Luciana
Dalla-Vechia
a,
Benedikt
Reichart
b,
Toma
Glasnov
b,
Leandro S. M.
Miranda
a,
C. Oliver
Kappe
*bc and
Rodrigo O. M. A.
de Souza
*a
aBiocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, CEP 22941 909, Rio de Janeiro, Brazil. E-mail: rodrigosouza@iq.ufrj.br; Fax: (+55)-212-5627001
bChristian Doppler Laboratory for Microwave Chemistry (CDLMC) and Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, A-8010 Graz, Austria. E-mail: oliver.kappe@uni-graz.at; Fax: (+43)-316-3809840; Tel: (+43)-316-3805352
cChemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
First published on 14th August 2013
The development of multistep continuous flow reactions for the synthesis of important intermediates for the pharmaceutical industry is still a significant challenge. In the present contribution the biaryl-hydrazine unit of Atazanavir, an important HIV protease inhibitor, was prepared in a three-step continuous flow sequence in 74% overall yield. The synthesis involved Pd-catalyzed Suzuki–Miyaura cross-coupling, followed by hydrazone formation and a subsequent hydrogenation step, and additionally incorporates a liquid–liquid extraction step.
In this context we believe that a continuous flow synthesis of the protease inhibitorAtazanavir (1) is of significant interest. Atazanavir, approved by the U.S. Food and Drug Administration (FDA) in 2003, is an antiretroviral drug that is used to treat infection with human immunodeficiency virus (HIV).7 It is estimated that nearly 7.7 million people in Africa need treatment for HIV and do not have access to appropriate medication.8 Notably, Brazil is a pioneer in providing free medication to HIV patients and in 2007 the Brazilian government spent 850 million USD treating 200000 HIV patients.9Atazanavir is one of the most prescribed protease inhibitors in Brazil (and worldwide) and thus a sufficient and cost effective supply of Atazanavir is of prime importance.
The general retrosynthetic analysis of the Atazanavir (1) molecule is shown in Fig. 1 and reveals an assembly of three different building blocks.10 The subject of the work described herein is the synthesis of the biaryl building block 310via a three step continuous flow protocol that avoids the isolation and (off-line) purification of any intermediates.
Fig. 1 General retrosynthetic analysis for Atazanavir (1). |
Scheme 1 Synthetic route developed by Bristol–Meyers–Squibb (BMS) for the N-Boc hydrazine biaryl intermediate 3. Reagents and conditions: (a) 1.9 equiv. 5, 0.2 mol% Pd(PPh3)4, aqueous 3 M Na2CO3, toluene–ethanol (4:3), reflux 20 h, 80% yield. (b) N-Boc hydrazine (1 equiv.), toluene–2-propanol (4:3), reflux 2 h and then 22 °C for 16 h, 85% yield. (c) 1 mol% Pd/C (10%), HCO2Na (0.8 equiv.), ethanol–water (5.5:1), 57 °C for 1.5 h, 78% yield (overall yield 53%). |
We envisaged that the biphasic conditions proposed for the Suzuki–Miyaura cross coupling reaction (Scheme 1) could be a good starting point for our investigations since the desired biaryl product 7 could potentially be separated from the reaction mixture by simple phase separation, without involving complicated purification steps. The Suzuki–Miyaura cross-coupling employing 0.2 mol% Pd(PPh3)4 was therefore performed under sealed vessel microwave heating using essentially the BMS conditions described in Scheme 1. By applying 150 °C for 20 min, the conversion to product was higher than 99% as indicated in Table 1 (entry 1). Despite showing high conversion, this procedure was not considered economical owing to the large excess (1.9 equiv.) of boronic acid5 used. In our hands, attempts to reduce the amount of boronic acid were unsuccessful, leading to reduced conversion and/or selectivity.
Entry | Boronic acid (equiv.) | Pd(PPh3)4 (mol%) | Base/additive | Solvent | Conversionb (%) | |
---|---|---|---|---|---|---|
Product | Side products | |||||
a Reagents and conditions: organic phase: 2-bromo-pyridine 6 (0.23 mmol, 1 equiv.), 4-formyl-phenylboronic acid5, and Pd(PPh3)4 as a catalyst in 2.3 mL of organic solvent. Aqueous phase: a solution of the base in the indicated concentration (350 μL). Microwave heating was performed at 150 °C for 20 min. b HPLC peak area integration at 254 nm. | ||||||
1 | 1.9 | 0.2 | Na2CO3 (3.3 M) | Toluene–EtOH (4:3) | 99.2 | 0.8 |
2 | 1.0 | 0.3 | K3PO4 (3.3 M) TBAB (10 mol%) | Toluene–EtOH (4:3) | 94.2 | 2.5 |
3 | 1.0 | 0.3 | K3PO4 (3.3 M) | Toluene–EtOH (4:3) | 96.5 | 2.3 |
4 | 1.2 | 0.3 | K3PO4 (3.3 M) | Toluene–EtOH (4:3) | 96.7 | 2.1 |
5 | 1.2 | 0.3 | K3PO4 (1.6 M) | Toluene–EtOH (4:3) | 97.2 | 2.5 |
In a recent publication, Buchwald and co-workers have reported a very efficient biphasic continuous flow carbon–nitrogen coupling protocol where the addition of tribasic potassium phosphate and tetrabutylammonium bromide (TBAB) to the aqueous phase enhanced the conversion to the biaryl product.12 Evaluating these coupling conditions for the Suzuki–Miyaura batch coupling reaction described herein provided 94% conversion to the desired biaryl product 7 using only 1 equiv. of the boronic acid and 0.3 mol% of the Pd catalyst (Table 1, entry 2). Cross-coupling without the addition of TBAB also resulted in good conversion and high selectivity to the desired biaryl product 7, demonstrating that no phase-transfer catalyst (PTC) is required under the high-temperature microwave conditions (Table 1, entry 3). Increasing the boronic acid concentration to 1.2 equiv. and reducing the amount of base slightly increased the product conversion (Table 1, entries 4 and 5). The desired biaryl derivative 7 was isolated in 91% yield from the latter experiment.
The microwave batch conditions were then translated to a continuous flow protocol11 utilizing an ASIA microreactor employing a two-feed (or a three-feed in the case of hydrazone formation) set-up with preheated 4 mL stainless steel coils (additional 325 μl preheating zone volume and 2175 μl cooling zone volume) with an inner diameter of 0.02 inch (∼0.5 mm). After some experimentation and minor modifications to reagent stoichiometry, a 95% conversion at 150 °C and 20 min residence time was achieved (Scheme 2). It is important to note that using a fixed bed reactor filled with 60–125 μm stainless steel beads of identical geometry as described by Buchwald12 did not improve the conversion or selectivity in this reaction compared to the coil reactor (data not shown). The conversion could also not be improved by increasing reaction temperature or residence time.
Scheme 2 Suzuki–Miyaura cross coupling under continuous flow conditions. |
We subsequently evaluated the hydrazone formation 7 → 8 (Scheme 1) under microwave conditions, using the crude reaction mixture containing biaryl7 obtained after a simple phase separation (organic phase, toluene–ethanol) from the microwave-assisted biphasic Suzuki–Miyaura cross-coupling described above. The hydrazone formation was initially tested using ∼1.0 equiv. tert-butyl carbazate without the addition of acid as described in the BMS procedure,10 but in our hands it proved to be rather unreactive under these conditions (after 25 min of reaction at 120 °C the conversion was only 10%, Table 2, entry 5). Addition of various acids to the reaction mixture in most instances significantly increased the rate of hydrazone formation (Table 2, entries 1–4). However, in most cases the formation of the hydrazone was accompanied by salt precipitation and therefore was considered unsuitable for continuous flow processing. In order to avoid the formation of a precipitate, 0.5 equiv. of trimethylsilyltriflate (TMSOTf) was used as a Lewis acid. Gratifyingly, under these conditions excellent conversion (98%) to the corresponding hydrazone8 was obtained after 5 min at 50 °C without the formation of any precipitate (Table 3, entry 4).
Entry | Acid | Conditions | Conversionb (%) |
---|---|---|---|
a Reagents and conditions: crude organic phase from the Suzuki–Miyaura reaction (∼2.3 mL, theoretical concentration of 0.1 M, see Table 1, entry 5), t-butyl carbazate (0.23 mmol, 1 equiv.) and 5 equiv. acid concentration unless otherwise stated. b Conversion to product, HPLC peak area integration at 254 nm. | |||
1 | HCl | 50 °C/5 min | 98 |
2 | AcOH | 50 °C/5 min | 78 |
3 | CF3SO3H (0.5 eq.) | 50 °C/5 min | 97 |
4 | TMSOTf (0.5 eq.) | 50 °C/5 min | 98 |
5 | No acid | 120 °C/25 min | 10 |
Entry | Feed A | Feed B | Feed C | t R | Conversionb (%) |
---|---|---|---|---|---|
a Flow reactions were performed in a Syrris Asia system by applying reagent loop technology, T-mixers and a reaction coil made from stainless steel. For further details, see the Experimental section. b Conversion to product. HPLC peak area integration at 254 nm. | |||||
1 | 0.1 M crude 7 | 0.05 M TMSOTf | — | 5 | 87.8 |
0.1 M carbazate | 0.5 equiv. | ||||
400 μL min−1 | 400 μL min−1 | ||||
2 | 0.1 M crude 7 | 0.075 M TMSOTf | — | 5 | 100 |
0.1 M carbazate | 0.75 equiv. | ||||
400 μL min−1 | 400 μL min−1 | ||||
3 | 0.075 M TMSOTf | 0.2 M carbazate | 5 | 80.5 | |
0.1 M crude 7 | |||||
0.75 equiv. | 1.0 equiv. | ||||
320 μL min−1 | |||||
320 μL min−1 | 160 μL min−1 | ||||
4 | 0.075 M TMSOTf | 0.24 M carbazate | 5 | 97.4 | |
0.1 M crude 7 | |||||
0.75 equiv. | 1.2 equiv. | ||||
320 μL min−1 | |||||
320 μL min−1 | 160 μL min−1 | ||||
5 | 0.075 M TMSOTf | 0.24 M carbazate | |||
0.1 M crude 7 | 8 | 99.0 | |||
0.75 equiv. | 1.2 equiv. | ||||
200 μL min−1 | |||||
200 μL min−1 | 100 μL min−1 |
Again, the reaction conditions optimized under microwave heating were then translated to a continuous flow protocol. To the crude reaction mixture obtained from the continuous flow Suzuki–Miyaura protocol (organic phase,13 theoretical concentration of 0.1 M biaryl 7), 1.0 equiv. of tert-butyl carbazate was manually added to evaluate the optimum concentration of TMSOTf that would allow efficient hydrazone formation. Performing a two feed continuous flow hydrazone formation at 50 °C and a residence time of 5 min with 0.5 equiv. and 0.75 equiv. of TMSOTf (feed B) led to conversions of 87% and 100%, respectively (Table 3, entries 1 and 2). In order to develop a fully continuous two-step synthesis a three feed concept was developed in which the stream from the continuous flow Suzuki–Miyaura coupling (after phase separation, toluene layer,13 see Scheme 2) was initially mixed with a 0.075 M solution of TMSOTf in toluene–ethanol (4:3). tert-Butyl carbazate (0.2 M in toluene–ethanol (4:3)) was then added via a third pump before the reaction mixture was flowed through a stainless steel coil heated to 50 °C. Fine-tuning residence times and reagent stoichiometries using this approach ultimately allowed achieving high overall conversion and selectivity for hydrazone formation (Table 3, entries 3–5).
Finally, hydrazonehydrogenation was performed directly under continuous flow conditions using an H-Cube Pro reactor and a Pd/C catalyst cartridge.14 For optimization purposes pure hydrazone8 dissolved in a 4:3 mixture of toluene and ethanol (0.1 M) was processed through the reactor at a flow rate of 1 mL min−1 and atmospheric hydrogen pressure. Full conversion was achieved for hydrogenations carried out at 40 °C (Table 4, entry 4). When using the acidic crude hydrazone mixture (see above), prior neutralization of the acid was required since the acidic medium apparently poisons the Pd catalyst or otherwise inhibits the reduction process. Both triethylamine and K2CO3 were successfully tested as bases (Table 4, entries 5 and 6). Since a 0.5 M aqueous solution of K2CO3 provided better results (i.e., less side products), this was the preferred base for all further studies. Neutralization of the crude reaction mixture was first performed off-line in batch mode to demonstrate proof-of-principle. In the overall continuous flow process this extraction was performed in-line using a membrane-based liquid/liquid flow extraction module (FLLEX,15 see the Experimental section for details).
Entry | Temp (°C) | Hydrazine 3b (%) | Side productsb (%) | Remaining hydrazone8b (%) |
---|---|---|---|---|
a Experiments were performed in an H-Cube Pro flow reactor with the following conditions: flow rate 1 mL min−1, 10% Pd/C cartridge and atmospheric pressure of H2. For further details see the Experimental section. b HPLC peak area integration at 254 nm. | ||||
Pure hydrazone in toluene–ethanol (0.1 M) | ||||
1 | 10 | 78.2 | 0 | 21.8 |
2 | 20 | 99.5 | 0 | 0.5 |
3 | 30 | 99.6 | 0 | 0.4 |
4 | 40 | 100 | 0 | 0 |
Crude hydrazone after neutralization with TEA | ||||
5 | 40 | 85.0 | 15.0 | 0 |
Crude hydrazone after washing with 0.5 M K2CO3 (FLLEX) | ||||
6 | 40 | 94.6 | 5.4 | 0 |
The neutralization of the reaction mixture with TEA leads to the formation of large amounts of a side product (Table 4, entry 5). In order to overcome the formation of this side product we decided to make a phase extraction of the acid using a basic solution of potassium carbonate (0.5 M). Gratifyingly, after washing with aqueous base the formation of this side-product was minimized and a good conversion was achieved. Manual washing with aqueous phase was then replaced by extraction on a flow liquid–liquid extractor (FLLEX),15 leading to high conversions without side products (Table 4, entry 6).
An overview of the three-step continuous flow process is depicted in Fig. 2. The Suzuki–Miyaura reaction takes place by pumping an organic mixture (toluene–ethanol 4:3) containing the boronic acid5, 2-bromo-pyridine (6) and the palladiumcatalyst through one pump, and an aqueous tribasic potassium phosphate solution through a second pump. In the first 4 mL stainless steel coil the reaction mixture is heated to 150 °C with a residence time of 20 min and the product stream collected. Phase separation of the triphasic product stream provides a biphasic toluene–ethanol mixture, containing biaryl intermediate 7 which is injected through a third pump to a second coil. Simultaneously, solutions of TMSOTf and tert-butyl-carbazate in toluene–ethanol (4:3) are pumped into the coil using two separate pumps. Hydrazone (8) formation takes place in this second coil at 50 °C and a residence time of 8 min. The outgoing stream is then pumped through a continuous flow liquid/liquid extractor (FLLEX)15 together with an aqueous potassium carbonate solution to eliminate the acid from the previous step. After the extraction, crude hydrazone8 is pumped through a flow hydrogenation unit applying a 10% Pd/C cartridge to provide the final biaryl product 3 in 74% overall yield after chromatographic purification.
Fig. 2 Overview of the three-step continuous flow synthesis of biaryl hydrazine intermediate of Atazanavir (3). |
Purification: the biphasic reaction mixture was separated and the organic layer was evaporated and subjected to silica gel column chromatography using a mixture of petroleum ether, ethyl acetate and triethylamine as an eluent (1:2:0.03). Yield under optimized conditions: 38.5 mg of 7 (91%).
Purification: the reaction mixture was neutralized with triethylamine and the solvent was evaporated. The crude residue was subjected to silica gel column chromatography using a mixture of petroleum ether, ethyl acetate and triethylamine as an eluent (1:2:0.03). Yield of 8 under optimized conditions: 61.5 mg (90%).
The reduction of the hydrazone to the corresponding hydrazine was directly tested in the H-Cube Pro flow reactor (ThalesNano Inc., Budapest, Hungary).
Purification: The reaction mixture obtained from H-Cube Pro was evaporated and purified by silica gel column chromatography using a mixture of petroleum ether, ethyl acetate and triethylamine (5:5:0.1) as an eluent. This procedure afforded the pure hydrazine3 in 74% (124 mg, 0.415 mmol) overall yield (for the three steps and the liquid–liquid extraction under continuous flow conditions after the 2nd step). The yield was calculated considering full conversion in the 1st step (of the 2-bromo-pyridine substrate into the 4-(pyridin-2-yl)benzaldehyde product) and the volume of the collected fraction (steady state fraction: 5 mL of a 0.1 M reaction mixture; 0.5 mmol). All further calculations on the overall yield have been defined by this sampling; thus the overall yield refers to a theoretical yield of 0.5 mmol of final product 3 (N-1-(tert-butyloxycarbonyl)-N-2-[4-(pyridine-2yl)benzylidene]-hydrazine).
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