Synthesis and Biological Activity of Hydroxamic Acid-Derived Vasopeptidase Inhibitor Analogues

Article abstract of DOI:10.1021/ol025896m

(Equation Presented) Syntheses of novel hydroxamic acid-derived azepinones containing pendant mercaptoacyl groups or formyl hydroxamates are described. These new analogues of therapeutically important ACE and NEP inhibitors include unprecedented changes a

Full text of DOI:10.1021/ol025896m

ORGANIC
LETTERS
2002
Vol. 4, No. 12
2047-2050
Synthesis and Biological Activity of
Hydroxamic Acid-Derived Vasopeptidase
Inhibitor Analogues
Andrew J. Walz and Marvin J. Miller*
Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall,
UniVersity of Notre Dame, Notre Dame, Indiana 46556
marVin.j.miller.2@nd.edu
Received March 19, 2002
ABSTRACT
Syntheses of novel hydroxamic acid-derived azepinones containing pendant mercaptoacyl groups or formyl hydroxamates are described.
These new analogues of therapeutically important ACE and NEP inhibitors include unprecedented changes at the previously assumed essential
acid component.
Inhibition of the zinc-containing metalloproteases, angio-
tensin-converting enzyme (ACE) and neutral endopeptidase
(NEP), is of considerable interest for the development of
therapeutically useful antihypertensive agents.1-10 ACE,
through the formation of angiotensin II (AII) from angio-
tensin I (AI) and the release of aldosterone, increases blood
pressure. Atrial natriuretic peptide (ANP) is a vasodilator
and a substrate for the protease NEP. Thus, inhibition of
both ACE and NEP by vasopeptidase inhibitors serves to
synergistically decrease the production of the vasoconstrictor
AII and increase the lifetime of the vasodilator ANP.
Recently, scientists at Bristol-Myers Squibb (BMS) published
a report on the synthesis and biological activity of substituted
azepinones in mercaptoacyl dipeptides (Figure 1).9 These
compounds, with geminal and spirocyclic substitutions at R
and R′, demonstrated good to excellent ACE and NEP
inhibition. The proposed structural properties of these
molecules necessary for activity are also shown in Figure 1.
Another paper reported that replacement of the thio (mer-
capto) group with an N-formyl hydroxylamine provided
additional potent peptide-based vasopeptidase inhibitors.10
(1) Delaney, N. G.; Barrish, J. C.; Neubeck, R.; Natarajan, S.; Cohen,
M.; Rovnyak, G. C.; Huber, G.; Murugesan, N.; Girotra, R.; Sieber-
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Waldron, T.; Petrillo, E. W. Bioorg. Med. Chem. Lett. 1994, 4, 1783-
1788.
(2) Robl, J. A.; Simpkins, L. M.; Stevenson, J.; Sun, C.-Q.; Murugesan,
N.; Barrish, J. C.; Asaad, M. M.; Bird, J. E.; Schaeffer, T. R.; Trippodo, N.
C.; Petrillo, E. W.; Karanewsky, D. S. Bioorg. Med. Chem. Lett. 1994, 4,
1789-1794.
(3) Robl, J. A.; Simpkins, L. M.; Sulsky, R.; Sieber-McMaster, E.;
Stevenson, J.; Kelly, Y. F.; Sun, C.-Q.; Misra, R. N.; Ryono, D. E.; Asaad,
M. M.; Bird, J. E.; Trippodo, N. C.; Karanewsky, D. S. Bioorg. Med. Chem.
Lett. 1994, 4, 1795-1800.
(4) Robl, J. A.; Sun, C.-Q.; Simpkins, L. M.; Ryono, D. E.; Barrish, J.
C.; Karanewsky, D. S.; Asaad, M. M.; Schaeffer, T. R.; Trippodo, N. C.
Bioorg. Med. Chem. Lett. 1994, 4, 2055-2060.
(5) Das, J.; Robl, J. A.; Reid, J. A.; Sun, C.-Q.; Misra, R. N.; Brown, B.
R.; Ryono, D. E.; Asaad, M. M.; Bird, J. E.; Trippodo, N. C.; Petrillo, E.
W.; Karanewsky, D. S. Bioorg. Med. Chem. Lett. 1994, 4, 2193-2198.
(6) Slusarchyk, W. A.; Robl, J. A.; Taunk, P. C.; Asaad, M. M.; Bird, J.
E.; Dimarco, J.; Pan, Y. Bioorg. Med. Chem. Lett. 1995, 5, 753-758.
(7) Robl, J. A.; Cimarusti, M. P.; Simpkins, L. M.; Brown, B.; Ryono,
D. E.; Bird, J. E.; Asaad, M. M.; Schaeffer, T. R.; Trippodo, N. C. J. Med.
Chem. 1996, 39, 494-502.
(8) Robl, J. A.; Sun, C.-Q.; Stevenson, J.; Ryono, D. E.; Simpkins, L.
M.; Cimarusti, M. P.; Dejneka, T.; Slusarchyk, W. A.; Chao, S.; Stratton,
L.; Misra, R. N.; Bednarz, M. S.; Asaad, M. M.; Cheung, H. S.; Abboa-
Offei, B. E.; Smith, P. L.; Mathers, P. D.; Fox, M.; Schaeffer, T. R.;
Seymour, A. A.; Trippodo, N. C. J. Med. Chem. 1997, 40, 1570-1577.
(9) Robl, J. A.; Sulsky, R. A.; Sieber-McMaster, E.; Ryono, D. E.;
Cimarusti, M. P.; Simpkins, L. M.; Karanewsky, D. S.; Chao, S.; Asaad,
M. M.; Seymour, A. A.; Fox, M.; Smith, P. L.; Trippodo, N. C. J. Med.
Chem. 1999, 42, 305-311.
(10) Robl, J. A.; Simpkins, L. A.; Asaad, M. M. Bioorg. Med. Chem.
Lett. 2000, 10, 257-260.
10.1021/ol025896m CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/14/2002

Figure 1. Vasopeptidase inhibitors developed by Bristol-Myers
Squibb.
Despite considerable structure-activity relationship (SAR)
studies that defined apparently essential features shown in
the generalized structure in Figure 1, the literature reports
of these vasopeptidase inhibitors did not present any data
pertaining to structural variation of the ionizable acid
functionality. During our studies related to the synthesis and
biological activity of mycobactins (mycobacterial iron-
sequestering agents) and analogues,11 we noted the similarity
of the terminal cyclolysine (cobactin component) to the
synthetic BMS vasopeptidase inhibitors. This resemblance
and earlier work in our labs related to the syntheses of novel
N-hydroxy-based â-lactam antibiotics (oxamazins)12 as well
as the related development of monobactams (i.e., aztreonam)
and monosulfactams at BMS13 suggested that incorporation
of the iron-binding, lysine-derived cyclic hydroxamic acid
(cobactin) substructure of the mycobactins into the vasopep-
tidase inhibitor framework might allow preparation of
analogues with novel and variable ionizable groups (Figure
2). Analogues 1 and 2 would provide acidic functionalities
with altered pKa values relative to the inhibitors shown in
Figure 1. The cobactin-related component of analogue 1 also
contains a metal-binding, O-unsubstituted cyclic hydroxamic
acid that could provide interesting additional metal coordina-
tion favorable toward inhibition of the target metallopro-
teases. Furthermore, target 3 would be derived from a
recently synthesized cobactin analog,11 while compound 4
incorporates the N-terminal formyl hydroxamic acid devel-
oped by workers at BMS and shown in Figure 1.
Figure 2. Mycobactins, monocyclic â-lactams, and proposed
hydroxamic acid-containing analogues of vasopeptidase inhibitors.
DMAP, and DMAP(HCl) followed by protection of the
hydroxamic acid with TBDPSCl.14 Subsequently, Kropp
reported a direct, oxone-mediated, microwave-assisted amine
to hydroxylamine oxidation, including the oxidation of the
ꢀ-amino group of R-protected lysines.15
An alternative procedure that generates stable, storable
nitrone intermediates is shown in Scheme 1.
Scheme 1a
a Reagents and conditions: (a) (1) PhCHO, KOH, MS, MeOH,
rt, 16 h; (2) m-CPBA, MeOH, from 0 °C to room temperature, 4
h; (3) TFA, CH2Cl2, rt, 1 h; (4) PhCHO, EtOAc, from 0 °C to
room temperature, 66% overall. (b) (1) NH2OH(HCl), MeOH, 65
°C, 20 min; (2) EDC, HOAt, NaHCO3, CH3CN, DMF, rt, 48 h,
(55%). (c) Ethyl bromoacetate, K2CO3, THF, H2O, rt, 24 h, 97%.
A previously reported synthesis of the desired L-lysine-
derived cyclic hydroxamic acid (N-hydroxy-3-amino-azepin-
2-one, the iron binding portion of cobactin) from these
laboratories involved the oxidation of Z-L-lysine with di-
methyldioxirane (DMD) in acetone followed by hydroxy-
lamine formation. Cyclization of the intermediate hydroxy-
lamine was accomplished with a 5-fold excess of DCC,
Thus, using chemistry recently employed in our group,16
R-Cbz-L-lysine was treated with benzaldehyde under basic
conditions to provide the corresponding ꢀ-imine that was
immediately used in subsequent reactions. Oxidation of the
imine, with dry m-CPBA was followed by TFA-promoted
isomerization of the intermediate oxaziridine to nitrone 5.
(11) Vergne, A. F.; Walz, A. J.; Miller, M. J. Nat. Prod. Rep. 2000, 17,
99-116.
(12) (a) Woulfe, S. R.; Miller, M. J. Tetrahedron Lett. 1984, 25, 3293-
3296. (b) Woulfe, S. R.; Miller, M. J. J. Med. Chem. 1985, 28, 1447-
1453. (c) Boyd, D. B.; Eigenbrot, C.; Indelicato, J. M.; Miller, M. J.; Pasini,
C. E.; Woulfe, S. R. J. Med. Chem. 1987, 30, 528 and references therein.
(d) Miller, M. J. Acc. Chem. Res. 1986, 19, 49-56.
(13) (a) Slusarchyk, W. A.; Dejneka, T.; Gordon, E. M.; Weaver, E. R.;
Koster, W. H. Heterocycles 1984, 21, 191. (b) Cimarusti, C. M.; Sykes, R.
B. Med. Res. ReV. 1984, 4, 1.
(14) Hu, J.; Miller, M. J. J. Am. Chem. Soc. 1997, 119, 3462-3470.
(15) Fields, J. D.; Kropp, P. J. J. Org. Chem. 2000, 65, 5937-5941.
(16) Lin, Y.-M.; Miller, M. J. J. Org. Chem. 1999, 64, 7451-7458.
2048
Org. Lett., Vol. 4, No. 12, 2002

Experimentally, it was found that treatment of the reaction
mixture with additional benzaldehyde at this stage improved
the yield of the nitrone, perhaps by reacting with small
amounts of prematurely released hydroxylamine. Conversion
of the nitrone to the hydroxylamine by an exchange reaction
with hydroxylamine hydrochloride was followed by EDC/
HOAt-mediated cyclization to hydroxamic acid 6a. Similar
to our earlier syntheses of oxamazins,12 treatment of 6a with
ethyl bromoacetate provided 6b in excellent yield.
Scheme 3a
With the syntheses of the desired cyclolysine components
6a,b complete, attention was focused on the preparation and
incorporation of the N-terminal residues responsible for metal
binding. The synthesis of dicyclohexylamine salt of (S)-2-
(acetylthio)benzenepropanoic acid is shown in Scheme 2.
a Reagents and conditions: (a) 5% KHSO4, EtOAc. (b) H2, 10%
Pd-carbon, MeOH, room temperature and pressure. (c) EDC,
HOAt, DMF, rt, 3 h, 85% from 6a, 75% from 6b. (d) NaOH,
MeOH, from 0 °C to room temperature, 6-12h, 1 (87%), 2 (87%).
of alkylated hydroxamic acid 6b was also successfully
coupled to the (S)-2-(acetylthio)benzenepropanoic acid under
the same conditions to provide protected analogue 9b. Final
deprotection of 9a,b was accomplished with NaOH in
methanol under an inert atmosphere to generate analogues
1 and 2.
Scheme 2a
The synthesis of formylated hydroxylamine analogue 3
began with the formylation of the cobactin analogue 10 with
CDI/formic acid to give 11.21 After purification of 11,
hydrogenolytic removal of the benzyl protecting group gave
the free formyl hydroxamate 3 in excellent overall yield from
1022 (Scheme 4).
a Reagents and conditions: (a) KBr, 48% HBr, NaNO2, H2O,
from -13 °C to room temperature, 24 h, 85%. (b) (1) CsSAc, DMF,
rt, 24 h; (2) dicyclohexylamine, Et2O, rt, 48 h, 65%.
Scheme 4a
The literature reports the synthesis of this salt9 and the free
acid17 without experimental detail. Workers at BMS reported
the use of Kellogg’s methodology for the synthesis of
optically active thiols.18
The requisite acid 8 was synthesized by adaptation of the
separate procedures of Petit and Maimind for conversion of
R-amino acids to R-bromoacids with retention of configu-
ration.19 Treatment of D-phenylalanine with sodium nitrite
in a HBr/KBr solution provided bromo acid 7 in good yield.
Treatment of this acid with 1.5 equiv of the cesium salt of
thioacetic acid in DMF, followed by addition of dicyclo-
hexylamine after workup, provided acid salt 8 in moderate
yield.
The coupling of the carboxylates of amino acid derivatives
to amines in the presence of unprotected hydroxamic acids
has been previously demonstrated by our group.20 The same
conditions (EDC/HOAt/DMF) proved to be successful in the
coupling of the free amine of 6a with the liberated acid of
8 to give protected analogue 9a (Scheme 3). The free amine
a Reagents and conditions: (a) CDI, HCOOH, THF, 0 °C, 30
min, and then 10, 0 °C, 87%. (b) H2, 10% Pd-carbon, MeOH,
room temperature and pressure, 97%.
Preparation of the â-N-hydroxy amino acid-containing
analogue 4 involved the synthesis of known benzylated
hydroxamic acid 13 and â-lactam 14.23 The desired (S)-
enantiomer of acid 12 was obtained through precedented
alkylation of BOMCl with the enolate of a dihydrocinnamic
acid-derived chiral oxazolidinone amide.22,24,25 Subsequent
reaction of the known chiral acid 12 with BnONH2 in the
presence of EDC led to the benzylated hydroxamic acid 13
in excellent yield and was followed by cyclization to give
(21) Krishnamurty, H. G.; Prasad, J. S. Tetrahedron Lett. 1977, 3071-
3072.
(17) Spaltenstein, A.; Leban, J. J.; Furfine, E. S. Tetrahedron Lett. 1993,
34, 1457-1460.
(18) Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664-3671.
(19) (a) Petit, Y.; Larcheveque, M. Org. Synth. 1997, 75, 37-43. (b)
Maimind, V. I.; Ermolaev, K. M.; Shemyakin, M. M. Zh. Obshch. Chim.
1956, 26, 2313.
(22) Prepared from the corresponding serine-derived â-lactam by methods
similar to those previously described (ref 20).
(23) Jin, Y.; Kim, D. H. Synlett 1998, 1189-1190.
(24) Crawforth, J. M.; Rawlings, B. J. Tetrahedron Lett. 1995, 35, 6345-
6346.
(25) Nakano, M.; Atsuumi, S.; Koike, Y.; Tanaka, S.; Funabashi, H.;
Hashimoto, J.; Ohkubo, M.; Morishima, H. Chem. Lett. 1990, 505-508.
(20) Xu, Y.; Miller, M. J. J. Org. Chem. 1998, 63, 4314-4322.
Org. Lett., Vol. 4, No. 12, 2002
2049

Compounds 1-4 were subjected to primary ACE and NEP
inhibition assays at PanLabs of Taiwan (MDS Pharma
Services) with the results summarized in Table 1. These
Scheme 5a
Table 1. ACE/NEP Inhibitory Activity of Compounds 1-4
compound
enzyme
concentration
% inhibition
1
1
2
2
3
3
4
4
ACE (rabbit)
NEP (human)
ACE (rabbit)
NEP (human)
ACE (rabbit)
NEP (human)
ACE (rabbit)
NEP (human)
10 µM
10 µM
10 µM
10 µM
10 µM
10 µM
10 µM
10 µM
63%
93%
76%
95%
5%
12%
5%
99%
assays give the percent inhibition of the target enzymes upon
treatment with the potential inhibitors at a concentration of
10 µM. Under these assay conditions, compound 1 inhibited
63% of ACE and 93% of NEP. Compound 2 inhibited 76%
of ACE and 95% of NEP. Not surprisingly based on a
previous SAR study at BMS, 3 only poorly inhibited ACE
(5%) and NEP (12%), but 4, with the apparently key aryl
substituent, was remarkably selective with barely detectable
ACE inhibition (5%) but outstanding inhibition of NEP
(99%). Thus, it appears that attachment of the acid func-
tionality to the hydroxamic acid group of the cyclolysine
component of ACE/NEP inhibitors is not necessary for
activity.
a Reagents and conditions: (a) BnONH2(HCl), EDC, DMAP,
H2O, DMF, rt, 16 h, 80%. (b) PPh3, CCl4, TEA, CH3CN, rt, 16 h,
83%. (c) (1) 4 N HCl, 1,4-dioxane, 60 °C, 1 h; (2) deprotected 6a,
EDC, HOAt, DMAP, DMF, rt, 48 h, 33%. (d) CDI, HCOOH, THF,
0 °C, 30 min, and then 14, 0 °C, 16 h, 90%. (e) H2, 10% Pd-
carbon, MeOH, room temperature and pressure, 97%.
â-lactam 14 (Scheme 5).26 An initial attempt at the synthesis
of the penultimate protected hydroxylamine 15 utilized a
cyanide anion â-lactam activation for the formation of the
amide bond. Thus, treatment of the amine derived from
hydrogenolysis of 6a and â-lactam 14 with TMSCN/TBAF27
led to the desired coupled product 15 but as a mixture of
diastereomers that were chromatographically inseparable.
This loss of optical integrity was surprising and encouraged
us to first hydrolyze â-lactam 1422 and couple the resulting
free carboxylic acid to the amine of the cobactin component
again obtained by hydrogenolysis of 6a, a process we had
previously used successfully in the synthesis of cobactin
analogues.19 The reaction proceeded in moderate overall yield
to provide 15 with no evidence for the presence of any
Four new vasopeptidase inhibitors 1-4 were synthesized
and found to have variable but promising and/or selective
ACE and NEP inhibition. The therapeutic importance of
ACE/NEP inhibition indicates that these new compounds
may have significant utility.
Acknowledgment. We gratefully acknowledge support
for the mycobactin research from the NIH that led to the
consideration of the syntheses of the novel vasopeptidase
inhibitors described. We acknowledge Don Schifferl and Dr.
Jaroslav Zajicek for NMR assistance and Dr. Bill Boggess
and Nonka Sevova for mass spectrometry. Special thanks
are extended to Maureen Metcalf for her assistance with this
manuscript.
1
undesired diastereomer in the H and 13C NMR spectra.
Formylation of 15 provided protected analogue 16 unevent-
fully. Hydrogenolytic removal of the benzyl protecting group
gave analogue 4 in good yield.
Supporting Information Available: Experimental pro-
cedures and characterization data for products 1, 2, 4, 5, 6a,
6b, 7, 8, 9a, 9b, 11, and 13-16. This material is available
free of charge via the Internet at http://pubs.acs.org.
(26) Miller, M. J.; Mattingly, P. G.; Morrison, M. A.; Kerwin, J. F. J.
Am. Chem. Soc. 1980, 102, 7026-7032.
(27) Soli, E. D.; Manoso, A. S.; Patterson, M. C.; DeShong, P.; Favor,
D. A.; Hirschmann, R.; Smith, A. B., III. J. Org. Chem. 1999, 64, 3171-
3177.
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Org. Lett., Vol. 4, No. 12, 2002