Designed spiro-bicyclic analogues targeting the ribosomal decoding center

Article abstract of DOI:10.1002/cbic.201000591

The bacterial ribosome represents the confirmed biological target for many known antibiotics that interfere with bacterial protein synthesis. Aminoglycosides represent a lead paradigm in RNA molecular recognition and constitute ideal starting points for t

Full text of DOI:10.1002/cbic.201000591

DOI: 10.1002/cbic.201000591
Designed Spiro-Bicyclic Analogues Targeting the
Ribosomal Decoding Center
Thomas Cottin, Constantina Pyrkotis, Christos I. Stathakis, Ioannis Mavridis,
Ioannis A. Katsoulis, Panoula Anastasopoulou, Georgia Kythreoti, Alexandros L. Zografos,
Victoria R. Nahmias, Athanasios Papakyriakou, and Dionisios Vourloumis*[a]
The bacterial ribosome represents the confirmed biological
target for many known antibiotics that interfere with bacterial
protein synthesis. Aminoglycosides represent a lead paradigm
in RNA molecular recognition and constitute ideal starting
points for the design and synthesis of novel RNA binders. Pre-
vious rational design approaches of RNA-targeting small mole-
cules have been mainly concentrated on direct functionaliza-
tion of aminoglycosidic substructures. Herein, we successfully
designed and synthesized rigid spirocyclic scaffolds locked in a
predicted ribosome-bound “bioactive” conformation. These an-
alogues are able to mimic many of the interactions of the nat-
ural products for the A-site, as proven by their obtained bind-
ing affinities. The development of an optimized approach for
their synthesis and their potential to inhibit protein production
in vitro are presented. Our results could be further utilized for
the development of analogues with improved antibiotic pro-
files.
Introduction
The discovery of antibiotics, small molecules of natural and
synthetic origin that specifically interfere with biological pro-
cesses in bacteria, has been one of the major breakthroughs of
medicine in the 20th century, allowing the treatment of life-
threatening infectious diseases. Early after their discovery, the
emergence of resistance mechanisms has been observed in
bacteria, which counteract their action. The challenge of antibi-
otic resistance is particularly severe because most antibacterial
drug classes used today have been in the clinic for more than
three decades. The initial broad stream of novel antibiotics dis-
covered from both natural and synthetic sources has been nar-
rowed down to a trickle, with only two new classes of antibac-
terials, fluoroquinolones and oxazolidinones, developed over
the last 30 years.[1] Novel potent antibiotics are thus required
in addition to the currently used drugs. In many bacteria, in-
cluding important pathogens, rrn operons that encode rRNA
are present in multiple copies, thus rendering resistance devel-
opment against ribosome-targeted antibiotics by mutations in
the rRNA an unlikely event.[2]
dues, which are involved in contacts with the mRNA–tRNA
hybrid, from the deep groove of the decoding-site rRNA. The
resulting conformational change leads to reduced discrimina-
tion against noncognate tRNAs and subsequently decreased
translational accuracy. Despite their undesirable pharmacologi-
cal profiles and resistance development limiting their antibiotic
utility,[11] their capacity to bind with high affinity to the bacteri-
al decoding site and several other RNA targets[12] renders them
a lead paradigm in RNA molecular recognition.[13,14] Therefore,
aminoglycosides and fragments thereof constitute ideal start-
ing points for the design and synthesis of novel RNA binders.
Three-dimensional structures of aminoglycosides in complex
with decoding-site oligonucleotides and whole 30S ribosomal
subunits provide us important information regarding the mo-
lecular recognition principals of the decoding site by antibiotic
ligands and the mechanics of translational fidelity.[10,15,16] Im-
portantly, these studies underline a key role for the 2-deoxy-
streptamine (2-DOS, Scheme 1) moiety in RNA recognition.[17]
In all cases, the cyclohexitol interacts with two consecutive
base pairs of the decoding-site RNA, C1407–G1494 and the
non-Watson–Crick pair U1406–U1495 (Scheme 1). Importantly,
these base pairs are conserved in both the bacterial and eu-
The bacterial ribosome represents the confirmed biological
target for many known antibiotics that interfere with bacterial
protein synthesis.[3] Crystallographic analyses of ribosomal sub-
units and domains thereof in complex with antibiotics have
demonstrated that the natural products interact almost exclu-
sively with ribosomal RNA components (rRNA),[4] a result that
lends support to earlier biochemical findings[5] and emphasizes
the importance of RNA as a drug target.[6,7] Specifically for nat-
ural aminoglycosides, such as paromomycin, neomycin B, and
neamine (Scheme 1), this is accomplished through their inter-
action with the decoding-site (A-site) ribosomal RNA[5,8]
(Scheme 1) and disruption of the mRNA-decoding fidelity.[9,10]
Upon binding, aminoglycosides displace two key adenine resi-
[a] Dr. T. Cottin, Dr. C. Pyrkotis, Dr. C. I. Stathakis, I. Mavridis, Dr. I. A. Katsoulis,
P. Anastasopoulou, Dr. G. Kythreoti, Dr. A. L. Zografos, Dr. V. R. Nahmias,
Dr. A. Papakyriakou, Dr. D. Vourloumis
Laboratory of Chemical Biology of Natural Products and Designed Mole-
cules
Institute of Physical Chemistry, N.C.S.R. “Demokritos”
15310 Ag. Paraskevi Attikis, Athens (Greece)
Fax: (+30)210-6511766
E-mail: vourloumis@chem.demokritos.gr
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000591.
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71

D. Vourloumis et al.
Herein, by concentrating our efforts on the principles of struc-
tural electrostatic complementarity, we formulate an approach
based on two major considerations. Firstly, the flexibility of the
glycosidic bond in the natural products, although limited,
might be responsible for the target variability demonstrated in
their action. Specifically, comparison of the oligosaccharide
conformations recognized by the bacterial ribosome and the
enzymes responsible for the antibiotic inactivation, presents
remarkable differences.[26] Thus, new chemical entities locked
in the ribosome-bound “bioactive” conformation in which the
glycosidic bond has been replaced by a rigid quaternary
center, were targeted (I, Scheme 1).[27]
Secondly, the conservation of established contacts between
natural aminoglycosides and the A-site RNA[28] represents a
major priority of our design. Specifically, key characteristic in-
teractions that we attempted to maintain (shown in Figure 1A
for neamine) are: 1) The formation of a pseudo base pair with
A1408; 2) The contacts with O4 of U1495; 3) The contact with
N7 of G1494; and 4) The contacts with the phosphate group
of A1492, A1493, and G1494. Consequently, small-molecule
rigid scaffolds, like compound 2 (Scheme 1), were modeled in
the bacterial A-site, exhibiting their oxa-spiro scaffold in the
orientation shown in Figure 1C, relatively to neamine. Briefly,
each designed compound was docked by using AUTO-
DOCK 3[29] into an energy-minimized average model of the bac-
terial A-site, which was calculated from several crystallographic
coordinates of RNA–aminoglycoside complexes.[28,30] The pre-
dicted RNA–scaffold conformation was selected among the
top-ranked solutions by virtue of the estimated binding affinity
and the specific intermolecular contacts. Energy minimization
was performed for the final complexes by using AMBER 9[31]
with the corresponding force fields for nucleic acids and or-
ganic molecules.
Scheme 1. Secondary structure of the bacterial decoding-site, depicting the
internal loop (A-site) in 16S rRNA. The four base-changes of the eukaryotic
sequence are indicated by arrows. The recognition site for the 2-DOS moiety
of aminoglycosides is boxed; structures of neomycin B and paromomycin,
neamine (neomycin A) and paromamine, 2-deoxy-streptamine (2-DOS) and
the conformationally restricted analogues (general structure I, and specific
constructs 1, 2 and 3) designed and synthesized in this study.
karyotic decoding sites. Examination of structurally character-
ized aminoglycoside complexes indicates that although the rel-
ative orientation of rings I and II (Scheme 1) is very similar, the
conformation adopted by rings III and IV is significantly varia-
ble depending on the RNA target.[18] Consequently, the molec-
ular discrimination observed in aminoglycosides for the bacte-
rial target is accomplished by the strategic placement of sugar
substituents at the 4- and 5- or the 4- and 6-positions of the
2-DOS moiety. The apparent rigidity of the neamine system
(rings I/II, Scheme 1) underlines the importance of this module
for RNA recognition because both rings (I and II, Scheme 1)
contribute in key interactions that are responsible for target
binding in decoding-site complexes with aminoglycosides.[18]
The target variability of the aminoglycosides has been attrib-
uted to two major factors: 1) Their highly charged nature, dic-
tated by the number of amino-functionalities, which is respon-
sible for the electrostatic nature of their RNA-binding mode
and 2) their conformational adaptability, which provides limit-
ed flexibility and is responsible for the formation of well-de-
fined drug complexes that are distinct from non-specific inter-
actions of nucleic acids with flexible polyamines such as sper-
midine. The term structural electrostatic complementarity has
been assigned to this promiscuous, yet target-specific binding
of aminoglycosides to RNA.[13]
Results and Discussion
Retrosynthetic analysis and synthesis
From a retrosynthetic perspective (Scheme 2), general struc-
ture I could be the outcome of an olefin-functionalization reac-
Rational design approaches of RNA-targeting small mole-
cules have been previously reported, mainly concentrating on
individually functionalized aminoglycosidic structural compo-
nents, explicitly 2-deoxystreptamine (2-DOS)[19] and glucosa-
mine,[20,21] as well as neamine[22,23] and paromamine.[24,25]
Scheme 2. Retrosynthetic analysis of general structure I. A) Olefin functionali-
zation and final deprotections; B) Ring-closing metathesis retron; C) Allyla-
tion; D) Organometallic addition; E) Appropriate protecting strategy, degra-
dation, oxidation.
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Designed Spiro-Bicyclic Analogues
and the variable reactivity characteristics, which are exhibited
from intramolecular interactions.
Synthesis of key intermediate ketone 6 (retron E)
The synthesis of the desired spiro ethers commenced with
alcohol 5, a common precursor to all of our analogues
(Scheme 3). This adduct was readily available from the mono-
ketal of tetra-benzyloxycarbonyl (Cbz)-protected neamine[23]
4
Scheme 3. Reagents and conditions: a) neamine (1.0 equiv), Cbz-Cl
(7.5 equiv), NaHCO3 (17.0 equiv), H2O/dioxane (2:1 v/v), 12 h, 08C!258C;
b) Dimethoxycyclohexane (7.5 equiv), p-TsOH (0.1 equiv), DMF (0.08m), 5 h,
508C, 95% over two steps; c) NaIO4 (6.0 equiv), MeOH (0.012m), 08C!258C,
14 h; d) Et3N (2.5 equiv), MeOH (0.015m), 408C, 14 h, 94% over two steps;
e) DMP (4.0 equiv), NaHCO3 (6.0 equiv), CH2Cl2 (0.07m), 508C, 1 h, 90%. DM
[O]=Dess–Martin oxidation; CbzCl=benzyl chloroformate; p-TsOH=p-tolue-
nesulfonic acid; DMF=dimethylformamide; DMP=Dess–Martin periodinane.
Figure 1. A) The contacts between neamine and the bacterial A-site RNA, ex-
tracted from the crystal structure with PDB ID 2ET8. Key conserved interac-
tions among several aminoglycosides are marked in red. B) Simulated inter-
actions between compound 2 and the bacterial A-site. W1 and W2 water
molecules were modeled in proximity to conserved hydration sites of rRNA–
aminoglycoside complexes. C) Predicted binding orientation of compound I
(carbon atoms in yellow) superimposed with the crystallographic position of
neamine (cyan carbons) into the bacterial A-site. Nitrogen atoms are colored
blue, oxygen atoms are red, and phosphorus atoms are brown.
upon oxidative cleavage of its 1,2-diol and subsequent chro-
matographic separation (Scheme 3). Initial attempts to oxidize
alcohol 5 to ketone 6, under relatively mild conditions with
oxalyl chloride/DMSO/Et3N (Swern)[32] or I2/KI[33] were not suc-
cessful, and only the starting material could be recovered. A
reliable solution proved to be the Dess–Martin periodinane,[34]
which upon reflux with alcohol 5 furnished the desired ketone
cleanly.
tion from intermediate II, which in turn could result from a
ring-closing metathesis transformation of diene III. The specific
diene could be formed through an allylation reaction of ter-
tiary alcohol IV, the product of an organometallic addition to
ketone V. Depending on the size of the organometallic re-
agent, vinyl, allyl, or homoallyl, the specific approach could
provide access to a series of designed spiro-analogues with
variations on the size of the newly formed ring (five, six, and
seven). Finally, the corresponding ketone would be the out-
come of a series of transformations, including appropriate pro-
tections of the functionalities present, degradation of neamine
and oxidation of the resulting chiral 2-DOS derivative. Our ap-
proach towards the designed analogues was expected, and
eventually proven to be chemically challenging due to the
high density of polar functionalities present in their structures
Organometallic additions to ketone 6 (retron D)
Based on our original design, ketone 6 would react with a
Grignard reagent to afford the corresponding tertiary alcohol
intermediate. Our initial attempts involved the addition of
vinyl magnesium chloride to the electrophilic carbonyl and
resulted in the isolation of the corresponding tertiary alcohol
7 along with oxazolidinone 10 (Scheme 4). The latter was
formed presumably from the attack of the neighboring oxyan-
ion to the carbonyl of the Cbz-group, with concomitant release
of benzyl alcohol, which was in turn identified in the reaction
products (6!VI!VII, Scheme 4). Similar results were obtained
for the allylic and homoallylic organometallic reagents that
form oxazolidinones 11 and 12, respectively. Products 10–12
were also synthesized by conventional methods (NaH, DMF) in
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D. Vourloumis et al.
Alkylation of tertiary alcohols 7–9 (retron C)
Our attempts to etherify tertiary alcohols 7–9 with
NaH resulted yet again in the formation of the oxazo-
lidinone byproducts 10–12, by the aforementioned
putative mechanism (Scheme 4). Alternatively, when
other basic conditions were employed (Ag2O, iPr2EtN,
lithium bis(trimethylsilyl)amide (LiHMDS)), with tetra-
butylammonium iodide (TBAI) as catalyst and allyl-
bromide as the electrophilic reagent, only the un-
reacted alcohol was recovered. The trichloroacetimi-
date allylic moiety was also assayed under mildly
acidic conditions, but still the desired etherification
could not be affected.
Careful examination of alternative routes, prompt-
ed us to remove the Cbz groups under strongly basic
conditions because a hydrogenation could affect the
existing double bonds. The specific transformation
was successfully accomplished by the use of KOH, re-
sulting in the synthesis of the desired diamines in
high yields. Three different protecting strategies were
selected at this stage for our synthesis, based on the
Scheme 4. Reagents and conditions: a) organometallic bromide or chloride (3.2 equiv),
THF (0.1m), ꢀ78!258C, 4 h, 32% for oxazolidinone 10, 35% for oxazolidinone 11, 37%
for oxazolidinone 12; b) TMSCl (3.2 equiv), organometallic bromide or chloride (3.2
equiv), THF (0.1m), ꢀ78!258C, 4 h, 36% for tertiary alcohol 7 (2.5:1 dr), 25% for tertiary
TMS ether TMS-7 (1:1.7), 68% for tertiary alcohol 8 (4:1 dr), 60% for tertiary alcohol 9
(7:1 dr). THF=tetrahydrofuran; TMS=trimethylsilyl.
high purity and yields for direct comparisons (Supporting In-
formation).
ability of the protecting group to withstand the reaction condi-
tions of the following steps, as well as our potential to remove
them towards the end of the synthesis, without affecting the
functionalities present in the final molecules. Initially, the dia-
mine was successfully protected as the di-trichloroacetamide
that further reacted with sodium hydride and allylic bromide[35]
toward the desired allylic ether 13. The more stable acetyl
group was also selected for the protection of the diamines as
the di-acetamides 14–16. Its etherification proceeded smoothly
to yield dienes 17–19 with high efficiency. Chromatographic
separation of the two diastereomers was successfully realized
only for diene 17, leading to the characterization of 17a
(major isomer, vinyl group in equatorial position) and 17b
(minor isomer). In the course of our synthetic route we also ex-
perimented with the potential benefit of using an azide to
mask the amines. Through a similar reaction sequence, diazide
20 became readily available and was further subjected to allyl-
bromide addition toward ether 21 (Scheme 5).
The undesired outcome was circumvented through the in-
clusion of trimethylsilyl chloride (TMSCl) in the reaction mix-
ture. The oxyanion was trapped in situ as the corresponding
TMS-ether, which could be easily cleaved during the final
workup, thus preventing the sequential formation of the oxa-
zolidinone byproduct. Consequently, all three required alcohols
7–9 (vinyl, allyl, and homoallyl) were successfully synthesized
(Scheme 4).
Regarding the stereoselectivity of the specific addition, it ap-
pears that the determining factor is the size of the organome-
tallic reagent. Thus, alcohol 7 was isolated as a 2.5:1 mixture of
isomers, alcohol 8 as a 4:1 mixture and alcohol 9 as a 7:1 mix-
1
ture, as determined by HPLC analysis and H NMR spectrosco-
py (Scheme 4). In all cases the major product was bearing the
olefinic substituent in the equatorial position (anti to the adja-
cent NHR and OR’ functionalities, C4-S), as it was unequivocally
established in the proceeding steps. Specifically regarding the
vinylic addition, the corresponding equatorial (inversed stereo-
selectivity) TMS-ether TMS-7 was also isolated in 25% yield, in-
dicating different hydrolysis rates of the ethers; the axial TMS
hydrolyzed faster than the equatorial TMS group. Thus, the
actual selectivity of the isolated nucleophilic attack in this case
was calculated to be 1.3:1.
Ring-closing metathesis (RCM) transformation for the
synthesis of the spiro-skeleton (retron B)
Dienes 17–19 were subjected to a ring-closing metathesis with
Grubbs’ second-generation catalyst[36] to furnish the corre-
sponding spiro ethers 22–26 as single isomers, after careful
chromatographic separation (Scheme 6). The minor isomer of
the seven-membered spiro-analogue was never observed, due
to the very small quantities present. The di-trichloroacetamide
diene 13 as well as the diazide analogue 21 were subjected to
the same reaction conditions, but to our disappointment the
desired products were formed only in trace amounts and were
never isolated. Upon deprotection of the ketal functionality
under acidic conditions diols 27–31 were isolated in high
yields. Furthermore, diol 28 produced crystallographic data
Further trials to perform the observed in situ trapping of the
tertiary oxyanion with allylbromide proved unsuccessful. Obvi-
ously in the specific case, the nature of the electrophile affects
the organometallic addition directly, leading either to a com-
plex reaction mixture through partial and uncontrolled remov-
al of the protecting groups, or to the formation of the same
oxazolidinone products 10–12.
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Designed Spiro-Bicyclic Analogues
Scheme 5. Reagents and conditions: a) KOH (aq), MeOH/H2O (1:1 v/v),
1008C, 14 h; b) Cl3CC(O)CCl3 (10 equiv), CHCl3, 258C, 12 h; then NaHCO3 (aq),
MeOH/H2O (1:1 v/v), 258C, 45 min, 37% for two steps; c) NaH (1.2 equiv),
DMF (0.1m), allylbromide (1.3 equiv), 0!258C, 10 h; d) Ac2O (5.0 equiv), Et3N
(9.0 equiv), CH2Cl2, 258C, 2 h; then K2CO3 (2.0 equiv), MeOH, 258C, 3 h, 50–
72% for two steps; e) TfN3 (10 equiv), CuSO4 (0.15 equiv), Et3N (6.0 equiv),
MeOH/H2O (4:1), 258C, 20 h, 88%; Tf=triflic=trifluoromethylsulfonic.
Scheme 6. Reagents and conditions: a) RCM: Grubbs 2nd generation
(0.1 equiv), CH2Cl2 (0.005m), 258C, 18 h, 60–88% for the two isomers;
b) AcOH/THF/H2O (3:3:1 v/v/v), 408C, 18 h, 75–95%; c) H2, Pd/C (cat.), MeOH,
258C, 18 h; d) KOH (70 equiv), H2O (1m), 120–1508C, 14–48 h.
that confirmed the relative stereochemistry of this substrate
(Scheme 6).
tion!dihydroxylation), confirmed the significant improvement
in the stereoselection of the final product (Scheme 7). The final
deprotection of the acetamide was accomplished with an
aqueous solution of KOH and heating up to 1308C overnight.
Attempts to remove the acetates with controlled microwave
heating (1708C, 10 min) were unsuccessful (incomplete re-
actions and mixtures were obtained). Purification of the final
product 2 by flash chromatography and repeated co-evapora-
tions to dryness with a 0.1m aqueous solution of HCl, fur-
nished its di-hydrochloride salt that was delivered for immedi-
ate biological evaluation. The indicated stereochemistry of di-
amine 2 was assigned from the NMR spectroscopic data
(NOESY, COSY). Strong NOE interactions were measured
among characteristic hydrogen atoms of the bicyclic system
(Scheme 7).
For the purpose of direct biological comparison, hydrogena-
tion of the double bond in 27–28 followed by hydrolysis of
the acetates furnished saturated spiro ethers 32 and 33
(Scheme 6). Cleavage of the N3 acetate in 33 could not be ach-
ieved even at high temperatures or extended reaction times.
The analogous unsaturated compounds were also synthesized
by basic treatment, as presented in Scheme 6. The order of the
two final transformations could be inverted, as exemplified for
7-membered spiro ether 37 from diacetate 29.
Dihydroxylation of olefins and final deprotections (retron A)
Dihydroxylation of olefin 28 under the standard Upjohn condi-
tions[37] furnished a mixture of diastereomeric vicinal diols in
1:9 ratio (Scheme 7). Based on literature reports[38] regarding
the selectivity of this reaction, we would expect it to proceed
predominantly toward product 39 (addition of OsO4 from the
less-hindered site) because the stereochemically encumbered
side is that facing the two hydroxyl groups of the 2-DOS-com-
ponent. To test this hypothesis, the dihydroxylation was also
assayed on the precursor acetal 23. The optimal experimental
conditions toward an enantiopure diol were found to be 08C
for 3 h (98:2 dr). Further deprotection to the tetraol 39 and
A similar synthetic pathway was also pursued for the more
polar, minor spiro ether epimer 31, which was dihydroxylated
with catalytic amounts of osmium tetroxide to furnish tetraols
41 in 2:1 ratio (Scheme 7). Attempts to improve the diastereo-
selectivity in this case were not successful because, similarly to
the previous rational, the reacting double bond is exposed to
less steric hindrance than the epimeric spiro ether 28. Thus,
the stereocontrol elements that favor attack of the oxidizing
agent from a certain direction are not in place, and a mixture
of products is formed. The two tetraols 41 could be separated
to the individual diastereomeric components by careful chro-
1
comparison of its H NMR spectrum with material obtained by
the aforementioned reverse synthetic protocol (ketal deprotec-
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D. Vourloumis et al.
(Scheme 9). In this case, dihydroxylation of olefin 24,
under standard conditions,[37] furnished a 2:3 mixture
of diastereomeric vicinal diols. Attempts to improve
the stereoselectivity of the reaction for the specific
substrate, even by the use of AD-mix-a, were not
successful. The differential topology of the double
bond and the higher flexibility of the seven-mem-
bered ring, in comparison to its five- and six-mem-
bered counterparts, might be responsible for the
observed outcome.
Removal of the ketal functionality in 46 and 47
under acidic conditions produced the corresponding
tetraols 48 and 49 respectively. Two-dimensional
NMR spectroscopy experiments at this stage allowed
us to assign the absolute stereochemistry of the two
compounds, as presented in Scheme 9. Final hydroly-
sis of the acetamides, followed by careful chromato-
graphic separation, afforded the desired spiro ethers
50 (minor) and 3 (major).
Due to the highly polar nature of our final prod-
ucts in combination with the relatively harsh experi-
mental conditions during the last steps of the synthe-
sis, the inorganic salt, as well as the silica gel content
of our samples represented a major consideration
because they could directly affect the validity of the
produced biological results. Thus, the final com-
pounds were routinely purified with sephadex col-
umns. Specifically, for compound 50 and only for the
direct comparison of its biological potency regarding
Scheme 7. Reagents and conditions: a) OsO4 (0.1 equiv), NMO (3.0 equiv), acetone/H2O
(1:1 v/v), 08C or 258C, 3 h, 78–90%; b) AcOH/THF/H2O (3:3:1 v/v/v), 408C, 18 h, 75–95%;
c) KOH (70 equiv), H2O (2 mL, 1m), 1308C, 15 h, 85–90%. NMO=N-methylmorpholine-N-
oxide.
matographic purification. Furthermore, tetraols 41 were sub-
jected to hydrolysis to furnish diamines 42. Because biological
evaluation of these isomers indicated significantly reduced po-
tency to inhibit protein production in bacteria in comparison
to 2, as will be presented in the following section, assignment
of their absolute stereochemistry was not pursued further.
For the more sterically hindered olefin 22 (Scheme 6) the
standard Upjohn conditions proved ineffective, resulting in
complete recovery of the starting material. The same result
was also obtained for diol 27. With stoichiometric amounts of
osmium tetroxide though, olefin 27 was converted to the cor-
responding osmate ester 43, which was isolated and further
Scheme 8. a) OsO4 (1.2 equiv), TMEDA (1.2 equiv), CH2Cl2, ꢀ788C!258C, 3 h,
75%, b) MeOH, HCl (cat.), 258C, 15 min, quant., c) KOH (70 equiv), H2O
(2 mL, 1m), 1408C, 48 h, 20%.
analyzed by two-dimensional NMR spectroscopy and NOE
measurements (Scheme 8). These experiments indicated that
the stereoselectivity of the hydroxylation in this case is oppo-
site to that obtained for the six-membered analogue 39. Appa-
rently, the immediate proximity of the 5-OH to the double
bond directs the oxidizing agent through a pre-coordination
effect.[39] Hydrolysis of the ester with catalytic amounts of hy-
drochloric acid furnished tetraol 44, which was further convert-
ed to diamine 1. Noticeably, removal of the acetates in this
case required higher temperatures and prolonged reaction
times. Similarly, spiro-olefin 30 produced final diamine 45
(Scheme 8).
its overall purity, a “chemical purification” method was applied.
Thus, 50 was re-protected as the di-Cbz carbamate, then puri-
fied and deprotected by a catalytic hydrogenation reaction
(Scheme 8). The potencies of the two samples, regarding their
binding affinities for the RNA A-site construct were evaluated
and the obtained values were identical. Additionally, quantifi-
cation of the substances delivered for biological evaluation
was performed by NMR spectroscopy. The method utilized is
adequately described in the literature[40] and has been success-
fully employed by us in the past.[41] It utilizes as an internal
An analogous synthetic pathway was applied for the forma-
tion of the seven-membered spiro derivatives 3 and 50
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Designed Spiro-Bicyclic Analogues
pounds able to interfere with protein synthesis inhib-
it enzyme production, and therefore reduce lumines-
cence (Figure 2D).
Aminoglycosides are also known to interfere with
eukaryotic protein synthesis. Some of them have the
ability to induce mammalian read-through stop-
codon mutations and generate fully functional pro-
teins in several genetic disorders.[43] Therefore, we
investigated the performance of these compounds
towards eukaryotic protein translation inhibition in
vitro. Similarly to the bacterial IVT, luciferase is ex-
pressed from a plasmid containing the encoding
gene under the command of T7 promoter, in a mix-
ture that combines RNA polymerase, reticulocyte
lysate solution and nucleotides. Compounds that in-
terfere with protein synthesis cause a decrease in the
observed luminescence (Table 1, Eukaryotic IVT).
As indicated in Table 1 and Figure 2, many of our
synthetic analogues exhibited satisfactory binding
affinities for the bacterial decoding center, with the
6,6- and 6,5- scaffolds producing the best results. In-
terestingly, a significant variability was observed re-
Scheme 9. Reagents and conditions: a) OsO4 (0.1 equiv), NMO (3.0 equiv), acetone/H2O
(1:1 v/v), 08C or 258C, 3 h, 70%; b) TFA/THF/H2O (3:3:1 v/v/v), 508C, 18 h, 75%; c) KOH
(70 equiv), H2O (2 mL, 1m), 1208C, 14 h, 70%; d) Cbz-Cl (4.0 equiv), NaHCO3 (8.0 equiv),
H2O/dioxane (2:1 v/v), 14 h, 08C!258C; e) Pd/C, H2, MeOH, 20 h.
Table 1. Biological activities for natural aminoglycosides and spiro deriva-
tives.
standard 2,5-dimethylfuran (DMFu) to assess the amount of
substance contained in the NMR spectroscopic solution by
direct integration of the appropriate peaks. All our measure-
ments indicated an excellent agreement between the sample’s
weight and its spectroscopic quantitation; this suggests the
complete removal of all impurities during the selected purifica-
tion process.
Compound
RNA EC50 [mm][b]
BIVTIC50 [mm][a]
EIVTIC50 [mm][c]
neomycin B
neamine
0.007
9.5
0.032
5
252
120
2
42
35
33
50
3
6.5
9.3
3.2[d]
18[d]
74
141
11.3
174
no activity
no activity
118
no activity
no activity
no activity
no activity
130
Biological evaluation of synthetic analogues
168
160
36
37
1
34
45
32
8.4[d]
no binding
1.5[d]
1.7[d]
2.4[d]
0.358[d]
no activity
no activity
187[d]
no activity
no activity
no activity
no activity
no activity
no activity
no activity
no activity
no activity
To evaluate the affinity of our synthetic analogues for the bac-
terial decoding-site, an RNA fluorescence assay was employed.
A model RNA oligonucleotide was used in which the adenine
at position 1492 has been replaced by a fluorescent probe, 2-
aminopurine (2AP).[42] The probe’s fluorescence is quenched
when it is found stacked with other bases, but it increases
upon compound binding and its exposure to solvent. We per-
formed therefore titrations of compounds to the 2AP-labelled
RNA double stranded construct, and increase in fluorescence
intensity was monitored (Table 1, RNA specific assay). As a con-
trol, compounds were also titrated to 2AP-labeled single-
stranded oligonucleotide with which no significant changes in
fluorescence were observed. Representative plots are present-
ed in Figure 2A–C.
[a] RNA EC50 values were determined by decoding-site RNA fluorescence
assay and calculated as the average of three replicate experiments for
each compound (ꢁ10%); [b] BIVTIC50 values were determined by coupled
transcription–translation assay by using E. coli extract, calculated as the
average of three replicate experiments for each compound (ꢁ15%);
[c] EIVTIC50 values were determined by TNT quick-coupled transcription–
translation assay, as the average of three replicate experiments per com-
pound (ꢁ15%); [d] Low efficacy in effect measured.
The biological activity of the compounds, as functional bac-
terial-translation inhibitors, was evaluated in a coupled in vitro
garding the efficacy of the fluorescent measurement. For com-
pounds 2, 42, 50, and 3 that exclusively generated an effective
fluorescent signal upon binding, these affinities are also sup-
ported by their potencies as inhibitors of bacterial protein pro-
duction. Especially for compound 2 (EC50 =6.5 mm), the ob-
served values are of great importance because they are direct-
ly comparable to neamine, despite the fact that the designed
analogue possesses half the electrostatic load present in the
natural product (EC50 =9.5 mm), with only two amino groups
transcription-translation assay (IVT; Table 1, Bacterial IVT).[25]
A
gene encoding for firefly luciferase, as the reporter enzyme,
was expressed in vitro by using bacterial S30 extract (Promega)
in a mixture of nucleotide triphosphates, amino acids, and
polymerase. The produced enzyme was quantified by subse-
quent addition of luciferin substrate, giving rise to a lumines-
cence value relative to the amount of enzyme present. Com-
ChemBioChem 2011, 12, 71 – 87
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
77

D. Vourloumis et al.
Figure 2. Normalized fluorescence signal at 370 nm during titration of neamine and A) 6,6-spiro compounds 2, 42, 35, and 33, B) 7,6-spiro ligands 50, 3, 36,
37, and C) 5,6-spiro analogues 1, 34, 45, 32 to RNA construct probed with 2AP label at residue 1492. D) Normalized Luminescence Units, corresponding to
bacterial in vitro transcription-translation, versus log(c) plots of neomycin and synthetic compounds 2, 42, 3, 50, and 1 by fitting to a variable slope dose–
response equation.
embedded in its structure. The ability of 7-membered counter-
parts 50 and 3 to maintain similar values between bacterial
and eukaryotic protein synthesis inhibition is also noticeable.
Regarding compounds 1, 32–35, and 45, the obtained EC50
values were calculated based on lower-efficacy fluorescent
measurements, as presented in Figure 2. We speculate that
this observation could be consistent with a smaller conforma-
tional change of the RNA target, which overall cannot suffi-
ciently alter the mechanism of protein production.
The hydroxyls O5 and O6 were found to exhibit major contacts
with C1407 and A1408, whereas O3’ forms direct contacts with
the phosphate of A1492. In the most favorable orientation, the
ether moiety of 2 is predicted to be stacked onto G1491 in the
same manner as ring I of all natural aminoglycosides. The con-
served interaction with A1408 is mediated by O5 of 2 and
probably a water molecule (W1 in Figure 1B), which was mod-
eled at the position occupied by N6’ of neamine’s ring I (Fig-
ure 1A). A second water molecule (W2 in Figure 1B) substitut-
ing O4’ of neamine, could potentially mediate a hydrogen
bond with O2P of A1493.
The similarities observed in the values of most analogues in
conjugation with our molecular modeling analysis lead to a
conserved, yet not fully optimized, binding orientation, in
which important interactions are preserved. For example, in
the simulated complex of 2 with the A-site (Figure 1B), N1 of 2
interacts with U1495 and G1494, whereas N3 is involved in
electrostatic interactions with the phosphate group of A1493.
Conclusions
By diverging from previous research efforts, which focused on
the direct modification of natural aminoglycosides and simpli-
78
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 71 – 87

Designed Spiro-Bicyclic Analogues
Rf =0.51 in 5% MeOH/CH2Cl2; 1H NMR (500 MHz, [D6]DMSO): d=
7.48 (d, J=8.0 Hz, 1H), 7.41–7.24 (m, 20H), 7.23 (d, J=7.7 Hz, 1H),
7.01 (brs, 1H), 6.56 (brs, 1H), 5.13–5.07 (m, 2H), 5.06–4.97 (m, 6H),
4.97–4.90 (m, 2H), 4.89–4.85 (brs, 1H) 3.74–3.62 (m, 4H), 3.58 (ddd,
J=8.8, 9.0, 9.0 Hz, 1H), 3.49–3.34 (m, 4H), 3.28–3.19 (m, 1H), 3.13
(brs, 1H), 1.93–1.82 (m, 1H), 1.59–1.28 (m, 10H), 1.28–1.15 ppm
(m, 1H); 13C NMR (125 MHz, [D6]DMSO): d=156.6, 156.0, 155.6,
155.4, 137.1, 137.0, 136.9 (2C), 128.3 (4C), 128.2, 127.8, 127.7 (2C),
127.6 (3C), 127.5, 110.8, 96,1, 80.2, 77.8, 76.1, 71.4, 70.4, 70.0, 65.4,
65.3 (3C), 55.5, 50.8, 48.3, 41.9, 36.1, 35.6, 24.5, 23.3, 23.1 ppm;
HRMS-ESI: m/z: calcd for C50H58N4NaO14: 961.3847 [M+Na]+, found:
961.3840.
fied analogues, we successfully constructed compounds that
are able to mimic the bonding interactions of the natural prod-
ucts for the ribosomal decoding center. Specifically, rigid spiro-
cyclic scaffolds have been designed and the development of
an optimized synthetic route for their production is described.
All final products were tested for their ability to bind to the
bacterial decoding center, as well as for their potential to
inhibit protein production in vitro. Our results formulate a pre-
liminary SAR study for the identification of important interac-
tions and support the development of analogues with im-
proved antibiotic profiles. Six-membered spirocyclic scaffold 2
represents the champion of the present effort and is able to
maintain the desired activity with half the number of the noto-
rious amino moieties present in neamine. Also, direct compari-
son between the 6,6- and 7,6-analogues introduces the possi-
bility of controlling the selectivity of action between bacteria
and eukaryotic organisms through fine tuning of structural ele-
ments. Additional examples, co-crystallographic studies and
biological evaluations are currently underway in our laboratory.
Synthesis of benzyl (3aS,4R,5S,7R,7aS)-4-hydroxyhexahydro-
spiro[benzo[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyldicarbamate
(5): The monoketal 4 (15.0 g, 16.0 mmol) was suspended in MeOH
(800 mL) and cooled to 08C. NaIO4 (20.5 g, 95.9 mmol) was added,
the reaction flask was wrapped with aluminum foil, and the mix-
ture was stirred vigorously overnight. The solids were filtered off,
and residual solvent was removed at high vacuum overnight.
Quick purification by column chromatography (CH2Cl2!10%
MeOH/CH2Cl2) afforded the corresponding dialdehyde as a white
amorphous solid (15.0 g, quant.; Rf =0.7 in 5% MeOH/CH2Cl2),
which was used directly in the next step.
Experimental Section
The corresponding dialdehyde (0.44 g, 0.47 mmol) was dissolved in
MeOH (33 mL), Et3N (0.16 mL, 1.2 mmol) was added, and the reac-
tion mixture was stirred vigorously at 408C for 13 h. The mixture
was concentrated under reduced pressure, and the residue was
purified by column chromatography (20!60% EtOAc/hexane) to
afford product 5 as a white amorphous solid (226 mg, 94% yield).
If necessary, an additional recrystallization by using EtOAc/n-
Unless otherwise noted, solvents and reagents for organic synthe-
sis were obtained from commercial suppliers and were used with-
out further purification. Tetrahydrofuran (THF), toluene and diethyl
ether were distilled from sodium benzophenone, and dichlorome-
thane (CH2Cl2) was distilled from calcium hydride. All reactions
were carried out under a dry argon atmosphere with anhydrous,
freshly distilled solvents under anhydrous conditions unless other-
wise noted. All reactions were magnetically stirred with Teflon stir
bars, and temperatures were measured externally. Reactions requir-
ing anhydrous conditions were carried out in oven dried (1208C,
24 h) or flame dried (vacuum <0.5 Torr) glassware. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous
materials. All reactions were monitored by thin layer chromatogra-
phy (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-
254) by using UV light as visualizing agent and 7% ethanolic phos-
phomolybdic acid or p-anisaldehyde solution and heat as develop-
ing agents. E. Merck silica gel (60, particle size 0.040–0.063 mm)
was used for flash column chromatography. Nuclear magnetic reso-
nance (NMR) spectra were recorded on a Bruker AM-250 or a
Bruker Avance DRX-500 instrument as noted individually. Unless
stated otherwise, NMR spectroscopy experiments were performed
at 258C. Chemical shifts are measured in parts per million (d) rela-
tive to the deuterated solvent used in the experiment.
hexane could be performed. Rf =0.69 in 35% EtOAc/CH2Cl2; [a]D22
=
ꢀ5.68 (c=0.6, MeOH); 1H NMR (500 MHz, CD3OD): d=7.33–7.27
(m, 10H), 5.06 (brs, 4H), 3.78 (brs, 1H), 3.57–3.53 (m, 2H), 3.44–
3.42 (m, 2H), 2.15 (ddd, J=4.4, 4.4, 13.3 Hz, 1H), 1.65–1.60 (m, 8H),
1.40–1.28 ppm (m, 3H); 13C NMR (125 MHz, CD3OD): d=158.6,
158.2, 138.2 (2C), 129.4 (2C), 128.9 (2C), 128.8 (2C), 128.8 (2C),
113.0, 82.0, 79.7, 73.9, 67.5 (2C), 54.9, 50.4, 37.5, 37.3, 37.1, 26.1,
24.7 ppm (2C) ; HRMS-ESI: m/z: calcd for C28H34N2NaO7: 533.2264
[M+Na]+, found: 533.2257.
Dess–Martin oxidation for the synthesis of benzyl
(3aR,5S,7R,7aS)-4-oxohexahydrospiro[benzo[d][1,3]dioxole-2,1’-
cyclohexane]-5,7-diyldicarbamate (6): The precursor alcohol 5
(1.25 g, 2.50 mmol) was dissolved in CH2Cl2 (37.5 mL) and NaHCO3
(1.26 g, 15.0 mmol) was added. The suspension was allowed to stir
for 10 min, the Dess–Martin periodinane (4.16 g, 10.0 mmol) was
added and the mixture warmed to 508C for 1 h. A sat. aq solution
of Na2S2O3 (18 mL) was added, and the mixture was vigorously
stirred until it became transparent. It was then extracted with
NaHCO3 (3ꢁ15 mL), dried (MgSO4), and concentrated in vacuo. The
solid crude product was purified by flash column chromatography
on silica gel (CH2Cl2!3% acetone/CH2Cl2) to afford ketone 6 as a
white amorphous solid. (1.13 g, 90% yield); Rf =0.9 in 5% acetone/
CH2Cl2; 1H NMR (500 MHz, CDCl3): d=7.41–7.27 (m, 10H), 4.56–
4.50 (m, 1H), 5.15–5.06 (m, 4H), 4.93–4.85 (m, 1H), 4.51–4.42 (m,
1H), 4.32 (d, J=10.2 Hz, 1H), 4.25–4.16 (m, 1H), 3.67–3.55 (m, 1H),
3.09–2.27 (m, 1H) 1.76–1.44 (m, 9H), 1.43–1.28 ppm (m, 3H);
13C NMR (125 MHz, [D6]DMSO): d=198.4, 155.8, 136.2, 133.2, 131.3,
129.3, 128.6, 128.3, 114.1, 80.2, 67.2, 55.1, 49.4, 37.3, 36.4, 35.7,
24.8, 23.5 ppm; IR (neat): n˜ =3032, 2938, 1704, 1526, 1250,
1,3,2’,6’-Tetra-Cbz-5,6-O-cyclohexylidine neamine 4: Tetra-Cbz-
neamine (22.4 g, 26.1 mmol) and p-toluenesulfonic acid (p-TsOH;
554 mg, 2.91 mmol) were dissolved in dry DMF (150 mL). While
heating the solution at 508C, 1,1-cyclohexanone dimethyl ketal
(29.8 mL, 195 mmol) was added. The solution was stirred at 508C
for 5 h. At this point the starting material was consumed and a
mixture of mono- and diketal was formed (Rf =0.5 and 0.85 respec-
tively, 5% MeOH/CH2Cl2). MeOH (10 mL) was added, and the mix-
ture was stirred for 1 h, at 508C while monitored by TLC. Et3N
(9.0 mL) was finally added, and the reaction mixture was cooled to
RT. H2O (500 mL) was added slowly to the stirred solution leading
to the formation of a precipitate. The solids were filtered off and
washed successively with H2O (250 mL) and ice-cold Et2O (2ꢁ
250 mL). The white solid was dried under reduced pressure and
finally overnight on high vacuum to obtain 4 (23.2 g, 95% yield);
1058 cmꢀ1
[M+Na]+, found: 531.2103.
; HRMS-ESI: m/z: calcd for C28H32N2NaO7: 531.2107
ChemBioChem 2011, 12, 71 – 87
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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79

D. Vourloumis et al.
Vinyl-Grignard addition to ketone 6. Synthesis of benzyl
(3aR,5S,7R,7aS)-4-hydroxy-4-vinylhexahydrospiro[benzo[d][1,3]
dioxole-2,1’-cyclohexane]-5,7-diyldicarbamate (7): At ꢀ788C,
ketone 6 (587 mg, 1.15 mmol) was dissolved in anhyd THF (8.7 mL)
and TMSCl (0.45 mL, 3.50 mmol) was added. A solution of vinyl-
magnesium chloride was added dropwise (1.6m in THF, 2.2 mL,
3.5 mmol) and the stirred solution was allowed to warm to RT over
4 h. Then a sat. aq solution of NH4Cl (4.0 mL) was added, and the
mixture was extracted with EtOAc (3ꢁ8.0 mL). The organic solution
was dried (MgSO4), concentrated in vacuo, and purified by flash
chromatography on silica gel (CH2Cl2!5% acetone/CH2Cl2) to
obtain the corresponding allylic alcohols 7 (225 mg, 36% yield,
2.5:1 dr; Rf =0.32 in 5% acetone/CH2Cl2) as a white solid and the
TMS ethers 7a (154 mg, 25% yield, 1:1.7 dr; Rf =0.68 in 5% ace-
major), 3.76 (brs, 3H; minor), 3.49 (d, J=7.0 Hz, 1H; major), 3.42
(d, J=6.5 Hz, 1H; minor), 2.47–2.32 (m, 6H), 2.31–2.19 (m, 2H),
2.00–1.79 (m, 4H), 1.78–1.63 (m, 20H), 1.60–1.52 ppm (m, 2H);
13C NMR (125 MHz, CDCl3): d=156.3, 156.2, 138.8, 136.8, 129.0,
128.6, 128.5, 115.5, 115.2, 112.0, 81.2, 80.6, 76.0, 74.4, 67.3, 67.2,
52.5, 52.1, 49.9, 36.8, 36.7, 36.1, 35.9, 35.6, 28.7, 25.4, 25.2, 24.1,
24.0, 23.9, 23.8 ppm; HRMS-ESI: m/z: calcd for C32H40N2NaO7:
587.2733 [M+Na]+; found: 587.2724.
Synthesis of N,N’-((3aR,5S,7R,7aS)-4-allyl-4-(allyloxy)hexahydro-
spiro[benzo[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyl)bis(2,2,2-tri-
chloroacetamide) (13): Alcohol 9 (0.12 g, 0.21 mmol) and KOH
(0.50 g, 8.9 mmol) were dissolved in H2O (1.0 mL) and MeOH
(1.0 mL) in a round-bottomed flask. The solution was warmed at
1008C for 14 h and subsequently concentrated in vacuo to dry-
ness. The solid residue was purified by flash chromatography on
silica gel (MeOH!10% NH4OH/MeOH) to furnish the correspond-
ing epimeric (at the quaternary center, 7:1 dr) diamines (52 mg,
85%).
1
tone/CH2Cl2) as colorless oils; 7: H NMR (500 MHz, CDCl3): d=7.30
(m, 10H), 5.86 (dd, J=11.0, 17.1 Hz, 1H; major), 5.69 (d, J=16.3 Hz,
1H; minor), 5.48 (J=10.6 Hz, 1H; minor), 5.39 (d, J=17.3 Hz, 1H;
major), 5.24 (J=11.0 Hz, 1H; major) 5.06–5.03 (m, 4H+1H minor),
3.82 (m, 2H), 3.65 (m, 1H), 3.47 (m, 1H), 2.73 (brs, 1H), 2.32 (t, J=
6.5 Hz, 1H), 1.84 (m, 1H), 1.65–1.35 ppm (m, 10H); 13C NMR
(125 MHz, CDCl3): d=156.9, 156.8, 156.0, 155.8, 139.7, 138.3, 136.3,
136.2, 136.1, 136.0, 129.0, 128.4, 128.0, 121.1, 116.4, 112.3, 111.7,
83.1, 81.3, 80.1, 76.3, 75.3, 74.8, 67.2, 67.1, 66.8, 66.6, 55.8, 52.5,
50.0, 49.4, 36.4, 36.2, 36.1, 35.9, 35.0, 29.6, 26.9, 24.9, 23.6,
23.5 ppm; IR (neat): n˜ =3339, 2945, 1682, 1507, 1454, 1342, 1253,
At 258C, under an argon atmosphere, the diamines (28 mg,
0.094 mmol) were dissolved in dry CHCl3 (1 mL) containing hexa-
chloroacetone (0.15 mL, 0.99 mmol) and allowed to stir for 12 h.
The solvents were removed in vacuo and a solution of NaHCO3
(20 mg, 0.24 mmol) in MeOH/H2O (1:1, v/v) was added and allowed
to vigorously stir for 45 min. The solvents were removed in vacuo
and the mixture was purified by flash chromatography on silica gel
(CH2Cl2 to 10% MeOH/CH2Cl2) to obtain the corresponding trichlor-
oacetamides (20 mg, 37% yield; Rf =0.46 in 10% MeOH/CH2Cl2);
1084, 1037, 697 cmꢀ1
; HRMS-ESI: m/z: calcd for C30H37N2O7:
1
537.2595 [M+H]+, found: 537.2593; 7a: H NMR (500 MHz, CDCl3):
d=7.34 (m, 10H), 5.94 (dd, J=10.9, 17.6 Hz, 1H; minor), 5.85 (m,
1H; major), 5.58 (d, J=17.6 Hz, 1H; major), 5.51 (d, J=17.5 Hz, 1H;
minor), 5.46 (d, J=10.7 Hz, 1H; major), 5.25 (d, J=11.2 Hz, 1H;
minor), 5.14–5.05 (m, 4H), 4.87 (d, J=10.0, Hz, 1H; minor), 4.77 (m,
1H; major), 3.81–3.52 (m, 4H), 2.40 (m, 1H; major), 2.34 (t, J=
6.8 Hz, 1H; minor), 1.65–1.20 (m, 11H), 0.17–0.15 ppm (m, 9H);
13C NMR (125 MHz, CDCl3): d=156.1, 155.7, 137.3, 136.4, 132.9,
128.5, 128.1, 128.0, 121.0, 117.8, 111.8, 111.5, 82.8, 80.7, 78.6, 78.3,
75.8, 75.6, 67.3, 66.7, 66.6, 56.6, 54.6, 49.9, 48.3, 36.4, 36.3, 35.9,
35.0, 25.0, 23.8, 23.8, 23.6, 23.4, 2.6, 1.9 ppm; IR (neat): n˜ =3327,
2937, 1700, 1516, 1453, 1251, 1046, 844, 738, 697 cmꢀ1; HRMS-ESI:
m/z: calcd for C33H44N2O7Si: 608.2912 [M+H]+, found: 608.2913.
1
[a]2D2 =ꢀ69.88 (c=0.8, CHCl3); H NMR (500 MHz, CDCl3): d=6.9 (m,
1H), 5.98 (m, 1H), 5.21 (d, J=17 Hz, 1H), 5.10 (d, J=10 Hz, 1H),
4.12–4.08 (m, 2H), 3.52 (m, 1H), 3.14 (m, 1H), 2.96–2.72 (brs, 1H),
2.47 (m, 1H), 2.45 (m, 2H), 2.04 (m, 1H), 1.82 (m, 1H), 1.81–1.65 (m,
10H), 1.52 ppm (m, 1H); 13C NMR (125 MHz, CDCl3): d=161.8,
138.3, 115.0, 112.0, 92.3, 80.9, 74.6, 73.0, 51.7, 50.6, 36.3, 36.2, 35.2,
34.9, 28.3, 25.0, 23.7, 22.7 ppm.
At 08C, under an argon atmosphere, the corresponding alcohol
(20 mg, 0.034 mmol) was dissolved in dry DMF (1.0 mL) and NaH
(1.5 mg, 0.037 mmol, 60% w/w) was slowly added. The mixture
was allowed to stir at this temperature for 30 min, then allyl bro-
mide (5.0 mL, 0.040 mmol) was added, and the solution was slow
warmed to 258C over 10 h. A sat. aq solution of NH4Cl (2 mL) was
added, and the mixture was extracted with EtOAc (2ꢁ5 mL). The
organic solution was washed with H2O (1 mL) and brine (1 mL),
dried (MgSO4) and concentrated in vacuo. The mixture was purified
by flash chromatography on silica gel (CH2Cl2!10% MeOH/CH2Cl2)
to obtain the corresponding ether 13 (9.0 mg, 43% yield); Rf =0.67
Allyl-Grignard addition to ketone 6. Synthesis of benzyl
(3aR,5S,7R,7aS)-4-allyl-4-hydroxyhexahydrospiro[benzo[d][1,3]di-
oxole-2,1’-cyclohexane]-5,7-diyldicarbamate (8): Similarly to 7
and with allylmagnesium bromide as the nucleophile, homoallylic
alcohols 8 were isolated as a white solid (281 mg, 68% yield, 4:1
dr); Rf =0.24 in 40% EtOAc/hexanes; 1H NMR (500 MHz, CDCl3):
d=7.55–7.37 (m, 10H), 6.05 (m, 1H), 5.44–5.11 (m, 6H), 3.98–3.78
(m, 4H), 3.49 (m, 1H), 2.65 (brs, 1H), 2.54–2.41 (m, 3H), 1.85–
1.44 ppm (m, 11H); 13C NMR (125 MHz, CDCl3): d=155.8, 136.4,
132.3, 128.5, 128.4, 128.2, 120.1, 111.5, 80.6, 77.4, 76.9, 73.3, 67.0,
66.8, 58.8, 51.1, 49.6, 40.7, 36.4, 35.2, 25.0, 23.8, 23.7 ppm; IR (neat):
1
in 10% MeOH/CH2Cl2; H NMR (500 MHz, CDCl3): d=5.87 (m, 2H),
5.19–4.95 (m, 5H), 4.80 (m, 1H), 4.11–4.03 (m, 1H), 3.32 (m, 1H),
3.64 (m, 1H), 3.39 (m, 1H), 2.51–2.11 (m, 5H), 1.91–1.59 ppm (m,
11H); 13C NMR (125 MHz, CDCl3): d=162.5, 155.3, 138.2, 133.7,
116.4, 114.9, 81.8, 71.2, 56.7, 50.9, 46.9, 39.9, 36.8, 36.4, 35.8, 27.4,
26.2, 24.9, 23.7, 23.5 ppm.
n˜ =3284, 3076, 2933, 2855, 1639, 1545, 1448, 1371, 1040, 920 cmꢀ1
;
HRMS-ESI: m/z: calcd for C31H38N2NaO7: 573.2577 [M+Na]+, found:
573.2571.
Synthesis of N,N’-((3aR,5S,7R,7aS)-4-hydroxy-4-vinylhexahydro-
spiro[benzo[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyl)diaceta-
mide (14): The Cbz-protected amine 7 (0.22 g, 0.41 mmol) was dis-
solved in H2O/MeOH (4.0 mL, 1:1 v/v) and KOH (0.92 g, 16 mmol)
was added. The solution was heated to 1008C for 14 h, and the
solvent was removed in vacuo. The crude product was purified by
flash column chromatography on silica gel (MeOH!10% NH4OH/
MeOH) to furnish the free amines (61 mg, 55% yield). The amines
(85 mg, 0.32 mmol) were dissolved in CH2Cl2 (1.0 mL) and cooled
to 08C. Et3N (0.40 mL, 2.8 mmol) and Ac2O (0.15 mL, 1.6 mmol)
Homoallyl-Grignard addition to ketone 6. Synthesis of benzyl
(3aR,5S,7R,7aS)-4-(but-3-enyl)-4-hydroxyhexahydrospiro[benzo-
[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyldicarbamate (9): Similar-
ly to 7 and with 3-butenylmagnesium bromide as the nucleophile,
homoallylic alcohols 9 were isolated (7:1 mixture of diastereoiso-
mers, 0.24 g, 60% yield) as a white foam; Rf =0.24 in 40% EtOAc/
hexanes; H NMR (500 MHz, CDCl3): d=7.49–7.39 (m, 20H), 5.97–
5.82 (m, 1H; major), 5.81–5.78 (m, 1H; minor), 5.31 (d, J=9.5 Hz,
2H), 5.26–5.14 (m, 8H), 5.09 (d, J=10.0 Hz, 2H), 3.89 (brs, 3H;
1
80
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 71 – 87

Designed Spiro-Bicyclic Analogues
were added. The solution was stirred at RT for 4 h, and the solvent
was removed in vacuo. The crude product was purified by flash
column chromatography on silica gel (CH2Cl2!7% MeOH/CH2Cl2)
to furnish the acetates 14 (88 mg, 79% yield; Rf =0.38 in 10%
MeOH/CH2Cl2) as a white solid. 1H NMR (500 MHz, CD3OD): d=
6.00 (dd, J=11.0, 17.0 Hz, 1H; minor), 5.84 (dd, J=10.8, 17.3 Hz,
1H; major), 5.62 (d, J=17.0 Hz, 1H; minor), 5.48 (d, J=11.0 Hz, 1H;
minor), 5.41 (d, J=17.5 Hz, 1H; major), 5.18 (d, J=11.0 Hz, 1H;
major), 4.10–3.93 (m, 3H; major+2H; minor), 3.70–3.63 (m, 2H;
minor), 3.54 ppm (d, J=9.0 Hz, 1H; major); 13C NMR (125 MHz,
CD3OD): d=172.4, 171.4, 171.4, 171.1, 138.9, 132.5, 119.3, 114.6,
111.3, 110.8, 83.3, 81.7, 76.3, 76.2, 75.1, 74.3, 54.4, 51.0, 47.7, 46.5,
36.1, 35.9, 35.9, 35.7, 34.2, 33.3, 24.7, 24.6, 23.3, 23.1, 21.3, 21.2,
21.1 ppm; IR (neat): n˜ =3283, 2937, 1653, 1554, 1448, 1281, 1118,
920 cmꢀ1; HRMS-ESI: m/z: calcd for C18H29N2O5: 353.2071 [M+H]+,
found: 353.2070.
135.8, 134.7, 120.1, 115.6, 111.8, 81.8, 78.9, 75.6, 67.1, 52.4, 48.8,
36.7, 36.2, 34.4, 25.1, 23.8, 23.8, 23.6, 23.4 ppm; IR (neat): n˜ =3230,
2934, 1636, 1541, 1373, 1116, 1083, 1037, 914 cmꢀ1; HRMS-ESI: m/z:
calcd for C21H33N2O5: 393.2384 [M+H]+, found: 393.2384. Minor
isomer (17b): Rf =0.61 in 10% MeOH/CH2Cl2; 1H NMR (500 MHz,
CDCl3): d=6.56 (brs, 1H), 5.92–5.79 (m, 3H), 5.62–5.48 (m, 2H),
5.23 (d, J=15.5 Hz, 1H), 5.05 (d, J=10.5 Hz, 1H), 4.23 (m, 1H), 4.14
(dd, J=4.5, 13.5 Hz, 1H), 4.04–4.02 (m, 2H), 3.74–3.66 (m, 2H), 2.30
(d, J=11.5 Hz, 1H), 1.95 (s, 3H), 1.94 (s, 3H), 1.60–1.30 ppm (m,
11H); 13C NMR (125 MHz, CDCl3): d=170.4, 169.9, 135.5, 131.1,
121.7, 115.1, 111.9, 81.9, 80.0, 77.1, 65.5, 51.7, 48.6, 36.5, 36.2, 35.2,
25.1, 23.9, 23.7, 23.5, 23.4 ppm; IR (neat): n˜ =3284, 2937, 1653,
1558, 1373, 1131, 1083, 917 cmꢀ1
; HRMS-ESI: m/z: calcd for
C21H33N2O5: 393.2384 [M+H]+, found: 393.2383.
Allylation of tertiary alcohols 15 and synthesis of N,N’-
((3aR,5S,7R,7aS)-4-allyl-4-(allyloxy)hexahydrospiro[benzo[d][1,3]
dioxole-2,1’-cyclohexane]-5,7-diyl)diacetamide (18): Similarly to
17, diene 18 was isolated as a white solid that contained both epi-
mers (at the quaternary center) in a ratio of 4:1 (58 mg, 90%
yield); Rf =0.31 in 10% MeOH/CH2Cl2; 1H NMR (500 MHz, CDCl3).
Major isomer: d=7.40 (d, J=7.4 Hz, 1H; NH), 7.28 (d, J=6.6 Hz,
1H; NH), 5.86 (m, 2H), 5.43–5.16 (m, 4H), 4.65 (dd, J=4.2, 12.8 Hz,
1H), 4.36–4.29 (m, 1H), 4.25–4.13 (m, 1H), 4.09–3.93 (m, 2H), 3.56
(d, J=9.1 Hz, 1H), 2.89 (dd, J=8.5, 12.8 Hz, 1H), 2.49–2.39 (m, 1H),
2.36–2.27 (m, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 1.84–1.49 ppm (m,
11H); 13C NMR (125 MHz, CDCl3): d=169.8, 169.0, 135.7, 135.3,
132.0, 129.1, 125.4, 120.0, 115.4, 111.6, 80.7, 77.9, 75.4, 65.4, 49.1,
48.9, 36.8, 36.3, 36.0, 34.8, 34.3, 25.1, 23.9, 23.8, 23.5, 23.4 ppm;
HRMS-ESI: m/z: calcd for C22H34N2NaO5: 429.2365 [M+Na]+, found:
429.2362.
Synthesis of N,N’-((3aR,5S,7R,7aS)-4-allyl-4-hydroxyhexahydro-
spiro[benzo[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyl)diaceta-
mide (15): Synthesis of the corresponding diacetates was per-
formed from 8 as described for 14, producing 15 as a white foam
(48 mg, 72% yield); Rf =0.28 in 10% MeOH/CH2Cl2; 1H NMR
(500 MHz, CDCl3). Major isomer: d=6.71 (d, J=5.5 Hz, 1H), 6.46 (d,
J=8.8 Hz, 1H), 5.94–5.82 (m, 1H), 5.18–5.07 (m, 2H), 4.07–3.91 (m,
2H), 3.83 (dd, J=9.6, 10.0 Hz, 1H), 3.36 (d, J=9.1 Hz, 1H), 2.37 (d,
J=7.3 Hz, 2H), 2.27–2.19 (m, 1H), 2.00 (s, 3H), 1.97 (s, 3H), 1.64–
1.34 ppm (m, 11H); 13C NMR (63 MHz, CDCl3): d=170.6, 170.0,
132.5, 111.2, 80.5, 75.0, 73.3, 49.4, 48.2, 40.6, 36.4, 36.2, 34.3, 25.0,
23.8, 23.3, 23.0 ppm; HRMS-ESI: m/z: calcd for C19H30N2NaO5:
389.2052 [M+Na]+, found: 389.2045.
Synthesis of N,N’-((3aR,5S,7R,7aS)-4-(but-3-enyl)-4-hydroxyhexa-
hydrospiro[benzo[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyl)diace-
tamide (16): Synthesis of the corresponding diacetates was per-
formed from 9 as described for 14, producing 16 as a white foam
(36.3 mg, 50%); Rf =0.28 in 10% MeOH/CH2Cl2; 1H NMR (CDCl3,
500 MHz): d=5.84–5.74 (m, 1H), 5.01 (d, J=17.0 Hz, 1H), 4.91 (d,
J=10.5 Hz, 1H), 4.02 (d, J=10.0 Hz, 1H), 3.95 (brs, 2H), 3.44 (d, J=
7.5 Hz, 1H), 2.30–2.20 (m, 1H), 2.15–2.05 (m, 1H), 1.97 (s, 3H), 1.94
(s, 3H), 1.94–1.88 (m, 1H), 1.90–1.80 (m, 2H), 1.63 (brs, 8H), 1.50–
1.36 ppm (m, 2H); 13C NMR (CD3OD, 125 MHz): d=171.2, 158.9,
137.5, 115.8, 112.9, 82.4, 78.5, 75.7, 56.3, 47.8, 37.1, 36.5, 33.2, 25.3,
24.1, 23.9, 23.3, 18.7 ppm; HRMS-ESI: m/z: calcd for C20H32N2NaO5:
403.2209 [M+Na]+, found: 403.2204.
Allylation of tertiary alcohols 16 and synthesis of N,N’-
((3aR,5S,7R,7aS)-4-(allyloxy)-4-(but-3-enyl)hexahydrospiro[benzo-
[d][1,3]dioxole-2,1’-cyclohexane]-5,7-diyl)diacetamide (19): Simi-
larly to 17, dienes 19 were isolated as a white solid (both epimers
at the quaternary center) in a ratio of 7:1 (50 mg, 70% yield); Rf =
0.31 in 10% MeOH/CH2Cl2; 1H NMR (500 MHz, CDCl3): d=6.02–5.82
(m, 2H), 5.32 (d, J=16.0 Hz, 1H), 5.22 (d, J=9.0 Hz, 1H), 5.14 (d,
J=17.0 Hz, 1H), 5.05 (d, J=12.5 Hz, 1H), 4.65–4.46 (m, 1H), 4.30–
4.13 (m, 2H), 4.09–3.90 (m, 2H), 3.60–3.48 (m, 1H), 2.47–2.32 (m,
1H), 2.30–2.10 (m, 5H), 2.05 (d, J=4.5 Hz, 3H), 1.80–1.60 (m, 10H),
1.58–1.53 (m, 1H), 1.52–1.36ppm (m, 2H); 13C NMR (125 MHz,
CDCl3): d=170.2, 169.4, 138.4, 136.1, 135.7, 134.8, 117.3, 116.2,
115.7, 115.5, 115.0, 111.9, 81.5, 81.2, 78.2, 75.9, 75.7, 65.4, 65.3, 49.9,
49.7, 49.5, 49.2, 37.0, 36.8, 36.5, 36.4, 36.3, 34.7, 29.8, 28.6, 25.4,
24.0 ppm; HRMS-ESI: m/z: calcd for C23H36N2NaO5: 443.2522
[M+Na]+; found: 443.2515.
Allylation of the tertiary alcohols 14 and synthesis of N,N’-
((3aR,5S,7R,7aS)-4-(allyloxy)-4-vinylhexahydrospiro[benzo[d][1,3]
dioxole-2,1’-cyclohexane]-5,7-diyl)diacetamide (17): Alcohol 14
(0.23 g, 0.64 mmol) was dissolved in DMF (7.0 mL) and cooled to
08C. NaH (31 mg, 0.77 mmol, 60% w/w) was added, and the solu-
tion was stirred at 08C for 45 min. Freshly distilled allyl bromide
(72 mL, 0.83 mmol) was added in one portion, and stirring was con-
tinued at RT for 24 h. A sat. aq solution of NH4Cl (4.5 mL) was then
added, and the mixture was extracted with CH2Cl2 (3ꢁ10 mL). The
organic solution was dried (MgSO4) and concentrated in vacuo.
The crude product was purified by flash column chromatography
on silica gel (CH2Cl2!10% MeOH/CH2Cl2) to furnish alkenes 17a
(76 mg) and 17b (46 mg) as white solids (122 mg, 48% overall
Synthesis of (3aR,5S,7R,7aS)-4-allyl-5,7-diazidohexahydrospiro-
[benzo[d][1,3]dioxole-2,1’-cyclohexan]-4-ol (20): At 08C, a bipha-
sic mixture of NaN3 (33 mg, 0.49 mmol) in H2O (0.50 mL) and
CH2Cl2 (0.50 mL) was treated with triflic anhydride (Tf2O; 42 mL,
0.25 mmol) dropwise and allowed to warm to RT, with continuous
stirring for another 2 h. A sat. aq solution of NaHCO3 (0.60 mL) was
added, and the mixture was extracted with CH2Cl2 (2ꢁ1 mL). This
solution of TfN3 was added dropwise to a mixture of the diamine
(14 mg, 0.049 mmol) with CuSO4·5H2O (2.0 mg, 0.007 mmol) in
MeOH (2 mL), H2O (0.50 mL) and Et3N (40 mL, 0.29 mmol) and
allowed to stir vigorously for 20 h. The solvents were partially
removed in vacuo (CAUTION: danger of explosion if the sample
reaches complete dryness) and the crude product was purified by
flash column chromatography on silica gel (hexanes!30% EtOAc/
hexanes) to furnish diazides 20 (14 mg, 88% yield); Rf =0.68 in
30% EtOAc/hexanes; 1H NMR (500 MHz, CDCl3): d=5.84 (m, 1H),
1
yield); Major isomer (17a): Rf =0.69 in 10% MeOH/CH2Cl2; H NMR
(500 MHz, CDCl3): d=5.90–5.85 (m, 3H), 5.78 (dd, J=11.3, 17.8 Hz,
1H), 5.58 (d, J=17.5 Hz, 1H), 5.40 (d, J=11.0 Hz), 5.21 (d, J=
17.0 Hz, 1H), 5.12 (d, J=10.0 Hz, 1H), 4.36 (dd, J=5.3, 13.3 Hz, 1H),
4.12 (dd, J=5.0, 13.5 Hz, 1H), 4.02–3.98 (m, 2H), 3.90 (t, J=9.8 Hz,
1H), 3.68 (d, J=9.0 Hz, 1H), 2.70–2.40 (m, 1H), 1.96 (m, 6H), 1.67–
1.44 ppm (m, 1H); 13C NMR (125 MHz, CDCl3): d=170.0, 169.3,
ChemBioChem 2011, 12, 71 – 87
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81

D. Vourloumis et al.
5.26–5.19 (m, 2H), 3.92 (t, J=9.7 Hz, 1H), 3.54 (td, J=4.2, 11.0 Hz,
1H), 3.19 (d, J=9.7 Hz, 1H), 3.13 (dd, J=4.3, 11.0 Hz, 2H), 2.59–
2.48 (m, 2H), 2.21 (m, 1H), 1.96 (q, J=11.9 Hz, 1H), 1.73–1.51 ppm
(m, 10H); 13C NMR (125 MHz, CDCl3): d=131.6, 120.6, 112.8, 79.1,
76.0, 73.9, 59.6, 57.8, 40.5, 36.3, 35.9, 30.8, 24.9, 23.6 ppm; IR (neat):
6.53 (brs, 1H), 6.31 (d, J=5.5 Hz, 1H), 5.78 (m, 1H), 4.65 (d, J=
13.0 Hz, 1H), 4.52 (d, J=13.0 Hz, 1H), 4.17 (m, 1H), 4.05 (m, 1H),
3.61 (m, 2H), 2.29 (m, 1H), 1.95 (s, 3H), 1.91 (s, 3H), 1.58–1.53 (m,
8H), 1.39–1.33 ppm (m, 3H); 13C NMR (125 MHz, CD3OD): d=
170.8, 170.7, 134.5, 122.5, 112.3, 94.0, 82.0, 77.8, 75.8, 52.6, 48.6,
36.7, 36.5, 36.0, 27.4, 25.4, 24.0, 23.8, 23.7 ppm; IR (neat): n˜ =3274,
2937, 1635, 1548, 1374, 1067, 1040, 914 cmꢀ1; HRMS-ESI: m/z:
calcd for C19H29N2O5: 365.2071 [M+H]+, found: 365.2069.
n˜ =3436, 2937, 2861, 2098, 1264, 1115, 921, 736 cmꢀ1
.
Allylation of tertiary alcohols 20 and synthesis of
(3aR,5S,7R,7aS)-4-allyl-4-(allyloxy)-5,7-diazidohexahydrospiro-
[benzo[d][1,3]dioxole-2,1’-cyclohexane] (21): At 08C, a solution of
homoallylic alcohol 20 (56 mg, 0.17 mmol) in dry DMF (3.5 mL) was
treated with NaH (9.4 mg, 0.23 mmol, 60% w/w), and the mixture
was allowed to stir for 15 min. This reaction mixture was cooled to
ꢀ108C, then allyl bromide (23 mL, 0.27 mmol) was added dropwise
and the temperature was slowly increased to 08C over 3 h. Sat. aq
NH4Cl (4 mL) was added, and the mixture was extracted with
EtOAc (3ꢁ10 mL), the organic solution dried (MgSO4), and the sol-
vents removed in vacuo. The crude product was purified by flash
column chromatography on silica gel (hexanes!20% EtOAc/hex-
anes) to furnish the diazide 21 (55 mg, 88% yield); Rf =0.53 in 10%
EtOAc/hexanes; 1H NMR (500 MHz, CDCl3): d=5.88 (m, 1H), 5.76
(m, 1H), 5.28 (d, J=10.3 Hz, 1H), 5.24 (d, J=10.0 Hz, 1H), 5.18 (d,
J=10.0 Hz, 1H), 5.13 (dd, J=1.4, 10.3 Hz, 1H), 4.45 (dd, J=5.0,
13.0 Hz, 1H), 3.96 (t, J=9.7 Hz, 1H), 3.51 (m, 1H), 3.24 (d, J=
9.7 Hz, 1H), 2.93 (dd, J=5.0, 11.0 Hz, 1H), 2.88 (dd, J=7.0, 13.0 Hz,
1H), 2.61 (dd, J=8.2, 13.0 Hz, 1H), 2.16–2.09 (m, 2H), 1.64–
1.41 ppm (m, 10H); 13C NMR (62 MHz, CDCl3): d=134.9, 131.8,
120.4, 115.4, 112.4, 79.3, 78.3, 76.3, 65.2, 58.8, 58.5, 36.2, 34.4, 30.2,
Ring-closing metathesis for the synthesis of N,N’-
((2’S,3aR,5S,7R,7aS)-2,2-cyclohexyl-3’,5,6,6’,7,7a-hexahydro-3aH-
spiro[benzo[d][1,3]dioxole-4,2’-pyran]-5,7-diyl)diacetamide (23)
and its 2’R isomer (26): According to the synthesis of 22 and 25,
spiro-olefins 23 (18 mg) and 26 (3.0 mg) were isolated from dienes
18 in 70% overall yield; Major isomer (23): Rf =0.46 in 10% MeOH/
CH2Cl2; [a]2D2 =ꢀ12.08 (c=0.2, CH2Cl2); 1H NMR (500 MHz, CDCl3):
d=5.94–5.79 (m, 2H), 5.76 (d, J=6.6 Hz, 1H), 5.71 (d, J=9.9 Hz,
1H), 4.60 (d, J=15.4 Hz, 1H), 4.09 (d, J=15.4 Hz, 1H), 4.04–3.86 (m,
3H), 3.38 (d, J=9.13 Hz, 1H), 2.39–2.24 (m, 2H), 1.98 (brs, 6H),
1.70–1.36 ppm (m, 12H); 13C NMR (125 MHz, CDCl3): d=169.9,
169.7, 124.6, 122.4, 111.8, 85.3, 74.8, 72.5, 64.0, 51.7, 49.0, 36.7, 36.1,
34.1, 28.6, 25.1, 23.9, 23.8, 23.6, 23.4 ppm; IR (neat): n˜ =3259, 2934,
2859, 1635, 1545, 1371, 1126, 1080, 733 cmꢀ1; HRMS-ESI: m/z: calcd
for C20H30N2NaO7: 401.2052 [M+Na]+, found 401.2050; Minor
isomer (26): Rf =0.29 in 10% MeOH/CH2Cl2; [a]2D2 =+3.08 (c=0.2,
1
CH2Cl2); H NMR (500 MHz, CDCl3): d=6.09 (d, J=7.8 Hz, 1H), 6.01
(d, J=6.1 Hz, 1H), 4.31 (m, 2H), 4.17 (m, 1H), 3.92 (m, 1H) 3.62–
3.52 (m, 2H), 2.57 (d, J=18.0 Hz, 1H), 2.41 (m, 1H), 2.08 (d, J=
18.0 Hz, 1H), 1.96 (s, 3H), 1.90 (s, 3H), 1.74–1.53 (m, 8H), 1.36 ppm
(m, 3H); 13C NMR (125 MHz, CDCl3): d=170.4, 169.8, 126.4, 122.0,
111.8, 83.5, 75.7, 73.8, 63.1, 54.3, 48.4, 36.4, 36.1, 35.8, 25.0, 23.6,
23.6, 23.4, 23.1, 22.1 ppm; IR (neat): n˜ =3287, 2935, 2861, 1653,
1558, 1448, 1375, 1133, 1089 cmꢀ1; HRMS-ESI (m/z) calcd for
C20H30N2NaO7: 401.2052 [M+Na]+, found: 401.2056.
24.9, 23.7 ppm; IR (neat): n˜ =2936, 2099, 1253, 1113, 916, 848 cmꢀ1
;
HRMS-ESI: m/z: calcd for C18H27N6O3: 375.2139 [M+H]+, found:
375.2136.
Ring-closing metathesis for the synthesis of N,N,’-
((2’S,3aR,5S,7R,7aS)-2,2-cyclohexyl-4’,5,6,7,7a,7’-hexahydro-
3aH,3’H-spiro[benzo[d][1,3]dioxole-4,2’-oxepine]-5,7-diyl)diaceta-
mide (22): Grubbs’ second-generation catalyst (16 mg,
0.019 mmol) was added to a stirred solution of diene 17a (76 mg,
0.19 mmol) in CH2Cl2 (39 mL) at RT, and the solution was allowed
to stir for 24 h (the color changed from green to dark brown). The
solvent was removed in vacuo and the crude product was purified
by flash chromatography on silica gel (CH2Cl2!10% MeOH/CH2Cl2)
to furnish spiro ether 22 (62 mg, 88% yield) as a light-brown solid;
Rf =0.31 in 7% MeOH/CH2Cl2; [a]2D2 =ꢀ34.08 (c=0.5, CH2Cl2);
1H NMR (500 MHz, CDCl3): d=6.21 (brs, 1H), 6.06 (d, J=6.0 Hz,
1H), 5.85 (d, J=9.0 Hz, 1H), 5.57 (d, J=6.0 Hz, 1H), 4.70 (d, J=
12.5 Hz, 1H), 4.65 (d, J=12.5 Hz, 1H), 4.28 (m, 1H), 4.03 (m, 1H),
3.88 (t, J=10.0 Hz, 1H), 3.50 (d, J=9.0 Hz, 1H), 2.34 (m, 1H), 1.98
(s, 3H), 1.91 (s, 3H), 1.60–1.24 ppm (m, 11H); 13C NMR (125 MHz,
CDCl3): d=170.4 (2C), 129.4, 127.2, 111.6, 92.2, 80.7, 76.9, 75.8,
49.7, 48.3, 36.6, 35.9, 34.7, 25.1, 23.7 (2C), 23.2, 22.9 ppm; IR (neat):
n˜ =3278, 2937, 1652, 1558, 1374, 1117, 1036 cmꢀ1; HRMS-ESI: m/z:
calcd for C19H29N2O5: 365.2071 [M+H]+, found: 365.2073.
Ring-closing metathesis for the synthesis of N,N’-
((2’S,3aR,5S,7R,7aS)-2,2-cyclohexyl-5,6,7,7a-tetrahydro-3aH,5’H-
spiro[benzo[d][1,3]dioxole-4,2’-furan]-5,7-diyl)diacetamide (24):
According to the synthesis of 22 and 25, spiro-olefin 24 (17 mg,
60% yield) was isolated as a dark-yellow solid from dienes 19. The
minor isomer could not be isolated due to its very small amount;
1
Rf =0.25 in 10% MeOH/CH2Cl2; H NMR (500 MHz, CDCl3): d=6.19
(d, J=9.5 Hz, 1H), 5.97 (d, J=7.0 Hz, 1H), 5.81 (brs, 1H), 5.72 (brs,
1H), 4.17 (dd, J=5.0, 16.0 Hz, 2H), 4.13–4.06 (m, 1H), 4.00 (t, J=
10.0 Hz, 1H), 3.83 (dd, J=6.5, 13.5 Hz, 1H), 3.50 (d, J=9.0 Hz, 1H),
2.78 (appt, J=13.0 Hz, 1H), 2.65 (appt, J=14.0 Hz, 1H), 2.45–2.35
(m, 1H), 2.34–2.28 (m, 2H), 2.08 (s, 3H), 2.07 (s, 3H), 1.98–1.94 (dd,
J=4.0, 15.0 Hz, 1H), 1.88–1.80 (m, 1H), 1.80–1.62 (m, 8H), 1.63–
1.54 ppm (m, 1H); 13C NMR (125 MHz, CDCl3): d=170.2, 169.4,
131.4, 129.2, 111.8, 84.2, 75.4, 64.5, 54.0, 49.0, 36.9, 36.4, 35.8, 33.8,
26.3, 25.4, 24.1, 23.9, 23.8 ppm; HRMS-ESI: m/z: calcd for
C21H32N2NaO5: 415.2209 [M+Na]+, found: 415.2205.
Ring-closing metathesis for the synthesis of N,N’-
((2’R,3aR,5S,7R,7aS)-2,2-cyclohexyl-5,6,7,7a-tetrahydro-3aH,5’H-
spiro[benzo[d][1,3]dioxole-4,2’-furan]-5,7-diyl)diacetamide (25):
Grubbs’ second-generation catalyst (9.5 mg, 0.011 mmol) was
added to a stirred solution of diene 17b (44 mg, 0.11 mmol) in
CH2Cl2 (23 mL) at RT, and the solution was allowed to stir for 18 h
(color change from green to dark brown). The solvent was re-
moved in vacuo and the crude product was purified by flash chro-
matography on silica gel with a gradient elution system (CH2Cl2!
10% MeOH/CH2Cl2) to furnish spiro ether 25 (28 mg, 69% yield) as
a light-brown solid; Rf =0.32 in 7% MeOH/CH2Cl2; [a]2D2 =ꢀ12.98
(c=1.0, CH2Cl2); 1H NMR (500 MHz, CD3OD): d=7.33 (brs, 1H),
Cleavage of the ketal and synthesis of N,N’-((5S,6S,8R,9S,10R)-
9,10-dihydroxy-1-oxaspiro[4.5]dec-3-ene-6,8-diyl)diacetamide
(27): A solution of spiro-olefin 22 (30 mg, 0.082 mmol) in a mixture
of HOAc/THF/H2O (3:3:1 v/v/v, 17 mL) was heated to 458C for 3 h.
The solvent was removed in vacuo, and the crude product was
purified by flash chromatography on silica gel (5!20% MeOH/
CH2Cl2) to furnish diol 27 (22 mg, 94% yield) as a white solid; Rf =
1
0.23 in 15% MeOH/CH2Cl2; [a]2D2 =ꢀ51.78 (c=1.0, MeOH); H NMR
(500 MHz, CD3OD): d=7.80 (d, J=9.5 Hz, 1H), 6.02 (d, J=6.0 Hz,
1H), 5.52 (m, 1H), 4.71–4.64 (m, 2H), 4.06 (m, 1H), 3.77 (m, 1H),
3.53 (t, J=10.0 Hz, 1H), 3.42 (d, J=9.5 Hz, 1H), 1.95 (s, 3H), 1.86 (s,
3H), 1.81 (dt, J=4.4, 12.4 Hz, 1H), 1.72 ppm (q, J=12.4 Hz, 1H);
82
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 71 – 87

Designed Spiro-Bicyclic Analogues
13C NMR (125 MHz, CD3OD): d=171.9, 171.2, 128.5, 127.6, 95.2,
76.6, 75.3, 73.3, 50.2, 49.2, 32.0, 21.3, 21.0 ppm; IR (neat): n˜ =3267,
2932, 1627, 1546, 1373, 1084, 1018, 741 cmꢀ1; HRMS-ESI: m/z:
calcd for C13H21N2O5: 285.1445 [M+H]+, found: 285.1444.
Rf =0.62 in 25% MeOH/CH2Cl2; [a]2D2 =+11.88 (c=0.12, MeOH);
1H NMR (500 MHz, D2O): d=3.87 (m, 2H), 3.40 (t, J=10.0 Hz, 1H),
3.27 (d, J=9.5 Hz, 1H), 2.95–2.92 (m, 2H), 2.09 (m, 1H), 1.96–1.88
(m, 3H), 1.80 (m, 1H), 1.52 ppm (q, J=12.5 Hz, 1H); 13C NMR
(125 MHz, D2O): d=86.4, 76.4, 73.1, 71.6, 51.9, 50.7, 32.5, 30.8,
Cleavage of the ketal and synthesis of N,N’-((6S,7S,9R,10S,11R)-
10,11-dihydroxy-1-oxaspiro[5.5]undec-3-ene-7,9-diyl)diaceta-
mide (28): According to the synthesis of 27, diol 28 was isolated
as a white solid (28 mg, 95% yield); Rf =0.63 in 30% MeOH/CH2Cl2;
26.0 ppm; IR (neat): n˜ =3258, 2890, 1635, 1540, 1100, 616 cmꢀ1
;
HRMS-ESI: m/z: calcd for C9H19N2O3: 203.1390 [M+H]+, found:
203.1391.
1
[a]2D2 =ꢀ54.48 (c=0.36, MeOH); H NMR (500 MHz, CDCl3): d=6.23
Synthesis of N-((6S,7S,9R,10S,11R)-9-amino-10,11-dihydroxy-1-
oxaspiro[5.5]undecan-7-yl)acetamide (33): According to the se-
quence described for 32, 6-membered spirocyclic analogue was
isolated as a colorless solid (8.6 mg, 73% yield); Rf =0.41 in 15%
MeOH/CH2Cl2; [a]2D2 =ꢀ6.88 (c=0.5, MeOH); 1H NMR (500 MHz,
CD3OD): d=4.27 (m, 1H), 3.74–3.68 (m, 2H), 3.61 (t, J=9.8 Hz, 1H),
3.34–3.27 (m, 2H), 2.21–2.10 (m, 2H), 1.97 (s, 3H), 1.94 (s, 3H),
1.78–1.50 ppm (m, 6H); 13C NMR (62.9 MHz, CD3OD): d=171.9,
171.4, 81.6, 74.4, 72.7, 64.0, 51.5, 50.5, 31.9, 29.1, 24.7, 21.3, 21.1,
19.7 ppm; IR (neat): n˜ =3289, 2940, 1637, 1431, 1204, 1138, 1059,
(d, J=5.1 Hz, 1H), 6.00 (d, J=4.7 Hz, 1H), 5.92–5.76 (m, 2H), 4.61
(d, J=15.7 Hz, 1H), 4.10 (d, J=15.5 Hz, 1H), 3.94–3.76 (m, 2H), 3.71
(dd, J=9.5, 9.5 Hz, 1H), 3.47–3.67 (m, 1H), 2.30–2.15 (m, 2H), 1.99
(s, 3H), 1.98 (s, 3H), 1.88 (m, 1H), 1.70 ppm (ddd, J=12.1, 12.1,
12.2 Hz, 1H); 13C NMR (125 MHz, CDCl3): d=172.2, 170.1, 125.2,
123.6, 81.3, 74.0, 73.7, 63.8, 51.8, 51.1, 32.5, 28.5, 23.5, 23.4 ppm; IR
(neat): n˜ =3285, 3078, 2927, 1696, 1635, 1558, 1541, 1086,
764 cmꢀ1
; HRMS-ESI: m/z: calcd for C14H22N2NaO5: 321.1426
[M+Na]+, found: 321.1421.
723 cmꢀ1
; HRMS-ESI: m/z: calcd for C14H24N2NaO5: 323.15774
Cleavage of the ketal and synthesis of N,N’-((5R,6S,8R,9S,10R)-
9,10-dihydroxy-1-oxaspiro[4.5]dec-3-ene-6,8-diyl)diacetamide
(30): According to the synthesis of 27, diol 30 (17 mg, 73% yield)
was isolated as a white solid; Rf =0.18 in 20% MeOH/CH2Cl2;
1H NMR (500 MHz, CD3OD): d=6.19 (d, J=6.5 Hz, 1H), 5.63 (m,
1H), 4.55 (d, J=12.6 Hz, 1H), 4.39 (d, J=12.6 Hz, 1H), 4.01 (dd, J=
3.9, 13.2 Hz, 1H), 3.71 (m, 1H), 3.38 (d, J=9.7 Hz, 1H), 3.26 (t, J=
10.0 Hz, 1H), 1.84 (s, 3H), 1.82 (m, 1H), 1.69 (s, 3H), 1.33 ppm (q,
J=12.8 Hz, 1H); 13C NMR (63 MHz, CD3OD): d=173.4, 173.0, 134.5,
123.7, 97.5, 79.1, 77.1, 75.3, 52.2, 52.1, 34.5, 23.0, 22.9 ppm; HRMS-
ESI: m/z: calcd for C13H20N2NaO5: 307.1270 [M+Na]+, found:
307.1272.
[M+Na]+, found: 323.15754.
A solution of the acetate (7 mg, 0.02 mmol) and KOH (92 mg,
1.6 mmol) in H2O (1.7 mL) was heated in a sealed pressurized tube
to 1408C for 16 h, and afterwards the solvent was evaporated. The
crude product was purified by column chromatography on silica
gel (MeOH!25% NH4OH/MeOH) to obtain amine 33 as a colorless
solid. (2.0 mg, 40%); Rf =0.48 in 25% NH4OH/MeOH; [a]2D2 =ꢀ6.98
1
(c=0.072, H2O); H NMR (500 MHz, D2O): d=4.07 (m, 1H), 3.86 (m,
1H), 3.76 (dd, J=4.5, 17.0 Hz, 1H), 3.55 (t, J=9.5 Hz, 1H), 3.35 (m,
1H), 2.85 (m, 1H), 2.07 (s, 3H), 1.78–1.57 ppm (m, 8H); 13C NMR
(125 MHz, D2O): d=166.1, 80.3, 76.1, 74.7, 65.4, 53.0, 51.8, 32.6,
29.4, 24.7, 22.7, 19.8 ppm; IR (neat): n˜ =3372, 2934, 1653, 1588,
1472, 1356, 1371 1083, 1056 cmꢀ1; HRMS-ESI: m/z: calcd for
C12H23N2O4: 259.16523 [M+H]+, found: 259.16507.
Cleavage of the ketal and synthesis of N,N’-((6R,7S,9R,10S,11R)-
10,11-dihydroxy-1-oxaspiro[5.5]undec-3-ene-7,9-diyl)diaceta-
mide (31): According to the synthesis of 27, diol 31 was isolated
as a white solid (7.0 mg, 85% yield); Rf =0.18 in 20% MeOH/
CH2Cl2; [a]2D2 =ꢀ2.88 (c=0.7, MeOH); 1H NMR (250 MHz, CD3OD):
d=5.86 (m, 1H), 5.76 (m, 1H), 4.23 (m, 2H), 4.18 (dd, J=4.4,
12.8 Hz, 1H), 3.75 (m, 1H), 3.37 (d, J=9.9 Hz, 1H), 3.23 (d, J=
9.9 Hz, 1H), 2.54 (dt, J=2.7, 18.0 Hz, 1H), 2.06 (m, 1H), 1.94 (s, 3H),
1.87 (s, 3H), 1.83 (dt, J=4.6, 13.0 Hz, 1H), 1.46 ppm (q, J=13.0 Hz,
1H); 13C NMR (63 MHz, CD3OD): d=175.4, 172.6, 126.5, 123.8,
80.47, 76.7, 73.7, 63.9, 54.2, 51.8, 34.8, 23.1, 22.9, 22.6 ppm; IR
Synthesis of (5S,6R,7S,8R,10S)-8,10-diamino-1-oxaspiro[4.5]dec-
3-ene-6,7-diol (34): A solution of 27 (10 mg, 0.037 mmol) and KOH
(0.14 g, 2.6 mmol) in H2O (2.8 mL) was heated to 1408C for 48 h.
The solvent was removed in vacuo and the crude product was pu-
rified by flash chromatography on silica gel (MeOH!17% NH4OH/
MeOH) to obtain spiro ether 34 as a white solid. (3.6 mg, 50%
yield): Rf =0.48 in 25% NH4OH/MeOH; [a]2D2 =+6.58 (c=0.34,
MeOH); 1H NMR (500 MHz, D2O): d=6.15 (d, J=5.5 Hz, 1H), 5.53
(m, 1H), 4.61 (m, 2H), 3.38 (d, J=9.5 Hz, 1H), 3.29 (t, J=10.0 Hz,
1H), 2.90 (dd, J=4.0, 12.5 Hz, 1H), 2.74 (m, 1H), 1.88 (m, 1H),
1.37 ppm (q, J=12.5 Hz, 1H); 13C NMR (62.5 MHz, D2O): d=131.1,
126.5, 95.6, 77.7, 75.0, 74.5, 50.6, 50.5, 33.7 ppm; IR (neat): n˜ =3348,
2860, 1570, 1472, 1379, 1109, 1076, 990, 744 cmꢀ1; HRMS-ESI: m/z:
calcd for C9H17N2O3: 201.1234 [M+H]+, found: 201.1236.
(neat): n˜ =3289, 2921, 1643, 1553, 1095, 1036 cmꢀ1
.
Synthesis of (5S,6R,7S,8R,10S)-8,10-diamino-1-oxaspiro[4.5]dec-
ane-6,7-diol (32): Palladium on activated C (10 wt%, 2.0 mg,
0.0015 mmol) was added to a solution of 27 (8.5 mg, 0.030 mmol)
in EtOAc/MeOH (2.2 mL, 10:1, v/v). The suspension was stirred
under an H2 atmosphere at RT for 18 h and then filtered trough
Celite. The solvent was evaporated and the hydrogenated diace-
tate (8.5 mg, quant.) was obtained without further purification as a
white solid; Rf =0.49 in 25% MeOH/CH2Cl2; [a]2D2 =ꢀ20.98 (c=0.8,
Synthesis of (6S,7R,8S,9R,11S)-9,11-diamino-1-oxaspiro[5.5]un-
dec-3-ene-7,8-diol (35): A solution of the acetate 28 (21 mg,
0.070 mmol) and KOH (0.27 g, 4.9 mmol) in H2O (5.0 mL) was
heated in a sealed tube at 1508C for 24 h, then the solvent was
evaporated. The crude product was purified by column chroma-
tography on silica gel (MeOH!25% NH4OH/MeOH) to obtain dia-
mine 35 as a colorless solid. (7.0 mg, 47%); Rf =0.57 in 25%
1
MeOH); H NMR (500 MHz, CD3OD): d=3.99–3.92 (m, 3H), 3.72 (td,
J=4.0, 12.0 Hz, 1H), 3.49 (t, J=10.0 Hz, 1H), 3.25 (d, J=9.5 Hz,
1H), 2.12 (m, 1H), 1.94 (m, 7H), 1.80–1.71 ppm (m, 3H), 1.66 ppm
(q, J=12.3 Hz, 1H); 13C NMR (125 MHz, CD3OD): d=171.8, 171.3,
86.4, 77.5, 73.6, 70.3, 50.3 (2C), 32.9, 30.5, 26.2, 21.3, 21.0 ppm; IR
(neat): n˜ =3276, 1653, 1559, 1375, 1086 cmꢀ1
.
1
NH4OH/MeOH; [a]2D2 =ꢀ2.08 (c=0.5, H2O); H NMR (500 MHz, D2O):
A solution of the diacetate (8.5 mg, 0.030 mmol) and KOH (0.12 g,
2.1 mmol) in H2O (2.3 mL) was heated to 1408C for 48 h. The sol-
vent was removed in vacuo, and the crude product was purified
by flash chromatography on silica gel (MeOH!20% NH4OH/
MeOH) to obtain spiro ether 32 as a white solid (1.3 mg, 21%);
d=5.83 (m, 2H), 4.33 (d, J=15.5 Hz, 1H), 4.14 (d, J=15.0 Hz, 1H),
3.72 (t, J=10.0 Hz, 1H), 3.27 (d, J=9.0 Hz, 1H), 3.19 (m, 1H), 3.08
(m, 1H), 2.33 (d, J=18.0 Hz, 1H), 2.17 (d, J=17.5 Hz, 1H), 2.06 (m,
1H), 1.92 ppm (m, 1H); 13C NMR (125 MHz, D2O): d=126.3, 122.3,
77.9, 73.0, 69.7, 63.3, 53.3, 50.5, 27.6, 27.0 ppm; IR (neat): n˜ =3245,
ChemBioChem 2011, 12, 71 – 87
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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83

D. Vourloumis et al.
uct 40 (40 mg, 78% yield); Rf =0.50 in 20% MeOH/CH2Cl2; [a]D22
ꢀ32.08 (c=0.7, MeOH); 1H NMR (500 MHz, CDCl3): d=6.00 (m,
1H), 5.52 (m, 1H), 4.41 (m, 1H), 4.28 (d, J=12.1 Hz, 1H), 4.02 (m,
1H), 3.91–3.81 (m, 4H), 3.35 (d, J=9.3 Hz, 1H), 2.25 (m, 1H), 2.02
(s, 3H), 1.98 (s, 3H), 1.80–1.38 ppm (m, 13H); 13C NMR (63 MHz,
CD3OD): d=173.2, 172.9, 112.6, 86.6, 79.9, 76.7, 69.6, 69.2, 67.0,
53.4, 37.6, 37.2, 34.4, 33.6, 26.2, 25.0, 22.8, 22.6 ppm; IR (neat): n˜ =
3272, 2922, 2855, 1653, 1522, 1456, 1084, 801, 620 cmꢀ1; HRMS-ESI:
m/z: calcd for C20H32N2NaO7: 435.2107 [M+Na]+, found: 435.2111.
1626, 1525, 1437, 1202, 1086, 616 cmꢀ1; HRMS-ESI: m/z: calcd for
C10H19N2O3: 215.13902 [M+H]+, found: 215.13890.
=
Cleavage of the ketal and hydrolysis for the synthesis of
(1R,2S,3R,5S,6S)-3,5-diamino-7-oxaspiro[5.6]dodec-9-ene-1,2-diol
(36): According to the synthesis of 27, diol 29 (28 mg, 75% yield,
Rf =0.48 in 10% MeOH/CH2Cl2) was isolated as a white solid and
immediately used for the next step. According to the hydrolysis
step described for 32, 7-membered unsaturated analogue 36 was
isolated after flash column chromatography on silica gel (MeOH!
30% NH4OH/MeOH) as a white solid (14 mg, 53% yield); Rf =0.73
Synthesis of final product (3S,4R,6S,7R,8S,9R,11S)-9,11-diamino-
1-oxaspiro[5.5]undecane-3,4,7,8-tetraol (2): The tetraol diaceta-
mide 39 (6.0 mg, 0.024 mmol) was dissolved in H2O (2.0 mL) and
KOH(s) (0.10 g, 1.7 mmol) was added. The mixture was warmed at
1308C for 15 h and the solvent was removed in vacuo. The crude
product was purified by flash column chromatography on silica gel
(10!50% NH4OH/MeOH) to furnish the final product diamine 2, in
the form of a white solid (4.0 mg, 90% yield); Rf =0.31 in 30%
NH4OH/MeOH; [a]2D2 =ꢀ4.68 (c=0.4, MeOH); 1H NMR (500 MHz,
D2O): d=4.52 (brs, 1H), 4.25 (d, J=11.0 Hz, 1H), 3.87–3.74 (m, 2H),
3.83 (dd, J=9.9, 9.9 Hz, 1H), 3.56–3.43 (m, 2H), 3.31–3.22 (m, 1H),
2.32–2.26 (m, 1H), 2.12 (ddd, J=12.0, 12.2, 12.3 Hz, 1H), 2.05–1.95
(m, 1H), 1.91–1.82 ppm (m, 1H); 13C NMR (125 MHz, D2O): d=78.8,
69.9, 67.8, 66.4, 65.2, 52.5, 50.4, 31.6, 27.7 ppm; IR (neat): n˜ =3440,
1
in 10% NH4OH/MeOH; [a]D22 =+0.78 (c=1.1, MeOH); H NMR (D2O;
500 MHz): d=5.74 (brs, 1H), 5.60 (brs, 1H), 4.31 (d, J=16.4 Hz,
1H), 4.21 (d, J=16.4 Hz, 1H), 4.04 (brs, 1H), 3.87 (brs, 1H), 3.73
(brs, 1H), 3.55 (brs, 1H), 3.46 (brs, 1H), 3.28 (brs, 1H), 2.20 (brs,
1H), 2.15 (d, J=14.5 Hz, 2H), 2.07–2.03 (m, 1H), 1.95–1.89 (m, 1H),
1.86–1.81 (m, 2H), 1.53 ppm (brs, 1H); 13C NMR (D2O, 125 MHz):
d=131.8, 128.3, 78.4, 73.1, 72.3, 60.5, 48.7, 47.3, 33.0, 23.6,
20.8 ppm; HRMS-ESI: m/z: calcd for C11H20N2O3: 228.1474 [M+H]+,
found: 228.1471.
Synthesis of (1R,2S,3R,5S,6S)-3,5-diamino-7-oxaspiro[5.6]do-
decane-1,2-diol (37): Hydrolysis of 29 followed by hydrogenation,
according to the procedure described for 32, produced 7-mem-
bered unsaturated analogue 37, which was isolated after flash
column chromatography on silica gel (MeOH!30% NH4OH/MeOH)
as a white amorphous solid. (21 mg, 69% yield); Rf =0.71 in 10%
NH4OH/MeOH; [a]2D2 =ꢀ0.68 (c=2.1, MeOH); 1H NMR (500 MHz,
D2O): d=4.12 (t, J=11.6 Hz, 1H), 3.65 (t, J=9.9 Hz, 1H), 3.41–3.03
(m, 3H), 2.37–2.15 (m, 1H), 2.10–1.87 (m, 2H), 1.82 (brs, 2H), 1.78–
1.74 (d, J=11.6 Hz, 1H), 1.70–1.29 (m, 3H), 1.24–1.09 ppm (m, 2H);
13C NMR (125 MHz, D2O): d=77.2, 76.8, 70.4, 67.6, 55.4, 50.7, 33.5,
31.7, 29.9, 28.0, 25.1 ppm; HRMS-ESI: m/z: calcd for C11H22N2O3:
230.1630 [M+H]+, found: 230.1627.
3166, 2954, 1645, 1042, 738 cmꢀ1
; HRMS-ESI: m/z: calcd for
C10H20N2NaO5: 271.1270 [M+Na]+, found: 271.1266.
Upjohn dihydroxylation of minor six-membered spiro analogue
31 and synthesis of N,N’-((6R,7S,9R,10S,11R)-3,4,10,11-tetrahy-
droxy-1-oxaspiro[5.5]undecane-7,9-diyl)diacetamide (41): N-
Methylmorpholine N-oxide (6 mg, 0.05 mmol) and then OsO4
(2.5 wt% in tBuOH, 17 mL, 1.7ꢁ10ꢀ3 mmol, 9 mol%) were added to
a stirred solution of the minor olefin (5 mg, 0.02 mmol) in a mix-
ture of acetone/H2O (1:1 (v/v), 2.0 mL), and the solution was al-
lowed to stir at 08C for 4 h. The solvent was removed in vacuo,
and the crude product was purified by flash column chromatogra-
phy on silica gel (CH2Cl2!50% MeOH/CH2Cl2) to furnish the white
crystalline mixture of diastereomeric products (2:1 dr; 4.6 mg, 82%
overall yield). 41 major product: Rf =0.25 in 40% MeOH/CH2Cl2;
1H NMR (500 MHz, CD3OD): d=4.25 (m, 1H), 3.93 (m, 1H), 3.85–
3.72 (m, 4H), 3.68 (m, 2H), 3.35 (m, 2H), 2.14 (dd, J=4.7, 14.3 Hz,
1H), 1.99 (dt, J=4.5, 12.0 Hz, 1H), 1.94 (s, 3H), 1.93 (s, 3H), 1.82
(dd, J=6.9, 14.3 Hz, 1H), 1.34 ppm (q, J=12.0 Hz, 1H).
Upjohn dihydroxylation of olefins 28 and synthesis of N,N’-
((3S,4R,6S,7S,9R,10S,11R)-3,4,10,11-tetrahydroxy-1-oxaspiro[5.5]
undecane-7,9-diyl)diacetamide (38) and its 3R,4S-isomer (39): N-
Methylmorpholine N-oxide (11 mg, 0.094 mmol) was added to a so-
lution of olefin 28 (10 mg, 0.033 mmol) in acetone/H2O (1:1 (v/v),
2 mL), then OsO4 (2.5 wt% in tBuOH, 0.035 mL, 3.4ꢁ10ꢀ3 mmol,
10 mol%) was added, and the solution was stirred at RT for 3 h.
The solvent was removed in vacuo, and the crude residue was pu-
rified by flash column chromatography on silica gel (CH2Cl2!30%
MeOH/CH2Cl2) to furnish tetraols 38 and 39 (1:9 dr; 9.8 mg, 90%
Synthesis of final epimeric products (6R,7S,9R,11S)-9,11-diami-
no-1-oxaspiro[5.5]dodecane-3,4,7,8-tetraol (42): Diacetamides 41
(4.0 mg, 0.012 mmol) were dissolved in H2O (2.0 mL), and KOH(s)
(0.12 g, 2.1 mmol) was added. The mixture was warmed at 1208C
for 17 h, and the solvent was removed in vacuo. The crude product
was purified by flash column chromatography on silica gel (10!
50% NH4OH/MeOH) to furnish the final diastereomeric diamines
42 (2:1 dr), as a white solid. (2.5 mg, 85% yield); 42 major product:
Rf =0.32 in 30% NH4OH/MeOH; 1H NMR (500 MHz, D2O): d=4.41
(m, 1H), 4.27 (d, J=12.1 Hz, 1H), 3.86 (d, J=12.1 Hz, 1H), 3.81 (m,
1H), 3.59–3.51 (m, 2H), 3.22–3.18 (m, 2H), 2.21 (m, 1H), 1.78–1.71
(m, 2H), 1.64 ppm (t, J=12.4 Hz, 1H); 13C NMR (63 MHz, D2O): d=
81.5, 78.3, 73.4, 70.0, 68.2, 66.6, 54.8, 52.1, 28.0, 27.0 ppm; HRMS-
ESI: m/z: calcd for C10H20N2NaO5: 271.1270 [M+Na]+, found:
271.1265.
1
overall yield); 38 (minor): H NMR (500 MHz, CD3OD): d=4.11 (m,
1H), 4.06–3.94 (m, 2H), 3.90–3.74 (m, 2H), 3.71 (m, 1H), 3.44 (m,
2H), 2.23 (m, 1H), 2.09 (m, 1H), 1.99 (s, 3H), 1.98 (s, 3H), 1.81 (m,
1H), 1.61 ppm (m, 1H).
39 (major): Rf =0.27 in 30% MeOH/CH2Cl2; 1H NMR (500 MHz,
CD3OD): d=4.50 (m, 1H), 4.44 (d, J=11.8 Hz, 1H), 3.83–3.69 (m,
5H), 3.59 (dd, J=9.9, 9.9 Hz, 1H), 1.98 (s, 3H), 1.95 (s, 3H), 1.91 (m,
1H), 1.80 (dd, J=4.7, 4.7 Hz, 1H), 1.71 (m, 1H), 1.63 ppm (dd, J=
5.9, 12.9 Hz, 1H).
Upjohn dihydroxylation of olefin 23 and synthesis of N,N’-
((2’S,3aR,4’S,5S,5’R,7R,7aS)-2,2-cyclohexyl-4’,5’-dihydroxyoctahy-
dro-3aH-spiro[benzo[d][1,3]dioxole-4,2’-pyran]-5,7-diyl)diaceta-
mide (40): N-Methylmorpholine N-oxide (44 mg, 0.37 mmol), and
then OsO4 (2.5 wt% in tBuOH, 0.12 mL, 0.012 mmol) were added to
a stirred solution of olefin 23 (47 mg, 0.12 mmol) in a mixture of
acetone/H2O (1:1 (v/v), 8 mL), and the solution was allowed to stir,
at 08C, for 3 h. The solvent was removed in vacuo, and the crude
product was purified by flash column chromatography on silica gel
(CH2Cl2!30% MeOH/CH2Cl2) to furnish the white crystalline prod-
Synthesis of N,N’-((3S,4S,5S,6S,8R,9S,10R)-3,4,9,10-tetrahydroxy-
1-oxaspiro[4.5]decane-6,8-diyl)diacetamide (44): A solution of 27
(11 mg, 0.039 mmol) and TMEDA (6.9 mL, 0.046 mmol) in CH2Cl2
(4.8 mL) was cooled to ꢀ788C, and OsO4 (2.5 wt% in tBuOH,
0.58 mL, 0.046 mmol) was added. The solution was stirred for 3 h
84
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 71 – 87

Designed Spiro-Bicyclic Analogues
while slowly warming to RT. The solvent was removed in vacuo,
and the crude product was purified by flash column chromatogra-
phy on silica gel (CH2Cl2!20% MeOH/CH2Cl2) to furnish osmate
ester 43 (19 mg, 75% yield) as a yellow solid; Rf =0.13 in 7%
MeOH/CH2Cl2.
1.69 (m, 4H), 1.68–1.50 (m, 18H), 1.49–1.31 ppm (m, 4H); 13C NMR
(125 MHz, CDCl3): d=171.9, 171.8, 171.7, 171.6, 111.3, 84.0, 83.8,
77.9, 75.5, 75.2, 73.6, 72.7, 71.7, 65.2, 64.7, 54.7, 54.0, 48.0, 36.4,
36.3, 34.5, 34.2, 29.1, 26.8, 26.3, 25.1 (2C), 23.8 (2C), 21.8,
21.7 ppm; HRMS-ESI: m/z: calcd for C21H34N2NaO7: 449.2264
[M+Na]+, found: 449.2263.
The product was dissolved in MeOH (1.0 mL), and concd HCl (1
drop) was added. The solution was stirred for 15 min until a yellow
precipitate was observed. The solvent was removed in vacuo, and
the crude product was purified by flash chromatography on silica
gel (20!50% MeOH/CH2Cl2) to furnish diol 44 (9.2 mg, quant.) as
a white solid; Rf =0.52 in 50% MeOH/CH2Cl2; [a]2D2 =+65.08 (c=0.2,
MeOH); 1H NMR (500 MHz, CD3OD): d=7.93 (d, J=9.2 Hz, 1H),
4.13–4.08 (m, 3H), 3.96 (m, 1H), 3.88 (d, J=10.0 Hz, 1H), 3.71 (m,
1H), 3.56–3.50 (m, 2H), 1.95 (s, 6H), 1.81 (m, 1H), 1.65 ppm (q, J=
12.5 Hz, 1H); 13C NMR (125 MHz, CD3OD): d=172.1, 171.9, 85.9,
74.3, 73.6, 73.3, 72.9, 71.4, 49.8, 48.7, 32.9, 21.3, 21.1 ppm; IR (neat):
n˜ =3363, 1635, 1559, 1376, 1036, 527 cmꢀ1; HRMS-ESI: m/z: calcd
for C13H23N2O7: 319.1500 [M+H]+, found: 319.1498.
Removal
of
the
ketal
and
synthesis
of
N,N’-
((1S,3R,4S,5R,6S,9S,10R)-4,5,9,10-tetrahydroxy-7-oxaspiro[5.6]do-
decane-1,3-diyl)diacetamide (48) and its 9R,10S isomer (49): The
aforementioned diols 46–47 (23 mg, 0.054 mmol) were dissolved
in a mixture of TFA/THF/H2O (3:3:1 (v/v/v), 0.6 mL) and the mixture
was stirred at 508C for 18 h. The mixture was then concentrated
under reduced pressure, and the residue was purified by flash
column chromatography on silica gel (10%!30% MeOH/CH2Cl2)
to afford the corresponding tetraols 48–49 (14 mg, 75% yield) as a
white solid (2:3 mixture of stereoisomers); Rf =0.63 in 30% MeOH/
CH2Cl2; 1H NMR (500 MHz, CD3OD): d=4.55 (dd, J=4.0, 13.5 Hz,
1H; major), 4.29 (dd, J=8.0, 12.0 Hz, 1H; minor), 3.85 (dd, J=3.5,
12.0 Hz, 1H), 3.78–3.63 (m, 2H), 3.61–3.53 (m, 2H), 3.35–3.30 (m,
1H), 3.26 (d, J=9.5 Hz, 1H; major), 3.17 (d, J=9.5 Hz, 1H; minor),
2.41–2.19 (m, 1H), 2.16–2.03 (m, 1H), 1.97 (d, J=4.5 Hz, 3H), 1.95
(s, 3H), 1.82–1.70 (m, 2H), 1.70–1.55 (m, 4H), 1.34–1.17 ppm (m,
2H); 13C NMR (125 MHz, CD3OD): d=172.3, 171.3, 79.1, 75.8, 73.7,
73.3, 73.0, 65.2, 54.2, 50.7, 33.0, 28.9, 27.3, 21.8, 21.7 ppm; HRMS-
ESI: m/z: calcd for C15H26N2NaO7: 369.1638 [M+Na]+, found:
369.1633.
Synthesis of final product (3S,4S,5S,6R,7S,8R,10S)-8,10-diamino-
1-oxaspiro[4.5]decane-3,4,6,7-tetraol (1): Tetraol diacetamide 44
(12 mg, 0.036 mmol) was dissolved in H2O (2.8 mL) and KOH(s)
(0.14 g, 2.5 mmol) was added. The mixture was warmed at 1408C
for 48 h and the solvent was removed in vacuo. The crude product
was purified by flash column chromatography on silica gel
(MeOH!25% NH4OH/MeOH) to furnish the final product diamine
1, as a white solid (1.7 mg, 20% yield); Rf =0.46 in 25% NH4OH/
MeOH; 1H NMR (500 MHz, D2O): d=4.45 (d, J=5.5 Hz, 1H), 4.21
(m, 1H), 3.95–3.90 (m, 2H), 3.58–3.55 (m, 2H), 3.33–3.29 (m, 1H),
3.15 (m, 1H), 2.21 (m, 1H), 1.74 ppm (m, 1H); 13C NMR (125 MHz,
D2O): d=77.7, 75.0, 72.4, 71.3, 71.2, 70.4, 50.0, 49.8, 30.2 ppm; IR
(neat): n˜ =3222, 1627, 1436, 1185, 1048, 864, 585 cmꢀ1; HRMS-ESI:
m/z: calcd for C9H19N2O5: 235.1289 [M+H]+, found: 235.1289.
Synthesis of (1R,2S,3R,5S,6S,9R,10S)-3,5-diamino-7-oxaspiro[5.6]
dodecane-1,2,9,10-tetraol (3) and its 9S,10R isomer (50): Tetraols
48--49 (6.0 mg, 0.024 mmol) were dissolved in H2O (2.0 mL), KOH
(0.10 mg, 1.7 mmol) was added, and the mixture was stirred for
14 h at 1208C. Subsequently, the reaction mixture was concentrat-
ed under reduced pressure and purified by flash column chroma-
tography on silica gel (10%!30% NH4OH/MeOH) to afford 50
(2.3 mg, 28% yield) and 3 (3.4 mg, 42% yield) as white solids.
Synthesis of (5R,6R,7S,8R,10S)-8,10-diamino-1-oxaspiro[4.5]dec-
3-ene-6,7-diol (45): According to the hydrolysis step described for
32, 5-membered unsaturated analogue 45 was isolated (5 mg,
quant.). Rf =0.15 in 30% NH4OH/MeOH; 1H NMR (500 MHz, D2O):
d=6.37 (d, J=6.2 Hz, 1H), 5.75 (m, 1H), 4.71 (m, 2H), 3.71 (m, 1H),
3.48 (d, J=10.0 Hz, 1H), 3.23 (t, J=9.8 Hz, 1H), 2.92 (d, J=12.3 Hz,
1H), 2.81 (m, 1H), 2.01 (m, 1H), 1.38 ppm (q, J=12.7 Hz, 1H);
13C NMR (125 MHz, D2O): d=133.4, 121.3, 97.9, 77.0, 76.7, 76.1,
52.5, 50.9, 35.4 ppm; HRMS-ESI: m/z: calcd for C9H16N2NaO3:
223.1059 [M+Na]+, found: 223.1057.
50: Rf =0.63 in 30% NH4OH/MeOH; [a]2D2 =+2.88 (c=0.2, MeOH);
1H NMR (D2O; 500 MHz): d=4.28 (d, J=13.4 Hz, 1H), 3.87 (dd, J=
5.2, 13.4 Hz, 1H), 3.84 (d, J=3.4 Hz, 1H), 3.82 (d, J=5.2 Hz, 1H),
3.78–3.74 (dd, J=3.4, 11.0 Hz, 1H), 3.51–3.41 (m, 1H), 3.27 (d, J=
9.6 Hz, 1H), 2.80–2.73 (m, 1H), 2.31–2.19 (m, 1H), 2.12–2.03 (m,
1H),1.82–1.55 ppm (m, 4H); 13C NMR (D2O, 125 MHz): d=79.8,
78.5, 75.2, 74.6, 73.1, 64.8, 54.2, 51.1, 28.7, 26.4, 22.5 ppm; IR (neat):
n˜ =3120, 3030, 2837, 1589, 1496, 1438, 1042 cmꢀ1; HRMS-ESI: m/z:
calcd for C11H22N2NaO5: 285.1426 [M+Na]+, found: 285.1417.
Upjohn dihydroxylation of minor seven-membered spiro ana-
logue 24 and synthesis of N,N’-((2’S,3aR,5S,5’R,6’S,7R,7aS)-2,2-
cyclohexyl-5’,6’-dihydroxytetrahydro-3aH-spiro[benzo[d][1,3]di-
oxole-4,2’-oxepane]-5,7-diyl)diacetamide (46) and its 5’S,6’R
isomer (47): Spiroalkene 24 (45 mg, 0.11 mmol) was dissolved in a
mixture of acetone/H2O (1:1 (v/v), 4.6 mL). N-Methylmorpholine N-
oxide (41 mg, 0.34 mmol) and OsO4 (0.11 mL, 0.011 mmol, 2.5% (w/
w)) were added, and the reaction mixture was stirred vigorously at
RT. After 13 h, the mixture was concentrated under reduced pres-
sure, and the residue was purified by flash column chromatogra-
phy on silica gel (CH2Cl2!10% MeOH/CH2Cl2) to afford the corre-
sponding diastereomeric vicinal diols 46–47 (36 mg, 70% yield) as
a yellowish solid (2:3 mixture of diastereoisomers); Rf =0.26 in 30%
3: Rf =0.31 in 30% NH4OH/MeOH; [a]2D2 =ꢀ2.68 (c=0.1, MeOH);
1H NMR (D2O; 500 MHz): d=4.49 (d, J=14.6 Hz, 1H), 3.97–3.84 (m,
2H), 3.84 (d, J=10.2 Hz, 1H), 3.82–3.74 (m, 1H), 3.48–3.43 (dd, J=
3.2, 10.2 Hz, 1H), 3.38 (d, J=10.2 Hz, 1H), 3.34–3.27 (m, 1H), 2.52
(t, J=13.4 Hz, 1H), 2.30–2.21 (m, 1H), 1.94–1.68 (m, 4H); 13C NMR
(D2O, 125 MHz): d=78.2, 77.1, 75.1, 72.2, 70.8, 65.5, 55.9, 51.1,
28.7, 27.7, 27.3; IR (neat): n˜ =3118, 3028, 1641, 1442, 1392,
1013 cmꢀ1
; HRMS-ESI: m/z: calcd for C11H22N2NaO5: 285.1426
[M+Na]+, found: 285.1419.
Bacterial decoding site construct: To investigate specific confor-
mational events in solution, an RNA construct with 2-aminopurine
(2AP) instead of adenine 1492 was utilized. The construct compris-
es the bacterial A-site sequence and flanking auxiliary base pairs
designed to form a double helix (Scheme 1). Gel-purified and
desalted oligonucleotides were purchased from Dharmacon Inc.
(Lafayette, CO, USA). Double-stranded constructs were obtained by
annealing complementary strands in sodium cacodylate buffer
(30 mm, pH 6.8) at 658C for 3 min and snap cooling on ice.
1
MeOH/CH2Cl2; H NMR (500 MHz, CD3OD): d=4.29 (d, J=13.5 Hz,
1H; major), 4.24 (d, J=9.6 Hz, 1H; minor), 4.00–3.90 (m, 4H), 3.86
(dd, J=4.2, 13.5 Hz, 2H), 3.76 (brs, 1H; minor), 3.73 (brs, 1H;
major), 3.67–3.60 (m, 2H), 3.55 (d, J=9.6 Hz, 2H), 3.44 (d, J=
8.7 Hz, 1H; minor), 3.40 (d, J=8.7 Hz, 1H; major), 2.15–2.00 (m,
4H), 1.97 (d, J=8.0 Hz, 6H), 1.94 (s, 6H), 1.93–1.85 (m, 2H), 1.84–
ChemBioChem 2011, 12, 71 – 87
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
85

D. Vourloumis et al.
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Acknowledgements
This research is supported by Marie Curie Actions, Excellence
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would also like to thank Dr. I. Mavridis (NCSR “Demokritos”) for
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Keywords: aminoglycosides · antibiotics · ribosomal A-site ·
rigid scaffolds · RNA recognition
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