Novel peptidyl phosphorus derivatives as inhibitors of human calpain I

Article abstract of DOI:10.1021/jm980325e

Dipeptidyl phosphorus compounds were synthesized as potential bioisosteric mimics of peptide α-ketoesters and α-ketoacids. α- Ketophosphonate Cbz-Leu-Leu-P(O)(OCH₃)₂ (1b), containing an α-ketoester bioisostere, inhibits human calpain

Full text of DOI:10.1021/jm980325e

3912
J . Med. Chem. 1998, 41, 3912-3916
Novel P ep tid yl P h osp h or u s Der iva tives a s In h ibitor s of Hu m a n Ca lp a in I
Ming Tao,* Ron Bihovsky, Gregory J . Wells, and J ohn P. Mallamo
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245
Received May 26, 1998
Dipeptidyl phosphorus compounds were synthesized as potential bioisosteric mimics of peptide
R-ketoesters and R-ketoacids. R-Ketophosphonate Cbz-Leu-Leu-P(O)(OCH3)2 (1b), containing
an R-ketoester bioisostere, inhibits human calpain I with an IC50 ) 0.43 µM. The potency of
1b compares very favorably with that of R-ketoester Cbz-Leu-Leu-CO2Et (IC50 ) 0.60 µM).
Monomethyl ketophosphonate Cbz-Leu-Leu-P(O)(OH)(OCH3) (1a , IC50 ) 5.2 µM), an R-ketoacid
mimic, is less potent. Dibutyl and dibenzyl R-ketophosphonates 1c,e,f are much less potent
calpain inhibitors than dimethyl R-ketophosphonate 1b. R-Ketophosphinate 1g (IC50 ) 0.37
µM) and R-ketophosphine oxide 1h (IC50 ) 0.35 µM) are also potent calpain inhibitors.
In tr od u ction
R-hydroxy dimethyl phosphonate 6b was monodemethy-
lated with lithium bromide in acetonitrile to monom-
ethyl phosphonate 7.5 Dess-Martin oxidation of 7
afforded monomethyl R-ketophosphonate 1a .
Alternatively, as shown in Scheme 2, the dipeptide
aldehyde Cbz-Val-Phe-H was directly converted to pep-
tidyl R-hydroxy phosphonates 6d -f in good yield (62-
97%) with dialkyl phosphite and KF. Subsequent
oxidation afforded R-ketophosphonates 1d-f. No epimer-
ization in the synthesis of compounds 1a -i was ob-
served by 1H NMR analysis. The diastereomeric purity
Calpains, calcium-dependent cysteine proteases,1 are
nonlysosomal protein-degrading enzymes found in most
mammalian cells. µ-Calpain, the more calcium-sensi-
tive form, may play critical roles in several pathophysi-
ological processes such as stroke, Alzheimer’s disease,
amyotrophy, motor neuron damage, and muscular dys-
trophy.2
A number of peptide-based reversible and irreversible
calpain inhibitors have been reported including alde-
hydes, R-ketoacids, R-ketoamides, R-ketoesters, R-dike-
tones, halomethyl ketones, epoxysuccinates, diazometh-
yl ketones, acyloxymethyl ketones, azapeptide esters,
and R-keto heterocycles.3,4 Reversible, substrate-based
calpain inhibitors usually contain an electrophilic car-
bonyl group in place of the scissile amide moiety of the
substrate. For example, R-ketoacids,3f R-ketoamides,3f,g,i
and R-ketoesters3e are potent calpain inhibitors. Breuer
et al. have shown that the carbonyl groups in nonpep-
tidic â-amino-R-ketophosphonates are electrophilic.5
Therefore, in search of novel calpain inhibitors, we
introduced R-ketophosphonates, -phosphinates, and
-phosphine oxides as bioisosteric replacements of the
R-dicarbonyl electrophilic center. We describe here the
synthesis and evaluation of R-ketophosphonates, -phos-
phinates, and -phosphine oxides 1 as calpain inhibitors.
of 1d was explicitly confirmed by synthesis of its P1
D
diastereomer. Thus, diastereomerically pure Cbz-Val-
D-Phe-H was condensed with dimethyl phosphite. Sub-
sequent Dess-Martin oxidation afforded diastereomer-
ically pure Cbz-Val-D-Phe-P(O)(OCH3)2, which is spectro-
scopically distinct from 1d . 1H NMR spectroscopy
disclosed no contamination of 1d with this P1 D diaste-
reomer.
Resu lts a n d Discu ssion
Peptidyl phosphorus compounds 1a -i were evaluated
as inhibitors of recombinant human calpain I at 20 °C,
using Suc-Leu-Tyr-NMA as the substrate, as previously
described.8 The inhibitory activities of these com-
pounds, as well as reference inhibitors Cbz-Leu-Leu-
CO2Et and Cbz-Leu-Leu-COOH, are shown in Table 1.
The Cbz-Leu-Leu and Cbz-Val-Phe dipeptide sequences
were selected as suitable P1-P2 calpain inhibitor ad-
dresses based upon the good affinity which these
sequences displayed when incorporated into other re-
versible calpain inhibitors, including aldehydes and
R-ketoamides.3
Ch em istr y
The synthesis of the peptidyl phosphonates, phosphi-
nates, and phosphine oxide is illustrated in Schemes 1
and 2. The reaction of Boc-Leu-H (2) with dialkyl
phosphites or dialkylphosphine oxide 3 and potassium
fluoride in DMF at room temperature afforded diaster-
eomeric mixtures of R-hydroxy phosphonate or phos-
phine oxide 4 in 75-92% yield.6 Deprotection of 4 and
coupling with Cbz-Leu-OH using BOP or DCC and
HOBt activation gave dipeptide R-hydroxy phosphonate
and phosphine oxide 6 in 14-71% yield after flash
chromatography. Oxidation of 6 with Dess-Martin
reagent7 in methylene chloride gave R-ketophosphonates
1b,c and phosphine oxide 1h . Compounds 1g,i were
prepared by the same procedure as 1c,h but Cbz-Leu-H
was utilized as the starting material and deprotection
was accomplished by hydrogenolysis over Pd/C. The
Dimethyl R-ketophosphonate 1b, the bioisosteric re-
placement of a peptidyl R-ketoester, exhibits potent
calpain inhibitory activity (IC50 ) 0.43 µM), comparable
with that of the peptide R-ketoester Cbz-Leu-Leu-CO2-
Et (IC50 ) 0.60 µM). However, monomethyl R-keto-
phosphonate 1a is almost 10 times less potent (IC50
)
5.2 µM), indicating that the free phosphonic acid
functionality is poorly tolerated by the active site of
calpain. In contrast, R-ketoacid Cbz-Leu-Leu-COOH is
nearly 100 times more potent than R-ketoester Cbz-Leu-
Leu-COOEt. Since R-ketoacids generally inhibit calpain
S0022-2623(98)00325-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/29/1998

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3913
Sch em e 1a
a
Reagents: (a) KF/DMF; (b) HCl/dioxane; (c) Cbz-Leu-OH, HOBt/BOP/NMM or DCC/HOBt/i-Pr2NEt/DMF; (d) Dess-Martin, CH2Cl2;
(e) LiBr/CH3CN; (f) Dess-Martin, CH2Cl2.
Sch em e 2a
greater potency of R-ketoacids compared to their esters
might be ascribed to Coulombic attraction with the
histidine residue of the catalytic triad. Perhaps the
tetrahedral geometry of phosphorus in R-ketophospho-
nate 1a , in contrast to the planar-trigonal geometry of
the carboxylate of an R-ketoacid, confines the anionic
oxygen of the phosphonic acid to a position in which
favorable interaction with the active site histidine is
impossible. Variation of the peptide address had a
dramatic effect on the inhibitory potencies of R-ketophos-
phonates 1b,d toward calpain. Cbz-Val-Phe-P(O)(OCH3)2
(1d ) is ∼17 times less potent than Cbz-Leu-Leu-P(O)-
(OCH3)2 (1b).
Dibutyl R-ketophosphonates 1c,e are essentially inac-
tive, and dibenzyl R-ketophosphonate 1f is a weak
inhibitor, indicating unfavorable P′ interactions. In
contrast, butyl and benzyl R-ketoamides and R-ke-
toesters, as well as analogues with more sterically
demanding P′ substituents, retain potency as calpain
inhibitors.3f,g,i
a
Reagents: (a) KF/DMF; (b) Dess-Martin, CH2Cl2.
Ta ble 1. Inhibition of Calpain I by Peptidyl Phosphonates,
Phosphinates, and Phosphine Oxidesa
The R-ketophosphine oxide functionality of 1h was
envisioned as a bioisostere of the R,â-diketone group,
known to be equipotent with R-ketoesters.3e R-Keto-
phosphinate 1g (IC50 ) 0.37 µM) and R-ketophosphine
oxide 1h (IC50 ) 0.35 µM) are potent calpain inhibitors.
Despite their aromatic substituents, inhibitors 1g,h
evidently do not produce unfavorable steric interactions
with calpain. In contrast, dimethyl R-ketophosphine
oxide 1i (IC50 > 10 µM) is essentially inactive.
In summary, R-ketophosphonate 1b, R-ketophosphi-
nate 1g, and R-ketophosphine oxide 1h , R-ketoester
bioisosteres, inhibit calpain with somewhat greater
potency than the corresponding R-ketoester Cbz-Leu-
Leu-CO2Et. These compounds constitute a novel and
interesting class of R-dicarbonyl mimic inhibitors of
calpain.
compd
peptidyl phosphorus compound
IC50 (µM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
Cbz-Leu-Leu-P(O)(OH)OCH3
Cbz-Leu-Leu-P(O)(OCH3)2
Cbz-Leu-Leu-P(O)(OC4H9)2
Cbz-Val-Phe-P(O)(OCH3)2
Cbz-Val-Phe-P(O)(OC4H9)2
Cbz-Val-Phe-P(O)(OCH2C6H5)2
Cbz-Leu-Leu-P(O)(Ph)OEt
Cbz-Leu-Leu-P(O)(C6H4-p-Cl)2
Cbz-Leu-Leu-P(O)(CH3)2
Cbz-Leu-Leu-CO2Et
5.2
0.43
>10
7.4
>10
6.0
0.37
0.35
>10
0.60
0.007
Cbz-Leu-Leu-COOH
a
n g 3 in all cases. Replicate determinations of IC50 agree
Exp er im en ta l Section
within 25%.
Gen er a l Asp ects. NMR spectra were recorded in CDCl3
solvent at 300 MHz using a General Electric QE Plus-300
instrument. Electrospray mass spectra were recorded on a
VG platform II instrument (Fisons Instruments). Elemental
analyses were performed by Quantitative Technologies Inc. of
more potently than their esters,3f the diminished affinity
of monomethyl R-ketophosphonate 1a compared with
dimethyl R-ketophosphonate 1b was unanticipated. The

3914 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Notes
Whitehouse, NJ . High-resolution mass spectroscopy was
performed by M-Scan Inc. of West Chester, PA.
To a solution of N-(benzyloxycarbonyl)-L-leucine (Cbz-Leu-
OH) (2.0 mmol) in DMF (4 mL) was added dimethyl 2-amino-
1-hydroxy-4-methylpentylphosphonate HCl salt (2.0 mmol)
(5b), i-Pr2NEt or NMM (4.0 mmol), HOBt (2.0 mmol), and DCC
or BOP (2.0 mmol) at 0 °C. The reaction mixture was stirred
at 0 °C for 5 min and for 2-24 h at room temperature.
Dicyclohexylurea was filtered off (when DCC was used), and
the solvent was evaporated. The residue was dissolved in ethyl
acetate (20 mL) and washed with 3% citric acid, 5% NaHCO3,
and brine. The organic layer was dried over MgSO4. Purifica-
tion by flash chromatography (5% MeOH in CH2Cl2) gave 0.65
g (69%) of 6b: 1H NMR δ 0.90 (t, 12H), 1.41-1.80 (m, 6H),
3.80 (d, 6H), 4.02 (m, 2H), 4.22 (m, 1H), 4.95 (m, 1H), 5.10 (s,
2H), 5.39 (d, 1H), 6.92 (d, 1H), 7.39 (s, 5H); MS m/z 473 (M +
H)+.
The following compounds were prepared according to the
representative procedures above. The starting materials,
reaction times, purification methods, and yields are given in
abbreviated format.
Dibu tyl N-(ben zyloxyca r bon yl)-L-leu cyl-(2S)-2-a m in o-
1-h yd r oxy-4-m eth ylp en tylp h osp h on a te (6c): method B,
N-(benzyloxycarbonyl)-L-leucine (Cbz-Leu-OH), dibutyl (2S)-
2-amino-1-hydroxy-4-methylpentylphosphonate HCl salt (5c),
36 h, flash chromatograghy (40% hexane in EtOAc); yield, 71%;
MS m/ z 557 (M + H)+, 579 (M + Na)+.
Dim eth yl N-(ben zyloxyca r bon yl)-L-va lyl-(2S)-2-a m in o-
1-h yd r oxy-3-p h en ylp r op ylp h osp h on a te (6d ): method A,
N-(benzyloxycarbonyl)-L-valyl-L-phenylalaninal (Cbz-Val-Phe-
H), dimethyl phosphite, 24 h; yield, 97%; 1H NMR δ 0.70-
0.94 (m, 6H), 2.08 (m, 1H), 3.04 (m, 2H), 3.63-4.21 (m, 9H),
5.12 (s, 2H), 5.38 (m, 1H), 5.43 (d, 1H), 7.02 (d, 1H), 7.16-
7.35 (m, 10H); MS m/z 515 (M + Na)+.
Dibu tyl N-(ben zyloxyca r bon yl)-L-va lyl-(2S)-2-a m in o-
1-h yd r oxy-3-p h en ylp r op ylp h osp h on a t e (6e): method A,
N-(benzyloxycarbonyl)-L-valyl-L-phenylalaninal (Cbz-Val-Phe-
H), dibutyl phosphite, 24 h; yield, 84%; 1H NMR δ 0.90 (m,
16H), 1.36 (m, 3H), 1.63 (m, 3H), 2.12 (m, 1H), 3.04 (m, 1H),
3.90-4.34 (m, 5H), 5.10 (m, 3H), 5.34 (d, 1H), 6.76 (d, 1H),
7.27-7.36 (m, 10H); MS m/z 577 (M + H)+.
N-(ter t-Bu toxyca r bon yl)-L-leu cin a l (2, Boc-Leu -H) was
prepared from (tert-butoxycarbonyl)-L-leucine N-methyl-O-
methylcarboxamide with lithium aluminum hydride in ether
according to Goel’s procedure.9a The 1H NMR spectrum agrees
with the literature spectrum.9a The compound was stored
under N2 in a freezer prior to use.
Ethyl N-(benzyloxycarbonyl)-L-leucyl-L-leucinecarboxylate
(Cbz-Leu-Leu-CO2Et), prepared by the lithiated ethyl vinyl
ether procedure,3e was hydrolyzed to N-(benzyloxycarbonyl)-
L-leucyl-L-leucinecarboxylic acid (Cbz-Leu-Leu-CO2H) with
NaOH in methanol.
N-(Ben zyloxycar bon yl)-L-valyl-L-ph en ylalan in al (9, Cbz-
Va l-P h e-H)3e,n was prepared by coupling N-(benzyloxycarbo-
nyl)-L-valine and L-phenylalanine N-methyl-O-methylamide
HCl salt with isobutyl chloroformate and N-methylmorpholine
in DMF as previously described.9b The resulting N-(benzyl-
oxycarbonyl)-L-valyl-L-phenylalanine N-methyl-O-methyla-
mide was reduced with lithium aluminum hydride in ether
according to Goel’s procedure.9a Crystallization from EtOAc/
hexane gave one pure isomer, N-(benzyloxycarbonyl)-L-valyl-
L-phenylalaninal (9): 1H NMR δ 0.87 (d, J ) 6 Hz, 3H), 0.94
(d, J ) 6 Hz, 2H), 2.11 (septet, J ) 6 Hz, 1H), 3.11 (d, 2H),
4.01 (t, 2H), 4.74 (q, 1H), 5.08 (s, 2H), 5.22 (d, 1H), 6.34 (d,
1H), 7.1-7.4 (m, 5H), 9.60 (s, 1H).
1H NMR spectroscopy revealed that this sample was dia-
stereomerically pure by comparison with N-(ben zyloxyca r -
bon yl)-L-va lyl-D-p h en yla la n in a l (Cbz-Va l-D-P h e-H): 1H
NMR δ 0.83 (d, J ) 6 Hz, 3H), 0.88 (d, J ) 6 Hz, 2H), 2.09
(septet, J ) 6 Hz, 1H), 3.11 (d, 2H), 4.02 (t, 2H), 4.75 (q, 1H),
5.08 (s, 2H), 5.20 (d, 1H), 6.35 (d, 1H), 7.1-7.4 (m, 5H), 9.60
(s, 1H). In pH 7.0 phosphate buffer (50 mM), Cbz-Val-Phe-H
epimerizes to Cbz-Val-D-Phe-H with a half-life of 4 days at 32
°C.
Rep r esen ta tive P r oced u r es for th e P r ep a r a tion of
Com p ou n d s 4 a n d 6d -f. Meth od A: Dim eth yl (2S)-2-
[(ter t-Bu t oxyca r b on yl)a m in o]-1-h yd r oxy-4-m et h ylp en -
tylp h osp h on a te (4b). 4b was prepared from N-(tert-butox-
ycarbonyl)-L-leucinal (Boc-L-Leu-H) (2.15 g, 10 mmol) and
dimethyl phosphite (1.10 g, 10 mmol) with KF (2.91 g, 50
mmol) in DMF at room temperature for 24 h according to the
procedure described by Texier-Boullet et al.6 Purification by
flash chromatography (10% MeOH in CH2Cl2) afforded 3.0 g
(92%) of 4b: 1H NMR δ 0.94 (d, 6H), 1.42 (s, 9H), 1.68 (m,
1H), 3.82 (m, 7H); MS m/z 348 (M + Na)+.
Dibu tyl (2S)-2-[(ter t-bu toxycar bon yl)am in o]-1-h ydr oxy-
4-m eth ylp en tylp h osp h on a te (4c): N-(tert-butoxycarbonyl)-
L-leucinal (Boc-Leu-H), dibutyl phosphite, 24 h; yield, 42%; 1H
NMR δ 0.93 (m, 12H), 1.30-1.77 (m, 11H), 1.42 (s, 9H), 3.95
(m, 1H), 4.15(m, 4H), 4.42 (m, 1H); MS m/z 410 (M + H)+.
Eth yl (2S)-[2-[(ben zyloxyca r bon yl)a m in o]-1-h yd r oxy-
4-m eth ylp en tyl](p h en yl)p h osp h in a te (4g): N-(benzyloxy-
carbonyl)-L-leucinal (Cbz-Leu-H), ethyl phenylphosphinate,10
23 h; yield, 75%; MS 420 m/z (M + H)+, 442 (M + Na)+.
(2S)-[2-[(ter t-Bu toxycar bon yl)am in o]-1-h ydr oxy-4-m eth -
ylp en tyl]bis(4-ch lor op h en yl)p h osp h in e oxid e (4h ): N-
(tert-butoxycarbonyl)-L-leucinal (Boc-Leu-H), bis(4-chlorophen-
yl)phosphine oxide (Organometallics, Inc.), 2.5 h; yield, 83%;
MS m/z 486 (M + H)+.
(2S)-[2-[(ter t-Bu toxycar bon yl)am in o]-1-h ydr oxy-4-m eth -
ylp en tyl](d im eth yl)p h osp h in e oxid e (4i): N-(tert-Butoxy-
carbonyl-L-leucinal (Boc-Leu-H), dimethylphosphine oxide,11
2.5 h; yield, 75%; MS m/z 316 (M + Na)+.
Rep r esen ta tive P r oced u r es for th e P r ep a r a tion of
Com pou n ds 5 an d 6b,c,g-i. Meth od B: Dim eth yl N-(Ben -
zyloxyca r bon yl)-L-leu cyl-(2S)-2-a m in o-1-h yd r oxy-4-m e-
th ylp en tylp h osp h on a te (6b). Dimethyl (2S)-2-[(tert-butox-
ycarbonyl)amino]-1-hydroxy-4-methylpentylphosphonate (4b)
was treated with 4 N HCl in dioxane for 2 h. The solvent was
evaporated in vacuo, and the residue was triturated with
diethyl ether. The crude white solid, dimethyl (2S)-2-amino-
1-hydroxy-4-methylpentylphosphonate HCl salt (5b), was used
directly for the next step.
Diben zyl N-(ben zyloxyca r bon yl)-L-va lyl-(2S)-2-a m in o-
1-h yd r oxy-3-p h en ylp r op ylp h osp h on a te (6f): method A,
N-(benzyloxycarbonyl)-L-valyl-L-phenylalaninal (Cbz-Val-Phe-
1
H), dibenzyl phosphite, 24 h; yield, 62%; H NMR δ 0.82 (d,
3H), 0.84 (d, 3H), 2.03 (m, 1H), 2.98 (m, 2H), 3.97 (m, 2H),
4.22 (m, 1H), 4.67 (m, 1H), 4.92-5.20 (m, 6H), 5.25 (d, 1H),
6.75 (d, 1H), 7.05-7.34 (m, 20H); MS m/z 645 (M + H)+. Anal.
(C36H41N2O7P) C, H, N.
Eth yl [N-(ben zyloxyca r bon yl)-L-leu cyl-(2S)-2-a m in o-1-
h yd r oxy-4-m eth ylp en tyl](p h en yl)p h osp h in a te (6g): hy-
drogenation over 10% Pd/C in ethanol; yield, 81%; product,
5g; MS m/z 286 (M + H)+. Method B: N-(benzyloxycarbonyl)-
L-leucine (Cbz-Leu-OH), ethyl (2S)-(2-amino-1-hydroxy-4-meth-
ylpentyl)(phenyl)phosphonate (5g), 4 h, flash chromatography
(ethyl acetate); yield, 67%; product, 6g; MS m/z 533 (M + H)+,
555 (M + Na)+.
[(Ben zyloxycar bon yl)-L-leu cyl-(2S)-2-am in o-1-h ydr oxy-
4-m eth ylpen tyl]bis(4-ch lor oph en yl)ph osph in e oxide (6h ):
method B, (2S)-[2-[(tert-butoxycarbonyl)amino]-1-hydroxy-4-
methylpentyl]bis(4-chlorophenyl)phosphine oxide (4h ) was
treated with 25% trifluoroacetic acid in dichloromethane for
1 h; yield, 97%; product, 5h ; MS m/z 386 (M+H)+. N-
(Benzyloxycarbonyl)-L-leucine (Cbz-Leu-OH), [(2S)-2-amino-1-
hydroxy-4-methylpentyl]bis(4-chlorophenyl)phosphine oxide
(5h ), 14 h, flash chromatography (50% ethyl acetate/hexane);
yield, 48%; product, 6h ; MS m/z 633 (M + H)+, 655 (M + Na)+.
[N-(Ben zyloxyca r b on yl)-L-leu cyl-(2S)-2-a m in o-1-h y-
d r oxy-4-m et h ylp en t yl](d im et h yl)p h osp h in e oxid e (6i):
hydrogenation over 10% Pd/C in ethanol; product, 5i; MS m/z
194 (M + H)+. N-(Benzyloxycarbonyl)-L-leucine (Cbz-Leu-OH),
[(2S)-2-amino-1-hydroxy-4-methylpentyl](dimethyl)phos-
phine oxide (5i), 14 h, flash chromatography (50% ethyl
acetate/hexane); yield, 41%; product, 6i; MS m/z 463 (M +
Na)+.
Met h yl H yd r ogen N-(Ben zyloxyca r b on yl)-L-leu cyl-

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3915
(2S)-2-a m in o-1-h yd r oxy-4-m eth ylp en tylp h osp h on a te (7).
A solution of dimethyl N-(benzyloxycarbonyl)-L-leucyl-(2S)-2-
amino-1-hydroxy-4-methylpentylphosphonate (6b) (0.3 mmol,
142 mg) in CH3CN (1.2 mL) was stirred at room temperature
under Ar as lithium bromide (0.95 mmol, 25 mg) was added.
The reaction mixture was stirred overnight. The solvent was
removed, and the crude product 7 was used directly for the
next step.
Meth yl h yd r ogen N-(ben zyloxyca r bon yl)-L-leu cyl-L-
leu cylph osph on ate (1a): method C, methyl hydrogen N-(ben-
zyloxycarbonyl)-L-leucyl-(2S)-2-amino-1-hydroxy-4-methylpen-
tylphosphonate (7), Dess-Martin periodinane, 16 h; yield,
65%; mp 57 °C dec; 1H NMR δ 0.92 (m, 6H), 1.40-1.78 (m,
6H), 3.81 (m, 6H), 4.02 (m, 1H), 4.22 (m, 1H), 5.12 (s, 2H),
5.40 (m, 1H), 7.38 (s, 5H); MS m/z 455 (M - H)-. Anal.
(C21H33N2O7P) C, H, N.
Dim eth yl N-(Ben zyloxycar bon yl)-L-leu cyl-L-leu cylph os-
p h on a te (1b). Meth od C: A solution of N-(benzyloxycarbo-
nyl)-L-leucyl-(2S)-2-amino-1-hydroxy-4-methylpentylphospho-
nate (6b) (94.4 mg, 0.2 mmol) and t-BuOH (34.5 mg, 0.41
mmol) in CH2Cl2 (5 mL) was stirred as Dess-Martin perio-
dinane (169.6 mg, 0.4 mmol) was added. The reaction was
stirred at room temperature for 2-3 h. The reaction progress
was followed by TLC. The reaction mixture was diluted with
CH2Cl2 and filtered through Celite. The filtrate was washed
with 10% Na2S2O3 and dried over MgSO4 to afford 80.0 mg
(85%) of 1b: 1H NMR δ 0.90 (t, 12H), 1.41-1.80 (m, 6H), 3.80
(d, 6H), 4.22 (m, 1H), 4.86 (m, 1H), 5.10 (m, 3H), 6.51 (d, 1H),
7.34 (s, 5H); MS m/z 471 (M + H)+; FAB m/z (M + H)+ calcd
for (C22H36N2O7P) 471.2260, observed m/z 471.2274.
NMR δ 0.92 (m, 12H), 1.40 (t, 3H), 1.40-1.80(m, 6H), 4.20
(m, 3H), 5.21 (m, 3H), 6.58 (d, 1H), 6.82 (d, 1H), 7.18-7.88
(m, 10H); MS m/z 531 (M + H)+, 553 (M + Na)+. Anal.
(C28H39Cl2N2O6P‚0.3 H2O) C, H, N.
[(Benzyloxycarbonyl)-L-leucyl-L-leucyl]bis(4-chlorophen-
yl)p h osp h in e oxid e (1h ): method C, [N-(benzyloxycarbonyl)-
L-leucyl-(2S)-2-amino-1-hydroxy-4-methylpentyl]bis(4-chlo-
rophenyl)phosphine oxide (6h ), Dess-Martin periodinane, 1
h; yield, 30%; 1H NMR δ 0.90 (dd, 6H), 1.21-1.75 (m, 6H),
4.10 (m, 1H), 5.02 (m, 3H), 7.20-7.65 (m, 13H); MS m/z 631
(M + H)+, 652, (M + Na)+. Anal. (C32H37Cl2N2O5P‚1.5H2O)
C, H, N.
[N-(Ben zyloxyca r bon yl)-L-leu cyl-L-leu cyl](d im eth yl)-
p h osp h in e oxid e (1i): method C, [N-(benzyloxycarbonyl)-L-
leucyl-(2S)-2-amino-1-hydroxy-4-methylpentyl](dimethyl)phos-
phine oxide (6i), Dess-Martin periodinane, 3 h; yield, 87%;
1H NMR δ 0.91 (bs, 12H), 1.41-1.75 (m, 12H), 4.12 (m, 1H),
4.57 (m, 1H), 5.15 (s, 2H), 5.32 (d, 1H), 6.42 9d, 1H), 7.33 (s,
5H). MS m/z 461 (M + Na)+. Anal. (C22H35Cl2N2O5P‚0.5H2O‚
0.2CH2CH2) C, H, N.
Ack n ow led gm en t. The authors acknowledge the
encouragement and support of Dr. J effry Vaught and
thank Mark A. Ator, Shobha E. Senadhi, Seetha Mur-
thy, Satish Mallya, Donna Bozyczko-Coyne, and Robert
Siman for biochemical assays. We also thank Dr. Manoj
Das for preparing Cbz-Leu-Leu-CO2Et and Cbz-Leu-
Leu-COOH and Kurt J osef for preparing Cbz-Val-D-Phe-
H. We acknowledge SmithKline Beecham for partial
support of this research.
Dibu tyl N-(ben zyloxyca r bon yl)-L-leu cyl-L-leu cylp h os-
p h on a te (1c): method C, dibutyl N-(benzyloxycarbonyl)-L-
leucyl-(2S)-2-amino-1-hydroxy-4-methylpentylphosphonate (6c),
Dess-Martin periodinane; 5 h, yield, 13% (after preparative
Refer en ces
1
HPLC); H NMR δ 0.90 (m, 22H), 1.42 (m, 5H), 1.70 (m, 5H),
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1H), 7.40 (s, 5H); MS m/z 555 (M + H)+, 577 (M + Na)+. Anal.
(C28H47N2O7P‚0.6H2O) C, H, N.
Dim eth yl N-(ben zyloxyca r bon yl)-L-va lyl-L-p h en yla la -
n ylp h osp h on a te (1d ): method C, dimethyl N-(benzyloxycar-
bonyl)-L-valyl-(2S)-2-amino-1-hydroxy-3-phenylpropylphospho-
nate (6d ), Dess-Martin periodinane, 2 h; yield, 88%; 1H NMR
δ 0.79 (d, J ) 6.5 Hz, 6H), 0.87 (d, J ) 6.5 Hz, 6H), 2.07 (m,
1H), 3.01 (dd, 1H), 3.30 (dd, 1H), 3.82 (d, J ) 4.5 Hz, 3H),
3.85 (d, J ) 4.5 Hz, 3H), 4.05 (m, 1H), 5.06 (d, 2H), 5.12 (m,
1H), 5.41 (d, NH), 6.88 (d, 1H), 7.11-7.40 (m, 10H); MS m/z
513 (M + Na)+. Anal. (C24H31N2O7P) C, H, N.
Dim eth yl N-(ben zyloxyca r bon yl)-L-va lyl-D-p h en yla la -
n ylp h osp h on a te was prepared by the same procedure from
N-(benzyloxycarbonyl)-L-valyl-D-phenylalaninal (Cbz-Val-D-
Phe-H): 1H NMR δ 0.74 (d, J ) 6.8 Hz, 6H), 0.82 (d, 6.8 Hz,
6H), 2.03 (m, 1H), 3.05 (dd, 1H), 3.31 (dd, 1H), 3.82 (d, J )
6.0 Hz, 3H), 3.86 (d, J ) 6.0 Hz, 3H) 4.02 (m, 1H), 5.09 (s,
2H), 5.16 (m, 1H), 6.41 (d, 1H), 7.15-7.38 (m, 11H).
Dib u t yl N-(b en zyloxyca r b on yl)-L-va lyl-L-p h en yla la -
n ylp h osp h on a te (1e): method C, dibutyl N-(benzyloxycar-
bonyl)-L-valyl-(2S)-2-amino-1-hydroxy-3-phenylpropylphospho-
nate (6e), Dess-Martin periodinane, 14 h; yield, 84%; 1H NMR
δ 0.88 (m, 12H), 1.40 (m, 4H), 1.62 (m, 4H), 2.03 (m, 1H), 3.20
(ABq, 2H), 4.12 (m, 5H), 4.98 (m, 1H), 5.22 (m, 2H), 5.55 (d,
1H), 6.80 (d, 1H), 7.11-7.40 (m, 10H); MS m/z 577 (M + H)+,
599 (M + Na)+. Anal. (C30H439N2O7P‚0.4CH2Cl2) C, H, N.
Diben zyl N-(ben zyloxyca r bon yl)-L-va lyl-L-p h en yla la -
n ylp h osp h on a te (1f): method C, dibenzyl N-(benzyloxycar-
bonyl)-L-valyl-(2S)-2-amino-1-hydroxy-3-phenylpropylphospho-
nate (6f), Dess-Martin periodinane, 1 h; yield, 62%; 1H NMR
δ 0.76 (d, 2H), 0.855 (d, 2H), 2.04 (m, 1H), 3.11 (ABq, 2H),
3.95 (m, 1H), 5.16 (m, 5H), 6.34 (d, 1H), 7.03 (d, 1H), 7.20-
7.33 (m, 10H); MS m/z 643 (M + H)+, 665 (M + Na)+. Anal.
(C36H39N2O7P‚1.5H2O) C, H, N.
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3916 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
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