"Reverse" carbocyclic fleximers: Synthesis of a new class of adenosine deaminase inhibitors

Article abstract of DOI:10.1080/15257770.2013.771187

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported "fleximers" from our laboratory, these analogues have the connectivity of the heterocyclic base system "reversed", where the pyri

Full text of DOI:10.1080/15257770.2013.771187

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“Reverse” Carbocyclic Fleximers:
Synthesis of a New Class of Adenosine
Deaminase Inhibitors
Sarah C. Zimmermann a , Joshua M. Sadler a , Peter I. O’Daniel b
Nathaniel T. Kim a & Katherine L. Seley-Radtke a
,
a Department of Chemistry and Biochemistry, University of Maryland,
Baltimore, Maryland
b Department of Chemistry and Biochemistry, University of Notre
Dame, Notre Dame, Indiana
To cite this article: Sarah C. Zimmermann , Joshua M. Sadler , Peter I. O’Daniel , Nathaniel T. Kim
& Katherine L. Seley-Radtke (2013): “Reverse” Carbocyclic Fleximers: Synthesis of a New Class of
Adenosine Deaminase Inhibitors, Nucleosides, Nucleotides and Nucleic Acids, 32:3, 137-154
To link to this article: http://dx.doi.org/10.1080/15257770.2013.771187
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Nucleosides, Nucleotides and Nucleic Acids, 32:137–154, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.771187
“REVERSE” CARBOCYCLIC FLEXIMERS: SYNTHESIS OF A NEW
CLASS OF ADENOSINE DEAMINASE INHIBITORS
Sarah C. Zimmermann,1 Joshua M. Sadler,1 Peter I. O’Daniel,2
Nathaniel T. Kim,1 and Katherine L. Seley-Radtke1
1Department of Chemistry and Biochemistry, University of Maryland, Baltimore, Maryland
2Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana
A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In
2
contrast to previously reported “fleximers” from our laboratory, these analogues have the connectivity
of the heterocyclic base system “reversed”, where the pyrimidine ring is attached to the sugar moiety,
rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their
inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase
the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition
of adenosine deaminase, despite their lack of resemblance to adenosine.
Keywords Fleximers; carbocyclic nucleosides; adenosine deaminase; 5-substituted
uracils; pyrimidine based inhibitors
INTRODUCTION
The development of drug resistance to currently used therapeutics is one
of the major challenges medicinal chemists are facing today. One common
resistance mechanism arises when mutations occur within the active site of
a target enzyme. A single point mutation of an amino acid residue within
the binding pocket can result in an unfavorable steric or electronic clash
and prevent an inhibitor from binding properly.[1] Recent reports have
shown that flexible inhibitors can overcome resistance mechanisms and
retain activity by utilizing secondary residues within the binding site not
previously involved in the enzyme’s mechanism of action.[2–5] Additionally,
Received 29 December 2012; accepted 25 January 2013.
We thank Professors Sunny Zhou (Northeastern University) for assistance with the SAHase and
ADA assay. This work was supported by the National Institutes of Health (NIH) (R01 CA97634 to KSR,
NIH T32 GM066706 CBI Fellowship (SCZ). We are also grateful to Dr. Phil Mortimer (Johns Hopkins
Mass Spectrometry Facility) for his invaluable assistance with the HRMS analysis.
Address correspondence to Katherine L. Seley-Radtke, Department of Chemistry and Biochemistry,
University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA. E-mail:
kseley@umbc.edu
137

138
S. C. Zimmermann et al.
the flexibility of the inhibitor may allow it to “masquerade” as a different
compound, which would slow the onset of resistance that can arise from
repeated exposure to a particular chemotherapeutic agent.[2,3]
One focus of our research has been to impart flexibility to the nucle-
obase scaffold to potentially increase inhibitor function and recognition.
The purine scaffold of the “fleximer” has been split into the individual imi-
dazole and pyrimidine rings that remain attached via a single carbon–carbon
bond.[6–9] As a result, the nucleobase retains the requisite hydrogen bonding
and aromatic features necessary for recognition, while gaining the flexibility
to adapt to an enzyme’s binding site and potential mutations. In that regard,
we have previously reported that a flexible guanosine nucleoside (1, Figure
1), served as an inhibitor of S-adenosylhomocysteine hydrolase (SAHase), an
adenosine-metabolizing enzyme.[10] The flexibility of the base unit yielded
rotational and conformational changes that allowed the guanosine analogue
(Flex-G) to mimic adenosine.[10] In addition to this, our investigations with
GTP fucose pyrophosphorylase (GFPP) have shown that the triphosphate
of Flex-G (Flex-GTP, 2) was preferred over the natural substrate, guanosine
triphosphate (GTP).[11] The ability of Flex-GTP to interact with secondary
amino acids within the binding site led to more favorable binding interac-
tions, thereby increasing the affinity in GFPP when compared to GTP.[11,12]
In addition to our findings, groups such as Hudson et al. have pursued a
series of analogues using click chemistry in which they have found inter-
esting fluorescent properties.[13] It is important to note that, to date, no
toxicity has been observed with any of the members of this interesting class
of nucleosides.
As an extension of our initial studies with the ribose and 2ꢁ-deoxyribose
fleximers,[14] we have designed a series of “reverse” fleximers,[15] where the
purine base scaffold is connected to the sugar moiety at the N-3 of the
pyrimidine ring rather than the N-9 of the imidazole ring, as shown in
Figure 2 (3a–c and 4a–c). This reverses the conformation of the split purine
base, which can also be viewed as a C5-substituted pyrimidine. This type
of connectivity is also found in Isoadenosine (IsoA)[16,17] and while IsoA
FIGURE 1 Flex-G and Flex-GTP.

“Reverse” Carbocyclic Fleximers
139
FIGURE 2 “Reverse” carbocyclic targets.
exhibited interesting biological properties, it was not extensively pursued
because of instability to both acidic and basic conditions, resulting in a 1,3-
migration to afford adenosine.[17]
Earlier efforts in our laboratory to address the stability issues of IsoA
prompted us to the employ the more stable carbocyclic sugar scaffold.[18]
This structural change imparts increased stability by rendering the labile
glycosidic hemiaminal bond a tertiary amine.[19,20] In addition, carbocyclic
nucleosides such as aristeromycin (Ari), 5ꢁ-norAri, Neplanocin A (NpcA),
and their truncated derivatives are potent inhibitors of SAHase; thus, the
carbocyclic modification was considered important for the design of the
reverse fleximers.[20–22]
Several ribose compounds structurally similar to our target compounds
have been reported. Tor et al. utilized substituted pyrimidine analogues
as a fluorescent bioprobe to study DNA helical structure.[23] Herdewijn
et al. developed a series of C5 substituted pyrimidine analogues that proved
active against HSV-1 due to phosphorylation by the viral thymidine ki-
nase (TK).[24–26] In contrast, our analogues would not be expected to
be phosphorylated by TK, since they lack the requisite 5ꢁ-hydroxyl that
would undergo phosphorylation, however, could inhibit other nucleoside-
metabolizing enzymes such as the aforementioned SAHase. Moreover, it
was speculated that they may be able to serve as inhibitors of both purine
metabolizing enzymes, such as SAHase, but might also prove to be in-
hibitors of pyrimidine metabolizing enzymes as was noted with Herdewijn’s
analogues.
As described herein, these novel pyrimidine nucleosides were not recog-
nized by SAHase as anticipated, but rather, by adenosine deaminase (ADA),
something we did not anticipate given their lack of resemblance to adeno-
sine. Although the exact mechanism of action has yet to be elucidated, spec-
ulation is that the compounds have a similar effect as exhibited by inosine
and xanthosine, which act as product inhibitors. Moreover, given the re-
ports that nucleoside ADA inhibitors can exhibit activity against lymphomas

140
S. C. Zimmermann et al.
and leukemias due to elevated levels of ADA present in malignant human
lymphocytes, this finding may prove significant.[27–29]
RESULTS AND DISCUSSION
Chemistry
In considering the design of the target compounds, the truncated systems
were initially chosen due to the toxicity exhibited by Ari and NpcA as a
result of phosphorylation at the 5ꢁ-hydroxyl.[30] Additionally, the thiophene,
furan, and thiazole substituents were chosen instead of the more traditional
imidazole due to the higher activity exhibited by Herdewijn’s analogues,
as well as the reported inability to remove the N-methyl group from the
imidazole analogue.[31]
The synthesis of 3a–c and 4a–c was envisioned starting from a well-
known enone intermediate,[32] which can undergo a Luche reduction[33] to
form the allylic alcohol (5, Figure 3) that can then be coupled using Mit-
sunobu conditions[34] to attach the pyrimidine. Subsequent organometallic
coupling, to the various 5-membered ring systems, would then produce the
desired targets.[26,31]
As outlined in Scheme 1, enone 6, which is synthesized from d-ribose,[35]
was stereospecifically reduced down to allylic alcohol 5, using Luche reduc-
tion conditions[36] and then coupled to either N3-benzoyl-5-bromouracil or
N3-benzoyluracil[37] through the use of the Mitsunobu[34] reaction to afford
7 and 8, respectively.
It should be noted that the most obvious route to the saturated carbo-
cycles 3a–c involved reducing the double bond in the cyclopentenyl ring
of products 4a–c; however, the presence of the C5 substituents prohibited
this approach, due to unwanted side reactions that would result from a pal-
ladium catalyzed hydrogenation. In addition, reduction prior to that point
would have removed the halide on the pyrimidine ring. Once the benzoyl
FIGURE 3 Retrosynthetic approach to targeted fleximers.

“Reverse” Carbocyclic Fleximers
141
SCHEME 1 Synthesis of truncated substituted uracil carbocycles. Conditions: (a) NaBH4, MeOH,
CeCl3·7H2O, r.t., 1 hour, yield: 93%; (b) N3-benzoyluracil or N3-benzoyl-5-bromouracil, DPPE, DIAD,
acetonitrile, r.t., 18 hours, for 7 yield: 78%; for 8 yield: 70%; and (c) methanolic ammonia, r.t., 12 hours,
for 9 yield: 62%; for 10 yield: 99%.
protected products, 7 and 8 were obtained, they were deblocked using mildly
basic conditions to yield 9 and 10.
Synthetic efforts then shifted to the saturated carbocyclic series (3a–c)
shown in Scheme 2, which could be obtained from intermediate 10. To
accomplish this, two additional steps were required to install the necessary
bromine functionality. Compound 10 was first reduced using catalytic hy-
drogenation conditions with Pd/C to afford 11 in quantitative yield, which
was subsequently brominated at the C5 position of the uracil ring using NBS
in dimethoxyethane and water to afford 12.[38]
As shown below in Scheme 3, intermediates 9 and 12 were then subjected
to Stille coupling methodology using the corresponding tin reagents of the
thiazole, thiophene, and furan. These are commercially available or readily
constructed using literature procedures.[39,40] These tin reagents can be cou-
pled using PdCl2(PPh3)2 or Pd(PPh3)4,[24,41,42] then subsequent deblocking
of the 2ꢁ,3ꢁ-diol of each of the compounds was readily achieved through the
use of mildly acidic conditions employing trifluoroacetic acid and water to
provide 3a–c or 4a–c in good yields.
SCHEME 2 Synthesis of saturated carbocyclic C5-bromouracil. Conditions: (a) Pd/C, MeOH, H2 25 psi,
5 hours, quantitative and (b) NBS, NaN3, DME, H2O, r.t., 24 hours, yield: 48%.

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S. C. Zimmermann et al.
SCHEME 3 Synthesis of reverse fleximer truncated carbocycles. Conditions: (a) for 3a and 4a, 12 or 9
respectively, tributylstannyl thiophene, dioxane, PdCl2(PPh3)2, reflux; for 3b and 4b, 12 or 9, respectively,
tributylstannyl furan, dioxane, PdCl2(PPh3)2, reflux for 3c and 4c, 12 or 9, respectively, tributylstannyl
thiazole, dioxane, Pd(PPh3)4, reflux; (b) TFA:H2O (2:1) in THF, r.t., 6 hours. Yield over two steps: for
3a yield: 90%; for 3b yield: 90%; for 3c yield: 89%; for 4a yield: 55%; for 4b yield: 58%; for 4c yield: 42%.
Assays
Given our initial goal of developing SAHase inhibitors, we then screened
the reverse fleximers against SAHase using a standard assay previously re-
ported by us and others.[43,44] The SAHase assay is a dual enzyme assay that
monitors the rate of hydrolysis of SAH by SAHase to homocysteine and
adenosine. This reaction utilizes adenosine deaminase (ADA) to monitor
the conversion of adenosine to inosine, which results in a decrease of ab-
sorbance at 265 nm. Initially the compounds appeared to inhibit SAHase,
however, upon further investigation it was revealed the compounds were not
inhibiting SAHase, but instead ADA (Table 1). ADA converts adenosine to
inosine, and is linked to the deactivation of various nucleoside drugs such
as AraA through deamination via ADA.[45–47]
Due to compounds 3a–c and 4a–c structural similarity to inosine, the
deaminated product of adenosine following metabolism by ADA, computa-
tional studies were conducted to uncover possible reasons for this unusual
activity. The structures were docked using Gold 4.1.2[48] in bovine ADA
[pdb file: 1KRM] that had been crystallized with the transition state ana-
logue, 6-hydroxy-1,6-dihydropurine ribose.[49] Each molecule was docked to
the binding site and scored. The top three results of each docked inhibitor

“Reverse” Carbocyclic Fleximers
143
TABLE 1 Percent inhibition and fitness of analogues in the active site of ADA
Compounda
Percent inhibitionb
Fitness scorec
Inosine
3a
3b
3c
4a
31%
36%
35%
28%
48%
55%
49%
50.00
50.90
48.70
47.29
54.78
53.62
51.90
4b
4c
aCompounds were dissolved in DMSO and tested at 100 μM for analysis.
bCalculated as: [1-(reaction rate of inhibited ADA/reaction rate of uninhibited ADA)]∗100%. Rates
were determined by monitoring the decrease in absorbance at 265 nm from the conversion of adenosine
to inosine.
cAnalogues were built in Chem3D then transferred to Sybyl before analysis. Fitness score is the relative
affinity of the ligand to the binding site.
were selected for analysis. Of the six inhibitors, four scored higher than
inosine (Table 1).
Upon further inspection of the ADA binding site, a large binding pocket
adjacent to the adenosine binding site was discovered. This adjacent pocket
can accommodate the pendant heteroaromatic rings as depicted in Figure 4
with inosine and 4b. Furan analogue 4b exhibited the best inhibition as
well as fitness score as shown in Table 1. Thus, it appears that the new
bonding interactions provided by the added flexibility and length of the C5
substitute pendant ring are likely assisting in the recognition and binding.
Interestingly, the more potent inhibitors exhibited a distinct perpendicular
conformation for the pendant and pyrimidine rings, while the remainder of
the nucleoside scaffold overlaps with inosine.
In comparing the computational results shown in Table 1 for the six in-
hibitors, several conclusions can be drawn. One, the unsaturated analogues
seem to be better inhibitors than the saturated series, which coincides with
the results observed for the inhibition. Also in agreement is that the thio-
phene and furan pendant systems have similar activities that are expected to
be potentially better than the thiazole pendant system due to their computa-
tional fitness. We are now focused on exploring additional heterocyclic and
aromatic C5 substituents and exploring the incorporation of linkers between
the pyrimidine and C5 substituent to lengthen the “reach” of the substituent
into the adjacent pocket, in hopes of increasing the affinity of the enzyme
for these nucleoside analogues.
The compounds were also subjected to broad screen antiviral testing
by Professor Jan Balzarini at the Katholieke Universiteit (Leuven, Belgium)
against viruses such as HSV-1, HSV-2, vaccinia and influenza types A and
B. Disappointingly, none of the compounds showed activity comparable to
Ribavirin.

144
S. C. Zimmermann et al.
FIGURE 4 Reverse fleximer 4b (purple) and inosine (cyan) in the active site of Bovine ADA (Color
figure available online).
CONCLUSION
In summary, a novel series of carbocyclic “reverse fleximer” nucleosides
was successfully synthesized and resulted in a new type of adenosine
deaminase inhibitor. These findings serve as further evidence that flexible
inhibitors can be recognized in atypical enzymatic systems, thus confor-
mational adaptability may prove to be beneficial for future therapeutics.
Current efforts are underway to synthesize the corresponding Ari and NpcA
analogues to see if the addition of the 4ꢁ-hydroxymethyl imparts recognition
by viral TK as was observed with Herdewijn’s series, as well as possible
recognition by other enzymes that require the 5ꢁ-hydroxyl. The results of
those studies will be reported elsewhere as they become available.

“Reverse” Carbocyclic Fleximers
145
EXPERIMENTAL SECTION
General
All chemicals were obtained from commercial sources and used with-
out further purification unless otherwise noted. Anhydrous DMF, MeOH,
DMSO, and toluene were purchased from Fisher Scientific (Pittsburgh, PA,
USA). Anhydrous THF, acetone, CH2Cl2, CH3CN and ether were obtained
using a solvent purification system (mBraun Labmaster 130, Stratham, NH,
USA). Melting points are uncorrected. NMR solvents were purchased from
Cambridge Isotope Laboratories (Andover, MA, USA). All 1H and 13C NMR
spectra were obtained on a JEOL ECX 400 MHz NMR, operated at 400 and
100 MHz and referenced to internal tetramethylsilane (TMS) at 0.0 ppm.
The spin multiplicities are indicated by the symbols s (singlet), d (dou-
blet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and b
(broad). Reactions were monitored by thin-layer chromatography (TLC) us-
ing 0.25 mm Whatman Diamond silica gel 60-F254 precoated plates. Column
chromatography was performed using silica gel (63–200 μ) from Dynamic
Adsorptions Inc. (Norcross, GA, USA), and eluted with the indicated solvent
system. Yields refer to chromatographically and spectroscopically (1H and
13C NMR) homogeneous materials. Mass spectra were recorded at the Johns
Hopkins Mass Spectrometry Facility (Baltimore, MD, USA).
General Procedure for Mitsunobu Coupling of Protected Uracils to
Cyclopentenol
To a stirred suspension of 5 (1.06 g, 6.80 mmol), protected uracil
(10.2 mmol), and 1,2-bis(diphenylphosphino)ethane (1.49 g, 3.37 mmol)
in acetonitrile (75 mL) was added dropwise DIAD (2.0 mL, 10.2 mmol) at
0◦C. The reaction mixture was allowed to stir at room temperature (r.t.)
for 18 hours. The solvent was then removed under reduced pressure and
the resulting residue purified using silica gel chromatography eluting with
hexanes:EtOAc (1:2).
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-3-benzoyl-5-bromouracil (7)
Off-white crystalline solid (4.02 g, 9.28 mmol, 78%). 1H NMR (CDCl3):
δ 1.34 (s, 3H), 1.41 (s, 3H), 4.62 (d, 1H, J = 14.9 Hz), 5.34 (dd, 1H, J =
14.8, 3.5 Hz), 5.40 (d, 1H, J = 3.5 Hz), 5.78 (dt, 1H, J = 13.8 Hz), 6.32 (dt,
1H, J = 3.4, 13.8 Hz), 7.41 (s, 1H), 7.48 (t, 2H, J = 18.3 Hz), 7.64 (t, 1H,
J = 18.3 Hz), 7.90 (d, 2H, J = 17.2 Hz). 13C NMR (CDCl3): δ 22.7, 25.6, 70.0,
83.5, 84.4, 96.8, 112.7, 128.8, 129.4, 130.7, 131.0, 135.5, 140.1, 140.7, 149.1,
156.5, 158.1, 167.5. HRMS calculated for C19H17BrN2O5 [M+H]+ 432.0320;
found, 432.0315.

146
S. C. Zimmermann et al.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-3-benzoyl-uracil (8)
Clear colorless foam (1.69 g, 4.77 mmol, 70%). 1H NMR (DMSO-d6): δ
1.33 (s, 3H), 1.41 (s, 3H), 4.71 (d, 1H, J = 13.7 Hz), 5.27 (s, 1H), 5.29 (d, 1H,
J = 11.5 Hz), 5.81 (d, 1H, J = 19.4 Hz), 5.84 (dd, 1H, J = 5.7, 13.7 Hz), 6.23
(dt, 1H, J = 4.6, 13.8 Hz), 7.51 (d, 1H, J = 19.5 Hz), 7.57 (t, 2H, J = 19.5 Hz),
7.78 (t, 1H, J = 19.5 Hz), 7.97 (d, 2H, J = 21.7 Hz). 13C NMR (DMSO-d6):
δ 24.4, 26.2, 69.1, 83.2, 84.6, 101.1, 111.8, 128.9, 129.0, 130.1, 131.6, 135.0,
139.0, 143.0, 149.9, 163.0, 168.8. HRMS calculated for C19H18N2O5 [M+H]+
355.1294; found, 355.1282.
General Procedure for the Removal of the Benzoyl Protecting
Group from Uracil Intermediates
To a stirred solution of benzoyl protected carbocyclic uracil (7.08 mmol)
in MeOH (10 mL) was added methanolic ammonia (100 mL) and allowed to
react for 18 hours. The reaction mixture was condensed under reduced pres-
sure and then re-evaporated from EtOH (3 × 100 mL). The crude residue
was purified by silica gel chromatography eluting with hexanes:EtOAc (1:4).
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-5-bromouracil (9)
1
Off-white solid (1.98 g, 5.75 mmol, 62%). H NMR (CDCl3) δ 1.35 (s,
3H), 1.45 (s, 3H), 4.56 (d, 1H, J = 14.9 Hz), 5.35 (dd, 1H, J = 3.5, 14.9 Hz),
5.46 (s, 1H), 5.76 (dd, 1H, J = 3.4, 13.8 Hz), 6.34 (dt, 1H, J = 4.6, 14.9 Hz),
7.28 (s, 1H), 8.67 (s, 1H). 13C NMR (CDCl3) δ 25.6, 27.3, 69.2, 83.7, 84.3, 97.0,
112.7, 129.0, 140.0, 140.5, 149.8, 158.7. HRMS calculated for C12H13BrN2O4
[M+H 79Br]+ 329.0137, [M+H 81Br]+ 331.0117; found, 329.0122, 331.0110.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-uracil (10)
1
Off-white solid (1.77 g, 7.07 mmol, 99%). H NMR (CDCl3): δ 1.31 (s,
3H), 1.41 (s, 3H), 4.56 (d, 1H, J = 14.9 Hz), 5.31 (dd, 1H, J = 2.3, 14.9), 5.41
(d, 1H, J = 4.6), 5.66 (d, 1H, J = 20.6 Hz), 5.72 (dd, 1H, J = 3.5, 13.7 Hz),
6.23 (d, 1H, J = 14.9 Hz), 6.98 (d, 1H, J = 20.6 Hz), 9.90 (s, 1H). 13C NMR
(CD3OD): δ 24.4, 26.2, 68.4, 83.4, 84.6, 101.2, 111.7, 129.3, 138.5, 142.5,
151.3, 165.0. HRMS calculated for C12H14N2O4 [M+H]+ 251.1032; found,
251.1026.
Preparation of 1-[(2ꢀ,3ꢀ-O-Isopropylidene)-cyclopentan-1ꢀ-yl]-uracil (11)
A solution of 10 (1.77 g, 7.07 mmol) and Pd/C (150 mg) in MeOH
(100 mL) was hydrogenated for 3 hours at 25 psi. The reaction mixture was
filtered through a pad of celite and the solvent removed under vacuum to
give 11 as an off-white foam (1.54 g, 6.11 mmol, 86%). 1H NMR (CDCl3): δ
1.27 (s, 3H), 1.46 (s, 3H), 1.88–1.93 (m, 2H), 2.11–2.15 (m, 1H), 2.29–2.34
(m, 1H), 4.37–4.39 (m, 1H), 4.76 (dd, 1H, J = 6.9, 15.5 Hz), 4.80 (td, 1H,
J = 5.7, 14.9 Hz), 5.66 (d, 1H, J = 20.6 Hz), 7.07 (d, 1H, J = 20.7 Hz),

“Reverse” Carbocyclic Fleximers
147
8.24 (bs, 1H). 13C NMR (DMSO-d6): 24.4, 27.3, 29.1, 31.2, 63.9, 80.3, 84.4,
101.8, 110.0, 143.9, 152.6, 163.8. HRMS calculated for C12H16N2O4 [M+H]+
253.1183; found, 253.1189.
Preparation of 1-[(2ꢀ,3ꢀ-O-Isopropylidene)-cyclopentan-1ꢀ-yl]-5-bromouracil
(12)
To a stirred solution of 9 (1.19 g, 4.72 mmol) in 1,2-dimethoxyethane
(200 mL) was added a solution of sodium azide (920 mg, 14.15 mmol) and
NBS (1.01 g, 5.66 mmol) in water (5 mL) and stirred for 24 hours at r.t. The
reaction mixture was then concentrated in vacuo and the resulting residue
purified by silica gel chromatography eluting with hexanes:EtOAc (1:2) to
1
afford 12 as a white solid (750 mg, 2.27 mmol, 48%). H NMR (CDCl3): δ
1.30 (s, 3H), 1.49 (s, 3H), 1.92–1.97 (m, 2H), 2.14–2.19 (m, 1H), 2.32–2.36
(m, 1H), 4.41 (m, 1H), 4.77 (dd, 1H, J = 6.9, 15.5 Hz), 4.82 (td, 1H, J =
5.7, 14.9 Hz), 7.42 (s, 1H), 9.33 (s, 1H). 13C NMR (CDCl3): δ 15.2, 24.5,
28.7, 29.2, 65.1, 81.3, 85.2, 89.8, 110.9, 150.5, 165.2, 165.3. HRMS calculated
for C12H15BrN2O4 [M+H 79Br]+ 331.0293, [M+H 81Br]+ 333.0243; found,
331.0287, 333.0259.
General Procedure for the Stille Coupling with Stannyl Thiophene
and Stannyl Furan Reagents
To a stirred solution of the desired 5-bromo carbocyclic nucleoside
(1.74 mmol) and aryl tin (8.72 mmol) in dioxane (50 mL) was added
PdCl2(PPh3)2 (122 mg, 0.17 mmol) and the temperature held at 90◦C for
18 hours. The reaction mixture was then evaporated to dryness and the
residue purified by silica gel chromatography eluting with hexanes:EtOAc
(1:1).
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(thiophene-2-yl)-uracil
Off-white solid (343 mg, 1.03 mmol, 59%). 1H NMR (CDCl3) δ 1.36 (s,
3H), 1.46 (s, 3H), 4.63 (d, 1H, J = 14.9 Hz), 5.37 (dd, 1H, J = 2.3, 11.4 Hz),
5.50 (d, 1H, J = 4.6 Hz), 5.82 (dd, 1H, J = 3.4, 13.8 Hz), 6.34 (dt, 1H, J = 4.5,
14.9 Hz), 7.04 (dd, 1H, J = 9.2, 12.6 Hz), 7.29 (d, 1H, J = 3.4 Hz), 7.31 (s,
1H), 7.35 (dd, 1H, J = 3.4, 9.1 Hz), 9.18 (s, 1H). 13C NMR (CDCl3) δ 25.6,
27.3, 69.3, 83.7, 84.5, 112.6, 124.8, 125.8, 127.3, 129.3, 133.1, 136.2, 139.5,
141.1, 160.7, 172.1. HRMS calculated for C16H16N2O4S [M+H]+ 332.0830;
found, 332.0840.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(furan-2-yl)-uracil
Off-white solid (520 mg, 1.64 mmol, 63%). 1H NMR (CDCl3) δ 1.35 (s,
3H), 1.46 (s, 3H), 4.62 (d, 1H, J = 13.7 Hz), 5.41 (dd, 1H, J = 2.6, 12.6 Hz),
5.55 (d, 1H, J = 3.5 Hz), 5.81 (dt, 1H, J = 3.5, 13.8 Hz), 6.35 (dt, 1H, J = 3.4,
14.9 Hz), 6.44 (dd, 1H, J = 4.6, 8.0 Hz), 7.03 (d, 1H, J = 8.0 Hz), 7.34 (d, 1H,

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S. C. Zimmermann et al.
J = 4.5 Hz), 7.44 (s, 1H), 8.93 (s, 1H). 13C NMR (CDCl3) δ 22.0, 29.8, 67.1,
69.0, 83.8, 84.5, 109.7, 112.1, 128.5, 129.5, 134.7, 136.7, 139.4, 141.3, 149.8,
160.1. HRMS calculated for C16H16N2O5 [M]+ 316.1059; found, 316.1053.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-cyclopentan-1ꢀ-yl]-5-(thiophene-2-yl)uracil
Off-white solid (111 mg, 0.31 mmol, 99%).1H NMR (CDCl3): δ 1.32 (s,
3H), 1.51 (s, 3H), 1.95–2.05 (m, 2H), 2.21–2.25 (m, 1H), 2.39–2.41 (m, 1H),
4.44–4.49 (m, 1H), 4.83–4.85 (m, 1H), 4.88 (dd, 1H, J = 6.9, 14.8 Hz), 7.04
(dd, 1H, J = 9.2, 12.6), 7.29 (dd, 1H, J = 2.3, 12.6), 7.39 (dd, 1H, J = 2.3,
9.1 Hz), 7.43 (s, 1H), 8.49 (s, 1H). 13C NMR (CD3OD): δ 12.6, 23.4, 26.0,
26.6, 27.9, 28.9, 31.0, 66.7, 80.9, 85.5, 109.7, 111.4, 123.6, 125.3, 126.2, 139.3.
HRMS calculated for C16H18N2O4S [M]+ 334.0987; found, 334.0979.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-cyclopentan-1ꢀ-yl]-5-(furan-2-yl)-uracil
Off-white solid (100 mg, 0.31 mmol, 99%). 1H NMR (CDCl3) δ 1.32 (s,
3H), 1.51 (s, 3H), 1.97–2.01 (m, 2H), 2.19–2.24 (m, 1H), 2.38–2.42 (m, 1H),
4.55–4.59 (m, 1H), 4.81 (dd, 1H, J = 5.7, 14.9 Hz), 4.87 (td, 1H, J = 5.7,
14.8 Hz), 6.46 (dd, 1H, J = 4.6, 9.2 Hz), 7.02 (d, 1H, J = 8.0 Hz), 7.36 (d,
1H, J = 4.5 Hz), 7.57 (s, 1H), 8.17 (s, 1H). 13C NMR (CD3OD): δ 13.7, 17.4,
24.5, 27.0, 28.4, 29.2, 31.6, 66.9, 80.7, 84.7, 109.5, 112.1, 136.5, 141.3, 149.5,
159.6. HRMS calculated for C16H18N2O5 [M]+ 318.1216; found, 318.1219.
General Procedure for the Stille Coupling with Stannyl Thiazole
Reagents
To a stirred solution of the desired 5-bromo carbocyclic nucleoside
(1.10 mmol) and 5-(tributylstannyl)thiazole (900 mg, 2.41 mmol) in dry
THF (50 mL) was added Pd(PPh3)4 (50 mg) and refluxed under nitro-
gen for 72 hours. The reaction mixture was then evaporated to dryness
and the resulting residue purified by silica gel chromatography eluting with
EtOH:CH2Cl2 (5:95).
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(thiazol-5-yl)-uracil
1
Off-white solid (100 mg, 0.30 mmol, 52%). H NMR (CD3OD) δ 1.36
(s, 3H), 1.42 (s, 3H), 4.72 (d, 1H, J = 13.7 Hz), 5.38 (dd, 1H, J = 2.3, 14.9
Hz), 5.42 (s, 1H), 5.85 (dd, 1H, J = 4.6, 14.9 Hz), 6.26 (dt, 1H, J = 4.6,
13.7 Hz), 7.83 (s, 1H), 8.17 (s, 1H), 8.90 (s, 1H). 13C NMR (CD3OD) δ 24.5,
26.3, 70.02, 83.3, 84.9, 106.1, 111.7, 129.1, 129.5, 138.4, 138.7, 139.2, 150.3,
153.9, 162.1. HRMS calculated for C15H15N3O4S [M+H]+ 334.0862; found,
334.0855.
1-[(2ꢀ,3ꢀ-O-Isopropylidene)-cyclopentan-1ꢀ-yl]-5-(thiazol-5-yl)-uracil
Off-white solid (96.2 mg, 0.29 mmol, 95%). 1H NMR (DMSO-d6): δ 1.21
(s, 3H), 1.39 (s, 3H), 1.67–1.74 (m, 1H), 1.95–1.99 (m, 1H), 2.10–2.19 (m,

“Reverse” Carbocyclic Fleximers
149
2H), 4.56–4.60 (m, 1H), 4.73–4.77 (m, 1H), 4.81 (dd, 1H, J = 0.8, 14.9 Hz),
8.22 (s, 1H), 8.28 (s, 1H), 8.96 (s, 1H), 11.78 (bs, 1H). 13C NMR (CD3OD):
δ 12.7, 23.5, 26.1, 29.0, 29.5, 30.9, 31.8, 67.0, 80.8, 84.4, 111.4, 128.7, 130.8,
132.5, 140.7. HRMS calculated for C15H17N3O4S [M+H]+ 336.1018; found,
336.1010.
General Procedure for the Removal of the 2ꢀ,3ꢀ-isopropylidene
Protecting Group
A solution of 2ꢁ,3ꢁ-protected carbocyclic nucleoside (0.22 mmol) in
TFA:H2O (9 mL, 2:1) was stirred at r.t. for 18 hours. The reaction mix-
ture was evaporated under reduced pressure and the resulting residue
was dissolved in EtOH (3 × 10 mL) and concentrated three times un-
der vacuum. The crude product was purified over silica gel eluting with
EtOAc:acetone:MeOH:H2O (8:1:1:0.5).
1-[(2ꢀ,3ꢀ-Dihydroxy)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(thiophene-2-yl)-uracil (4a)
1
Yellow solid (60.3 mg, 0.21 mmol, 94%). λmax = 259 nm. H NMR
(DMSO-d6): δ 4.07 (q, 1H), 4.42 (bs, 1H), 4.81 (d, 1H, J = 13.8 Hz), 4.99
(d, 1H, J = 17.2 Hz), 5.36 (d, 1H, J = 14.9 Hz), 5.89 (dd, 1H, J = 4.5, 14.9
Hz), 6.08 (dt, 1H, J = 5.8, 16.0 Hz), 7.01 (dd, 1H, J = 9.1, 12.6 Hz), 7.42
(d, 1H, J = 12.6 Hz), 7.44 (dd, 1H, J = 2.3, 9.1 Hz), 7.74 (s, 1H), 11.94 (s,
1H). 13C NMR (DMSO-d6): δ 19.1, 31.5, 67.3, 72.9, 76.2, 119.1, 123.5, 126.3,
126.9, 133.1, 137.2, 162.0, 181.0. HRMS calculated for C13H12N2O4S [M]+
292.0518; found, 292.0515.
1-[(2ꢀ,3ꢀ-Dihydroxy)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(furan-2-yl)-uracil (4b)
1
Off-white foam (20.0 mg, 0.07 mmol, 92%). λmax = 256 nm. H NMR
(DMSO-d6): δ 3.93 (q, 1H, J = 13.7 Hz), 4.40 (m, 1H), 4.85 (d, 1H, J = 14.9
Hz), 5.00 (d, 1H, J = 16.1 Hz), 5.39 (dt, 1H, J = 3.4, 10.3 Hz), 5.89 (dd, 1H,
J = 4.6, 14.9 Hz), 6.13 (dt, 1H, J = 5.8, 16.0 Hz), 6.49 (dd, 1H, J = 4.6,
9.2 Hz), 6.83 (d, 1H, J = 6.9 Hz), 7.52 (s, 1H), 7.60 (dd, 1H, J = 2.3, 4.6
Hz), 11.60 (s, 1H). 13C NMR (acetone-d6): 67.5, 73.4, 76.4, 106.2, 108.2,
111.5, 132.2, 135.2, 137.9, 141.2, 146.8, 150.3, 160.1 HRMS calculated for
C13H12N2O5 [M+H]+ 277.0825; found, 277.0821.
1-[(2ꢀ,3ꢀ-Dihydroxy)-4ꢀ-cyclopenten-1ꢀ-yl]-5-(thiazol-5-yl)-uracil (4c)
1
Off-white foam (71.7 mg, 0.22 mmol, 81%). λmax = 260 nm. H NMR
(acetone-d6): δ 4.19 (q, 1h, J = 13.7 Hz), 4.25–4.27 (m, 1H), 4.68–4.69 (m,
1H), 5.52–5.54 (m, 1H), 6.02 (dd, 1H, J = 5.7, 16.0 Hz), 6.21 (dt, 1H, J = 5.7,
10.3 Hz), 7.00 (dd, 1H, J = 9.2, 12.6 Hz), 7.36 (dd, 1H, J = 3.4, 13.7 Hz), 7.41
(dd, 1H, J = 2.3, 9.2 Hz), 7.73 (s, 1H), 10.32 (bs, 1H). 13C NMR (acetone-d6):
δ 13.5, 24.5, 64.0, 71.0, 75.5, 106.0, 129.2, 139.1, 140.1, 159.3, 152.9. HRMS
calculated for C12H11N3O4S [M+H]+ 294.0549; found 294.0547.

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1-[(2ꢀ,3ꢀ-Dihydroxy)-cyclopentan-1ꢀ-yl]-5-(thiophene-2-yl)-uracil (3a)
1
Off-white foam (5.7 mg, 0.02 mmol, 91%). λmax = 260 nm. H NMR
(acetone-d6): δ 1.65–1.68 (m, 1H), 1.86–1.89 (m, 1H), 2.09–2.16 (m, 2H),
3.83 (bs, 1H), 4.09–4.11 (m, 1H), 4.25 (bs, 1H), 4.42 (q, 1H, J = 11.5 Hz),
4.70–4.79 (m, 1H), 7.01 (dd, 1H, J = 9.1, 12.6 Hz), 7.35 (dd, 1H, J = 2.3,
12.6 Hz), 7.47 (dd, 1H, J = 2.3, 9.1 Hz), 8.01 (s, 1H), 10.28 (s, 1H). 13C NMR
(acetone-d6): δ 12.7, 23.8, 64.1, 70.7, 74.3, 107.1, 128.7, 131.0, 138.6, 141.1,
158.9, 160.9, 161.2. HRMS calculated for C13H14N2O4S [M+H]+ 295.0753;
found, 295.0747.
1-[(2ꢀ,3ꢀ-Dihydroxy)-cyclopentan-1ꢀ-yl]-5-(furan-2-yl)-uracil (3b)
1
White solid (20.0 mg, 0.07 mmol, 91%). λmax = 255 nm. H NMR
(acetone-d6): δ 1.63–1.66 (m, 1H), 1.87–1.90 (m, 1H), 2.11–2.17 (m, 2H),
3.83 (bs, 1H), 4.08–4.11 (m, 1H), 4.25 (s, 1H), 4.42 (q, 1H, J = 11.4 Hz),
4.72–4.78 (m, 1H), 6.47 (dd, 1H, J = 3.5, 8.1 Hz), 6.93 (d, 1H, J = 6.8 Hz),
7.50 (d, 1H, J = 3.4 Hz), 7.95 (s, 1H), 10.24 (bs, 1H). 13C NMR (acetone-
d6): δ 12.5, 23.68, 64.3, 71.0, 74.6, 106.9, 127.6, 131.3, 137.9, 141.4, 158.6,
161.1, 161.7. HRMS calculated for C13H14N2O5 [M+H]+ 279.0981; found,
279.0952.
1-[(2ꢀ,3ꢀ-Dihydroxy)-cyclopentan-1ꢀ-yl]-5-(thiazol-5-yl)-uracil (3c)
White solid (25.2 mg, 0.09 mmol, 94%). λmax = 260 nm. 1H NMR (DMSO-
d6): δ 1.51–1.54 (m, 1H), 1.67–1.70 (m, 1H), 1.95–2.21 (m, 2H), 3.90 (bs,
1H), 4.20–4.24 (m, 1H), 4.59 (d, 1H, J = 6.9 Hz), 4.68 (q, 1H, J = 22.9
Hz), 4.96 (d, 1H, J = 16.1 Hz), 8.33 (s, 1H), 8.34 (s, 1H), 8.95 (s, 1H),
11.72 (s, 1H). 13C NMR (DMSO-d6): δ 24.8, 29.0, 62.5, 70.7, 75.3, 105.8,
129.3, 139.3, 140.5, 150.8, 154.1, 161.8. HRMS calculated for C12H13N3O4S
[M+H]+ 296.0705; found, 296.0703.
SAHase Assay
The enzyme-coupled continuous assay in the hydrolysis direction was
performed as previously reported.[43] Briefly, SAH is hydrolyzed to homocys-
teine and adenosine, which is subsequently converted by adenosine deami-
nase into ammonia and inosine, a process associated with a decrease of ab-
sorbance at 265 nm. Each assay was conducted in thermostatted 1 cm quartz
cuvettes at 37◦C maintained by a Peltier unit on a Cary 100 ultraviolet-visible
spectrophotometer. Enzyme assay solution typically contained 50 mM potas-
sium phosphate at pH 7.4, 0.39 units of adenosine deaminase (Worthington
Biochemical Corp., Lakewood, NJ, USA), 0.132 nM of SAHase (provided
by Dr. Lynne Howell), 10 μM of SAH and various concentrations of in-
hibitors in a total volume of 1000 μL. The reactions were initiated by the
addition of SAH. In all cases, we ascertained that SAH hydrolysis catalyzed
by SAHase was rate limiting under testing conditions (data not shown). The

“Reverse” Carbocyclic Fleximers
151
kinetic data were analyzed using KaleidaGraph 4.0 (Synergy Inc., Reading,
PA, USA). Based on a competitive inhibition mechanism, the Ki value was
determined using the equation, v = kcat × [S] × [E]/{Km × (1 + [I]/Ki)
+ [S]}; where v, kcat, [S], [E], Km, [I], and Ki stand for the initial reac-
tion rates, rate constant, substrate concentration, enzyme concentration,
Michaelis–Menten constant, inhibitor concentration, and dissociation con-
stant of the enzyme-inhibitor complex, respectively. A previously reported
Km value of 7.9 μM for SAH in the hydrolysis direction was used for all
calculations.[50]
ADA Assay
The ADA assay was performed in a similar manner to the SAHase as-
say conducted in thermostatted 1 cm quartz cuvettes at 37◦C maintained
by a Peltier unit on a Cary 100 ultraviolet-visible spectrophotometer. ADA
mitigates the reaction of adenosine to inosine, which can be monitored by
measuring the decrease in absorbance at 265 nm. Assay solutions contained
50 mM potassium phosphate at pH 7.4, 0.001 units of adenosine deaminase
(Worthington Biochemical Corp.), 3 μM of adenosine at a total volume of
1000 μL. Inosine, 3a–c and 4a–c (100 μM) were monitored for the con-
sumption of substrate. All reactions were performed in triplicate, and then
the rates were averaged. Relative Inhibition was determined by dividing the
rate of the inhibited reaction by the rate of the reaction with no inhibitor
present. Bovine adenosine deaminase was used due to similarity to human
adenosine deaminase and its availability.
Docking
Six compounds were devised for binding to Bovine ADA (Protein Data
Bank ID: 1KRM).[49] Each derivative was built in Chem3D and then trans-
ferred to Sybyl. The derivatives were assigned with Gasteiger–Huckel charges
and were energy minimized in Sybyl using the Powell method with 1000 it-
erations. The Tripos Mol2 files generated in Sybyl were merged into one file
and transferred to Gold 4.1.2.[48] Each derivative was then docked to Bovine
ADA as the receptor.
The crystal structure of Bovine ADA included a bound inosine, zinc, car-
bon dioxide, and an amine. The inosine, carbon dioxide and the amine were
removed electronically. After protonation of the N-terminus and deproto-
nation of the C-terminus, the charges of the protein functionalities were
determined as described for the above derivatives. The inosine was used for
the receptor-grid generation and the final grid volume corresponding to the
˚
active site of the protein was 20 × 20 × 20 A. Standard precision docking was
applied with flexibility of ring flips, twists, and internal hydrogen bonding
in the ligands and no positional constraints were applied to the ligands.

152
S. C. Zimmermann et al.
The structures were docked using Gold 4.1.2[48] in Bovine ADA (Protein
Data Bank ID: 1KRM).[49] Each molecule was docked to the binding site and
scored. For each docked inhibitor, the top three results were selected for
analysis. From the analysis, one structure for each inhibitor was selected and
of the six inhibitors, four scored higher than the natural substrate.
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