Synthesis and Evaluation of Macrocyclic Transition State Analogue Inhibitors for α-Chymotrypsin

Article abstract of DOI:10.1021/jo034837z

Lactams 1 and 2 are readily formed from acyclic precursors in the presence of trypsin and chymotrypsin, respectively, identifying the macrocyclic ring system as a potential motif for constrained transition state analogue inhibitors of the serine peptidases. Ketone 3 was synthesized and shown to be a modest inhibitor of chymotrypsin (K_i = 220 μM), albeit 4-fold more potent than the acyclic hydroxy acid 25 (K_i = 1.5 mM as a mixture of epimers). A precursor (31) to the amino boronic acid 4 was also prepared; although this derivative was a potent inhibitor of chymotrypsin (K_i = 130 nM) by virtue of the boronic acid moiety, it showed no advantage over the des-amino analogue 32 (K_i = 120 nM), which is not capable of cyclizing.

Full text of DOI:10.1021/jo034837z

Syn th esis a n d Eva lu a tion of Ma cr ocyclic Tr a n sition Sta te
An a logu e In h ibitor s for r-Ch ym otr yp sin
Kristina K. Hansen, Benjamin Grosch, Sorana Greiveldinger-Poenaru, and Paul A. Bartlett*
Center for New Directions in Organic Synthesis,† Department of Chemistry, University of California,
Berkeley, California 94720-1460
paul@fire.cchem.berkeley.edu
Received J une 16, 2003
Lactams 1 and 2 are readily formed from acyclic precursors in the presence of trypsin and
chymotrypsin, respectively, identifying the macrocyclic ring system as a potential motif for
constrained transition state analogue inhibitors of the serine peptidases. Ketone 3 was synthesized
and shown to be a modest inhibitor of chymotrypsin (Ki ) 220 µM), albeit 4-fold more potent than
the acyclic hydroxy acid 25 (Ki ) 1.5 mM as a mixture of epimers). A precursor (31) to the amino
boronic acid 4 was also prepared; although this derivative was a potent inhibitor of chymotrypsin
(Ki ) 130 nM) by virtue of the boronic acid moiety, it showed no advantage over the des-amino
analogue 32 (Ki ) 120 nM), which is not capable of cyclizing.
Design of In h ibitor s
macrocyclic transition state analogue.7 For synthetic
expediency, an isobutyl group representing a leucine side
chain was incorporated at the P1 position in place of the
phenylalanine benzyl group, even though this replace-
ment was expected to reduce the binding affinity ap-
proximately 16-fold (based on the rates of deacylation for
a series of acyl-R-chymotrypsins).8,9 Finally, compound
4 was synthesized to explore the possibility that, with a
favorable ring conformation, a cyclic boronate ester could
be formed in the enzyme active site and lead to even more
potent inhibition.10
In the preceding paper, we describe the application of
an on-bead, enzyme-catalyzed cyclization process as a
way to screen for macrocyclic frameworks of potential
relevance in the design of conformationally constrained
peptidase inhibitors.1 In this report, we present the
synthesis and evaluation of some transition state ana-
logue inhibitors based on a macrocyclic motif identified
in this manner. The ring system represented by lactams
1 and 2 is one of the simplest of those whose formation
is readily catalyzed by trypsin and R-chymotrypsin. Thus,
to assess the validity of this approach for the identifica-
tion of effective inhibitor scaffolds, ketone 3 and boronic
acid 4 were synthesized and evaluated as inhibitors of
R-chymotrypsin.
Syn th esis of In h ibitor s
La ct a m 2. PEGA (poly(ethylene glycol)-polyacryl-
amide) beads bearing a photocleavable linker11 were
subjected to four cycles of standard solid-phase peptide
synthesis to afford resin-linked NR-Boc-Nâ-Fmoc-(S)-R,â-
diaminopropanoyl-Leu-Gly-Gly, 5 (Scheme 1).12 Depro-
tecting the â-amino group followed by acylation with
N-Cbz-threonine gave alcohol 6, which was acylated with
N-Fmoc-L-aspartic acid 1-allyl ester to give ester 7. After
cleavage of the allyl protecting group,13 the resulting acid
The substrate-like lactam 2 was included among the
analogues to be synthesized and evaluated because of the
precedents of naturally occurring cyclic depsipeptides as
potent inhibitors of serine proteases (see, for example,
the cyanopeptolins).2 Peptidyl boronic acids are among
the most potent inhibitors of serine proteases known.3-6
The ketomethylene derivative 3 was prepared as a stable,
(3) Katz, B. A.; Finer-Moore, J .; Mortezaei, R.; Rich, D. H.; Stroud,
R. M. Biochemistry 1995, 34, 8264.
(4) Kettner, C. A.; Shenvi, A. B. J . Biol. Chem. 1984, 259, 15106-
15114.
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18289-18297.
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Chang, C. W.; Kettner, C. A. Biochemistry 1995, 34, 3750-3757.
(7) J etten, M.; Peters, C. A. M.; Visser, A.; Grootenhuis, P. D. J .;
van Nipsen, J . W.; Ottenheijm, H. C. J . Bioorg. Med. Chem. 1995, 3,
1099-1114.
(8) Ingles, D. W.; Knowles, J . R. Biochem. J . 1967, 104, 369-377.
(9) Mader, M. M.; Bartlett, P. A. Chem. Rev. 1997, 97, 1281-1301.
(10) Tian, Z. Q.; Brown, B. B.; Mack, D. P.; Hutton, C. A.; Bartlett,
P. A. J . Org. Chem. 1997, 62, 514-522.
(11) Holmes, C. P. J . Org. Chem. 1997, 62, 2370-2380.
(12) Fields, G. B.; Noble, R. L. Int. J . Pept. Protein Res. 1990, 35,
161-214.
† The Center for New Directions in Organic Synthesis is supported
by Bristol-Myers Squibb as a sponsoring member and Novartis Pharma
as supporting member.
(1) Hansen, K. K.; Hansen, H. C.; Clark, R. C.; Bartlett, P. A. J .
Org. Chem. 2003, 68, 8459-8464.
(2) Bonjouklian, R.; Smitka, T. A.; Hunt, A. H.; Occolowitz, J . L.;
Perun, T. J . J .; Doolin, L.; Stevenson, S.; Knauss, L.; Wijayaratne, R.;
Szewczyk, S.; Patterson, G. M. L. Tetrahedron 1996, 52, 395-404.
10.1021/jo034837z CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/08/2003
J . Org. Chem. 2003, 68, 8465-8470
8465

Hansen et al.
SCHEME 1
SCHEME 2
to the sequence outlined below for the leucine analogue
(see Scheme 2). However, at the stage of intermediate
16, we were unable to effect hydrogenation of the nitrile
group without concomitant reduction of the phenyl ring.
Although in hindsight we should have carried the result-
ing cyclohexylalanine derivative forward,15 we elected to
prepare the leucine analogue 3 instead.
was activated and coupled with L-phenylalanine methyl
ester to provide compound 8. The succinyl-valyl moiety
was then introduced in a stepwise fashion, and the Boc
protecting group was removed from the R-position group
of the diaminopropanoyl (Dap) unit prior to photolytic
cleavage from the resin. Cyclization of the amino ester
was accomplished in high yield in acetonitrile/water with
R-chymotrypsin.
Ma cr ocyclic Keton e 3. Prior to carrying out the
synthesis of macrocycle 3, with a leucine side chain at
the P1 position, we undertook to synthesize the phenyl-
alanine analogue 10. Two routes were explored, but the
difficulties encountered in each led us to abandon this
target. In the first route, we prepared the ketone 11
described by De´ziel et al. as a 5:1 mixture of diastereo-
mers.14 After several steps involving protecting group
manipulation, we obtained the carboxylic acid 12. Un-
fortunately, attempts to introduce the side chain amino
group by Curtius rearrangement and trapping of the
isocyanate intermediate with a suitable alcohol to give
carbamate 13 were sidetracked by formation of the
lactam 14 or dihydrouracil 15 instead. An alternative,
non-stereoselective route was then pursued, analogous
(13) Kates, S. A.; Daniels, S. B.; Sole´, N. A.; Barany, G.; Albericio,
F. Proc. American Pept. Symp. 13th 1994.
(14) De´ziel, R.; Plante, R.; Caron, V.; Grenier, L.; Llinas-Brunet, M.;
Duceppe, J .-S.; Malenfant, E.; Moss, N. J . Org. Chem. 1996, 61, 2901-
2903.
The key component required for synthesis of ketone 3
was a suitably protected ketomethylene analogue of
8466 J . Org. Chem., Vol. 68, No. 22, 2003

Macrocyclic Transition State Analogue Inhibitors
N-(NR-leucyl-R,â-diaminopropanoyl)leucinamide (Leu-(S)-
Dap-Leu-NH2). This moiety was constructed as the
reduced amino alcohol 21, as shown in Scheme 2.
Alkylation of the Boc-leucine bromomethyl ketone 1716
with methyl cyanoacetate afforded cyano ketone 18,
which possesses the carbon skeleton of the Leu-Dap
dipeptide analogue. To avoid problems anticipated on
generation of a primary amine in proximity to the ketone
and the methyl ester, 18 was reduced with NaBH4 in
MeOH and the resulting mixture of hydroxy ester and
lactone 19 was converted entirely to the latter with
DMAP in 52% overall yield.17 Although four diastereo-
mers of lactone 19 were present, separation was not
attempted at this stage since one of the stereocenters was
to be removed later and because separation and assign-
ment of the two final isomers were more readily ac-
complished when the macrocyclic materials were in hand.
The lactone of 19 served as an activated form of the
carboxyl group, coupling with leucinamide in the pres-
ence of stannous acetate in DMF to give amide 20 in good
yield. Hydrogenation of the nitrile moiety over PtO2 in
acetic acid, followed by neutralization with an anion-
exchange resin, provided the key intermediate 21 in 85%
yield.
The remaining steps in elaboration of macrocyclic
ketone 3 began with acylation of the primary amine in
21 with an N,O-diprotected threonine derivative.18 The
yield in this straightforward reaction was reduced by
competing relactonization, which led to loss of the leu-
cinamide moiety. SO3/pyridine oxidation19 of the free
hydroxyl group in the acylated product gave keto amide
22, and removal of the silyl protecting group20 and
esterification with the â-carboxyl group of N-Alloc-L-
aspartate R-tert-butyl ester21 then provided compound 23
in good yield. Cleavage of the tert-butyl ester and N-Boc
protecting group followed by cyclization of the resulting
amino acid with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HATU)22 and
DIEA in DMF provided macrocycle 24 as a mixture of
diastereomers in 88% yield.23 The diastereomeric prod-
ucts were separated with difficulty by silica gel chroma-
tography, providing small amounts (∼8%) of each epimer
in ca. 80% purity. Distinctive NOESY NMR spectra were
obtained for each diastereomer, but the interactions
observed were not conclusive in assigning the stereo-
chemistry of each compound. The remaining synthetic
steps were carried out on each diastereomer separately,
and the S- and R-configurations at C-5 were assigned at
a later stage based on their activity as inhibitors of
chymotrypsin.
palladium(0) and diethylamine as the allyl scavenger.
Boc-valine was coupled to each amino terminus and
deprotected with TFA in CH2Cl2. Finally, succinic anhy-
dride, DMAP, and pyridine in CH2Cl2 provided the
succinylated compounds 3-S and 3-R in 16-17% yield
over the four steps.
Acyclic Keton e 25. To provide a comparison com-
pound for the macrocyclic ketone 3-S, the lactone linkage
was cleaved with aq K2CO324 to give the hydroxy acid 25.
These conditions also caused epimerization at the ste-
reocenter adjacent to the ketone to give a 60/40 mixture
of diastereomers.
Bor on ic Acid 4. Synthesis of the boronic acid ana-
logue 4, in the form of precursor 31, began with prepara-
tion of the (+)-pinanediol boronate analogue of L-phenyl-
alanine, 28. This material was prepared in enantio-
merically pure form using the procedure of Matteson and
Sadhu (Scheme 3).25,26 Benzylboronic acid was formed
SCHEME 3
from the Grignard reagent and trimethyl borate and
esterified with (+)-pinanediol in the presence of MgSO4.
The product 26 was homologated with dichloromethyl-
lithium (generated at -100 °C) in the presence of zinc
chloride to give the (S)-1-chloro-2-phenylethyl boronate
diastereomer 27. We found the stereoselectivity of this
transformation to be very sensitive to moisture; with
strictly anhydrous ZnCl2, diastereomerically pure mate-
rial was obtained, but without rigorous care to exclude
water, 1:1 diastereomeric mixtures resulted. The chloride
was displaced with inversion with lithium hexamethyl-
disilazide in THF at -78 °C, and the crude product as a
solution in hexane was converted to the hydrochloride
28 with HCl in dioxane, again at -78 °C. Although we
did not optimize the procedures, and thus obtained the
final product in only a modest overall yield of 45%, we
include the experimental details in the Supporting
Information, since we do not believe they have been
reported previously for this analogue.
The Alloc protecting group was removed from macro-
cycles 24-S and 24-R with tetrakis(triphenylphosphine)-
(15) J ones, J . B.; Kunitake, T.; Niemann, C.; Hein, G. E. J . Am.
Chem. Soc. 1965, 87, 1777.
(16) Rotella, D. P. Tetrahedron Lett. 1995, 36, 5453-5456.
(17) Tao, J .; Hoffman, R. V. J . Org. Chem. 1997, 62, 6240-6244.
(18) Kurokawa, N.; Ohfune, Y. Tetrahedron 1993, 49, 6195-6222.
(19) Parikh, J . R.; Doering, W. V. E. J . Am. Chem. Soc. 1967, 89,
5505-5507.
(20) Corey, E. J .; Venkateswarlu, A. J . Am. Chem. Soc. 1972, 94,
6190-6191.
(24) Kaestle, K. L.; Anwer, M. K.; Audhya, T. K.; Goldstein, G.
Tetrahedron Lett. 1991, 32, 327-330.
(21) Unverzagt, C.; Kunz, H. Bioorg. Med. Chem. 1994, 2, 1189-
1201.
(25) Matteson, D. S.; Sadhu, K. M. J . Am. Chem. Soc. 1981, 103,
5241-524.
(22) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber,
H.; Carpino, L. A.; Bienert, M. J . Org. Chem. 1996, 61, 8831-8838.
(23) Wipf, P.; Uto, Y. J . Org. Chem. 2000, 65, 1037-1049.
(26) Matteson, D. S.; Sadhu, K. M. Organometallics 1984, 3, 614-
618.
J . Org. Chem, Vol. 68, No. 22, 2003 8467

Hansen et al.
SCHEME 4
case. The inclusion of scavengers (anisole, thioanisole,
thiophenol, or triisopropylsilane/water) did not suppress
these side reactions. The sensitivity of 30a was not a
function of the resin-depsipeptide component, since re-
moval of the Boc group from the precursor 7 could be
effected cleanly under a variety of conditions. Nor was
the sensitivity due to the R-acyl boro-Phe unit alone; the
N-acetyl derivative of boro-Phe pinanyl ester was per-
fectly stable to all of the Boc-deprotection conditions
evaluated. While the Achilles heel of 30a was not
identified with certainty, the need to replace the Boc
group with a protecting group that could be removed
under different conditions was clearly indicated.
The instability of boro-Phe derivative 28 to DIEA led
us to worry that the conditions for removal of the Fmoc
protecting group from the aspartic acid unit might also
be incompatible with the boronate ester. Unfortunately,
these concerns were justified, as deboronated compounds
were the major products resulting from treatment of 30a
with 20% piperidine in DMF for 20 min. Replacement of
the Fmoc group of 7 with an acetyl moiety prior to
coupling with boro-Phe proceeded without complication,
but the coupled product (28a , with P G2 ) Ac) was formed
as a mixture of diastereomers, presumably having epimer-
ized at the Asp R-position during activation.
The difficulties encountered with the Boc and Fmoc
protecting groups were avoided by repeating the synthe-
sis of the depsipeptide precursor, incorporating 2-(tri-
methylsilyl)ethoxycarbonyl (Teoc) and methoxycarbonyl
(Meoc) groups, respectively, in intermediate 9. We an-
ticipated that the Teoc group could be removed from the
Dap unit with fluoride under alkaline or mildly acidic
conditions, and the Meoc group was to be retained in the
final product.27 With the optimized coupling conditions
described above, the boro-Phe pinanyl ester was coupled
to the Asp carboxyl group after deallylation to give 30b
in 55-63% yield and without any deboronated product.
Ultimately, Teoc proved to be the right protecting
group but for the wrong reason. Attempts to effect
cleavage with fluoride ion also resulted in significant
deboronation, whether under basic (TBAF), slightly acidic
(HF/pyridine), or neutral conditions (tris(dimethylamino)-
sulfonium difluorotrimethyl silicate, TAS-F).28,29 Fortu-
nately, however, the Teoc group was sufficiently acid
labile that it could be removed cleanly with 20% TFA in
dichloromethane, with no significant amount of debor-
onation. We do not have a good explanation for why the
Teoc group, but not the Boc group, could be removed
cleanly under essentially the same conditions.
Subsequent steps in the synthesis of boronate 31
followed closely the route described for the lactam
precursor above (Scheme 4). After deallylation of depsi-
peptide intermediate 7, considerable effort was required
to optimize the coupling reaction between the resin-bound
acid and the boro-Phe derivative. Conventional coupling
conditions, such as DIC/HOBt, PyBOP, or BOP and
DIEA, with or without HOBt, produced significant
amounts of the deboronated 2-(phenylethyl)amide as a
contaminant. This side reaction appeared to be a function
of the DIEA; indeed, when the boro-Phe component was
treated with the base prior to addition of the other
reagents, deboronated material was the major product.
Conversely, when boro-Phe was added 10 min after all
the other components were combined and the resin-bound
acid had presumably been activated, the desired product
30a was formed in acceptable yield (ca. 65%) with only a
trace of the contaminant.
Conditions required for removal of either the Boc or
the Fmoc protecting groups of 30a proved to be incom-
patible with the boronate moiety, at least in the context
of these complex analogues. A number of acid reagents
were explored for cleavage of the Boc group, including
TFA/CH2Cl2 mixtures, HCl or H2SO4 in ether or dioxane,
or TMSCl/phenol, but loss of the boronate ester function
and formation of a plethora of products resulted in every
After the conditions had been optimized for the solid-
phase synthesis of the boronate ester 31, a preparative-
scale synthesis was carried out and material was isolated
and purified after photolytic detachment from the resin
at 366 nm. Photolysis was carried out with three cycles
of irradiation for 4 h, at which point no further material
was isolated from the resin. The crude product was
purified by reverse-phase HPLC to give the final product
in 16% isolated yield over the 16 steps of the synthesis.
To assess whether any potential cyclic boronate-
enzyme adducts would result in enhanced binding affin-
ity, the comparison compound 32 lacking the nucleophilic
amino group was also synthesized. The route followed
that outlined in Scheme 4, except for the simplification
8468 J . Org. Chem., Vol. 68, No. 22, 2003

Macrocyclic Transition State Analogue Inhibitors
TABLE 1. In h ibition of r-Ch ym otr yp sin by Ma cr ocyclic
CHART 1
Tr a n sition Sta te An a logu esa
compound
lactam 2
ketone 3-S
ketone 3-R
acyclic ketone 25
boronate 31
boronate 32
Ki (µM)
comments
slow substrate
configuration assigned on
basis of affinity
as mixture of epimers
pinanediol esters hydrolyze to
boronic acids in buffer
1100
220
1700
1500
0.13
0.12
a
Inhibition was determined at 25 °C in pH 7.8 Tris, 0.10 M
CaCl2 buffer or pH 7.0 HEPES buffer (for boronates 3 and 16)
with succinyl-Ala-Ala-Pro-Phe p-nitroanilide as substrate.
gauged by Km values) by about 5-fold31,32 and their
transition state affinity (as gauged by kcat/Km) by 40-fold.33
Thus, even though the ketone in macrocycle 4-S is not
particularly electron deficient, it nonetheless enhances
the binding affinity significantly over what would be
expected for an analogue of lactam 2 with leucine at the
P1 position.
afforded by replacement of the protected R,â-Dap moiety
with 3-(Fmoc-amino)propanoic acid.
Boronic acid peptide analogues readily form enzyme
adducts that mimic the tetrahedral intermediate of the
deacylation step in the mechanism of serine proteases
and, as a consequence, are among the most potent
reversible inhibitors of these enzymes. High affinity was
therefore expected for the boronic acids from hydrolysis
of 31 and 32. Although these inhibitors are bound more
weakly than analogues that occupy extended binding
sites,4 they are comparable to other analogues limited to
the S1 and S2 sites (Chart 1).10 However, the similar
affinity of the amino boronate 31 and the des-amino
analogue 32 suggests that the former does not form a
macrocyclic adduct with the enzyme (e.g., 33) that would
mimic the tetrahedral intermediate of the acylation step
of the enzyme mechanism. These results are reminiscent
of our earlier attempts to find evidence for such cyclic
adducts with the simpler analogues, 34; these derivatives
also had similar inhibition constants (70-130 nM),
irrespective of the presence or absence of a nucleophilic
group on the P1′ residue.10
In h ibition of r-Ch ym otr yp sin
Lactam 2, the two diastereomers of macrocyclic ketone
3, and boronate 31 were evaluated as competitive inhibi-
tors of R-chymotrypsin, along with acyclic ketone 25
(mixture of epimers) and boronate 32 as comparison
compounds; the results are listed in Table 1. The relative
affinities of the two diastereomers of ketone 3 were used
to assign their configurations. These isomers differ at the
center equivalent to the R-carbon of the P1′ residue; the
“natural” S-configuration was therefore assigned to the
higher affinity isomer (Ki ) 220 µM). Comparison to the
acyclic hydroxy acid 25 indicates that the macrocyclic
structure of 3-S enhances the affinity by about a factor
of 4, assuming that the inhibition observed for 25
emanates from only one of the two epimers present. The
binding enhancement due to macrocyclization in this case
is less than what we have observed for other macrocyclic
transition state analogues.30 Either the macrocyclic
ketone 3-S retains considerable flexibility which must be
lost on binding, or the conformation it adopts in the
enzyme active site is significantly higher in energy than
the low-energy solution conformation(s).
Comparison of 3-S to lactam 2 suggests that the ketone
moiety in 3-S provides an important enhancement in
affinity over the amide unit of the substrate analogue.
Lactam 2 is weakly bound to the enzyme (Ki ) 1.1 mM)
and slowly turned over as a substrate. The ketone 3-S
differs from the lactam 2 in having a leucine side chain
at the P1 position, instead of the phenylalanine residue
of the lactam. This substitution typically reduces the
ground state affinity of R-chymotrypsin substrates (as
Con clu sion
In summary, we have synthesized substrate and
transition state analogues corresponding to a macrocyclic
motif identified from the screening strategy discussed in
the preceding paper. The macrocyclic lactam proved to
be a weak substrate of R-chymotrypsin, while the mac-
rocyclic ketone was a modest inhibitor. Comparison of
the cyclic ketone with an acyclic analogue showed that
the macrocyclic structure was advantageous, but not to
the extent seen in other cases. Although greater affinity
would be anticipated on incorporation of a phenylalanine
residue at P1 or a more electrophilic difluoroketone
moiety, the potential advantage of the macrocyclic frame-
work did not appear to translate to the boronic acid class
of inhibitors.
Ack n ow led gm en t. This work was supported by a
grant from the National Institutes of Health (Grant No.
GM30759) and by fellowships from the Studienstiftung
des Deutschen Volkes and the Bayerische Begabten-
(27) Procedures for the preparation of NR-Teoc-Nâ-Fmoc-R,â-
diaminopropanoic acid and N-methoxycarbonyl-L-aspartic acid 1-allyl
ester are provided in the Supporting Information.
(28) Scheidt, K. A.; Chen, H.; Follows, B. C.; Chemler, S. R.; Coffey,
D. S.; Roush, W. R. J . Org. Chem. 1998, 63, 6436-6437.
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1997, 119, 11311-11312.
(30) (a) Morgan, B. P.; Holland, D. R.; Matthews, B. W.; Bartlett,
P. A. J . Am. Chem. Soc. 1994, 116, 3251-3260. (b) Meyer, J . H.;
Bartlett, P. A. J . Am. Chem. Soc. 1998, 120, 4600-4609. (c) Smith,
W. W.; Bartlett, P. A. J . Am. Chem. Soc. 1998, 120, 4622-4628 and
references therein.
(31) Bauer, C. A.; Thompson, R. C.; Blout, E. R. Biochemistry 1976,
15, 1296-1299.
(32) Neil, G. L.; Niemann, C.; Hein, G. E. Nature 1966, 210, 903-
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(33) Schellenberger, V.; Braune, K.; Hofmann, H. J .; J akubke, H.
D. Eur. J . Biochem. 1991, 199, 623-636.
J . Org. Chem, Vol. 68, No. 22, 2003 8469

Hansen et al.
sis of the inhibitors and intermediates. This material is
available free of charge via the Internet at http://pubs.acs.org.
fo¨rderung (to B.G.) and the Ligue Nationale Contre le
Cancer (to S.G.-P.).
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the synthesis, characterization, and enzymatic analy-
J O034837Z
8470 J . Org. Chem., Vol. 68, No. 22, 2003