# Ultrasonic-Assisted Synthesis of Twot-Butoxycarbonylamino Cephalosporin Intermediates on SiO2

Authors: Feng Xue; Yibin Wei; Shengui Ju; Weihong Xing
Journal: Journal of Chemistry
 (2016)

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## Abstract

Herein, we describe a facile and high efficient strategy for the synthesis of two forms of the 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylates using ultrasonic irradiation. By SiO2 as weak Lewis acid catalyst, 4-methoxybenzyl 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-carboxylate (Boc-ACLE) and benzhydryl 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylate (Boc-ACLH) were successfully synthesized through the efficient protection of theN-t-butoxycarbonyl (N-Boc), and the reactions occurred at low temperature requiring short reaction times and exhibiting excellent isolated yields (96% and 96.2%, resp.). The advantages of this reaction route including the usage of economical reagents and mild reaction conditions and high isolated yield make the two significantt-butoxycarbonylamino cephalosporin intermediates possible in large-scale production.


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## Body

## 1. Introduction

7β-t-Butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylates serving as the extremely important intermediates are employed to synthesize the commonly used antibiotics, cephalosporin. As shown in Figure 1, the chemical structure of cephalosporin intermediate contains a chemically active 3-chloromethyl-group at 3-side chain, which can be easily coupled with drug-active functional groups forming cephalosporin derivatives, especially the fourth-generation cephalosporins Cefoselis sulfate [1, 2]. However, owing to their presence in various biological environments, the amine functionality at 7 positions needs to be protected which is one of the most challenging issues in this synthetic chemistry. Among the current amine protection methods, thet-butoxycarbonyl (Boc) protection has been considered as the useful one because of its excellent stability regarding catalytic hydrogenation and extreme resistance to underlying basic or nucleophilic reactions [3].Figure 1
General structure of 3-chloromethyl cephalosporin.In the past decade, multiple processes have been available to introduce the Boc protecting group using Boc2O (di-t-butyl dicarbonate) to synthesize 4-methoxybenzyl 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-carboxylate (Boc-ACLE). The synthesis of Boc-ACLE has been reported by Lee et al. [4] that they dissolved 4-methoxybenzyl 7β-amino-3-chloromethyl-3-cephem-carboxylate (ACLE·HCl) into CH2Cl2 solvent first. Next, the mixture was reacted with di-t-butyldicarbonate (Boc2O) in the presence of a catalyst ofN-(trimethylsilyl) acetamide (NSA). However, the catalyst of NSA has numerous drawbacks for the practical reaction such as its high costs, sensitivity to moisture, the low yield (51%), and other further troubles from processing (number 1, Table 1). Recently, Du [5] used tetrabutylammonium bromide (TEBA) as phase transfer catalyst to synthesize Boc-ACLE compound, where ACLE.HCl was reacted with Boc2O in weak basic aqueous solution at room temperature. However, Boc2O was easily decomposed under basic aqueous solution environment in which Boc2O was largely lost, and the reaction required long reaction times (10 h) giving a yield of 80% (number 2, Table 1). In 2010, Lin [6] reported that the Boc-ACLE could be synthesized via a catalyst-free method starting from the reaction of ACLE·HCl and Boc2O in tetrahydrofuran (THF). Using triethylamine as acid binding agent, they finally got a high yield of 87.21%. Unfortunately, the reproducibility seems not good and we determined that the yield was only 15% (number 3, Table 1).Table 1
Comparison of three methods for the synthesis of Boc-ACLE.
Number
Catalyst
Reaction time
Solvent
Yield%
1
NSA
12 h
CH2Cl2
51 [4]
2
TEBA
10 h
Dioxane/H2O
80 [5]
3∗
Catalyst-free
8 h
THF
15 [6]
∗The HPLC assay indicated the yield was 15% according to the reported methods.Anothert-butoxycarbonylamino cephalosporin intermediate, benzhydryl 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylate (Boc-ACLH), was typically synthesized by Dai’s group using 7-aminocephalosporanic acid (7-ACA) as starting material following the approach outlined in Scheme 1 [7]. Key intermediate1 was obtained when 7-ACA was hydrolyzed in basic solution at −30°C. Sequentially, compound1 reacted with Boc2O to protect amine group catalyzed by TEBA in NaCO3-H2O-acetone solution to produce compound2, which was then treated with diphenyldiazomethane inn-hexane forming compound3. In the last step, the hydroxyl group of compound3 was substituted by chlorine in presence of phosphorus pentachloride and pyridine in dichloromethane at −45°C to get the desired product4b (Boc-ACLH). It is apparent that this synthetic route includes tedious steps, complex experimental operations, and harsh conditions. Alternative synthetic strategies with advantages such as short steps, mild conditions, and high efficiency are keen to be developed.Scheme 1
Synthetic route of Boc-ACLH.It can be deduced from the above-mentioned cases that the choice of raw materials, catalyst, solvent, and the reaction conditions play significant roles in synthesizing the twot-butoxycarbonylamino cephalosporin intermediates. Generally, in terms of producing theseN-Boc derivatives, there are two kinds of catalytic methods (i.e., using base catalyst and Lewis acid catalyst). There have been plenty of base-catalyzed Boc-protection studies reported by using Boc2O/DMAP system; however, the high toxicity of DMAP was not neglectable [8]. What is worse, the base-catalyzed reactions reported often resulted in the generation of byproducts like isocyanate, urea, andN,N-di-Boc derivatives [9, 10]. Other catalytic approaches involving Lewis acids, such as La(NO3)3-6H2O, ZrCl4, LiClO4, Cu(BF4)2-9H2O, Zn(ClO4)2-6H2O, Yttria-Zirconia, HClO4/SiO2, montmorillonite K-10, amberlyst-15, H3PW12O40, sulfamic acid, I2, and hexafluoroisopropanol (HFIP), have been attempted [11–15]. As the increasing demand of mild reaction conditions encourages the development of greener route to achieve these significant synthetic works in recent years, the sonochemistry has offered a solution that a large variety of organic/medical transformations succeeded using this more efficient and facile method [16]. For instance, Amira et al. [17] have applied ultrasonic irradiation technology for theN-Boc protection which not only eliminated the harsh reaction conditions but also assisted in impressive yield. Dighe and Jadhav [18] found microwave assisted chemoselective method could shorten the reaction time for the generation ofN-Boc products with excellent isolated yield. It is worth mentioning that solvent-free synthetic strategies using ionic liquid catalyst have emerged which show great potential forN-Boc formation [19, 20].In this paper, we aim at proposing an optimum route for the synthesis of the twot-butoxycarbonylamino cephalosporin intermediates based upon the synergetic effect of ultrasonic assistance and SiO2 catalyst. The efficiency of our route forN-Boc formation of our targeted product was analyzed and the mechanism of the proposed ultrasonic-assisted strategy forN-Boc protection of amines was investigated. In addition, the products were confirmed by spectrometric methods. In comparison to conventional methods, our route may offer a highly efficient and methodologically simple catalytic process to introduceN-Boc protecting group into our desired products.


## 2. Experimental Section

### 2.1. General Information

ACLE·HCl and ACLH·HCl were purchased from Shanghai Arbor Chemical Co., Ltd. The other reagents and solvents were obtained from Sigma-Aldrich and used as received without any further purification. All reactions were monitored by thin-layer chromatography (TLC) using commercial silica gel plates. JL-120DTH ultrasonic bath was purchased from Shanghai Jnlsh Testmart Co., Ltd. The purity was measured by high performance liquid chromatography (HPLC) on Agilent 1,100 series. The liquid chromatographic system was equipped with an ODS-3 C18 column (GL Science Co. Ltd., 180 × 4.6 mm i.d., 5.0μm). Melting points were observed on YRT-3 Melting Point Tester and were uncorrected. NMR spectra were determined on Bruker AV300 in DMSO-d
6 with TMS as internal standard for 1H NMR (300 MHz) and 13C NMR (75 MHz), respectively. HRMS were carried out on an Agilent 6230-LC/TOF MS mass spectrometer.


### 2.2. General Procedure fort-Butoxycarbonylamino Cephalosporin Intermediates

ACLE·HCl (5a) or ACLH·HCl (5b) as starting reagent was added to CH2Cl2 (200 mL) firstly; triethylamine was then introduced into the mixture to neutralize the pH at 7-8 at 0–5°C. Then, appropriate amounts of SiO2 and Boc2O were added to the mixture for the appropriate time of bath ultrasonication at 0–5°C (Table 2). After completion of the reaction indicated by thin-layer chromatography (TLC), the mixture was poured into water and the organic phase was separated followed by washing with brine water and drying with anhydrous sodium sulfate under vacuum evaporation. The crude product was purified by column chromatography over silica gel to yield the desired compounds.Table 2
The effect of SiO2 and ultrasound on the yields of the Boc-ACLE.
Number
SiO2
Ultrasound
Reaction time
Yield%
1
✕
✕
6 h
15
2
✓
✕
6 h
35
3
✕
✓
6 h
40
4
✓
✓
30 min
96
Note:✕: no; ✓: yes.4-Methoxyphenyl 7β-t-Butyl-carbonylamino-3-chloromethyl-3-cephem-4-carboxylate (
4a
).4a: Solid, m.p. 63–65°C. The yield was 96.0%. HPLC assay confirmed the purity of4a was 98.5%. The column oven temperature was set at 295 K. The mobile phase consisted of methanol and deionized water (75 : 25, v/v) and flowed through the column in 15 min with the flow rate of 1.0 mL/min. Photodiode array detection was used to detect the Boc-ACLE at the wavelength of 254 nm. 1H and 13C NMR refer to Tables 3 and 4. ESI-HRMS: calcd. for C21H24ClN2O6S ([M−H]−) 467.1049, found 467.1045.Table 3
Comparison of1H NMR data (4a).
Lee et al. reported (500 MHz, CDCl3)a
Synthetic4a (300 MHz, DMSO-d6)b
1.45 (s, 9H)
1.37 (s, 9H)
3.47 (d, 1H,J= 18.2 Hz),
3.48 (d, 1H,J = 17.91 Hz)
3.66 (d, 1H,J= 18.2 Hz)
3.64 (d, 1H,J = 9.65 Hz)
3.73 (s, 3H)
4.55 (d, 1H,J= 12.1 Hz)
4.41 (d, 1H,J = 11.31 Hz)
4.49 (d, 1H,J = 11.07 Hz)
4.95 (d, 1H,J= 4.6 Hz)
5.07 (d, 1H,J = 3.93 Hz)
5.23 (s, 2H)
5.12 (d, 1H,J = 12.09 Hz)
5.61 (dd, 1H,J= 5.0, 9.5 Hz)
5.19 (d, 1H,J = 11.73 Hz)
5.48 (s, 2H)
6.88–6.91 (d
t
a
p
p, 2H)
6.89–6.92 (d
t
a
p
p, 2H)
7.33–7.36 (d
t
a
p
p, 2H)
7.32–7.34 (d
t
a
p
p, 2H)
aData taken from [4]; bthis work.Table 4
Comparison of13C NMR data (4a).
Lee et al. reported (125 MHz, CDCl3)a
Synthetic4a (75 MHz, DMSO-d6)b
—
170.86
165.6
165.01
161.4
161.06
160.2
159.34
154.7
135.70
149.6
130.25
131.0
128.16
126.8
126.84, 126.43
125.9
125.29, 124.83
114.2
113.77
81.5
—
68.5
67.28
61.2
59.18
58.3
57.96, 57.86
55.5
55.06
43.6
43.72, 40.33
28.4
28.93
27.4
26.09
aData taken from [4]; bthis work.Benzhydryl 7β-t-Butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylate (
4b
).(4b): Solid, m.p. 140–142°C (decomposed). The yield was 96.2%. (HPLC assay of4b was 98.1%. The HPLC method of4b was similar to the4a.) 1H NMR refer to Table 5. 13C NMR (75 MHz, DMSO-d6): δ 170.86, 165.01, 161.06, 159.34, 135.70, 130.25, 28.93, 128.16, 126.84, 126.43, 125.29, 124.83, 113.77, 67.28, 59.18, 57.96, 57.86, 55.06, 43.72, 41.55, 40.33, 26.09. ESI-HRMS: calcd. for C26H27ClN2O5NaS ([M+Na]+) 537.1221, found 537.1243.Table 5
Comparison of1H NMR data (4b).
Dai et al. reported (500 MHz, CDCl3)a
Synthetic4b (300 MHz, DMSO-d6)b
1.49 (s, 9H)
1.38 (s, 9H)
3.49, 3.57 (d, 2H,J = 14 Hz)
3.52 (d, 1H,J = 1.38 Hz)
3.66 (d, 1H,J = 17.97 Hz)
4.98 (d, 1H,J = 4 Hz)
5.12 (d, 1H,J = 4.14 Hz)
5.21 (d, 1H,J = 8 Hz)
—
5.65 (dd, 1H,J = 4, 8 Hz)
5.56 (s, 1H)
6.98 (s, 1H)
6.93 (s, 1H)
7.3–7. 5 (m, 10H)
7.27–7.49 (d
t
a
p
p, 10H)
7.38 (s, 1H)
8.01–8.04 (d, 1H,J = 8.49)
aData taken from [7]; bthis work.


## 2.1. General Information

ACLE·HCl and ACLH·HCl were purchased from Shanghai Arbor Chemical Co., Ltd. The other reagents and solvents were obtained from Sigma-Aldrich and used as received without any further purification. All reactions were monitored by thin-layer chromatography (TLC) using commercial silica gel plates. JL-120DTH ultrasonic bath was purchased from Shanghai Jnlsh Testmart Co., Ltd. The purity was measured by high performance liquid chromatography (HPLC) on Agilent 1,100 series. The liquid chromatographic system was equipped with an ODS-3 C18 column (GL Science Co. Ltd., 180 × 4.6 mm i.d., 5.0μm). Melting points were observed on YRT-3 Melting Point Tester and were uncorrected. NMR spectra were determined on Bruker AV300 in DMSO-d
6 with TMS as internal standard for 1H NMR (300 MHz) and 13C NMR (75 MHz), respectively. HRMS were carried out on an Agilent 6230-LC/TOF MS mass spectrometer.


## 2.2. General Procedure fort-Butoxycarbonylamino Cephalosporin Intermediates

ACLE·HCl (5a) or ACLH·HCl (5b) as starting reagent was added to CH2Cl2 (200 mL) firstly; triethylamine was then introduced into the mixture to neutralize the pH at 7-8 at 0–5°C. Then, appropriate amounts of SiO2 and Boc2O were added to the mixture for the appropriate time of bath ultrasonication at 0–5°C (Table 2). After completion of the reaction indicated by thin-layer chromatography (TLC), the mixture was poured into water and the organic phase was separated followed by washing with brine water and drying with anhydrous sodium sulfate under vacuum evaporation. The crude product was purified by column chromatography over silica gel to yield the desired compounds.Table 2
The effect of SiO2 and ultrasound on the yields of the Boc-ACLE.
Number
SiO2
Ultrasound
Reaction time
Yield%
1
✕
✕
6 h
15
2
✓
✕
6 h
35
3
✕
✓
6 h
40
4
✓
✓
30 min
96
Note:✕: no; ✓: yes.4-Methoxyphenyl 7β-t-Butyl-carbonylamino-3-chloromethyl-3-cephem-4-carboxylate (
4a
).4a: Solid, m.p. 63–65°C. The yield was 96.0%. HPLC assay confirmed the purity of4a was 98.5%. The column oven temperature was set at 295 K. The mobile phase consisted of methanol and deionized water (75 : 25, v/v) and flowed through the column in 15 min with the flow rate of 1.0 mL/min. Photodiode array detection was used to detect the Boc-ACLE at the wavelength of 254 nm. 1H and 13C NMR refer to Tables 3 and 4. ESI-HRMS: calcd. for C21H24ClN2O6S ([M−H]−) 467.1049, found 467.1045.Table 3
Comparison of1H NMR data (4a).
Lee et al. reported (500 MHz, CDCl3)a
Synthetic4a (300 MHz, DMSO-d6)b
1.45 (s, 9H)
1.37 (s, 9H)
3.47 (d, 1H,J= 18.2 Hz),
3.48 (d, 1H,J = 17.91 Hz)
3.66 (d, 1H,J= 18.2 Hz)
3.64 (d, 1H,J = 9.65 Hz)
3.73 (s, 3H)
4.55 (d, 1H,J= 12.1 Hz)
4.41 (d, 1H,J = 11.31 Hz)
4.49 (d, 1H,J = 11.07 Hz)
4.95 (d, 1H,J= 4.6 Hz)
5.07 (d, 1H,J = 3.93 Hz)
5.23 (s, 2H)
5.12 (d, 1H,J = 12.09 Hz)
5.61 (dd, 1H,J= 5.0, 9.5 Hz)
5.19 (d, 1H,J = 11.73 Hz)
5.48 (s, 2H)
6.88–6.91 (d
t
a
p
p, 2H)
6.89–6.92 (d
t
a
p
p, 2H)
7.33–7.36 (d
t
a
p
p, 2H)
7.32–7.34 (d
t
a
p
p, 2H)
aData taken from [4]; bthis work.Table 4
Comparison of13C NMR data (4a).
Lee et al. reported (125 MHz, CDCl3)a
Synthetic4a (75 MHz, DMSO-d6)b
—
170.86
165.6
165.01
161.4
161.06
160.2
159.34
154.7
135.70
149.6
130.25
131.0
128.16
126.8
126.84, 126.43
125.9
125.29, 124.83
114.2
113.77
81.5
—
68.5
67.28
61.2
59.18
58.3
57.96, 57.86
55.5
55.06
43.6
43.72, 40.33
28.4
28.93
27.4
26.09
aData taken from [4]; bthis work.Benzhydryl 7β-t-Butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylate (
4b
).(4b): Solid, m.p. 140–142°C (decomposed). The yield was 96.2%. (HPLC assay of4b was 98.1%. The HPLC method of4b was similar to the4a.) 1H NMR refer to Table 5. 13C NMR (75 MHz, DMSO-d6): δ 170.86, 165.01, 161.06, 159.34, 135.70, 130.25, 28.93, 128.16, 126.84, 126.43, 125.29, 124.83, 113.77, 67.28, 59.18, 57.96, 57.86, 55.06, 43.72, 41.55, 40.33, 26.09. ESI-HRMS: calcd. for C26H27ClN2O5NaS ([M+Na]+) 537.1221, found 537.1243.Table 5
Comparison of1H NMR data (4b).
Dai et al. reported (500 MHz, CDCl3)a
Synthetic4b (300 MHz, DMSO-d6)b
1.49 (s, 9H)
1.38 (s, 9H)
3.49, 3.57 (d, 2H,J = 14 Hz)
3.52 (d, 1H,J = 1.38 Hz)
3.66 (d, 1H,J = 17.97 Hz)
4.98 (d, 1H,J = 4 Hz)
5.12 (d, 1H,J = 4.14 Hz)
5.21 (d, 1H,J = 8 Hz)
—
5.65 (dd, 1H,J = 4, 8 Hz)
5.56 (s, 1H)
6.98 (s, 1H)
6.93 (s, 1H)
7.3–7. 5 (m, 10H)
7.27–7.49 (d
t
a
p
p, 10H)
7.38 (s, 1H)
8.01–8.04 (d, 1H,J = 8.49)
aData taken from [7]; bthis work.


## 3. Results and Discussion

In present work, as described in Scheme2, we selected inexpensive ACLE·HCl (5a) or benzhydryl 7β-amino-3-chloromethyl-3-cephem-carboxylate (ACLH·HCl,5b) as starting materials. They were neutralized by triethylamine in CH2Cl2 solvent at 0–5°C for 30 min, and then the free base (ACLE or ACLH) was reacted with Boc2O byN-acylation reaction at presence of SiO2 under ultrasonic irradiation for 30 min to form 7β-t-butoxycarbonylamino-3-chloromethyl-3-cephem-4-carboxylates (4a, Boc-ACLE, or4b, Boc-ACLH).Scheme 2
Ultrasonic synthesis of Boc-ACLE and Boc-ACLH.To overcome the exothermicity of the reaction while carrying out the reaction on a large-scale operation, the reaction temperature should be controlled and determining an appropriate solvent which depends on substrate reactivity and solubility seems very important [14]. As the exothermic phenomenon was found in ourN-acylation reaction, therefore, to maintain the stability of thermos-sensitive cephalosporin compounds, we chose dichloromethane as solvent and kept the reaction temperature below 10°C.The effect of SiO2 and ultrasound on the yields of the formation of Boc-ACLE was recorded in Table 2. The proposed route without both catalyst and ultrasonic irradiation took 6 hours to achieve the reaction but the resulting isolated yield was only 15%. When Lewis acid catalyst SiO2 was added to the route, the yield of the reaction was improved up to 35%. To find the synergic effect of ultrasound and SiO2, another control reaction using ultrasound alone was performed under the same reaction time without SiO2 that a low yield of 40% was found. Notably, when the Lewis acid catalyst of SiO2 and ultrasonic power were utilized together, an overwhelming yield of 96% was discovered after 30 mins. The similar impressive result also happened to synthesize Boc-ACLH (the yield is 96.2%) using both ultrasound and SiO2 which implied these results were attributed to the synergy effect of ultrasonic irradiation and SiO2.To explain this mechanism, we believe that, as a result of the electronic and steric molecular structure of 7β-amino-3-chloromethyl-3-cephem-4-carboxylates, the amine group is low nucleophilic which could be activated by the weak Lewis acid support body of SiO2 under ultrasonic irradiation. Figure 2 describes the structure of SiO2 surface. Tran et al. [21] believed that the adjacent hydroxyl and double hydroxyl groups of SiO2 surface showing weak acidity could activate the carbonyl compounds. We hypothesize that the surface of SiO2 support body formed the polyhydroxyl matrix and the (Boc)2O was attacked by the free base (ACLE or ACLH) on the matrix which was consistent with the conclusion of Sunseri et al. [22]. Ultrasonic vibrations enable the formation of intermolecular extrusions and confusions which has a significant influence on carbon dioxide generated from mono-t-butyl carbonate (compound6). We believe there are two reasonable mechanisms of facilitating the nucleophilic attack of amine group on the carbonyl group involved during the process; the former is that with the bubble formation and breakdown; the cavitation contributes to the efficient generation of theN-Boc amide (Boc-ACLE or BOC-ACLH); the latter is based on the reaction equilibrium that the continuous escape of CO2 results in the reaction preferring a forward tendency (Scheme 3).Scheme 3
The proposed ultrasonic-assisted synthetic mechanism ofN-Boc protection for amines.Figure 2
The structure of silicon dioxide surface (note: 1: silicon oxygen group; 2: isolated hydroxyl group; 3: adjacent hydroxyl group; 4: double hydroxyl group).


## 4. Conclusions

In summary, the ultrasonic-assisted approach catalyzed by SiO2 for the synthesis of twot-butoxycarbonylamino compounds at low temperature was investigated. By means of the synergetic effect of the ultrasound and SiO2, the designed reaction system showed a high efficiency on the desired products without any harsh conditions. Additionally, compared with existing production processing of the twot-butoxycarbonylamino cephalosporin intermediates, the work described in this study allows for stabilization of the real-time reaction temperature and low cost for the reaction. Importantly, this study greatly advances not only the practical cephalosporin production field but also the field of other synthetic drugs and their derivatives requiring Boc protection of amines.



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*Source: 9734108-2016-08-23.xml*