Efficient resolution of cis -(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate with soluble Candida antarctica lipase B (CAL B)

Article abstract of DOI:10.1021/op5003424

(2S,3R)-Dimethyl 1-acetylpiperidine-2,3-dicarboxylate, a valuable intermediate for moxifloxacin, was prepared by CAL B catalyzed kinetic resolution of the racemic diester. The reaction has an optimum temperature of 45-50 °C and an optimum pH of 7.5. It follows typical Michaelis-Menten kinetics with V_max,_obsd = 0.061 ± 0.008 M/h/g, K_m,obsd = 0.2 ± 0.045 M at 45°C. The reaction is highly enantioselective with an enantioselectivity E value of 80 at 45°C and pH 7.5. Preparative scale enzymatic resolution was carried out in 0.01 M sodium phosphate buffer, pH 7.5 at 45°C using a substrate loading of 8% (w/w) and a substrate-to-enzyme ratio of 2:1 (w/w). The reaction was complete in 16 h. The enantiomerically pure diester (ee >99%) is obtained in 47.5% yield (95% of theoretical). The unwanted hydrolyzed half ester (ee 87%) was easily converted back to racemic diester in 5 simple steps and 75% overall yield.

Full text of DOI:10.1021/op5003424

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Full Paper
Efficient resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-
dicarboxylate with soluble Candida antarctica lipase B (CAL B)
Nitin Wasantrao Fadnavis, Perla Ramesh, and Tirunagari Harini
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op5003424 • Publication Date (Web): 14 Dec 2014
Downloaded from http://pubs.acs.org on December 21, 2014
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Efficient resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate
with soluble Candida antarctica lipase B (CAL B)
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Perla Ramesh, Tirunagari Harini, and Nitin W Fadnavis*
Natural Products Chemistry Division, Indian Institute of Chemical Technology, Uppal Road,
Hyderabadꢀ500007, India.
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ABSTRACT:
(2S,3R)ꢀDimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate,
a
valuable intermediate for
Moxifloxacin was prepared by CAL B catalyzed kinetic resolution of the racemic diester. The
reaction has optimum temperature of 45ꢀ50 °C and optimum pH of 7.5. It follows typical
MichaelisꢀMenten kinetics with Vmax obsd = 0.061 ± 0.008 M/h/g, Km,obsd = 0.2 ± 0.045 M at
,
45 °C. The reaction is highly enantioselective with enantioselectivity E value of 80 at 45 °C
and pH 7.5. Preparative scale enzymatic resolution was carried out in 0.01 M sodium
phosphate buffer, pH 7.5 at 45 °C using substrate loading of 8% (w/w) and substrate to
enzyme ratio of 2:1 (w/w). The reaction was complete in 16 h. The enantiomerically pure
diester (ee > 99%) is obtained in 47.5% yield (95% of theoretical). The unwanted hydrolyzed
half ester (ee 87%) was easily converted back to racemic diester in 5 simple steps and 75%
overall yield.
INTRODUCTION
Moxifloxacin is a broadꢀspectrum fluoroquinolone antibacterial agent used for
treating respiratory infections such as pneumonia, chronic sinusitis and chronic bronchitis.1
The market demand of this antibiotic is increasing every year and developing better and
greener technologies for its synthesis is of great interest. (S,S)ꢀ2,8ꢀdiazobicyclo [4.3.0]
nonane 5 is a key chiral intermediate of moxifloxacin. Although several synthetic routes to 5
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have been reported,2 two of the most economically attractive routes start from pyridineꢀ2,3ꢀ
dicarboxylic acid 1 as raw material (Scheme 1). The first route comprises of reaction of
pyridineꢀ2,3ꢀdicarboxylic acid anhydride with benzyl amine to give Nꢀbenzylꢀ2,3ꢀpyridineꢀ
dicarboxamide 2. Hydrogenation of the pyridine ring gives cisꢀ8ꢀbenzylꢀ7,9ꢀdioxoꢀ2,8ꢀ
diazabicyclo[4.3.0]nonane 3. The dioxoꢀintermediate is reduced to bicyclic 4 with LiAlH4 or
NaBH4/AlCl3. Reduction of the dioxo groups has also been reported using sodium bis(2ꢀ
methoxyethoxy)aluminium hydride (Vitride).3 The racemic mixture of bicyclic nonane 4 is
resolved with Lꢀtartaric acid and hydrogenated to remove the benzyl group to obtain required
nonane 5.4 Alternatively, the optical resolution of the intermediate cisꢀ8ꢀbenzylꢀ7,9ꢀdioxoꢀ
2,8ꢀdiazabicyclo[4.3.0]nonane 3 is resolved with an amine resolving agent such as Lꢀtartaric
acid,4 camphor sulfonic acid or (ꢀ)ꢀ2,3:4,6ꢀdiꢀOꢀisopropylidineꢀ2ꢀketoꢀLꢀgulonic acid.5 The
unwanted isomer after resolution can be converted back into racemic 3 via Nꢀchlorination
with sodium dichloroisocyanurate bihydrate (NaDCC) followed by dehydrochlorination with
triethylamine.6 In an alternate route, 2,3ꢀdichloromethyl pyridine 7, prepared from either 6 or
10 is converted to Nꢀtosyl derivative 8 which is hydrogenated to obtain 9.7 The isomers of
racemic 9 are separated by resolution with (S)ꢀ(+)ꢀmandelic acid or (D)ꢀ(+)ꢀO,Oꢀ
dibenzoyltartaric acid.8 Although the first route is economically most attractive, it involves a
reduction step where hazardous reagents like LiAlH4 or AlCl3 are used. The second route
does not involve use of such reagents but it is difficult racemize the unwanted isomer of 9.
Both the routes involve the resolution step at a late stage which is also not a preferred choice.
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Scheme 1. Routes to large scale preparation of (S,S)ꢀ2,8ꢀdiazobicyclo [4.3.0] nonane 5.
Recently, an enantioselective synthesis of 5 using using (R)ꢀ2ꢀaminoꢀ2ꢀphenylethanol
as chiral induction reagent has been reported (Scheme 2).2 Similarly, an asymmetric synthesis
using (R)ꢀphenylethylamine has also been reported9 but their use in industrial scale
production has yet to be demonstrated as a viable alternative.
Scheme 2. Enantioselective synthesis of (S,S)ꢀ2,8ꢀdiazobicyclo [4.3.0] nonane.
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Scheme 3. Asymmetric synthesis of (S,S)ꢀ2,8ꢀdiazobicyclo [4.3.0] nonane.
Direct enzymatic resolution of nonane 5 using Novozyme 435 (lipase B from Candida
antarctica, CAL B immobilized on polystyrene support) via selective acetylation has been
claimed in a patent by Bayer.10 However, the reaction proceeds very slowly, requiring 14
days at a temperature of 40 0C.
A chemoꢀenzymatic strategy for 5 involves chiral intermediate (ꢀ)ꢀ26 prepared via
kinetic resolution of the racemic cisꢀ(±)ꢀdimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate 24
(Scheme 4).11
Scheme 4. CAL B mediated resolution of cisꢀ(±)ꢀdimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate.
The procedure described in the patent11 discloses the use of covalently immobilized
lipase B from Candida antarctica (CAL B). The hydrolytic reaction was carried out with 80 g
substrate and 40 g immobilized enzyme in an aqueous buffer at pH 6. The enantioselectivity
of the reaction was excellent (E > 2000) and the enzyme could be reused, the reaction period
(140 h) was however very long. Although the global trend is to use immobilized enzymes
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wherever possible, maintaining pH during hydrolytic reactions with immobilized enzymes is
cumbersome. The reactor needs to be specially designed to prevent attrition of the
immobilization matrix while providing proper stirring.12ꢀ15, In addition, ester hydrolysis
reactions are accompanied by pHꢀdrop due to formation of corresponding acid and it is
necessary to maintain the pH of the reaction medium at an optimum, generally near pH 7.5 to
8.0, by addition of an alkali solution. During this operation, it is necessary to ascertain that
the added alkali is efficiently distributed since localization of the alkali would result in pH
increase beyond 10 and cause enzyme denaturation. Such a dedicated system requires capital
investment which is a deterrent for most medium and small scale manufacturing units. Thus
the process economics prompted us to examine the reaction parameters using commercially
available enzyme solution. In this case a simple stirred tank reactor with pH and temperature
control would suffice.
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RESULTS AND DISCUSSION
Substrate Selectivity
Racemic cisꢀ(±)ꢀdimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate 24 is prepared in 3 simple
steps. Pyridine 2,3ꢀdicarboxylic acid 1 is esterified with methanol to 6, hydrogenated over
PdꢀC and acetylatated with acetic anhydride.11 Other derivatives of the substrate such as
ethyl, nꢀpropyl and nꢀbutyl diesters as well as Nꢀbenzoyl and Nꢀphenylacetyl derivatives of
the dimethyl ester were also prepared to study the scope of CAL B catalyzed reaction on the
piperidine 2,3ꢀdicarboxylic acid template. Surprisingly, only the Nꢀacetyl dimethyl ester 24
was accepted by the enzyme as a substrate. Even the dimethyl ester without Nꢀacetylation
was not accepted by the enzyme. Similarly, when the substrate 24 was subjected to CAL B
catalyzed hydrolysis, only the monoester 25 was formed. HPLC analysis of the reaction
mixture did not show formation of Nꢀdeacetylated diester or the corresponding diacid which
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is not surprising since CAL B is an esterase and not an amidase. The enzymatic reaction
appears to be completely stereo and regioselective.
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Effect of Temperature.
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Usually, lipase catalyzed hydrolysis reactions are carried out in dilute aqueous buffers and the
pH is continuously adjusted with NaOH. In case of liquid substrates, such reactions can be
carried out as a dispersion of the ester in aqueous buffer. However, mass transfer limitations
due to poor solubility can be an issue for lipases and this can cause the process to be very
volume intensive for the reaction and subsequent extraction, hence it is not a preferred option
for scaleꢀup for low cost API manufacture.
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Racemic cisꢀ(±)ꢀdimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate 24 is prepared in 3 simple
steps. Pyridine 2,3ꢀdicarboxylic acid 1 is esterified with methanol to 6, hydrogenated over
PdꢀC and acetylatated with acetic anhydride.11 The racemic ester 24 is a low melting solid
(Mp 64 ꢀ 66 °C) and is partially soluble in aqueous buffer. The solubility increases with
temperature and at 50 °C a clear ester solution with composition of 8 % (w/v) can be
obtained in 0.01 M sodium phosphate buffer (pH 7.5). On cooling to 25 °C, the ester
remained in an emulsion state and did not precipitate. It was sufficient to stir the reaction
mixture at 200 rpm to get reproducible results. Increasing the stirring speed up to 300 rpm did
not change the reaction rates showing that mass transfer limitations do not play a major role
above stirring speed of 200 rpm.
o
As expected, the rate of enzymatic hydrolysis increases with temperature from 30 C to
o
50 C. The enzyme itself is stable at 45 ꢀ 50 °C for more than 72 h but loses its activity
rapidly at 60 °C within 30 min (Figure 1). The enantioselectivity E of the reaction at different
temperatures was determined from the equation:16
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where 'c' is the conversion. eep and ees denote enantiomeric excess of substrate and product
respectively.
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Interestingly, the E values were not greatly affected by temperature and remained at 80 ± 5 in
the temperature range of 30 to 50 °C. Based on these results, 45 oC was chosen as the optimal
temperature for further studies.
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Enantioselectivity
E
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Enantiomeric excess
(% e.e.) of diester 26
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Temperature oC
Figure 1. Effect of temperature on CAL B catalyzed enantioselective hydrolysis of (24). Reaction conditions:
0.16 M [24] in sodium phosphate buffer (0.01 M, pH 7.5), 50 mL; CAL B 150 mg (750 units). Reaction period
20 h.
Kinetic Parameters.
Effect of substrate concentration on the initial reaction rate was investigated at a fixed
amount of enzyme. The kinetic parameters obtained from nonꢀlinear analysis were: Vmax obsd
,
= 305 ± 42 µM/h/g, Km,obsd = 0.2 ± 0.045 M, n = 9, Correlation coeff. r = 0.986 (Figure 2).
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0
0.0
0.1
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[Ester], M
Figure 2. Effect of substrate concentration on the initial reaction rate of CAL B catalyzed hydrolysis of (24).
Reaction conditions: sodium phosphate buffer (100 mL, 0.01 M, pH 7.5), CAL B 500 mg (2500 units), 45 C.
Vmax obsd = 305 ± 42 µM/h/g, Km,obsd = 0.2 ± 0.045 M, n = 9, Correlation coeff. r = 0.986.
o
,
Considering the active enzyme content of 40 mg/g and molecular mass of 33273, the kcat
value from results in Fig.2 is calculated to be 4.23 minꢀ1.
Effect of Enzyme Loading.
In order to reduce reaction period to an acceptable level, the effect of enzyme concentration
on conversion was studied. At a fixed substrate concentration, the conversion increased with
increased enzyme loading. At a substrate to enzyme ratio of 2:1 (w/w), the reaction was
complete (ee > 99%) in 16 h. Thus enzymatic reaction was performed with 0.32 M (8 %
(w/v)) substrate on 1 L scale using substrate:enzyme ratio of 2:1 (w/w) at 45 °C. The required
enantiomer (ꢀ)ꢀ26 (45 % yield) and the monoester (52 % yield) were easily recovered.
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[CAL B], mg/mL
Figure 3. Effect of CAL B concentration on hydrolysis of (±)ꢀ(24). Reaction conditions: [24] = 0.32 M in
sodium phosphate buffer (50 mL, 0.01 M, pH 7.5), 45 oC. Reaction period 16 h.
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Recycling of 25.
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Once an efficient enzymatic kinetic resolution was developed, recycling of the remaining
enantiomer 25 was explored. The unwanted isomer was recycled in 5 steps by the method of
Pallavicini and coꢀworkers.17 Thus the compound 25 was completely hydrolyzed to 27 by
refluxing with aqueous hydrochloric acid and reꢀesterified to obtain 28 with free amino
group. NꢀChlorination of 28 with sodium dichloroisocyanurate bihydrate (NaDCC) in
tolueneꢀwater followed by dehydrochlorination of the intermediate 29 with triethyl amine
gave the unsaturated piperidine derivative 30. Hydrogenation over PdꢀC and acetylation with
Ac2O gave racemic 31 (Scheme 5).
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Scheme 5. Recycling of unwanted isomer.a
aReagents and conditions: a) 6N HCl, reflux, 8 h; b) SOCl2, MeOH; 0ꢀ70 oC, 12 h; c) NaDCC, TolueneꢀH2O, rt,
o
30 min then Et3N, 1 h (80% in three steps); d) Pd/C, H2 (10 bars), Toluene, 60 C, 4h; e) Ac2O, TEA, DMAP,
Toluene, rt, 3 h (94% in two steps).
CONCLUSION
Enantioselective hydrolysis of cisꢀ(±)ꢀdimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate
catalyzed by CAL B has been efficiently carried out. Compared to immobilized enzyme of
similar activity, the reaction with soluble enzyme is completed in 16 h instead of 140 h.
Although the enzyme cannot be recovered and reused, the savings on time and investment on
enzyme purchase far outweighs the advantages of using immobilized enzyme. The
enantioselectivity E of 80 is quite acceptable (eediester > 99 % at 53 % conversion) and the
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unwanted isomer can be easily recycled using inexpensive reagents. We have performed the
reaction at 80 g scale. The overall process has an excellent potential for industrial scale
production and studies on scaleꢀup are in progress.
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EXPERIMENTAL SECTION
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General.
Addzyme CAL B (Lipase B from Candida Antarctica, activity 5000 TBU) was obtained from
Advanced Enzyme Technologies Ltd, Thane, India (www.enzymeindia.com). cisꢀ(±)ꢀ
dimethyl 1ꢀacetylpiperidineꢀ2,3ꢀdicarboxylate was prepared from pyridine 2,3ꢀdicarboxylic
acid as described in literature.11 The product was finally crystallized from nꢀheptane to obtain
white crystals (Mp 64ꢀ66 °C). All other chemicals were purchased from SD Fine Chem,
India. IR spectra were recorded on a PerkinꢀElmer RXꢀ1 FTꢀIR system. 1H NMR (300 MHz)
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and C NMR (75 MHz) spectra were recorded on a Bruker Avanceꢀ300 MHz spectrometer.
For low (MS) and high resolution mass spectra (HRMS) ion mass/charge (m/z) ratios were
reported as values in atomic mass units. Chiral HPLC analyses were carried out on Shimadzu
HPLC Unit LC 20 AD with diode array detector. HPLC columns were obtained from
Phenomenex, USA and Daicel Chemical Corporation Japan.
Enzymatic Reaction.
The reaction was carried out in a double walled vessel equipped with mechanical stirrer, pH
and temperature control. Substrate 24 (80 g) was suspended in 0.01 M sodium phosphate
o
buffer (1 L) and heated to 45 C to get clear solution. The enzyme CAL B (40 g) was added
and the reaction mixture was stirred mechanically at 200 rpm. The temperature was
maintained at 45 °C and pH was maintained at 7.5 with continuous addition of 2N NaOH.
The reaction was followed by reverse phase HPLC along with consumption of NaOH. At the
end of reaction (16 h), 80 mL of NaOH were consumed and the HPLC showed acid and ester
concentrations of 45 and 55% respectively (based on area %). This ratio did not change over
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2 more hours. HPLC analysis on chiral stationary phase showed that the ee of unreacted
diester 26 was >99%. At this point, disodium hydrogen phosphate (141 g) was added to
saturate the reaction mixture and was extracted with diethyl carbonate (3 x 1 L). The solvent
extracts were dried over MgSO4 and evaporated on rotavapor to give compound 26 (37 g,
46%, 92.5% of theoretical, ee > 99%). [α]D25 = − 114.8 (c = 1, CH3OH).
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The aqueous layer was acidified to pH 2.5 with conc. HCl and the monoester 25 was
extracted with CH2Cl2 (3 X 1 L). The organic extracts were dried over MgSO4 and the
solvent was evaporated on rotavapor to obtain a mixture of monoesters 25a and 25b (41.6 g,
52% yield). [α]D25 = 113.2 (c = 1, CH3OH). The migration of methyl group from carboxylic
group at position 2 to that at position 3 has been reported earlier.11 For determination of
enantioselectivity of the reaction, the monoester was converted to diester by esterification
with methanolꢀthionyl chloride as described for compound 29 and enantiomeric purity of this
diester was determined by chiral HPLC analysis (ee 85%).
Reverse phase HPLC analysis.
The hydrolysis of diester 24 was followed by reverse phase HPLC. Column Cꢀ8 (250 x 5
mm), Chrompack, The Netherlands. Mobile phase, 30 % methanol in water containing 0.1%
perchloric acid. Flow rate, 0.7 mL/min. Detection wavelength, 220 nm. Retention times: Nꢀ
acetyl piperidine 2,3ꢀdicarboxylic acid (Nꢀacetyl diacid) 4.3 min; Monoester 25: 10.3 min;
Diester 24: 17.9 min.
HPLC analysis on chiral stationary phase.
Analysis was carried out on Chiralcel ODꢀH column (250 x 5 mm), Daicel Chemical
Industries, Japan. Mobile phase, 15 % 2ꢀpropanol in hexane. Flow rate, 1 mL/min. Detection
wavelength 220 nm. Retention times (+)ꢀ(2R,3S)ꢀ24: 10.8; (ꢀ)ꢀ(2S,3R)ꢀ26: 13.6 min.
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[α]D25 = − 114.8 (c = 1, CH3OH). 1H NMR (300MHz, CDCl3): δ [minor rotamer in brackets]
1.37ꢀ1.52 (m, 1H), 1.63ꢀ1.71 (m, 1H), 1.77ꢀ1.84 (m, 2H), 2.10ꢀ2.15 (m, 1H), 2.16 (s, 3H,
[2.21, s]), 2.53ꢀ2.57 (dt, 1H, [2.60ꢀ2.64, dt]), 3.05ꢀ3.11 (dt, 1H, [2.35ꢀ2.41, dt]), 3.70 (s, 3H,
[3.74, s]), 3.71 (s, 3H, [3.74, s]), 6.0 (d, J = 4.8 Hz, 1H, [5.03, d]) ppm. 13C NMR (75MHz,
CDCl3): δ [minor rotamer in brackets] 171.2, 169.5, 169.0 [169.7], 52.6 [57.7], 51.9 [52.2],
51.4 [51.6] 43.3 [42.9], 42.3 [38.2], 24.3 [23.5], 22.0, 21.1 ppm. ESIMS: m/z 244 (M+1)+,
266 (M+Na)+. HRMS calculated for C11H17O5NNa [M+Na]+ 266.09989, found 266.09996.
(2R,3S)-1-acetyl-3-(methoxycarbonyl)piperidine-2-carboxylic acid 25a and (2R,3S)-1-
acetyl -2-(methoxycarbonyl)piperidine-3-carboxylic acid 25b.11
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[α]D25 = 113.2 (c = 1, CH3OH). H NMR (300MHz, CDCl3): δ [minor rotamer in brackets]
1.41ꢀ1.86 (m, 4H), 2.09ꢀ2.16 (m, 1H), 2.18 (s, 3H, [2.23, s]), 2.52ꢀ2.59 (dt, 1H, [2.64ꢀ2.69,
dt]), 3.03ꢀ3.12 (dt, 1H, [2.37ꢀ2.46, t]), 3.72 (s, 3H, [3.76, s]), 5.95 (d, J = 4.3 Hz, 1H, [5.07,
d]) ppm. 13C NMR (75MHz, CDCl3): δ 173.2, 173.1, 170.8, 170.4, 169.6, 169.1, 57.7, 52.8,
52.2, 51.9, 43.5, 42.8, 42.3, 38.4, 24.4, 23.5, 21.9 (2C), 20.9, 20.8 ppm. ESIMS: m/z 230
(M+1)+, 252 (M+Na)+. HRMS calculated for C10H15O5NNa [M+Na]+ 252.08424, found
252.08337.
(2R,3S)-piperidine-2,3-dicarboxylic acid hydrochloride 27.18
A solution of compound 25 (10 g) in 6N HCl (100 mL) was stirred under reflux for 8 h. The
reaction mixture was concentrated to obtain product 27 which was used in the next step
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without purification. H NMR (300MHz, D2O): δ 1.39ꢀ1.49 (m, 1H), 1.69ꢀ1.73 (m, 1H),
1.76ꢀ1.83 (m, 1H), 2.09ꢀ2.12 (m, 1H), 2.87ꢀ2.93 (m, 1H), 3.29ꢀ3.31 (m, 1H), 3.40 (q, J = 3.9
and 3.8 Hz, 1H), 4.01 (d, J = 3.8 Hz, 1H) ppm; 13C NMR (75MHz, D2O): δ 20.3, 26.1, 41.7,
46.0, 58.2, 172.3, 177.5 ppm; ESIMS: m/z 174 (M+1)+.
Dimethyl 1,4,5,6-tetrahydropyridine-2,3-dicarboxylate 30.17
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Thionyl chloride (12 mL) was added dropꢀwise to a stirred solution of crude product 27 in
MeOH (150 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and at 70 °C for another 10
h. The reaction mixture was concentrated under vacuum to give the product 28 which was
suspended in toluene (200 mL). Sodium dichloro isocyanurate bihydrate solution (10.7 g, 60
% w/w) in water (30 mL) was added at room temperature and the reaction mixture was stirred
for 30 min. The organic phase was separated and triethylamine (12 mL) was added to the
organic layer. After stirring for 1 h, the organic layer was washed with water, dried over
MgSO4 and concentrated under vacuum to afford compound 30 (6.95 g, 80% in three steps)
as a colourless oil. 1H NMR (300MHz, CDCl3): δ 1.80ꢀ1.85 (m, 2H), 2.38 (t, J = 6.2 Hz, 2H),
3.23ꢀ3.26 (m, 2H), 3.66 (s, 3H), 3.84 (s, 3H), 4.49 (s, br, 1H) ppm; 13C NMR (75MHz,
CDCl3): δ 20.3, 21.1, 40.8, 50.9, 52.7, 94.8, 145.8, 167.3, 167.7 ppm; ESIMS: m/z 200
(M+1)+, 222 (M+Na)+.
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The compound 30 (6.95 g), toluene (30 mL) and 10 % Pd/C (0.5 g) were charged in an
autoclave. The autoclave was heated to 60 °C and hydrogenation was carried out at 10 bar
pressure for 4 h. After cooling the the reaction mixture to room temperature, the catalyst was
filtered off. The filtrate containing compound 31 was used in the next step without any
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purification. H NMR (300MHz, CDCl3): δ 1.47ꢀ1.52 (m, 2H), 1.75ꢀ1.82 (m, 1H), 2.13ꢀ2.19
(m, 1H), 2.21 (s, br, 1H), 2.66ꢀ2.71 (m, 1H), 2.99ꢀ3.06 (m, 2H), 3.64 (d, J = 3.3 Hz, 1H), 3.70
(s, 3H), 3.74 (s, 3H) ppm; 13C NMR (75MHz, CDCl3): δ 23.3, 25.8, 42.1, 45.2, 51.8, 52.0,
59.2, 172.4, 173.1 ppm; ESIMS: m/z 202 (M+1)+, 224 (M+Na)+; HRMS calculated for
C9H16O4N [M+H]+ 202.10738, found 202.10741.
Dimethyl 1-acetylpiperidine-2,3-dicarboxylate 24.11
To a stirred solution of compound 31 in toluene (50 mL) was added triethyl amine (4.7 mL)
and catalytic amount of dimethylaminopyridine (DMAP) at room temperature. Acetic
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anhydride (3.4 mL) was added and stirring was continued for 3 h at room temperature. The
reaction mixture was quenched with 5 % NaHCO3 solution (2 mL). The organic layer was
washed with water (20 mL) and concentrated under reduced pressure to obtain compound 24
(7.5 g, 94 % in two steps). Mp 64 ꢀ 66 oC.
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ASSOCIATED CONTENT
Supporting Information
1H and 13C NMR data of compounds 25-27, 30 and 31 is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: fadnavis@iict.res.in; fadnavisnw@yahoo.com
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
We thank UGC New Delhi for grant of SRF to P. Ramesh, and CSIR New Delhi for financial
support under XII Five Year Plan CSC0108ꢀORIGIN.
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Graphical Abstract
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