Convergent, short synthesis of the muscarinic superagonist iperoxo

Article abstract of DOI:10.1016/j.tetlet.2010.04.130

Up to now the availability of iperoxo, an important superagonist of the muscarinic acetylcholine receptors, was limited because the synthesis of its precursor, the iperoxo base, was characterized by low yields, laborious chromatography and low reproducibility. Here we report a robust convergent three-step synthesis by means of a Mannich reaction and nucleophilic substitution at the 3-nitro-Δ²-isoxazoline. The new route combines short reaction time, high reproducibility and an overall yield increased from 12% to 42%.

Full text of DOI:10.1016/j.tetlet.2010.04.130

Tetrahedron Letters 51 (2010) 3470–3472
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Convergent, short synthesis of the muscarinic superagonist iperoxo
*
Jessica Kloeckner, Jens Schmitz, Ulrike Holzgrabe
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Up to now the availability of iperoxo, an important superagonist of the muscarinic acetylcholine recep-
tors, was limited because the synthesis of its precursor, the iperoxo base, was characterized by low yields,
laborious chromatography and low reproducibility. Here we report a robust convergent three-step syn-
Received 12 February 2010
Revised 14 April 2010
Accepted 19 April 2010
Available online 7 May 2010
thesis by means of a Mannich reaction and nucleophilic substitution at the 3-nitro-D
2-isoxazoline. The
new route combines short reaction time, high reproducibility and an overall yield increased from 12%
to 42%.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Iperoxo
Muscarinic superagonist
Convergent synthesis
Muscarinic receptors, belonging to the superfamily of G-pro-
tein coupled receptors and being part of the parasympathetic,
central nervous system are involved in the inhibitory and excit-
atory modulation of a large number of central and peripheral
physiological functions. Highly potent agonists are currently of
interest for the treatment of Alzheimer’s disease and pain. Re-
cently, the oxotremorine-M derivative iperoxo, [4-(4,5-dihydro-
isoxazole-3-yloxy)-but-2-ynyl]-N,N-dimethylamine, was reported
to have an activity which is more than two orders of magnitude
higher than the activity of the endogenous ligand acetylcholine.
The potency of receptor-mediated G-protein activation is indi-
nation of the mesylate with dimethylamine.1 Even though we
have improved each step of this synthesis, the reproducibility re-
mained low, because especially the last step may result in a dark
brown oil and no product formation. Thus, a better synthesis
pathway to achieve the iperoxo base is urgently needed. Eventu-
ally, iperoxo 4 is obtained by methylation of the tertiary amine 3
with methyliodide in quantitative yields.9
By application of isopentyl nitrite instead of propyl nitrite
3-nitro-D
2-isoxazoline 1 is formed in yields of 65% (Ref. 8: 45%).11
By means of a Mannich reaction with 2-propyn-1-ol, dimethyl-
ammonium hydrochloride and aqueous formaldehyde solution
in the presence of copper sulfate, the 4-dimethylamino-but-
2-yn-1-ol can be obtained in 80% yield (Ref. 12: 81%).13 In order
cated by [35S]GTP
cS binding to membranes from CHO cells stably
transfected with the human M2 receptor gene (see Fig. 1).1–4 Its
precursor, the iperoxo base, is an important building block for
the M2-subtype-selective dualsteric agonists such as hybrid 1
(Fig. 1).5–7
to combine the 3-nitro-D
2-isoxazoline and the 4-dimethylamino-
but-2-yn-1-ol, NaH is used for deprotonation and iperoxo is
obtained via a nucleophilic displacement reaction in 80% yield
(see Scheme 1).14 The spectroscopic data of iperoxo are in agree-
The synthesis of the key precursor, iperoxo base 3, is critical
because it consists of some steps which need laborious chro-
matographic purification procedures and, thus, results in a sub-
stantial loss of yield in addition to the need of huge amounts
of solvents. The four step synthesis is characterized by the con-
version of propyl nitrite with 1-bromo-3-chloropropane in the
1
ment with that reported in Ref.
Besides the fact that no laborious chromatography is needed
when following the procedure reported here, the overall yield
of iperoxo synthesis could be increased from 12% (if any product
could be isolated) to 42%. This can be attributed to the reduction
of the number of steps to three and the convergent synthesis
strategy. Furthermore, the reproducibility of the new synthesis
pathway is, compared to the procedure described in Ref. 1, very
high. Using the new synthesis, it is now possible to produce
iperoxo as a radioligand and further dualsteric agonists which
are necessary to establish structure–activity relationships and
to improve the pharmacological properties of this subtype-selec-
tive agonist.
presence of NaNO2,8 reaction of the resulting 3-nitro- 2-isoxaz-
D
oline 1 (see Scheme 1) with 2-butyne-1,4-diol in the presence
of NaH, mesylation of the primary alcohol and subsequent ami-
* Corresponding author. Tel.: +49 931 3185460.
E-mail address: u.holzgrabe@pharmazie.uni-wuerzburg.de (U. Holzgrabe).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.130

J. Kloeckner et al. / Tetrahedron Letters 51 (2010) 3470–3472
3471
O
N
O
O
N
N+
a
O
acetylcholine
3 (iperoxo-base)
O
N
I
O
N+
O
4 (iperoxo)
2 Br-
N
O
N
O
N+
N+
hybrid 1
O
b
ACh
Iperoxo
Hybrid1
1.00
0.75
0.50
0.25
0.00
-∞
-12 -11 -10 -9 -8 -7 -6 -5
test compound [log M]
Figure 1. (a) Structural formulae of acetylcholine (ACh), iperoxo and the dualsteric hybrid 1; (b) concentration–response curve of ACh, iperoxo and hybrid 1 showing the
agonistic potency of all compounds (n = 3–9).6,10 Maximum acetylcholine induced [35S]GTP
S binding is set as 1.0.
c
Cl
Br
+
ONO
HO
.
HN(CH3)2 HCl
CuSO4. 5 H2O
HCHO
NaNO2
N
N
NO2
O
+
HO
2 (80 %)
1 (65 %)
N
NaH
N
O
O
3 (80 %)
CH3I
N
O
N+
O
I
4 (iperoxo, quant.)
Scheme 1. New synthetic pathway to iperoxo base.

3472
J. Kloeckner et al. / Tetrahedron Letters 51 (2010) 3470–3472
stirred at room temperature for 24 h. The precipitate was filtered and washed
Acknowledgements
with cold diethyl ether to yield 99% of 4 (iperoxo) as a white solid.
10. Zahn, K.; Eckstein, N.; Tränkle, C.; Sadee, W.; Mohr, K. J. Pharmacol. Exp. Ther.
2002, 301, 720–728.
Thanks are due to the Deutsche Forschungsgemeinschaft (DFG,
Bonn) for the financial support (HO 1368/12-1) and Professor Dr. K.
Mohr, University of Bonn, Germany, for providing the biological
data of the agonists ACh, iperoxo and hybrid 1.
11. Optimized procedure for the synthesis of 3-nitro-
D
2-isoxazoline 1: NaNO2
(35.87 g, 0.52 mol) and isopentyl nitrite (30.5 g, 0.26 mol) were dissolved in
DMSO (250 mL) and 1-bromo-3-chloropropane (40 g, 0.25 mol) added
dropwise. After stirring for 24 h at room temperature the mixture was
poured into ice-water and extracted with dichloromethane. The organic layer
was dried over anhydrous Na2SO4 and the solvent removed in vacuo.
Distillation of the crude product at 2 Â 10À3 mbar and 73 °C yielded 65% of
References and notes
3-nitro-D
2-isoxazoline as a yellow oil.
1. Dallanoce, C.; Conti, P.; De Amici, M.; De Micheli, C.; Barocelli, E.; Chiavarini,
M.; Ballabeni, V.; Bertoni, S.; Impicciatore, M. Bioorg. Med. Chem. 1999, 7, 1539–
1547.
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C.; Impicciatore, M. Life Sci. 2000, 67, 717–723.
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13. Synthesis of 2: Dimethylammonium hydrochloride (8.72 g, 0.11 mol) was
dissolved in deionized water and 2 M aqueous NaOH solution added to
adjust pH 9. Aqueous formaldehyde solution (40%, 11.61 g, 0.15 mol), 2-
propyn-1-ol (5 g, 0.089 mol) and a solution of CuSO4Á5H2O (0.69 g in 4.46 mL
dem. water) were added and the pH-value adjusted to 8 using 2 M aqueous
NaOH solution. After heating to 80 °C for 1 h the mixture was poured into
30 mL of 25% aqueous ammonia solution. Continuous extraction with diethyl
ether for 12 h, drying of the organic layer over anhydrous MgSO4, and removal
of the solvent in vacuo yielded 80% of 2 as a yellow oil.
14. Synthesis of iperoxo base 3: 4-Dimethylamino-2-butyn-1-ol 2 (2 g, 0.0177 mol)
was suspended in 20 mL of abs THF under an inert atmosphere, NaH (60%
suspension in paraffine, 0.7 g, 0.018 mol) added and the mixture stirred at
room temperature for 1 h. 1 (2.04 g, 0.018 mol, dissolved in 10 mL of THF) was
added dropwise and refluxed for 3 h. The mixture was poured into deionized
water, extracted with chloroform, the organic layer dried over anhydrous
Na2SO4 and the solvent removed in vacuo. The crude product was purified via
column chromatography (MeOH/CHCl3 9:1) to yield 80% of 3 (iperoxo base) as
an orange oil. Microanalyses (C, H, N) of the oxalate (3ÁC2H4O4) agreed with the
theoretical value 0.3% (Anal. C11H16N2O6).
7. Mohr, K.; Traenkle, C.; Kostenis, E.; Barocelli, E.; De Amici, M.; Holzgrabe, U. Br.
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8. Wade, P. A. J. Org. Chem. 1978, 43, 2020–2022.
9. Synthesis of iperoxo 4: Iperoxo base 3 (0.42 g, 0.0023 mol) was dissolved in 7 mL
of abs chloroform, methyliodide (0.82 g, 0.0058 mol) added and the mixture