Concise synthesis of α-methylene-β-hydroxy-γ-carboxy- γ-lactams

Article abstract of DOI:10.1002/jhet.1578

A concise protocol for the synthesis of α-methylene-β-hydroxy- γ-carboxy-γ-lactams has been described via alkylation of amino acid derived iminoesters with α-bromomethylmethacrylate, followed by allylic hydroxylation. All the synthesized compounds have been evaluated for their cytotoxicity on multiple myeloma cancer cell lines.

Full text of DOI:10.1002/jhet.1578

July 2013
Concise Synthesis of a-Methylene-b-hydroxy-g-carboxy-g-lactams
Srinivas Tekkam,a Joseph L. Johnson,a Subash C. Jonnalagadda,b and Venkatram R. Mereddya,c
955
*
aDepartment of Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, Minnesota 55812
bDepartment of Chemistry and Biochemistry, Rowan University, Glassboro, New Jersey, 08028
cPharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, Duluth, Minnesota 55812
*
E-mail: vmereddy@d.umn.edu
Received August 27, 2011
DOI 10.1002/jhet.1578
Published online 24 June 2013 in Wiley Online Library (wileyonlinelibrary.com).
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A concise protocol for the synthesis of a-methylene-b-hydroxy-g-carboxy-g-lactams has been described
via alkylation of amino acid derived iminoesters with a-bromomethylmethacrylate, followed by allylic
hydroxylation. All the synthesized compounds have been evaluated for their cytotoxicity on multiple
myeloma cancer cell lines.
J. Heterocyclic Chem., 50, 955 (2013).
INTRODUCTION
establishing an a,b-unsaturated system in the pyroglutamate
could provide a reactive site for potential nucleo-
philic interaction with the proteasome. In fact, similar
phenomenon was observed for proteasome inhibition by
macrocyclic lactam natural product syringolin A (5),
wherein Thr1 of proteasome has been shown to undergo
1,4-addition with additional stabilization of the intermediate
oxyanion by the G47 nitrogen leading to irreversible
inhibition (Fig. 2) [4].
g-Carboxy-g-lactams (pyroglutamates) are important
structural components found in many biologically impor-
tant heterocyclic natural products [1]. The ester group as
well as the lactam ring in pyroglutamates could be further
manipulated to synthesize a variety of structurally complex
molecules, and hence they serve as important synthetic
intermediates in heterocyclic chemistry [1].
The clinical success of the proteasome inhibitor bortezomib
(Velcade, 1) led to the fast track approval of this drug in 2003
for treatment of multiple myeloma [2]. This molecule is also
undergoing clinical trials for several other cancers. However,
clinical use of this compound has revealed limitations such
as intrinsic and acquired resistance [2]. In this regard, some
of the pyroglutamate containing natural products such as
omuralide 2, lactacystin 3, and cinnabaramides 4 were found
to exhibit excellent anticancer activity by inhibiting the
enzyme proteasome (Fig. 1) [3]. Whereas bortezomib is a
reversible proteasome inhibitor, the corresponding natural
products mentioned above are irreversible proteasome
inhibitors. The mechanism of action for these molecules
involves the hydrolytic attack of the proteasome onto the
b-lactone unit resulting in the formation of a strong covalent
bond and the irreversible inhibition of the proteasome. One
potential problem with the use of these natural products as
therapeutic agents is their short half-life in solution or in
serum due to the highly unstable b-lactone unit and conse-
quent loss of activity.
We have recently reported concise and stereoselective
methodologies for the synthesis of functionalized
pyroglutamates [5]. In continuation of this work, we have
developed a novel protocol for the concise synthesis of
a-methylene-b-hydroxy-g-carboxy-g-lactams.
RESULTS AND DISCUSSION
We initiated the synthesis of the target compounds with
the esterification of representative amino acids valine 6a,
leucine 6b, and phenylalanine 6c as their corresponding
benzyl esters 7a–c. Imination of the a-amino esters 7a–c
was accomplished by reacting with p-chlorobenzaldehyde
in the presence of MgSO4 providing the imines 8a–c in
quantitative yield. These imines were utilized in the next
step without further purification. Lithium enolates of
imines 8a–c were generated using LiHMDS, and they were
reacted with a-bromomethylacrylate 9 [6].The acidic work
up resulted in the hydrolysis of imines, and subsequent
lactamization was realized upon refluxing the crude
reaction mixture in HCl and toluene to provide pure a-
methylenepyroglutamates 10a–c in good yield (Scheme 1) [5].
We envisaged that removing the labile b-lactone unit would
afford aqueous stability to the pyroglutamate ring and
© 2013 HeteroCorporation

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S. Tekkam, J. L. Johnson, S. C. Jonnalagadda, and V. R. Mereddy
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After obtaining the a-methylene pyroglutamates 10a–c,
we envisioned the introduction of b-hydroxyl group via
allylic oxidation with SeO2 [7]. Lactams 10a–c were treated
with catalytic selenium dioxide and tert-butyl hydroperoxide
t
in butanol. However, the reaction was very sluggish, and
negligible (<5%) amount of the product was observed.
Under these conditions, the benzyl ester underwent
trans-esterification to afford the t-butyl ester containing
pyroglutamates. Selenium dioxide and hydrogen peroxide in
various aprotic solvents such as toluene, dichloromethane,
and THF also proved futile. However, employing selenium
dioxide in refluxing acetic acid proved optimal for the reaction
as monitored by TLC. The reaction mixture was worked up
with water and then NaHCO3 and extracted with ethyl
acetate. Concentration of the organic layer provided a dark
colored product with many organoselenium impurities. The
crude product was further dissolved in THF and treated with
stoichiometric amount of hydrogen peroxide to reduce
the amount of selenium impurities. The crude reaction
product was purified by silica gel column chromatography
using hexane and ethyl acetate. The purified product
was further recrystallized in ethyl acetate to obtain pure
Figure 1. Reversible and irreversible proteasome inhibitors.
Figure 2. Mechanism of action for proteasome inhibition by syringolin A.
Scheme 1. Synthesis of a-methylene-b-hydroxypyroglutamates.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2013
Concise Synthesis of a-Methylene-b-hydroxy-g-carboxy-g-lactams
957
Scheme 2. Synthesis of a-methylene-b-hydroxypyroglutamates.
a-methylene-b-hydroxy-g-carboxy-g-lactams 11a–c in
modest yields (Scheme 2). High levels of diastereoselectivity
was observed in all the examples, and as expected, the 1,2-
trans isomer was obtained as the major product upon
rigorous silica gel column chromatography. Hydroxylation
of valine derived pyroglutamate 10a essentially provided
one diastereomer 11a and the corresponding phenylalanine,
and leucine based pyroglutamates afforded 20:1 dr (for
11b) and 18:1 dr (for 11c), respectively. The hydrolysis of
esters 11a–c was carried out using 0.1 N LiOH, and the
hydroxy acids 12aÀc were obtained in good yields
(68–79%) in all the cases (Scheme 2).
substrate controlled diastereoselective alkylation of a chiral
oxazole followed by allylic hydroxylation. As a representa-
tive example, the requisite chiral oxazole 15 was obtained
from L-threonine 13 in two steps involving esterification
and subsequent treatment with benzimidate [8]. The alkyl-
ation of oxazole 15 was realized via lithiation with LDA
followed by the addition of a-bromomethylmethacrylate
9 to furnish the a-methylenepyroglutamate 16 upon acidic
hydrolysis [9]. Allylic hydroxylation of 16 with SeO2 in
acetic acid furnished the requisite b-hydroxypyroglutamate
17 in excellent diastereoselectivity (dr 15:1) upon silica gel
column chromatography (Scheme 3).
This methodology was further extended toward the
enantioselective synthesis of a-methylene-b-hydroxy-g-
carboxy-g-lactams. For this purpose, we chose to utilize a
Owing to the biological relevance of pyroglutamates, all
the compounds synthesized were evaluated for their efficacy
as potential anticancer agents utilizing multiple myeloma
Scheme 3. Enantioselective synthesis of a-methylene-b-hydroxypyroglutamates.
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8.05 (br s, 1H), 7.78–7.76 (m, 2H), 7.46–7.27 (m, 8H), 5.93 (s,
1H), 5.73 (q, J = 6.5 Hz, 1H), 5.46 (s, 1H), 5.41 (d, J = 12.0 Hz,
1H), 5.33 (d, J = 12.0 Hz, 1H), 4.75 (d, J = 10.0 Hz, 1H), 4.64
(d, J = 9.5 Hz, 1H), 1.22 (d, J = 6.5 Hz, 3H); 13C-NMR
(125 MHz, DMSO-d6): d 170.0, 165.8, 165.6, 141.9, 134.9,
133.6, 130.1, 130.0, 129.4, 129.0, 128.8, 128.6, 121.5, 74.7,
73.7, 71.8, 68.6, 15.8.
cancer cell lines (RPMI-8226). Briefly, the cancer cells were
plated in 96 well plates and allowed to adhere for 3d. Cells
were then treated with each compound (50 mM) or with
DMSO alone for 24 h. MTS assay was used for determining
the number of remaining viable cells after exposure to com-
pounds. MTS (20 mL) was added to 100 mL culture medium
in each well. After incubation at 37ꢀC for 3 h, absorbance
was measured using an ELISA plate reader. Unfortunately,
none of the compounds showed any appreciable cytotoxicity
at this concentration. We believe that further studies are
required to understand the structure activity relationship to
identify a lead molecule.
Representative procedure for the preparation of hydroxy
acid: valine a-methylene-b-hydroxy lactam acid 12a.
To a
solution of the hydroxy lactam 11a (0.66 mmol) dissolved in
5 mL CH3OH was added the aqueous LiOH (0.1 N, 5 mL) and
stirred for 48 h at RT. It was then acidified with 2 M HCl and
concentrated in vacuo. The crude product obtained was purified
by column chromatography (MeOH/CHCl3, 1:9) to obtain acid
12a (79% yield). 1H-NMR (500 MHz, DMSO-d6): d 8.44 (s,
1H), 5.81 (s, 1H), 5.47 (s, 1H), 4.49 (s, 1H), 2.29 (m, 1H), 0.87
(d, J = 6.5 Hz, 3H), 0.70 (d, J = 6.5 Hz, 3H); 13C-NMR
(125 MHz, DMSO-d6): d 172.9, 168.8, 145.5, 117.7, 72.9,
72.5, 33.5, 18.9, 16.7; ESI–MS: m/z 222 (M + Na+) for
C9H13NO4Na.
CONCLUSIONS
In conclusion, we have developed a concise protocol for
the synthesis of a-methylene-b-hydroxy-g-carboxy-g-lac-
tams via iminoester alkylation followed by allylic hydroxyl-
ation with selenium dioxide. All the compounds synthesized
were evaluated for their cytotoxity against multiple myeloma
cancer cell lines. Owing to the importance of pyroglutamates
in organic and medicinal chemistry, we anticipate this meth-
odology to be useful for the synthetic community.
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EXPERIMENTAL
General methods. All operations were carried out under an
inert nitrogen atmosphere. THF, CH2Cl2, toluene, and ethyl
acetate were purchased as anhydrous solvents and used as such.
The 1H- and 13C-NMR spectra were plotted on a Varian
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methylene-b-hydroxy-g-carboxy-g-lactam
11a.
SeO2
(18.53 mmol) was added to the valine lactam 10a (26.46 mmol)
dissolved in glacial acetic acid (80 mL) and heated to reflux for
1 h. The reaction mixture was filtered, added with water
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J = 12 Hz, 1H), 5.16 (d, J = 12 Hz, 1H), 4.69 (d, J = 9.5 Hz, 1H),
4.49 (d, J = 9 Hz, 1H), 2.40 (m, 1H), 0.78 (d, J = 7 Hz, 3H), 0.66
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Threonine a-methylene-b-hydroxy lactam 17.
Procedure
1
was as described above for 11a. H-NMR (500 MHz, CDCl3): d
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet