Scale-up synthesis of a deuterium-labeled cis-cyclobutane-1,3-Dicarboxylic acid derivative using continuous photo flow chemistry

Article abstract of DOI:10.1016/j.tet.2018.12.037

Continuous photo flow synthesis of tert-butyl 3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-6-carboxylate (2b) from tert-butyl 2-oxo-2H-pyran-5-carboxylate (1b) has been investigated for scale-up synthesis of cis-3-(tert-butoxycarbonyl)-2,3,4-d₃-cyclobuta

Full text of DOI:10.1016/j.tet.2018.12.037

Accepted Manuscript
Scale-up synthesis of a deuterium-labeled cis-cyclobutane-1,3-Dicarboxylic acid
derivative using continuous photo flow chemistry
Toshiro Yamashita, Hitoaki Nishikawa, Tetsuji Kawamoto
PII:
S0040-4020(18)31515-1
https://doi.org/10.1016/j.tet.2018.12.037
TET 30023
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 November 2018
Revised Date: 14 December 2018
Accepted Date: 18 December 2018
Please cite this article as: Yamashita T, Nishikawa H, Kawamoto T, Scale-up synthesis of a deuterium-
labeled cis-cyclobutane-1,3-Dicarboxylic acid derivative using continuous photo flow chemistry,
Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2018.12.037.
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Graphical Abstract
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Scale-up Synthesis of a Deuterium-labeled cis-Cyclobutane-
1,3-Dicarboxylic Acid Derivative using Continuous Photo
Flow Chemistry
Leave this area blank for abstract info.
Toshiro Yamashita,* Hitoaki Nishikawa, and Tetsuji Kawamoto*
Chemistry Research Division, Axcelead Drug Discovery Partners,
26-1 Muraoka-Higashi2-chome, Fujisawa, Kanagawa 251-0012, Japan

ACCEPTED MANUSCRIPT
Scale-up Synthesis of a Deuterium-labeled cis-Cyclobutane-1,3-Dicarboxylic Acid
Derivative using Continuous Photo Flow Chemistry
Toshiro Yamashita,* Hitoaki Nishikawa, and Tetsuji Kawamoto*
Chemistry Research Division, Axcelead Drug Discovery Partners,
26-1 Muraoka-Higashi2-chome, Fujisawa, Kanagawa 251-0012, Japan
Abstract: Continuous photo flow synthesis of tert-butyl 3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-
6-carboxylate (2b) from tert-butyl 2-oxo-2H-pyran-5-carboxylate (1b) has been investigated for
scale-up synthesis of cis-3-(tert-butoxycarbonyl)-2,3,4-d3-cyclobutanecarboxylic acid (3c) as a
useful building block for preparation of various biologically active compounds and those of material
sciences containing cyclobutane ring system(s) labeled with deuterium atoms. Optimization of
reaction conditions and modification of the photo flow reaction system have brought about the
production of cis-3-(tert-butoxycarbonyl)-2,3,4-d3-cyclobutanecarboxylic acid (3c) with excellent
deuterium content in 3.6 g through the continuous photo flow synthesis for 22 h followed by
hydrogenation with deuterium gas.
Also, application of the product to synthesis of
cis-3-((benzyloxycarbonyl)methyl-d2)cyclobutane-1,2,4-d3-carboxylic acid (11) is described for
preparation of internal standards of drug candidate compounds in quantitative mass spectrometry
analyses in nonclinical and clinical pharmacokinetic studies.
Keywords
Flow synthesis; Photocycloaddition; Cyclobutane-1,3-dicarboxylic acid; Deuterium-labeled
molecules; Internal standards
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1. Introduction
Recently, deuterium-labeled molecules1 have attracted considerable attention especially in
pharmaceutical industry because of their application to drug discovery research by improving
pharmacokinetic properties (ADMET),2 proteomics,3 metabolomics,4 and medical imaging,5 as well
as internal standards in quantitative mass spectrometry analyses6 in nonclinical and clinical
pharmacokinetic studies.
Furthermore, deuterium-labeled molecules are commonly used as tools for reaction mechanism
studies7 and as functional materials.8 Therefore, efficient and regioselective incorporation of
deuterium atom(s) into a variety of compounds has been of great synthetic interest.9
Cyclobutane10 ring system is widely found in various biologically active compounds11 and in
recent drug discovery research, 1,3-cyclobutylene unit has been reported to act as bio-isosteres of
vinylene12 and 1,4-phenylene13 groups to give rise to drug candidate compounds. From these points
of view, it should be intriguing to introduce deuterium atom(s) onto their cyclobutane ring system not
only for improvement of their pharmacokinetic properties (ADMET)14 but preparation of their
analytical standards in mass spectrometry. However, practical syntheses of deuterium-labeled
cyclobutane derivatives15 have rarely been reported and their application to drug discovery research
and material sciences are remained to be explored.
Among synthetic building blocks containing cyclobutane ring system(s) cis-cyclobutane-1,3-
dicarboxylic acid derivatives16 have been used popularly for preparation of various biologically
active compounds.11 The present paper describes scale-up synthesis of a deuterium-labeled
cis-cyclobutane-1,3-dicarboxylic acid derivative, cis-3-(tert-butoxycarbonyl)-2,3,4-d3-cyclobutane-
carboxylic acid (3c), through investigation of continuous photo flow synthesis of tert-butyl
3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-6-carboxylate (2b) from tert-butyl 2-oxo-2H-pyran-5-
carboxylate (1b) and its following hydrogenation with deuterium gas.
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Also, an application of 3c to synthesis of cis-3-((benzyloxycarbonyl)methyl-d2)cyclobutane-
1,2,4-d3-carboxylic acid tert-butyl ester (11) is described for preparation of analytical standards in
mass spectrometry of drug candidate compounds.
2. Results and discussion
2.1. Investigation of continuous photo flow synthesis of 2 from 1
Baran reported new synthetic method of 1,3-cyclobutane dicarboxylate16a,b from methyl coumalate
(1a) by intramolecular [2+2] photocycloaddition reaction followed by hydrogenation reaction in the
presence of Pt/C (Scheme 1). The synthetic scheme involves a potential intermediate 2a for 3a18
labeled with three deuterium atoms on the cyclobutane ring system by hydrogenation with deuterium
gas. Generally intramolecular [2+2] photocycloaddition reactions of methyl coumalate in
batch16a,b,19 are carried out under diluted conditions to avoid side reactions which would not be
suitable for practical syntheses of 2a and 3a for research and development of new drugs and
materials. To solve the problems of the photo reaction in conventional batch process application of
continuous flow chemistry to the photocycloaddition reaction was investigated.
Scheme 1. Synthesis of 1,3-cyclobutanedicarboxylate 3a reported by Baran.
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A photo flow reactor20 (Fig. 1) was set up for investigations: FEP capillary tube (inner diameter =
0.75 mm, length = 8 m) was coiled around a Pyrex immersion well (a winding core, outer diameter
(D) = 53 mm) and high pressure Hg lamp (400 or 450 W) was placed at the center of the core.
Continuous stream of the solution of 1a (5 x 10-2 – 1 x 10-1 (M)) was provided into the flow reactor
by syringe pump at varying flow rates (0.1 - 0.5 (mL/min)). The ratio of 1a, 2a, and by-products in
reaction solution was measured by means of an HPLC and the results are shown in Table 1.
Fig. 1. Flow photocycloaddtion reaction system with FEP-capillary coiled around a high pressure
mercury lamp.
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Table 1
Feasibility study of continuous intramolecular [2+2] photocycloaddition reaction of 1a into 2a.a
τ (min)b
entry
1a (M)
flow rate (mL/min)
HPLC area (%)
By-productsc
1a
76
58
30
0
2a
24
1
2
1 x 10-1
1 x 10-1
1 x 10-1
1 x 10-1
5 x 10-2
5 x 10-2
6 x 10-2
5 x 10-2
5 x 10-2
5 x 10-1
3 x 10-1
2 x 10-1
1 x 10-1
2 x 10-1
3 x 10-1
2 x 10-1
2 x 10-1
7.1
0
3
11.8
17.7
35.3
17.7
11.8
17.7
17.7
39
3
59
11
18
0
4
82
5
0
100
> 99
> 99
100
61
6
< 1
< 1
0
0
7
0
8d
9e
0
Reaction in batch
0
39
a At 6 – 8 °C, in toluene, under N2, irradiated by 450 W high pressure Hg lamp.
b Residence time.
c Total unidentified by-products.
d Continuous stream of solution of 1a was provided for 6.7 h.
e Reaction time, 2 h.
The conversion of 1a into 2a increased as flow rate of solution 1a decreased (entries 1 – 4)
concomitant with formation of by-products and 1a was consumed completely on irradiation for 35.3
minutes at a flow rate of 0.1 mL/min (entry 4).
Considering these results, continuous flow reactions with lower concentration and higher flow rate
than those shown in entry 4 were investigated to optimize the reaction condition (entries 5 – 7).
And it has been found that full conversion of 1a into 2a was observed without significant formation
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of by-products under reaction condition shown in entry 5. Higher flow rate than that in entry 5
resulted in recovery of small amount of 1a (entry 6). Thus, continuous stream of a solution of 1a
was provided into the flow reactor for 6.7 h (total 616 mg) under the same reaction condition as entry
5 (entry 8) and obtained 2a was subjected to hydrogenation in the presence of Pt/C to give rise to 3a
(631 mg) almost quantitatively. In contrast to the results of continuous photo flow reactions, the
corresponding photocycloaddition reaction in conventional batch process brought about full
conversion of 1a on irradiation for 2 h to give 2a with significant formation of by-products (entry 9).
These results strongly support that continuous photo flow synthesis of 2a would be suitable for
scale-up synthesis of 3a and that labeled with deuterium atoms.
Next, optimization of the continuous photocycloaddition reaction condition was investigated using
tert-butyl 2-oxo-2H-pyran-5-carboxylate (1b) as a substrate which would give rise to a more
versatile cyclobutane building block cis-3-(tert-butoxycarbonyl)-2,3,4-d3-cyclobutane-carboxylic
acid (3c) via tert-butyl 3-oxo-2- oxabicyclo[2.2.0]hex-5-ene-6-carboxylate (2b) (Table 2).
It has been found that continuous stream of solution of 1b (total 780 mg) into the photo flow
reactor under same condition as Table 1. entry 8 and following hydrogenation of 2b afforded 3b (678
mg, 85% in 2 steps) without significant formation of by-products, though 1b was not completely
consumed (entry 1). The photo flow reactor coiled with FEP capillary tube with larger inner
diameter (ID = 1.59 mm) (entry 2) and that with double-coil of FEP capillary tube (ID = 0.75 mm)
(entry 3) gave similar conversion of 1b to that in entry 1 but significant formations of by-products
(data not shown) were observed probably due to longer residence time.
Thus, continuous stream
of solution 1b (total 13.5 g) was provided at higher flow rate than in entry 3 into the photo flow
reactor with double-coil of FEP capillary tube for 76 h and obtained reaction solution was
hydrogenated in the presence of Pt/C to afford 3b (11.1 g, 81% in 2 steps) without significant
formation of by-products (entry 4).
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Table 2
Effects of FEP capillary tube coil on intramolecular [2+2] photocycloaddition reaction of 1b into 2b.a
entry FEP capillary tube flow rate
Operation Conversion yield of yield of
τ
(mL/min) (min)b time (h)c of 1b (%)d
2be
3b
(g/h)
(g)f
IDg
Lh
Type
(mm) (m) of coil
1
0.75
1.59
8
5
single 2 x 10-1
single 2 x 10-1
17.7
6.7
92
0.10
0.68
(85%)
ND
2
3
4
49.7
59.7
39.8
1.0
1.2
76
91
94
86
ND
ND
0.75 27 double 2 x 10-1
0.75 25 double 3 x 10-1
ND
0.15
11.1
(81%)
a [1b] = 5 x 10-2 (M), in toluene, at 6 – 8 °C, under N2, irradiated by 450 W high pressure Hg lamp.
b Residence time.
c Time for continuous delivery of a stream of solution of 1b into the reactor.
d Determined by an HPLC.
e Estimated from yield of 3b and operation time.
f Yield from 1b (for 2 steps).
g Inner diameter of FEP capillary tube.
h Length of the coil.
Furthermore, modification of the photo flow reactor and effect of reaction solvent was investigated
to provide higher yield of 2b (Table 3). The photo flow reactor composed of 400 W high pressure
Hg lamp and Pyrex immersion well with shorter diameter (D = 43 mm) has been found to give 2b in
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better yield (0.18 g/h) (entry 2) than that in entry 1 (0.10 g/h) composed of the 450 W lamp and the
well with longer diameter (D = 53 mm). The result suggests that change in distance between
radiation source and reaction solution rather than that in power of radiation source affects
significantly the yield of 2b.
Table 3
Effects of modification of the photo flow reactor and solvent on intramolecular [2+2]
photocycloaddition reaction of 1b into 2b.a
entry
number
of
High
D
solvent
flow rate
Conversion yield of
τ
pressure (mm)b
(mL/min) (min)c of 1b (%)d
2b
reactor
Hg
lamp
(W)
(g/h )e
1f
2
3
4
5
1
1
1
1
2
450
400
400
400
400
53
43
43
43
43
Toluene
Toluene
Toluene
MeCN
2 x 10-1
3 x 10-1
5 x 10-1
5 x 10-1
7.5 x 10-1
17.7
11.8
12.9
12.9
10.0
92
92
76
86
86
0.10
0.18
ND
0.24
0.36
MeCN
a [1b] = 5 x 10-2 (M), at 6 – 8 °C, under N2.
b Outer diameter of Pyrex immersion well.
c Residence time.
d Determined by an HPLC.
e Estimated from yield of 3b.
f Data from Table 2 entry 1.
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Treatment of solution of 1b at higher flow rate than in entry 2 resulted in lower conversion of 1b
(entry 3). Other solvents (THF, EtOAc, DMF, and MeCN) than toluene were tested for the reaction
under the same condition as entry 3 and MeCN has been found to afford better yield of 2b (0.24 g/h)
(entry 4). The two photo flow reactors connected in series afforded 2b in 0.36 g/h (entry 5) which
was not twice as much as that in entry 4.
2.2. Synthesis of cis-3-(tert-butoxycarbonyl) -2,3,4-d3-cyclobutanecarboxylic acid (3c)
It is well understood that the internal standards in quantitative mass spectrometry analysis6 in
nonclinical and clinical pharmacokinetic studies are necessary to contain at least 3 deuterium atoms
and no parent compounds without deuteration. Synthesis of 3c with excellent deuterium atom
content was investigated by hydrogenation with deuterium gas of 2b obtained by continuous photo
flow synthesis from 1b under the reaction condition shown in Table 3. entry 2.
Continuous flow hydrogenation of 2b with deuterium gas by using H-Cube® treated with D2O9a)
was investigated first using a Pt/C catalyst cartridge. It has been found that compound 3c with
deuterium atom content21 (d0 : d1 : d2 : d3 = 1.7 : 10.7: 48.7 : 100)22 insufficient for analytical
standards in mass spectrometry was obtained in 73% probably due to contamination of D2O with
moisture in the air. Therefore, compound 2b was hydrogenated with deuterium gas in MeOD in
batch carefully to afford 3c in 89% with excellent deuterium atom content (d0 : d1 : d2 : d3 = 0 : 1:
18 : 100). Thus, scale-up synthesis of compound 3c (3.6 g, 89%) has successfully been achieved
through continuous photo flow synthesis of 2b from 1b (3.9 g) for 22 h followed by hydrogenation
with deuterium gas.
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2.3. Syntheses of analytical standards in mass spectrometry of drug candidates from 3c
Recently, compounds 523a and 623b bearing a cis-1,3-cyclobutylene unit have been developed as
candidate drugs (Fig. 2). Their analytical standards in mass spectrometry are essential for their
research and development and they would be synthesized most conveniently from 7 labeled with
more than 3 deuterium atoms6 and corresponding amines. It is plausible that compound 7 labeled
with more than 3 deuterium atoms on the cyclobutanemethylene moiety would be prepared from 3c
by a homologation reaction of the carboxylic acid group.
Fig. 2. Drug candidate compounds bearing cis-1,3-cyclobutylene unit and a common key
intermediate 7 to be labeled with deuterium atoms.
Thus, diazoacetyl compound 8 was synthesized from 3c and a mixture of 8 and benzyl alcohol was
treated with MeOD before Wolff rearrangement reaction to afford 10 with an excellent deuterium
atom content (d0 : d1 : d2 : d3 : d4 : d5 = 0 : 0: 0.7 : 9 : 47 : 100).24 Interestingly enough, another 2
deuterium atoms have been found to be introduced into 10 in the homologation reaction, suggesting
that 9 was obtained by an H/D exchange of 825 in MeOD.
Removal of the tert-butyl group of 10 with TFA afforded 11 almost quantitatively without loss of
deuterium atom content. 1H NMR spectra of 11 revealed all the deuterium atoms adopt syn
orientation regarding cyclobutane ring system suggesting that hydrogenation of 2b into 3b takes
place only on its one face (Schemes 1 and 2).
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Condensation of 11 with the corresponding amine followed by hydrogenation would afford an
analytical standard of 5. Also, removal of the benzyl group of 10 by hydrogenation would give a
building block for synthesis of an analytical standard of 6.
Scheme 2. Synthesis of compound 11 from 3c.
CO2t-Bu
D
CO2H
CO2t-Bu
CO2t-Bu
CO2t-Bu
D
D
D
D
D
O
D
O
D
D
a, b
c
e
d
D
D
D
D
D
D
D
D
CO2H
N2
N2
CO2Bn
CO2Bn
D
D
D
9
3c
8
10
11
d0:d1:d2:d3 = 0:1:18:100
d0:d1:d2:d3:d4:d5 = 0:0:0.7:9:47:100
Reagents and conditions: (a) ClCO2Et, Et3N, - 15 °C, 2 h; (b) trimethylsilyldiazomethane, - 15 °C, 16
h, 24% (for 2 steps); (c) BnOH, MeOD, rt, 0.5 h (total); (d) AgOBz, Et3N, rt, 0.5 h then 50 °C, 1 h,
55.5% (for 2 steps) ; (e) TFA, rt, 3 h, quant.
3. Conclusions
Continuous photo flow synthesis of 2b from 1b has been investigated for scale-up synthesis of 3c
as a useful building block for preparation of various biologically active compounds and those of
material sciences containing cyclobutane ring system(s) labeled with deuterium atoms.
Optimization of reaction conditions and modification of the photo flow reaction system has led to
scale-up synthesis of 2b without significant formation of by-products found in corresponding batch
reactions. And hydrogenation of 2b with deuterium gas gave rise to 3c with excellent deuterium
atom content. Application of 3c as a building block to synthesis of 11 has been demonstrated for
preparation of internal standards of drug candidate compounds in quantitative mass spectrometry
analysis in nonclinical and clinical pharmacokinetic studies.
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The above results also suggest that the present method could give
a
cis-cyclobutane-1,3-dicarboxylate fully labeled with deuterium atoms from 2-oxo-2H-pyran-5-
carboxylate-3,5,6-d3 which would be more useful for research and development of drugs and new
materials.
In conclusion, the present studies affect significantly not only development of continuous photo
flow chemistry for practical syntheses but also research and development of drugs and new materials
containing cyclobutane ring system labeled with deuterium atoms.
4. Experimental section
4.1. General methods and materials
13
1H NMR spectra were recorded on Bruker AVANCE III (300 MHz) and C NMR spectra were
recorded on Bruker Avance II+ 600 (600 MHz), Chemical shifts are given in parts per million (ppm)
downfield from tetramethysilane (δ) as the internal standard in deuterated solvent and coupling
constants (J) are in Hertz (Hz). Data are reported as follows: chemical shift, integration, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, brs = broad
singlet) and coupling constants. All solvents and reagents were obtained from commercial suppliers
and used without further purification. Thin-layer chromatography (TLC) was performed on Merck
TLC silica gel plates 60F254 or Fuji Silysia Chromatorex TLC plates NH. Column 13
chromatography was performed on silica gel 60 (0.063–0.200 or 0.040–0.063 mm, Merck). LC-MS
analysis involving D content measurement was performed on a Waters, Agilent, or Shimadzu Liquid
Chromatography-Mass Spectrometer System, operating in APCI (+ or −) or ESI (+ or −) ionization
mode. Analytes were eluted using a linear gradient of 0.05% TFA containing water/acetonitrile,
0.1% TFA containing water/acetonitrile or 5 mM ammonium acetate containing water/acetonitrile
mobile phase. The purities of compounds were determined by LC-MS analysis (detection at 220 nm).
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High resolution MS analyses were carried out by Sumika Chemical Analysis Service, Ltd. Elemental
analyses were carried out by Torey Research Center Inc. Yields are not optimized.
4.2.Typical procedure for continuous photo flow synthesis 2 from 1
4.2.1
Typical
procedure
for
continuous
photo
flow
synthesis
of
methyl
3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-6-carboxylate (2a) (Table 1. entry 1)
A continuous stream of solution of methyl 2-oxo-2H-pyran-5-carboxylate 1a in toluene (1 x 10-1
(M)) (degassed prior to use) was provided by a syringe pump at flow rate of 5 x 10-1 (mL /min) to a
photo flow reactor composed of FEP capillary tube (Shimadzu GLC FEP, i.d. = 0.75 mm, L = 8 m),
high pressure Hg lamp (USHIO 450W UM452), and Pyrex emmersion well (outer diameter = 53
mm) to be irradiated at 6 - 8 ºC in EtOH/water bath. The collected reaction solution was subjected
to an HPLC analysis to determine the ratio of 1a, 2a, and by-products.
4.2.2.
Typical
procedure
for
continuous
photo
flow
synthesis
of
tert-butyl
3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-6-carboxylate (2b) (Table 2. entry 1)
Continuous stream of a solution of tert-butyl 2-oxo-2H-pyran-5-carboxylate 1b in toluene (5 x
10-2 (M)) (degassed prior to use) was provided by syringe pump at flow rate of 2 x 10-1 (mL/min) to
the same photo flow reactor described in 4.2.1. The collected solution was subjected to an HPLC
analysis to determine the ratio of 1b, 2b, and by-products.
4.3. Synthetic procedures
4.3.1. tert-Butyl 2-oxo-2H-pyran-5-carboxylate (1b)
To
a
solution of 2-oxo-2H-pyran-5-carboxylic acid (5.00 g, 35.69 mmol) and
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di-tert-butyldicarbonate (9.84 mL, 42.83 mmol) in tert-BuOH (40 mL), was added
4-dimethylaminopyridine (4.36 g, 35.69 mmol) all at once at 0 °C (Caution, vigorous evolution of
CO2 gas). The mixture was stirred at room temperature under N2 for 4 h and concentrated under
reduced pressure. The residue was purified by column chromatography on silica gel (eluted with a
gradient of 0 - 25% of EtOAc in hexane) to give 1b (3.00 g, 42.8 %) as a white amorphous powder.
1H NMR (600 MHz, CDCl3) δ 1.55 (s, 9H), 6.32 (dd, J = 9.9, 1.1 Hz, 1H), 7.75 (dd, J = 9.5, 2.6 Hz,
1H), 8.21 (dd, J = 2.6, 1.1 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 28.13, 82.70, 113.43, 115.00,
142.01, 157.62 ,160.18, 162.02. HRMS (m/z): [M-H]+ calcd. for C10H12O4, 197.0808; found
197.0811.
4.3.2. cis-3-(Methoxycarbonyl)cyclobutanecarboxylic acid (3a) (Table 1. entry 8)
Continuous stream of a solution of methyl 2-oxo-2H-pyran-5-carboxylate 1a (616 mg, 3.99 mmol)
in toluene (80 mL, degassed prior to use) was provided by syringe pump at flow rate of 0.2 mL/min
for 6.7 h to the above described photo flow reactor. The collected reaction solution was
concentrated under reduced pressure below 40 ºC to give crude methyl 3-oxo-2-
oxabicyclo[2.2.0]hex-5-ene-6-carboxylate 2a26) (616 mg, quant.). The crude 2a (616 mg, 3.99
mmol) was dissolved in THF (12 mL) and the solution was hydrogenated in the presence of 5%
platinum carbon (100 mg, 0.03 mmol) under atmospheric pressure at room temperature for 16 h. The
catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The
residue was dissolved in Et2O and extracted with aqueous saturated NaHCO3 solution. The
aqueous layer was acidified with aqueous 2 N HCl solution at pH = 2 under cooling and was
extracted back with EtOAc twice. The combined organic layer was washed with brine and
concentrated under reduced pressure to afford 3a18a (631 mg, quant.) as a colorless oil.
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4.3.3. cis-3-(tert-Butoxycarbonyl)cyclobutanecarboxylic acid (3b) (Table 2. entry 1).
Continuous stream of a solution of tert-butyl 2-oxo-2H-pyran-5-carboxylate 1b (780 mg, 3.98
mmol) in toluene (80 mL, degassed prior to use) was provided by syringe pump at flow rate of 0.2
mL/min for 6.7 h to the same photo flow reactor described above. The collected solution was
concentrated under reduced pressure below 40 ºC to give crude tert-butyl
3-oxo-2-oxabicyclo[2.2.0]hex-5-ene-6-carboxylate 2b27) (783 mg, quant.). The crude 2b (780 mg,
3.98 mmol) was dissolved in THF (12 mL) and the solution was hydrogenated in the presence of 5%
platinum carbon (100 mg, 0.03 mmol) under atmospheric pressure at room temperature for 16 h. The
catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The
residue was dissolved in Et2O and extracted with aqueous saturated NaHCO3 solution. The
aqueous layer was acidified with aqueous 2 N HCl solution at pH = 2 under cooling and was
extracted back with EtOAc twice. The combined organic layer was washed with brine and
1
concentrated under reduced pressure to afford 3b (678 mg, 85%) as a colorless oil. H NMR (600
MHz, CDCl3) δ 1.45 (s, 9H), 2.41-2.48 (m, 2H), 2.48-2.55 (m, 2H), 2.97 (quin, J = 9.0 Hz, 1H), 3.06
(quin, J = 9.1 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 28.04, 28.13, 33.54 , 34.93, 80.57,
173.49,180.02 . HRMS (m/z): [M-H]- calcd. for C10H16O4, 199.0976; found 199.0986.
4.3.4. cis-3-(tert-Butoxycarbonyl)cyclobutanecarboxylic acid-d3 (3c)
Continuous stream of a solution of tert-butyl 2-oxo-2H-pyran-5-carboxylate 1b (3.90 g, 19.88
mmol) in toluene (400 mL, degassed prior to use) was provided for 22.2 h by syringe pump at flow
rate of 0.3 mL/min to the photo flow reactor composed of FEP capillary tube (Shimadzu GLC FEP,
i.d. = 0.75 mm, L = 8 m), Pyrex emmersion well (inner diameter = 43 mm) and high pressure Hg
lamp (Sen 400W HL400B-8) to be irradiated at 6-8 ºC in EtOH/water bath. Collected reaction
solution was concentrated under reduced pressure below 40 ºC to give crude tert-butyl 3-oxo-2-
oxabicyclo[2.2.0]hex-5-ene-6-carboxylate 2b (3.90 g, quant.). Crude 2b was dried by azeotoropic
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distillation with MeOD (20 mL) three times before it was hydrogenated by Method A or B shown
below.
Method A: Continuous-flow hydrogenation of compound 2b by using H-Cube® treated with D2O.9b
Continuous stream of a solution of 2b (293 mg, 1.49 mmol) in THF (20 mL) was provided into
H-cube® (ThalesNano Inc.) treated with D2O (99.8%) with 10% Pt/C catalyst cartridge (70 mm) at
flow rate of 0.4 mL/min at room temperature for 50 min. The collected reaction solution was
concentrated under reduced pressure. To the residue was added aqueous saturated NaHCO3
solution and washed with Et2O. The aqueous layer was acidified with aqueous 2 N HCl at pH = 2
at 0 ºC and was extracted back with EtOAc twice. The combined extracts were washed with brine
and concentrated under reduced pressure to give 3c (220 mg, 72.5%).
To a solution of obtained 3c and 3-aminopyridine (93 mg, 0.98 mmol) in DMF (3 mL), was added
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (115 mg, 0.74 mmol) and 1-hydroxybenzotriazole
hydrate (113 mg, 0.74 mmol) at 0 °C. The mixture was stirred at room temperature under N2 for 2
h.
The obtained reaction solution of N-(3-aminopyridin-2-yl)-cis-3-(tert-butoxycarbonyl)-
2,3,4-d3-cyclobutanecarboxamide was subjected to an LCMS analysis to determine deuterium
content of 3c.
Method B: Hydrogenation with deuterium gas in batch.
A solution of crude 2b (3.90 g) in MeOD (40 mL) was hydrogenated by deuterium gas (99.96%)
in the presence of Pt/C (400 mg, 0.10 mmol) under atmospheric pressure at room temperature for 24
h. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure.
The residue was dissolved in IPE and extracted with aqueous saturated NaHCO3 solution. The
aqueous layer was acidified aqueous 2 N HCl solution at pH = 2 under cooling and was extracted
back with IPE twice. The combined organic layer was washed with brine to afford 3c28) (3.58 g,
16

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89 %) as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 1.45 (s, 9H), 2.49 (br d, J = 9.2 Hz, 2H),
3.05 (t, J = 9.2 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 27.69 (t, J = 21.60 Hz), 28.04, 33.29, 34.33
(t, J = 21.60 Hz), 80.55, 173.51, 180.03. HRMS (m/z): [M-H]- calcd. for C10H13D3O4 202.1164,
found 202.1171.
4.3.5. cis-3-((Benzyloxycarbonyl)methyl-d2)cyclobutane-1,2,4-d3-carboxylic acid tert-butyl ester
(10)
To a solution of cis-3-(tert-butoxycarbonyl)cyclobutanecarboxylic acid-d3 3c (1.29 g, 6.35 mmol)
and TEA (0.796 mL, 5.71 mmol) in dry THF (24 mL), was added a solution of ethyl chloroformate
(0.668 mL, 6.98 mmol) in THF (3.0 mL) dropwise at - 15 ºC. The mixture was stirred at - 15 ºC
under N2 for 2 h. To the mixture were added a solution of 0.6 M trimethylsilyldiazomethane solution
in hexane (26.4 mL, 15.87 mmol) and MeCN (15 mL) at - 15 ºC and the mixture was stirred at r.t. for
16 h.
The reaction mixture was concentrated and the residue was purified by column
chromatography on silica gel (eluted with a gradient of 0 - 30% of EtOAc in hexane) to give 8 (0.346
g, 24.0 %) as a pale yellow oil which was used for the subsequent step immediately.
A mixture of 8 (894 mg, 3.93 mmol) and benzyl alcohol (9.77 mL, 94.41 mmol) was treated with
MeOD (23.8 mL, 590.03 mmol) at room temperature for 10 min and was concentrated under reduced
pressure. The treatment was repeated 3 times before a solution of silver benzoate (360 mg, 1.57
mmol) in triethylamine (2.74 mL, 19.67 mmol) was added to the residue at 5 ºC. The mixture was
stirred at room temperature for 30 min, then at 50 ºC for 1 h. The reaction mixture was filtered by
Celite and the filtercake was washed with EtOAc. The filtrate was washed with aqueous saturated
NaHCO3 solution, aqueous saturated NH4Cl solution and brine, and then concentrated under reduced
pressure. The residue was purified by column chromatography on silica gel (eluted with a gradient
of 0 - 50% EtOAc in hexane) to give 1028) (676 mg, 55.5 %) as a colorless oil. 1H NMR (600 MHz,
CDCl3) δ 1.43 (s, 9H), 1.90 (br d, J = 9.2 Hz, 2H), 2.54-2.59 (m, 1H), 5.09 (s, 2H), 7.29-7.38 (m,
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13
5H). C NMR (151 MHz, CDCl3) δ 27.25, 28.08, 30.74 (t, J = 21.60 Hz), 35.11 (t, J = 21.00 Hz),
40.33 (quin, J = 18.80 Hz), 66.09, 80.00, 128.12, 128.17, 128.54, 136.03, 172.14, 174.41. HRMS
(m/z): [M+H]+ calcd. for C18H19D5O4, 310.2061; found 310.2020.
4.3.6. cis-3-((Benzyloxycarbonyl)methyl-d2)cyclobutane-1,2,4-d3-carboxylic acid (11)
A mixture of cis-3-((benzyloxycarbonyl)methyl-d2)cyclobutane-1,2,4-d3-carboxylic acid tert-butyl
ester (10) (1.28 g, 4.14 mmol) and TFA (15.94 mL, 206.84 mmol) was stirred at r.t. for 3 h and
concentrated. Removal of excess TFA by azeotropic distillation with toluene gave 1128) (1.050 g,
1
quant.) as a colorless oil. H NMR (600 MHz, CDCl3) δ 2.00 (br d, J = 9.2 Hz, 2H), 2.61-2.66 (m,
1H), 5.10 (s, 2H), 7.30-7.38 (m, 5H). 13C NMR (151 MHz, CDCl3) δ 27.39, 30.66 (t, J = 21.00 Hz),
33.77 (t, J = 20.50 Hz), 40.19 (quin, J = 20.50 Hz), 66.20, 128.17, 128.23, 128.56, 135.93, 171.97,
180.54. HRMS (m/z): [M-H]- calcd. for C14H11D5O4 , 252.1290; found 252.1281.
Acknowledgments
We would like to acknowledge and thank Mr. M. Iida and Ms. Y. Kumagai of SAMSA group in
1
Axcelead Drug Discovery Partners Inc. for measurements and analyses of H and 13C NMR of
compounds and helpful discussion for determination of structure of compound 11.
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1
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Structure of 3c depicted is estimated from the structure of 11 determined by H NMR
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Deuterium content of compound 3c was determined by the LC/MS analysis of
N-(3-aminopyridin-2-yl)-cis-3-(tert-butoxycarbonyl)-2,3,4-d3-cyclobutanecarboxamide
obtained by condensation of 3c with 3-aminopyridine.
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”
22.
23.
The ratio of “d0 : d1 : d2 : d3 in 3c means the ratio of “M(3b) : M(3b) + 1 : M(3b) + 2 :
M(3b) + 3” determined by LCMS.
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24.
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The ratio of “d0 : d1 : d2 : d3 : d4 : d5 in 10 means the ratio of “M(7) : M(7) + 1 : M(7) + 2 :
M(7) + 3 : M(7) + 4 : M(7) + 5” determined by LCMS.
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26.
27.
28.
2a: 1H NMR (300 MHz, CDCl3) δ 3.83 (s, 3 H), 4.49 (dd, J = 1.89, 1.13 Hz, 1H), 5.52 (dd, J
= 4.15, 1.89 Hz, 1H), 7.32 (dd, J = 4.34, 0.94 Hz, 1H).
2b: 1H NMR (300 MHz, CDCl3) δ 1.52 (s, 9H), 4.42 (dd, J = 1.70, 0.94 Hz, 1H), 5.44 - 5.49
(m, 1H), 7.20 (d, J = 4.48 Hz, 1H).
1
Copies of the H NMR spectra of compounds 3c, 10, and 11are available in the supporting
information file.
23