Amino acid derived enamides: Synthesis and aminopeptidase activity

Article abstract of DOI:10.1021/ol7025729

Recently developed copper-catalyzed coupling methodology has been applied to the synthesis of amino acid derived enamides. Bond formation proved to be strongly influenced by protection strategy and vinyl iodide substitution while tolerant of limited side chain functionality. Assessment of aminopeptidase activity revealed a preference for (E)-1,2-disubstituted constructs.

Full text of DOI:10.1021/ol7025729

ORGANIC
LETTERS
2007
Vol. 9, No. 26
5617-5620
Amino Acid Derived Enamides:
Synthesis and Aminopeptidase Activity
Richard R. Cesati III,* Greg Dwyer, Reinaldo C. Jones, Megan P. Hayes,
Padmaja Yalamanchili, and David S. Casebier
Pharmaceutical Research and DeVelopment, Bristol-Myers Squibb Medical Imaging,
North Billerica, Massachusetts 01862
richard.cesati@bms.com
Received October 22, 2007
ABSTRACT
Recently developed copper-catalyzed coupling methodology has been applied to the synthesis of amino acid derived enamides. Bond formation
proved to be strongly influenced by protection strategy and vinyl iodide substitution while tolerant of limited side chain functionality. Assessment
of aminopeptidase activity revealed a preference for (E)-1,2-disubstituted constructs.
Interest in the enamide functionality has increased exponen-
tially in recent years due primarily to the expanding number
of biologically relevant molecules in which it is found.1 The
chemo- and stereoselectivity challenges associated with
enamide synthesis in these complex molecular frameworks
has underscored the limitations of conventional methods,
particularly the acylation of imines and ketoximes,2 direct
condensation of aldehydes and amides,3 Curtius rearrange-
ment of R,â-unsaturated acyl azides,4 dehydration of hemi-
aminals,5 and a variety of olefination processes.6 In response,
the synthetic community has rapidly developed a variety of
mild, metal-mediated C-N bond-forming processes,7 includ-
ing Ag-8 and Ru-catalyzed hydroamidation,9 Pd-catalyzed
oxidative amidation of olefins,10 and direct Pd-11 and Cu-
catalyzed12 coupling of amides with vinyl (pseudo) halides.13
Our own interest evolved from a desire to integrate an
alternative pro-drug system within a substrate peptide
designed to target matrix metalloproteinases (MMPs).14 In
(6) (a) Peterson: (i) Hudrlick, P. F.; Hudrlick, A. M.; Rona, R. J.; Misra,
R. N.; Withers, G. P. J. Am. Chem. Soc. 1977, 99, 1993. (ii) Cuevas, J.-C.;
Patil, P.; Snieckus, V. Tetrahedron Lett. 1989, 30, 5841. (iii) Palomo, C.;
Aizpurua, J. M.; Legido, M.; Picard, J. P.; Dunogues, J.; Constantieux, T.
Tetrahedron Lett. 1992, 33, 3903. (iv) Furstner, A.; Brehm, C.; Cancho-
Grande, Y. Org. Lett. 2001, 3, 3955. (b) Horner-Wittig: Couture, A.;
Deniau, E.; Grandclaudon, P. Tetrahedron Lett. 1993, 34, 1479. (c)
Wadsworth-Emmons: (i) Paterson, I.; Cowden, C.; Watson, C. Synlett
1996, 209. (ii) Villa, M. V. J.; Targett, S. M.; Barnes, J. C.; Whittingham,
W. G.; Marquez, R. Org. Lett. 2007, 9, 1631.
(7) For a recent review, see; Dehli, J. R.; Legros, J.; Bolm, C. Chem.
Commun. (Cambridge, UK) 2005, 973.
(8) Sun, J.; Kozmin, S. A. Angew. Chem., Int. Ed. 2006, 45, 4991.
(9) (a) Kondo, T.; Tanaka, A.; Kotachi, S.; Watanabe, Y. J. Chem. Soc.,
Chem. Commun. 1995, 413. (b) Goossen, L. J.; Rauhaus, J. E.; Deng, G.
Angew. Chem., Int. Ed. 2005, 44, 4042.
(1) For selected total syntheses of the salicylate enamide macrolides,
see: (a) Furstner, A.; Dierkes, T.; Thiel, O. R.; Blanda, G. Chem. Eur. J.
2001, 7, 5286. (b) Nicolaou, K. C.; Kim, D. W.; Baati, R. Angew. Chem.,
Int. Ed. 2002, 41, 3701. (c) Wang, X.; Porco, J. A., Jr. J. Am. Chem. Soc.
2003, 125, 6040. For a recent review, see: Yet, L. Chem. ReV. 2003, 103,
4283.
(2) Recent examples: (a) Sun, C.; Weinreb, S. M. Synthesis 2006, 3585.
(b) Savarin, C. G.; Boice, G. N.; Murry, J. A.; Corley, E.; DiMichele, L.;
Hughes, D. Org. Lett. 2006, 8, 3903. For a recent application in the synthesis
of salicylihalamide A, see: Smith, A. B., III; Zheng, J. Tetrahedron 2002,
58, 6455.
(3) (a) Zeeza, C. A.; Smith, M. B. Synth. Commun. 1987, 17, 729. (b)
Kiefel, M. J.; Maddock, J.; Pattenden, G. Tetrahedron Lett. 1992, 33, 3227.
(4) (a) Brettle, R.; Mosedale, A. J. J. Chem. Soc., Perkin. Trans. 1 1988,
2, 185. (b) Snider, B. B.; Song, F. Org. Lett. 2000, 2, 407. (c) Stefanuti, I.;
Smith, S. A.; Taylor, R. J. K. Tetrahedron Lett. 2000, 41, 3735. (d)
Kuramochi, K.; Watanabe, H.; Kitahara, T. Synlett 2000, 397. For a recent
application in the total synthesis of salicylihalamide A, see: Wu, Y.; Liao,
X.; Wang, R.; Xie, X.-S.; De Brabander, J. K. J. Am. Chem. Soc. 2002,
124, 3245.
(10) (a) Hosokawa, T.; Takano, M.; Kuroki, Y.; Murahashi, S.-Y.
Tetrahedron Lett. 1992, 33, 6643. (b) Brice, J. L.; Meerdink, J. E.; Stahl,
S. S. Org. Lett. 2004, 6, 1845. (c) Hansen, A. L.; Skrydstrup, T. J. Org.
Chem. 2005, 70, 5997. (a) Lee, J. M.; Ahn, D.-S.; Jung, D. Y.; Lee, J.; Do,
Y.; Kim, S. K.; Chang, S. J. Am. Chem. Soc. 2006, 128, 12954.
(11) (a) Kozawa, Y.; Mori, M. Tetrahedron Lett. 2002, 43, 111. (b)
Wallace, D. J.; Klauber, D. J.; Chen, C.-y.; Volante, R. P. Org. Lett. 2003,
5, 4749. (c) Willis, M. C.; Brace, G. N.; Holmes, I. P. Synthesis 2005,
3229. (d) Klapars, A.; Campos, K. R.; Chen, C.-y.; Volante, R. P. Org.
Lett. 2005, 7, 1185. (e) Barluenga, J.; Moriel, P.; Aznar, F.; Valdes, C.
Org. Lett. 2007, 9, 275.
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10.1021/ol7025729 CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/28/2007

principle, the enamide function was expected to append the
localization sequence in such a manner that the combination
of endo- and aminopeptidase (APM) action would reveal a
carbonyl fragment on the drug substance (Figure 1). In this
completely regioselective formation of the expected enamide
derivative of Boc-Leu-NH2 (entry 1, Table 1). Reaction of
Table 1. Effect of Protection Strategy on Coupling Efficiency
Figure 1. Enamide-based pro-drug strategy.
regard, a principal requirement of the design was compat-
ibility with essential enzyme systems such that maximum
turnover in the target tissue was achieved. We therefore
required a fast and flexible synthesis of amino acid derived
enamides15 in order to evaluate factors influencing the
enzyme kinetics. As such, we decided to investigate the
utility of modern Cu-catalyzed amidation methodology16 and
report herein our results on the scope and limitations in the
C-N bond-forming process and include an evaluation of
proteolysis activity of these constructs with aminopeptidase
M.
Our study commenced with an evaluation of the impact
of protection strategy on the efficiency and chemoselectivity
of C-N bond formation. Using the standard conditions
reported by Buchwald17 and the model vinyl iodide, (E)-4-
iodooct-4-ene, we were pleased to observe the efficient and
(12) (a) Ogawa, T.; Kiji, T.; Hayami, K.; Suzuki, H. Chem. Lett. 1991,
1443. (b) Shen, R.; Porco, J. A., Jr. Org. Lett. 2000, 2, 1333. (c) Jiang, L.;
Job, G. E.; Klapars, A.; Buchwald, S. L. Org. Lett. 2003, 5, 3667. (d) Lam,
P. Y. S.; Vincent, G.; Bonne, D.; Clark, C. G. Tetrahedron Lett. 2003, 44,
4927. (e) Coleman, R. S.; Liu, P.-H. Org. Lett. 2004, 6, 577. (f) Han, C.;
Shen, R.; Su, S.; Porco, J. A., Jr. Org. Lett. 2004, 6, 27. (g) Pan, X.; Cai,
Q.; Ma, D. Org. Lett. 2004, 6, 1809. (h) Hu, T.; Li, C. Org. Lett. 2005, 7,
2035. (i) Lu, H.; Li, C. Org. Lett. 2006, 8, 5365. (j) Martin, R.; Rivero, M.
R.; Buchwald, S. L. Angew. Chem., Int. Ed. 2006, 45, 7079. (k) Yuan, X.;
Xu, X.; Zhou, X.; Yuan, J.; Mai, L.; Li, Y. J. Org. Chem. 2007, 72,
1510.
a Conditions: 5 mol % of CuI, 10 mol % of N,N′-dimethylethylenedi-
amine, 2 equiv of amide, 1.5 equiv of Cs2CO3, 0.5 M THF, 70 °C, 16 h.
Reaction times not optimized. Average of at least two runs. b Isolated yield
after silica gel chromatography. c Conversion determined by HPLC analysis
of the crude reaction mixture. d 1 equiv of amide. e Isolated 26% of the
derived hydantoin. f 0.25 M. g Determined by chiral GLC analysis (see the
Supporting Information for details).
(13) For closely related methods which involve the preparation of
ynamides and their subsequent conversion to enamides, see: (a) Witulski,
B.; Buschmann, N.; Bergstrasser, U. Tetrahedron 2000, 56, 8473. (b)
Tanaka, R.; Hirano, S.; Urabe, H.; Sato, F. Org. Lett. 2003, 5, 67. (c) Dunetz,
J. R.; Danheiser, R. L. Org. Lett. 2003, 5, 4011. (d) Zhang, X.; Zhang, Y.;
Huang, J.; Hsung, R. P.; Kurtz, K. C. M.; Oppenheimer, J.; Petersen, M.
E.; Sagamanova, I. K.; Shen, L.; Tracey, M. R. J. Org. Chem. 2006, 71,
4170 and references therein. For catalytic olefin isomerization methodology,
see: (e) Trost, B. M.; S. J.-P. Angew. Chem., Int. Ed. 2001, 40, 1468. (f)
Sergeyev, S. A.; Hesse, M. HelV. Chim. Acta 2003, 86, 750. For a unique
rearrangement of R-silyl amides, see: (g) Lin, S.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2002, 41, 512. (h) Zhang, X.; Houck, K. N.; Lin, S.;
Danishefsky, S. J. J. Am. Chem. Soc. 2003, 125, 5111.
(14) Harris, T. D.; Yalamanchili, P.; Cesati, R. R., III. Compounds
Containing Matrix Metalloproteinase Substrates and Methods of their use.
EP1691845 A1, August 23, 2006.
(15) Several papers addressing enamide formation in the context of amino
amides have recently appeared: (a) Yuen, J.; Fang, Y.-Q.; Lautens, M.
Org. Let. 2006, 8, 653. (b) He, G.; Wang, J.; Ma, D. Org. Lett. 2007, 9,
1367. (c) Toumi, M.; Couty, F.; Evano, G. Angew. Chem., Int. Ed. 2007,
46, 572. (d) Toumi, M.; Couty, F.; Evano, G. J. Org. Chem. 2007, 72,
9003.
the analogous Cbz-protected material, however, resulted in
formation of a 2:1 mixture of the expected product and its
derived hydantoin (entry 2).18 Importantly, we obserVed that
protection of the R-nitrogen was not required, as the free
amino enamide could be isolated as the exclusiVe product,
in accordance with conversion (entries 3 and 5). Equally
impressive was reaction with the dipeptide fragment H-Leu-
Ala-NH2, which revealed complete chemoselectiviy of C-N
bond formation (entry 6). In contrast, very low conversion
was noted with the corresponding (Z)-4-iodooct-4-ene (entry
4).19
Our next concern was to address the influence of vinyl
iodide substitution and stereochemistry on coupling ef-
(17) (a) Reference 12c. (b) For purposes of comparison, we also evaluated
the methods of Porco12b (98% conversion and 70% yield with 20 mol % of
CuTC) and Ma12g (88% conversion and 73% yield with 10 mol % of CuI
and 20 mol % N,N′-dimethylglycine; 37% conversion and 28% yield with
5 mol % of CuI and 10 mol % of N,N′-dimethylglycine) in the reaction of
H-Leu-NH2 with (E)-4-iodooct-4-ene (e.g., entry 3, Table 1).
(18) Control experiments indicate hydantoin formation occurs after the
cross-coupling event.
(16) Given the requirement of activating functionality in current Pd-
catalyzed processes, we opted for the Cu-catalyzed amidation methods such
that our drug substance would be released with minimal trace of the enamide
linkage. It should be noted, however, that Willis and co-workers have
recently reported the Pd-catalyzed amidation of simple cyclic pseudohalides
(ref 11c); extension to acyclic substrates has yet to be reported.
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Org. Lett., Vol. 9, No. 26, 2007

ficiency. Using four model vinyl iodides, we noted a dramatic
enhancement in reaction rate with the (E)-1,2-disubstituted
derivative (entry 1, Table 2). As comparison of the data in
Table 3. Amino Acid Substitution and Hydrolysis Rate
entry
side chaina
yield (convn) (%)
rate (µmol/min‚U)e
1
2
3
4
5
6
7
8
9
Gly (12)
Ala (13)
Val (14)
Leu (15)
Ile (16)
Met (17)
Glnb (18)
Phe (19)
Tyrb (20)
Trp (21)
32 (53)
82 (98)
58 (80)
87 (98)
63 (91)
77 (98)
31 (95)c
87 (98)
30 (78)
50 (98)d
<0.050
1.83 (0.950)
<0.050
0.411 (0.401)
<0.050
0.846 (0.711)
0.245 (0.252)
0.416 (0.350)
0.396 (0.353)
<0.05
Table 2. Affect of Vinyl Iodide Substitution and
Stereochemistry
10
a Conditions: 5 mol % of CuI, 10 mol % of N,N′-dimethylethylenedi-
amine, 1 equiv of 8, 2 equiv of amino amide, 1.5 equiv of Cs2CO3, THF,
70 °C, 16 h. Average of at least two runs. b 0.5 M DMF, 50 °C, 1 h. c Also
isolated 7% γ-lactam- and 7% derived dimeric-coupled products. d Also
isolated 10% indole-coupled and 20% dimeric products. e See Table 4 for
assay details.
solution. The dramatic rate acceleration observed with this
solvent system resulted in an overall reduction in coupling
efficiency20 (e.g., entries 7 and 9). Noteworthy among these
examples was the chemoselectivity in the coupling of H-Gln-
NH2, where a 20:1 preference for the main chain amide was
observed; perhaps reflecting a directing influence of the free
R-nitrogen. With regard to other competing side chain fun-
ctionality, we observed significant reaction at the indole
nitrogen (entry 10) as well as a distinct preference for coup-
ling at the phenol of H-Tyr-NH2 (entry 9). Other amino
amides which proved problematic in this regard include
H-Arg-NH2, H-His-NH2, and H-Glu-NH2 (data not shown).
The measured enantiomeric ratio for several enamide
contructs revealed little to no loss of stereochemical intergrity
during the coupling reaction (cf. Tables 1 and 2). That said,
the derived hydantoin product of 2 was shown to be nearly
racemic. We did, however, note an erosion of enantiomeric
ratio at elevated concentration, where coupling of H-Leu-
Ala-NH2 required more dilute conditions (0.25 M), as
reaction at 0.50 M produced a 4:1 mixture of diastereomeric
products at the alanine R-carbon. It is interesting to note that
no stereomutation was ever observed at the leucine residue
of this dipeptide.
a Conditions: 5 mol % of CuI, 10 mol % of N,N′-dimethylethylenedi-
amine, 2 equiv of H-Leu-NH2, 1.5 equiv of Cs2CO3, THF, 70 °C, 16 h.
Average of at least two runs. b Mass balance consists of a 1:1 mixture of
imidazolidinone diastereomers resulting from cyclization of the parent
enamide. c Determined by chiral GLC analysis (see the Supporting Informa-
tion for details).
Table 2 reveals, alternative substitution patterns on the vinyl
iodide typically resulted in reduced conversion; coupling
efficiency did not, however, markedly decrease. During the
reaction with 2,2-disubstitutued vinyl iodide 10, we observed
formation of the derived imidazolidinone in addition to the
expected enamide product (entry 3, Table 2), a side reaction
unique to this vinyl iodide substitution pattern.
In the design of our pro-drug linker, the C-terminal amino
acid serves as part of the peptide substrate as well as the
release mechanism and must therefore be tolerated by both
the endo- and aminopeptidases. In this context, we decided
to evaluate several amino acid derivatives of our (E)-1,2-
disubstituted vinyl iodide 8 in order to identify a series of
residues which maintain high aminopeptidase activity and
provide diversity in selection of the MMP substrate. Hence,
based on known substrate specificity of the target MMPs, a
series of amino amides were selected and coupled, without
protecting groups, to 8 using the standard Buchwald condi-
tions described above.
With a series of amino acid derived enamides in hand,
we turned our attention to the determination of proteolysis
rates with aminopeptidase M (Tables 3 and 4). In the
experiment, substrates were incubated with two unique
enzyme concentrations (25 min at 37 °C) and the change in
peak area monitored by HPLC. Rates were calculated
according to following equation
As the results in Table 3 indicate, a variety of side chain
functionality was well tolerated by the catalyst system.
Overall reaction rates and yields were highest with lipophilic
side chains, including methionine (entries 2-6), although
â-substitution resulted in a moderate reduction of isolated
yield (e.g., entries 3 and 5). In certain cases limited solubility
of the amino amide required the reaction to be run in DMF
k ) {(% hydrolyzed/100)*[S]}/[E]*[time]
where
(19) (Z)-4-Iodooct-4-ene was prepared from 4-octyne according to a
popular published method: Kamiya, N.; Chikami, Y.; Ishii, Y. Synlett 1990,
675. We noted, however, the stereochemistry of HI addition was incorrectly
assigned in this report; see the Supporting Information for complete details.
(20) For comparison, the product of 8 and H-Phe-NH2 was isolated in
only 54% yield using these conditions.
S ) test substrate concentration in µmol
E ) aminopeptidase concentration in U/mL
k ) µmol substrate hydrolyzed/minute/U
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Org. Lett., Vol. 9, No. 26, 2007

For purposes of comparison, we first determined the
hydrolysis rate of the N-terminal residue on dipeptide
enamide 7 (entry 1, Table 4).21 Subsequent evaluation of the
hydrolysis (cf. entries 2-4 and 8). Extension to the disub-
stituted analogues 15 and 22-24 revealed (E)-1,2-susbtitu-
tion to be well tolerated, while a dramatic reduction in rate
was observed for the (Z)-1,2- and 2,2-disubstituted deriva-
tives; reflecting changes in rotamer population as a conse-
quence of developing allylic strain in these systems (e.g.,
entries 5-7). A trend that may limit the scope of carbonyl
functionality on our drug substance.
Table 4. Hydrolysis of N-Terminal Residue by APM
Subsequent study of the relationship between side-chain
functionality and aminopeptidase activity revealed that
aliphatic side chains which do not contain a â-substituent
are in general well-tolerated by the enzyme system (e.g.,
entries 1-6, Table 3). Also worthy of note was the dramatic
enhancement in hydrolysis rate upon substitution of Ala for
Gly, where hydrolysis of the alanine enamide occurs with
nearly the same facility as the peptide bond in 7 (entries
1-2); the facility of this process augurs well for our pro-
drug application. Interestingly, in the series of aromatic
amino enamides, both Phe and Tyr were well tolerated while
very little hydrolysis was noted with the Trp derivative.
Taken together, these results expose several options for
application as the C-terminal residue in our MMP substrate
peptides.
In conclusion, we have described an extension of the Cu-
catalyzed coupling of amides and vinyl iodides to include
amino acid derived products. The expanded understanding
of protecting and side chain functional group tolerance of
the method, revealed herein, should find widespread ap-
plication in a variety of synthetic endeavors. Further exten-
sion of the utility of an enamide-derived pro-drug strategy
is a subject of continued investigation.
Acknowledgment. We are grateful to Wendy Mello, John
Pietryka, Valerie Letsou, and Peta Ryan of the BMS-MI
Pharmacy and Anaytical Development group for NMR and
MS assistance, respectively.
a The APN assay was performed at three enzyme concentrations: 0, 6.5
× 10-4 and 15.0 × 10-3 U. The rate data are given at the 6.5 × 10-4
U
concentration. The value obtained at 15.0 × 10-3 U is listed in parentheses.
Enzyme was denatured with MeCN due to the acid-sensitivity of the
substrate. Average of two runs. R ) (CH2)4(2-furan).
Supporting Information Available: Full experimental
procedures and characterization data for all new compounds
described in this study. This material is available free of
charge via the Internet at http://pubs.acs.org.
mono amino acid enamides derived from trisubstituted vinyl
iodides revealed significantly reduced hydrolysis rates; in
fact, only the alanine derivative 5 displayed measurable
(21) For comparison to literature values, see: Niven, G. W.; Holder, S.
A.; Strøman, P. Appl. Microbiol. Biotechnol. 1995, 44, 100.
OL7025729
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