Recent advances in the synthesis of conformationally locked nucleosides and their success in probing the critical question of conformational preferences by their biological targets

Article abstract of DOI:10.1081/NCN-120021954

The present work describes some recent approaches to the syntheses of three classes of locked-North nucleosides: β-D-ribo-, β-D-deoxyribo-, and β-D-dideoxy-ribonucleosides. The method developed for the latter class permitted access to a novel bicyclo[3.1.

Full text of DOI:10.1081/NCN-120021954

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Recent Advances in the Synthesis of Conformationally
Locked Nucleosides and Their Success in Probing the
Critical Question of Conformational Preferences by
Their Biological Targets
Yongseok Choi a , Hyung R. Moon a , Yuichi Yoshimura a & Victor E. Marquez a b
a Laboratory of Medicinal Chemistry , Center for Cancer Research , National Cancer Institute
at Frederick , NIH, HHS, Frederick, Maryland, USA
b Laboratory of Medicinal Chemistry , Center for Cancer Research , National Cancer Institute
at Frederick , NIH, HHS, 376 Boyles St., Frederick, MD, 21702, USA
Published online: 31 Aug 2006.
To cite this article: Yongseok Choi , Hyung R. Moon , Yuichi Yoshimura & Victor E. Marquez (2003) Recent Advances in the
Synthesis of Conformationally Locked Nucleosides and Their Success in Probing the Critical Question of Conformational
Preferences by Their Biological Targets, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 547-557, DOI: 10.1081/
NCN-120021954
To link to this article: http://dx.doi.org/10.1081/NCN-120021954
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 547–557, 2003
Recent Advances in the Synthesis of Conformationally
Locked Nucleosides and Their Success in Probing the
Critical Question of Conformational Preferences
by Their Biological Targets
Yongseok Choi, Hyung R. Moon, Yuichi Yoshimura,
*
and Victor E. Marquez
Laboratory of Medicinal Chemistry, Center for Cancer Research, National
Cancer Institute at Frederick, NIH, HHS, Frederick, Maryland, USA
ABSTRACT
The present work describes some recent approaches to the syntheses of three
classes of locked-North nucleosides: b-D-ribo-, b-D-deoxyribo-, and b-D-dideoxy-
ribonucleosides. The method developed for the latter class permitted access to
a novel bicyclo[3.1.0]hexene-type nucleosides structurally similar to D4T and
carbovir. A structural analysis and biological activities are discussed.
Key Words: Bicyclo[3.1.0]hexane and bicyclo[3.1.0]hexene; Conformationally
locked carbocyclic nucleosides; Pseudorotational analysis; D4T and carbovir ana-
logues; Intramolecular cyclopropanation; Lipase-catalyzed resolution.
*Correspondence: Victor E. Marquez, Laboratory of Medicinal Chemistry, Center for Cancer
Research, National Cancer Institute at Frederick, NIH, HHS, 376 Boyles St., Frederick, MD
21702, USA; E-mail: marquez@dc37.nci.nih.gov.
547
DOI: 10.1081/NCN-120021954
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

548
Choi et al.
Figure 1. Fixed location of (N)- and (S)-methanocarba nucleosides in the pseudorotational
cycle (nucleoside numbering).
INTRODUCTION
Conventional nucleosides or nucleotides equilibrate rapidly in solution
between two extreme forms of ring pucker: (1) The North conformation with
3
3
pseudorotational P values ranging between 342ꢀ to 18ꢀ (2E ! T2 ! E) and (2) the
2
antipodal South conformation with values of P between 162ꢀ to 198ꢀ (2E ! T3 ! 3E)
(Fig. 1).[1,2] Preference for any of these specific conformations in solution is deter-
mined by the interplay of important interactions such as anomeric and gauche
effects.[2] However, when a nucleoside or nucleotide binds to its target enzyme, only
one form is present at the active site. In order to query these enzymes for their
conformational preference we have designed and synthesized conformationally
locked nucleosides that reside strictly in the normal range of either North or South
conformations.
The bicyclo[3.1.0]hexane scaffold provides a convenient way to lock the confor-
mation of carbocyclic nucleosides which are known generically as a methanocarba
(MC) nucleosides. Because of its exclusive pseudoboat conformation, a rigid North
envelope (2E) conformation can be constructed when the cyclopropane ring appears
0
fused between carbons C4 and C5 . Conversely, fusion of the cyclopropane ring
0
0
between carbons C1 and C5 provides a rigid South (3E) envelope conformation
0
(Fig. 1).

Conformationally Locked Nucleosides
RESULTS AND DISCUSSION
The North Scaffold as a Promising Template for Drug Development
549
North- and South-MC nucleosides with the same nucleobase are very nicely dis-
criminated by specific enzymes and thus function as target-selective agents. Some of
the most important compounds with potential as drug candidates are in the North
hemisphere. (N)-MC thymidine (1) is a very selective antiviral agent which has
shown greater potency against HSV-1 and HSV-2 infection and less toxicity than
the standard ganciclovir.[3,4] This compound is also effective in vivo against murine
MC38 tumors transduced with the herpes thymidine kinase gene.[5] MRS2279 (2), a
30,50-biphosphate purine analogue from Dr. Jacobson’s laboratory is a very potent
and selective high affinity P2Y1 receptor agonist which does not to block nucleotide
signaling at most of the other known P2Y receptor subtypes.[6]
SYNTHETIC METHODS
Most of the published methods for (N)-MC nucleosides rely on a critical hydro-
xyl-directed cyclopropanation step (Sch. 1) from the allylic position on a cyclo-
pentenol precursor.[7–9] Although the cyclopropanation step works well, the synthesis
of the starting carbocycle is quite laborious and requires expensive starting materials.
The resulting pseudosugars can be converted to purine or pyrimidine (N)-MC
nucleosides by convergent approaches (from 4) or by linear approaches (from 6).
Scheme 1.

550
Choi et al.
Scheme 2.
Normally, a convergent Mitsunobu coupling with the preformed nucleobase works
better with purines. On the other hand, for pyrimidines, the formation of abundant
O-alkylation products makes the linear approach the preferred route.
In the past couple of years we have developed some new approaches using
more accessible and less expensive starting materials. At the core of these approaches
lies a critical intramolecular cyclopropanation step from a carbene intermediate
engendered from a diazo b-keto ester. The retrosynthetic analysis depicted in Sch. 2
illustrates the general strategy.
Steps Ia to VIIa describe the approach to ribo-like, (N)-MC nucleosides,
whereas steps Ib to VIIb correspond to the deoxyribo series. The syntheses of the
precursor diazo b-keto esters IIIa and IIIb are simple and straightforward, and after
metal-catalyzed thermolysis the carbene intermediates IVa and IVb fold intramole-
cularly into the desired bicyclo[3.1.0]hexane scaffold. The top part in Sch. 2 depicts
the desired b-D-like configuration, achieved via a stereospecific synthesis from a
chiral precursor (Ia). For the bottom part, the process leading to intermediate VIb
is achiral, and thus the racemic mixture is resolved at this stage via a lipase-catalyzed
asymmetric acetylation.

Conformationally Locked Nucleosides
551
A. Enantiospecific Synthesis of Ribo-type,
North-Methanocarba Nucleosides
Starting with inexpensive and plentiful D-isoascorbic acid, the chiral precursor
[10,11] was obtained in 4 steps and 60% overall yield (Sch. 3). After the stepwise oxi-
7
dation of 7, first under Swern conditions and then with sodium chlorite, Dieckmann
condensation of the activated acid 8 with ethyl 2-lithioacetate afforded b-keto esters
9 (8%) and 10 (59%) as a mixture of separable diastereoisomers. Following diazo
transfer with tosyl azide and triethylamine (TEA), b-keto ester 10 was converted
to diazo compound 11, which underwent metal-catalyzed thermolysis to gene-
rate the desired bicyclo[3.1.0]hexane scaffold via a carbenoid intermediate (IVa,
Sch. 2). Comparisons with authentic compounds previously made from different
sources,[12] confirmed that the major isomer, 12, had the desired stereochemistry.
Reduction of the carbonyl moiety gave diol 14, which can be protected. In the case
of benzyl-protected 15, acid-catalyzed removal of the acetonide and reaction of 16
with thionyl chloride produced the cyclic sulfite 17. This compound reacted well with
sodium azide to give 82% of 18 and 10% of the alternative regioisomer. Azide 18 was
efficiently reduced to amine 19 (overall yield from D-isoascorbic acid ¼ 2%) which is
a direct precursor of purine and pyrimidine analogues using well-known linear
approaches.[12]
Scheme 3.

552
Choi et al.
B. Chemico-Enzymatic Approaches to Deoxyribo-type,
North-Methanocarba Nucleosides
In this case the process is basically achiral (Ib ! VIIb, Sch. 2). A final resolution
step using adenosine deaminase worked well only for purines, particulary for the
guanosine analogue, which was obtained directly from the deamination of the
2,6-diaminopurine nucleoside.[13] To overcome this limitation, a resolution step
prior to the incorporation of the nucleobase was recently accomplished via an
economical lipase-catalyzed asymmetric acetylation performed on diol VIb
(Sch. 2).[14]
Commercially available lipase PS-C in the presence of a large excess of vinyl
acetate effectively discriminated between the enantiomers of rac-20 (VIb,
R ¼ SiPh2CMe3, Schs. 2 and 4) producing diacetate 21 (57%) and monoacetate 22
(42%) after 64 h. Using chiral HPLC, the enantiomeric excess of monoacetate 22
was estimated to be 96% and, although the enantiomeric excess of 21 should have
been over 98%, it contained 13% of an impurity (210). Removal of this impurity
was important since diacetate 21 corresponded to the enantiomer with the desired
D-like configuration.[14] The structure of 210 was that of an acetal arising from the
reaction of the unrecognized enantiomer 22 with excess acetaldehyde—generated
during the reaction—followed by further enzymatic acetylation. Treatment of con-
taminated 21 with ammonium hydrogenfluoride in aqueous DMF (50ꢀC, overnight)
gave an easily separable mixture of 23 and diol 12 in 79% and 11% yields, respec-
Scheme 4.

Conformationally Locked Nucleosides
553
tively. The optical purity of 23, was estimated to be 99% ee by HPLC after its con-
version back to 21 with tert-butyldiphenylsilyl chloride.[14] Completing the synthesis
of deoxyribo-type, (N)-MC nucleosides from diacetate 23 was simple and straight-
forward.
C. Enantiospecific Synthesis of Dideoxyribo-type and Dideoxy-
didehydroribo-type, North-Methanocarba Nucleosides. Synthesis
of Analogues of D4T and Carbovir
0
The attempt to remove the secondary, C3 -OH (nucleoside numbering) under
radical deoxygenation conditions resulted in the destruction of the fused cyclopro-
pane ring, which collapsed after formation of a radical intermediate at the adjacent
carbon. To circumvent this problem, deoxygenation was performed prior to the
formation of the bicyclo[3.1.0]hexane template (Sch. 5) starting from a compound
readily available in our laboratory, such as 24.[8] The corresponding thiocarbonyl-
imidazolide (25) gave under radical deoxygenation conditions an inseparable mixture
of cyclic allylic (26) and homoallylic (27) benzoate esters. The mixture of alcohols (28
and 29) obtained after hydrolysis of the benzoate esters was cyclopropanated to give
isomeric bicyclo[3.1.0]hexanols, 30 and 31, that were easily separable by column
chromatography. With the exception of optical rotation, the spectral properties
of 30 matched exactly those of its previously reported racemate (rac-30).[15] From
compound 30, purine nucleosides are readily accessible in a convergent manner by
Mitsunobu coupling with the corresponding nucleobase. However, coupling of
rac-30 with pyrimidines, such as N3-benzoylthymine, was reported to give rather low
yields of the desired N-alkylated product.[15]
To improve the coupling yields for pyrimidine nucleosides we postulated that a
bicyclo[3.1.0]hexenol, such as 35, would react more readily (Sch. 6). The synthesis of
0
this precursor required the elimination of the C3 -OH (nucleoside sugar numbering)
from a compound, such as 33, which was easily obtained from 32[9] in 3 steps.
Scheme 5.

554
Choi et al.
Scheme 6.
When the secondary alcohol was converted to the mesylate ester 34 for a possi-
ble base-catalyzed E2 elimination, only the hydrolyzed product was obtained.
Because of the constrained nature of the bicyclo[3.1.0]hexane template, a more
favorable anti-elimination was sought by attempting to invert the configuration of
the leaving group after treatment with I2=Ph3P=imidazole. However, the constrained
nature of the system forced a double inversion to give instead the thermodynamic
product 36 with the iodine axially disposed. Still, elimination occurred by refluxing
36 in toluene in the presence of DBU to produce the desired bicyclo[3.1.0]hexene
pseudosugar 37 in 51% yield. Base-catalyzed hydrolysis of the benzoate ester
afforded the key intermediate 35. As predicted, coupling of 35 with N3-benzoyl-
thymine under Mitsunobu conditions gave the corresponding N-alkylated product
as the major isomer in 43% yield. The final D4T analogue 38 was obtained after
treatment with NH4OH and final removal of the TBDPS group. A similar approach
was used for the synthesis of the carbovir analogue 39. Catalytic hydrogenation of 38
and 39 afforded the corresponding dideoxyribo-type analogues in excellent yields.
STRUCTURAL ANALYSIS AND STRUCTURE-ACTIVITY
RELATIONSHIP
The use of bicyclo[3.1.0]hexane templates has already allowed us to determine
conformational preference for enzymes such as adenosine deaminase,[9] HIV reverse

Conformationally Locked Nucleosides
555
Table 1. Pseudorotational parameters for 38, D4T (40), and Carbovir (41a and 41b). Num-
bers represent degrees (ꢀ).
n0
n1
n2
n3
n4
P
nmax
w
g
38
40
6.56 ꢁ5.83
0.20
2.67
0.19
1.68 ꢁ5.20 293.14
6.81 ꢁ119.12
0.61 ꢁ100.82
56.17
52.80
ꢁ0.51
ꢁ0.49
0.60
25.68
26.98
72.08
92.23 27.98
89.55 28.91
41a ꢁ26.47
17.89 ꢁ1.09 ꢁ16.02
17.21
0.23 ꢁ17.56
ꢁ86.24 ꢁ177.76
41b ꢁ27.22
ꢁ52.63
ꢁ82.90
transcriptase,[16] DNA (cytosine-C5) methyl transferase,[17] and several subtypes of
adenosine receptors.[7] A common structural characteristic of D4T (40) and carbovir
(41) is a double bond, a feature that imparts a certain level of rigidity to the five
member ring. Thus, compounds 38 and 39 represent hybrid molecules that incorpo-
rate in their bicyclo[3.1.0]hexene template a similar double bond. Compound 38 pro-
vided adequate crystals for X-ray analysis. At first glance, a comparison between the
X-ray structure of 38 and that of D4T (40) obtained from the Cambridge database
revealed little differences. Indeed, the superposition of both structures shows a RMS
˚
deviation of only 0.039 A. On the other hand, the pseudorotational parameters for
these molecules are quite different, particularly with respect to the value of P
(Table 1). Both structures are in the North hemisphere, but they are 140ꢀ apart from
each other, separated from a perfect North (P ¼ 0ꢀ) by an almost equal number of
degrees (ca. 70ꢀ) towards the West and East, respectively. Also, the ring of D4T
(40) is far more planar (nmax ¼ 0.61ꢀ) with a mean deviation from planarity of only
0.0025 A. The ring of 38 is more puckered (nmax ¼ 6.81ꢀ) with a mean deviation from
˚
˚
planarity of 0.025 A.
Although we did not obtain a crystal structure of 39, the rigid bicyclo[3.1.0]-
hexene ring is expected to be similar to that of 38 with identical pseudorotational
parameters. In contrast, the pseudorotational parameters for the two molecules
present in the unit cell of a carbovir X-ray structure are quite different from those
of 39 (Table 1). The main differences found are first the degree of flatness (see nmax
)
which shows that the five-member ring of 38 (and by analogy that of 39) is much
flatter than that of carbovir. Secondly, the value of P places carbovir in an almost
perfect East conformation, 157ꢀ away from 38 or 39. These conformational differ-
ences may be associated with some unique biological activity for compounds 38
and 39, which is currently under investigation.
REFERENCES
1. Saenger, W. Chapter 2, Defining terms for the nucleic acids. In Principles in
Nucleic Acid Structure; Springer-Verlag: New York, NY, 1984.
2. Thibaudeau, C.; Chattopadhyaya, J. Stereoelectronic effects in nucleoside and
nucleotides. In Stereoelectronic Effects in Nucleosides and Nucleotides and Their
Structural Implications; Uppsala University Press: Uppsala, Sweden, 1999.
3. Marquez, V.E.; Siddiqui, M.A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner,
R.W.; Matteucci, M. Nucleosides with a twist. Can fixed forms of sugar ring

556
Choi et al.
pucker influence biological activity in nucleosides and oligonucleotides?
J. Med. Chem. 1996, 39, 3739–3747.
4. Zalah, L.; Huleihel, M.; Manor, E.; Konson, A.; Ford, H., Jr.; Marquez, V.E.;
Johns, D.G.; Agbaria, R. Metabolic pathways of N-methanocarbathymidine,
a novel antiviral agent in native and herpes simplex virus-infected vero cells.
Antiviral Res. 2002, 55, 63–75.
5. Noy, R.; Ben-Zvi, Z.; Manor, E.; Candotti, F.; Morris, J.C.; Ford, H., Jr.;
Marquez, V.E.; Johns, D.G.; Agbaria, R. Antitumor activity and metabolic
activation of (N)-methanocarbathymidine, a novel thymidine analog with a
pseudosugar moiety rigidly fixed in the northern conformation, in murine colon
cancer cells expressing herpes simplex virus thymidine kinase. Mol. Cancer
Therap. 2002, 1, 585–593.
6. Boyer, J.L.; Adams, M.; Ravi, R.G.; Jacobson, K.A.; Harden, T.K. 2-Chloro
N6-methyl-(N)-methanocarba-20-deoxyadenosine-30,50-bisphosphate is a selec-
tive high affinity P2Y1 receptor antagonist. Brit. J. Pharmacol. 2002, 135,
2004–2010.
7. Siddiqui, M.A.; Ford, H., Jr.; George, C.; Marquez, V.E. Synthesis, conforma-
tional analysis, and biological activity of a rigid carbocyclic analogue of 20-
deoxyaristeromycin built on a bicyclo[3.1.0]hexane template. Nucleosides
Nucleotides 1996, 15, 235–250.
8. Ezzitouni, A.; Russ, P.; Marquez, V.E. (IS,2R)-[(Benzyloxy)methyl]cyclopen-
tenol. A versatile synthon for the preparation of 40,10a-methano and 10,10a-
methanocarbocylic nucleosides. J. Org. Chem. 1997, 62, 4870–4873.
9. Marquez, V.E.; Russ, P.; Alonso, R.; Siddiqui, M.A.; Hernandez, S.; George,
C.; Nicklaus, M.C.; Dai, F.; Ford, H., Jr. Synthesis of conformationally
restricted carbocyclic nucleosides. The role of the O40-oxygen in the key hydra-
tion step of adenosine deaminase. Helv. Chim. Acta 1999, 82, 2119–2129.
10. Cohen, N.; Banner, B.L.; Laurenzano, A.J.; Carozza, L. 2,3-O-Isopropylidene-
D-erythronolactone. Org. Syn. 1985, 63, 127–135.
11. Rao, B.V.; Lahiri, S. Stereoselective transformations leading to pentono-1,4-
lactones. J. Carbohydr. Chem. 1996, 15, 975.
12. Moon, H.R.; Kim, H.O.; Chun, M.W.; Jeong, L.S.; Marquez, V.E. Synthesis of
cyclopropyl-fused carbocyclic nucleosides via the regioselective opening of cyc-
lic sulfites. J. Org. Chem. 1999, 64, 4733–4741.
13. Moon, H.R.; Ford, H., Jr.; Marquez, V.E. A remarkably simple chemico enzy-
matic approach to structurally complex bicyclo[3.1.0]hexane carbocyclic
nucleosides. Org. Lett. 2000, 2, 3793–3796.
14. Yoshimura, Y.; Moon, H.R.; Choi, Y.S.; Marquez, V.E. Enantioselective
synthesis of bicyclo [3.1.0]hexane carbocyclic nucleosides via a lipase-catalyzed
asymmetric acetylation. Characterization of an unusual acetal byproduct.
J. Org. Chem. (in press).
15. Rodriguez, J.B.; Marquez, V.E.; Nicklaus, M.C.; Mitsuya, H.; Barchi, J.J.
Conformationally locked nucleoside analogues. Synthesis of dideoxycarbo-
cyclic nucleoside analogues structurally related to neplanocin C. J. Med. Chem.
1994, 37, 3389–3399.
16. Marquez, V.E.; Ezzitouni, A.; Russ, P.; Siddiqui, M.A.; Ford, H., Jr.; Feld-
man, R.J.; Mitsuya, H.; George, C.; Barchi, J.J., Jr. HIV-1 reverse transcriptase

Conformationally Locked Nucleosides
557
can discriminate between two conformationally locked carbocyclic AZT-tri-
phosphate analogues. J. Amer. Chem. Soc. 1998, 120, 2780–2789.
17. Wang, P.; Nicklaus, M.C.; Marquez, V.E.; Brank, A.S.; Christman, J.K.;
Banavali, N.K.; MacKerell, A.D., Jr. Use of oligodeoxyribonucleotides with
conformationally constrained abasic sugar targets to probe the mechanism of
base flipping by HhaI DNA (cytosine C5)-methyltransferase. J. Amer. Chem.
Soc. 2000, 122, 12,422–12,434.