Synthesis and pharmacological characterization of new tetrahydrofuran based compounds as conformationally constrained histamine receptor ligands

Article abstract of DOI:10.1039/c3ob40441b

A series of tetrahydrofuran based compounds with a bicyclic core that provides conformational restriction were synthesized and investigated by radioligand displacement studies and functional [³⁵S]GTPγS binding assays at the human histamine receptor (hHR) subtypes. The amines 8a and 8b ((1S,3R,5S,6R)- and ((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicyclo[3.1.0] hexan-6-yl)methanamine), exhibited submicromolar K_i values at the hH₃R with 10-fold higher affinities than their corresponding (6S)-epimers and 25- and >34-fold selectivity over the hH₄R, respectively. Both compounds act as neutral antagonists at the hH₃R with K_B values of 181 and 32 nM, respectively. The cyanoguanidines of the imidazole series and the oxazole analogues turned out to be inactive at all hHR subtypes. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40441b

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Synthesis and pharmacological characterization of new
tetrahydrofuran based compounds as conformationally
constrained histamine receptor ligands†
Cite this: DOI: 10.1039/c3ob40441b
Julian Bodensteiner,a Paul Baumeister,b Roland Geyer,b Armin Buschauer*b and
Oliver Reiser*a
A series of tetrahydrofuran based compounds with a bicyclic core that provides conformational restric-
tion were synthesized and investigated by radioligand displacement studies and functional [35S]GTPγS
binding assays at the human histamine receptor (hHR) subtypes. The amines 8a and 8b ((1S,3R,5S,6R)-
and ((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicyclo[3.1.0]hexan-6-yl)methanamine), exhibited submicro-
molar Ki values at the hH3R with 10-fold higher affinities than their corresponding (6S)-epimers and
25- and >34-fold selectivity over the hH4R, respectively. Both compounds act as neutral antagonists at the
hH3R with KB values of 181 and 32 nM, respectively. The cyanoguanidines of the imidazole series and the
oxazole analogues turned out to be inactive at all hHR subtypes.
Received 4th March 2013,
Accepted 17th April 2013
DOI: 10.1039/c3ob40441b
www.rsc.org/obc
Alzheimer’s disease, attention-deficit/hyperactivity disorder
(ADHD), epilepsy, migraine, narcolepsy, obesity, schizophrenia
Introduction
Histamine is a biogenic amine that mediates its multiple physio- and depression.10
logical effects by the interaction with four known histamine
Recently, the H3R antagonist pitolisant (tiprolisant) has
receptor (HR) subtypes, termed H1R, H2R, H3R and H4R, all been introduced as an orphan drug for the treatment of narco-
belonging to rhodopsin-like family A of G-protein-coupled lepsy.11 In the years 2000 and 2001, the H4R was identified and
receptors (GPCRs).1
cloned independently by several research groups.12 The H4R is
Activation of the H1R has long been known to be associated mainly expressed in blood forming organs and immunocytes
with allergic conditions.1a,2 Antagonists of this receptor such as mast cells, basophils, eosinophils, monocytes, T-lym-
subtype (popularly referred to as antihistamines) are used as phocytes and dendritic cells.13 It is considered as a new thera-
anti-allergic drugs since the 1940s.2 The H2R plays a pivotal peutic target for the modulation of various inflammatory and
role in gastric acid secretion.3 H2R antagonists became immunological processes and disorders including bronchial
blockbuster drugs for the treatment of gastric and duodenal asthma, atopic dermatitis, allergic rhinitis, pruritus, colitis,
ulcer and gastroesophagal reflux disease. The histamine H3R pain, cancer, rheumatoid arthritis and multiple sclerosis.14
is located predominantly in the central nervous system (CNS)
Due to the significant sequence homology of the H4R with
and acts both as a presynaptic autoreceptor4 modulating hista- the H3R (about 40% overall sequence identity and about 60%
mine release and as an inhibitory heteroreceptor5 regulating within the transmembrane domains),12 many of the reported
the release of multiple neurotransmitters, such as acetyl- H3R agonists and antagonists showed considerable H4R
choline,6 dopamine,7 noradrenaline8 and serotonin.9 H3R activity as well.15 However, the development of more selective
antagonists are being investigated as potential drugs for thera- ligands remains indispensable in order to further elucidate
peutic applications against a variety of CNS disorders such as the (patho-) physiological roles of H3R and H4R, which might
offer new opportunities for the therapy of several diseases.
Since endogenous ligands such as histamine possess flexible
structures owing to rotations around single bonds, a reason-
able concept to improve potency and subtype selectivity is to
restrict the conformational flexibility.16 The affinity at the
respective receptor subtypes increases if such conformationally
restricted analogues superimpose the bioactive conformation
of the natural ligand. In the case of membrane-bound proteins
where structural information is not known precisely, this
aInstitut für Organische Chemie, Universität Regensburg, Universitätsstr. 31,
93053 Regensburg, Germany. E-mail: oliver.reiser@chemie.uni-regensburg.de
bInstitut für Pharmazie, Universität Regensburg, Universitätsstr. 31, 93053
Regensburg, Germany. E-mail: armin.buschauer@chemie.uni-regensburg.de;
Fax: +49 941 943 4820; Tel: +49 941 943 4827; Fax: +49 941 943 4121;
Tel: +49 941 943 4631
†Electronic supplementary information (ESI) available: 1H and 13C spectra for all
new synthesized compounds, HPLC data. See DOI: 10.1039/c3ob40441b
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Scheme 1 Retrosynthetic analysis of the target compounds.
Fig. 1 Conformationally restricted histamine receptor ligands.
pharmacological evaluation of potential HR ligands 6–9
containing a modified tetrahydrofuran spacer with a confor-
mationally constrained scaffold and diverse spatial orien-
tations (Scheme 1). The core structure consists of a fused ring
system and is formed by an asymmetric cyclopropanation reac-
tion, which gives rise to the bicyclic building block 10. The for-
mation of the imidazole moiety represents a key step in the
synthetic route and is realized by the conversion of aldehyde
11 via the TosMIC strategy. Finally, the amino group of 8
and the cyanoguanidino group of 6 are introduced by further
functional group interconversions including a Mitsunobu-type
Gabriel reaction. In parallel, analogues 9 and 7 with an
oxazole moiety as a potential bioisostere are synthesized and
pharmacologically characterized. All target compounds are
accessible as both enantiomers depending on the choice of
the respective chiral ligand in the asymmetric cyclopropana-
tion step.
strategy can be extended by the variation of stereochemistry
to explore the right spatial orientation and stereochemical
requirements of the pharmacophoric elements in the binding
pocket and to refine the models of ligand–receptor interaction.
This approach has been successfully applied for the devel-
opment of selective H3R and H4R ligands. Fig. 1 shows some
examples of histamine analogues comprising rigid carbo- and
heterocyclic cores. From a series of cyclopropane-based con-
formationally constrained histamine analogues with diverse
stereochemistry, Shuto et al. identified the “folded” cis-
analogue (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopro-
pane (1, AEIC) to be the most potent agonist at the hH3R (Ki =
1.3 nM, EC50 = 10 nM) which had virtually no effect on the
H4R subtype.17
Yamatodani et al. synthesized all imifuramine (2) stereo-
isomers and its corresponding cyanoguanidine analogues and
examined the binding affinity and functional activity at the
human H3 and H4 receptors by in vitro studies.18 Replacement
of the amino group by the cyanoguanidine moiety, which is
uncharged at physiological pH, resulted in a decrease in agon-
istic activity at the hH3R. In contrast, the potencies and intrin-
sic activities increased at the hH4R for most isomers. As a
result, OUP-16 (3) was identified as the first selective H4R
agonist.
Recently, we explored rigidified cyanoguanidine-type HR
ligands having a phenylene or a 1,4-cyclohexylene linker.19
While the phenylene linker yielded only very weakly active
compounds at both hH3R and hH4R, the less rigid 1,4-cyclo-
hexylene linker gave cis- and trans-configured molecules reveal-
ing EC50 or KB values ≥110 nM at the hH3R and hH4R. The
cis-configured isomers 4 preferred the hH4R and were partial
agonists, whereas the trans-isomers 5 were antagonists at the
hH4R. At the hH3R the trans-diastereomers 5 were superior to
the cis-isomers 4 by a factor of 10. Thus, previous results
suggest that the variation of conformational constraints and
stereochemical properties is a promising approach to further
explore the structure–activity and structure–selectivity relation-
ships of HR ligands.
Results and discussion
Chemistry
In the course of synthesizing various γ-butyrolactone contain-
ing natural products20 we had previously developed the
asymmetric cyclopropanation of furan-2-carboxylic acid ester
12 derived from commercially available 2-furoic acid
(Scheme 2).21 The reaction proceeded with ethyl diazoacetate
in the presence of catalytic copper(I)-isopropyl bis(oxazoline)
with high enantio- and diastereoselectivity to give bicyclic com-
pound 10. The double bond in 10 was hydrogenated accord-
ingly using palladium on charcoal in EtOAc.22 The
hydrogenation proceeds via syn-addition exclusively from the
less hindered convex face of the bicyclic framework to form 13
as a single stereoisomer after recrystallization. A directed
thus highly chemoselective reduction of the methyl ester to
alcohol 14, being assisted by the adjacent furan ring oxygen,
was followed by a Dess–Martin oxidation to give aldehyde 15.
Subsequent base-induced [3 + 2]cycloaddition with p-toluene-
sulfonylmethyl isocyanide (TosMIC) afforded tosyloxazoline 16
as a mixture of diastereomers.23 The conversion to the corres-
ponding imidazole 17 by treatment with ammonia according
to Horne et al. turned out to be not feasible.24 Also several
As part of our efforts to develop further potent and selective
H3 and H4 receptor ligands that may serve as pharmacological
tools and unravel the interactions within the binding
pockets, we herein describe the enantioselective synthesis and
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Scheme 2 Reagents and conditions: (a) ref. 21; (b) ref. 22; (c) LAH, THF, 0 °C, 45 min, 87%; (d) Dess–Martin periodinane, DCM, rt, 1 h, 88%; (e) TosMIC, NaCN,
EtOH, rt, 1 h, 70%; R = OEt, OMe, NH2.
other TosMIC based methods25 starting from aldehyde 15 were displace the ethyl ester group by a benzyl ether protecting
not successful. Any unexpected ring-opening reaction was group which is inert under the prevailing basic conditions. For
ruled out by different test reactions. However, it could not be this reason, the primary alcohol of compound 14 was TBS-
totally excluded that the ester moiety was interfering with the protected (Scheme 3). The ester group of intermediate 18 was
imidazole forming reaction. To circumvent the difficulties con- reduced with LiAlH4 and the resulting primary alcohol 19 was
cerning the introduction of the imidazole ring we decided to protected with benzyl bromide. After TBAF-mediated cleavage
Scheme 3 Reagents and conditions: (a) NEt3, TBSCl, DMAP, DCM, rt, 18 h, 95%; (b) LAH, THF, 0 °C, 45 min, 95%; (c) NaH, BnBr, DMF, 0 °C to rt, 2 h, 85%; (d) TBAF,
THF, rt, 13 h, 95%; (e) Dess–Martin periodinane, DCM, rt, 2 h, 90%; (f ) TosMIC, NaCN, EtOH, rt, 2 h, 77%; (g) NH3 saturated in MeOH, 95 °C, sealed pressure tube,
16 h, (24a : 24b = 5 : 1); (h) ethyl chloroformate, pyridine, DMAP, benzene, 50 °C, 10 min, 73%; (i) Pd(OH)2–C, cyclohexene, EtOH, reflux, 1 h, 73%; ( j) PPh3, phthali-
mide, DIAD, THF, rt, 18 h, 29%; (k) hydrazine hydrate, EtOH, reflux, 1 h, 77%; (l) (i) dimethyl N-cyanodithioiminocarbonate, MeOH, rt, 18 h; (ii) MeNH2 in EtOH, rt,
18 h, 69% over two steps; (m) PPh3, phthalimide, DIAD, THF, rt, 18 h, 27%; (n) hydrazine hydrate, EtOH, reflux, 1.5 h, 68%; (o) (i) dimethyl N-cyanodithioiminocarbo-
nate, MeOH, rt, 18 h; (ii) MeNH2 in EtOH, rt, 18 h, 64% over two steps.
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of the silylether, oxidation of alcohol 21 by means of Dess– excess of dimethyl N-cyanodithioiminocarbonate in MeOH to
Martin periodinane afforded aldehyde 22 which underwent furnish an isothiourea compound which was then directly con-
cycloaddition with TosMIC.23 The resulting oxazoline diaster- verted without purification to the desired cyanoguanidine 6a
eomers 23 were treated with a solution of ammonia in MeOH by adding an ethanolic solution of MeNH2.26
at elevated temperature in a sealable pressure tube giving rise
The respective (3S)-configured target compounds, amine 8b
to the desired imidazole 24 in up to 68% yield.24 Besides the and cyanoguanidine 6b, were derived from the corresponding
expected imidazole isomer 24a, epimer 24b was identified as (3S)-configured alcohol 26b running through an analogous
well due to the basic and high temperature conditions applied. synthetic pathway via phthalimide 27b. Consequently, the
Best results (24a : 24b = 5 : 1) were obtained by heating at (6R)-configured target molecules, amines 6a and 6b and the
95 °C, while higher temperatures caused increased epimeriza- cyanoguanidines 8a and 8b could be prepared. Additionally,
tion. Following the protocol of Harusawa et al., imidazole 24 by employing the (R,R)-isopropyl bis(oxazoline) ligand in
was converted to its base-sensitive carbamate-protected deriva- the cyclopropanation step, the respective (6S)-enantiomers,
tive 25 using ethyl chloroformate to improve the solubility pro- amines 8c and 8d and cyanoguanidines 6c and 6d, were
perties and to facilitate the separation of the epimers at a later accessible.
stage of the reaction sequence.26 Cleavage of the benzyl ether
As a putative bioisostere of the imidazole, an oxazole
to give alcohol 26 was realized by catalytic transfer hydrogen- moiety was introduced by elimination of p-toluene sulfinic
ation using palladium hydroxide on carbon and cyclohexene acid from tosyloxazoline 16 accompanied by transesterification
as the hydrogen donor.27 At this point, separation of the of the ethyl ester group to give compound 33 (Scheme 5).23
isomers was necessary since the next step provided several Subsequent reduction with LiAlH4 furnished alcohol 34. The
side-products, which were otherwise tedious to separate and to hydroxy group was converted into a phthalimide moiety via a
characterize. To displace the hydroxyl group of the (3R)- Mitsunobu reaction.28 In comparison to the similar transfor-
isomer 26a with an amino moiety, a phthaloylimination under mation of 26a to 27a described above, the reaction proceeded
Mitsunobu conditions and subsequent hydrazinolysis were with the formation of smaller amounts of ring opening pro-
performed.28 By treating 26a with phthalimide in the presence ducts and this with higher yield to the desired 35 (55% vs.
of PPh3 and DIAD, the desired phthalimide 27a was obtained 29% for 27a) for reasons that are not clear. Hydrazinolysis gave
in low yield. Further ring-opening gave phthalimides 28 as a rise to the amine 9a. The corresponding cyanoguanidine 7a
mixture of epimers due to a cyclopropylcarbinyl–homoallylic was readily obtained by converting the amino group with
rearrangement (Scheme 4).29 Diene 29 was observed as well dimethyl N-cyanodithioiminocarbonate to isothiourea 36 fol-
but was not separable from the triphenylphosphine oxide lowed by treatment with MeNH2 in EtOH.26 In turn, the appli-
byproduct. In order to optimize the conditions for the prepa- cation of the enantiomeric isopropyl bis(oxazoline) ligand in
ration of the desired phthalimide 27a different conditions the asymmetric cyclopropanation reaction provided access to
were screened using model compound 19. However, the the enantiomeric target compounds, 9b and 7b, as well.
change of the addition order of the reagents, variation in the
Pharmacology
relative reagent concentrations and different reaction temp-
eratures and solvents did not lead to a significantly improved
ratio of products. Cleavage of the phthalimide moiety of com-
pound 27a by means of hydrazinolysis proceeded smoothly
with simultaneous removal of the base-sensitive carbamate
protection group at the imidazole ring to give the desired
target compound amine 8a (Scheme 3). The conversion to the
analogous cyanoguanidine-containing compound 6a required
two additional steps. First, amine 8a was treated with an
All the synthesized target compounds depicted in Fig. 2 were
investigated at the hH1R, hH2R, hH3R and hH4R in radioligand
binding assays using membrane preparations of Sf9 insect
cells coexpressing the hH1R + RGS4, hH2R−Gsαs fusion
protein, hH3R + Gαi2 + Gβ1γ2 or hH4R + Gαi2 + Gβ1γ2, respect-
ively. Those compounds having submicromolar Ki values were
investigated for agonism or antagonism at hH3R and hH4R
subtypes in functional [35S]GTPγS assays using the above
Scheme 4 Mitsunobu reaction.
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Scheme 5 Reagents and conditions: (a) K2CO3, MeOH, reflux, 0.5 h, 31%; (b) LAH, THF, 0 °C, 0.5 h, 71%; (c) PPh3, phthalimide, DIAD, THF, 0 °C, 0.5 h, 55%; (d)
hydrazine hydrate, EtOH, reflux, 1.5 h, 72%; (e) dimethyl N-cyanodithioiminocarbonate, EtOH, rt, 18 h, quant.; (f) MeNH2 in EtOH, rt, 18 h, 90%.
Fig. 2 Synthesized target compounds.
mentioned membrane preparations. Intrinsic activities (α) >34, >4 and 3-fold higher than at the hH4R subtype, respect-
refer to the maximal response induced by the standard agonist ively. Additionally, 8a–d were devoid of activity at the hH1R
histamine (α = 1.0). Compounds identified to be inactive as and hH2R. 8a and 8b were investigated for their functional
agonists (α < 0.13 or negative values, respectively, determined activity at the hH3R. In contrast to imifuramine and its stereo-
in the agonist mode) were investigated in the antagonist isomers, which were all reported to act as full agonists at the
mode. The corresponding KB values of neutral antagonists H3R,18 8a and 8b turned out to be almost neutral antagonists
were determined from the concentration-dependent inhibition with KB values of 181 and 32 nM, respectively. The elongated
of the histamine-induced increase in [35S]GTPγS binding.
spacer and the different spatial arrangement of the pharmaco-
In agreement with the findings of Hashimoto et al., the phoric elements were tolerated to a certain extent for the con-
amines 8a–d exhibited significantly stronger binding affinities formationally constrained amines compared to Hashimoto’s
at the hH3R than at the hH4R (Table 1). At the hH3R the (6R)- THF-based ligands. At both HR subtypes comparable Ki values
configured eutomers 8a and 8b showed submicromolar Ki were determined, especially at the hH3R, but the quality of
values. Both compounds were about 10-fold more potent than action was different.
their (6S)-configured distomers 8c and 8d. At the hH4R, com-
In contrast, the cyanoguanidines 6a–d turned out to be
pounds 8a and 8d, having the (3R)-configuration, exhibited inactive (Ki > 10 000 nM) at all four HR subtypes, notably at the
weak binding affinities with low micromolar Ki values. In con- H4R. In this case, the orientation of the pharmacophoric
trast to this, the respective (3S)-configured epimers 8b and 8c elements, provided by the bicyclic core, is detrimental for
did not show any significant binding (Ki > 10 000 nM) at this receptor binding. An increase in hH4R affinity by replacement
receptor subtype. An unambiguous preference for either the of the amino group with a cyanoguanidino moiety – as
folded isomers ((3R,6R)-cis-8a and (3S,6S)-cis-8d) or the observed for the imifuramine based compounds18 – was not
extended analogues ((3S,6R)-trans-8b and (3R,6S)-trans-8c) was achieved.
not observed at both receptor subtypes. As a result, binding
The synthesized oxazoles 9a, 9b, 7a and 7b were investi-
affinities for the amines 8a, 8b, 8c and 8d at the hH3R were 25, gated in radioligand binding assays at the hH1R and hH2R
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Table 1 Affinities, potencies and efficacies of the synthesized amines and cyanoguanidines at the hHR subtypes determined in radioligand binding studiesa and
functional [35S]GTPγS assaysb
hH1R
hH2R
hH3R
hH4R
Ki (nM)
Ki (nM)
Compound
Histaminec
Imifuramine (2)d
OUP-16 (3)d
8a
Config.
Ki (nM)
200
N
Ki (nM)
5 × 104
—
N
(KB or EC50 b (nM))
α
N
(KB or EC50 b (nM))
α
N
10
(5.0)
229
(45)
2188
(3261)
231 106
(181 119)
295 154
(32 17)
2326 982
2818 1823
Inactive
Inactive
Inactive
Inactive
(Inactive)
(Inactive)
(Inactive)
(Inactive)
7.9(13)
1.00
1.04
0.79
1.00
0.70
0.99
—
891
(1995)
126
(78)
5787 853
—
—
3R,6R
3S,6R
Inactive
Inactive
2
2
Inactive
Inactive
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
−0.10
−0.12
8b
>10 000
8c
8d
6a
6b
6c
6d
9a
9b
7a
7b
3S,6S
3R,6S
3R,6R
3S,6R
3S,6S
3R,6S
3R,6R
3S,6S
3R,6R
3S,6S
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
2
2
2
2
2
2
2
2
2
2
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
Inactivee
2
2
2
2
2
2
2
2
2
2
>10 000
8415 417
Inactive
Inactive
Inactive
2
2
2
2
2
2
2
2
2
2
Inactive
−0.08
−0.06
0.07
(Inactive)
(Inactive)
(Inactive)
(Inactive)
0.02
0.13
−0.03
−0.06
−0.07
a Determination of hH1R binding by displacement of [3H]pyrilamine (5 nM) from Sf9 cell membranes expressing the hH1R + RGS4, hH2R
binding by displacement of [3H]UR-DE257 (30 nM) from Sf9 cell membranes expressing the hH2R−Gsαs, hH3R binding by displacement of [3H]
Nα-methylhistamine (3 nM) or [3H]histamine (15 nM) from Sf9 cell membranes expressing the hH3R + Gαi2 + Gβ1γ2 or hH4R binding by
displacement of [3H]histamine (15 nM) from Sf9 cell membranes expressing the hH4R + Gαi2 + Gβ1γ2 was determined as described in the
Pharmacology section. b Functional [35S]GTPγS binding assays with membrane preparations of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2 or the
hH4R + Gαi2 + Gβ1γ2 were performed as described in the Pharmacology section. a,bLigands concentrations from 1 nM to 1 mM as appropriate to
generate saturated concentration/response curves. Compounds showing no effect in this range were referred to as inactive. N gives the number of
independent experiments performed in triplicate each. The intrinsic activity (α) of histamine was set to 1.00 and α values of other compounds
were referred to this value. The α values of neutral antagonists and inverse agonists were determined at a concentration of 10 μM. The KB values
of neutral antagonists were determined in the antagonist mode versus histamine (100 nM) as the agonist. c Values taken from Igel et al.30 d Values
for hH3R and hH4R taken from Hashimoto et al.18 e Measured at a ligand concentration of 100 µM.
subtypes and in functional [35S]GTPγS assays at the hH3R and the imidazole compounds the introduction of the aromatic
hH4R subtypes but did not reveal any activity at all receptor heterocyles by TosMIC chemistry gave rise to additional
subtypes. Even oxazole 9a, whose imidazole analogue 8a epimers. As a result, different isomers with distinct stereoche-
exhibited submicromolar affinities at the hH3R, was inactive at mical orientations could be achieved. Pharmacological investi-
the hH3R. Thus, in this class of HR ligands, independently gations at the human HR subtypes revealed affinities of the
from the other structural modifications, the oxazole ring amines 8 at the hH3R in a comparable range as reported for
proved to be inappropriate as a bioisostere of an imidazole the imifuramine derived stereoisomers, but with different
ring. This is presumably due to a different H-bond donor– qualities of action. Especially 8b showed high subtype selecti-
acceptor interaction pattern and the reduced basicity of the vity for the hH3R with no affinity for the hH4R. In agreement
oxazole moiety compared to the imidazole ring.
with these findings and in contrast to the cyanoguanidine 3
bearing the tetrahydrofuran moiety, the cyanoguanidines 6
were devoid of activity at the hH4R as well. These results
suggest that the 2-oxabicyclo[3.1.0]hexane framework used in
this study might be a promising scaffold for the hH3R selective
Conclusion
In conclusion, we have synthesized and pharmacologically ligands. Replacement of the imidazole ring by an oxazole
characterized a set of bicyclic imifuramine analogues as poten- substituent as a potential bioisostere led to loss of activities
tial histamine receptor ligands. In the imidazole series, the at HR subtypes. Apparently, the H-bond donor and acceptor
conformationally constrained amines and cyanoguanidines properties and the basicity of the oxazole ring are
were obtained in 15 and 17 steps, respectively, starting from inappropriate.
commercially available furan-2-carboxylic acid. The oxazole
These findings contribute to the objective of further eluci-
analogues could be realized in 10 and 12 steps, respectively. dating the structural requirements of selective histamine H3
The synthetic pathways deliver valuable information about and H4 receptor ligands that may help to enable the synthesis
the scope and limitations of the rigid bicyclic core in terms of tailored compounds as novel pharmacological tools and
of chemical transformations of its substituents. In the case of potential drugs with the intended quality of action.
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Acetonitrile was removed from the eluates under reduced
pressure at 45 °C prior to lyophilization.
Preparation of 1-(((1S,3R,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabi-
Experimental
Chemistry
Analytical HPLC analysis was performed with a system from cyclo[3.1.0]hexan-6-yl)methyl)-2-cyano-3-methylguanidine (6a).
Merck (Darmstadt, Germany) consisting of a L-5000 controller, A solution of compound 8a (5.0 mg, 0.028 mmol) and
a 655A-12 pump, a 655A-40 autosampler and a L-4250 UV-VIS dimethyl N-cyanodithioiminocarbonate (9.9 mg, 0.067 mmol,
detector on a Eurospher-100 C18 column (250 × 4 mm, 5 μm, 2.4 equiv.) in anhydrous MeOH (0.55 mL) was stirred at room
Knauer, Berlin, Germany) at a flow rate of 0.8 mL min−1. Mix- temperature for 18 h. Then a 33% solution of MeNH2 in EtOH
tures of acetonitrile and 0.05% aq. TFA were used as the (0.52 mL) was added and stirred for 18 h at room temperature.
mobile phase. Helium degassing was used throughout. Com- The solvent was evaporated to give a residual oil that was puri-
pound purities were calculated as the percentage peak area of fied by column chromatography (EtOAc–MeOH 4 : 1) to give
the analyzed compound by UV detection at 210 nm. HPLC con- compound 6a (5.0 mg, 0.019 mmol, 69%) as a colorless oil. For
ditions, retention times (tR), capacity factors (k′ = (tR − t0)/t0) pharmacological testing the product was further purified by
and purities of the synthesized compounds are listed in the preparative HPLC (YMC-Triat column, mobile phase: MeCN,
ESI.†
All reactions were carried out in oven-dried glassware under
0.1% aq. NH3).
Rf = 0.19 (EtOAc–MeOH 4 : 1); [α]2D0 = +18.2 (MeOH, c = 0.2);
atmospheric conditions unless otherwise stated. All solvents 1H-NMR (300 MHz, MeOD): δH = 7.62 (d, J = 1.0 Hz, 1H), 6.96
were dried and distilled prior to use. Thin layer chromato- (s, 1H), 5.38 (t, J = 7.9 Hz, 1H), 3.89 (dd, J = 6.4, 1.1 Hz, 1H),
graphy (TLC) was performed using silica gel 60 F254 alu- 3.05 (dd, J = 14.3, 6.9 Hz, 1H), 2.91 (dd, J = 14.3, 7.9 Hz, 1H),
minium plates (Merck). Eluted plates were visualized using a 2.79 (s, 3H), 2.55 (dt, J = 12.8, 7.4 Hz, 1H), 2.06 (ddd, J = 12.8,
254 nm UV lamp and/or by treatment with a suitable stain fol- 8.1, 1.9 Hz, 1H), 1.74–1.63 (m, 1H), 1.46–1.36 (m, 1H);
lowed by heating. Column chromatography was performed on 13C-NMR (75 MHz, MeOD): δC = 161.96 (Cq), 140.02 (Cq),
silica gel 60 (0.063–0.200 mm, Merck). 1H- and 13C-NMR 136.79 (+), 120.08 (Cq), 117.42 (+), 83.90 (+), 65.43 (+), 42.58
spectra were recorded on a Bruker Avance 300 (300 MHz for (−), 36.41 (−), 33.19 (+), 28.67 (+), 24.09 (+); IR (ATR): ˜ν (cm−1
)
1H, 75 MHz for 13C), Bruker Avance III 400 “Nanobay” = 3268 (br), 2928, 2160, 1729, 1575, 1485, 1448, 1404, 1369,
(400 MHz for 1H, 101 MHz for 13C) or Avance III 600 (600 MHz 1247, 1174, 1097, 1066, 1030, 988, 838, 752, 716, 618, 570; MS
for 1H, 151 MHz for 13C) FT-NMR-spectrometer. Chemical (ESI): m/z (%) = 163.1 [M+ΔC3H5N4] (60), 261.1 [MH+] (100);
shifts are reported in parts per million (ppm). ATR-IR HRMS (ESI): calcd for C12H17N6O [MH+] 261.1458, found
spectroscopy was carried out on a Biorad Excalibur FTS 3000 261.1458.
spectrometer equipped with a Specac Golden Gate Diamond
Preparation of 1-(((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabi-
Single Reflection ATR-system. Optical rotations were measured cyclo[3.1.0]hexan-6-yl)methyl)-2-cyano-3-methylguanidine (6b).
on a P8000T polarimeter (Kruess) at a wavelength of 589 nm in A solution of compound 8b (3.0 mg, 0.017 mmol) and
a 5 cm cell of 0.7 mL volume in the specified solvent. Con- dimethyl N-cyanodithioiminocarbonate (8.2 mg, 0.05 mmol,
centrations are indicated in [g/100 mL]. The melting points 3.0 equiv.) in anhydrous MeOH (0.34 mL) was stirred at
were measured on a Büchi SMP-20 apparatus in a silicon oil room temperature for 18 h. Then a 33% solution of MeNH2
bath. Values thus obtained were not corrected. Mass spec- in EtOH (0.31 mL) was added. The resulting mixture
trometry was performed on a Varian MAT 311A, Finnigan MAT was stirred for 18 h at room temperature. The solvent was evap-
95, Thermoquest Finnigan TSQ 7000 or Agilent Technologies orated to give a residual oil that was purified by column
6540 UHD Accurate-Mass Q-TOF LC/MS. The percentage set chromatography (EtOAc–MeOH 4 : 1) to give compound 6b
in brackets gives the peak intensity related to the basic peak (2.8 mg, 0.011 mmol, 64%) as a colorless oil. For pharmaco-
(I = 100%). High resolution mass spectrometry (HRMS): the logical testing the product was further purified by preparative
molecular formula was proven by the calculated precise mass. HPLC (YMC-Triat column, mobile phase: MeCN, 0.1% aq.
Elemental analyses (C, H, N, Heraeus Elementar Vario EL III) NH3).
were performed by the Analytical Department of the University
of Regensburg.
Rf = 0.19 (EtOAc–MeOH 4 : 1); [α]2D0 = +36.7 (MeOH, c = 0.1);
1H-NMR (600 MHz, MeOD): δH = 7.62 (s, 1H), 7.00 (s, 1H), 4.76
Preparative HPLC was performed at room temperature with (dd, J = 8.7, 7.7 Hz, 1H), 3.94 (dd, J = 5.7, 1.1 Hz, 1H), 3.07 (dd,
a system from Knauer (Berlin, Germany) consisting of two J = 14.3, 6.9 Hz, 1H), 2.97 (dd, J = 14.3, 7.7 Hz, 1H), 2.81 (s,
K-1800 pumps, a K-2001 detector (UV detection at 220 nm) 3H), 2.29 (dd, J = 12.4, 7.1 Hz, 1H), 2.25–2.19 (m, 1H),
and a RP-column (VP Nucleodur 100-5 C18 ec, 250 × 21 mm, 1.62–1.59 (m, 1H), 1.59–1.54 (m, 1H); 13C-NMR (151 MHz,
5 μm, Macherey Nagel, Düren, Germany) at a flow rate of MeOD): δC = 162.01 (Cq), 136.88 (+), 120.08 (Cq), 63.74 (+),
15 mL min−1 or a RP-column (YMC-Triat C18, 150 × 20.0 m, 49.57 (+), 42.71 (−), 35.51 (−), 28.69 (+), 22.28 (+), 21.94 (+), Im-
5 µm, YMC Europe GmbH, Dinslaken, Germany) at a flow rate C5 and Im-C4 signals too weak to be observed; IR (ATR):
of 10 mL min−1. Mixtures of acetonitrile and 0.1% aq. TFA ˜ν (cm−1) = 2934 (br), 2163, 1582, 1486, 1410, 1372, 1322, 1175,
were used as the mobile phase in the case of the Nucleodur 1121, 1100, 922, 892, 833, 689, 617; MS (ESI): m/z (%) = 261.1
column and mixtures of acetonitrile and 0.1% aq. NH3 were [MH+] (100), 521.2 [2MH+] (15); HRMS (ESI): calcd for
used as the mobile phase in the case of the YMC-Triat column. C12H17N6O [MH+] 261.1458, found 261.1457.
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Preparation of 2-cyano-1-methyl-3-(((1S,3R,5S,6R)-3-(oxazol- refluxed for 1.5 h. The solvent was removed under reduced
5-yl)-2-oxabicyclo[3.1.0]hexan-6-yl)methyl) guanidine (7a). pressure and the residue was purified by column chromato-
Compound 36 (26 mg, 0.09 mmol) was dissolved in a 33% graphy (MeOH–saturated NH3 in MeOH 97 : 3) to afford com-
solution of MeNH2 in EtOH (2 mL) and stirred for 18 h at pound 8b (5.1 mg, 0.028 mmol, 68%) as
a colorless
room temperature. The solvent was evaporated under reduced amorphous solid. For pharmacological testing the product was
pressure. Purification by column chromatography (DCM then further purified by preparative HPLC (Nucleodur column,
DCM–MeOH 9 : 1) afforded compound 7a (22 mg, 0.08 mmol, mobile phase: MeCN, 0.1% aq. TFA).
90%) as a colorless oil.
8b·2TFA: Rf = 0.20 (MeOH–saturated NH3 in MeOH 95 : 5);
Rf = 0.32 (DCM–MeOH 9 : 1); [α]2D0 = +18.9 (DCM, c = 1.0); [α]D20 = +5.5 (DCM, c = 0.2); 1H-NMR (600 MHz, MeOD): δH
=
1H-NMR (400 MHz, CDCl3): δH = 7.84 (s, 1H), 6.96 (s, 1H), 5.64 8.88 (d, J = 1.3 Hz, 1H), 7.50 (d, J = 0.9 Hz, 1H), 4.94 (dd, J =
(s, 1H), 5.43 (dd, J = 8.3, 7.4 Hz, 1H), 5.20 (s, 1H), 3.95 (dd, J = 8.9, 7.5 Hz, 1H), 4.14 (dd, J = 5.8, 1.2 Hz, 1H), 2.82 (dd, J =
6.3, 1.0 Hz, 1H), 3.24–3.13 (m, 1H), 2.95–2.86 (m, 1H), 2.85 (d, 13.4, 8.0 Hz, 1H), 2.77 (dd, J = 13.4, 7.8 Hz, 1H), 2.51 (dd, J =
J = 4.9 Hz, 3H), 2.62 (ddd, J = 13.1, 8.6, 7.0 Hz, 1H), 2.14 (ddd, 12.7, 7.4 Hz, 1H), 2.22 (ddd, J = 12.8, 9.1, 5.6 Hz, 1H),
J = 13.1, 7.0, 1.4 Hz, 1H), 1.73–1.67 (m, 1H), 1.37 (tdd, J = 8.0, 1.83–1.79 (m, 1H), 1.56 (tdd, J = 7.9, 3.9, 1.1 Hz, 1H). 13C-NMR
4.0, 1.0 Hz, 1H); 13C-NMR (101 MHz, CDCl3): δC = 160.65 (Cq), (151 MHz, MeOD): δC = 162.80 (Cq, TFA), 162.56 (Cq, TFA),
151.72 (Cq), 151.44 (+), 124.01 (+), 118.53 (Cq), 78.46 (+), 65.08 136.08 (Cq), 134.59 (+), 119.04 (+, TFA), 117.45 (+), 117.11
(+), 42.03 (−), 33.91 (−), 30.74 (+), 28.57 (+), 23.15 (+); IR (ATR): (+, TFA), 72.81 (+), 64.08 (+), 40.76 (−), 35.51 (−), 22.52 (+),
˜ν (cm−1) = 3292 (br), 2954, 2929, 2165, 1583, 1507, 1453, 1409, 20.51 (+); IR (ATR): ˜ν (cm−1) = 3240 (br), 2935, 2873, 2627,
1370, 1174, 1103, 1028, 963, 838, 717; MS (ESI): m/z (%) = 1580, 1492, 1420, 1372, 1312, 1180, 1101, 899, 840, 630, 540;
262.1 (25) [MH+], 523.2 (100) [2MH+]; HRMS (EI): calcd for MS (ESI): m/z (%) = 180.0 (100) [MH+], 359.2 (20) [2MH+];
C12H15N5O2 [M+˙] 261.1226, found 261.1222.
HRMS (ESI): calcd for C9H14N3O [MH+] 180.1131, found
Preparation of ((1S,3R,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicy- 180.1133.
clo[3.1.0]hexan-6-yl)methanamine (8a). A solution of phthali-
Preparation of ((1S,3R,5S,6R)-3-(oxazol-5-yl)-2-oxabicyclo-
mide 27a (30 mg, 0.079 mmol) and hydrazine hydrate (21 µL, [3.1.0]hexan-6-yl)methanamine (9a). A solution of phthali-
0.43 mmol, 5.4 equiv.) in anhydrous EtOH (1.6 mL) was mide 35 (60 mg, 1.19 mmol) and hydrazine hydrate (48 mg,
refluxed for 1 h. The solvent was removed under reduced 0.97 mmol, 5 equiv.) in EtOH (4 mL) was refluxed for 1.5 h and
pressure and the residue was purified by column chromato- then cooled in an ice bath. The white precipitate was removed
graphy (MeOH–saturated NH3 in MeOH 95 : 5) to afford com- by filtration through a Celite pad. The filtrate was concentrated
pound 8a (11 mg, 0.061 mmol, 77%) as a colorless amorphous in vacuo. Column chromatography (DCM–saturated NH3 in
solid. For pharmacological testing the product was further MeOH 20 : 1) afforded compound 9a (25 mg, 0.10 mmol, 72%)
purified by preparative HPLC (Nucleodur column, mobile as a colorless solid.
phase: MeCN, 0.1% aq. TFA).
Mp = 51 °C; Rf = 0.32 (DCM–saturated NH3 in MeOH 9 : 1);
1
Rf = 0.20 (MeOH–saturated NH3 in MeOH 95 : 5); [α]D20
=
[α]2D0 = +36.2 (DCM, c = 1.0); H-NMR (400 MHz, CDCl3): δH
=
1
+36.4 (MeOH, c = 0.5); H-NMR (300 MHz, MeOD): δH = 7.61 7.80 (s, 1H), 6.93 (s, 1H), 5.41 (dd, J = 8.1, 7.4 Hz, 1H), 3.82
(d, J = 1.0 Hz, 1H), 6.95 (s, 1H), 5.38 (t, J = 7.9 Hz, 1H), 3.82 (dd, J = 6.3, 1.2 Hz, 1H), 2.58 (ddd, J = 12.9, 8.6, 7.0 Hz, 1H),
(dd, J = 6.4, 1.2 Hz, 1H), 2.63–2.49 (m, 1H), 2.38 (d, J = 7.3 Hz, 2.51–2.39 (m, 2H), 2.11 (ddd, J = 12.9, 6.9, 1.5 Hz, 1H),
2H), 2.04 (ddd, J = 12.7, 8.0, 1.9 Hz, 1H), 1.61 (tdd, J = 6.2, 3.9, 1.59–1.51 (m, 1H), 1.28–1.22 (m, 1H), 1.25 (br s, 2H); 13C-NMR
1.9 Hz, 1H), 1.26 (tdd, J = 7.4, 4.0, 1.1 Hz, 1H); 13C-NMR (75 MHz, CDCl3): δC = 152.16 (Cq), 151.25 (+), 123.68 (+), 78.37
(75 MHz, MeOD): δC = 136.71 (+), 117.55 (+), 83.87 (+), 65.46 (+), 65.19 (+), 42.33 (−), 34.78 (−), 34.12 (+), 22.68 (+); IR (ATR):
(+), 42.61 (−), 36.63 (−), 36.07 (+), 23.99 (+), Im-Cq-signal too ˜ν (cm−1) = 3356 (br), 3127, 2949, 1636, 1567, 1508, 1482, 1427,
weak to be observed; IR (ATR): ˜ν (cm−1) = 3094 (br), 2937, 2869, 1377, 1318, 1180, 1103, 1027, 980, 955, 849, 723, 646, 610; MS
2625, 1573, 1454, 1414, 1361, 1306, 1177, 1098, 1067, 1028, (ESI): m/z (%) = 181.0 (7) [MH+], 222.0 (100) [MH+MeCN];
980, 912, 841, 632, 540, 497; MS (ESI): m/z (%) = 163.1 (100) HRMS (ESI): calcd for C9H13N2O2 [MH+] 181.0972, found
[MH+ΔNH3], 180.1 (19) [MH+], 359.2 (11) [2MH+]; HRMS (ESI): 181.0969.
calcd for C9H14N3O [MH+] 180.1131, found 180.1130.
Preparation of (1S,3R,5S,6S)-ethyl 3-(hydroxymethyl)-2-oxabi-
8a·2TFA: 1H-NMR (600 MHz, MeOD): δH = 8.83 (d, J = cyclo[3.1.0]hexane-6-carboxylate (14). To a stirred ice-cooled
1.0 Hz, 1H), 7.45 (s, 1H), 5.51 (t, J = 7.4 Hz, 1H), 4.10 (dd, J = solution of 13 (2.45 g, 11.4 mmol) in anhydrous THF (45 mL)
6.3, 0.7 Hz, 1H), 2.90–2.63 (m, 3H), 2.14 (ddd, J = 13.1, 7.0, under a nitrogen atmosphere, a suspension of LAH (260 mg,
1.5 Hz, 1H), 1.92–1.87 (m, 1H), 1.31–1.26 (m, 1H); 13C-NMR 6.87 mmol, 0.6 equiv.) in anhydrous THF (5 mL) was added
(151 MHz, MeOD): δC = 163.10 (Cq, TFA), 162.87 (Cq, TFA), dropwise within 10 min. The reaction mixture was stirred for
136.63 (+), 135.90 (Cq), 119.17 (+, TFA), 117.23 (+, TFA), 116.76 45 min at 0 °C. After dropwise addition of water (260 µL) the
(+), 79.55 (+), 65.65 (+), 40.61 (−), 35.88 (−), 29.34 (+), 24.45 (+). mixture was stirred for another 30 min. Then a 15% aqueous
Preparation of ((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicy- NaOH solution (260 µL) was added, followed by water (780 µL).
clo[3.1.0]hexan-6-yl)methanamine (8b). A solution of phthali- The mixture was warmed to room temperature, treated
mide 27b (16 mg, 0.042 mmol) and hydrazine hydrate (11 µL, with MgSO4 and filtered through a Celite pad. The solvent was
0.23 mmol, 5.4 equiv.) in anhydrous EtOH (0.85 mL) was evaporated under reduced pressure. The crude product was
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purified by chromatography (PE–EtOAc 1 : 1) to obtain com- diastereomeric mixture of compound 16 (2.10 g, 5.54 mmol,
pound 14 (1.85 g, 9.94 mmol, 87%) as a colorless oil.
70%) as a yellowish foam.
Rf = 0.34 (PE–EtOAc 1 : 1), 0.49 (EtOAc); [α]2D0 = +63.7 (DCM,
Major: Rf = 0.29 (PE–EtOAc 1 : 1); 1H-NMR (400 MHz,
c = 1.0); 1H-NMR (300 MHz, CDCl3): δH = 4.60–4.50 (m, 1H), CDCl3): δH = 7.80 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H),
4.18 (d, J = 5.9 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.58 (ddd, J = 7.01 (s, 1H), 4.91 (dd, J = 5.8, 4.4 Hz, 1H), 4.86 (dd, J = 5.9,
11.9, 6.0, 3.2 Hz, 1H), 3.41–3.31 (m, 1H), 2.37 (ddd, J = 13.1, 1.7 Hz, 1H), 4.60–4.52 (m, 1H), 4.17 (d, J = 6.0 Hz, 1H), 4.09 (q,
8.8, 7.0 Hz, 1H), 2.26–2.11 (m, 1H), 2.14 (br s, 1H), 1.81 (ddd, J = 7.1 Hz, 2H), 2.51–2.41 (m, 1H), 2.44 (s, 3H), 2.27–2.20 (m,
J = 13.1, 7.7, 1.1 Hz, 1H), 1.72 (dd, J = 3.8, 0.8 Hz, 1H), 1.22 (t, 1H), 1.88 (ddd, J = 13.6, 8.2, 1.2 Hz, 1H), 1.73 (d, J = 3.8, 1H),
J = 7.1 Hz, 3H); 13C-NMR (75 MHz, CDCl3): δC = 170.50 (Cq), 1.23 (t, J = 7.1, 3H); 13C-NMR (75 MHz, CDCl3): δC = 169.87
87.00 (+), 67.37 (+), 64.91 (−), 60.59 (−), 33.39 (+), 30.31 (−), (Cq), 159.32 (+), 145.83 (Cq), 132.87 (Cq), 129.97 (+), 129.63 (+),
27.56 (+), 14.33 (+); IR (ATR): ˜ν (cm−1) = 3460 (br), 2978, 2939, 86.60 (+), 85.71 (+), 79.54 (+), 67.06 (+), 60.71 (−), 33.52 (+),
2880, 1713, 1454, 1407, 1386, 1309, 1269, 1175, 1111, 1074, 30.01 (−), 26.70 (+), 21.82 (+), 14.29 (+).
1048, 980, 878, 851, 808, 712; MS (ESI): m/z (%) = 186.9 (40)
Minor: Rf = 0.29 (PE–EtOAc 1 : 1); 1H-NMR (400 MHz,
[MH+], 228.0 (100) [MH+MeCN], 373.1 (40) [2MH+], 390.0 (30) CDCl3): δH = 7.79 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H),
[2MNH4 ]; HRMS (ESI): calcd for C9H15O4 [MH+] 187.0965, 7.00 (s, 1H), 4.99 (dd, J = 6.4, 1.7 Hz, 1H), 4.88–4.81 (m, 1H),
+
found 187.0966.
4.63–4.55 (m, 1H), 4.15 (d, J = 6.0 Hz, 1H), 4.07 (q, J = 7.1 Hz,
Preparation of (1S,3R,5S,6S)-ethyl 3-formyl-2-oxabicyclo- 2H), 2.57–2.49 (m, 1H), 2.44 (s, 3H), 2.27–2.20 (m, 1H), 2.12
[3.1.0]hexane-6-carboxylate (15). Dess–Martin periodinane (ddd, J = 13.4, 8.2, 1.0 Hz, 1H), 1.73, (d, J = 3.8, 1H), 1.21 (t, J =
(4.24 g, 10.0 mmol, 1.05 equiv.) was added to a solution of 7.1, 3H); 13C-NMR (75 MHz, CDCl3): δC = 170.04 (Cq), 159.15
alcohol 14 (1.77 g, 9.52 mmol) in DCM (95 mL) at room temp- (+), 145.79 (Cq), 133.02 (Cq), 129.97 (+), 129.53 (+), 87.27 (+),
erature and stirred for 1 h. After completion the reaction was 86.65 (+), 68.76 (+), 67.33 (+), 60.64 (−), 33.15 (+), 30.58 (−),
quenched with a mixture of saturated aqueous Na2S2O3 solu- 27.11 (+), 21.82 (+), 14.29 (+).
tion (50 mL) and saturated aqueous NaHCO3 solution (50 mL).
Data for the isomeric mixture: IR (ATR): ˜ν (cm−1) = 2978,
The mixture was stirred for 15 min; afterwards the organic 2936, 1716, 1618, 1453, 1408, 1387, 1306, 1177, 1149, 1108,
layer was separated and the aqueous layer was extracted with 1086, 1075, 975, 934, 852, 813, 707, 668; Elemental analysis
DCM (2 × 50 mL). The combined organic layers were washed calcd (%) for C16H21NO6S·1.2H2O: C 53.91, H 5.88, N 3.49, S
with brine (1 × 50 mL), dried over MgSO4 and evaporated 8.00, found C 53.83, H 5.93, N 3.34, S 7.92.
in vacuo. The crude product was purified by chromatography
(PE–EtOAc 1 : 1) to give compound 15 (1.54 mg, 8.37 mmol, oxy)methyl)-2-oxabicyclo[3.1.0]hexane-6-carboxylate (18). To a
88%) as a yellowish oil. stirred solution of alcohol 14 (2.53 g, 13.6 mmol) in DCM
Preparation of (1S,3R,5S,6S)-ethyl 3-((tert-butyldimethyl-silyl-
Rf = 0.31 (PE–EtOAc 1 : 1); [α]2D0 = +44.8 (DCM, c = 0.5); (45 mL) under a nitrogen atmosphere, anhydrous NEt3
1H-NMR (300 MHz, CDCl3): δH = 9.59 (s, 1H), 4.64 (dd, J = 10.6, (2.8 mL, 20 mmol, 1.5 equiv.), TBSCl (2.48 g, 16.5 mmol,
3.8 Hz, 1H), 4.34 (dd, J = 5.7, 0.8 Hz, 1H), 4.08 (q, J = 7.1 Hz, 1.2 equiv.) and DMAP (83 mg, 0.68 mmol, 0.05 equiv.) were
2H), 2.51 (ddd, J = 13.4, 10.6, 5.7 Hz, 1H), 2.36 (dd, J = 13.3, added successively. The reaction mixture was stirred for 18 h at
3.9 Hz, 1H), 2.20 (td, J = 5.5, 3.9 Hz, 1H), 1.46 (dd, J = 3.8, room temperature and then quenched with a saturated
1.0 Hz, 1H), 1.23 (t, J = 7.1 Hz, 3H), 1.26–1.19 (m, 1H); 13C-NMR aqueous NH4Cl solution (40 mL). The layers were separated
(75 MHz, CDCl3): δC = 203.36 (+), 170.20 (Cq), 85.29 (+), 67.22 and the aqueous layer was extracted with DCM (2 × 20 mL).
(+), 60.85 (−), 30.26 (−), 27.54 (+), 25.21 (+), 14.31 (+); IR (ATR): The combined organic phases were dried over MgSO4, filtered
˜ν (cm−1) = 3435 (br), 2984, 1712, 1451, 1411, 1385, 1323, 1298, and concentrated in vacuo. Purification by column chromato-
1273, 1177, 1107, 1051, 1035, 976, 926, 870, 849, 796, 749, 702; graphy (PE–EtOAc 5 : 1) afforded compound 18 (3.88 g,
MS (CI): m/z (%) = 185.0 (15) [MH+], 202.1 (100) [MNH4 ]; 12.9 mmol, 95%) as a colorless oil.
+
HRMS (ESI): calcd for C9H13O4 [MH+] 185.0808, found
185.0808.
Rf = 0.52 (PE–EtOAc 5 : 1); [α]2D0 = +35.0 (DCM, c = 1.0);
1H-NMR (300 MHz, CDCl3): δH = 4.48 (ddt, J = 9.1, 7.0, 4.1 Hz,
Preparation of (1S,3R,5S,6S)-ethyl 3-(4-tosyl-4,5-dihydrooxa- 1H), 4.13 (d, J = 5.9 Hz, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.54 (dd,
zol-5-yl)-2-oxabicyclo[3.1.0]hexane-6-carboxylate (16). Finely J = 11.0, 4.0 Hz, 1H), 3.45 (dd, J = 11.0, 4.2 Hz, 1H), 2.33 (ddd,
powdered NaCN (70 mg, 1.42 mmol, 0.18 equiv.) was added in J = 13.0, 9.2, 6.9 Hz, 1H), 2.21–2.11 (m, 1H), 1.91 (ddd, J = 13.0,
one portion to a stirred solution of TosMIC (1.70 g, 8.70 mmol, 6.9, 0.8 Hz, 1H), 1.87 (dd, J = 3.9, 0.9 Hz, 1H), 1.21 (t, J =
1.1 equiv.) and aldehyde 15 (1.46 g, 7.91 mmol) in anhydrous 7.1 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H); 13C-NMR
EtOH (80 mL) at room temperature under a nitrogen atmos- (75 MHz, CDCl3): δC = 170.74 (+), 86.16 (+), 67.32 (+), 65.31 (−),
phere. After 1 h, the solvent was evaporated under reduced 60.22 (−), 32.22 (+), 30.13 (−), 27.42 (+), 25.94 (+), 18.38 (Cq),
pressure. The residue was dissolved in CHCl3 (100 mL) 14.23 (+), −5.35 (+), −5.43 (+); IR (ATR): ˜ν (cm−1) = 2955, 2931,
and washed with a saturated aqueous NaHCO3 solution 2858, 1720, 1463, 1408, 1309, 1256, 1176, 1112, 1096, 1054,
(1 × 100 mL). The aqueous layer was extracted with CHCl3 979, 839, 778; MS (ESI): m/z (%) = 301.0 (100) [MH+]; HRMS
(1 × 40 mL) and the combined organic layers were dried (EI): calcd for C15H28SiO4 [M+˙] 300.1757, found 300.1760.
over MgSO4, filtered and concentrated in vacuo. Purification
Preparation of ((1S,3R,5S,6R)-3-((tert-butyldimethylsilyloxy)-
by column chromatography (PE–EtOAc 1 : 1) afforded a 2 : 1 methyl)-2-oxabicyclo[3.1.0]hexan-6-yl)-methanol (19). To
a
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stirred ice-cooled solution of 18 (3.88 g, 12.9 mmol) in anhy- 2932, 2858, 1461, 1380, 1254, 1182, 1132, 1091, 1009, 840,
drous THF (50 mL) under a nitrogen atmosphere, a suspension 778, 738, 697; MS (ESI): m/z (%) = 241.1 (100) [M+ΔC7H7O],
of LAH (412 mg, 10.9 mmol, 0.84 equiv.) in anhydrous THF 349.1 (15) [MH+], 366.1 (65) [MNH4+], 714.5 (20) [2MNH4 ];
+
(5 mL) was added dropwise within 10 min. The reaction HRMS (ESI): calcd for C20H33O3Si [MH+] 349.2193, found
mixture was stirred for 45 min at 0 °C. After dropwise addition 349.2197.
of water (0.41 mL) the mixture was stirred for another 30 min.
Preparation of ((1S,3R,5S,6R)-6-(benzyloxymethyl)-2-oxabicy-
Then a 15% aqueous NaOH solution (0.41 mL) was added, fol- clo[3.1.0]hexan-3-yl)methanol (21). To a solution of compound
lowed by water (1.24 mL). The mixture was warmed to room 20 (3.03 mg, 8.69 mmol) in anhydrous THF (60 mL) a solution
temperature, treated with MgSO4 and filtered through a Celite of TBAF·3H2O (4.11 mg, 13.0 mmol, 1.5 equiv.) in anhydrous
pad. The solvent was evaporated under reduced pressure. THF (30 mL) was added and stirred for 13 h at room tempera-
The crude product was purified by column chromatography ture. After evaporating the solvent the crude product was puri-
(PE–EtOAc 3 : 1 then 1 : 1) to obtain compound 19 (3.17 g, fied by column chromatography (EtOAc) to give 21 (1.94 mg,
12.3 mmol, 95%) as a colorless oil.
8.28 mmol, 95%) as a colorless oil.
Rf = 0.30 (PE–EtOAc 1 : 1); [α]2D0 = +44.6 (DCM, c = 1.0);
Rf = 0.42 (EtOAc); [α]D20 = +47.2 (DCM, c = 1.0); 1H-NMR
1H-NMR (300 MHz, CDCl3): δH = 4.43 (tt, J = 7.9, 4.9 Hz, 1H), (300 MHz, CDCl3): δH = 7.39–7.23 (m, 5H), 4.59–4.49 (m, 1H),
3.73 (dd, J = 6.3, 1.1 Hz, 1H), 3.46 (d, J = 4.9 Hz, 2H), 3.37–3.21 4.48 (d, J = 2.2 Hz, 2H), 3.78 (dd, J = 6.2, 1.1 Hz, 1H), 3.56 (ddd,
(m, 2H), 2.23 (ddd, J = 12.8, 8.3, 7.2 Hz, 1H), 2.21 (br s, 1H), J = 11.4, 5.4, 3.2 Hz, 1H), 3.42–3.31 (m, 1H), 3.26 (dd, J = 10.5,
1.68 (ddd, J = 12.8, 7.6, 1.5 Hz, 1H), 1.51–1.43 (m, 1H), 7.0 Hz, 1H), 3.16 (dd, J = 10.5, 7.1 Hz, 1H), 2.26 (ddd, J = 12.8,
1.21–1.13 (m, 1H), 0.85 (s, 9H), 0.01 (s, 6H); 13C-NMR (75 MHz, 8.1, 7.3 Hz, 1H), 2.07 (br s, 1H), 1.69 (ddd, J = 12.8, 8.0, 1.6 Hz,
CDCl3): δC = 87.94 (+), 66.01 (−), 64.33 (+), 62.32 (−), 34.78 (+), 1H), 1.55 (dddd, J = 7.6, 6.0, 4.0, 1.6 Hz, 1H), 1.20 (tdd, J = 7.1,
31.54 (−), 26.01 (+), 21.98 (+), 18.45 (Cq), −5.25 (+), −5.27 (+); 4.0, 1.2 Hz, 1H); 13C-NMR (75 MHz, CDCl3): δC = 138.35 (Cq),
IR (ATR): ˜ν (cm−1) = 3386 (br), 2953, 2929, 2858, 1463, 1410, 128.51 (+), 127.78 (+), 127.74 (+), 88.08 (+), 72.67 (−), 69.47 (−),
1254, 1130, 1095, 1023, 837, 777, 669; MS (ESI): m/z (%) = 65.36 (−), 64.71 (+), 32.57 (+), 31.17 (−), 22.51 (+); IR (ATR):
241.0 (78) [MH+ΔH2O], 259.0 (55) [MH+], 276.1 (20) [MNH4+], ˜ν (cm−1) = 3421 (br), 3027, 2924, 2862, 1497, 1454, 1414, 1360,
300.0 (100) [MH+MeCN], 481.2 (35) [2MH+Δ2H2O], 499.2 (85) 1180, 1087, 1071, 1028, 987, 844, 810, 739, 698, 614; MS (ESI):
[2MH+ΔH2O], 517.2 (50) [2MH+]; HRMS (ESI): calcd for m/z (%) = 235.0 (5) [MH+], 469.0 (25) [2MH+], 486.1 (75)
C13H27O3Si [MH+] 259.1724, found 259.1731.
[2MH4 ], 491.1 (100) [2MNa+]; HRMS (ESI): calcd for
+
Preparation of (((1S,3R,5S,6R)-6-(benzyloxymethyl)-2-oxabi- C14H18NaO3 [MNa+] 257.1148, found 257.1153.
cyclo[3.1.0]hexan-3-yl)methoxy)(tert-butyl)-dimethylsilane (20).
Preparation of (1S,3R,5S,6R)-6-(benzyloxymethyl)-2-oxabicy-
To a solution of alcohol 19 (1.00 g, 3.87 mmol) in anhydrous clo[3.1.0]hexane-3-carbaldehyde (22). To a stirred solution of
DMF (25 mL), NaH (309 mg, 60 wt% in mineral oil, alcohol 21 (4.35 g, 18.6 mmol) in DCM (150 mL) was added in
7.74 mmol, 2.0 equiv.) was added in one portion at 0 °C under one portion Dess–Martin periodinane (8.66 g, 20.4 mmol, 1.1
a nitrogen atmosphere. The resulting suspension was stirred at equiv.) at room temperature. After 2 h, a saturated aqueous
0 °C for 10 min, and then benzyl bromide (919 µL, 7.74 mmol, NaHCO3 (60 mL) and a saturated aqueous Na2S2O3 (60 mL)
2.0 equiv.) was added dropwise at 0 °C. The mixture was were added. The mixture was stirred for another 15 min. After
allowed to warm to room temperature and stirred for 2 h. completion the reaction was quenched with a mixture of satu-
MeOH (5 mL) was added carefully to quench the reaction. The rated aqueous Na2S2O3 solution (60 mL) and saturated
solvent was evaporated under reduced pressure. The residue aqueous NaHCO3 solution (60 mL). The mixture was stirred for
was diluted in DCM and washed with a saturated aqueous 15 min, then the organic layer was separated and the aqueous
NH4Cl solution (20 mL). The aqueous phase was extracted with layer was extracted with DCM (2 × 50 mL). The combined
DCM (3 × 20 mL). The organic layers were combined, dried organic layers were washed with brine (1 × 50 mL), dried over
over MgSO4 and concentrated in vacuo. The resulting residue MgSO4 and evaporated in vacuo. Purification by column
was purified by column chromatography (PE–EtOAc 9 : 1) to chromatography (PE–EtOAc 1 : 1) afforded compound 22
obtain compound 20 (1.14 g, 3.28 mmol, 85%) as a colorless (3.87 g, 16.7 mmol, 90%) as a colorless oil.
oil.
Rf = 0.31 (PE–EtOAc 1 : 1); [α]2D0 = +57.5 (DCM, c = 1.0);
Rf = 0.22 (PE–EtOAc 9 : 1), 0.53 (PE–EtOAc 3 : 1); [α]2D0 = +22.5 1H-NMR (300 MHz, CDCl3): δH = 9.57 (d, J = 0.8 Hz, 1H),
(DCM, c = 1.0); 1H-NMR (300 MHz, CDCl3): δH = 7.38–7.22 7.39–7.21 (m, 5H), 4.58 (ddd, J = 10.2, 3.9, 0.7 Hz, 1H), 4.47 (d,
(m, 5H), 4.46 (ddd, J = 9.8, 8.1, 4.9 Hz, 3H), 3.75 (dd, J = 6.2, J = 1.1 Hz, 2H), 3.95 (dd, J = 5.9, 1.3 Hz, 1H), 3.30 (dd, J = 10.5,
1.1 Hz, 1H), 3.50 (d, J = 5.0 Hz, 2H), 3.35 (dd, J = 10.6, 6.6 Hz, 6.7 Hz, 1H), 3.17 (dd, J = 10.5, 7.1 Hz, 1H), 2.40 (ddd, J = 13.0,
1H), 3.09 (dd, J = 10.6, 7.6 Hz, 1H), 2.27 (ddd, J = 12.8, 8.3, 7.2 10.3, 5.9 Hz, 1H), 2.26 (ddd, J = 13.0, 4.0, 0.6 Hz, 1H), 1.53
Hz, 1H), 1.74 (ddd, J = 12.8, 7.5, 1.5 Hz, 1H), 1.61–1.45 (tdd, J = 5.8, 4.0, 0.6 Hz, 1H), 0.97 (tdd, J = 6.9, 3.9, 1.3 Hz, 1H);
(m, 1H), 1.23 (dddd, J = 7.7, 6.7, 4.0, 1.2 Hz, 1H), 0.90 (s, J = 2.9 13C-NMR (75 MHz, CDCl3): δC = 204.38 (+), 138.19 (Cq), 128.54
Hz, 9H), 0.06 (d, J = 1.0 Hz, 6H); 13C-NMR (75 MHz, CDCl3): δC (+), 127.81 (+), 127.77 (+), 86.02 (+), 72.77 (−), 69.06 (−), 64.86
= 138.50 (Cq), 128.49 (+), 127.75 (+), 127.68 (+), 87.80 (+), 72.50 (+), 31.08 (−), 25.84 (+), 20.30 (+); IR (ATR): ˜ν (cm−1) = 3031,
(−), 69.61 (−), 66.20 (−), 64.62 (+), 31.99 (+), 31.66 (−), 26.10 2942, 2860, 1730, 1497, 1455, 1422, 1362, 1091, 1076, 1030,
(+), 22.45 (+), 18.54 (Cq), −5.18 (+); IR (ATR): ˜ν (cm−1) = 3038, 988, 738, 699; MS (EI): m/z (%) = 91.1 (100) [C7H7 ], 231.1 (<1)
+
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[M˙ΔH+]; HRMS (ESI): calcd for C14H20NO3 [MNH4 ] 250.1438,
found 250.1439.
24a: Rf = 0.22 (DCM–saturated NH3 in MeOH 9 : 1); 1H-NMR
(300 MHz, CDCl3): δH = 8.28 (br s, 1H), 7.48 (s, 1H), 7.38–7.22
+
Preparation of 5-((1S,3R,5S,6R)-6-(benzyloxymethyl)-2-oxabi- (m, 5H), 6.83 (s, 1H), 5.42 (t, J = 7.5 Hz, 1H), 4.47 (d, J = 1.7 Hz,
cyclo[3.1.0]hexan-3-yl)-4-tosyl-4,5-dihydro-oxazole (23). Finely 2H), 3.86 (dd, J = 6.2, 1.2 Hz, 1H), 3.29–3.15 (m, 2H), 2.64–2.51
powdered NaCN (28 mg, 0.57 mmol, 0.22 equiv.) was added in (m, 1H), 2.15 (ddd, J = 12.8, 7.0, 1.4 Hz, 1H), 1.66–1.57 (m,
one portion to a stirred solution of TosMIC (555 mg, 1H), 1.45–1.34 (m, 1H); 13C-NMR (75 MHz, CDCl3): δC = 139.57
2.84 mmol, 1.1 equiv.) and aldehyde 22 (600 mg, 2.58 mmol) (Cq), 138.27 (Cq), 135.36 (+), 128.54 (+), 127.87 (+), 127.80 (+),
in anhydrous EtOH (25 mL) at room temperature under a 116.10 (+), 81.53 (+), 72.69 (−), 69.71 (−), 64.45 (+), 35.21 (−),
nitrogen atmosphere. The reaction mixture was stirred for 2 h. 30.89 (+), 22.67 (+).
The solvent was evaporated under reduced pressure. The
24b: Rf = 0.22 (DCM–saturated NH3 in MeOH 9 : 1); 1H-NMR
residue was dissolved in CHCl3 (30 mL) and washed with a (300 MHz, CDCl3): δH = 8.28 (br s, 1H), 7.55 (s, 1H), 7.38–7.22
saturated aqueous NaHCO3 solution (30 mL). The aqueous (m, 5H), 6.90 (s, 1H), 4.76 (t, J = 8.2 Hz, 1H), 4.50 (d, J = 2.6 Hz,
layer was extracted with CHCl3 (2 × 15 mL) and the combined 2H), 3.90–3.78 (m, 1H), 3.42–3.31 (m, 1H), 3.23–3.11 (m, 1H),
organic layers were dried over MgSO4, filtered and con- 2.38–2.23 (m, 2H), 1.59–1.48 (m, 1H), 1.19–1.05 (m, 1H);
centrated in vacuo. Purification by column chromatography 13C-NMR (75 MHz, CDCl3): δC = 138.22 (Cq), 137.81 (Cq),
(PE–EtOAc 1 : 1) afforded a 3 : 2 diastereomeric mixture of com- 135.62 (+), 128.54 (+), 127.86 (+), 127.80 (+), 115.90 (+), 74.14
pound 23 (854 mg, 2.00 mmol, 77%) as yellowish foam.
(+), 72.72 (−), 69.90 (−), 62.56 (+), 34.54 (−), 21.40 (+),
Major: Rf = 0.41 (PE–EtOAc 1 : 1); 1H-NMR (300 MHz, 20.80 (+).
CDCl3): δH = 7.81 (d, J = 8.3 Hz, 2H), 7.39–7.33 (m, 2H),
Data for the isomeric mixture: IR (ATR): ˜ν (cm−1) = 3090
7.34–7.20 (m, 5H), 7.00–6.96 (m, 1H), 4.94–4.91 (m, 1H), (br), 2936, 2858, 1716, 1670, 1496, 1453, 1362, 1313, 1273,
4.92–4.89 (m, 1H), 4.62–4.52 (m, 1H), 4.51–4.45 (m, 2H), 3.79 1087, 1071, 1027, 839, 738, 698, 626; MS (ESI): m/z (%) = 271.0
(dd, J = 6.2, 0.9 Hz, 1H), 3.31 (dd, J = 10.5, 6.5 Hz, 1H), 3.11 (100) [MH+], 312.1 (30) [MH+MeCN], 541.2 (40) [2MH+]; HRMS
(dd, J = 10.5, 7.3 Hz, 1H), 2.44 (s, 3H), 2.40–2.20 (m, 1H), 1.81 (ESI): calcd for C16H19N2O2 [MH+] 271.1441, found 271.1446.
(ddd, J = 13.3, 8.8, 1.3 Hz, 1H), 1.65–1.53 (m, 1H), 1.30–1.18
Preparation of ethyl 5-((1S,3R,5S,6R)-6-(benzyloxymethyl)-2-
(m, 1H); 13C-NMR (75 MHz, CDCl3): δC = 159.28 (+), 145.62 oxabicyclo[3.1.0]hexan-3-yl)-1H-imidazole-1-carboxylate (25a)
(Cq), 138.15 (Cq), 133.09 (Cq), 129.89 (+), 129.60 (+), 128.47 (+), and ethyl 5-((1S,3S,5S,6R)-6-(benzyloxymethyl)-2-oxabicyclo-
127.75 (+), 127.71 (+), 86.80 (+), 86.36 (+), 79.63 (+), 72.70 (−), [3.1.0]hexan-3-yl)-1H-imidazole-1-carboxylate (25b). A solution
69.00 (−), 64.69 (+), 33.03 (+), 30.85 (−), 22.12 (+), 21.79 (+).
of a 3 : 1 epimeric mixture of imidazole 24a and 24b (1.10 g,
Minor: Rf = 0.41 (PE–EtOAc 1 : 1); 1H-NMR (300 MHz, 4.06 mmol), ethyl chloroformate (733 µL, 7.72 mmol,
CDCl3): δH = 7.80 (d, J = 8.3 Hz, 2H), 7.39–7.33 (m, 2H), 1.9 equiv.), anhydrous pyridine (623 µL, 7.72 mmol, 1.9 equiv.)
7.34–7.20 (m, 5H), 7.00–6.96 (m, 1H), 4.98 (dd, J = 6.2, 1.7 Hz, and DMAP (79 mg, 0.65 mmol, 0.16 equiv.) in benzene (80 mL)
1H), 4.84 (dd, J = 6.2, 3.4 Hz, 1H), 4.58–4.50 (m, 1H), 4.47–4.42 was stirred for 10 min at 50 °C. After the addition of water
(m, 2H), 3.76 (dd, J = 6.3, 0.8 Hz, 1H), 3.26 (dd, J = 10.5, 6.8 Hz, (5 mL), the solvent was evaporated. A saturated aqueous NH4Cl
1H), 3.14 (dd, J = 10.5, 7.2 Hz, 1H), 2.44 (s, 3H), 2.43–2.33 (m, solution (50 mL) was added and extracted with DCM (3 ×
1H), 2.01 (ddd, J = 13.0, 8.4, 1.6 Hz, 1H), 1.65–1.53 (m, 1H), 25 mL). The extract was washed with brine, dried over MgSO4,
1.30–1.18 (m, 1H); 13C-NMR (75 MHz, CDCl3): δC = 159.35 (+), filtered and concentrated in vacuo. The residual oil was puri-
145.62 (Cq), 138.19 (Cq), 133.09 (Cq), 129.89 (+), 129.52 (+), fied by column chromatography (PE–EtOAc 1 : 1) to give a 3 : 1
128.45 (+), 127.75 (+), 127.71 (+), 87.66 (+), 87.31 (+), 79.13 (+), epimeric mixture of compounds 25a and 25b (1.01 g,
72.63 (−), 69.04 (−), 64.92 (+), 32.83 (+), 31.19 (−), 22.12 (+), 2.95 mmol, 73%) as a colorless oil.
1
21.79 (+).
25a: Rf = 0.26 (PE–EtOAc 1 : 1); H-NMR (300 MHz, CDCl3):
Data for the isomeric mixture: IR (ATR): ˜ν (cm−1) = 3033, δH = 8.06 (d, J = 1.3 Hz, 1H), 7.37–7.27 (m, 5H), 7.28 (t, J =
2948, 2861, 1616, 1597, 1486, 1455, 1362, 1319, 1304, 1292, 1.2 Hz, 1H), 5.38 (ddd, J = 8.4, 6.7, 0.9 Hz, 1H), 4.48 (d, J =
1148, 1108, 1086, 1071, 1028, 939, 848, 813, 739, 700, 664, 651, 3.8 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 3.88 (dd, J = 6.1, 1.2 Hz,
587, 533.
1H), 3.33 (dd, J = 10.5, 6.7 Hz, 1H), 3.13 (dd, J = 10.6, 7.4 Hz,
Preparation of 5-((1S,3R,5S,6R)-6-(benzyloxymethyl)-2-oxabi- 1H), 2.61 (ddd, J = 12.8, 8.6, 6.9 Hz, 1H), 2.15 (ddd, J = 12.8,
cyclo[3.1.0]hexan-3-yl)-1H-imidazole (24a) and 5-((1S,3S ,5S,6R)- 6.7, 1.4 Hz, 1H), 1.67–1.57 (m, 1H), 1.47–1.39 (m, 1H), 1.42 (t,
6-(benzyloxymethyl)-2-oxabicyclo[3.1.0]hexan-3-yl)-1H-imidazole J = 7.1 Hz, 3H); 13C-NMR (75 MHz, CDCl3): δC = 148.63 (Cq),
(24b). In a sealable pressure tube, oxazoline 23 (1.70 g, 145.81 (Cq), 138.32 (Cq), 137.17 (+), 128.41 (+), 127.70 (+),
3.98 mmol) and a saturated solution of NH3 in anhydrous 127.26 (+), 113.26 (+), 81.75 (+), 72.52 (−), 69.50 (−), 64.68 (+),
MeOH (40 mL, 70 equiv.) were heated at 95 °C for 16 h. 64.45 (−), 43.88 (−), 30.63 (+), 22.66 (+), 14.21 (+).
Within this time the solution turned red. After cooling,
25b: Rf = 0.24 (PE–EtOAc 1 : 1), 0.54 (EtOAc); 1H-NMR
the solvent was removed under reduced pressure. The (300 MHz, CDCl3): δH = 8.07 (d, J = 1.3 Hz, 1H), 7.35–7.26 (m,
residue was purified by column chromatography (DCM– 5H), 7.33–7.31 (m, 1H), 4.72 (dd, J = 8.8, 7.4 Hz, 1H), 4.49 (d,
saturated NH3 in MeOH 9 : 1) to give a 5 : 1 epimeric mixture J = 3.1 Hz, 2H), 4.43 (q, J = 7.1 Hz, 2H), 3.91 (dd, J = 5.5,
of compounds 24a and 24b (726 mg, 2.69 mmol, 68%) as a 1.6 Hz, 1H), 3.38 (dd, J = 10.5, 6.2 Hz, 1H), 3.13 (dd, J = 10.5,
colorless oil.
7.5 Hz, 2H), 2.35 (dd, J = 12.3, 7.3 Hz, 1H), 2.24 (ddd, J = 12.4,
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9.0, 5.0 Hz, 1H), 1.59–1.50 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H); vinyltetrahydrofuran-2-yl)-1H-imidazole-1-carboxylate
(28b).
13C-NMR (75 MHz, CDCl3): δC = 148.58 (Cq), 143.45 (Cq), DIAD (211 mg, 0.98 mmol, 1.5 equiv.) was added to a solution
138.34 (Cq), 137.26 (+), 128.44 (+), 127.72 (+), 127.67 (+), 113.85 of PPh3 (257 mg, 0.98 mmol, 1.5 equiv.) in anhydrous THF
(+), 74.88 (+), 72.58 (−), 69.71 (−), 64.49 (−), 62.91 (+), 34.52 (7 mL) at room temperature under a nitrogen atmosphere.
(−), 22.14 (+), 20.86 (+), 14.21 (+).
After stirring for 10 min phthalimide (144 mg, 0.98 mmol,
Data for isomeric mixture: IR (ATR): ˜ν (cm−1) = 3032, 2937, 1.5 equiv.) was added and stirred for another 10 min. After
2859, 1759, 1482, 1454, 1409, 1388, 1336, 1252, 1207, 1093, addition of alcohol 26a (165 mg, 0.65 mmol) in THF the reac-
1069, 1019, 843, 769, 740, 699, 607; MS (ESI): m/z (%) = 342.9 tion mixture was stirred overnight. The solvent was evaporated
(100) [MH+]; HRMS (ESI): calcd for C19H23N2O4 [MH+] under reduced pressure. The residue was purified by column
343.1652, found 343.1656.
chromatography (PE–EtOAc 5 : 1 to EtOAc) to obtain 28a
Preparation of ethyl 5-((1S,3R,5S,6R)-6-(hydroxymethyl)-2- (126 mg, 0.33 mmol, 51%), 28b (25 mg, 0.07 mmol, 10%) and
oxabicyclo[3.1.0]hexan-3-yl)-1H-imidazole-1-carboxylate (26a) 27a (72 mg, 0.19 mmol, 29%) as colorless oils.
and ethyl 5-((1S,3S,5S,6R)-6-(hydroxymethyl)-2-oxabicyclo-
28a: Rf = 0.37 (PE–EtOAc 1 : 1); [α]2D0 = −23.6 (DCM, c = 1.0);
[3.1.0]hexan-3-yl)-1H-imidazole-1-carboxylate (26b). A 3 : 1 epi- 1H-NMR (300 MHz, CDCl3): δH = 8.10 (d, J = 1.2 Hz, 1H), 7.85
meric mixture of compounds 25a and 25b (134 mg, (dd, J = 5.5, 3.0 Hz, 2H), 7.72 (dd, J = 5.5, 3.0 Hz, 2H), 7.40 (dd,
0.39 mmol), Pd(OH)2–C (20%, 95 mg) and cyclohexene J = 1.3, 0.7 Hz, 1H), 5.93 (d, J = 7.5 Hz, 1H), 5.86 (ddd, J = 17.1,
(1.6 mL, 16 mmol, 40 equiv.) in anhydrous EtOH (15 mL) was 10.3, 8.0 Hz, 1H), 5.50 (dd, J = 10.6, 4.9 Hz, 1H), 5.13 (dt, J =
refluxed for 1 h. After filtration through a Celite pad the 17.1, 1.2 Hz, 1H), 5.10–5.04 (m, 1H), 4.44 (q, J = 7.1 Hz, 2H),
solvent was evaporated. The residue was purified by column 4.01–3.85 (m, 1H), 2.65 (ddd, J = 12.2, 7.2, 5.0 Hz, 1H), 2.24
chromatography (EtOAc, then EtOAc–MeOH 19 : 1) to afford a (dt, J = 12.2, 11.3 Hz, 1H), 1.41 (t, J = 7.1 Hz, 3H); 13C-NMR
3 : 1 epimeric mixture of alcohol 26a and 26b (72 mg, (75 MHz, CDCl3): δC = 167.92 (Cq), 148.63 (Cq), 143.33 (Cq),
0.29 mmol, 73%) as a colorless foam. Separation of the 137.37 (+), 136.39 (+), 134.37 (+), 132.02 (Cq), 123.60 (+), 117.58
epimers by iterated chromatography.
(−), 114.40 (+), 85.06 (+), 76.33 (+), 64.59 (−), 46.68 (+), 39.34
26a: Rf = 0.38 (EtOAc–MeOH 19 : 1); [α]2D0 = −4.6 (DCM, c = (−), 14.27 (+); IR (ATR): ˜ν (cm−1) = 2985, 2927, 2853, 1760,
1
1.0); H-NMR (300 MHz, CDCl3): δH = 8.06 (d, J = 1.3 Hz, 1H), 1716, 1468, 1410, 1367, 1332, 1252, 1210, 1084, 1019 977, 919,
7.29 (m, 1H), 5.38 (ddd, J = 8.5, 6.6, 0.9 Hz, 1H), 4.45 (q, J = 891, 845, 769, 736, 721, 655, 611, 531; MS (ESI): m/z (%) =
7.1 Hz, 2H), 3.90 (dd, J = 6.2, 1.3 Hz, 1H), 3.40 (dd, J = 11.6, 381.9 (100) [MH+], 422.9 (45) [MH+MeCN], 763.2 (90) [2MH+];
7.4 Hz, 1H), 3.33 (dd, J = 11.6, 7.3 Hz, 1H), 2.61 (ddd, J = 12.8, HRMS (ESI): calcd for C20H20N3O5 [MH+] 382.1375, found
8.6, 6.9 Hz, 1H), 2.16 (ddd, J = 12.8, 6.7, 1.5 Hz, 1H), 1.75 (br s, 382.1365.
1H), 1.62 (tdd, J = 6.8, 4.0, 1.5 Hz, 1H), 1.50–1.43 (m, 1H), 1.42
28b: Rf = 0.33 (PE–EtOAc 1 : 1); [α]2D0 = +73.6 (DCM, c = 0.5);
(t, J = 7.1 Hz, 3H); 13C-NMR (75 MHz, CDCl3): δC = 148.69 (Cq), 1H-NMR (300 MHz, CDCl3): δH = 8.09 (d, J = 1.3 Hz, 1H), 7.83
145.65 (Cq), 137.31 (+), 113.54 (+), 81.81 (+), 64.60 (−), 64.51 (dd, J = 5.5, 3.0 Hz, 2H), 7.72 (dd, J = 5.5, 3.0 Hz, 2H),
(+), 62.61 (−), 34.83 (−), 33.45 (+), 22.44 (+), 14.30 (+); IR (ATR): 7.61–7.53 (m, 1H), 6.21 (d, J = 8.6 Hz, 1H), 5.69 (ddd, J = 17.5,
˜ν (cm−1) = 3373 (br), 2982, 2943, 2876, 1758, 1489, 1409, 1336, 10.2, 7.5 Hz, 1H), 5.20 (dt, J = 17.2, 1.3 Hz, 1H), 5.11–5.04 (m,
1253, 1176, 1103, 1068, 1018, 847, 768, 606; MS (ESI): m/z (%) 1H), 5.06–5.00 (m, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.56–3.41 (m,
= 252.9 (40) [MH+], 294.0 (15) [MH+MeCN], 505.1 (100) [2MH+]; 1H), 2.93–2.77 (m, 1H), 2.51–2.40 (m, 1H), 1.42 (t, J = 7.1 Hz,
HRMS (ESI): calcd for C12H17N2O4 [MH+] 253.1183, found 3H); 13C-NMR (75 MHz, CDCl3): δC = 167.77 (Cq), 148.78 (Cq),
253.1190.
143.44 (Cq), 136.78 (+), 134.30 (+), 133.84 (+), 131.98 (Cq),
26b: Rf = 0.36 (EtOAc–MeOH 19 : 1); [α]2D0 = +10.5 (DCM, c = 123.61 (+), 118.82 (−), 114.31 (+), 82.69 (+), 77.63 (+), 64.53 (−),
1
1.0); H-NMR (300 MHz, CDCl3): δH = 8.05 (d, J = 1.2 Hz, 1H), 47.93 (+), 36.93 (−), 14.33 (+); IR (ATR): ˜ν (cm−1) = 2985, 2955,
7.32–7.29 (m, 1H), 4.70 (dd, J = 8.6, 7.5 Hz, 1H), 4.42 (q, J = 2925, 1762, 1720, 1468, 1410, 1364, 1326, 1258, 1228, 1113,
7.1 Hz, 2H), 3.91 (dd, J = 5.5, 1.6 Hz, 1H), 3.41 (dd, J = 11.5, 1090, 1018, 901, 838, 792, 770, 722, 604, 530; MS (ESI): m/z (%)
6.8 Hz, 1H), 3.32 (dd, J = 11.5, 7.1 Hz, 1H), 2.32 (dd, J = 12.4, = 381.9 (100) [MH+], 763.2 (10) [2MH+]; HRMS (ESI): calcd for
7.2 Hz, 1H), 2.21 (ddd, J = 12.4, 9.0, 5.0 Hz, 1H), 1.58–1.47 (m, C20H20N3O5 [MH+] 382.1397, found 382.1403.
2H), 1.38 (t, J = 7.1 Hz, 3H); 13C-NMR (75 MHz, CDCl3): δC
=
27a: Rf = 0.27 (PE–EtOAc 1 : 1); [α]2D0 = −12.4 (DCM, c = 1.0);
148.66 (Cq), 143.43 (Cq), 137.38 (+), 113.98 (+), 75.10 (+), 64.64 1H-NMR (300 MHz, CDCl3): δH = 8.03 (d, J = 1.2 Hz, 1H), 7.83
(−), 62.86 (−), 62.42 (+), 34.59 (−), 24.90 (+), 20.54 (+), 14.30 (dd, J = 5.5, 3.0 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.24 (t,
(+); IR (ATR): ˜ν (cm−1) = 3349 (br), 2939, 2869, 1760, 1487, J = 1.0 Hz, 1H), 5.36 (t, J = 7.7 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H),
1409, 1342, 1254, 1123, 1018, 852, 768, 607; MS (ESI): m/z (%) 4.05 (dd, J = 6.2, 0.9 Hz, 1H), 3.51 (dd, J = 14.3, 7.1 Hz, 1H),
= 252.8 (100) [MH+], 505.1 (30) [2MH+]; HRMS (ESI): calcd for 3.38 (dd, J = 14.3, 8.2 Hz, 1H), 2.59 (ddd, J = 12.9, 8.4, 7.1 Hz,
C12H17N2O4 [MH+] 253.1183, found 253.1188.
1H), 2.07 (ddd, J = 12.7, 7.2, 1.6 Hz, 1H), 1.76 (ddd, J = 7.0, 3.9,
Preparation of ethyl 5-((1S,3R,5S,6R)-6-((1,3-dioxoisoindolin- 1.5 Hz, 1H), 1.57–1.48 (m, 1H), 1.40 (t, J = 7.1 Hz, 3H),
2-yl)methyl)-2-oxabicyclo[3.1.0]hexan-3-yl)-1H-imidazole-1-car- 1.30–1.20 (m, 1H); 13C-NMR (75 MHz, CDCl3): δC = 168.45 (Cq),
boxylate (27a), ethyl 5-((2R,4S,5R)-5-(1,3-dioxoisoindolin-2-yl)- 148.65 (Cq), 145.26 (Cq), 137.27 (+), 134.06 (Cq), 132.31 (+),
4-vinyltetrahydrofuran-2-yl)-1H-imidazole-1-carboxylate (28a) 123.37 (+), 113.43 (+), 82.36 (+), 65.09 (+), 64.54 (−), 38.00 (−),
and ethyl 5-((2R,4S,5S)-5-(1,3-dioxoisoindolin-2-yl)-4- 35.03 (−), 30.67 (+), 23.63 (+), 14.28 (+); IR (ATR): ˜ν (cm−1) =
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2977, 2931, 1760, 1711, 1467, 1433, 1409, 1391, 1357, 1336, 2.73 (dddd, J = 14.4, 10.4, 1.8, 0.7 Hz, 1H), 2.48 (dddd, J = 14.6,
1253, 1211, 1137, 1102, 1019, 950, 846, 769, 721, 614, 530; MS 7.3, 1.7, 0.6 Hz, 1H), 0.89 (s, 9H), 0.07 (s, 3H), 0.07 (s, 3H);
(ESI): m/z (%) = 381.9 (100) [MH+], 763.3 (75) [2MH+]; HRMS 13C-NMR (75 MHz, CDCl3): δC = 144.97 (+), 129.08 (+), 116.31
(ESI): calcd for C20H20N3O5 [MH+] 382.1397, found 382.1396.
(Cq), 109.70 (−), 83.00 (+), 65.64 (−), 30.71 (−), 26.01 (+), 18.50
Preparation of ethyl 5-((1S,3S,5S,6R)-6-((1,3-dioxoisoindolin- (Cq), −5.14 (+), −5.19 (+); IR (ATR): ˜ν (cm−1) = 2955, 2929, 2857,
2-yl)methyl)-2-oxabicyclo[3.1.0]hexan-3-yl)-1H-imidazole-1-car- 1641, 1472, 1463, 1253, 1105, 1006, 980, 834, 776, 667.
boxylate (27b). DIAD (54 mg, 0.25 mmol, 1.5 equiv.) was
31b: Rf = 0.40 (PE–EtOAc 5 : 1); [α]2D0 = +95.5 (DCM, c = 0.5);
added to a solution of PPh3 (65.5 mg, 0.25 mmol, 1.5 equiv.) in 1H-NMR (300 MHz, CDCl3): δH = 7.83 (dd, J = 5.6, 3.0 Hz, 2H),
anhydrous THF (1.3 mL) at room temperature under a nitrogen 7.72 (dd, J = 5.6, 3.0 Hz, 2H), 6.13 (d, J = 8.4 Hz, 1H), 5.62 (ddd,
atmosphere. After stirring for 10 min phthalimide (37 mg, J = 17.5, 10.2, 7.5 Hz, 1H), 5.15 (dt, J = 17.1, 1.4 Hz, 1H), 4.99
0.25 mmol, 1.5 equiv.) was added and stirred for another (ddd, J = 10.2, 1.5, 1.0 Hz, 1H), 4.18 (ddt, J = 10.7, 6.5, 5.2 Hz,
10 min. After addition of alcohol 26b (42 mg, 0.17 mmol) the 1H), 3.97 (dd, J = 10.5, 6.6 Hz, 1H), 3.82 (dd, J = 10.5, 4.9 Hz,
reaction mixture was stirred for 18 h. The solvent was evapor- 1H), 3.41–3.26 (m, 1H), 2.39 (dt, J = 12.1, 11.2 Hz, 1H), 2.13
ated under reduced pressure. The residue was purified by (ddd, J = 11.7, 7.6, 5.5 Hz, 1H), 0.89 (s, 9H), 0.07 (s, 3H), 0.06
column chromatography (PE–EtOAc 3 : 1 to 1 : 1) to obtain (s, 3H); 13C-NMR (75 MHz, CDCl3): δC = 167.89 (Cq), 134.26 (+),
compound 27b (17 mg, 0.04 mmol, 27%) as a colorless oil.
134.12 (+), 131.99 (Cq), 123.50 (+), 118.46 (−), 83.27 (+), 82.56
Rf = 0.43 (PE–EtOAc 1 : 3); [α]2D0 = +17.1 (DCM, c = 0.2); (+), 65.93 (−), 47.39 (+), 34.02 (−), 26.14 (+), 18.62 (Cq), −5.04
1H-NMR (400 MHz, CDCl3): δH = 8.07 (d, J = 1.3 Hz, 1H), (+), −5.11 (+); IR (ATR): ˜ν (cm−1) = 2956, 2928, 2857, 1787,
7.90–7.82 (m, 2H), 7.76–7.67 (m, 2H), 7.30 (s, 1H), 4.68 (t, J = 1772, 1720, 1470, 1416, 1370, 1351, 1327, 1255, 1117, 1101,
8.0 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 4.11 (dd, J = 5.7, 1.2 Hz, 1059, 1005, 924, 891, 838, 777, 720; MS (ESI): m/z (%) = 388.0
1H), 3.54 (dd, J = 14.2, 6.9 Hz, 1H), 3.42 (dd, J = 14.3, 7.8 Hz, (100) [MH+], 729.4 (15) [2MH+]; HRMS (ESI): calcd for
1H), 2.36–2.19 (m, 2H), 1.73–1.62 (m, 1H), 1.48–1.38 (m, 1H), C21H30NO4Si [MH+] 388.1939, found 388.1941.
1.41 (t, J = 7.1 Hz, 3H); 13C-NMR (101 MHz, CDCl3): δC = 168.50
31a: Rf = 0.38 (PE–EtOAc 5 : 1); [α]2D0 = −41.5 (DCM, c = 1.0);
(Cq), 148.68 (Cq), 143.44 (Cq), 137.38 (+), 134.12 (+), 132.33 1H-NMR (300 MHz, CDCl3): δH = 7.84 (dd, J = 5.6, 3.0 Hz, 2H),
(Cq), 123.43 (+), 113.93 (+), 75.15 (+), 64.59 (−), 63.38 (+), 38.09 7.72 (dd, J = 5.6, 3.0 Hz, 2H), 5.79 (ddd, J = 17.1, 10.2, 8.0 Hz,
(−), 34.48 (−), 21.89 (+), 21.56 (+), 14.30 (+); IR (ATR): ˜ν (cm−1
)
1H), 5.74 (d, J = 7.6 Hz, 1H), 5.09 (dt, J = 17.7, 1.4 Hz, 1H), 5.04
= 2978, 2936, 2873, 1758, 1707, 1467, 1391, 1336, 1251, 1139, (ddd, J = 7.9, 1.4, 0.9 Hz, 1H), 4.53 (dq, J = 5.2, 4.1 Hz, 6H),
1087, 1017, 944, 850, 769, 721, 611, 541, 501; MS (ESI): m/z (%) 3.87–3.73 (m, 7H), 3.73 (dd, J = 11.0, 4.2 Hz, 10H), 3.67 (dd, J =
= 381.9 (100) [MH+], 763.3 (10) [2MH+]; HRMS (ESI): calcd for 11.0, 4.4 Hz, 1H), 2.35 (ddd, J = 12.6, 7.6, 5.3 Hz, 1H), 1.89
C20H20N3O5 [MH+] 382.1397, found 382.1405.
(ddd, J = 12.2, 11.1, 10.1 Hz, 1H), 0.90 (s, 9H), 0.07 (s, 3H), 0.06
Preparation of 2-(((1S,3R,5S,6R)-3-((tert-butyldimethylsilyl- (s, 3H); 13C-NMR (75 MHz, CDCl3): δC = 167.95 (Cq), 136.90 (+),
oxy)methyl)-2-oxabicyclo[3.1.0]hexan-6-yl)-methyl)isoindoline- 134.31 (+), 132.06 (Cq), 123.57 (+), 117.25 (−), 85.23 (+), 80.85
1,3-dione (30), 2-((2R,3S,5R)-5-((tert-butyldimethylsilyloxy)- (+), 65.07 (−), 46.15 (+), 35.26 (−), 26.08 (+), 18.51 (Cq), −5.10
methyl)-3-vinyltetra-hydrofuran-2-yl)isoindoline-1,3-dione (31a), (+), −5.21 (+); IR (ATR): ˜ν (cm−1) = 2954, 2929, 2857, 1775,
2-((2S,3S,5R)-5-((tert-butyldimethylsilyloxy)-methyl)-3-vinyltetra- 1717, 1470, 1405, 1368, 1328, 1253, 1084, 996, 921, 872, 836,
hydrofuran-2-yl)isoindoline-1,3-dione (31b), and (R)-tert-butyl- 777, 718, 665, 530; MS (ESI): m/z (%) = 388.0 (70) [MH+], 405.0
+
+
dimethyl((4-vinyl-2,3-dihydrofuran-2-yl)methoxy)silane
(32). (70) [MNH4 ], 775.4 (20) [2MH+], 792.4 (100) [2MNH4 ]; HRMS
DIAD (1.58 g, 8.72 mmol, 1.5 equiv.) was added dropwise to a (ESI): calcd for C21H30NO4Si [MH+] 388.1939, found 388.1942.
solution of alcohol 19 (1.50 g, 5.82 mmol), PPh3 (2.29 g,
30: mp = 63–65 °C; Rf = 0.25 (PE–EtOAc 5 : 1); [α]2D0 = +28.9
1
8.72 mmol, 1.5 equiv.) and phthalimide (1.28 mg, 8.72 mmol, (DCM, c = 1.0); H-NMR (300 MHz, CDCl3): δH = 7.79 (dd, J =
1.5 equiv.) in anhydrous THF (116 mL) at 50 °C. After stirring 5.5, 3.0 Hz, 2H), 7.67 (dd, J = 5.4, 3.1 Hz, 2H), 4.46–4.34 (m,
at 50 °C for 1 h the mixture was cooled to room temperature 1H), 3.89 (dd, J = 6.2, 0.6 Hz, 1H), 3.47 (dd, J = 14.3, 6.9 Hz,
quenched with water (50 mL). The phases were separated and 1H), 3.41 (d, J = 4.9 Hz, 2H), 3.28 (dd, J = 14.3, 8.4 Hz, 1H),
the organic layer was extracted with DCM (3 × 25 mL). The 2.20 (ddd, J = 12.7, 8.1, 7.3 Hz, 1H), 1.70–1.53 (m, 2H),
combined organic layers were dried over MgSO4, filtered and 1.36–1.25 (m, 1H), 0.82 (s, 9H), −0.02 (s, 6H); 13C-NMR
concentrated in vacuo. The residue was purified by column (75 MHz, CDCl3): δC = 168.28 (Cq), 133.94 (+), 132.23 (Cq),
chromatography (PE–EtOAc 9 : 1, then 5 : 1) to obtain com- 123.22 (+), 87.79 (+), 65.97 (−), 64.86 (+), 37.87 (−), 31.45 (−),
pound 32 (100 mg, 0.42 mmol, 7%) as a colorless oil and com- 31.13 (+), 25.97 (+), 23.09 (+), 18.42 (Cq), −5.30 (+), −5.31 (+); IR
pound 31b (115 mg, 0.30 mmol, 5%), compound 31a (1.04 g, (ATR): ˜ν (cm−1) = 2955, 2929, 2856, 1772, 1712, 1468, 1433,
2.67 mmol, 46%) and compound 30 (665 mg, 1.72 mmol, 1391, 1356, 1330, 1253, 1188, 1137, 1088, 1007, 990, 950, 836,
29%) as colorless solids.
777, 720, 529; MS (ESI): m/z (%) = 388.1 (50) [MH+], 405.0 (100)
+
32: Rf = 0.59 (PE–EtOAc 9 : 1); [α]2D0 = −125.8 (DCM, c = 1.0); [MNH4 ]; HRMS (ESI): calcd for C21H30NO4Si [MH+] 388.1939,
1H-NMR (300 MHz, CDCl3): δH = 6.46 (ddd, J = 18.0, 11.0, found 388.1940.
0.6 Hz, 1H), 6.41 (m, 1H), 4.85 (dd, J = 10.7, 1.1 Hz, 1H),
Preparation of (1S,3R,5S,6S)-methyl 3-(oxazol-5-yl)-2-oxabicy-
4.85–4.76 (m, 1H), 4.70 (dddd, J = 10.5, 7.3, 5.9, 4.9 Hz, 1H), clo[3.1.0]hexane-6-carboxylate (33). To a solution of oxazoline
3.73 (dd, J = 10.9, 5.9 Hz, 1H), 3.65 (dd, J = 10.9, 4.8 Hz, 1H), 16 (396 mg, 1.04 mmol) in anhydrous MeOH (10 mL), K2CO3
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(289 mg, 2.09 mmol, 2 equiv.) was added under a nitrogen obtain compound 35 (51 mg, 0.17 mmol, 55%) as a colorless
atmosphere. The reaction mixture was refluxed for 30 min, solid.
quenched with water (15 mL) and extracted with DCM (3 ×
Mp = 83 °C; Rf = 0.51 (EtOAc); [α]2D0 = +18.9 (DCM, c = 1.0);
15 mL). The combined organic layers were dried over MgSO4, 1H-NMR (300 MHz, CDCl3): δH = 7.84 (dd, J = 5.5, 3.1 Hz, 2H),
filtrated and concentrated in vacuo. Purification by column 7.79 (s, 1H), 7.72 (dd, J = 5.5, 3.1 Hz, 2H), 6.92 (s, 1H),
chromatography (PE–EtOAc 1 : 1) afforded compound 33 5.45–5.37 (m, 1H), 4.08 (dd, J = 6.3, 1.0 Hz, 1H), 3.56 (dd, J =
(67 mg, 0.32 mmol, 31%) as a colorless solid.
14.4, 6.9 Hz, 1H), 3.36 (dd, J = 14.4, 8.4 Hz, 1H), 2.59 (ddd, J =
Mp = 61 °C; Rf = 0.36 (PE–EtOAc 1 : 3); [α]2D0 = +30.6 (DCM, 13.1, 8.6, 7.1 Hz, 1H), 2.09 (ddd, J = 13.1, 7.2, 1.4 Hz, 1H),
1
c = 1.0); H-NMR (300 MHz, CDCl3): δH = 7.84 (s, 1H), 6.97 (s, 1.84–1.75 (m, 1H), 1.59–1.51 (m, 1H); 13C-NMR (75 MHz,
1H), 5.47 (dd, J = 9.4, 6.1 Hz, 1H), 4.30 (dd, J = 5.9, 0.6 Hz, 1H), CDCl3): δC = 168.33 (Cq), 151.59 (Cq), 151.30 (+), 134.08 (+),
3.65 (s, 3H), 2.72 (ddd, J = 13.4, 9.5, 6.6 Hz, 1H), 2.37–2.30 (m, 132.18 (Cq), 123.87 (+), 123.36 (+), 78.48 (+), 65.23 (+), 37.73
1H), 2.26 (ddd, J = 13.5, 6.1, 0.7 Hz, 1H), 1.98 (dd, J = 3.9, 0.9 (−), 33.92 (−), 30.69 (+), 23.24 (+); IR (ATR): ˜ν (cm−1) = 3141,
Hz, 1H); 13C-NMR (75 MHz, CDCl3): δC = 170.80 (Cq), 151.63 3938, 1769, 1708, 1509, 1467, 1433, 1392, 1357, 1329, 1260,
(Cq), 151.51 (+), 124.18 (+), 77.16 (+), 67.36 (+), 51.89 (+), 33.00 1225, 1197, 1136, 1101, 1071, 1026, 950, 851, 798, 720, 645,
(−), 31.18 (+), 27.25 (+); IR (ATR): ˜ν (cm−1) = 3435 (br), 3129, 531; MS (EI): m/z (%) = 77.1 (8), 95.1 (100), 104.1 (6), 130.1 (6),
2954, 1716, 1507, 1440, 1394, 1311, 1274, 1198, 1171, 1107, 160.1 (18), 310.1 (1) [M+˙]; HRMS (EI): calcd for C17H14N2O4
1070, 963, 860, 715; MS (EI): m/z (%) = 95.0 (100), 180.1 (39) [M+˙] 310.0954, found 310.0956.
[M+ΔCHO], 209.1 (<1) [M+˙]; HRMS (EI): calcd for C10H11NO4
Preparation of methyl-N′-cyano-N-(((1S,3R,5S,6R)-3-(oxazol-5-
yl)-2-oxabicyclo[3.1.0]hexan-6-yl)methyl)carbamimido-thioate
[M+˙] 209.0688, found 209.0694.
Preparation of (1S,3R,5S,6R)-3-(oxazol-5-yl)-2-oxabicyclo- (36). A solution of aminooxazole 9a (19 mg, 0.11 mmol) and
[3.1.0]hexan-6-yl)methanol (34). To a stirred ice-cooled solu- dimethyl N-cyanodithioiminocarbonate (34 mg, 0.22 mmol,
tion of oxazole 33 (65 mg, 0.31 mmol) in anhydrous THF 2 equiv.) in EtOH was stirred at room temperature for 18 h.
(3 mL) under a nitrogen atmosphere, LAH (9.3 mg, 0.25 mmol, The solvent was evaporated under reduced pressure. The crude
0.8 equiv.) was added in small portions within 5 min. The reac- product was purified by column chromatography (DCM then
tion mixture was stirred for 30 min at 0 °C. After addition of DCM–MeOH 9 : 1) to afford compound 36 (29 mg, 0.11 mmol,
water (10 µL) the mixture was stirred for another 30 min. Then quantitative) as a colorless oil.
a 15% aqueous NaOH solution (10 µL) was added followed by
Rf = 0.51 (PE–EtOAc 9 : 1); [α]2D0 = +14.3 (DCM, c = 1.0);
water (30 µL). The mixture was warmed to room temperature, 1H-NMR (300 MHz, CDCl3): δH = 7.84 (s, 1H), 7.16 (s, 0.5H),
treated with MgSO4 and filtered through a Celite pad. The 6.96 (s, 1H), 6.53 (s, 0.5H), 5.43 (dd, J = 8.4, 7.2 Hz, 1H), 3.96
solvent was evaporated under reduced pressure. The crude (dd, J = 6.3, 0.9 Hz, 1H), 3.45–2.86 (m, signal broadening due
product was purified by column chromatography (EtOAc) to to rotamers, 2H), 2.73–2.32 (m, signal broadening due to rota-
obtain compound 34 (40 mg, 0.22 mmol, 71%) as a colorless mers, 3H), 2.62 (ddd, J = 13.0, 8.6, 7.0 Hz, 1H), 2.15 (dd, J =
solid.
13.0, 6.9 Hz, 1H), 1.77–1.68 (m, 1H), 1.50–1.37 (m, 1H);
Mp = 95 °C; Rf = 0.19 (EtOAc); [α]2D0 = +25.8 (DCM, c = 1.0); 13C-NMR (75 MHz, CDCl3): δC = 151.67 (Cq), 151.44 (+), 123.99
1H-NMR (400 MHz, CDCl3): δH = 7.81 (s, 1H), 6.93 (s, 1H), 5.41 (+), 78.34 (+), 64.99 (+), 44.01 (signal broadening due to rota-
(dd, J = 8.4, 6.9 Hz, 1H), 3.90 (dd, J = 6.2, 1.2 Hz, 1H), 3.39 (dd, mers, −), 33.82 (−), 30.09 (+), 23.30 (+), 14.64 (signal broaden-
J = 11.6, 7.2 Hz, 1H), 3.33 (dd, J = 11.5, 7.1 Hz, 1H), 2.59 (ddd, ing due to rotamers, +), CvN and CuN signals too weak to be
J = 13.0, 8.7, 6.9 Hz, 1H), 2.13 (ddd, J = 13.0, 6.7, 1.4 Hz, 1H), observed; IR (ATR): ˜ν (cm−1) = 3263 (br), 3126, 3011, 2939,
2.11 (br s, 1H), 1.69–1.62 (m, 1H), 1.40 (tdd, J = 7.1, 4.0, 1.2 Hz, 2174, 1716, 1554, 1511, 1430, 1357, 1285, 1182, 1104, 938, 846,
1H); 13C-NMR (75 MHz, CDCl3): δC = 152.18 (Cq), 151.31 (+), 645; MS (ESI): m/z (%) = 279.0 (30) [MH+], 296.0 (40) [MNH4 ],
+
123.62 (+), 78.11 (+), 64.70 (+), 62.12 (−), 33.85 (−), 33.45 (+), 557.1 (100) [2MH+]; HRMS (EI): calcd for C12H14N4O2S [M+˙]
22.08 (+); IR (ATR): ˜ν (cm−1) = 3369 (br), 3125, 2945, 2881, 278.0837, found 278.0833.
1508, 1461, 1414, 1353, 1262, 1176, 1106, 1026, 990, 966, 910,
Pharmacology
885, 846, 645; MS (CI): m/z (%) = 182.1 (99) [MH+], 199.1 (100)
+
[MNH4 ]; HRMS (LSI): calcd for C9H12NO3 [MH+] 182.0817, Histamine dihydrochloride was purchased from Alfa Aesar
found 182.0816.
Preparation of 2-(((1S,3R,5S,6R)-3-(oxazol-5-yl)-2-oxabicyclo- mine and [3H]histamine were from Perkin Elmer Life Sciences
[3.1.0]hexan-6-yl)methyl)isoindoline-1,3-dione (35). DIAD (Boston, MA). Guanosine diphosphate (GDP) was from Sigma-
GmbH & Co. KG (Karlsruhe, Germany). [3H]Nα-methylhista-
(96 mg, 0.45 mmol, 1.5 equiv.) was added dropwise to a solu- Aldrich Chemie GmbH (Munich, Germany), and unlabeled
tion of oxazole 34 (54 mg, 0.30 mmol), PPh3 (117 mg, GTPγS was from Roche (Mannheim, Germany). [3H]pyrilamine
0.45 mmol, 1.5 equiv.) and phthalimide (66 mg, 0.45 mmol, was purchased from Hartmann Analytic (Braunschweig,
1.5 equiv.) in anhydrous THF (6 mL) at 0 °C under a nitrogen Germany). [35S]GTPγS was from PerkinElmer Life Sciences
atmosphere. After stirring at 0 °C for 30 min the mixture (Boston, MA) or Hartmann Analytic GmbH (Braunschweig,
was allowed to warm to room temperature and the solvent Germany). [3H]UR-DE257 (N-(6-(3,4-dioxo-2-(3-(3-(piperidin-1-
was evaporated under reduced pressure. The residue was puri- ylmethyl) phenoxy)propylamino)-cyclobut-1-enylamino)hexyl)-
fied by column chromatography (PE–EtOAc 3 : 1 to EtOAc) to [2,3-3H]-propionamide) was synthesized in our laboratory.31
Org. Biomol. Chem.
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Paper
GF/C filters were from Whatman (Gaithersburg, USA). chosen ensured that not more than 10% of the total amount
For liquid scintillation counting was used: PerkinElmer of [35S]GTPγS added was bound to filters. Non-specific binding
MicroBeta2 2450 MicroplateCounter (Massachusetts, USA), was determined in the presence of 10 μM unlabeled GTPγS.
Brandel Harvester MWXRT-96TI, Brandel (Gaithersburg, USA).
Scintillation cocktail Rotiszint™ eco plus was from Carl Roth experiments performed in triplicate. Maximal responses
GmbH & Co KG (Karlsruhe, Germany). (intrinsic activities) were expressed as α-values. The α-value of
All data are presented as mean SEM of N independent
Radioligand binding experiments32 were performed on the histamine was set to 1.00; α-values of other compounds were
hH1R, hH2R, hH3R and hH4R as follows. H1R assays: Sf9 insect referred to this value. IC50 values were converted to Ki and KB
cell membranes expressing the hH1R + RGS4 were employed; values using the Cheng–Prussoff equation.36 Ki values were
H2R assays: Sf9 insect cell membranes expressing the hH2R analyzed by nonlinear regression and best fit to one-site
−Gsαs fusion protein were employed; H3R assays: Sf9 insect (monophasic) competition isotherms. pKB values from the
cell membranes coexpressing the hH3R, mammalian Gαi2 and functional [35S]GTPγS were analyzed by nonlinear regression
Gβ1γ2 were employed, H4R assays: Sf9 insect cell membranes and best fit to sigmoidal dose-response curves (GraphPad
coexpressing the hH4R, mammalian Gαi2 and Gβ1γ2 were Prism 5.0 software, San Diego, CA).
employed. The respective membranes were thawed and sedi-
mented by centrifugation at 4 °C and 13 000g for 10 min.
Membranes were resuspended in binding buffer (12.5 mM
MgCl2, 1 mM EDTA and 75 mM Tris/HCl, pH 7.4). Each well
Acknowledgements
(total volume 250 µL) contained 20 µg (hH1R), 28 µg (hH2R),
This work was supported by the Graduate Training Program
50 µg (hH3R), or 120 µg (hH4R) of membrane protein. Compe- (Graduiertenkolleg) GRK 760 “Medicinal Chemistry: Molecular
tition binding experiments were performed in the presence of Recognition – Ligand–Receptor Interactions” of the Deutsche
5 nM [3H]pyrilamine (hH1R), 30 nM [3H]UR-DE257 (hH2R), Forschungsgemeinschaft.
3 nM [3H]Nα-methylhistamine (hH3R) or 15 nM [3H]histamine
(hH3R and hH4R) and increasing concentrations of unlabeled
ligands. Incubations were conducted for 60 min at 25 °C and
shaking at 250 rpm. Bound radioligand was separated from
References
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bration phase of at least 12 h by liquid scintillation counting.
Functional [35S]GTPγS assays33 were performed as pre-
viously described for the H3R34 and H4R.35 H3R assays: Sf9
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Gαi2 and Gβ1γ2 were employed. H4R assays: Sf9 insect cell
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