DAST is not suitable for large scale due to
low temperature reaction conditions and thermally unstable/potentially explosive.16 Other
deoxyfluorination agents such as Ishikawa reagent17, Diethylaminosulfur trifluoride &
Morpholinoaminosulfurtrifluoride18a,b, Yarovenko reagent19, 2,2-difluoro-1,3-dimethyl
imidazolidine20, Perfluoroalkanesulfonylfluoride/base21, Sulfuryl fluoride22 are expensive,
highly reactive, involved complex work-up, racemisation and offer only marginal
improvements in chemo-selectivity. Therefore, the use of conventional fluorinating
agents in a large scale may result in severe handling (costs, time) and safety problems.
Unfortunately, the majority of synthetic methods for fluorination have poor green
chemistry metrics or lack practicality. Nucleophilic aliphatic substitution is more general
method through the displacement of a leaving group with a simple fluoride source.23 The
present study describes the use of inexpensive Nosyl fluoride as a fluorinating agent24a,b
which is safe, stable and easy to handle.
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Conventional methods for synthesis of 1 suffer with substantial amount of
elimination products. Hence the purification of optically active 1 from 4 (3,4 or 4,5-
dehydro impurities) is the major challenge in large scale. As outlined in Scheme-1,
literature synthesis of 1 is as follows
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Scheme-1. Literature synthesis of 1
F
F
b
c
O
OH
N
Boc
N
Boc
O
O
3
1
F
F
F
d,e
c
F
O
f,g
O
HO
OH
OH
OH
N
H
a
O
N
N
O
O
N
O
N
H
O
Boc
O
H
O
N
H
O
N
N
Boc
O
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8
Boc
O
6
Boc
O
5
1
3
2
4
F
F
i
h
OH
OH
N
F
N
Boc
O
NH2
10
Boc
O
c
OH
OH
1
N
N
Boc
O
Boc
O
F
1
9
j
OH
N
Boc
O
1
Reagents: (a) Fluorinating agent; (b) Column chromatography; (c) NaOH; (d) TFA;(e) Pd/C; (f) recrystalization; (g) Boc-anhydride; (h)
cylcohexylamine & recrystalization; (i) HCl; (j) NaOCl
a) Column chromatography purification of N-Boc-cis-4-fluoro-L-proline methyl
ester (3) followed by hydrolysis. b) Boc deprotection and reduction of double bond with
Pd/C of 3 & 4. Hydrolysis of 5 & 6 followed by hot ethanol/water treatment to get 4-
Fluoro-L-Proline (7)25 which on Boc protection affords 1. c) Hydrolysis of 3 & 4 to get 1
& 9. Obtained acid derivatives were converted to corresponding cyclohexylamine salts.
Pure 10 was obtained by recrystallization followed by acidification with HCl affords 1.26
d) Hydrolysis of 3 & 4 to get 1 & 9 followed by oxidization affords 1.20 For the pilot-
plant campaign, major issues need to address were economic synthesis, simple process
and most importantly isolation without chromatography.
We postulate that the polar diol derivatives can be easily removed by polar
solvents. As shown in Scheme 2, our initial approach was to prepare easily removable
polar derivatives of 4. Osmium tetroxide (OsO4) & Potassium permanganate (KMnO4)
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were found to be best among different oxidizing agents investigated for oxidation of
alkenes 4 to get corresponding diol 11. Multiple solvents and mixture of solvents were
tested to eliminate diol derivatives 11 while keeping product intact in Toluene.
Gratifyingly, 50% methanol in water was achieved complete elimination of 11 from 1.
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Scheme-2. Initial approach for synthesis of 1
F
OH
HO
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b
a
c
O
O
O
N
N
N
Boc
O
F
Boc
11
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Boc
O
O
O
3
3
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c
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Boc
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Boc
O
F
4
3
OH
d
OH
OH
N
N
N
Boc
O
Boc
O
Boc
O
9
1
Reagents: (a) KMnO4 or OsO4; (b) washing with mixture of solvents; (c) NaOH;
(d) hot toluene
However, these procedures were not taken forward for large scale as OsO4 is
highly toxic27 and expensive whereas KMnO4 reaction involved tedious work-up.
Therefore, we examined alternate suitable conditions for large scale synthesis.
Development of the crystallization of 1 was a major focus prior to the pilot-plant
campaign because this offered an excellent point in the synthesis sequence to purge
impurities and ensure that the final product 1 would be obtained with consistent purity.
We decided to do hydrolysis of 3 to utilize an aqueous workup to remove
impurities. The decision to isolate the carboxylic acid 1 was further supported by physical
properties and solubility studies. Crystalline 1 could be obtained from hot Toluene.
Serendipitously, simple hydrolysis of 3 & 4 using sodium hydroxide afforded 1 & 9,
where pH and temperature played crucial role during isolation. Loss of yield has been
observed in case of lower/higher pH with elevated temperature. A screening of solvents
showed hot Toluene was the most effective solvent to isolate 1 selectively. Optimized
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purification process was successfully applied to isolate 1 for popular reaction conditions
using deoxyfluorination agents such as DAST and Triflic anhydride/TBAF (1M
Tetrabutyl ammonium fluoride in THF).
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We also developed large scale suitable economical process for Nosyl fluoride (13).
Economical, easily recoverable and re-usable solvent such as Toluene was used instead
of Tetrahydrofuran28, Acetonitrile29 and Acetone30. Time of reaction was reduced by
elevated temperature to 55 oC from RT. It was observed that Toluene layer retain pure
product 13 whereas simple water work-up able to remove of associated impurities.
During lab scale reactions, 13 was obtained as a solid by concentration of Toluene
solution to dryness. In view of safety during pilot batch, a solution of 13 in Toluene layer
directly used for next reaction instead of solid isolation. Etching on glass assembly was
observed during lab reaction, it is highly recommended to wear appropriate personal
protective equipment (PPE) and adhere to the standard operating procedure (SOP) while
performing reaction.31
Ultimately, for the pilot-plant campaign, we chose to implement an inherently
safer process in which Toluene layer with 13 was obtained from inexpensive
commercially available Nosyl chloride (12) by treatment of Potassium fluoride (KF) in
biphasic media such as Toluene and water using 18-crown-6 as phase transfer catalyst at
50-55oC. Aqueous layer separated and Toluene layer with 13 was utilized for
deoxyfluorination of 2 to afford 3 & 4 using 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)
as base. After treatment with KHSO4 solution and NaHCO3 solution, Toluene layer was
hydrolysed by NaOH. Acid derivatives 1 & 9 were then isolated by acidification of
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aqueous layer with 50% HCl in water till pH ~3-4 at 0 C. Obtained solid was treated
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with Toluene at 80 oC. Pure 1 obtained by filtration (lab scale)/centrifugation (pilot scale)
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at RT as shown in Scheme-3 and Figure-1.
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Scheme-3. Pilot-plant synthesis of 1
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SO2F
SO2Cl
F
F
c
b
a
O
OH
O
N
N
N
Boc
O
Boc
O
Boc
O
NO2
13
NO2
12
1
4
3
Reagents: (a) KF, 18-crown-6, Toluene; (b) N-Boc-4-hydroxy-L-Proline methyl ester, DBU,
Toluene; (c) NaOH and hot Toluene
Figure-1. Flow diagram of 1
Use of single solvent was major advantage of developed process in case of
recovery and re-use of solvent. Current process established on pilot scale; however it can
be scaled up on commercial scale.
Conclusion
An economical process had been developed for the large scale synthesis of pure
N-Boc-4-Fluoro-L-Proline with moderate yield. The new process involved minimum
steps using plant feasible/re-usable single solvent and overcame purification problems
related to the synthesis of 1. In addition, cost-effective and safe process of Nosyl fluoride
as deoxyfluorinating agent on large scale was identified.
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Experimental Section
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N-Boc-cis-4-fluoro-L-proline methyl ester (3).
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Method-a (using DAST): To a stirred solution of 2 (200g, 0.812 mol) in DCM (2L),
DAST (129mL, 0.978mol) [Caution: highly toxic, extremely corrosive to skin and
readily etches glass. Must be handled with appropriate precautions!] was added
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drop-wise at below -15 C, during the addition, a slight exotherm was observed and
stirred for 4h at RT. After completion of the reaction, the reaction mixture allowed to
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cool to 0 C and quenched with 1.5L of saturated NaHCO3. Separated organic layer
washed with 1L of brine. Organic layer concentrated under reduced pressure. The crude
material (195g) dissolved in 4L acetone and water (1:1), MgSO4.7H2O (100g, 0.406 mol)
followed by addition of KMnO4 (64.14g, 0.406mol) portion-wise at 10-20 C and stirred
at RT for 2-3h. 100g of celite was added and stirred for 30min. The resulting light brown
suspension was filtered, washed with acetone (800mL); filtrate was concentrated under
reduced pressure and diluted with 800mL Toluene. Toluene layer washed with 3x200mL
of methanol: water (1:1) and concentrated under reduced pressure to afford 3 as thick oil
(133g, 66%).
Method-b (using Nosyl Fluoride): To a stirred solution of 2 (1Kg, 4.08 mol) in Toluene
(10 L), DBU (1.24Kg, 8.16mol) was added drop-wise and stirred for 30min at RT. Nosyl
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fluoride (1Kg, 4.89 mol) was added portion-wise and heated to 45 C for 10h. After
completion of the reaction, the reaction mixture allowed to cool to RT and washed with
5% aq. KHSO4 solution (2L). Separated Toluene layer washed with saturated NaHCO3
(2L) and concentrated under reduced pressure to get thick oily mass. The crude material
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(1.1Kg) dissolved in 4L acetone and water (1:1), MgSO4.7H2O (500g, 2.04mol) followed
by addition of KMnO4 (320g, 2.04 mol) portion-wise at 10-20 oC. Reaction mass allowed
to warm to RT and stirred for 2-3h. After completion of reaction, 500g of celite was
added and stirred for 10min. The resulting light brown suspension was filtered, washed
with acetone (500mL); filtrate was concentrated under reduced pressure and diluted with
4L of Toluene. Toluene layer washed with 3x1L of methanol:water (1:1) and
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concentrated under reduced pressure to afford 3 as thick oil (524.3g, 52%). H NMR (a
mixture of rotamers, 400 MHz, CDCl3) δ 1.43-1.49 (s, 9H), 2.29-2.52 (m, 2H), 3.61-3.69
(m, 1H), 3.75 (s, 3H), 3.79-3.86 (m, 1H), 4.42-4.44 (d, 0.5H), 4.54-4.56 (d, 0.5H), 5.15-
13
5.25 (br d, 1H); CNMR (125 MHz, CDCl3) δ 28.2, 28.4, 36.5, 36.7, 37.4, 37.5, 52.2,
52.3, 52.8, 53.0, 53.1, 53.3, 57.2, 57.6, 60.4, 76.9, 77.1, 77.4, 80.4, 90.4, 91.5, 91.9, 92.9,
153.6, 154.0, 171.9, 172.3; Mass: 248 as (M+1)+.
N-Boc-cis-4-fluoro-L-proline (1).
Method-a (using DAST): To a stirred solution of 2 (200g, 0.812 mol) in DCM (2L),
DAST (129mL, 0.978mol) [Caution: highly toxic, extremely corrosive to skin and
readily etches glass. Must be handled with appropriate precautions!] was added
o
drop-wise at below -15 C, during the addition, a slight exotherm was observed and
stirred for 4h at RT. After completion of the reaction, the reaction mixture allowed to
o
cool to 0 C and quenched with 1.5L of saturated NaHCO3. Separated organic layer
washed with 1L of brine. Solution of NaOH (65.3g, 1.63mol) in water (650mL) was
added to organic layer and heated to 40oC for 3h. After completion of reaction, DCM
layer was separated. Aqueous layer acidified with 50% HCl in water at 0oC till pH ~3-4.
Obtained solid was filtered, suspended in Toluene (600mL) and heated to 80 oC for 2h.
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Reaction mass allowed to cool to RT, filtered, washed with Toluene (200mL), dried to
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afford 1 as white solid (137g, 72%)
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Method-b (using triflic anhydride/TBAF): To a stirred mixture of 2 (50g, 0.204 mol)
and Triethyl amine (41.3g, 0.408 mol) in THF (500mL), triflic anhydride (70.52g,
0.25mol) was added drop-wise at 0-5 oC and stirred for 4h at RT. After completion of
reaction, 1M TBAF in THF (306mL) was added at 0-5 oC and stirred for 16h at RT. After
completion of reaction, reaction mass diluted with ice-cold water and extracted with
DCM. Solution of NaOH (16.2g, 2eq) in water (160mL) was added to DCM layer and
heated to 40 oC for 3h. DCM layer was separated. Aqueous layer was acidified with 50%
HCl in water at 0oC till pH ~3-4. Obtained solid was filtered, suspended in Toluene
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(150mL) and heated to 80 C for 2h. Reaction mass allowed to cool to RT, filtered,
washed with Toluene (50mL), dried to afford 1 as white solid (26.1g, 55%)
Method-c (using Nosyl fluoride): To a stirred solution of 4-nitrobenzene-1-sulfonyl
chloride (11) (8.89Kg, 40.11 mol) in Toluene (35L), solution of KF (11.55Kg, 199.1mol)
in water (35L) followed by 18-crown-6 (0.115kg, 0.44mol) were added and heated to 55
oC for 6h. After completion of reaction, reaction mass cooled to RT, filtered through
pressure nutsche filter (PNF) and washed with Toluene (5L). Separated Toluene layer
was added to stirred mixture of 2 (7.0 Kg, 28.57mol) and DBU (8.7 Kg, 57.16mol) in
Toluene (21L). Reaction mass heated to 50-55oC for 10h. After completion of reaction,
reaction mixture allowed to cool to RT and washed by 5% aq. KHSO4 solution (21L).
Separated Toluene layer was washed with saturated NaHCO3 (21L). Solution of NaOH
(2.4Kg, 60mol) in water (17L) was added to Toluene layer and heated to 45oC for 3h.
Toluene layer was separated and recovered. Aqueous layer acidified with 50% HCl (10L)
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in water at 0 oC till pH ~3-4. Obtained solid was filtered, suspended in Toluene (21L) and
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heated to 80 C for 2h. Reaction mass allowed to cool to RT, filtered, washed with
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Toluene (7L), dried to afford 1 as white solid (4.2Kg, 63%). H NMR (a mixture of
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rotamers, 400 MHz, DMSO-d6) δ 1.36 (s, 5H), 1.41 (s, 4H), 2.21-2.57 (m, 2H), 3.51-3.58
(m, 2H), 4.25-4.30 (m, 1H), 5.19-5.31 (br d, 1H); 13C NMR (125 MHz, DMSO-d6) δ 27.9,
28.1, 35.9, 36.0, 36.7, 36.8, 52.7, 52.9, 53.0, 53.1, 56.9, 57.2, 78.9, 79.0, 90.9, 92.0, 92.3,
93.4, 153.1, 153.2, 172.6, 172.8; Mass: 232 as (M-1)+; HPLC: 99.29%; SOR [α] 22/D:
−71.0 to -74.0 deg, c = 1 in chloroform; Melting point: 159-162 °C (D).
Supporting Information
Experimental procedures, copies of the NMR, Mass and HPLC spectra of all
intermediates and the final product.
AUTHOR INFORMATION
Corresponding Author
* Email: amerwade@wockhardt.com. Telephone: (0091) 2406694142
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Notes
The authors are employees of Wockhardt Research Centre, Aurangabad, India and
declare no competing financial interest.
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Acknowledgments
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We wish to thank Dr. Vipul Rane and Dr. Ravindra Yeole for analytical support as well
as pilot plant team for scale up support.
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