Azodicarboxamides: A New Class of Cysteine Proteinase Inhibitor for Hepatitis a Virus and Human Rhinovirus 3C Enzymes

Article abstract of DOI:10.1021/jo9915123

Hepatitis A virus (HAV) 3C and human rhinovirus (HRV) 3C cysteine proteinases have been shown to be essential for viral maturation and infectivity through the cleavage of a viral polyprotein precursor. A number of bis-hydrazides (e.g., 11-14), analogous to nanomolar inhibitors of cathepsin K, were synthesized and tested for effectiveness against HAV 3C and HRV 3C proteinases, but these compounds showed no significant inhibition of the viral enzymes. However, oxidation of the bis-hydrazides to the corresponding azodicarboxamides gave potent, irreversible inhibitors with IC₅₀'s in the low micromolar range. These compounds probably act by adding the active site thiol to the azo moiety in a Michael fashion to give a covalent complex, which was detected by electrospray mass spectrometry. Azodicarboxamide 16 was shown to have a rate constant (k_inact/k_I) of 35 644 M^-1 min^-1.

Full text of DOI:10.1021/jo9915123

9538
J . Org. Chem. 1999, 64, 9538-9546
Azod ica r boxa m id es: A New Cla ss of Cystein e P r otein a se In h ibitor
for Hep a titis A Vir u s a n d Hu m a n Rh in ovir u s 3C En zym es
Richard D. Hill and J ohn C. Vederas*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Received September 27, 1999
Hepatitis A virus (HAV) 3C and human rhinovirus (HRV) 3C cysteine proteinases have been shown
to be essential for viral maturation and infectivity through the cleavage of a viral polyprotein
precursor. A number of bis-hydrazides (e.g., 11-14), analogous to nanomolar inhibitors of cathepsin
K, were synthesized and tested for effectiveness against HAV 3C and HRV 3C proteinases, but
these compounds showed no significant inhibition of the viral enzymes. However, oxidation of the
bis-hydrazides to the corresponding azodicarboxamides gave potent, irreversible inhibitors with
IC50’s in the low micromolar range. These compounds probably act by adding the active site thiol
to the azo moiety in a Michael fashion to give a covalent complex, which was detected by electrospray
mass spectrometry. Azodicarboxamide 16 was shown to have a rate constant (kinact/kI) of 35 644
M-1 min-1
.
In tr od u ction
and mouth disease, and viral meningitis and are also
implicated in triggering various autoimmune diseases.2
Hepatitis A virus is the only known member of the genus
hepatovirus and causes an acute form of infectious
hepatitis.3 Hepatitis A is still encountered in parts of the
world with restricted access to safe drinking water
supplies and sporadic outbreaks still occur in the devel-
oped world.4 Human rhinovirus is the major cause of the
common cold in humans and comprises more than 100
serotypes, thereby making vaccine development difficult.
Picornaviral 3C cysteine proteinases are responsible for
co- and posttranslational processing of an initially pro-
duced polyprotein and are known to be essential for viral
infectivity and maturation.5 They have a unique active
site geometry,2 which suggests that specific and selective
inhibitors can be designed as therapeutic agents.
The role of cysteine proteinases in a variety of diseases
and physiological disorders makes them an attractive
target for the development of therapeutic agents. Inhibi-
tors of cysteine proteinases are currently being sought
as potential agents against viral and parasitic infections,
cancer, arthritis, and osteoporosis.1-14 Hepatitis A virus
(HAV) and human rhinovirus (HRV) belong to the
Picornaviridae family, which is a large family of positive-
sense, single-stranded RNA viruses with more than 200
known members.2 In addition to HAV and HRV, picor-
naviruses are the causative agents of poliomyeltis, foot
* Corresponding author. Tel (780)-492-5475, fax (780)-492-8231,
e-mail: john.vederas@ualberta.ca.
(1) Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133-171.
(2) Bergmann, E. M.; J ames, M. N. G. In Proteinases as Targets for
Therapy; von der Helm, K., Korant, B., Eds.; Springer: Heidelberg,
1999.
(3) Hollinger, F. B.; Ticehurst, J . R. In Fields Virology; Fields, B.
N., Knipe, D. M., Howley, P. M., Channock, R. M., Melnick, J . L.,
Monath, T. P., Roizmann, B. E., Straus, S. E., Eds.; Lippincott-Raven
Publishers: Philadelphia, 1996.
(4) Pebody, R. G.; Leino, T.; Ruuutu, P.; Kinnunen, L.; Davidkin,
I.; Nohynek, H.; Leinikki, P. Epidemiol. Infect. 1998, 120, 55-59.
(5) Bergmann, E. M.; J ames, M. N. G. In Proteinases of Infectious
Agents; Dunn, B., Ed.; Academic Press: San Diego, CA, 1998.
(6) Using the notation of Schecter and Berger. Schecter, I.; Berger,
A. Biochem. Biophys. Res. Commun. 1967, 27, 157-162.
(7) (a) Shepherd, T. A.; Cox, G. A.; McKinney, E.; Tang, J .;
Wakulchik, M.; Zimmerman, R. E.; Villarreal, E. C. Bioorg. Med. Chem.
Lett. 1996, 6, 2893-2896. (b) Webber, S. E.; Okano, K.; Little, T. L.;
Reich, S. H.; Xin, Y.; Fuhrman, S. A.; Matthews, D. A.; Love, R. A.;
Hendrickson, T. F.; Patick, A. K.; Meador, J . W., III; Ferre, R. A.;
Brown, E. L.; Ford, C. E.; Binford, S. L.; Worland, S. T. J . Med. Chem.
1998, 41, 2786-2805. (c) Malcolm, B. A.; Lowe, C.; Shechosky, S.;
McKay, R. T.; Yang, C. C.; Shah, V. J .; Simon, R. J .; Vederas, J . C.;
Santi, D. V. Biochemistry 1995, 34, 8172-8179.
Although the picornaviral 3C enzymes are cysteine
proteinases, they are structurally similar to the chymo-
trypsin-like serine proteinases. The active site cysteine
(Cys 172 in HAV 3C) is in close proximity to a histidine
residue (His 44 in HAV 3C), which acts as a general base
to form the thiolate; an ordered water molecule is thought
(13) (a) Dragovich, P. S.; Prins, T. J .; Zhou, R.; Webber, S. E.;
Marakovits, J . T.; Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Lee,
C. A.; Ford, C. E.; Burke, B. J .; Rejto, P. A.; Hendrickson, T. F.;
Tuntland, T.; Brown, E. L.; Meador, J . W., III; Ferre, R. A.; Harr, J .
E. V.; Kosa, M. B.; Worland, S. T. J . Med. Chem. 1999, 42, 1213-
1224 and references therein. (b) Kong, T. S.; Venkatraman, S.; Furness,
K.; Nimkar, S.; Shepherd, T. A.; Wang, Q. M.; Aube, J .; Hanzlik, R. P.
J . Med. Chem. 1998, 41, 2579-2587. (c) Roush, W. R.; Gwaltney, S.
L., II; Cheng, J .; Scheidt, K. A.; McKerrow, J . H.; Hansell, E. J . Am.
Chem. Soc. 1998, 120, 10994-10995.
(14) (a) Yamashita, D. S.; Smith, W. W.; Zhao, B.; J anson, C. A.;
Tomaszek, T. A.; Bossard, M. J .; Levy, M. A.; Oh, H.-J .; Carr, T. J .;
Thompson, S. K.; Ijames, C. F.; Carr, S. A.; McQueney, M.; D′Alessio,
K. J .; Amegadzie, B. Y.; Hanning, C. R.; Abdel-Meguid, S.; DesJ arlais,
R. L.; Gleason, J . G.; Veber, D. F. J . Am. Chem. Soc. 1997, 119, 11351-
11352. (b) Thompson, S. K.; Halbert, S. M.; Bossard, M. J .; Tomaszek,
T. A.; Levy, M. A.; Zhao, B.; Smith, W. W.; Abdel-Meguid, S. S.; J anson,
C. A.; D’Alessio, K. J .; McQueney, M. S.; Amegadzie, B. Y.; Hanning,
C. R.; DesJ arlais, R. L.; Briand, J .; Sarkar, S. K.; Huddleston, M. J .;
Ijames, C. F.; Carr, S. A.; Garnes, K. T.; Shu, A.; Heys, J . R.; Bradbeer,
J .; Zembryki, D.; Lee-Rykaczewski, L.; J ames, I. E.; Lark, M. W.;
Drake, F. H.; Gowen, M.; Gleason, J . G.; Veber, D. F. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 14249-14254.
(8) Xing, R.; Hanzlik, R. P. J . Med. Chem. 1998, 41, 1344-1351.
(9) Wang, Q. M.; J ohnson, R. B.; J ungheim, L. N.; Cohen, J . D.;
Villarreal, E. C. Antimicrob. Agents Chemother. 1998, 42, 916-920.
(10) Morris, T. S.; Frormann, S.; Schechosky, S.; Lowe, C.; Lall, M.
S.; Gauss-Muller, V.; Purcell, R. H.; Emerson, S. U.; Vederas, J . C.;
Malcolm, B. A. Bioorg. Med. Chem. 1997, 5, 797-807.
(11) McKendrick, J . E.; Frormann, S.; Luo, C.; Semchuck, P.;
Vederas, J . C.; Malcolm, B. A. Int. J . Mass Spectrometry 1998, 176,
113-124.
(12) Lall, M. S.; Karvellas, C.; Vederas, J . C. Org. Lett. 1999, 1, 803-
806.
10.1021/jo9915123 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/07/1999

Azodicarboxamides as Cysteine Proteinase Inhibitors
J . Org. Chem., Vol. 64, No. 26, 1999 9539
these enzymes. To the best of our knowledge, azodicar-
boxamides have not previously been investigated as
cysteine proteinase inhibitors, although certain arylazo-
formates (e.g., 3) have been examined as cleavable
substrates for metalloproteinases and serine protein-
ases.18 Related compounds bearing electron-withdrawing
groups have recently been shown to react with cysteine
and thiol-containing enzymes.19 The parent azodicar-
boxamide, azodicarbonamide 4, inhibits HIV-1 replication
and is currently in clinical trials in Europe for the
treatment of advanced AIDS.20 Azodicarbonamide is
believed to interact with the zinc finger motifs of the
nucleocapsid protein and cause extrusion of zinc.
Resu lts a n d Discu ssion
F igu r e 1.
Syn th esis of In h ibitor s. The initial target, bis-
hydrazide 13, is readily synthesized by the method
outlined in Scheme 1. Michael addition of hydrazine to
N,N-dimethylacrylamide using the procedure of Huang,16
followed by diprotection using benzyl chloroformate gives
5 in 59% yield. Further protection of the terminal
nitrogen with a tert-butoxycarbonyl group affords 6, and
removal of the Cbz groups by catalytic hydrogenation
provides the selectively protected hydrazide 8. Reaction
of 8 with diphenyl hydrazodicarboxylate 1021 generates
bis-hydrazide 11 in moderate (43%) yield. Substitution
of the phenoxy group with phenethylamine in the pres-
ence of triethylamine provides the initial target 13.
Since bis-hydrazide 13 and its precursor 11 show no
inhibition of HAV 3C proteinase at 100 µM (see below),
both compounds were oxidized to their azo derivatives.
Oxidation of 13 using N-bromosuccinimide and pyridine21
leads to azodicarboxamide 15 in 71% yield. This com-
pound displays good inhibition of HAV 3C proteinase
with an approximate IC50 of 23 µM. Following these
results, a small number of analogues of 15 were prepared
in which the Boc protecting group, dimethylpropanamide
side chain and phenethyl moiety are individually altered.
Cleavage of the Boc group with trifluoroacetic acid
followed by attempted reprotection with an acetyl group
is troublesome, possibly due to the sensitivity of unpro-
tected hydrazines to aerobic oxidation.22 The acetyl group
is therefore introduced earlier in the synthesis (5 f 7),
and the remainder of the transformations to azodicar-
boxamide 16 are done in a manner directly analogous to
that outlined above (Scheme 1).
to complete the catalytic triad.2 Key substrate recognition
is partly provided by another histidine residue (His 191
in HAV 3C) which hydrogen bonds to the side chain
carbonyl of the P1 residue, which is glutamine in the
majority of the picornaviral 3C proteinases.
6
Various classes of inhibitors have been reported for
HAV 3C/HRV 3C proteinases. These include peptide
aldehydes,7 azapeptides,8 homophthalimides,9 halomethyl
ketones,10 iodoacetamides,11 â-lactones,12 and R,â-unsat-
urated compounds.13 Recently, a novel symmetric bis-
hydrazide 1 was reported14 as a potent and selective
inhibitor of cathepsin K, a cysteine proteinase of the
papain superfamily implicated in the process of bone
resorption. This compound is able to span the enzyme
active site and acts by adding the cysteine thiol to the
central urea carbonyl. Bis-hydrazide 1 showed a Ki of 0.7
nM against cathepsin K.14 We therefore chose to inves-
tigate this type of functionality for potential inhibitors
of HAV 3C and HRV 3C proteinases.
Although HAV 3C proteinase is known to show a
binding preference for a glutamine residue at P1, replace-
ment of the glutamine side chain with N,N-dimethyl-
glutamine does not greatly reduce inhibition but simpli-
fies synthetic strategies.15 Hence, we targeted synthesis
of nonsymmetrical bis-hydrazides in which one of the
hydrazide moieties bears a N,N-dimethylpropanamide
side chain to mimic the glutamine required at P1,
whereas the other bears a phenethylaminocarbonyl side
chain to mimic the phenylalanine at P2′. Incorporation
of hydrazine moieties into peptide backbones is well
established for the generation of proteinase inhibitors,
and we have recently reported the synthesis and testing
of azaglutamine haloacetyl derivatives such as 2 as
inhibitors of HAV 3C proteinase.16 Other substituted
hydrazides as glutamine mimics have also been reported
as HRV 3C proteinase inhibitors.17
HRV 3C proteinase may not tolerate dimethylglutamine
analogues at P1 as well as HAV 3C does, but a sulfone
moiety is accepted as a glutamine mimic.7a Therefore the
sulfone azodicarboxamide 23 appeared an attractive
target. The selectively protected sulfone 20 is available
by a process similar to that used for 8; Michael addition
of hydrazine to vinyl methyl sulfone followed by Cbz
In the present study, a number of bis-hydrazides are
synthesized and tested for inhibition of HAV 3C and HRV
3C proteinases but show disappointing activity. However,
oxidation of these compounds to the corresponding azodi-
carboxamides leads to potent irreversible inhibitors of
(18) (a) Mock. W. L.; Stanford, D. J . Biochemistry 1996, 35, 7369-
7377. (b) Mock, W. L.; Xu, D. Bioorg. Med. Chem. Lett. 1999, 9, 187-
192
(19) Antonini, G.; Pitar, G.; Caccuri, A. M.; Ricci, G.; Boschi, D.;
Frittero, R.; Gasco, A.; Ascenzi, P. Eur. J . Biochem. 1997, 245, 6663-
667.
(15) J ewell, D. A.; Swietnicki, W.; Dunn, B. M.; Malcolm, B. A.
Biochemistry 1992, 31, 7862-7869.
(20) (a) Vandevelde, M.; Witvrouw, M.; Schmit, J . C.; Sprecher, S.;
De Clercq, E.; Tassignon, J .-P. AIDS Res. Hum. Reteroviruses 1996,
12, 567-568. (b) Rice, W. G.; Turpin, J . A.; Huang, M.; Clanton, D.;
Buckheit, R. W.; Covell, D. G.; Wallqvist, A.; McDonnell, N. B.;
DeGuzman, R. N.; Summers, M. F.; Zalkow, L.; Bader, J . P.; Haugwitz,
R. D.; Sausville, E. A. Nature Medicine 1997, 3, 341-345.
(21) Harris, J . M.; McDonald, R.; Vederas, J . C. J . Chem. Soc., Perkin
Trans. 1 1996, 2669-2674.
(16) Huang, Y.; Malcolm, B. A.; Vederas, J . C. Bioorg. Med. Chem.
1999, 7, 607-619.
(17) (a) Venkatraman, S.; Kong, J .-S.; Nimkar, S.; Wang, Q. M.;
Aube, J .; Hanzlik, R. P. Bioorg. Med. Chem. Lett. 1999, 9, 577-580.
(b) Kati, W. M.; Sham, H. L.; McCall, J . O.; Montgomery, D. A.; Wang,
G. T.; Rosenbrook, W.; Miesbauer, L.; Buko, A.; Norbeck, D. W. Arch.
Biochem. Biophys. 1999, 362, 363-375.
(22) Ciufolini, M. A.; Xi, N. Chem. Soc. Rev. 1998, 27, 437-445.

9540 J . Org. Chem., Vol. 64, No. 26, 1999
Hill and Vederas
Sch em e 1
Sch em e 2
protection gives bis-Cbz protected sulfone 17 along with
some of the monoprotected compound 18 (Scheme 2). Boc
protection of 17 followed by cleavage of the Cbz groups
generates compound 20 in 97% yield. Reaction of 20 with
diphenyl hydrazodicarboxylate 10, as described for the
previous series of compounds, did not yield any of the
desired product. However, reaction with bis-pentafluo-
rophenyl carbonate21 affords 21 in good yield. Substitu-
tion of the pentafluorophenoxy group with hydrazine
followed by subsequent reaction with phenethyl isocy-
anate gives the desired bis-hydrazide 22. Oxidation to
azodicarboxamide 23 proceeds quantitatively using N-
bromosuccinimide and pyridine.
It was also decided to alter the phenethylamine side
chain on the P′ side, which was present as a phenylala-
nine surrogate. It seemed that the introduction of a
phenylalanamide (i.e., insertion of CONH2) at this posi-
tion might reduce hydrophobicity and enhance recogni-
tion. Attempted reaction of bis-hydrazide 11 with either
phenylalanine or phenylalanamide using similar condi-
tions to those described earlier fails to generate the
desired product, as does variation of the base and solvent
employed. Treatment of 11 with benzyl phenylalaninate
in chloroform in the presence of triethylamine gives the
desired benzyl ester 24 albeit in poor yield (16%). The
major product of this reaction is the hydantoin 25, formed
by intramolecular cyclization (Scheme 3). However, it is
possible to convert hydantoin 25 into the desired primary
amide 26 by treatment with concentrated ammonium
hydroxide in 2-propanol. This can be oxidized to the
azodicarboxamide 27 using N-bromosuccinimide and
pyridine, but in considerably lower yield.
En zym e In h ibition . Bis-hydrazides (11-14, 22, and
26) and azodicarboxamides (15-16, 23, and 27) were
tested for inhibition of cloned and overexpressed HAV
3C and HRV 3C proteinases. The HAV 3C proteinase
assays (enzyme concentration approximately 0.1 µM)
employ a C24S mutant in which the nonessential surface
cysteine is replaced with serine and which displays
catalytic parameters indistinguishable from those of the
wild-type enzyme.23 HRV assays use the 3C proteinase
from serotype 14 and are done using an enzyme concen-
tration of approximately 0.4 µM. For HAV 3C proteinase,
enzyme activity is conveniently monitored using a fluo-
rometric assay similar to one described for HRV 3C
(23) Malcolm, B. A.; Chin, S. M.; J ewell, D. A.; Stratton-Thomas, J .
R.; Thudium, K. B.; Rosenberg, S. Biochemistry 1992, 31, 3358-3363.

Azodicarboxamides as Cysteine Proteinase Inhibitors
J . Org. Chem., Vol. 64, No. 26, 1999 9541
Sch em e 3
by structurally related hydrazides against mammalian
cysteine proteinase, cathepsin K.14 Representative ex-
amples of these bis-hydrazides were retested against
HAV 3C with preincubation times of 15 min but this had
no effect on enzyme activity. The corresponding azodi-
carboxamides display much stronger inhibition and have
IC50’s in the range 4-62 µM.25 Table 1 shows that the
activity of each azo compound is comparable for both
enzymes, with the biggest difference being for sulfone 23,
which displays an 8-fold difference in inhibition of HRV
3C proteinase over HAV 3C proteinase. For the com-
pound which shows the best inhibition of HAV 3C,
azodicarboxamide 16, a rate constant (kinact/kI) of 35 644
M-1min-1 was calculated using the method of Kitz and
Wilson26 at six different inhibitor concentrations. Azodi-
carboxamide 15 was also tested for inhibition of HAV 3C
proteinase in the presence of additional thiols. When
N-acetylcysteine or dithiothreitol (DTT) is added to the
assay mixtures in excess or stoichiometric quantities, no
enzyme inhibition by 15 is observed. This suggests that
added thiol reacts rapidly with the azodicarboxamide
moiety in a fashion analogous to that seen with azodi-
carboxylates,27 namely Michael addition on the azo
nitrogen. Model experiments with inhibitor 15 and N-
acetylcysteamine in the absence of enzyme show that the
azo chromophore disappears instantly upon exposure to
thiol and that N-acetylcysteamine disulfide as well as
thiol-azo adducts27 can be seen by mass spectrometry.
However, following inactivation of HAV 3C with azodi-
carboxamide 15, it was not possible to regenerate enzyme
activity by the addition of up to a 4-fold excess of
N-acetylcysteamine. Since disulfide is readily formed in
the model reaction and the hydrazo compound 13 which
is produced as a product is a weak competitive inhibitor
of HAV 3C, this suggests that the structural framework
of the protein hinders attack by extraneous thiol (N-
acetylcysteamine) on the enzyme-azodicarboxamide ad-
duct. Although the reactivity of azodicarboxamides with
extraneous thiols would initially seem to preclude the in
vivo use of such molecules as drugs, it is important to
note that the parent compound, azodicarbonamide 4, is
being clinically tested for the treatment of AIDS.20 Thus,
despite apparent reactivity to thiols, inhibitors such as
15 are attractive leads for the development of antiviral
agents.
Ta ble 1.25
approximate IC50 values,a µM
HAV 3Ca
compound
HRV 3Cb
15
16
23
27
23
10
32
62
10
12
4
Mech a n ism of In h ibition . To confirm the hypothesis
that enzyme inhibition is due to covalent modification of
the active site thiol via Michael addition onto the azo
moiety following the formation of an initial enzyme-
inhibitor complex, the C24S HAV 3C mutant enzyme was
incubated with a solution of azodicarboxamide 16 in
DMSO for 15 min at 4 °C. Dialysis of the mixture followed
by electrospray mass spectrometry shows peaks at 23881
((12) for the enzyme alone and 24252 ((41) for the
enzyme inhibitor complex (increase of ca. 371 mass
units), which corresponds to the addition of azodicar-
boxamide 16 (MW 376) (Figure 2a). In a control experi-
ment in which DMSO containing no inhibitor is added,
a peak for the enzyme is observed at 23881 ((34) (Figure
nd
a
0.1 µM HAV 3C, 10 µM Dabsyl-GLRTQSFS-Edans, 2 mM
b
EDTA, 0.1 mg/mL BSA, 100 mM K3PO4, 1% DMF, pH 7.5; 0.4
µM HRV 3C, 10 µM EALFn-pNA, 50 mM HEPES, 150 mM NaCl,
1 mM, EDTA, 1% DMF, pH 7.5. nd ) not determined.
proteinase.24 The HRV 3C proteinase activity could be
readily assayed in the presence of EALFQ-pNA as a
chromogenic substrate. Assays are initiated by the ad-
dition of enzyme and no preincubation time is used. The
inhibition results for the azodicarboxamides are sum-
marized in Table 1. All of the bis-hydrazides show no
inhibition of HAV 3C or HRV 3C at concentrations of 100
µM with the exception of 13 and 22 which give 19 and
43% inhibition, respectively, against HRV 3C proteinase.
This contrasts the nanomolar inhibition constants achieved
(25) IC50 values were all determined using at least five different
inhibitor concentrations. In each case assays were initiated by the
addition of enzyme, and the first 3 min of data were used for calculation
purposes.
(26) Kitz, R.; Wilson, I. B. J . Biol. Chem. 1962, 237, 3245-3249.
(27) For leading references see: Klinge, M.; Vederas, J . C. Di-tert-
butyl Azodicarboxylate. In Encyclopedia of Reagents for Organic
Synthesis; Coates, R. M., Ed.; Wiley: Chichester, UK, 1995; Vol. III,
pp 1586-1590.
(24) Wang, Q. M.; J ohnson, R. B.; Cohen, J . D.; Voy, G. T.;
Richardson, J . M.; J unghein, L. N. Antiviral Chem. Chemother. 1997,
8, 303-310.

9542 J . Org. Chem., Vol. 64, No. 26, 1999
Hill and Vederas
F igu r e 3.28,29 Molecular model of azodicarboxamide 16 fol-
lowing Michael addition with Cys 172 in the active site of HAV
3C proteinase. For the inhibitor, carbon atoms are shown
green, nitrogen atoms blue, and oxygen atoms red. The protein
side chains are colored by type and the backbone is gray. Key
hydrogen-bonds predicted between the inhibitor and the
protein are shown as dotted yellow lines.
of the target cysteine proteinases. Molecular modeling
studies suggest that these compounds are potentially
useful tools for cocrystallization studies since recognition
elements can be built onto either side of the azo moiety,
thereby spanning both the P and P′ recognition sites of
the active site. Presently most cysteine proteinase inhibi-
tors utilize recognition on only one side of the active site.
Additional studies to improve recognition and specificity
of inhibition are in progress.
F igu r e 2. (a) Mass spectrum of HAV 3C C24S mutant +
azodicarboxamide 16 in DMSO. (b) Mass spectrum of HAV 3C
C24S mutant + DMSO. Enzyme was incubated for 15 min at
4 °C with a solution of 16 in DMSO or DMSO alone.
Exp er im en ta l Section
Gen er a l Meth od s a n d En zym e Assa ys. Most general
procedures and instrumentation have been previously de-
scribed.30 Inhibitors were screened for effectiveness against
HAV 3C proteinase and HRV 3C proteinase. Both enzymes
were expressed and purified according to previously described
procedures.7c,31 HAV 3C proteinase assays employed a C24S
mutant in which the nonessential surface cysteine was re-
placed with serine and which displays catalytic parameters
indistinguishable from those of the wild-type enzyme.23 HRV
3C assays used the 3C proteinase from serotype 14. Purity of
the enzyme samples was greater than 90% as determined by
SDS-PAGE analysis. Proteinase concentrations were deter-
mined spectrophotometrically using ꢀ280 ) 1.2 mg/mL. En-
zymes were dialyzed against reaction buffer to remove DTT
immediately prior to use.
2b). This supports the proposal that the mode of inhibi-
tion for the azodicarboxamides involves the formation of
a covalent complex. Figure 3 shows compound 16 mod-
eled28 into the active site of HAV 3C based upon a
cocrystal structure of HAV 3C with a covalent addition
of acetyl-Val-Phe-amide to Cys 172.29 The carbonyl which
hydrogen-bonds to the backbone nitrogen of residue 170
does not fall into the oxyanion hole canonically and does
not appear to hydrogen-bond to the nitrogen of Cys 172.
Furthermore, owing to the rigidity of the inhibitor
relative to a peptide, the phenyl ring is not apparently
able to fully enter the S2′ pocket.
In the case of HAV 3C proteinase, enzyme activity was
monitored using a fluorometric assay similar to one described
for HRV 3C proteinase.24 Assays were done at pH 7.5 (100 mM
KH2PO4/K2HPO4, 20 mM, EDTA) at 25 °C. Bovine serum
albumin (BSA) was added to give a concentration of 0.1 mg/
mL. Inhibitor stock solutions were prepared at 10 mM in DMF
and serial dilutions made in DMF. Inhibitor concentrations
were varied from 100 µM to 1 µM. Dabcyl-GLRTQSFS-Edans
(Bachem) was prepared as a stock solution in DMF and a final
concentration of 10 µM used in each assay. Assays were
initiated by the addition of enzyme (0.1 µM), and the increase
in fluorescence (λex 336 nm, λem 472 nm) was monitored with
a Shimadzu RF5301 spectrofluorophotometer. In the case of
Su m m a r y
The present work describes the syntheses of a number
of unsymmetrical bis-hydrazides and azodicarboxamides.
In contrast to the cysteine proteinase cathepsin K, HAV
3C and HRV 3C proteinases are not effectively inhibited
by bis-hydrazides. However, oxidation of these com-
pounds to the corresponding azodicarboxamides results
in compounds which are potent, irreversible inactivators
(28) Model created by Dr. J onathan Parrish, Biochemistry Depart-
ment, University of Alberta, using InsightII (MSI corporation, San
Diego, CA) and X-plor (Brunger, A. T. X-PLOR: A System for X-ray
Crystallography and NMR; Yale University Press: New Haven, 1992).
The figure created using BOBscript (Bacon, D. J .; Anderson, W. F. J .
Mol. Graphics 1988, 6, 219-220, and Merritt, E. A.; Murphy, M. E. P.
Acta Crystallogr. 1994, D50, 869-873).
(29) Bergmann, E. M.; Cherney, M. M.; McKendrick, J .; Frormann,
S.; Luo, C.; Malcolm, B. A.; Vederas, J . C.; J ames, M. Virology 1999,
in press.
(30) (a) Harris, J . M.; Bolessa, E. A.; Mendonca, A. J .; Feng, S.-C.;
Vederas, J . C. J . Chem. Soc., Perkin Trans. 1 1995, 1945-1950. (b)
Merkler, D. J .; Glufke, U.; Ritenour-Rodgers, K. J .; Baumgart, L. E.;
DeBlassio, J . L.; Merkler, K. A.; Vederas, J . C. J . Am. Chem. Soc. 1999,
121, 4904-4905.
(31) Birch, G. M.; Blac, T.; Malcolm, S. K.; Lai, M. T.; Zimmerman,
R. C.; J askunas, S. R. Protein Expression Purif. 1995, 6, 609-618.

Azodicarboxamides as Cysteine Proteinase Inhibitors
J . Org. Chem., Vol. 64, No. 26, 1999 9543
HRV 3C proteinase, EALFQ-pNA (Bachem) (10 µM) was
incubated at 30 °C with inhibitor (100 µM to 1 µM in DMSO)
at pH 7.5 (HEPES 50 mM, NaCl 150 mM, EDTA 1 mM). HRV
3C proteinase (0.4 µM) was added and the increase at λmax 405
nm monitored using a GBC Cintra 40 UV spectrometer.
In both cases the data for the initial 3 min of the assay were
used for calculation of inhibition. In all cases control assays
were performed using enzyme and solvent alone. IC50 values
were calculated for compounds which showed a value <100
µM (as determined by an initial assay at 100 µM) and were
determined using at least five different inhibitor concentra-
tions. The rate of inactivation for azodicarboxamide 16 was
determined using the method of Kitz and Wilson,26 using six
different inhibitor concentrations (1-50 µM) and six incubation
times at each concentration. To see if the addition of a low
molecular weight thiol could regenerate enzyme activity
following enzyme inactivation by an azodicarboxamide, HAV
3C was inactivated by 15 (50 or 100 µM) as described above.
A solution of N-acetylcysteamine in DMF (100-200 µM) was
then added, and the fluorescence spectrum was monitored over
15-30 min. No recovery of enzyme activity was observed
during any of the experiments.
Hz), 2.78 (6 H, br s), 3.66 (1 H, ddd, J ) 7.1, 7.1, 14.7 Hz),
3.82 (1 H, ddd, J ) 7.1, 7.1, 14.4 Hz), 5.05 (2 H, br s), 5.22 (2
H, s) and 7.35 (10 H, br s); MS (+ve ES) 905.3 (M2Na+, 13%),
464.1 (MNa+, 21%) and 442.3 (MH+, 100%). Anal. Calcd for
C23H27N3O6: C, 62.57, H, 6.16, N, 9.52%. Found: C, 62.46; H,
6.17; N, 9.41%.
3-[N2-(ter t-Bu toxyca r bon yl)h yd r a zin o]-N,N-d im eth yl-
p r op a n a m id e (8). Hydrazide (6) (4.51 g, 9.03 mmol) was
dissolved in MeOH (100 mL) in the presence of 10% Pd/C (450
mg, 10% w/w) under an argon atmosphere. The suspension
was stirred under a hydrogen atmosphere until the uptake of
hydrogen ceased (approximately 4 h). Filtration through Celite,
followed by removal of the solvent in vacuo, gave the title
compound as a colorless oil (2.02 g, 97%); IR (CHCl3 cast) 3295,
2976, 2933, 1709, and 1641 cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.45 (9 H, s), 2.46 (2 H, t, J ) 6.5 Hz), 2.92 (3 H, s), 2.93 (3
H, s), 3.10 (2 H, t, J ) 6.5 Hz), 4.11 (1 H, br s), and 6.13 (1 H,
br s); 13C NMR (75.5 MHz, CDCl3) δ 28.33, 31.59, 35.32, 37.16,
47.91, 80.40, 156.67, and 171.45; MS (+ve ES) 254.1 (MNa+,
100%). Anal. Calcd for C10H21N3O3: C, 51.93; H, 9.15; N,
18.17%. Found: C, 51.97; H, 8.98; N, 17.94%.
3-(N2- Acetylh ydr azin o)-N,N-dim eth ylpr opan am ide (9).
This was prepared using the same method as that described
for the preparation of 8. A solution of hydrazide (7) (157 mg,
0.36 mmol) in MeOH (10 mL) was stirred under a hydrogen
atmosphere in the presence of 10% Pd/C (15 mg) until the
uptake of hydrogen gas ceased (20 min) and then for a further
2 h. The suspension was filtered through Celite and the solvent
removed in vacuo to yield a white solid which was recrystal-
lized from EtOAc to give a white solid (56 mg, 90%); mp 112-
3-[N1,N2-Bis(ca r boben zyloxy)h yd r a zin o]-N,N-d im eth -
ylp r op a n a m id e (5). This was prepared by a modification of
the procedure of Huang.16 To a solution of hydrazine mono-
hydrate (1.20 mL, 25.0 mmol) in MeOH (20 mL) at room
temperature was added N,N-dimethylacrylamide (2.50 mL,
25.0 mmol) over a 10 min period. The reaction mixture was
stirred for 2 h, dried (MgSO4), and concentrated in vacuo. The
residual oil was dissolved in diethyl ether (60 mL) and cooled
to 0 °C. To this solution was added 1 N NaOH (50 mL) and
water (15 mL), followed after 5 min by benzyl chloroformate
(7.15 mL, 50 mmol). The reaction mixture was stirred for 1 h
and diluted with EtOAc (100 mL), and the aqueous layer was
washed with EtOAc (3 × 100 mL). The combined organic
extracts were dried (MgSO4) and concentrated to give a yellow
oil which crystallized on standing. Purification by column
chromatography on silica gel eluting with 5% MeOH in EtOAc
gave the desired product as a white crystalline solid (5.91 g,
59%). A sample was recrystallized from EtOAc for analysis.
mp 106-108 °C; IR (CHCl3 cast) 3228, 2949, 1715, and 1630
cm-1; 1H NMR (300 MHz, CDCl3) δ 2.66 (2 H, br s), 2.68 (6 H,
br s), 3.80 (2 H, t, J ) 6.3 Hz), 5.11 (4 H, br s), and 7.29 (10 H,
br s); 13C NMR (75.5 MHz, CDCl3) δ 31.66, 35.34, 37.22, 47.52,
67.62, 128.13, 128.31, 128.56, 129.15, 135.77, 136.02, 155.76,
and 155.99; MS (+ve ES) 422.2 (MNa+, 100%) and 400.2 (MH+,
44%). Anal. Calcd for C21H25N3O5: C, 63.15; H, 6.31; N, 10.52%.
Found: C, 63.26; H, 6.40; N, 10.48%.
1
113 °C; IR (CHCl3 cast) 3264, 2933, and 1635 cm-1; H NMR
(300 MHz, CDCl3) δ 1.90 (3 H, s), 2.47 (2 H, t, J ) 6.4 Hz),
2.91 (3 H, s), 2.97 (3 H, s), 3.12 (2 H, t, J ) 6.4 Hz), 4.48 (1 H,
br s), and 7.95 (1 H, br s); 13C NMR (75.5 MHz, CDCl3) δ 19.38,
21.11, 30.80, 32.04, 35.41, 37.12, 37.25, 47.65, 48.31, 169.21,
and 171.61; MS (+ve ES) 196.1 (MNa+, 17%), 174.1 (MH+,
94%), and 130.1 (M+ - CH3CO, 100%). Anal. Calcd for
C7H15N3O2: C, 48.54; H, 8.73; N, 24.26%. Found: C, 48.53; H,
8.74; N, 24.02%.
3-[N1-P h en oxyca r bon ylh yd r a zid o][N2-(ter t-bu toxyca r -
bon yl)h yd r a zin o]-N,N-d im eth ylp r op a n a m id e (11). To a
solution of Boc-hydrazide (8) (750 mg, 3.24 mmol) in chloroform
(35 mL) at room temperature, was added diphenyl hydrazodi-
carboxylate (10)21 (971 mg, 3.57 mmol) and the resulting
suspension was stirred for 5 min. Triethylamine (0.45 mL, 3.24
mmol) was added and the suspension was heated to reflux at
which point all of the solid dissolved. Heating was maintained
for 3 h, the solution was cooled to room temperature and the
solvent was removed in vacuo. Purification by column chro-
matography on silica gel eluting with 5% MeOH in EtOAc gave
the desired product as a white solid which was recrystallized
from EtOAc/hexane (567 mg, 43%); mp 149-151 °C; IR
(microscope) 3337, 3226, 3003, 2979, 1770, 1727, 1664, and
1637 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.46 (9 H, s), 2.71 (2
H, br s), 3.01 (6 H, 2 × s), 3.72 (2 H, br s), 6.87 (1 H, s), 7.12-
7.35 (5 H, m), and 7.63 (2 H, br s); 13C NMR (75.5 MHz, CDCl3)
δ 28.20, 32.38, 35.59, 46.70, 82.29, 121.48, 125.66, 129.35,
150.81, 155.19, 155.92, 157.35 and 172.13; MS (+ve ES) 432.2
(MNa+, 100%), 410.2 (MH+, 25%), 354.2 (M+ - C4H9, 29%),
332.2 (M+ - C6H5, 47%), and 310.2 (M+ - Boc). Anal. Calcd
for C18H27N5O6: C, 52.80; H, 6.65; N, 17.10%. Found: C, 52.82;
H, 6.67; N, 16.87%.
3-[N1-(Ca r boben zyloxy)- N2-(ca r boben zyloxy,ter t-bu -
toxyca r bon yl)h yd r a zin o]-N,N-d im eth ylp r op a n a m id e (6).
To a solution of hydrazide (5) (5.36 g, 13.43 mmol) in aceto-
nitrile (75 mL) at room temperature, under an argon atmo-
sphere, was added (dimethylamino)pyridine (DMAP) (164 mg,
1.34 mmol) followed, 5 min later, by a solution of di-tert-butyl
dicarbonate (3.22 g, 14.8 mmol) in acetonitrile (50 mL). The
reaction mixture was stirred overnight at room temperature
under argon. The solvent was removed in vacuo, and the
residual oil was purified by column chromatography on silica
gel eluting with (2:1) EtOAc/hexane to give the desired
compound as a clear colorless oil (5.07 g, 76%); IR (CHCl3 cast)
1804, 1766, 1725, and 1651 cm-1; 1H NMR (500 MHz, DMSO-
d6, 120 °C) δ 1.40 (9 H, s), 2.58 (2 H, t, J ) 7.3 Hz), 2.93 (6 H,
s), 3.76 (2 H, m), 5.12 (2 H, br s), 5.20 (2 H, s) and 7.35 (10 H,
br s); MS (+ve ES) 522.2 (MNa+, 76%), 500.2 (MH+, 7%) and
422.1 (M+ - Boc, 100%). Anal. Calcd for C26H33N3O7: C, 62.51;
H, 6.66; N, 8.41%. Found: C, 62.50; H, 6.77; N, 8.45%.
3-[N1-(Car boben zyloxy)-N2-(car boben zyloxy)-N2-acetyl-
h yd r a zin o]-N,N-d im eth ylp r op a n a m id e (7). Hydrazide (5)
(200 mg, 0.50 mmol) and DMAP (12 mg, 0.10 mmol) were
suspended in acetic anhydride (5 mL) and heated at 80 °C for
4 h. The reaction was cooled to room temperature and purified
by column chromatography on silica gel eluting with EtOAc.
The product was obtained as a colorless oil (191 mg, 86%); IR
3-[N1-P h en oxycar bon ylh ydr azido][N2-acetylh ydr azin o]-
N,N-d im eth ylp r op a n a m id e (12). This was prepared using
the same method as that described for the preparation of 11.
Hydrazide (9) (350 mg, 2.02 mmol) in chloroform (15 mL) was
treated with triethylamine (0.42 mL, 3.04 mmol) and diphenyl
hydrazodicarboxylate (10) (825 mg, 3.04 mmol) at reflux for 6
h. Purification by column chromatography on silica gel eluting
with 5% MeOH in CH2Cl2 gave 12 as a white solid (192 mg,
27%); mp 139-141 °C; IR (CHCl3 cast) 3243, 3008, 1959, 1771,
1
1750, 1733, 1683, 1675, and 1652 cm-1; H NMR (300 MHz,
CDCl3) δ 2.04 (3 H, s), 2.70 (2 H, br s), 2.91 (3 H, s), 2.96 (3 H,
s), 3.75 (2 H, br s), 6.90 (<1 H, br s), 7.11-7.29 (5 H, m), 7.60
(<1 H, br s) and 8.82 (<1 H, br s); 13C NMR (75.5 MHz, CD3-
1
(CHCl3 cast) 1754, 1721, 1647, and 1402 cm-1; H NMR (500
MHz, DMSO-d6, 120 °C) 2.42 (3 H, s), 2.55 (2 H, t, J ) 7.1

9544 J . Org. Chem., Vol. 64, No. 26, 1999
Hill and Vederas
OD) δ 20.89, 32.72, 35.69, 37.71, 47.01, 61.53, 122.65, 126.61,
130.35, 152.46, 157.59, 159.38, 172.75 and 173.38; MS (+ve
ES) 374.1 (MNa+, 38%), 352.1 (MH+, 100%); MS (+ve ES)
Found 352.162389. C15H22N5O5 (MH+) requires 352.162094.
3-[N1-P h en eth yla m id oh yd r a zid o][(N2-ter t-bu toxyca r -
bon yl)h yd r a zin o]-N,N-d im eth ylp r op a n a m id e (13). To a
solution of phenoxy bis-hydrazide (11) (389 mg, 0.95 mmol) in
chloroform (25 mL) was added phenethylamine (0.126 mL, 1.0
mmol) followed by triethylamine (0.139 mL, 1.0 mmol) and
the solution heated at reflux for 7 h. Removal of the solvent
in vacuo gave a white solid which was purified by column
chromatography on silica gel eluting with 5% MeOH in EtOAc.
The title compound was collected as a white solid which was
recrystallized from MeOH/EtOAc (331 mg, 80%); mp 179-180
°C; IR (microscope) 3357, 3305, 3269, 2981, 2922, 1706, and
1626 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.44 (9 H, s), 2.70 (2
H, br s), 2.75 (2 H, t, J ) 7.6 Hz), 2.93 (3 H, s), 3.00 (3 H, s),
3.43 (2 H, t, J ) 7.6 Hz), 3.80 (2 H, br s), 7.13-7.29 (5 H, m),
7.50 (1 H, br s, exchanges with D2O), and 8.04 (1 H, br s,
exchanges with D2O); 13C NMR (75.5 MHz, CDCl3) δ 28.22,
32.42, 35.61, 36.31, 37.41, 41.55, 46.55, 82.50, 126.26, 128.48,
128.86, 139.26, 156.06, 157.31, 159.18, and 172.20; MS (+ve
ES) 475.3 (MK+), 459.3 (MNa+, 100%), 437.3 (MH+, 64%),
359.2 (MNa+ - Boc, 37%) and 337.2 (MH+ - Boc, 20%). Anal.
Calcd for C20H32N6O5: C, 55.03; H, 7.39; N, 19.25%. Found:
C, 55.08; H, 7.50; N, 19.39%.
residue was purified by column chromatography on silica gel
eluting with 5% MeOH in EtOAc to give the title compound
which was recrystallized from EtOAc to give yellow needles
(21 mg, 81%); mp 137-138 °C; IR (CHCl3 cast) 3243, 3025,
1728, 1630, and 1498 cm-1; UV (CH3CN) 239 (ꢀ 1879) and 410
1
(ꢀ 47) nm; H NMR (300 MHz, CDCl3) δ 1.89 (3 H, s), 2.83 (2
H, br s), 2.94 (5 H, m), 3.00 (3 H, s), 3.73 (2 H, dt, J ) 6.7, 6.8
Hz), 3.88 (2 H, br s), 6.59 (1 H, br t), 7.26 (5 H, m), and 8.91
(1 H, s); MS (+ve ES) 775.2 (M2Na+, 49%), 399.1 (MNa+, 46%)
and 377.2 (MH+, 62%). Anal. Calcd for C17H24N6O4: C, 54.25;
H, 6.43; N, 22.33%. Found: C, 53.94; H, 6.40; N, 21.91%.
2-[(N1,N2-Bis(ca r boben zyloxy)h yd r a zin o]eth yl Meth yl
Su lfon e (17) a n d 2-[N1 -(Ca r boben zyloxy)h yd r a zin o]-
eth yl Meth yl Su lfon e (18). This was prepared by a method
similar to that described for the preparation of 5. A solution
of hydrazine monohydrate (0.49 mL, 10.0 mmol) in MeOH (10
mL) was treated with vinyl methyl sulfone (0.88 mL, 10.0
mmol) in MeOH (5 mL), followed by 1 N NaOH (22 mL) and
benzyl chloroformate (2.86 mL, 20.0 mmol). After aqueous
workup, purification by column chromatography on silica gel
eluting with EtOAc gave bis-Cbz sulfone (17) as a white solid
which was recrystallized from EtOAc/hexane to give white
needles (1.558 g, 38%). The monoprotected sulfone (18) was
also isolated as a white solid (884 mg, 33%).
Da ta for bis-Cbz su lfon e (17): IR (CHCl3 cast) 3300, 3031,
2930, 1718, and 1302 cm-1; 1H NMR (300 MHz, CDCl3) δ 2.87
(3 H, br s), 3.32 (2 H, br s), 4.02 (2 H, br s), 5.12 (4 H, br s)
and 7.25 (10 H, br s); 13C NMR (75.5 MHz, CDCl3) δ 41.12,
44.75, 52.03, 68.08, 68.62, 128.25, 128.49, 128.57, 128.63,
128.67, 135.33, 135.45, and 155.50; MS (+ve ES) 429.2 (MNa+,
37%), 407.4 (MH+, 52%), and 363.4 (M+ - CO2, 100%). Anal.
Calcd for C19H22N2O6S: C, 56.15; H, 5.46; N, 6.89%. Found:
C, 56.34; H, 5.27; N, 6.73%.
3-[N1-(P h en eth ylam idoh ydr azido)-N2-acetylh ydr azin o]-
N,N-d im eth ylp r op a n a m id e (14). This was prepared using
the same method as that described for the preparation of 13.
A solution of phenoxy bis-hydrazide (12) (192 mg, 0.55 mmol)
in chloroform (15 mL) was treated with triethylamine (0.083
mL, 0.60 mmol) and phenethylamine (0.075 mL, 0.60 mmol)
at reflux for 6.5 h. Purification by column chromatography
eluting with 5% MeOH in CH2Cl2 gave the desired compound
as a white solid (98 mg, 48%); mp 112-115 °C; IR (CHCl3 cast)
3257, 2934, 1665, and 1547 cm-1; 1H NMR (300 MHz, CDCl3)
δ 2.02 (3 H, s), 2.63 (2 H, br s), 2.79 (2 H, t, J ) 7.2 Hz), 2.88
(3 H, s), 2.95 (3 H, s), 3.37 (2 H, dt, J ) 6.0, 6.0 Hz), 3.77 (2 H,
br s), 6.06 (1 H, br s), 6.86 (1 H, br s), 7.12-7.27 (5 H, m), 8.06
(1 H, br s), and 9.55 (1 H, br s); 13C NMR (75.5 MHz, CD3OD)
δ 20.95, 32.57, 35.70, 37.29, 37.70, 42.62, 46.97, 127.22, 129.45,
129.85, 140.67, 159.39, 161.73, 173.19, and 173.33; MS (+ve
ES) 757.4 (M2H+, 56%), 401.2 (MNa+, 63%), and 379.2 (MH+,
100%); MS (+ve ES) Found: 401.191141. C17H26N6O4Na
(MNa+) requires 401.191323.
Da ta for m on o-Cbz su lfon e (18): mp 88-90 °C; IR (CHCl3
cast) 3345, 2927, 1694, 1497, and 1290 cm-1 1H NMR (300
;
MHz, CDCl3/D2O) δ 2.86 (3 H, s), 3.28 (2 H, t, J ) 6.5 Hz),
3.90 (2 H, t, J ) 6.5 Hz), 5.13 (2 H, s), and 7.33 (5 H, m); 13C
NMR (75.5 MHz, CDCl3) δ 40.74, 44.56, 51.79, 68.11, 128.18,
128.38, 128.58, 128.65, 135.86, and 157.00; MS (+ve ES) 295.4
(MNa+, 23%) and 273.5 (MH+, 100%). Anal. Calcd for
C
11H16N2O4S: C, 48.52; H, 5.92; N, 10.29%. Found: C, 48.42;
H, 6.02; N, 10.16%.
2-[N1-(Ca r boben zyloxy)-N2-(ca r boben zyloxy)-N2-(ter t-
b u t oxyca r b on yl)h yd r a zin o]et h yl Met h yl Su lfon e (19).
This was prepared by a method similar to that described for
the preparation of 6. To a solution of bis-Cbz sulfone (17) (1.40
g, 3.45 mmol) in acetonitrile (25 mL) were added DMAP (42
mg, 0.35 mmol) and di-tert-butyl dicarbonate (828 mg, 3.79
mmol), and the reaction mixture was stirred overnight.
Purification by column chromatography on silica gel, eluting
with (1:1) EtOAc/hexane, gave the desired product as a
colorless oil (1.75 g, quantitative); IR (CHCl3 cast) 1805, 1766,
Azo-N1-(ph en eth ylfor m a m ide)- N1′-[N,N-d im eth ylpr op-
d im eth ylp r op a n a m id e-ter t-bu toxyca r bon yl h yd r a zin o-
for m a m id e] (15). This was prepared by an adaptation of the
method of Harris.21 To a solution of bis-hydrazide (13) (48 mg,
0.11 mmol) in CH2Cl2 (5 mL) at room temperature were added
pyridine (0.012 mL, 0.12 mmol) and N-bromosuccinimide (19
mg, 0.11 mmol). The resulting orange solution was stirred at
room-temperature overnight, diluted with CH2Cl2 (20 mL), and
washed with water and brine. The organic layer was dried
(MgSO4), and the solvent was removed in vacuo. The title
compound was purified by column chromatography on silica
gel eluting with EtOAc to give an orange solid (34 mg, 71%);
mp 125-126 °C; IR (CHCl3 cast) 3252, 2933, 1738, 1732, 1715,
and 1651 cm-1; UV (CH3CN) 273 (ꢀ 1777) and 414 (ꢀ 38) nm;
1H NMR (300 MHz, CDCl3) δ 1.36 (9 H, s), 2.81 (2 H, s), 2.90-
2.94 (5 H, m), 2.99 (3 H, s), 3.70 (2 H, dt, J ) 6.7, 6.8 Hz,
becomes t in D2O), 3.92 (2 H, br s), 6.52 (1 H, br s, exchanges
in D2O), 7.27 (5 H, m) and 7.73 (1 H, br s, exchanges in D2O);
MS (+ve ES) 457.1 (MNa+, 43%), 435.2 (MH+, 45%) and 379.1
(M+ - C4H8, 100%); MS (+ve ES) Found: 457.216799.
1
1727, and 1309 cm-1; H NMR (500 MHz, DMSO-d6, 120 °C)
δ 1.39 (9 H, s), 2.95 (3 H, s), 3.34 and 4.00 (2 H, 2 × t, J ) 7.5
Hz), 3.92 (2 H, m), 5.13 (2 H, m), 5.25 (2 H, m), and 7.35 (10
H, m); MS (-ve ES) 405.6 (M - Boc-, 34%) and 371.7 (M -
CO2CH2Ph-, 100%); MS (+ve ES) 529.3 (MNa+, 11%). Anal.
Calcd for C24H36N2O5S: C, 56.91; H, 5.97; N, 5.53%. Found:
C, 57.18; H, 6.03; N, 5.47%.
2-[N2-(ter t-Bu toxycar bon yl)h ydr azin o]eth yl Meth yl Su l-
fon e (20). This was prepared by a method similar to that
described for the preparation of 8. A solution of sulfone (19)
(1.677 g, 3.31 mmol) in MeOH (50 mL) was stirred under a
hydrogen atmosphere in the presence of 10% Pd/C (160 mg)
until the uptake of hydrogen gas ceased (20 min) and then for
a further 1 h. The suspension was filtered through Celite and
the solvent removed in vacuo to yield a white solid (769 mg,
97%). A sample was recrystallized from EtOAc/hexane for
analysis. mp 99-100 °C; IR (CHCl3 cast) 3334, 3321, 3308,
2928, 1687, and 1287 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.43
(9 H, s), 2.98 (3 H, s), 3.17 (2 H, dt, J ) 0.6, 6.3 Hz), 3.36 (2 H,
t, J ) 6.3 Hz), 4.25 (<1 H, br t, J ) 0.6 Hz, exchanges with
D2O), and 6.12 (<1 H, br s, exchanges with D2O); 13C NMR
(75.5 MHz, CDCl3) δ 28.33, 41.98, 45.69, 45.96, 75.81, and
156.88; MS (+ve ES) 261.5 (MNa+, 52%), 183.6 (M+ - C4H9,
C
C
20H30N5O5Na (MNa+) requires 457.217538. Anal. Calcd for
20H30N6O5: C, 55.29; H, 6.96; N, 19.34%. Found: C, 55.09;
H, 7.00; N, 19.00%.
Azo-N1-(p h en et h ylfor m a m id e)-N1′-[N,N-d im et h ylp r o-
p a n a m id e-a cetyl h yd r a zin ofor m a m id e] (16). This was
prepared using a method similar to that described for the
preparation of 15. Bis-hydrazide (14) (26 mg, 0.07 mmol) in
CH2Cl2 (2 mL) was treated with pyridine (8 µL, 0.08 mmol)
and N-bromosuccinimide (13 mg, 0.08 mmol) at room temper-
ature for 5 h. The solvent was removed in vacuo, and the

Azodicarboxamides as Cysteine Proteinase Inhibitors
100%) and 139.6 (M+
C8H18N2O4S: C, 40.32; H, 7.61; N, 11.76%. Found: C, 40.37;
H, 7.80; N, 11.63%.
J . Org. Chem., Vol. 64, No. 26, 1999 9545
-
Boc, 3%). Anal. Calcd for
heated at reflux for 6 h, cooled, and stirred at room-temper-
ature overnight. Removal of the solvent in vacuo followed by
purification by column chromatography on silica gel, eluting
with 5% MeOH in EtOAc, yielded benzyl ester (24) as an oil
(77 mg, 16%) and hydantoin (25) as a white solid (197 mg,
51%).
2-[N1-(P en ta flu or op h en oxyca r bon yl)-N2-(ter t-bu toxy-
ca r bon yl)h yd r a zin o]eth yl Meth yl Su lfon e (21). To a solu-
tion of hydrazide (20) (332 mg, 1.40 mmol) in THF (10 mL) at
room temperature under an argon atmosphere was added
pyridine (0.138 mL, 1.40 mmol). After stirring for 2 min, bis-
pentafluorophenyl carbonate21 (500 mg, 1.27 mmol) was added
and the stirring continued overnight. The solvent was removed
in vacuo and the residue purified by column chromatography
on silica gel eluting with a gradient of (2:1) hexane/EtOAc to
(1:1) hexane/EtOAc. The product was collected as a white solid
which was recrystallized from hexane/EtOAc (490 mg, 86%);
mp 132-133 °C; IR (CHCl3 cast) 3316, 2983, 2935, 1758, and
1523 cm-1; 1H NMR (300 MHz, CDCl3) δ 1.48 (9 H, s), 2.98 (3
H, s), 3.41 and 3.47 (2 H, br t and br s, J ) 6.0 Hz), 4.10 and
4.23 (2 H, br s and br t, J ) 6.0 Hz), and 6.98 (<1 H, br s,
exchanges with D2O); MS (+ve ES) 471.0 (MH+, 100%), 393.0
(M+ - C4H7, 36%), 349.0 (M+ - Boc, 30%). Anal. Calcd for
Da ta for ester (24): [R]26 -5.8° (c 1, MeOH); IR (CHCl3
D
cast) 2977, 2938, 1736, 1679, and 1497 cm-1
;
1H NMR (300
MHz, CDCl3) δ 1.45 (9 H, s), 2.65 (2 H, br s), 2.68 (3 H, s),
2.95 (3 H, s), 3.07 (2 H, d, J ) 6.4 Hz), 3.65 (2 H, br s), 4.75 (1
H, dd, J ) 6.4, 14.0 Hz), 5.10 (2 H, ABq, J ) 12.4 Hz), 6.06 (1
H, d, J ) 7.3 Hz), 6.21 (1 H, s), 7.05-7.61 (10 H, m), and 7.64
(1 H, br s); 13C NMR (75.5 MHz, CDCl3) δ 28.18, 32.23, 35.53,
37.30, 38.26, 46.53, 54.19, 66.87, 82.27, 126.75, 128.24, 128.35,
128.40, 128.48, 128.93, 129.23, 129.45, 135.40, 136.27, 155.82,
157.58, 158.42, 171.96, and 172.04; MS (+ve ES) 593.3 (MNa+,
75%), 571.3 (MH+, 100%), 515.2 (M - C4H7, 16%), and 417.2
(M - Boc, 16%); MS (+ve ES) Found: 593.269838. C28H38N6O7-
Na (MNa+) requires 593.269968.
Da ta for h yd a n toin (25): mp 89-93 °C; [R]26 -53.3° (c
D
1.06, CHCl3); IR (CHCl3 cast) 3274, 1741, 1629, and 1498 cm-1
;
C
15H17F5N2O6S: C, 40.18; H, 3.82; N, 6.25%. Found: C, 40.11;
1Η NMR (300 MHz, CDCl3) δ 1.46 (9 H, s), 2.65 (2 H, br s),
2.84 (1 H, dd, J ) 9.9, 13.9 Hz), 2.90 (3 H, s), 2.95 (3 H, s),
3.32 (1 H, br d, J ) 13.9 Hz), 3.50-4.00 (<2 H, br s), 4.27 (1
H, br d, J ) 6.6 Hz), 5.86 (1 H, br s, exchanges with D2O),
7.18-7.32 (5 H, m), and 7.56 (1 H, br s, exchanges with D2O);
13C NMR (75.5 MHz, CDCl3) δ 28.10, 32.01, 35.61, 37.29, 38.15,
46.68, 57.28, 82.35, 127.40, 129.00, 129.24, 135.59, 154.79,
170.83, and 171.86; MS (+ve ES) 947.5 (M2Na+, 13%), 485.2
(MNa+, 27%), 463.2 (MH+, 100%), and 407.1 (M+ -C4H7, 20%);
MS (+ve ES) Found: 485.212846. C21H30N6O6Na (MNa+)
requires 485.212453.
H, 3.71; N, 6.16%.
2-[N1-P h en eth yla m id oh yd r a zid o] 2-[N2-(ter t-bu toxy-
ca r bon yl)h yd r a zin o]eth yl Meth yl Su lfon e (22). To a solu-
tion of pentafluorophenoxy hydrazide (21) (162 mg, 0.36 mmol)
in THF (25 mL) at room temperature under an argon atmo-
sphere was added hydrazine monohydrate (0.053 mL, 1.09
mmol) followed by triethylamine (0.151 mL, 1.09 mmol). After
2.5 h, phenethyl isocyanate (0.243 mL, 1.81 mmol) was added
followed by a further portion of triethylamine (0.151 mL, 1.09
mmol), and a white suspension formed. After stirring at room-
temperature overnight, the solvent was removed in vacuo, and
the residue was diluted with EtOAc (50 mL) and filtered. The
filtrate was concentrated in vacuo and purified by column
chromatography on silica gel eluting with 5% MeOH in EtOAc
to yield a white solid (75 mg, 47%); mp 79-82 °C; IR (CHCl3
ter t-Bu toxyca r bon yl-N,N-d im eth yla za glu ta m in e-a za -
glycin e-(2S)-p h en yla la n a m id e (26). To a solution of hy-
dantoin (25) (47 mg, 0.10 mmol) in 2-propanol (1 mL) at room
temperature was added concentrated ammonium hydroxide
(0.25 mL) and the mixture stirred overnight. TLC analysis
indicated that unreacted starting material remained, a further
portion of NH4OH (0.25 mL) was added, and stirring was
continued. After 4 h a final portion of NH4OH (0.40 mL) was
added and the reaction stirred overnight. Removal of the
solvents in vacuo followed by purification by column chroma-
tography eluting with 5-10% MeOH in EtOAc gave 26 which
was recrystallized from MeOH/EtOAc (18 mg, 37%); IR
(microscope) 3449, 3334, 3283, 3213, 3024, 2980, 1709, 1681,
1642, 1608, and 1553 cm-1; 1H NMR (300 MHz, CD3OD) δ 1.49
(9 H, s), 2.68 (2 H, t, J ) 7.0 Hz), 2.92 (4 H, m), 3.04 (3 H, s),
3.22 (1 H, dd, J ) 4.4, 14.1 Hz), 3.71 (2 H, br s), 4.45 (1 H, dd,
J ) 4.8, 9.4 Hz), and 7.23 (5 H, m); MS (+ve ES) 502.2 (MNa+,
100%), 480.3 (MH+, 65%), and 380.2 (M+ - Boc, 20%); MS (+ve
ES) Found: 502.239497. C21H33N7O6Na (MNa+) requires
502.239002.
Azo-N1-(ca r boxy-(2S)-p h en yla la n a m id e)- N1′-[N,N-d im -
et h ylp r op a n a m id e-ter t-b u t oxyca r b on yl h yd r a zin ofor -
m a m id e] (27). This was prepared using a method similar to
that described for the preparation of 15. Amide (26) (14 mg,
0.03 mmol) in CH2Cl2 (5 mL) was treated with pyridine (3 µL,
0.03 mmol) and N-bromosuccinimide (6 mg, 0.03 mmol) at
room temperature for 2.5 h during which time a red/orange
color developed. The solvent was removed in vacuo and the
residue purified by column chromatography on silica gel
eluting with 5% MeOH in CH2Cl2 to give 26 as a yellow oil
(2.5 mg, 18%); 1H NMR (300 MHz, CDCl3) showed the presence
of a 4:1 mixture of conformers, data for major compound: δ
1.36 (9 H, s), 2.81 (2 H, br s), 2.95 (3 H, s), 3.00 (3 H, s), 3.09
(1 H, dd, J ) 8.2, 13.8 Hz), 3.26 (1 H, dd, J ) 5.8, 13.8, Hz),
3.92 (2 H, br s), 4.65 (1 H, ddd, J ) 5.7, 8.0, 10.1 Hz), 5.39 (1
H, br s), 5.50 (1 H, br s), 7.25 (5 H, m), and 7.75 (1 H, br s);
MS (+ve ES) 500.2 (MNa+, 100%) and 478.2 (MH+, 15%); MS
(+ve ES) Found: 500.222980. C21H31N7O6Na (MNa+) requires
500.223352.
cast) 3320, 2979, 2931, 1717, 1671, 1546, and 1291 cm-1; H
1
NMR (300 MHz, CDCl3) δ 1.43 (9 H, s), 2.77 (2 H, t, J ) 7.6
Hz), 2.92 (3 H, s), 3.37 (4 H, br d, J ) 6.3 Hz), 4.00-4.50 (<2
H, br s), 6.00 (<1 H, br s), 6.83 (<1 H, br s), 7.13-7.27 (5 H,
m), 8.35 (1 H, s), and 8.39 (1 H, s); 13C NMR (75.5 MHz, CDCl3)
δ 28.25, 36.27, 40.88, 41.62, 43.42, 51.84, 82.59, 126.37, 128.53,
128.82, 138.97, 155.04, 158.39, and 159.76; MS (+ve ES) 887.5
(M2H+, 14%), 466.1 (MNa+, 100%), and 444.1 (MH+, 8%); MS
(+ve ES) Found: 466.173494. C18H29N5O6SNa (MNa+) requires
466.173626.
Azo-N1-(p h en et h ylfor m a m id e)-N1′-[2-(N2-ter t-b u t oxy-
ca r bon yl)h yd r a zin o]et h yl Met h yl Su lfon e F or m a m id e
(23). This was prepared using a method similar to that
described for 15. Bis-hydrazide (22) (50 mg, 0.11 mmol) in CH2-
Cl2 (5 mL) was treated with pyridine (0.012 mL, 0.12 mmol)
and N-bromosuccinimide (22 mg, 0.12 mmol) under an argon
atmosphere for 2 h. Removal of the solvent in vacuo followed
by purification by column chromatography on silca gel, eluting
with EtOAc, yielded 23 as a yellow solid (50 mg, quantitative).
A sample was recrystallized from EtOAc/hexane; mp 111-112
°C; IR (CHCl3 cast) 3300, 2981, 2933, 1732, and 1369 cm-1
;
1
UV (CH3CN) 250 (ꢀ 2326) and 419 (ꢀ 50) nm; H NMR (300
MHz, CDCl3) δ 1.38 (9 H, s), 2.93 (2 H, t, J ) 7.1 Hz), 3.00 (3
H, s), 3.45 (2 H, t, J ) 6.4 Hz), 3.73 (2 H, dt, J ) 6.3, 13.3 Hz),
4.16 (2 H, m), 6.68 (1 H, br s), and 7.25 (6 H, m); 13C NMR
(75.5 MHz, CDCl3) δ 27.96, 35.24, 41.34, 42.22, 44.43, 51.52,
126.84, 128.68, 128.84; MS (+ve ES) 480.2 (MK+, 20%), 464.2
(MNa+, 100%) and 459.2 (MNH4+, 23%); MS (+ve ES) Found:
464.157347. C18H27N5O6SNa (MNa+) requires 464.157975.
Anal. Calcd for C18H27N5O6S: C, 48.97; H, 6.16; N, 15.86%.
Found: C, 49.12; H, 6.40; N, 14.72%.
ter t-Bu toxycar bon yl-N,N-(dim eth yl)azaglu tam in e-aza-
glycin e-(2S)-p h en yla la n in e Ben zyl Ester (24) a n d N-(4S-
Ben zyl-2,5-d ioxoim id a zolid in -1-yl)-N-(ter t-bu toxyca r bo-
n ylh yd r a zin o)-N,N-d im eth ylp r op a n a m id e Ur ea (25). To
a solution of L-phenylalanine benzyl ester (264 mg, 1.04 mmol)
in chloroform (10 mL) at room temperature was added tri-
ethylamine (0.26 mL, 1.86 mmol) followed by phenoxy bishy-
drazide (11) (339 mg, 0.83 mmol). The resulting solution was
Ack n ow led gm en t. The authors thank Dr. Bruce
Malcolm (Schering Plough), Dr. David Wishart, and

9546 J . Org. Chem., Vol. 64, No. 26, 1999
Hill and Vederas
Scott Watson (Faculty of Pharmacy, University of
Alberta) for enzyme preparation, Manjinder S. Lall for
assistance with enzyme assays, and Dr. J onathan
Parrish and Dr. Michael J ames (Biochemistry Depart-
ment, University of Alberta) for molecular modeling and
helpful discussion. We are indebted to Dr. J ohn McK-
endrick and Constantine Karvellas for extensive pre-
liminary studies on other types of azo and hydrazo
compounds as potential enzyme inhibitors. These in-
vestigations were supported by the U.S. National In-
stitutes of Health (Grant AI 38249) and the Natural
Sciences and Engineering Research Council of Canada.
Su p p or tin g In for m a tion Ava ila ble: Su p p or tin g In -
for m a tion Ava ila ble: 1H and 13C NMR spectra of selected
hydrazides and azodicarboxamides. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O9915123