Efficient continuous flow synthesis of hydroxamic acids and suberoylanilide hydroxamic acid preparation

Article abstract of DOI:10.1021/jo900144h

A continuous flow tubing reactor can be used to readily transform methyl or ethyl carboxylic esters into the corresponding hydroxamic acids. Flow rate, reactor volume, and temperature were optimized for the preparation of a small collection of hydroxamic acids. Synthetic advantages were identified as an increased reaction rate and higher product purity. This method was also successfully applied to the multistep preparation of suberoylanilide hydroxamic acid, a potent HDAC inhibitor used in anticancer therapy.

Full text of DOI:10.1021/jo900144h

Efficient Continuous Flow Synthesis of
Hydroxamic Acids and Suberoylanilide
Hydroxamic Acid Preparation
with performing reactions under micro/meso continuous flow
have been demonstrated for a number of common organic
transformations, ranging from liquid-liquid to solid-liquid-gas
4
systems. Established advantages of continuous flow chemistry
processes include precise control of variables such as temper-
ature, pressure, concentration, residence time, and heat transfer.
All of these aspects significantly affect the reaction outcome,
†
‡
§,‡
Elena Riva, Stefania Gagliardi, Caterina Mazzoni,
†
‡
‡
Daniele Passarella, Anna Rencurosi, Daniele Vigo, and
,
‡
Marisa Martinelli*
5
improving yield and selectivity. Moreover, the possibility of
Dipartimento di Chimica Organica e Industriale, UniVersita`
degli Studi di Milano, Via Venezian 21, 20133 Milan, Italy,
and NiKem Research Srl, Via Zambeletti 25,
carrying out reactions in superheated solvents allows novel
thermal regimes previously inaccessible within conventional
apparatus. By rapid and efficient heat dispersion, large exo-
6
2
0021 Baranzate, Milan, Italy
therms can be minimized, producing safer and more selective
7
processes.
marisa.martinelli@nikemresearch.com
Here we report a general and efficient procedure for the
conversion of esters into the corresponding hydroxamic acids
with good yields and purities using a commercially available
ReceiVed January 23, 2009
8
continuous flow reactor.
Hydroxamic acids occur in several molecules with a wide
9
spectrum of biological activities such as antibacterial, antifun-
gal, antiinflammatory, antiasthmatic, and anticancer properties.
In particular this moiety is present in potent matrix metallo-
1
0
11
proteinase and hystone deacetylase inhibitors because hy-
droxamic acids are strong bidentate metal-ion-chelating agents
that interact with zinc(II)-containing proteins. Given the im-
portance of this functionality, the development of new meth-
odologies for a general and efficient synthesis are still of great
interest, although several methods have been developed and
A continuous flow tubing reactor can be used to readily
transform methyl or ethyl carboxylic esters into the corre-
sponding hydroxamic acids. Flow rate, reactor volume, and
temperature were optimized for the preparation of a small
collection of hydroxamic acids. Synthetic advantages were
identified as an increased reaction rate and higher product
purity. This method was also successfully applied to the
multistep preparation of suberoylanilide hydroxamic acid, a
potent HDAC inhibitor used in anticancer therapy.
12
published so far. As a part of a medicinal chemistry project,
a simple conversion of ester into hydroxamic acid was envisaged
as a suitable and convenient synthetic method for the preparation
of a collection of compounds featuring this particular moiety.
We first investigated the use of methylbenzoate (1a) to optimize
the parameters (flow rate, residence time, and temperature). A
mixture of 1a (0.5 M in MeOH) and hydroxylamine (1:10 ratio)
was simultaneously pumped into the flow reactor with a solution
of MeONa (0.5 M in MeOH) of MeONa, allowing efficient
mixing in the PTFE tubing at the selected temperature. The
optimization of the experimental parameters was systematically
In recent years pharmaceutical and biotech companies are
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well-differentiated drugs with a reduced cost both for discovery
and development. With the aim at increasing the productivity
of original and highly pure molecules as potential modulators
of therapeutic targets, different and novel technologies (related
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P. H Chem.-Eur. J. 2006, 12, 8434–8442.
1
to synthesis, workup, and isolation) were developed. Among
these new technologies, continuous flow organic synthesis, after
an extensive investigation at the academic level, is now being
applied in fine chemistry with the transfer of many classes of
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2
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reaction successfully reported. More recently pharmaceutical
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G. Synthesis 2003, 18, 2827–2830. (b) Zhang, X.; Stefanick, S.; Villani, F. J.
Org. Process Res. DeV. 2004, 8, 455–460.
(8) The process makes use of the combination of R-2 Pump Module and
R-4 Reactor Module, a commercially available Vapourtec system.
3
batch techniques. Theoretical and practical benefits associated
(
9) (a) Weber, G. Cancer Res. 1983, 43, 3466–3492. (b) Miller, M. J. Chem.
†
Universit a` degli Studi di Milano.
NiKem Research Srl.
Present address: Ansaldo Nucleare S. p. A., C.so Perrone 25, 16152 Genova,
ReV. 1989, 89, 1563–1579.
‡
(10) (a) Brown, P. D.; Davidson, A. H.; Gearing, A. J. H.; Whittaker, M. In
Metalloproteinase Inhibitor in Cancer Chemotherapy; Clendininn, N. J., Appelt,
K., Eds.; Humana Press: Totowa, NJ, 2001; pp 113-142. (b) Jung, M. Curr.
Med. Chem. 2001, 8, 1505–1511. (c) Breslow, R.; Belvedere, S.; Gershell, L.
HelV. Chim. Acta 2000, 83, 1685–1692. (d) Whittaker, M.; Floyd, C. D.; Brown,
P.; Gearing, J. H. Chem. ReV. 1999, 99, 2735–2776.
§
Italy.
(
1) (a) Chighine, A.; Seche, G.; Bradley, M. Drug DiscoVery Today 2007,
1
2
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(
2) (a) Wiles, C.; Watts, P Eur. J. Org. Chem. 2008, 10, 1655–1671. (b)
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3
540 J. Org. Chem. 2009, 74, 3540–3543
10.1021/jo900144h CCC: $40.75  2009 American Chemical Society
Published on Web 04/07/2009

TABLE 1. Optimization of Experimental Parameters
TABLE 3. Conversion of Esters into Hydroxamic Acids under
Flow Conditions
flow rate
(mL/min)
residence
time (min)
conversion
a
entry
T (°C)
(%)
1
2
3
4
5
6
1
1
1
0.5
0.5
0.5
5
5
5
20
20
30
50
70
80
60
70
70
52
65
58
76
74
80
b
a
b
Conversion was determined by LC/MS at 215 nM. Corresponding
carboxylic acid was present as byproduct.
TABLE 2. Comparison of Reaction Carried out in Batch, under
Microwave Irradiation, and Using the Flow Reactor
a
entry
experimental condition
conversion (%)
1
2
3
batch reaction
sealed tube with MW irradiation
flow system
58
72
80
a
Conversion was determined by LC/MS at 215 nM.
investigated by varying flow rate, temperature, and reactor
volume. This process was greatly facilitated by continuous flow
conditions and a significant number of runs were rapidly
conducted in a sequential manner (Table 1).
The temperature of 70 °C resulted in the best compromise to
have good conversion without the formation of the correspond-
ing carboxylic acid. This byproduct was significantly present
when the reaction was performed at 80 °C. The conversion into
the hydroxamic acid was improved by prolonging the residence
time by lowering the flow rate and increasing the volume of
the reactor. When the reaction was performed at 70 °C for 30
min, 80% conversion was achieved by LC/MS, resulting in 82%
yield of isolated 2a (entry 6). For comparative purposes the
reaction was run both as a standard batch process and in a sealed
tube under microwave irradiation, applying the same conditions
of temperature, concentration of reagents, and reaction time as
optimized in the flow protocol (Table 2).
We found that such conditions gave moderate conversion
using conventional equipment (58%, entry 1) and comparable
result under microwave irradiation (72%, entry 2). The precise
control of temperature distribution and the efficient heating and
mixing associated with flow technique minimized the formation
of the corresponding carboxylic acid as a byproduct and ensured
that this simple chemical transformation proceeded at faster rates
compared to the batch system.
a
Isolated yields; purity >95% (LC/MS).
To verify the effectiveness and reproducibility of the opti-
mized reaction conditions, the synthesis of a small collection
of hydroxamic acids was undertaken. The employed flow
protocol was based upon the optimized conditions described
for the synthesis of 2a without further optimization (i.e., 70
°
C, flow rate 0.5 mL/min, residence time 30 min) in a 250 mg
scale. All of the desired hydroxamic products were obtained in
good to excellent yields (Table 3) with no detection of the
corresponding carboxylic acids byproduct.
The data reported in Table 3 clearly show that the formation
of hydroxamic acids from esters is quite general and occurs
smoothly in the continuous flow system. In fact simple aryl or
alkylaryl esters (entries 1 and 2), simple amino esters (entry 3)
and Boc-protected (entries 7-9) amino esters, sulfonamido-ester
(entry 4), and heteroaryl esters (entries 5 and 6) were suitable
substrates for the reaction. Moreover the optimized reaction
conditions were successfully applied to enantiomerically pure
esters without loss of stereochemical integrity. The specific
optical rotation for the hydroxamic acid of N-Boc-alanine methyl
ester 2g was found to be identical to that reported in literature
(
12) Preparation of hydroxamic acids. From hydroxylamine and ester: (a)
Mordini, A.; Reginato, G.; Russo, F.; Taddei, M. Synthesis 2007, 20, 3201–
204. (b) Hauser, C. R.; Renfrow, W. B., Jr. Organic Synthesis; Wiley: New
York, 1943; Collect. Vol. II, pp 67-68. Cleavage of an ester on a solid support:
c) Thouin, E.; Lubell, W. Tetrahedron Lett. 2000, 41, 457–460. From carboxylic
3
(
acids and amine using coupling reagents: (d) De Luca, L.; Giacomelli, G.; Taddei,
M. J. Org. Chem. 2001, 66, 2534–2537. (e) Giacomelli, G.; Porcheddu, A.;
Salaris, M. Org. Lett. 2005, 5, 2715–2717. (f) Ech-Chahad, A.; Minassi, A.;
Berton, L.; Appendino, G. Tetrahedron Lett. 2005, 46, 5113–5115. (g) Katritzky,
A. R.; Kirichenko, N.; Rogovoy, B. V. Synthesis 2003, 18, 2777–2780. (h) Bail e` n,
M. A.; Chinchilla, R.; Dodsworth, D. J.; N a` jera, C. Tetrahdron Lett. 2001, 42,
20
20
for the enantiopure compound {[R]
D
-28; lit. [R]
D
-29 (c
1
2a
1
, MeOH)}.
With compounds 1b, 1d, and 1j the obtained results show
an improvement if compared to the microwave-assisted process
5
013–5016. (i) Sekar Reddy, A.; Suresh Kumar, M.; Ravindra Reddy, G.
Tetrahedron Lett. 2000, 41, 6285–6288. From N-acyloxazolidinones: (l) Sibi,
M. P.; Hasegawa, H.; Ghorpade, S. R. Org. Lett. 2002, 4, 3343–3346. From
esters and O-Bn hydroxylamine: (m) Pirrung, M. C.; Chau, G. H. L. J. Org.
Chem. 1995, 60, 8084–8085. (n) Gissot, A.; Volonterio, A.; Zanda, M. J. Org.
Chem. 2005, 70, 6925–6928. Angelini-Rimini’s reaction on solid support: (o)
Porcheddu, A.; Giacomelli, G. J. Org. Chem. 2006, 71, 7057–7059.
1
2a
described in the literature. Noticeably for compound 1d the
isolated yield was increased from 33% to 95%; in the other
cases an average improvement of about 20% was observed.
J. Org. Chem. Vol. 74, No. 9, 2009 3541

SCHEME 1. Two-Step Synthesis of Suberoylanilide
Hydroxamic Acid (SAHA, 5)
TABLE 4. First Step (Scheme 1) Optimization Results
flow rate residence
entry [A] (M) [B] (M) (mL/min) time (min) T (°C) 4 (%)
a
1
2
0.05
1
0.05
1
2
3.5
2.5
1.4
rt
rt
>95
>95
a
Conversion was determined by LC/MS at 215 nM.
TABLE 5. Second Step (Scheme 1) Optimization Results
MeONa flow rate residence
entry (equiv) (mL/min) time (min) T (°C) 4 (%) 5 (%) 6 (%)
a
a
a
1
2
3
4
1
1
2
2
0.33
0.33
0.33
0.4
30
30
30
50
70
90
90
90
73
39
17
3
19
49
70
83
8
12
13
15
Simple scale-up makes the continuous flow technique very
advantageous when compared to MW systems, as up to now
MW heating has proved to be unsuitable for large scale
processes. Moreover the risks associated with failing to scale-
up a process are limited using continuous flow. The reaction
condition setup on the microreactor can be directly transferred
to production on larger scale without the need for reoptimization
by simply using the flow reactor for an extended time or by
employing multichannel parallel reactors (numbering-up pro-
a
Conversion was determined by LC/MS at 215 nM.
by the transformation of ester by aqueous hydroxylamine in
presence of sodium methoxide (Scheme 1).
In the first step a solution of suberoyl chloride in THF and a
2
mixture of aniline and sodium carbonate in THF/H O were
simultaneously pumped at room temperature into the reactor.
In the optimized protocol the conversion of the acyl chloride
into the corresponding amide resulted in clean and quantitative
yield in less than 2 min (Table 4).
No product isolation at this intermediate stage was necessary:
the output of the reaction was evaporated, and the obtained
amido ester was dissolved in methanol and used directly in the
second step without further purification.
Unfortunately the optimized reaction conditions to obtain
hydroxamic acid described above gave only low conversion to
(SAHA). A new optimization process was necessary (Table
), and the best conditions were achieved by flowing a solution
1
3
cess).
A demonstration of this preparative capability was readily
obtained. Applying the optimized conditions and doubling the
concentration of the starting materials, 4.3 g of N-hydroxy-2-
phenylacetamide 2b was straightforwardly produced after 1.5 h
(
output 2.9 g/h). Yield and purity were similar to that of the
smaller scale, proving that the reaction conditions identified for
the production of a few milligrams can be transferred to a larger
scale without any further optimization.
5
5
In addition to the easy scalability of the protocol, the careful
control of temperature exotherms and the small volumes
employed by flow reactor allow the safer use of the highly toxic
of the crude methyl suberanilate and aqueous hydroxylamine
in methanol together with a stream of 2 equiv of sodium
methoxide in MeOH at 90 °C for 50 min.
1
4
and potentially explosive hydroxylamine.
On the basis of the good results obtained in the development
of the continuous flow synthesis of hydroxamic acids, this new
methodology was applied to the synthesis of suberoylanilide
hydroxamic acid (SAHA, Scheme 1). This compound, one of
the early histone deacetylase (HDAC) inhibitors discovered by
With the aim of avoiding a time-consuming workup procedure
and extensive manual purification of the final compound, an
integrated sequential flow synthetic pathway was set up,
18
employing an immobilized scavenger. The reaction stream was
19
then directly passed through a short packed column containing
1
5
20
Breslow and colleagues, was approved by the U.S. FDA in
October, 2006 for the treatment of patients with cutaneous T-cell
silica-supported quaternary amine (ISOLUTE PE-AX) for the
selective removal of the carboxylic acid byproduct 6 (8-oxo-
8-(phenylamino)octanoic acid). The solution containing the
product and traces of the starting material was collected, and
after solvent evaporation, crystallization from MeOH afforded
SAHA in 84% yield and 99% purity (80% yield over two steps).
In conclusion, we have developed a simple and high-yielding
method for the synthesis of hydroxamic acids directly from the
corresponding carboxylic esters using a flow reactor. The
synthetic protocol is amenable both for preparation of compound
collections and for the scale-up of single derivatives. Various
reaction conditions can be screened very easily and rapidly to
set up a more efficient protocol by optimizing temperature,
1
6
lymphoma (CTCL). Despite the several synthetic procedures
1
7
for the preparation of SAHA that have been reported, as far
as we know, none of them described the synthesis under flow
conditions. Our two-step sequence entails the conversion of the
commercially available methyl suberoyl chloride 3 into methyl
suberanilate 4 under Schotten-Baumann conditions, followed
(
13) (a) Anderson, N. G. Org. Process Res. DeV. 2001, 5, 613–621. (b)
Wheeler, R. C.; Benali, O.; Deal, M.; Farrant, E.; MacDonald, S. J. F.;
Warrington, B. H. Org. Process Res. DeV. 2007, 11, 704–710.
(
14) Hydroxylamine Hazard Code: Xn, N; Risk Statement 5-22-37/38-41-
4
3-48/22-50.
15) Breslow, R.; Marks, P. A.; Jursic, B. PTC Int. Appl. WO 93/07148,
April 15, 1993.
(
(
16) (a) FDA approves vorinostat for CTC: http://www.fda.gov/. (b) Grant,
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Ladlow, M.; Tapolczay, D. J.; Vickerstaffe, E. J. Comb. Chem. 2007, 9, 422–
430. (b) Baxendale, I. R.; Griffiths-Jones, C. M.; Ley, S. V.; Tranmer, G. K.
Synlett 2006, 3, 427–430. (c) Baumann, M.; Baxendale, I. R.; Ley, S.; Smith,
C. D.; Tranmer, G. K. Org. Lett. 2006, 23, 5231–5234. (d) Smith, C. D.;
Baxendale, I. R.; Lanner, S.; Hayward, J. J.; Smith, S. C.; Ley, S. V. Org. Biomol.
Chem. 2007, 5, 1559–1561.
(19) Commercially available Omnifit glass chromatography columns with
adjustable height endpieces.
(20) ISOLUTE PE-AX Sorbent is commercially available.
S.; Easley, C.; Kirkpatrick, P. Nat. ReV. Drug DiscoVery 2007, 6, 21–22. (c)
Butler, L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose, A.; Cordon-Cardo,
C.; Thaler, H. T. Cancer Res. 2000, 60, 5165–5170.
(
17) (a) Gediya, L. K.; Chopra, P.; Purushottamachar, P.; Maheshwari, N.;
Njar, V. C. O. J. Med. Chem. 2005, 48, 5047–5051. (b) Stowell, J. C.; Huot,
R. I.; Van Voast, L. J. Med. Chem. 1995, 38, 1411–1413. (c) Mai, A.; Esposito,
M.; Sbardella, G.; Massa, S. OPPI Briefs 2001, 33, 391–394. (d) Curtin, M. L.
Curr. Opin. Drug DiscoVery DeV. 2004, 7, 848–868. (e) PTC Int. Appl. WO
2
006/127321 A2, November 30, 2006.
3
542 J. Org. Chem. Vol. 74, No. 9, 2009

stoichiometry, and residence time. Continuous operations
provide an attractive approach for handling hazardous processes
and reagents in a safer manner. Remarkably, the method was
successfully employed with enolizable esters, such as chiral
amino acids, with no trace of racemization at the R carbon.
Furthermore once the optimum reaction conditions are estab-
lished the reaction can be scaled up to provide virtually any
amount of product by running the apparatus for longer periods
of time. In particular we have demonstrated the possibility of
preparing 2.9 g/h of 2b. Moreover the two-step synthesis of
SAHA was developed and the purification of final product was
achieved using immobilized scavengers.
both solutions were transferred at a constant flow rate (3.5 mL/
min) inside a PTFE tubing reactor (reactor volume 10 mL) at room
temperature. The reaction took place into the reactor, and the
product was collected in a suitable product stock bottle. The solvent
was evaporated, and the crude of methyl suberanilate was used for
the next step without further purification.
Second Step. Preparation of Suberoylalanide Hydroxamic
Acid (SAHA). Two tubing reactors (10 mL each) were installed
and connected together in series. The second reactor was connected
to a packed column with solid-supported scavenger. Reagent stock
bottle C: methyl suberanilate (255 mg, 0.97 mmol, 1 equiv); 50%
aq hydroxylamine (0.58 mL, 9.7 mmol, 10 equiv), 0.5 M solution
in MeOH. Reagent stock bottle D: MeONa (105 mg, 1.95 mmol,
2
equiv), 1 M solution in MeOH. Using the automated injection
system, both solutions were transferred at a constant flow rate (0.2
mL/min) into preheated PTFE tubing reactors (total reactor volume
Experimental Section
2
0 mL) maintained at 90 °C. The tubing reactor was connected to
The flow reactor was configured using a combination of an R-2
Pump Module and R-4 Reactor Module.
an Omnifit glass column (6.6 mm i.d.) packed with silica-supported
quaternary amine (1.5 g) to trap the carboxylic acid byproduct.
8
20
General Reaction Procedure for the Synthesis of
Hydroxamic Acids. Reagent stock bottle A: ester (1 equiv); 50%
aq hydroxylamine (10 equiv), 0.5 M solution in MeOH. Reagent
stock bottle B: MeONa (1 eq), 0.5 M solution in MeOH. Using
the automated injection system, both solutions were transferred at
a constant flow rate (0.166 mL/min) into a preheated PTFE tubing
reactor (reactor volume 10 mL) maintained at 70 °C. The reaction
took place inside the reactor, and the product was collected in an
appropriate product stock bottle. The solvent was evaporated, and
the product was purified by a suitable method. The reactions were
performed on a 250 mg scale.
After this purification step, the product was collected in an
appropriate product stock bottle. The solvent was evaporated, water
(
10 mL) was added, and pH was adjusted to 7 with acetic acid.
The precipitated solid was filtered and dissolved in hot MeOH, and
then the solution was cooled to 0 °C. The formed solid was filtered
to give 219 mg of suberoylalanide hydroxamic acid. Overall yield
8
0%.
Acknowledgment. The authors gratefully acknowledge Dr.
Andrea Colombo for helpful discussion on SAHA preparation
and Dr. Sergio Menegon for NMR characterization of products.
Synthesis of Suberoylalanide Hydroxamic Acid. First Step.
Preparation of Suberanilic Acid Methyl Ester. Reagent stock
bottle A: methyl suberoyl chloride (200 mg, 0.97 mmol, 1 equiv)
Supporting Information Available: Experimental proce-
dures and characterization. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
0
M solution in THF. Reagent stock bottle B: aniline (62.4 mg,
.97 mmol, 1 equiv); NaHCO (81.2 mg, 0.97 mmol, 1 eq), 1 M
O. Using the automated injection system,
3
solution in 1:1 THF/H
2
JO900144H
J. Org. Chem. Vol. 74, No. 9, 2009 3543