Synthesis and conformational analysis of locked carbocyclic analogues of 1,3-diazepinone riboside, a high-affinity cytidine deaminase inhibitor

Article abstract of DOI:10.1021/jo901127a

(Chemical Equation Presented) Cytidine deaminase (CDA) catalyzes the deamination of cytidine via a hydrated transition-state intermediate that results from the nucleophilic attack of zinc-bound water at the active site. Nucleoside analogues where the leav

Full text of DOI:10.1021/jo901127a

pubs.acs.org/joc
Synthesis and Conformational Analysis of Locked Carbocyclic Analogues
of 1,3-Diazepinone Riboside, a High-Affinity Cytidine Deaminase
Inhibitor
Olaf R. Ludek,† Gottfried K. Schroeder,‡,§ Chenzhong Liao,† Pamela L. Russ,†
Richard Wolfenden,‡ and Victor E. Marquez*,†
†Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, Maryland 21702, and Department of Biochemistry and Biophysics,
‡
University of North Carolina, Chapel Hill, North Carolina 27599.
§Current address: University of Texas at Austin, Division of Medicinal Chemistry, College of Pharmacy,
1 University Station C0850, Austin, TX 78712.
marquezv@mail.nih.gov
Received June 2, 2009
Cytidine deaminase (CDA) catalyzes the deamination of cytidine via a hydrated transition-state
intermediate that results from the nucleophilic attack of zinc-bound water at the active site.
Nucleoside analogues where the leaving NH3 group is replaced by a proton and prevent conversion
of the transition state to product are very potent inhibitors of the enzyme. However, stable
carbocyclic versions of these analogues are less effective as the role of the ribose in facilitating
formation of hydrated species is abolished. The discovery that a 1,3-diazepinone riboside (4)
operated as a tight-binding inhibitor of CDA independent of hydration provided the opportunity
to study novel inhibitors built as conformationally locked, carbocyclic 1,3-diazepinone nucleosides
to determine the enzyme’s conformational preference for a specific form of sugar pucker. This work
describes the synthesis of two target bicyclo[3.1.0]hexane nucleosides, locked as north (5) and south
(6) conformers, as well as a flexible analogue (7) built with a cyclopentane ring. The seven-membered
1,3-diazepinone ring in all the three targets was built from the corresponding benzoyl-protected
carbocyclic bis-allyl ureas by ring-closing metathesis. The results demonstrate CDA’s binding
preference for a south sugar pucker in agreement with the high-resolution crystal structures of other
CDA inhibitors bound at the active site.
Introduction
(ts) intermediate (Figure 1).1 The spontaneous deamination
of cytidine is very slow and proceeds with a rate constant
around 10-10 s-1; however, the enzyme is able to accelerate
the rate of deamination by an impressive 12-14 orders
of magnitude.2 This tremendous enhancement reflects
an extraordinary affinity for the hydrated intermediate
Cytidine deaminase (CDA; EC 3.5.4.5) is a zinc-dependent
enzyme that catalyzes the deamination of cytidine to uridine
via the formation of an unstable, hydrated transition-state
(1) Cacciamani, T.; Vita, A.; Cristalli, G.; Vincenzetti, S.; Natalini, P.;
Ruggieri, S.; Amici, A.; Magni, G. Arch. Biochem. Biophys. 1991, 290, 285–
292.
(2) Schroeder, G. K.; Wolfenden, R. Biochemistry 2007, 46, 13638–13647.
6212 J. Org. Chem. 2009, 74, 6212–6223
Published on Web 07/20/2009
DOI: 10.1021/jo901127a
r
2009 American Chemical Society

Ludek et al.
JOCArticle
FIGURE 1. Transition state for the hydrolytic deamination of cytidine.
(Kts ∼10-16 M).3a Formation of such a hydrated intermedi-
ate has been confirmed biochemically3b, 3c and by the crystal
structure of the inhibitor zebularine (1) bound to CDA,
which revealed the formation of the 3,4-double-bond
hydrate (2) at the active site.3d Inhibition of CDA is of
particular interest therapeutically to enhance the activity of
readily deaminated antitumor nucleosides such as cytosine
arabinoside and 5-aza-20-deoxycytidine.4
was indeed wholly independent from a hydration mechanism
and showed instead that the strong binding at the active site
resulted from the edge-on π-π interaction between the dia-
zepinone ring’s double bond and Phe137.7
For these three molecules, zebularine (1), THU (3), and
diazepinone riboside (4), it is clear that the interactions of the
nucleobase with the active site are critical for strong binding,
and not surprisingly, the majority of the structure-activity-
relationship (SAR) studies reported to date for CDA deal
almost exclusively with changes in the heterocyclic base. In
terms of the role of the sugar moiety, however, fewer studies
have been conducted. Even though the sugar ring has a small
effect on the rate of the spontaneous deamination reaction of
cytidine, it greatly enhances the reactivity of the enzymatic
reaction by increasing the effective concentration of the
altered substrate at the active site.8 Such an entropic advan-
tage is the direct result of the parts (base and ribose) being
properly connected to fit the active site.
The critical parameter of the ribose (or 20-deoxyribose)
that ensures that the interactions between the parts are
optimal is the sugar pucker, which is defined by the pseudor-
otational phase angle P.9 Normally, conventional nucleo-
sides in solution undergo rapid equilibration between ranges
of P defined by two main twist furanose puckering domains
3
2
centered around a T2 (north-type) and a T3 (south-type)
conformation. Despite the small difference in energy
between these two conformations, an emerging picture from
recent observations is that the majority of nucleoside(tide)
target enzymes, whether anabolic or catabolic, appear to
have strict conformational requirements for substrate bind-
ing, accepting the furanose ring only in a particular, well-
define shape.10 Because the value of P and the corresponding
glycosyl torsion angle χ are interdependent, with the latter
responding in a concerted manner to minimize steric clashes,
the value of χ is also of critical importance for the two
connecting pieces to provide optimal recognition.
In the specific case of CDA, the crystal structures of the
enzyme bound to zebularine hydrate (2), THU (3), and
diazepinone riboside (4) show the conformation of the sugar
ring in the south hemisphere, close to a 20-endo conforma-
tion. In particular, the 1.48 A resolution structure by
Kumakasa et al.5 shows the ribose ring of THU clearly in a
southconformationwiththe30-OH hydrogen bonded to the side
Another well-known inhibitor of CDA is tetrahydrouridine
(3, THU), which despite lacking the 5,6-double bond of the
hydrated zebularine ring possesses a hemiaminal structure that
embodies the properties of a powerful transition-state mimic.
Indeed, a recent, high-resolution crystal structure of mouse
CDA complexed with THU shows the active diastereoisomer
with the 4-OH group spatially oriented toward the zinc ion.5
An intriguing molecule, also displaying a strong inhibition
of CDA (Ki = 25 nM), but for which the mechanism of
action remained unexplained for many years, is the diazepi-
none ribose 4.6 This compound possesses a critical double
bond, but as opposed to the hydrated 3,4-double of zebular-
ine and the hemiaminal functionality of THU, it lacks any
possibility of interacting with CDA via zinc coordination.
Fourteen years later, after solving the crystal structure of human
CDA bound to the diazepinone ribose 4, Verdine et al. demon-
strated that the mode of action of this tight-binding inhibitor
(3) (a) Snider, M. J.; Wolfenden, R. Biochemistry 2001, 40, 11364–11371.
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mother. Pharmacol. 1992, 30, 7–11. (b) Lemaire, M.; Momparler, L. F.;
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Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2008; Chapter 12, pp
307-341 (ISBN 978-3-527-31820-9).
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CDA accepts and deaminates efficiently both ribo and
20-deoxyribonucleosides, we chose for simplicity to prepare
20-deoxy versions of these carbocyclic nucleosides. For com-
parison, the plain and flexible cyclopentane analogue (7) was
also synthesized; all of the compounds were evaluated as
CDA inhibitors and compared with the parent riboside (4).
Results
FIGURE 2. Retrosynthetic analysis of the target compounds 5-7
(PG = protecting group).
Synthesis. To accomplish the syntheses of these com-
pounds, we sought a linear strategy that could be adapted to
all three targets. This approach differs from the convergent
approachusedforthe synthesis of4 in order to avoid problems
with the likely formation N- and O-alkylated products.6 The
retrosynthetic analysis used in the current approach shows
how the 1,3-diazepin-2-one moiety is to be engendered by a
ring-closing metathesis (RCM) reaction on the bis-allyl ureas
(I) (Figure 2). These in turn could be accessed by the reaction
of allyl isocyanate with the monoalkylated amines (II) derived
from the appropriate carbocyclic primary amines (III).
The synthesis of the locked north-diazepinone nucleoside
started with the known bicyclo[3.10]hexanol 8.15 As shown in
Scheme 1, conversion of 8 to its methanosulfonyl ester 9 was
followed by nucleophilic attack with sodium azide to give 10 with
inversion of stereochemistry. Alternatively, alcohol 8was reacted
with diphenylphosphoryl azide (DPPA) in DMF to give directly
the desired azide 10.16 After hydrogenolysis over Lindlar’s
catalyst, the azide was quantitatively reduced to the key inter-
mediate amine 11, and alkylation of the primary amine with
allyl bromide in DMF, with either K2CO3 or Cs2CO3 as catalysts,
gave the monoalkylated amine 12 in 35 and 33% yield, respec-
tively, showing no clear advantage with the use of Cs2CO3.17
A change in strategy involving the reduction of the azide 11
in the presence of di-tert-butyl dicarbonate (Scheme 2) led to
the Boc-protected amine 14,18 which was selectively alkylated
with allyl bromide and potassium hexamethyldisilazide in
DMF to give 15. Removal of the Boc group with TFA led to
the monoallylated amine 12, which reacted with allylisocyanate
to form the desired diallylurea 16. Unfortunately, all attempts
to form the seven-membered cyclic urea 17 via RCM19 with
chains of highly conserved Asn54 and Glu56. Using our inter-
active tool, PROSIT,11 which calculates the pseudorotational
parameters of nucleosides and nucleotides bound to proteins,
we calculated a value ofP = 158.55° (20-endo) for THU which
is clearly in the south hemisphere. Other parameters calcu-
lated included the maximum puckering amplitude (νmax
=
36.68°) and the glycosyl torsion angle of the tetrahydrouracil
ring, which is in the anti range (χ = -136.51°). Similarly, the
conformational parameters for the bound diazepinone ribo-
side (4)7 calculated by PROSIT matched very closely those of
THU (P = 158.18°, νmax = 32.10°, and χ = -149.66°).
The unique preference of CDA for the south (20-endo)
conformation stands in sharp contrast with the related
purine deaminase, adenosine deaminase (ADA), which pre-
fers to bind both substrate and inhibitors in the antipodal
north (30-endo) conformation.12 Despite the similarity of the
mechanisms of deamination, which in both enzymes proceed
via analogous tetrahedral intermediates, the secondary and
tertiary structural motifs of the two enzymes are completely
unrelated and lack any evolutionary homology.13
With the aim of studying the impact that the sugar conforma-
tion has on CDA, and how it might impinge on the other critical
interactions with important residues at the active site that are
independent of zinc binding, and thus not controlled by the chemical
generation of an unstable, chiral hydrated intermediate, we chose
the diazepinone riboside 4 as a prototype. This diazepinone
riboside preserves all the other critical interactions with conserved
aminoacids,suchasGlu56andAsn64,plustheCONHportionof
the diazepinone ring forms a similar set of hydrogen bonds with
Ala66 and Glu67 at the active site that are seen with the
nucleobases of the hydrate of zebularine and with THU. The
only difference is the pronounced wrinkle of the seven-membered
ring in 4, which juts the double bond out of plane and allows it to
interact with the π-face of Phe137 via a canonical π/π-interaction.7
In the present work, we report on the construction of
conformationally locked north- (5) and south-diazepinone
(6) riboside analogues of 4 built on a stable carbocyclic
scaffold: the bicyclo[3.1.0]hexane template.14 Because
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10, 2723–2730. (b) Wolff, O.; Waldvogel, S. R. Synthesis 2004, 8, 1303–1305.
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Chem. Soc. 1995, 117, 2108–2109. (b) Martin, S. F.; Follows, B. C.;
Hergenrother, P. J.; Franklin, C. L. J. Org. Chem. 2000, 65, 4509–4514.
(c) Hoffmann, R. V.; Madan, S. J. Org. Chem. 2003, 68, 4876–4885.
(d) Winkler, J. D.; Asselin, S. M.; Shepard, S.; Yuang, J. Org. Lett. 2004,
6, 3821–3824. (e) Gracias, V.; Gasiecki, A. F.; Djuric, S. W. Org. Lett. 2005,
7, 3183–3186. (f) Dougherty, J. M.; Jimenez, M.; Hanson, P. R. Tetrahedron
2005, 61, 6218–6230. (g) Tao, Z.-F.; Chen, Z.; Bui, M.-H.; Kovar, P.;
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(13) Carter, C. W., Jr. Biochimie 1995, 77, 92–98.
(14) Part of this work was presented as a poster at the XVIII Interna-
tional Round Table for Nucleosides, Nucleotides and Nucleic Acids, Kyoto,
Japan, Sep 8-11, 2008: Ludek, O. R.; Schroeder, G. K.; Wolfenden, R.;
Marquez, V. E. Nucleic Acids Symp. Series No. 52, 2008, 659-660.
6214 J. Org. Chem. Vol. 74, No. 16, 2009

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SCHEME 1
SCHEME 2
Grubb’s second-generation catalysts at room temperature or
reflux in either CH2Cl2 or toluene failed and only starting
material was recovered.
We surmised that the failure of the metathesis reaction was
due to the unfavorable orientation of the allyl groups in 16,
which prevented the intramolecular condensation.19c At
6 mM substrate concentration no cross-metathesis products
were observed either. After considering the equilibrium
between the cis- and trans-configurations of the amide, we
realized that the expected, more stable trans-configuration
would indeed place the allyl groups away from each other
making ring closure impossible (Figure 3).
FIGURE 3. Equilibrium between the cis- and trans-conformations
of the amide bond in 16.
ꢀ
Since Kavalec et al. have demonstrated that the cis/trans-
isomerization barrier can be significantly lowered by alkyla-
tion of the amide,20 we considered that such equilibrium
could be tunable by using a suitably protected amide group.
As a first attempt, the amide moiety was silylated with
hexamethyldisilazane and ammonium sulfate as catalyst to
give the aza-enol ether 18 in quantitative yield, where the
bulky trimethylsily group was expected to favor a desirable
conformational change (Scheme 3). Because of the moisture
sensitivity expected for this class of silylated compounds, the
RCM reaction was performed immediately with Grubbs
second-generation catalyst under reflux in methylene chlor-
ide to give, according to TLC analysis, a single product after
30 min. Unfortunately, the expected seven-membered ring
was not formed and instead the six-membered urea 20 was
obtained in 75% yield. Although the formation of 20 was
ꢁ
ꢁ
(20) Kavalek, J.; Machacek, V.; Svoboda, G.; Sterba, V. Collect. Czech.
ꢀ
ꢀꢁ
ꢀ
Chem. Commun. 1987, 52, 1999–2004.
J. Org. Chem. Vol. 74, No. 16, 2009 6215

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SCHEME 3
SCHEME 4
disappointing, a result that most likely happened due to the
isomerization of the double bond to form the s-cis conju-
gated π-system,21 the isolation of a cyclic product proved
that a cis-geometry was essential for the success of the RCM
reaction.
Lindlar’s catalyst23 in the presence of Boc-anhydride gave
the Boc-protected amine 26b. Similarly, reaction of the
enantiomerically pure amine 2524 with Boc-anhydride gave
the protected amine 26a. From this point onward, the two
approaches converge and the reactions and yields obtained
for the penultimate intermediates 32a and 32b were essen-
tially identical to those obtained before. The final removal of
the benzyl protecting groups proceeded uneventfully in the
presence of BCl3 to give the target compounds 6 and 7.
Kinetic Analysis of Carbocyclic Diazepinones as Inhibitors
of Cytidine Deaminase. Cytidine deaminase activity was
measured spectrophotometrically by following the loss of
absorbance of cytidine at 282 nm (Δε = -3600) in 0.1 M
phosphate buffer (pH 6.8) at 25 °C. Concentrations of stock
solutions of the three carbocyclic diazepinones were deter-
mined by proton NMR (see the Supporting Information
Figures 1-3) in reference to a known concentration of
pyrazine (10-3 M) added as an integration standard. Initial
rates for the deamination of cytidine in the presence of all
three inhibitors were determined and plotted as a function of
[S] vs [S]/vi (Hanes plot, Figures 4 and 5). Inhibition con-
stants (Ki) for the three inhibitors (Table 1) were calculated
using the following equation
Encouraged by these results, we then investigated the
effect of a simple acyl group for the RCM reaction, which
would allow for the easy removal of all protecting groups in
one step. Therefore, urea 16 was acylated with benzoyl
chloride in pyridine to form the UV-active urea 21 in 89%
yield. Interestingly, the introduction of the benzoyl group led
to significant broadening of some signals in the proton and
carbon NMR spectra. The RCM reaction, performed under
the same conditions as with 18, smoothly afforded the
desired seven-membered ring in 92%, thus confirming the
role of the stereochemistry of the urea in the intramolecular
cyclization. Finally, after global deprotection of the acyl
groups, achieved with 1% NaOH solution in MeOH, the
target structure 5 was obtained in excellent yield (Scheme 4).
The optimized reaction conditions for the synthesis of the
conformationally locked north-diazepinone 5 were applied
to the syntheses of both south and flexible targets (6 and 7),
respectively (Scheme 5). Starting from enantiomerically pure
2322 for the flexible analogue, mesylation and subsequent
nucleophilic substitution with azide afforded compound 24
in 78% yield (two steps). Reduction of the azido group with
½iꢀ
Ki ¼
ðKMapp=KMÞ -1
where KaMpp is the apparent value of the Michaelis constant
(KM) when measured in the presence of an inhibitor. Values
(21) (a) Hong, S. H.; Sanders, D. P.; Lee, C. W.; Grubbs, R. H. J. Am.
Chem. Soc. 2005, 127, 17160–17161. (b) Schmidt, B. J. Mol. Catal. A: Chem.
2006, 254, 53–57. (c) Gimeno, N.; Formentın, P.; Steinke, J. H. G.; Vilar, R.
´
Eur. J. Org. Chem. 2007, 918–924. (d) Schmidt, B; Biernat, A. Org. Lett.
2008, 10, 105–108. (e) Krompiec, S.; Krompiec, M.; Penczek, R.; Ignasiak, H.
Coord. Chem. Rev. 2008, 252, 1819–1841.
(23) Reddy, P. G.; Pratap, T. V.; Kumar, G. D. K.; Mohanty, S. K.;
Baskaran, S. Eur. J. Org. Chem. 2002, 3740–3743.
(22) (a) Ludek, O. R.; Meier, C. Synthesis 2003, 13, 2101–2109. (b) Ludek,
€
O. R.; Balzarini, J.; Kramer, T.; Meier, C. Synthesis 2006, 8, 1313–1324.
(24) Ezzitouni, A.; Barchi, J. J., Jr.; Marquez, V. E. J. Chem. Soc., Perkin
Trans. 1 1997, 1073–1078.
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SCHEME 5
for KaMpp at various concentrations of inhibitor were esti-
mated from the Hanes plots (Figures 4 and 5), which have
-KM as the intercept on the [S] axis.
riboside 4 (PDB ID: 1MQ0).7 The structures of the locked
north- and south-diazepinone nucleosides (5 and 6) were
constructed by importing the bicyclo[3.1.0]hexane templates
from the crystal structures of north- (LETJEZ) and south-
(YESMIS) bicyclo[3.1.0]hexane thymidine nucleosides
which were downloaded from the Cambrige structural data-
base (CCDC ConQuest version 1.5). The diazepinone ring as
it appeared in the crystal structure of the CDA complexed
with the diazepinone riboside 4 was mounted on the two
bicyclo[3.1.0]hexane templates (see Figure 1 of the Support-
ing Information).
The diazepinone riboside 4 (Figure 6A) forms six hydro-
gen bonds (yellow dashed lines). Two hydrogen-bonding
interactions are between the diazepinone ring and Glu67
(subunit C) and Ala66 (subunit C). The ribose ring forms the
rest of the hydrogen bonds with Glu56 (subunit C), Asn54
Figure 4 shows that both south (6) and north (5) carbo-
cyclic diazepinones are competitive inhibitors of cytidine
deaminase (parallel lines) and that the south derivative 6 is
more potent than its north counterpart 5 (by a factor of ∼2).
Figure 5 shows that the flexible, carbocyclic diazepinone is
also a competitive inhibitor, which surprisingly, as indicated
in Table 1, is significantly more potent than either one of the
locked derivatives. This compound, however, is still 16-fold
less potent than the corresponding riboside 4.6
Molecular Modeling. Molecular dynamics calculations
and docking of the new carbocyclic diazepinone nucleosides
(5-7) at the active site of CDA were based on the available
crystal structure of human CDA bound to the diazepinone
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FIGURE 5. Hanes plot for cytidine deaminase activity at various
concentrations (defined in figure) of the flexible diazepinone in-
hibitor 7 (2). The parallel lines represent linear fits to the data and
are indicative of competitive inhibition.
TABLE 1. Inhibition Constants for Carbocyclic Diazepinones
compd
Ki (μM)
north-diazepinone (5)
south-diazepinone (6)
flexible-diazepinone (7)
70 ( 10
40 ( 10
0.4 ( 0.1
Discussion
FIGURE 4. Hanes plot for cytidine deaminase activity at various
concentrations (defined in the figure) of south/north carbocyclic
diazepinone inhibitors. The parallel lines represent linear fits to the
data and are indicative of competitive inhibition: (A) south-diaze-
pinone 6 (b); (B) north-diazepinone 5 (9).
The selection of the diazepinone riboside 4 as a prototype
to study the preferred conformation of CDA for a particular
sugar pucker was based on the premise that the newly
revealed edge-on π-π interaction between the diazepinone
ring’s double bond and Phe1377 provided a mechanism of
inhibition independent of the zinc-promoted hydration for
which the role of the sugar O40 oxygen would not be
considered critical. In the case of the mechanistically related
adenosine deaminase (ADA)26 and also for CDA,27 studies
with carbocyclic nucleosides in our hands have revealed that
the sugar moiety plays a significant role in facilitating zinc-
promoted hydration due to the more electronegative nature
of the sugar moiety and important orbital interactions
between the oxygen’s (O40) lone pair with the highest
p orbital component and the adjacent C-N glycosyl bond
through the anomeric effect. Thus, replacement of the ribose
or 20-deoxyribose ring of adenosine and cytidine with carbo-
cyclic pseudosugars removes any communication between
the sugar and the nucleobase and the rates of deamination
decrease.27 The diazepinone-based inhibitors (4-7), which
lack any possibility of interacting with CDA via zinc
coordination and work independently of hydration, are
therefore ideal candidates to study the effect of sugar (or
pseudosugar) ring puckering on the mechanism of CDA.
Because all the crystal structures of bound inhibitors to
CDA have shown the conformation of the sugar ring to be
in the south hemisphere,3c, 3d, 5, 7 close to a 20-endo confor-
mation, we hypothesized that the conformationally south-
locked diazepinone nucleoside 6 would be the most potent
inhibitor in comparison with the antipodal north-locked 5,
(subunit C), Ala58 (subunit D), and Tyr60 (subunit D). In
addition, there is the important canonical π/π interaction
with the π face of Phe137 (subunit A). The worst ligand, the
north-diazepinone 5 (Figure 6B), maintains the hydrogen-
bonding interactions of the diazepinone ring to Glu67 and
Ala66, but the north-pseudosugar moiety forces the equato-
rially disposed 30-OH to form a weaker hydrogen bond to
Glu56. The 50-OH cannot bind to Ala 58 or Tyr60 and
reaches to Leu142 to form a new weak hydrogen bond. In
addition, the interaction with Asn 54 is missing. The flexible
diazepinone 7 (Figure 6C) and the south-diazepinone 6
(Figure 6D) form almost identical interactions. However,
the hydrogen-bonding interactions of the 30-OH in the
flexible compound 7 (1.682 and 1.795 A) appear to be
stronger than those in the locked south-diazepinone 6
(1.751 and 1.930 A).
The major discrepancies in the hydrogen-bonding net-
work between these compounds (4-7) can be better under-
stood by viewing the superimposed structures and observing
the orientation of the critical OH groups (Figure 7). With the
exception of the equatorial 30-OH in north-diazepinone 5
(yellow), the axial orientations of the 30-OH in the rest of the
molecules remains in close proximity. There is good freedom
of rotation for the CH2OH group in 5, which allows it to
reach a new amino acid, Leu142, to compensate for the lack
of interaction with Ala58 and Tyr60 seen with the rest of the
compounds. The diazepinone ring appears perfectly super-
imposed in all four structures. The 20-OH is present only in
the parent riboside 4 but this group appears not to be
engaged in any important interaction, thus supporting the
equivalent affinity that CDA displays for both ribo and
20-deoxyribonucleosides (substrates or inhibitors).
(25) Macromodel 9.5, Schrodinger, LLC, New York, 2007.
(26) Marquez, V. E.; Russ, P.; Alonso, R.; Siddiqui, M. A.; Hernandez,
S.; George, C.; Nicklaus, M. C.; Dai, F.; Ford, H., Jr. Helv. Chim. Acta 1999,
82, 2119–2129.
(27) Marquez, V. E.; Schroeder, G. K.; Ludek, O. R.; Siddiqui, M. A.;
Ezzitouni, A.; Wolfenden, R. Nucleosides, Nucleotides Nucleic Acids, in
press.
6218 J. Org. Chem. Vol. 74, No. 16, 2009

Ludek et al.
JOCArticle
FIGURE 6. Monte Carlo/Stochastic Dynamics simulations in MacroModel 9.525 of four diazepinone compounds in human cytidine
deaminase: (A) diazepinone riboside (4, conformation in the crystal structure); (B) north-diazepinone (5); (C) flexible diazepinone (7);
(D) south-diazepinone (6). Hydrogen bonds appear as yellow dashed lines, and the canonical π/π interaction with the π-face of Phe137 is
represented by a group of purple dotted lines. As a reference point, the atom of zinc appears bonded to Cys65.
Surprisingly, the flexible cyclopentane diazepinone nucleo-
side 7 was more potent than the south-diazepinone 6 by a
factor of 100 (Table 1). However, relative to the normal
riboside (4), the flexible diazepinone 7 was only 16-fold less
potent. A likely explanation for the less than expected
performance of the south-diazepinone nucleoside 6 stems
from the fusion of the cyclopropane ring adjacent to the
glycosyl C-N bond. As the crystal structures of CDA in
complex with THU and diazepinone riboside 4 show, the
glycosyl torsion angles (χ = -136.51° and χ = -149.66°) are
clearly in the anti range. Unfortunately, as we have observed
FIGURE 7. Overlapping of CDA-bound conformation of 4 (khaki),
5 (yellow), 6 (cyan), and 7 (green).
with all pyrimidine nucleosides built on a south-bicyclo-
[3.1.0]hexane pseudosugar template, both single-crystal
X-ray structures and ab initio calculations have shown that
and the flexible diazepinone nucleoside 7. As mentioned
the glycosyl torsion angle is more stable in the syn region.28
before, the preference of CDA for the south (20-endo)
This means that the enzyme has to pay a significant penalty
conformation is in sharp contrast with that of ADA, which
to accommodate the inhibitor in the less stable anti con-
prefers to bind both substrate and inhibitors in the anti-
formation. Despite this shortcoming, the south-diazepinone
podal north (30-endo) conformation.12 This unique prefer-
6 was still able to inhibit CDA more effectively than its north
ence for an antipodal sugar puckering by CDA is another
counterpart. However, the penalty incurred in changing the
important element of differentiation from the seemingly
conformation from syn to anti inside a binding pocket that is
similar ADA.
completely sequestered from bulk solvent diminishes the
The biochemical results from this investigation indeed
confirm CDA’s preference for a south sugar conformation
since the south inhibitor 6 was more potent than its north
counterpart 5 albeit only by a factor of ∼2 (Table 1).
(28) Marquez, V. E.; Ben-Kasus, T.; Barchi, J. J., Jr.; Green, K. M.;
Nicklaus, M. C.; Agbaria, R. J. Am. Chem. Soc. 2004, 126, 543–549.
J. Org. Chem. Vol. 74, No. 16, 2009 6219

Ludek et al.
JOCArticle
capacity of CDA to discriminate effectively between the two
inhibitors on the basis of ring pucker.
The same argument can explain the 100-fold difference
between the flexible diazepinone 7 and the south-diazepi-
none 6, which as seen in parts C and D of Figure 6 fit almost
identically. The flexible diazepinone does not have a con-
strained syn glycosyl torsion angle since the plastic cyclo-
pentane ring can easily reach the P value observed for the
ribose ring in 4 (P = 158.18°) at the active site of CDA. If we
take as a reference the approximate 10-exo (P = 118.6°)
conformation found in the crystal structure of plain carbo-
cyclic thymidine,29 the cyclopentane ring in 7 has to change
its puckering 40° (pseudorotational degrees) to reach the
20-endo conformation. This change is very much achievable
in solution and with a much lower energy penalty when
compared to that required to rotate the glycosyl bond in 6.
This small penalty in changing ring puckering could very well
reflect the 16-fold lower potent inhibition of CDA by 7
relative to the parent ribose 4.
FIGURE 8. Ab initio potential energy scan (PES) of south-locked
diazepinone 6 and south-locked thymidine (six-membered ring).
To strengthen our argument that the effective north/south
discrimination by CDA was weakened by the syn-preference
of the south-locked diazepinone 6, we performed a confor-
mational analysis by sampling the entire conformational
space of χ from 0° to 360° (Figure 8) as we have done
previously for the locked north- and south-thymidine ana-
logues.28 For the locked south-thymidine analogue, which
has the same bicyclo[3.1.0]hexane scaffold of 6, a relative
energy barrier of ∼17 kcal/mol separated the most stable syn
conformer (χ = ∼60°) from the less stable anti conformer
(χ = ∼-120°). The energy barrier for the larger, bulkier
diazepinone ring was somewhat higher (∼18 kcal/mol kcal/
mol), reflecting a greater difficulty in achieving the anti
conformation demanded by the enzyme. Furthermore, the
anti conformer for 6 was significantly less stable than the
corresponding south-locked thymidine anti conformer. As in
the case of the south-locked thymidine analogue, the integ-
rity of the bicyclo[3.1.0]hexane system remained constant
during the calculations as indicated by the small variance of
P across the entire range (not shown).
In conclusion, the observed order of inhibitory potency
4 > 7 . 6 > 5 can be easily explained in terms of two
important factors: (1) the preferred sugar puckering of
CDA for the south conformation and (2) the preferred anti
disposition of the base bound at the active site. The small
∼2-fold difference between south-locked 6 and north-locked
5 suggests that the most efficient network of hydrogen bonds
provided by the south template allows CDA to overcome the
penalty incurred in rotating χ from syn to anti and differ-
entiate between the two. However, this penalty becomes an
important issue when comparing the south-locked 6 with the
flexible-diazepinone 7, for which such a penalty no longer
exists, allowing the ring to reach the required 20-endo pucker
with relative ease from its preferred 10-exo conformation.
These conformational issues could explain the 100-fold
separation in potency observed between these two com-
pounds. Finally, the diazepinone-riboside 4 also does not
incur the penalty of rotating χ, and by its very nature, the
ribose ring has a very small energy barrier separating the
30-endo from the precise 20-endo conformation required for
binding. We can therefore conclude that in accordance to the
structural information gathered from X-ray crystallography
our set of inhibitors prove that CDA indeed has a penchant
for the south conformation, which is opposite to that of
ADA. One final point of interest in cancer chemotherapy
regarding the combined use of antitumor nucleosides that
are readily deaminated by CDA is that the very stable and
quite potent carbocyclic diazepinone nucleoside (7) could
overcome many of the instability problems associated with
the use of tetrahydrouridine (THU, 3).30
Experimental Section
Molecular Modeling. Molecular dynamics calculations were
performed using the Monte Carlo/Stochastic Dynamics (MC/
SD) program available in MacroModel 9.525 to simulate
molecular movement over time using Newton’s equation of
motion. This program performs constant temperature calcula-
tions that take advantage of the strengths of the Monte Carlo
method for quickly introducing large changes in a few degrees of
freedom and stochastic dynamic for an effective local sampling
of collective motions. For these calculations, the OPLS_2005
force field was employed and water was as used as a solvent. To
force the system to meet the desired geometrical conditions and
to reduce the cost of computer time by eliminating calculations
expected to have little influence on the results, all residues that
do not have any atoms within 6 A of the ligands and the seven
atoms of the diazepinone ring were fixed. The maximum itera-
tions and convergence threshold were set to 5000 and 0.00,
respectively. The number of structures to sample was set to 10,
and the simulation time was 100 ps.
Ab Initio Potential Energy Scans (PES). The structure of the
south-locked thymidine analogue (entry code: YESMIS31) was
taken from the Cambridge Structural Database.32,33 Since YES-
MIS isa dimer containing two conformers, one of them was taken
for further ab initio calculations. The structure of compound 6
was built from two segments, the sugar ring extracted from
YESMIS and the diazepinone ring extracted from the PDB file
(30) Kelley, J. A.; Driscoll, J. S.; McCormack, J. J.; Roth, J. S.; Marquez,
V. E. J. Med. Chem. 1986, 29, 2351–2358.
(31) Altmann, K. H.; Imwinkelried, R.; Kesselring, R.; Rihs, G. Tetra-
hedron Lett. 1994, 35, 7625–7648.
(32) Allen, F. H. Acta Crystallogr B 2002, 58, 380–388.
(33) Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M.; Macrae, C. F.;
McCabe, P.; Pearson, J.; Taylor, R. Acta Crystallogr B 2002, 58, 389–397.
(29) Kalman, A.; Koritsanszky, T.; Beres, J.; Sagi, G. Nucleosides
Nucleotides 1990, 9, 235–243.
6220 J. Org. Chem. Vol. 74, No. 16, 2009

Ludek et al.
JOCArticle
1MQ0.7 The structure of 6 was first minimized, and then a
conformational search was performed employing the Systematic
Search method in MOE 2007.09.34 The identified global energy
minimum conformation was then used for ab initio calculations.
Both the south-locked thymidine and 6 were preoptimized at the
HF/3-21G leveloftheory and thenreoptimizedat the HF/6-311G
level in the program Gaussian 03, revision E01 (Gaussian, Inc.).
The two structures, which were assumed to be local or global
energy minima, were investigated by performing a PES of the
dihedral angle χ by rotating the base in steps of 15°. Using the
Gaussian HF method at the same 6-311G level of theory, each of
the geometries obtained were optimized for all internal coordi-
nates, except the dihedral angle χ.
Cytidine Deaminase Inhibition. Cytidine deaminase from
E. coli was purified as previously described.3a Enzymatic activity
(∼1 nM CDA) was measured spectrophotometrically in 1 mL
cuvettes by following the loss of absorbance of cytidine at
282 nm (Δε = -3600) in 0.1 M phosphate buffer (pH 6.8) at
25 °C. Concentrations of stock solutions of the three carbocyclic
diazepinones (5-7) were determined by proton NMR (see
Figures 2-4 in the Supporting Information) in reference to a
known concentration of pyrazine (10-3 M) added as an integra-
tion standard.
((1R,2S,4S,5S)-2-Acetoxy-4-azidobicyclo[3.1.0]hexan-1-yl)-
methyl Acetate (10) (Method A). To a stirred solution of alcohol 8
(850 mg, 3.72 mmol) in dry DMF (25 mL) were added diphenyl-
phosphoryl azide (DPPA, 2.40 mL, 11.2 mmol) and triethylamine
(1.80 mL, 13.0 mmol) under argon at room temperature. The
mixture was warmed to 60 °C and stirred for 20 h. The solvent was
evaporated, and the residue was partitioned between water (50
mL) and CH2Cl2 (50 mL). After phase separation, the aqueous
phase was extracted again with CH2Cl2 (2 ꢁ 50 mL), and the
combined extracts were dried (Na2SO4) and concentrated. The
crude material was purified by flash chromatography on silica gel
(EtOAc in hexanes 25-45%) to yield the azide 10 (706 mg, 75%)
as a colorless oil: [R]20D -57.71 (c 0.78, CHCl3); IR (neat) 3014,
2098, 1733, 1631, 1370, 1230, 1025, 973, 905, 835 cm-1; 1H NMR
(400 MHz, CDCl3) δ5.49 (app t, 1H, J ≈8.8 Hz, H-2), 4.22 (d, 1H,
J = 12.0 Hz, CHH-OAc), 3.97 (d, 1H, J = 12.0 Hz, CHH-OAc),
3.81 (d, 1H, J = 6.2 Hz, H-4), 2.25 (ddd, 1H, J = 14.8, 8.1, 1.0 Hz,
H-3a), 2.09 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.53 (dd, 1H, J = 8.6,
4.3 Hz, H-5), 1.41 (ddd, 1H, J = 14.8, 8.4, 6.2 Hz, H-3b), 0.84 (dd,
1H, J = 6.2, 4.3 Hz, H-6a), 0.79-0.75 (m, 1H, H-6b); 13C NMR
(100 MHz, CDCl3) δ 171.0, 70.8 (2 ꢁ CdO, Ac), 74.7 (C-2), 65.1
(CH2-OAc), 61.5 (C-4), 34.5 (C-3), 30.8 (C-1), 26.5 (C-5), 20.9,
20.8 (2 ꢁ CH3, Ac), 11.1 (C-6); FAB-MS m/z (relative intensity)
254 (45), 194 (100). Anal. Calcd for C11H15N3O4: C, 52.17; H,
5.97; N, 16.59. Found: C, 52.26; H, 6.01; N, 16.35.
((1R,2S,4S,5S)-2-Acetoxy-4-azidobicyclo[3.1.0]hexan-1-yl)methyl
Acetate (10) (Method B). The bicyclic hexanol 8(600 mg, 2.36 mmol)
was dissolved in dry CH2Cl2 (20 mL) under argon at 0 °C, and
triethylamine (1.10 mL, 7.89 mmol) and methansulfonyl chloride
(MsCl, 305 μL, 3.94 mmol) were slowly added. The reaction mixture
was stirred for 1 h at 0 °C and then poured into a mixture of ice-cold
phosphate buffer (pH, 7.2, 150 mL) and Et2O (150 mL). The
aqueous phase was extracted with Et2O (2 ꢁ 100 mL), and the
combined extracts were dried (MgSO4) and concentrated. The crude
mesylate 9 was used in the next substitution reaction without any
further purification (decomposition occurred during chromatogra-
phy on silica gel): IR (neat) 3019, 2941, 1733, 1446, 1351, 1237, 1171,
1032, 970, 922, 846 cm-1;1H NMR (400 MHz, CDCl3) δ5.20 -5.11
(m, 2H, H-2, H-4), 4.20 (d, 1H, J = 12.0 Hz, CHH-OAc), 3.78 (d,
1H, J= 12.0 Hz, CHH-OAc),2.94(s,3H,CH3-SO3), 2.63 (ddd, 1H,
J = 14.1, 7.8, 7.8 Hz, H-3a), 1.99 (s, 3H, OAc), 1.98 (s, 3H, OAc),
1.83 - 1.78 (m, 1H, H-3b), 1.25 (dd, 1H, J = 6.0, 4.3 Hz, H-6a),
0.81 (app t, 1H, J ≈ 7.0 Hz, H-6b); FAB-MS m/z (relative intensity)
307 (5), 247 (24), 211 (100).
The mesylate 9 (810 mg, 2.36 mmol) was dissolved in dry
DMF (20 mL), and sodium azide (1.70 g, 23.6 mmol) was added
in one portion under argon at room temperature. The mixture
was heated to 90 °C for 1 h and allowed to cool to room
temperature. The reaction was poured into a mixture of phos-
phate buffer (pH 7.2, 100 mL) and Et2O (100 mL), and the
aqueous phase was extracted with Et2O (2 ꢁ 100 mL). The
combined organic extracts were dried (MgSO4) and concen-
trated. Flash chromatography on silica gel (EtOAc in hexanes
25-45%) yielded the azide 10 (436 mg, 73%) as a colorless oil.
The spectral data were identical to those reported above.
((1R,2S,4S,5S)-2-Acetoxy-4-aminobicyclo[3.1.0]hexan-1-yl)-
methyl Acetate (11). Azide 10 (690 mg, 2.72 mmol) was dis-
solved in MeOH (25 mL) and CH2Cl2 (25 mL) under argon at
room temperature, and Lindlar’s catalyst (100 mg) was added.
The mixture was flushed with hydrogen and stirred at room
temperature overnight. The mixture was filtered over a short
pad of Celite, and the solvent was evaporated off. The crude
amine 11 (618 mg, 100%) was obtained as a colorless wax,
which was sufficiently pure to be used in the next step without
any further purification. An analytical sample was prepared by
flash chromatography on silica gel (MeOH in EtOAc 10-40%):
[R]20D -19.21 (c 0.36, CHCl3); IR (neat) 3332, 2947, 1729, 1654,
1548, 1370, 1238, 1025, 733 cm-1; 1H NMR (400 MHz, CDCl3)
δ 5.45 (app t, 1H, J ≈ 8.3 Hz, H-2), 4.17 (d, 1H, J = 11.6 Hz,
CHH-OAc), 3.92 (d, 1H, J = 11.6 Hz, CHH-OAc), 3.15 (d, 1H,
J = 6.0 Hz, H-4), 2.40-2.10 (bs, 2H, NH2), 1.94 (s, 3H, OAc),
1.92 (s, 3H, OAc), 1.74 (dd, 1H, J = 13.5, 8.0 Hz, H-3a), 1.30-
1.22 (m, 2H, H-3b, H-5), 0.72 (app t, 1H, J ≈ 4.8 Hz, H-6a), 0.62
(dd, 1H, J = 8.2, 5.5 Hz, H-6b); 13C NMR (100 MHz, CDCl3)
δ 171.5, 171.4 (2 ꢁ CdO, Ac), 75.9 (C-2), 66.4 (CH2-OAc), 51.2
(C-4), 36.2 (C-3), 30.2 (C-1), 29.5 (C-5), 19.5, 19.4 (2 ꢁ CH3, Ac),
10.4 (C-6); FAB-MS m/z (relative intensity) 228 (100), 186 (38).
Anal. Calcd for C11H17NO4 0.2H2O: C, 57.23; H, 7.60; N, 6.07.
3
Found: C, 57.08; H, 7.59; N, 5.86.
((1R,2S,4S,5S)-2-Acetoxy-4-allylaminobicyclo[3.1.0]hexan-1-yl)-
methyl Acetate (12) and ((1R,2S,4S,5S)-2-Acetoxy-4-diallylamino-
bicyclo[3.1.0]hexan-1-yl)methyl Acetate (13). The amine 11 (100 mg,
0.440 mmol) was dissolved in dry DMF (2.0 mL) at room
temperature, and potassium carbonate (anhydrous, 166 mg,
1.20 mmol) was added in one portion under argon. Allyl bromide
was slowly added at room temperature and the mixture was stirred
overnight. The solvent was evaporated off, and the residue was
chromatographed on silica gel (MeOH in EtOAc 0-10%) to yield
the title compound 12 (41.0 mg, 35%) as a colorless oil, together
with the dialkylated derivative 13 (33.8 mg, 25%).
Compound 12: [R]20 3.54 (c 0.58, CHCl3); IR (neat) 3323,
D
2946, 1731, 1643, 1457, 1370, 1238, 1111, 1024, 916, 759 cm-1
;
1H NMR (400 MHz, CDCl3) δ 5.84 (m, 1H, CHdCH2), 5.49
(app t, 1H, J ≈ 8.3 Hz, H-2), 5.13 (ddd, 1H, J = 17.1, 3.0, 1.6 Hz,
CHdCHH), 5.04 (ddd, 1H, J = 10.2, 3.0, 1.6 Hz, CHdCHH),
4.18 (d, 1H, J = 12.0 Hz, CHH-OAc), 3.99 (d, 1H, J = 12.0 Hz,
CHH-OAc), 3.25-3.15 (m, 2H, CH2-CHdCH2), 3.14 (d, 1H,
J = 6.2 Hz, H-4), 2.09 (dd, 1H, J = 14.3, 8.3 Hz, H-3a), 2.00 (s,
3H, OAc); 1.99 (s, 3H, OAc), 1.64-1.59 (bs, 1H, NH), 1.40 (dd,
1H, J = 8.4, 4.4 Hz, H-5), 1.26 (m, 1H, H-3b), 0.82 (dd, 1H, J =
5.6, 4.4 Hz, H-6a), 0.68 (dd, 1H, J = 8.4, 5.6 Hz, H-6b); 13C
NMR (100 MHz, CDCl3) δ 171.1 (2 ꢁ C = O, OAc), 136.6
(CHdCH2), 115.8 (CHdCH2), 76.0 (C-2), 66.2 (CH2-OAc),
57.5 (CH2-CHdCH2), 49.6 (C-4), 34.2 (C-3), 30.1 (C-1), 28.2
(C-5), 21.1, 20.9 (2 ꢁ CH3, OAc), 11.2 (C-6); FAB-MS m/z
(relative intensity) 268 (100), 208 (30). Anal. Calcd for
C14H21NO4: C, 62.90; H, 7.92; N, 5.24. Found: C, 62.81; H,
7.96; N, 5.02.
Compound 13: 1H NMR (400 MHz, CDCl3) δ 5.73-5.78 (m,
1H, CHdCH2-A), 5.72-5.68 (m, 1H, CHdCH2-B), 5.39 (app t,
(34) MOE, version 2007.09, Chemical Computing Group, Montreal,
Quebec, Canada. 2007.
J. Org. Chem. Vol. 74, No. 16, 2009 6221

Ludek et al.
JOCArticle
1H, J ≈ 8.3 Hz, H-2), 5.13 (dd, 1H, J = 3.0, 1.7 Hz, CHdCHH-
A), 5.08 (dd, 1H, J = 3.0, 1.6 Hz, CHdCHH-A), 5.05 (br dd, 1H,
CHdCHH-B), 5.02 (br dd, 1H, CHdCHH-B), 4.26 (d, 1H, J =
12.0 Hz, CHH-OAc), 3.89 (d, 1H, J = 12.0 Hz, CHH-OAc), 3.38
(d, 1H, J = 7.9 Hz, H-4), 3.20 (ddd, 2H, J = 14.0, 5.4, 1.5 Hz,
CH2-allyl-A), 2.80 (dd, 2H, J = 14.0, 7.3 Hz, CH2-allyl-B), 2.24
(dd, 1H, J = 15.1, 8.5 Hz, H-3a), 1.98 (s, 3H, OAc), 1.95 (s, 3H,
OAc), 1.34 (dd, 1H, J = 7.5, 5.4 Hz, H-5), 1.22-1.14 (m, 1H,
H-3b), 0.69-0.64 (m, 2H, 6-CH2); 13C NMR (100 MHz, CDCl3)
δ 170.9 (2 ꢁ CdO, OAc), 136.6 (2 ꢁ CHdCH2), 117.0 (2 ꢁ
CHdCH2), 76.4 (C-2), 66.1 (CH2-OAc), 60.8 (2 ꢁ CH2-CHd
CH2), 53.1 (C-4), 31.7 (C-3), 30.8 (C-1), 26.5 (C-5), 21.1, 20.8 (2 ꢁ
CH3, Ac), 11.4 (C-6); ESI-MS (m/z) 308.2 (M þ Hþ).
0.680 mmol) was dissolved in anhydrous CH2Cl2 (15 mL), and
TFA (2.5 mL) was added at 0 °C under argon. After the addition,
the mixture was continued to stir at 0 °C and frequently monitored
by TLC (CH2Cl2-MeOH 9:1). After the mixture was stirred for 3 h
at 0 °C, all starting material was consumed and satd NaHCO3
(50 mL) was carefully added while the mixture was vigorously
stirred. The layers were separated, and the aqueous phase was
extracted with CH2Cl2 (2 ꢁ 50 mL). The combined extracts were
dried (MgSO4) and concentrated. The crude product was suffi-
1
ciently pure (by TLC, H NMR) to be used without any further
purification in the next step. The spectroscopic data were identical
to those reported above.
((1R,2S,4S,5S)-2-Acetoxy-4-(1,3-diallylureido)bicyclo[3.1.0]-
hexan-1-yl)methyl Acetate (16). The allyl amine 12 (260 mg,
0.973 mmol) was dissolved in anhydrous CH2Cl2 (20 mL)
and cooled to -20 °C under argon. Allyl isocyanate (162 mg,
1.38 mmol) was slowly added, and the mixture was gradually
warmed to room temperature and stirred overnight. The solvent
was evaporated off under reduced pressure, and the residue was
purified by flash chromatography (silica gel, EtOAc in hexanes
60-80%) to yield the urea 16 (307 mg, 93%) as a colorless oil:
((1R,2S,4S,5S)-2-Acetoxy-4-(tert-butoxycarbonylaminobicyclo-
[3.1.0]hexan-1-yl)methyl Acetate (14). Azide 11 (1.20 g, 4.74 mmol)
was dissolved in CH2Cl2, and di-tert-butyl dicarbonate ((Boc)2O,
1.24 g, 5.69 mmol) was added at room temperature under argon.
The hydrogenation catalyst (Pd/C, 10%, 150 mg) was added, and
the mixture was flushed with H2 and stirred at room temperature
until all starting material was consumed by TLC analysis. The
solvent was filtered through a short pad of Celite, and the filtrate
was concentrated. The crude was purified by flash chromatogra-
phy on silica gel (EtOAc in hexanes 20-40%) to yield the
carbamate 14 (1.46 g, 94%) as a colorless oil: [R]20D 7.59 (c 0.83,
CHCl3); IR (neat) 3345, 2976, 1707, 1518, 1454, 1364, 1237, 1166,
1038, 974, 905, 853, 783 cm-1; 1H NMR (400 MHz, CDCl3) δ 5.47
(app t, 1H, J ≈ 8.4 Hz, H-2), 4.57 (br s, 1H, NH), 4.27 (d, 1H, J =
11.8 Hz, CHH-OAc), 4.04-3.98 (m, 1H, H-4), 3.92 (d, 1H, J =
11.8 Hz, CHH-OAc); 2.11 (dd, 1H, J = 14.4, 8.0 Hz, H-3a); 2.05
(s, 3H, OAc); 2.02 (s, 3H, OAc), 1.47-1.37 (m, 11H, H-3b, H-5,
t-Bu, Boc), 0.89 (dd, 1H, J = 6.0, 4.4 Hz, H-6a), 0.72 (dd, 1H,
J = 8.4, 6.0 Hz, H-6b); 13C NMR (100 MHz, CDCl3) δ 171.1,
170.9 (2 ꢁ CdO, OAc), 154.8 (CdO, Boc), 79.6 (C(CH3)3, Boc),
74.9 (C-2), 65.8 (CH2-OAc), 51.1 (C-4), 35.4 (C-1), 30.2 (C-3), 28.4
(C(CH3)3, Boc), 27.7 (C-5), 21.0, 20.9 (2 ꢁ CH3, OAc), 11.0 (C-6);
FAB-MS m/z (relative intensity) 328 (3.7), 212 (100). Anal. Calcd
for C16H25NO6: C, 58.70; H, 7.70; N, 4.28. Found: C, 58.25; H,
7.70; N, 4.19.
[R]20 4.94 (c 0.89, CHCl3); IR (neat) 3430, 3366, 3079, 2982,
D
1
1734, 1637, 1522, 1370, 1237, 1024, 915, 850 cm-1; H NMR
(400 MHz, CDCl3) δ 5.85-5.75 (m, 2H, 2 ꢁ CHdCH2), 5.49
(app t, 1H, J ≈ 8.5 Hz, H-2), 5.30-5.20 (m, 2H, 2 ꢁ CHdCHH),
5.10-5.00 (m, 2H, 2 ꢁ CHdCHH), 4.87 (d, 1H, J = 8.1 Hz,
H-4), 4.49 (t, 1H, J = 5.4 Hz, NH), 4.35 (d, 1H, J = 12.0 Hz,
CHH-OAc), 3.84-3.75 (m, 5H, CHH-OAc, 2 ꢁ CH2-CHd
CH2), 2.09 (dd, 1H, J = 15.5, 8.8 Hz, H-3a), 1.98 (s, 6H, 2 ꢁ
OAc), 1.66-1.58 (m, 1H, H-3b), 1.30 (dd, 1H, J = 8.8,
4.4 Hz, H-5), 0.77 (dd, 1H, J = 5.9, 4.4 Hz, H-6a), 0.70 (dd,
1H, J = 8.8, 5.9 Hz, H-6b); 13C NMR (100 MHz, CDCl3) δ
171.1, 170.7 (2 ꢁ CdO, Ac), 157.9 (CdO, urea), 135.7, 135.4
(2 ꢁ CHdCH2), 116.8, 115.4 (2 ꢁ CHdCH2), 75.3 (C-2), 65.9
(CH2-OAc), 55.7 (C-4), 45.6, 43.2 (2 ꢁ CH2-allyl), 36.3 (C-3),
32.2 (C-1),26.9 (C-5), 21.0, 20.8 (2 ꢁ CH3, Ac), 11.3 (C-6); FAB-
MS m/z (relative intensity) 351 (15), 291 (100). Anal. Calcd for
for C18H26N2O5: C, 61.70; H, 7.48; N, 7.99. Found: C, 61.60; H,
7.68; N, 7.86.
((10R,20S,40S,50S)-20-Acetoxy-40-(2-oxo-2,3-dihydropyrimidin-
1(6H)-yl)bicyclo[3.1.0]hexan-10-yl)methyl Acetate (20). Urea 16
(20.0 mg, 0.057 mmol) was dissolved in hexamethyldisilazane
(HMDS, 1.0 mL), and a catalytic amount of ammonium sulfate
was added. The mixture was heated to reflux for 2 h and cooled to
room temperature. The solvent was removed in vacuo, and
the residue was dissolved in anhydrous and deoxygenated
CH2Cl2 (5 mL). Grubbs second-generation catalyst (4.8 mg,
5.65 μmol) was added. The mixture was heated to reflux for 1 h
and cooled to room temperature. The solvent was evaporated off,
andthe residuewas purifiedbyflash chromatography onsilicagel
(EtOAc in hexanes, 60-80%) to yield the six-membered urea 20
(12.5 mg, 71%) as a brown foam. The sample was still contami-
nated with tricyclohexylphosphineoxide resulting from catalyst
decomposition: 1H NMR (400 MHz, CDCl3) δ 6.10-6.00 (br s,
1H, NH), 6.00-6.95 (m, 1H, H-4), 5.48 (app t, 1H, J ≈ 8.4 Hz, H-
20), 4.99 (d, 1H, J =8.2 Hz, H-40), 4.69-4.66 (m, 1H, H-5), 4.30 (d,
1H, J = 12.0 Hz, CHH-OAc), 3.98 (ddd, 1H, J = 14.3, 3.2, 1.3 Hz,
H-6a), 3.90 (d, 1H, J = 12.0 Hz, CHH-OAc), 3.79 (ddd, 1H, J =
14.3, 3.2, 1.7 Hz, H-6b), 2.14 (dd, 1H, J= 15.6, 8.4 Hz, H-30a), 1.99
(s, 3H, CH3, OAc), 1.98 (s, 3H, CH3, OAc), 1.64-1.56 (m, 1H, H-
30b), 1.30 (dd, 1H, J = 8.6, 4.4 Hz, H-50), 0.77-0.70 (m, 2H, H-60a,
b); 13C NMR (100 MHz, CDCl3) δ 171.2, 170.7 (2 ꢁ CdO, Ac),
153.4 (C-2), 124.8 (C-4), 96.6 (C-5), 75.8 (C-20), 65.8 (CH2-OAc),
54.1 (C-40), 41.2 (C-6), 34.1 (C-30), 32.0 (C-10), 25.8 (C-50), 21.0,
20.8 (2 ꢁ CH3, Ac), 11.0 (C-60); FAB-MS m/z (relative intensity)
309 (32), 249 (100), 297 (tricyclohexylphonsphineoxide, 71).
((1R,2S,4S,5S)-2-Acetoxy-4-(1,3-diallyl-3-benzoylureido)bicyclo-
[3.1.0]hexan-1-yl)methyl Acetate (21).Urea 16 (140 mg, 0.400 mmol)
((1R,2S,4S,5S)-2-Acetoxy-4-(allyl-tert-butoxycarbonylamino)-
bicyclo[3.1.0]hexan-1-yl)methyl Acetate (15). To a stirred solution
of carbamate 14 (350 mg, 1.07 mmol) in dry DMF (14.0 mL) were
added KHMDS (0.5 M in toluene, 2.57 mL, 1.28 mmol) and allyl
bromide (120 μL, 1.39 mmol) at 0 °C under argon. The mixture
was continued to stir for 5 h at 0 °C and then poured into a
mixture of phosphate buffer (100 mL, pH 7.2) and Et2O
(100 mL). The aqueous phase was further extracted with Et2O
(2 ꢁ 100 mL), and the combined organic phases were dried
(Na2SO4) and concentrated. The crude residue was purified by
flash chromatography (EtOAc in hexanes 20-40%) to yield the
title compound 15 (252 mg, 64%) as a colorless oil: [R]20 3.31
D
(c 1.16, CHCl3); IR (neat) 2976, 1736, 1687, 1454, 1364, 1330,
1
1237, 1170, 1143, 1023, 910, 776 cm-1; H NMR (400 MHz,
CDCl3) δ 5.81 (m, 1H, CHdCH2), 5.54 (app t, 1H, J ≈ 8.4 Hz,
H-2), 5.16-5.06 (m, 2H, CHdCH2), 4.16 (br s, 1H, H-4),
4.35 (d, 1H, J = 12.0 Hz, CHH-OAc), 3.89 (d, 1H, J=12.0 Hz,
CHH-OAc), 3.81 (br s, 2H, CH2-CHdCH2), 2.16 (dd, 1H, J =
15.3, 8.5 Hz, H-3a), 2.03 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.62-1.54
(m, 1H, H-3b), 1.41 (s, 9H, OtBu), 1.37 (dd, 1H, J = 8.8, 4.3 Hz,
H-5), 0.77 (dd, 1H, J = 5.8, 4.3 Hz, H-6a), 0.71 (dd, 1H, J = 8.8,
5.8 Hz, H-6b); 13C NMR (100 MHz, CDCl3) δ 171.1, 170.8 (2 ꢁ
CdO, Ac), 155.1 (CdO, BOC), 135.9 (CHdCH2), 115.2
(CHdCH2), 79.8 (O-C(CH3)), 75.5 (C-2), 65.9 (CH2-OAc), 56.3
(CH2-CHdCH2), 45.6 (C-4), 36.1 (C-3), 32.2 (C-1), 28.4 (O-C-
(CH3), 26.8(C-5), 21.0, 20.8(2ꢁ CH3, Ac), 11.2 (C-6); FAB-MS m/
z (relative intensity) 368 (5), 252 (100). Anal. Calcd for C19H29NO6:
C, 62.11; H, 7.96; N, 3.81. Found: C, 62.36; H, 7.89; N, 3.79.
((1R,2S,4S,5S)-2-Acetoxy-4-allylaminobicyclo[3.1.0]hexan-1-yl)-
methyl Acetate (12). The Boc-protected amine 15 (250 mg,
6222 J. Org. Chem. Vol. 74, No. 16, 2009

Ludek et al.
JOCArticle
was dissolved in dry pyridine (3.5 mL) at 0 °C under argon, and
triethylamine (168 μL, 1.20 mmol) and benzoyl chloride (140 μL,
1.20 mmol) were added dropwise. The mixture was allowed to warm
to room temperature and was stirred for 3 h. The solvent was
evaporated off, and the residue was partitioned between CH2Cl2
(50 mL) and 1 N HCl (50 mL). The aqueous phase was further
extracted with CH2Cl2 (2 ꢁ 50 mL), and the combined extracts were
dried (MgSO4) and concentrated. The crude was purified by flash
chromatography on silica gel (EtOAc in hexanes 50-70%) to yield
the N-acylurea 21 (162 mg, 89%) as a colorless syrup: [R]20D -6.57
(c 1.06, CHCl3); IR (neat) 3088, 2948, 2252, 1736, 1674, 1648, 1578
1412, 1369, 1238, 1195, 1025, 915, 795 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.62-7.58 (m, 2H, CH-arom), 7.47-7.42 (m, 1H, CH-
arom), 7.38-7.32 (m, 2H, CH-arom), 5.94-5.84 (m, 1H, CHdCH2-
A), 5.66-5.56 (m, 1H, CHdCH2-B), 5.41 (app t, 1H, J ≈ 8.3 Hz,
H-2), 5.30-5.10 (m, 4H, CHdCH2-A, CHdCH2-B), 4.45-4.15 (m,
4H, CH2-CH-A, CHH-OAc, H-4), 3.90-3.60 (m, 3H, CH2-CH-B,
CHH-OAc), 1.98 (s, 3H, CH3, OAc), 1.96 (s, 3H, CH3, OAc), 1.50-
1.30 (m, 1H, H-5), 0.65-0.59 (m, 2H, H-6a,b); 13C NMR (100 MHz,
CDCl3) δ 170.8, 170.6 (2 ꢁ CdO, Ac), 169.4 (CdO, Bz), 157.8
(CdO, urea), 134.9, 133.4 (2 ꢁ CHdCH2), 132.4, 131.6, 128.3, 128.1
(C-arom), 119.2, 117.7 (2 ꢁ CHdCH2),75.0(C-3),67.9(CH2-OAc),
65.4 (CH2-CH-a), 59.2 (C-1), 49.3 (CH2-CH-b), 35.6 (C-4),
32.8 (C-2), 25.5 (C-5), 20.9, 20.7 (2 ꢁ CH3, Ac), 11.1 (C-6); FAB-
MS m/z (relative intensity) 455 (14), 105 (100). Anal. Calcd
for C25H30N2O6: C, 66.06; H, 6.65; N, 6.16. Found: C, 65.95; H,
6.33; N, 6.08.
((10R,20S,40S,50S)-20-Acetoxy-40-(N-benzoyl-2-oxo-2,3,4,7-tetra-
hydro-1H-1,3-diazepin-1-yl)bicyclo[3.1.0]hexan-10-yl)methyl Acet-
ate (22). The N-benzoylurea 21 (150 mg, 0.330 mmol) was dissolved
in dry and deoxygenated CH2Cl2 (50 mL) under argon at room
temperature, and second-generation Grubbs catalyst (28.0 mg,
0.033 mmol) was added in one portion. The mixture was heated
to reflux for 1 h and cooled to room temperature again. The solvent
was evaporated off, and the crude was directly subjected to flash
chromatography on silica gel (EtOAc in hexanes 45-65%) to yield
the diazepinone 22 (130 mg, 92%) as a colorless foam: [R]20D 39.21
(c 1.00, CHCl3); IR (neat) 3037, 2960, 2883, 1732, 1677, 1461, 1364,
1235, 1192, 1024, 990, 848 cm-1; 1H NMR (400 MHz, CDCl3) δ
7.50-7.46 (m, 2H, CH-arom), 7.43-7.38 (m, 1H, CH-arom),
7.36-7.31 (m, 2H, CH-arom), 5.90-5.82 (m, 1H, H-6), 5.81-
5.70 (m, 1H, H-5), 5.54 (app t, 1H, J ≈ 8.4 Hz, H-20), 4.61 (d, 1H,
J = 8.1 Hz, H-40), 4.54 (br d, 1H, J = 18.5 Hz, H-4a), 4.44 (d, 1H,
J = 12.0 Hz, CHH-OAc), 4.29 (br d, 1H, J = 18.5 Hz, H-4b),
4.11-3.92 (m, 2H, H-7a,b), 3.79 (d, 1H, J = 12.0 Hz, CHH-
OAc), 2.06-2.01 (m, 1H, H-30a), 2.05 (s, 3H, OAc), 2.00 (s, 3H,
OAc), 1.58 (m, 1H, H-30b), 1.27 (dd, 1H, J = 8.3, 4.8 Hz,
H-50), 0.77-0.70 (m, 2H, H-60a,b); 13C NMR (100 MHz, CDCl3)
δ 171.1 (CdO, Bz), 170.8, 170.6 (2 ꢁ CdO, Ac), 158.8 (C-2), 135.2,
131.4, 128.93, 128.4 (C-arom), 127.1 (C-6), 123.8 (C-5), 74.8
(C-20),65.6 (CH2-OAc), 57.0 (C-40), 43.0 (C-4), 40.8 (C-7), 35.7
(C-10), 32.4(C-30), 26.5(C-50), 21.0, 20.9 (2 ꢁ CH3, Ac), 11.1(C-60);
HRMS-FAB m/z calcd for C23H27N2O6 (M þ Hþ) 427.1869,
found 427.1856.
1-((10S,20S,40S,50R)-40-Hydroxy-50-hydroxymethylbicyclo[3.1.0]-
hexan-20-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one (5). The fully
acylated nucleoside analogue 22 (50.0 mg, 0.117 mmol) was
dissolved in a 1% solution of NaOH in methanol (5.0 mL) and
stirred at room temperature for 10 min. The mixture was heated to
reflux for 1 h and cooled to room temperature again. After
neutralization with 2 N HCl, the solvent was evaporated off and
the crude was purified by flash chromatography on silica gel
(MeOH in EtOAc 5-15%) to yield the diazepinone nucleoside 5
(24.5 mg, 88%) as a colorless foam. The foam was dissolved in
acetonitrile/water (1:1, 3 mL) and was lyophyllized to yield a
hygroscopic and colorless amorphous semisolid: [R]20 16.23
D
(c 1.00, H2O); IR (neat) 3318, 2869, 1633, 1485, 1428, 1373, 1309,
1190, 1149, 1046, 1007, 846, 786 cm-1;1H NMR (400 MHz, D2O) δ
5.83-5.76 (m, 1H, H-6), 5.74-5.68 (m, 1H, H-5), 4.65 (t, 1H, J =
8.6 Hz, H-40), 4.27 (d, 1H, J = 8.3 Hz, H-20), 3.85 (d, 1H, J =
12.3 Hz, CHH-OH), 3.76 (m, 1H, H-7a), 3.66-3.51 (m, 3H, H-7b,
H-6a,b), 3.29 (d, 1H, J = 12.3 Hz, CHH-OH), 1.86 (dd, 1H, J =
15.3, 8.6 Hz, H-30a), 1.52 (m, 1H, H-30b), 1.17 (dd, 1H, J = 7.8,
5.1 Hz, H-10), 0.56-0.52 (m, 2H, H-60a,b); 13C NMR (100 MHz,
D2O) δ166.5 (C-2), 128.0 (C-6), 126.3 (C-5), 72.5 (C-40),62.9(CH2-
OH),57.7(C-20),42.2(C-7),41.1(C-50),37.þ4(C-4),36.3(C-3þ0),25.6
(C-10), 9.3 (C-60); ESI-MS m/z 239 (M þ H ), 261 (M þ Na ), 277
(M þ Kþ). Anal. Calcd for C12H18N2O3 1.0 H2O: C, 56.23; H,
3
7.87; N, 10.93. Found: C, 56.49; H, 7.51; N, 10.59.
Acknowledgment. This research was supported by the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research, and by Grant No.
GM-18325 (D.W. and G.K.S.).
Supporting Information Available: Superposition of north-
bicyclo[3.1.0]hexane thymidine and south-bicyclo[3.1.0]hexane
thymidine adopted in the crystal structures LETJEZ and YES-
MIS with docking conformations of 5 and 6; synthetic methods
for the compounds described in Scheme 5; proton NMR spectra
(500 MHz) of locked north- and south-diazepinones 5 and 6,
plus flexible diazepinone 7 in D2O together with internal
standard; and 1H and13C NMR spectra of new compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 74, No. 16, 2009 6223