Synthesis and pharmacological evaluation of conformationally restricted κ-opioid receptor agonists

Article abstract of DOI:10.1039/c6md00441e

In order to obtain novel polar κ agonists the κ-pharmacophoric ethylenediamine structural element was embedded in a rigid bicyclic scaffold. The pyridooxazine system was selected since it contains polar O- and N-atoms in the 1- and 7-positions, respectively. An axially oriented pyrrolidine ring was attached at the 5-position and the dichlorophenylacetyl moiety was introduced at N-4. The key steps of the 11-step synthesis are a double Henry reaction of iminodiacetaldehyde 7 with nitromethane and the introduction of the azido moiety of 13 by Mitsunobu reaction of the alcohol 11 with Zn(N₃)₂·(pyridine)₂. The X-ray crystal structure analysis of 17b shows a dihedral angle N(acyl)-C-C-N(pyrrolidine) of ?60.8(2)°, which is close to the postulated optimal angle. Moderate κ affinity was found for the secondary amine 17a (K_i = 132 nM) and the methyl derivative 17b (K_i = 266 nM). In the [³⁵S]GTPγS assay the secondary amine 17a showed 28% agonistic activity compared to U-69,593. Although 17a and 17b contain all crucial κ-pharmacophoric elements, their κ affinity is rather low, which might be attributed to the unfavorable cis-orientation of the pyrrolidine ring and the dichlorophenylacetamido moiety and/or the additional O- and N-atoms in the 1- and 7-positions.

Full text of DOI:10.1039/c6md00441e

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DOI: 10.1039/C6MD00441E
Synthesis and pharmacological evaluation of conformationally restricted
κꢀopioid receptor agonists†
Yvonne Wenker,a Michael Soeberdt,b Constantin Daniliuc,c Sonja Ständer,d Dirk
Schepmann,a Bernhard Wünsch*a,e
a Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelmsꢀ
Universität Münster, Corrensstraße 48, Dꢀ48149 Münster, Germany
Tel.: +49ꢀ251ꢀ8333311; Fax: +49ꢀ251ꢀ8332144; Eꢀmail: wuensch@uniꢀmuenster.de
b Dr. August Wolff GmbH & Co. KG Arzneimittel, Sudbrackstr. 56, Dꢀ33611 Bielefeld,
Germany
c
OrganischꢀChemisches Institut der Westfälischen WilhelmsꢀUniversität Münster,
Corrensstr. 40, Dꢀ48149 Münster, Germany
d
Center for Chronic Pruritus and Department of Dermatology, University Hospital
Münster, vonꢀEsmarchꢀStraße 58, Dꢀ48149 Münster, Germany
e
CellsꢀinꢀMotion Cluster of Excellence ((EXC 1003 – CiM), Westfälische Wilhelmsꢀ
Universität Münster, Germany
†The authors declare no competing interests.
Abstract
In order to obtain novel polar κ agonists the κꢀpharmacophoric ethylenediamine
structural element was embedded in a rigid bicyclic scaffold. The pyridooxazine
system was selected, since it contains polar Oꢀ and Nꢀatoms in 1ꢀ and 7ꢀposition,
respectively. An axially oriented pyrrolidine ring was attached at 5ꢀposition and the
dichlorophenylacetyl moiety was introduced at Nꢀ4. The key steps of the 11ꢀstep
synthesis are a double Henry reaction of iminodiacetaldehyde 7 with nitromethane
1

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and the introduction of the azido moiety of 13 by Mitsunobu reaction of the alcohol 11
.
with Zn(N3)2 (pyridine)2. The Xꢀray crystal structure analysis of 17b shows a dihedral
angle N(acyl)ꢀCꢀCꢀN(pyrrolidine) of ꢀ60.8(2)°, which is close to the postulated optimal
angle. Moderate κ affinity was found for the secondary amine 17a (Ki = 132 nM) and
the methyl derivative 17b (Ki = 266 nM). In the [35S]GTPγS assay the secondary
amine 17a showed 28 % agonistic activity compared to Uꢀ69,593. Although 17a and
17b contain all crucial κꢀpharmacophoric elements, their κ affinity is rather low, which
might be attributed to the unfavorable cisꢀorientation of the pyrrolidine ring and the
dichlorophenylacetamido moiety and/or the additional Oꢀ and Nꢀatom in 1ꢀ and 7ꢀ
position.
Key words: κꢀopioid receptor agonists; iminodiacetaldehyde; Henry reaction;
nitropiperidineꢀ3,5ꢀdiols, pyridooxazines; conformational restriction; dihedral angle;
receptor selectivity
1. Introduction
Strong analgesia is achieved by activation of ꢀꢀ, κꢀ and δꢀopioid receptors. Whereas
dangerous side effects, such as respiratory depression as well as physical and
psychical dependence, are mediated by typical ꢁ agonists, κ agonists do not produce
these side effects. However, activation of κꢀopioid receptors is associated with
centrally mediated sedation, dysphoria, and strong diuresis inhibiting the clinical use
of κ agonists so far.1ꢀ4
κꢀOpioid receptors are localized not only in the central nervous system, but also in
some tissues in the periphery, including the skin, bowels and joints. Activation of
2

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these peripherally localized receptors can be used for the treatment of inflammatory
and itching skin diseases (e.g. atopic dermatitis) and visceral pain.5ꢀ7 Therefore, we
are interested in κ agonists, which are able to activate κꢀopioid receptors with high
affinity and intrinsic activity, but cannot pass the bloodꢀbrainꢀbarrier and enter the
central nervous system. Due to this property they should stay in the periphery and
thus cannot induce centrally mediated side effects, as outlined above.
1
N
Cl
Cl
3
R
4
O
N
Cl
Cl
N
O
5
N
CH3
Cl
Cl
3
N
O
4
N
2
1
1
HN
Cl
Cl
4a
O
5
N
N
1
2
X
H3CO2C
4
N
4
N
Cl
Cl
4a
5
7
N
R
N
H3C
O
N
2
5 (X = NR, O)
Figure 1: Development of the novel κ agonists 5 from prominent lead compounds 1ꢀ
4. 1 (Uꢀ50,488): Ki = 0.34 nM; 2: Ki = 0.31 nM; 3a (R = CO2CH3): Ki = 9.7 nM; 4a (R =
CO2CH3): Ki = 0.35 nM.
In addition to morphinoids,8,9 peptides10,11 and natural products (salvinorins),12,13
ethylenediamines represent a privileged class of κ agonists.14 In order to obtain high
κ agonistic activity one Nꢀatom of the ethylenediamine substructure has to be
embedded in a pyrrolidine ring and the other Nꢀatom has to be acylated with the 2ꢀ
(3,4ꢀdichlorophenyl)acetyl moiety. Uꢀ50,488 (1) represents the first potent κ agonist
3

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(Ki = 0.34 nM) of this class. (Figure 1) In Uꢀ50,488 the κꢀpharmacophoric
ethylenediamine structure is part of a transꢀconfigured cyclohexane ring.15 However,
the ethylenediamine structure can also be part of a piperidine16 or piperazine ring.17,18
The piperazine derivative 2 shown in Figure 1 interacts in the subnanomolar range
(Ki = 0.31 nM) with κꢀopioid receptors.18
Combination of the cyclohexane ring of Uꢀ50,488 (1) with the piperazine ring of 2
resulted in the perhydroquinoxalines 3 and 4. The κ affinity of the trans,transꢀ
configured compounds 3 can be modulated by the substituent at the Nꢀatom (Nꢀ1)
outside the κꢀpharmacophore. The methoxycarbonyl derivative 3a (R = CO2CH3)
reveals a κ affinity of 9.7 nM. However, the 3ꢀpyridylmethyl derivative 3b (R = 3ꢀ
pyridylmethyl) interacts in the subnanomolar range with the κꢀopioid receptor (Ki =
0.13 nM). Both compounds 3a and 3b represent full κ agonists in the [35S]GTPγS
assay.19ꢀ21
In contrast to the trans,transꢀconfigured perhydroquinoxalines 3, the cyclohexane
and piperazine rings of 4 are cisꢀannulated. Changing of the configuration of the
perhydroquinoxaline ring system increased the κ affinity considerably. The κ affinity
of the racemic methoxycarbonyl derivative 4a (R = CO2CH3) is 0.35 nM, which
resides almost exclusively in the dextrorotatory (4aR,5S,8aS)ꢀconfigured enantiomer
(Ki = 0.25 nM).22
In this manuscript, we report on the synthesis and pharmacological evaluation of κꢀ
opioid receptor agonists of type 5. The scaffold of 5 consists of an annulated
piperazine (X = NR) or morpholine ring (X = O) and a piperidine ring. The κꢀ
4

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pharmacophoric ethylenediamine system is found in the substructure “N(acyl)ꢀCHꢀ
CHꢀN(pyrrolidine)”. In contrast to the κ agonists 2ꢀ4, ligands 5 have two positions in
the core system, which allow the modulation of the pharmacodynamic and
pharmacokinetic properties of the ligands: X in 1ꢀposition should be either a polar Oꢀ
or Nꢀatom increasing the polarity of the compounds. The additional Nꢀatom in 7ꢀ
position of the ring system contributes to the overall polarity of the ligands and
moreover, allows the introduction of various substituents. A high polarity of the κ
agonists is desired in order to minimize the penetration of the bloodꢀbrainꢀbarrier and
reduce centrally mediated κ agonist side effects.
2. Synthesis
For the synthesis of bicyclic κ agonists 5 a similar strategy was followed as for the
synthesis of perhydroquinoxalines 3.19,20 Thus, iminodiacetaldehydes 7 were
selected as starting materials, which were prepared by Malaprade cleavage of
pyrrolidineꢀ3,4ꢀdiols 6 with NaIO4.23 (Scheme 1) At first dialdehyde 7a was reacted
with nitromethane and NaOH in methanol under the same reaction conditions as
described for the transformation of glutaraldehyde.24,25 Under these reaction
conditions the double Henry reaction did not lead to a clean product, probably due to
hydrate, hemiacetal and/or acetal formation of dialdehyde 7a with water and/or
methanol. Therefore, the solvent methanol was replaced by tertꢀbutanol, THF and
ethyl acetate. The highest yield of 8a was obtained with NaOH in ethyl acetate. After
flash chromatographic purification, the achiral (3r)ꢀconfigured 4ꢀnitropiperidineꢀ3,5ꢀ
diol 8a with one center of pseudochirality was isolated in 59 % yield. The benzoyl
derivative 7b was reacted in a THF/tertꢀbutanol mixture affording 50 % yield of
nitrodiol 8b.
5

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OH
OH
OH
CH=O
(c)
(a)
(b)
NO2
OH
N
R
R N
N
CH=O
R
8a,b
7a,b
6a,b
6ꢀ8
a
R
Boc
b
C(=O)Ph
Cl
O
OH
O
OH
O
(e)
(d)
NH2
OH
NH
OH
N
N
H
N
N
OH
Boc
Boc
Boc
11
10
9
Scheme 1: Synthesis of bicyclic lactams 11.
Reagents and reaction conditions: (a) NaIO4, THF, rt, 24 h, 99 % (7a), 50 % (7b).23
(b) CH3NO2, NaOH, EtOAc or THF/tertꢀbutanol, rt, 12ꢀ20 h, 59 % (8a), 50 % (8b). (c)
H2, 1 bar (balloon), RaneyꢀNi, EtOH, rt, 5 h, 86 %. (d) ClCH2C(=O)Cl, THF, NEt3, rt, 1
h, 53 %. (e) Bu4NI, NaH, THF, rt, 48 h, 61 %. Only one enantiomer of racemate 11 is
shown in the Scheme.
In case of 2ꢀnitrocyclohexaneꢀ1,3ꢀdiols the OH moieties could be replaced by various
amino groups by simple stirring the nitrodiol with the respective primary or secondary
amine in water.19,20 However, in case of the piperidines 8a and 8b all reactions with
amines failed to give the corresponding nitrodiamines. Since it was hypothesized that
the sterically demanding and electron withdrawing Boc and benzoyl protective groups
of 8a and 8b were the origin of this problem, the Boc group was replaced by other
protective groups. Treatment of 8a with trifluoroacetic acid afforded the secondary
amine 8c, which was reacted with allyl bromide, benzaldehyde/NaBH(OAc)3, and 4ꢀ
methoxybenzaldeyhyde/NaBH(OAc)3 to provide the allyl, benzyl, and 4ꢀ
methoxybenzyl derivatives 8dꢀf, respectively. (Scheme 2) However, as reported for
the Boc and benzoyl derivatives 8a,b, neither the allyl (8d), benzyl (8e) nor the 4ꢀ
6

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methoxybenzyl (8f) derivatives reacted with various primary and secondary amines to
provide nitropiperidinediamines.
Scheme 2: Exchange of the NꢀBoc group of nitrodiol 8a by other protective groups.
Reagents and reaction conditions: (a) CF3CO2H, CH2Cl2, rt, 3 h, 62 %. (b)
Ph(C=O)Cl, NEt3, CH2Cl2, rt, 48 h, 43 % (8b). (c) allyl bromide, K2CO3, THF, rt, 24 h,
15 % (8d). (d) PhCH=O, NaBH(OAc)3, NEt3, CH3CN, rt, 20 h, 50 % (8e). (e) 4ꢀ
Methoxybenzaldehyde, NaBH(OAc)3, NEt3, THF, rt, 1 h, 38 % (8f).
Thus the nitrodiol 8a was reduced with H2 in the presence of the catalyst Raney Ni to
give the aminodiol 9. (Scheme 1) Reaction of 9 with chloroacetyl chloride provided
the amide 10, which was cyclized upon treatment with NaH and Bu4NI to afford the
morpholine annulated piperidine 11 in 61 % yield. This cyclization led to
desymmetrization of the system: whereas the achiral (4r)ꢀconfigured diols 8ꢀ10
contain a center of pseudochirality, the bicyclic compound 11 is chiral (racemic
mixture) with three centers of chirality.
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O
O
O
O
O
O
(d)
(b)
(a)
NH
NH
NH
11
HN
N
N
Boc
N
Boc
NR2
13: NR2 = NH2
N3
12
(c)
14: NR =
N
15a
2
(g)
O
O
O
O
(h)
(f)
NH
(e)
N
Cl
Cl
NH
R
R
R
N
N
N
O
N
N
N
15cꢀg
17aꢀg
16aꢀg
15ꢀ17
R
(i)
a
b
c
d
e
f
H
CH3
17f
17a
CH2CH3
CH2CH2CH2CH3
cylopentyl
CH2Ph
g
CH2(3ꢀpyridyl)
Scheme 3: Synthesis of arylacetamides 17.
.
Reagents and reaction conditions: (a) Zn(N3)2 (pyridine)2, PPh3, DIAD, THF, rt, 4.5 h,
70 %. (b) H2, 1 bar (balloon), Pd/C, CH3OH, rt, 18 h, 90 %. (c) 1,4ꢀdiiodobutane,
NaHCO3, CH3CN, reflux, 20 h, 55 %. (d) CF3CO2H, CH2Cl2, rt, 20 h, 18 %. (e)
RCH=O or cyclopentanone, NaBH(OAc)3, THF, rt, 50 min ꢀ 20 h, 74 ꢀ 95 % (from 14).
(f) AlCl3, LiAlH4 (1:3), THF, rt, 20 min, then addition of lactam 15, THF, 0 – 20 °C, 15
ꢀ 60 min. (g) AlCl3, LiAlH4 (1:3), THF, rt, 20 min, then addition of lactam 14, THF, 0 °C
for 45 min, then rt for 20 min, 99 % (16b). (h) 2ꢀ(3,4ꢀdichlorophenyl)acetyl chloride,
CH2Cl2, NaOH, 0 – 20 °C, 71 ꢀ 92 % (from 14 or 15); yield of 17g only 38 % due to
side reactions. (i) H2, 1 bar (balloon), Pd/C, THF/H2O, HCl, rt, 30 min, 90 %. Only one
enantiomer of racemic mixtures 12ꢀ17 is shown in the Scheme.
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In the next step the remaining OH moiety of 11 should be transformed into a
pyrrolidine ring. Mesylation of the secondary alcohol 11 and subsequent nucleophilic
substitution with pyrrolidine as well as oxidation followed by reductive amination of
the formed ketone did not lead to pyrrolidine derivatives. Therefore, the alcohol 11
.
was reacted in a Mitsunobu reaction with Zn(N3)2 (pyridine)2, PPh3 and DIAD26 to give
the azide 12. (Scheme 3) Reduction of the azido group with H2 and Pd/C provided
the primary amine 13, which reacted with 1,4ꢀdiiodobutane to afford the pyrrolidine 14
in 55 % yield.
Scheme 4: Synthesis of different (dichlorophenyl)acetamides.
Reagents and reaction conditions: (a) 1. AlCl3, LiAlH4 (1:3), THF, rt, 20 min, then
addition of lactam 15g, THF, 0 °C for 45 min, then rt for 20 min; 2. 2ꢀ(3,4ꢀ
dichlorophenyl)acetyl chloride, 1.4 equiv., CH2Cl2, NaOH, rt, 12 h, 20 % (from 15g).
(b) 1. CF3CO2H, CH2Cl2, rt, 4 h; 2. 2ꢀ(3,4ꢀdichlorophenyl)acetyl chloride, CH2Cl2,
NaOH, rt, 50 min, 68 % (from 14). Only one enantiomer of racemic mixtures 17h and
18 is shown in the Scheme.
In order to introduce diverse substituents at the Nꢀatom in 7ꢀposition of the ring
system outside the κ pharmacophore, the Boc group of 14 was removed with
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trifluoroacetic acid and the resulting secondary amine 15a was alkylated reductively
with various aldehydes and a ketone in the presence of NaBH(OAc)327 to provide the
tertiary amines 15cꢀg. Reduction of the lactams 15cꢀg with AlH3, which was prepared
in situ by mixing AlCl’3 and LiAlH4 in the ratio 1:3,28 led to the amines 16cꢀg. The
secondary amine 16a and the methylated amine 16b were obtained by reduction of
the lactams 15a and the Bocꢀderivative 14, respectively. In the last step the
secondary amines 16bꢀg were acylated with (dichlorophenyl)acetyl chloride to
produce
the
desired
(dichlorophenyl)acetamides
17bꢀg.
The
(dichlorophenyl)acetamide 17a with a proton at the Nꢀatom in 7ꢀposition was
obtained by hydrogenolytic removal of the benzyl group of 17f.
The yield of the pyridylmethyl derivative 17g was only 38 %, which is due to side
reactions during the AlH3 reduction of the pyridylmethyl derivative 15g. On the one
side the pyridine ring was reduced to give a piperidine ring, which was acylated by
(dichlorophenyl)acetyl chloride. On the other side the pyridylmethyl group was
cleaved off leading to 16a, which was acylated twice. Performing this reaction in a
larger scale allowed the isolation of the diacylated compound 17h in 20 % yield.
(Scheme 4) The (dichlorophenyl)acetamide 18 bearing the κꢀpharmacophoric
elements in a different arrangement at the ring system, was obtained by acylation of
the secondary amine 15a, which was prepared in situ by TFA cleavage of the Bocꢀ
group of 14. (Scheme 4)
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Figure 2: Xꢀray crystal structure of (dichlorophenyl)acetamide 17b (thermal ellipsoids
are shown at 15 % probability).
Elemental unit (a0 ≠ b0 ≠ c0, α = γ = 90°, β = 105.364(8)°) with the lattice constants
a = 14.1810(20) Å, b = 12.8440(9) Å und c = 11.3534(8) Å; the crystal has the space
group P21/c.
Recrystallization of the methylated derivative 17b led to crystals, which were suitable
for Xꢀray crystal structure analysis. (Figure 2) The crystal structure nicely confirms
the transꢀannulation of the piperidine and morpholine rings. The pyrrolidinyl group
adopts an axial orientation in a relative cisꢀconfiguration to the Nꢀacyl structural
element. The piperidine ring exists in a chair conformation, whereas the morpholine
ring adopts a twist conformation. The dihedral angle of the ethylenediamine κꢀ
pharmacophore (N1ꢀC10ꢀC9ꢀN21) was determined to be ꢀ60.8(2)°. This dihedral
angle is very close to the dihedral angle of the ethylenediamine substructure of Uꢀ
50,488 (60°), which has been postulated to be optimal for high κ affinity.29
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For the bicyclic system 18 bearing the required κꢀpharmacophoric elements at
different positions a similar dihedral angle N(acyl)ꢀCꢀCꢀN(pyrrolidine) of 63.7° was
calculated by MMX/AM1 method.
3. Pharmacological evaluation
The affinity of the pyridooxazines 17 and 18 towards the κꢀopioid receptor was
determined in competitive receptor binding studies. The tritium labeled potent and
selective κ agonist [3H]Uꢀ69,593 was used as radioligand and homogenates
prepared from guinea pig brain served as receptor material. A high concentration (10
ꢁM) of nonꢀlabeled Uꢀ69,593 was used to determine the nonꢀspecific binding.30,31
The Ki values of the reference compounds Uꢀ69,593, naloxone and morphine are
listed in Table 1 to demonstrate the validity of the affinity data.
In addition to the κ affinity the affinity towards ꢀꢀ and δꢀopioid receptors was
recorded, to determine the selectivity of the ligands over related opioid receptors.
Since small structural changes of potent κ ligands often leads to high σ1 and/or σ2
affinity, the affinity towards the σ receptors was also included into this study.32ꢀ34
The highest κ affinity was found for the secondary amine 17a showing a Ki value of
132 nM. Introduction of a methyl moiety decreased the κ affinity to a Ki value of 286
nM. A further increase of the size of the substituent at Nꢀ7 led to a further decrease of
κ affinity. Obviously, substituents larger than a methyl moiety are not tolerated at this
position, which is in contrast to the perhydroquinoxalines 3.
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Table 1: Receptor affinity of pyridooxazines 17 and 18 towards opioid and σ
receptors.
Κi ± SEM [nM]
κ
ꢀ
δ
σ1
σ2
compd.
R
[3H]ꢀ(+)ꢀ
[3H]Uꢀ69,593 [3H]DAMGO [3H]DPDPE
[3H]DTG
pentazocine
17a
17b
17c
17d
17e
17f
H
132 ± 44
286 ± 63
1300
>1000
>1000
>1000
502
>1000
>1000
>1000
>1000
717
>1000
>1000
391
>1000
>1000
102
CH3
CH2CH3
(CH2)3CH3 >1000
cyclopentyl >1000
CH2ꢀphenyl 912
192
316
447
181
45
261 ± 79
260
983
>1000
CH2ꢀ3ꢀ
>1000
pyridyl
17g
633
666
291
>1000
17h
18
DCPAa
>1000
>1000
0.97 ± 0.40
6.9 ± 0.5
35 ± 6.0
ꢀ
>1000
>1000
991
>1000
ꢀ
>1000
>1000
>1000
>1000
Uꢀ69,593
ꢀ
ꢀ
ꢀ
ꢀ
Naloxone
2.1 ± 0.5
2.4 ± 0.5
ꢀ
ꢀ
Morphine
3.9 ± 2.1
2.0 ± 0.3
ꢀ
ꢀ
(+)ꢀpentazocine
ꢀ
ꢀ
ꢀ
ꢀ
5.4 ± 0.5
6.6 ± 0.9
ꢀ
Haloperidol
ꢀ
78 ± 2.3
a) DCPA = dichlorophenylacetyl;
Ki values are given as mean values ± SEM of three experiments (n = 3); due to low
affinity, the Ki values of some compounds were recorded only once; if the reduction
of radioligand binding at a test compound concentration of 1 ꢁM is lower than 40 % a
value >1000 nM is given; the exact reduction of radioligand binding in % is given in
Table SI1 (Supporting Information);
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In the class of perhydroquinoxalines 3 a proton (3c: R = H) or a methyl moiety (3d: R
= CH3) at Nꢀ1 resulted in very high κ affinity with Ki values of 2.1 nM (3c) and 2.7 nM
(3d).20 Moreover, the pyridylmethyl group at Nꢀ1 led to extraordinarily high κ affinity
(3b: Ki = 0.13 nM).21 In the class of pyridooxazines 17 the ligands with a proton (17a)
and a methyl moiety (17b) show the highest κ affinity, but the affinity is 60ꢀ and 100ꢀ
fold lower than the κ affinity of the corresponding perhydroquinoxalines 3c and 3d,
respectively. The pyridylmethyl substituted pyridooxazine 17g was not tolerated by
the κꢀopioid receptor, it did not compete with the radioligand even at the very high
concentration of 1 ꢁM.
The reduced κ affinity of pyridooxazines 17 can be explained by an unfavorable
orientation
of
the
κꢀpharmacophoric
elements
pyrrolidine
and
(dichlorophenyl)acetylamido moiety. In the potent lead compounds 1, 3, and 4 these
pharmacophoric elements are transꢀoriented with respect to the cyclohexane ring,
which is in contrast to the cisꢀorientation in 17. However, the dihedral angle N(acyl)ꢀ
CꢀCꢀN(pyrrolidine) is ꢀ60.8(2)° according to the Xꢀray crystal structure analysis. This
value is close to the postulated value of 60° for Uꢀ50,488,29 which indicates that the
dihedral angle alone cannot be the origin of the low affinity.
Alternatively, the low κ affinity of pyridooxazines 17 could be explained by the Oꢀ
atom in 1ꢀposition replacing the (substituted) Nꢀatom in the lead compounds 2, 3, and
4. Although the Oꢀatom has quite different properties than the (substituted) Nꢀatom,
the observed effect should not result from this substitution.
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Finally, the Nꢀatom in 7ꢀposition of pyridooxazines 17 was postulated to be
responsible for low κ affinity. It could be possible that the high polarity of the Nꢀ
containing functional group together with large substituents at Nꢀ7 are not tolerated
by the κꢀopioid receptor. The Nꢀsubstituents will clash with lipophilic amino acid
residues in the κꢀopioid receptor binding site.
The negligible κ affinity of 18 was unexpected, since it contains all required κꢀ
pharmacophoric elements. Only the position of the (dichlorophenyl)acetyl moiety at
the bicyclic system is different from those in ligands 17.
In general the ꢁꢀ and δꢀopioid receptor affinity of the pyridooxazines 17 and 18 is
rather low. In particular the most potent κ ligands 17a and 17b did not compete with
the ꢁꢀ and δꢀselective radioligands [3H]DAMGO and [3H]DPDPE even at a
concentration of 1 ꢁM. Obviously, 17a and 17b show high selectivity over ꢁꢀ and δꢀ
opioid receptors.
Introduction of larger substituents at the Nꢀatom, such as a butyl (17d), cyclopentyl
(17e), benzyl (17f) or pyridylmethyl (17g) moiety led to moderate ꢁ affinity in the
range of 260ꢀ630 nM. With exception of the butyl derivative 17d, the δ affinity of
these ligands is in the same order (260ꢀ720 nM). However, 17dꢀg cannot be
regarded as potent ꢁ and/or δ ligands. The moderate interaction of the ligands 17dꢀg
with ꢁꢀ and δꢀopioid receptors was very surprising, since they contain the structural
features (dichlorophenylacetamide, pyrrolidine), which are responsible for high κ
affinity. This observation may be explained by the structural similarity of κꢀ, ꢁꢀ and δꢀ
opioid receptors belonging to the same class of receptors.
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Since small structural changes of κ agonists could result in potent σ ligands,32ꢀ34 the
affinity towards σ1 and σ1 receptors was included into this study. The most interesting
κ ligands 17a and 17b did not show any σ1 or σ2 affinity up to a concentration of 1 ꢁM,
indicating high selectivity over both σ receptor subtypes. However, the compounds
17d and 17e without κ affinity displayed moderate σ1 affinity of 192 nM and 181 nM,
respectively. For the cyclopentyl derivative 17e and the allyl derivative 17c σ2 affinity
of 45 nM and 102 nM was determined, respectively. It is assumed that large lipophilic
substituents at the Nꢀatom (e.g. butyl, cyclopentyl) increase the affinity of 17d and
17e towards σ1 and σ2 receptors. A similar observation was made in the class of
benzomorphans.33,34
In order to determine the κ agonistic activity, the most potent κ ligand 17a (Ki = 132
nM) of this series of compounds was investigated exemplarily in the [35S]GTPγS
assay.35,36 At a concentration of 1 ꢁM the secondary amine 17a showed 27 %
agonistic activity compared to the prototypical full κ agonist Uꢀ69,593. Obviously 17a
behaved in this assay as weak partial agonist.
The logP value of the most potent κ agonist 17a was calculated with different
software packages and the values were compared with the logP values calculated for
the lead compounds 1ꢀ4. The results are summarized in Table 2. It can be seen that
the calculated logP values of 17a are 1ꢀ2 orders of magnitude lower than the logP
values of the lead compounds 1ꢀ4, indicating an increased polarity for 17a. These
data indicate that the selected modifications introduced into 17a are useful to reduce
the lipophilicity, which may translate into reduced penetration into the central nervous
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system. In a model of the bloodꢀbrainꢀbarrier, it was shown that the
perhydroquinoxaline 3c could not pass the endothelial cell layer. Compared to 3c,
the new κ agonist 17a has a different stereochemistry, the NHꢀgroup is at a different
position, but most importantly, it has an additional Oꢀatom within the ring system. The
calculations of the logP values in Table 2 reveal an even higher polarity for 17a
compared to 3c and therefore a reduced penetration into the central nervous system
is expected.
Table 2: logP values of 1ꢀ4 and 17a calculated with different software packages.
compd.
clogPa)
clogP(H+)b) milogPc) milogP(H+)d)
1 (Uꢀ50,488)
4.91
3.73
4.14
4.14
2.67
5.36
4.09
4.50
4.50
3.16
4.57
2.80
3.70
3.70
2.29
1.41
ꢀ0.33
0.54
0.54
ꢀ0.87
2
3c (R = H)
4
17a
a) clogP = calculated logP value (ChemBioDraw 14.0.0.117).
b) clogP(H+) = calculated logP value of the monoprotonated species (ChemBioDraw
14.0.0.117).
c) milogP = calculated logP value (www.molinspiration.com v2014.11).
d) milogP(H+) = calculated logP value of the monoprotonated species
(www.molinspiration.com v2014.11 ).
4. Conclusion
In this project the κꢀpharmacophoric elements of the ethylenediamine class of κ
agonists were newly arranged at a novel heterocyclic system, i.e. the hydrogenated
pyridooxazine scaffold. In an elevenꢀstep synthesis the dichlorophenylacetyl moiety
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and the pyrrolidine ring were attached at the transꢀconfigured bicyclic ring system.
Although the dihedral angle of the pharmacophoric elements (N(acyl)ꢀCꢀCꢀ
N(pyrrolidine) was ꢀ60.8(2)° and thus close to the postulated optimum, the most
promising compound 17a reached only an κ affinity of 132 nM. Substituents larger
than a CH3 moiety were not tolerated at the additional Nꢀatom within the bicyclic ring
system. An unfavorable orientation of the pharmacophoric elements, the additional
Oꢀatom in 1ꢀposition and/or the substituted Nꢀatom in 7ꢀposition of the ring system
are discussed to be responsible for the moderate κ affinity. The secondary amine 17a
showing the highest κ affinity (Ki = 132 nM) in this series of ligands behaved as weak
partial agonist in the [35S]GTPγS assay.
5. Experimental, Chemistry
5.1. General
Unless otherwise noted, moisture sensitive reactions were conducted under dry
nitrogen. THF was dried with sodium/benzophenone and was freshly distilled before
use. Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck). Flash
chromatography (fc): Silica gel 60, 40–64 ꢁm (Merck); parentheses include: diameter
of the column, eluent, fraction size, Rf value. Melting point: Melting point apparatus
SMP 3 (Stuart Scientific), uncorrected. MS: MAT GCQ (ThermoꢀFinnigan); IR: IR
1
spectrophotometer 480Plus FTꢀATRꢀIR (Jasco). H NMR (400 MHz), 13C NMR (100
MHz): Unity Mercury Plus 400 spectrometer (Varian); δ in ppm related to
tetramethylsilane; coupling constants are given with 0.5 Hz resolution.
5.2. HPLC method for the determination of the purity
Merck Hitachi Equipment; UV detector: Lꢀ7400; autosampler:Lꢀ7200; pump: Lꢀ7100;
degasser: Lꢀ7614; column: LiChrospher® 60 RPꢀselect B (5 ꢁm), 250ꢀ4 mm; flow
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rate: 1.00 mL/min; injection volume: 5.0 ꢁL; detection at λ = 210 nm; solvents: A:
water with 0.05 % (v/v) CF3CO2H; B: CH3CN with 0.05 % (v/v) CF3CO2H: gradient
elution: (A %): 0ꢀ4 min: 90 % , 4ꢀ29 min: gradient from 90 % to 0 %, 29ꢀ31 min: 0 %,
31ꢀ31.5 min: gradient from 0 % to 90 %, 31.5ꢀ40 min: 90 %.
5.3. Xꢀray diffraction
Data sets were collected with a Nonius Kappa CCD diffractometer. Programs used:
data collection, COLLECT (R. W. W. Hooft, Bruker AXS, 2008, Delft, The
Netherlands); data reduction DenzoꢀSMN;37 absorption correction, Denzo;38 structure
solution SHELXSꢀ97;39 structure refinement SHELXLꢀ9740 and graphics, XP
(BrukerAXS, 2000). Rꢀvalues are given for observed reflections, and wR2 values are
given for all reflections.
5.4. General Procedures
General Procedure A for cleavage of the Bocꢀprotective group of 14 and
subsequent reductive alkylation of secondary amine 15a
Trifluoroacetic acid (30 eq) was added to a solution of carbamate 14 in CH2Cl2. The
mixture was stirred for 20 h and the solvent was removed in vacuum. The residue
was dissolved in CH2Cl2 and the pH value was adjusted to pH 7 using Merck® ion
exchanger III. The mixture was filtered and the solvent was removed under reduced
pressure. The residue was dissolved in THF, and the aldehyde (1.0 eq – 1.5 eq) or
ketone (1 eq) was added and the mixture was stirred for 15 min. In case of the
ketone trifluoroacetic acid was used to accelerate the conversion. NaBH(OAc)3 was
added and the mixture was stirred for 50 min ꢀ 18 h until the amine was converted
completely. A small amount of water was added and the mixture was stirred for
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additional 20 min. The solvent was removed under reduced pressure and the product
was purified.
General Procedure B for the preparation of alane
Under N2 atmosphere dry AlCl3 (45 mg, 0.33 mmol) was suspended in THF (2.5 mL)
and the mixture was stirred for 5 min at 0 °C. Then a solution of LiAlH4 in THF (1.0 M,
1.0 mL, 1.0 mmol) was added dropwise. The mixture was stirred for 20 min at rt. The
amount of AlH3 equivalents in the resulting suspension is 1.33 mmol.
5.5. Synthetic procedures
5.5.1. tertꢀButyl (4r)ꢀ3,5ꢀdihydroxyꢀ4ꢀnitropiperidineꢀ1ꢀcarboxylate (8a)
At 0 °C nitromethane (0.50 g, 8.2 mmol) and 15 % (m/m) NaOH (1 mL) were added
dropwise to a solution of iminodiacetaldehyde 7a (1.50 g, 7.5 mmol) in EtOAc (5 mL).
The mixture was stirred for 12 h at rt, then neutralized using Amberlite® ion exchange
resin (IRꢀ120) and stirred for 1 h. The mixture was filtered, concentrated under
reduced pressure and the residue was purified by fc (∅ = 5 cm, h = 16 cm,
CH2Cl2/ethyl acetate = 4/1, V = 65 mL, Rf = 0.22 (cyclohexane/ethyl acetate = 4/1)).
Colorless crystals,
mp 145 – 150 °C,
yield 1.17 g (59 %).
C10H18N2O6
(M = 262.3 g/mol). 1H NMR (d6ꢀDMSO): δ (ppm) = 1.41 (s, 9 H, C(CH3)3), 2.50 – 2.79
(m, 2 H, 2ꢀCH2, 6ꢀCH2), 3.69 – 3.76 (m, 2 H, 2 x CHOH), 3.90 – 4.10 (m, 2 H, 2ꢀCH2,
6ꢀCH2), 4.35 (t, 3J = 9.8 Hz, 1 H, CHNO2) 5.93 (d, 3J = 5.6 Hz, 2 H, 2 x OH). 13C NMR
(d6ꢀDMSO): δ (ppm) = 27.9 (3 C, C(CH3)3), 47.1 (NCH2), 48.0 (NCH2), 67.5
(2 x CHOH), 79.7 (C(CH3)3), 96.5 (CHNO2), 153.6 (C=O). MS (EM, ESI): m/z = calcd.
for C10H17N2O6 [M − H]− 261.1087, found 261.1053; calcd. for C10H15N2O5
[M − H2O − H]− 243.0981, found 243.0943. IR (neat): (cmꢀ1) = 3437/3378 (w, OꢀH),
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2996/2920/2866 (w, CꢀH), 1691 (s, C=Ocarbamate); 1554/1370 (s, CꢀNO2). Purity
(HPLC): 89 %, tR = 11.31 min.
5.5.2. (4r)ꢀ4ꢀNitropiperidineꢀ3,5ꢀdiol (8c)
Trifluoroacetic acid (0.12 mL, 1.52 mmol) was added to
a
solution of
nitropiperidinediol 8a (80.0 mg, 0.31 mmol) in CH2Cl2 (5 mL). The mixture was stirred
for 3 h at rt. The solvent was removed under reduced pressure and the product was
purified by fc (∅ = 2 cm, h = 16 cm, CH2Cl2/CH3OH/NH3 = 9.5/4.7/0.3, V = 10 mL,
Rf = 0.54 (CH3OH)). Yellow resin, yield 30.7 mg (62 %). C5H10N2O4
(M = 162.1 g/mol). 1H NMR (d6ꢀDMSO):
δ
(ppm) = 2.24 (dd, 2J = 12.6 Hz,
2
3
3J = 10.6 Hz, 2 H, 2ꢀCH2ax, 6ꢀCH2ax), 2.93 (dd, J = 12.7 Hz, J = 5.1 Hz, 2 H, 2ꢀ
3
CH2eq, 6ꢀCH2eq), 3.69 – 3.80 (m, 2 H, 2 x CHOH), 4.18 (t, J = 9.8 Hz, 1 H, CHNO2),
5.54 (s broad, 3 H, OH, NH). MS (EM, APCI): m/z = calcd. for C5H11N2O4 [M + H]+
163.0719, found 163.0707; calcd. for C5H9N2O3 [M + H – H2O]+ 145.0613, found
145.0605. IR (neat): (cmꢀ1) = 3283 (w, OꢀH), 2928/2827 (w, CꢀH), 1548/1372 (s, Cꢀ
NO2).
5.5.3. tertꢀButyl (4r) 4ꢀaminoꢀ3,5ꢀdihydroxypiperidineꢀ1ꢀcarboxylate (9)
A mixture of nitropiperidinediol 8a (5.65 g, 21.5 mmol) and RaneyꢀNi (12.0 g, 1 mL
precipitate
0.6 g) in ethanol (110 mL) was shaken for 5 h under H2 atmosphere
(1 bar, balloon). The mixture was filtered through Celite® and the solvent was
removed under reduced pressure. Rf = 0.35 (CH3OH). Yellow oil, yield 4.30 g (86 %).
C10H20N2O4 (M = 232.3 g/mol). 1H NMR (d6ꢀDMSO): δ (ppm) = 1.38 (s, 9 H, C(CH3)3),
3
2.33 (t, J = 9.1 Hz, 1 H, CHNH2), 2.35 – 2.55 (m, 2 H, 2ꢀCH2, 6ꢀCH2), 2.96 – 3.04
(m, 2 H, 2ꢀCH2, 6ꢀCH2), 3.88 (s broad, 2 H, 2 x CHOH), 5.06 (s broad, 2 H, 2 x OH).
1
A signal for NH2 protons is not observed in the H NMR spectrum. MS (ESI):
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m/z = 233 [M + H]+, 255 [M + Na]+, 465 [2 x M + H]+. IR (neat): (cmꢀ1) = 3350 (w, Oꢀ
H), 2928 (w, CꢀH), 1668 (s, C=Ocarbamate).
5.5.4.
tertꢀButyl
(4r)
4ꢀ(2ꢀchloroacetamido)ꢀ3,5ꢀdihydroxypiperidineꢀ1ꢀ
carboxylate (10)
Chloroacetyl chloride (1.47 mL, 18.5 mmol) dissolved in THF (75 mL) was added to
an iceꢀcooled solution of amine 9 (4.3 g, 18.5 mmol) and triethylamine (2.56 mL,
18.5 mmol) in THF (75 mL) dropwise within 2 h. The mixture warmed up to rt and
stirred for 1 h. The solvent was removed under reduced pressure and the product
was purified by fc (∅ = 8 cm, h = 18 cm, CH2Cl2/CH3OH = 9.7/0.3
→
CH2Cl2/CH3OH = 9.5/0.5, V = 65 mL, Rf = 0.15 (CH2Cl2/CH3OH = 9.5/0.5)). Yellow
1
resin, yield 3.0 g (53 %). C12H21ClN2O5 (M = 308.8 g/mol). H NMR (CD3OD): δ
(ppm) = 1.46 (s, 9 H, C(CH3)3), 2.53 – 2.70 (m, 2 H, 2ꢀCH2, 6ꢀCH2), 3.43 – 3.51 (m,
3
2 H, 2 x CHOH), 3.63 (d, J = 9.7 Hz, 1 H, CHNHCO), 4.11 (s, 2 H, CH2Cl), 4.13 –
1
4.23 (m, 2 H, 2ꢀCH2, 6ꢀCH2). A signal for NH proton is not observed in the H NMR
spectrum. 13C NMR (CD3OD): δ (ppm) = 28.6 (3 C, C(CH3)3), 43.5 (CH2Cl), 49.6
(NCH2), 50.5 (NCH2), 62.3 (CHNHCO), 69.1 (2 C, 2 x CHOH), 81.7 (C(CH3)3), 156.1
(C=Ocarbamate), 170.2 (C=Oamide). MS (EM, APCI): m/z = calcd. for C12H2235ClN2O5
[M + H]+ 309.1217, found 309.1174; calcd. for C8H1435ClN2O5 [M – C(CH3)3 + 2 H]+
253.0591, found 253.0564; calcd. for C7H1435ClN2O3 [M – OCOC(CH3)3 + 2 H]+
209.0693, found 209.0660. IR (neat): (cmꢀ1) = 3319 (m, OꢀH), 2983/2928/2857 (w,
CꢀH), 1701 (s, C=O), 1647 (s, C=O).
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5.5.5. tertꢀButyl (4aRS,8RS,8aSR)ꢀ8ꢀhydroxyꢀ2ꢀoxoꢀ2,3,4a,5,6,7,8,8aꢀoctahydroꢀ
1Hꢀpyridoꢀ[3,4ꢀb][1,4]oxazineꢀ6ꢀcarboxylate (11)
At 0 °C tetrabutylammonium iodide (1.25 g, 3.39 mmol) and NaH (119.1 mg,
2.83 mmol) were added to a solution of alcohol 10 (873 mg, 2.83 mmol) in THF
(20 mL). The reaction mixture was allowed to warm up and stirred for 2 d at rt. After
cooling to 0 °C the transformation was stopped by addition of a small amount of
water. The solution was neutralized with 1 M HCl and stirred for 30 min. The mixture
was extracted with CH2Cl2 (30 mL). The organic layer was washed with H2O (10 mL).
The aqueous layer was extracted with CH2Cl2 (3 x 10 mL). The combined organic
layer was washed with brine, dried (Na2SO4) and filtered. The filtrate was
concentrated in vacuum and the product was purified by fc (∅ = 8 cm, h = 17 cm,
CH2Cl2/CH3OH = 9.7/0.3, V = 65 mL, Rf = 0.35 (CH2Cl2/CH3OH = 9.5/0.5)). Colorless
solid, mp 181 – 182 °C, yield 466 mg (61 %). C12H20N2O5 (M = 272.3 g/mol). 1H NMR
(CDCl3): δ (ppm) = 1.45 (s, 9 H, C(CH3)3), 2.47 – 2.81 (m, 2 H, 5ꢀCH2, 7ꢀCH2), 3.21 (t,
3J = 9.4 Hz, 1 H, 8aꢀCH), 3.25 – 3.34 (m, 1 H, 4aꢀCH), 3.46 – 3.56 (m, 1 H, 8ꢀCH),
2
4.16 – 4.50 (m, 3 H, 5ꢀCH2, 7ꢀCH2, OH), 4.19 (d, J = 17.1 Hz, 1 H, 3ꢀCH2), 4.30 (d,
2J = 17.1 Hz, 1 H, 3ꢀCH2), 7.51 (s broad, 0.5 H, NH), 7.87 (s broad, 0.5 H, NH).
13C NMR (CDCl3): δ (ppm) = 28.4 (3 C, C(CH3)3), 46.1 (Cꢀ5 or Cꢀ7), 47.9 (Cꢀ7 or Cꢀ
5), 61.2 (Cꢀ8a), 67.7 (Cꢀ3), 68.1 (Cꢀ8), 72.1 (Cꢀ4a), 81.2 (C(CH3)3), 154.6
(C=Ocarbamate), 169.3 (C=Oamide). MS (EM, APCI): m/z = calcd. for C12H21N2O5
[M + H]+ 273.1451, found 273.1476; calcd. for C8H13N2O5 [M – C(CH)3 + 2 H]+
217.0825, found 217.0854. IR (neat): (cmꢀ1) = 3204 (w, OꢀH), 2962/2944/2832 (m,
CꢀH), 1690 (s, C=O), 1630 (s, C=O). Elemental analysis: calcd. C 52.93, H 7.40, N
10.29, found 52.47, H 7.57, N 9.61.
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5.5.6. tertꢀButyl (4aRS,8RS,8aRS)ꢀ8ꢀazidoꢀ2ꢀoxoꢀ2,3,4a,5,6,7,8,8aꢀoctahydroꢀ1Hꢀ
pyrido[3,4ꢀb][1,4]oxazineꢀ6ꢀcarboxylate (12)
Zn(N3)2ꢂ(pyridine)2 (185 mg, 0.6 mmol) and PPh3 (393 mg, 1.5 mmol) were added to
a solution of alcohol 11 (136 mg, 0.50 mmol) in THF (6 mL). DIAD (0.29 mL,
1.5 mmol) was added dropwise and the mixture was stirred for 4.5 h at rt. The solvent
was removed under reduced pressure and the product was purified by fc (∅ = 4 cm,
h = 16 cm, CH2Cl2/CH3OH = 9.85/0.15 → CH2Cl2/CH3OH = 9.8/0.2, V = 20 mL,
Rf = 0.53 (CH2Cl2/CH3OH = 9.5/0.5)). Colorless solid, mp 162 – 163 °C, yield 105 mg
1
(70 %). C12H19N5O4 (M = 297.3 g/mol). H NMR (CDCl3): δ (ppm) = 1.48 (s, 9 H,
C(CH3)3), 2.53 – 2.75 (m, 1 H, 5ꢀCH2 or 7ꢀCH2), 2.81 – 3.08 (m, 1 H, 7ꢀCH2 or 5ꢀ
3
3
CH2), 3.46 (dd, J = 9.2 Hz, J = 3.1 Hz, 1 H, 8aꢀCH), 3.61 – 3.70 (m, 1 H, 4aꢀCH),
3.88 – 3.91 (m, 1 H, 8ꢀCH), 4.15 – 4.30 (m, 2 H, 3ꢀCH2), 4.31 – 4.41 (m, 1 H, 5ꢀCH2
13
or 7ꢀCH2), 4.42 – 4.58 (m, 1 H, 7ꢀCH2 or 5ꢀCH2), 7.86 – 8.09 (m, 1 H, NH). C NMR
(CDCl3): δ (ppm) = 28.4 (3 C, C(CH3)3), 45.5 (Cꢀ5 or Cꢀ7), 45.9 (Cꢀ7 or Cꢀ5), 57.4 (Cꢀ
8a), 58.7 (Cꢀ8), 67.9 (Cꢀ3), 69.3 (Cꢀ4a), 81.3 (C(CH3)3), 154.7 (C=Ocarbamate), 170.1
(C=Oamid). MS (EM, APCI): m/z = calcd. for C12H20N5O4 [M + H]+ 298.1515, found
298.1503; calcd. for C8H12N5O4 [M – C(CH)3 + 2 H]+ 242.0889, found 242.0899. IR
(neat): (cmꢀ1) = 2970/2897 (w, CꢀH), 2118 (s, N3), 1705 (s, C=O), 1670 (s, C=O).
Elemental analysis: calcd. C 48.48, H 6.44, N 23, found 48.14, H 6.42, N 23.33.
5.5.7. tertꢀButyl (4aRS,8RS,8aRS)ꢀ8ꢀaminoꢀ2ꢀoxoꢀ2,3,4a,5,6,7,8,8aꢀoctahydroꢀ1Hꢀ
pyrido[3,4ꢀb][1,4]oxazineꢀ6ꢀcarboxylate (13)
Azide 12 (45.6 mg, 0.15 mmol) and Pd/C (20 % (m/m), 9.12 mg) were stirred for 18 h
under H2 atmosphere (1 bar, balloon) in CH3OH (3 mL). The mixture was filtered
through Celite® and the solvent was removed under reduced pressure. The product
was purified by fc (∅ = 2 cm, h = 20 cm, CH2Cl2/CH3OH = 9/1, V = 10 mL, Rf = 0.31).
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Colorless solid, mp 155 – 156 °C, yield 36.7 mg (90 %). C12H21N3O4 (M = 271.3
1
g/mol). H NMR (CD3OD): δ (ppm) = 1.47 (s, 9 H, C(CH3)3), 2.57 – 2.77 (m, 1 H, 5ꢀ
CH2 or 7ꢀCH2), 2.89 – 3.09 (m, 1 H, 7ꢀCH2 or 5ꢀCH2), 3.13 – 3.24 (m, 1 H, 8ꢀCH),
3
3
3.40 (dd, J = 9.6 Hz, J = 3.5 Hz, 1 H, 8aꢀCH), 3.69 – 3.78 (m, 1 H, 4aꢀCH), 4.10 –
4.18 (m, 1 H, 5ꢀCH2 or 7ꢀCH2), 4.20 (s, 2 H, 3ꢀCH2), 4.25 – 4.35 (m, 1 H, 7ꢀCH2 or 5ꢀ
1
CH2). Signals for NH2 and NH protons are not observed in the H NMR spectrum.
13C NMR (CD3OD): δ (ppm) = 28.6 (3 C, C(CH3)3), 46.7 (Cꢀ5 or Cꢀ7), 48.6 (Cꢀ7 or Cꢀ
5), 49.6 (Cꢀ8), 59.6 (Cꢀ8a), 68.4 (Cꢀ3), 69.9 (Cꢀ4a), 81.8 (C(CH3)3), 156.9
(C=Ocarbamate), 171.9 (C=Oamid). MS (EM, APCI): m/z = calcd. for C12H22N3O4 [M + H]+
272.1610, found 272.1572; calcd. for C8H14N3O4 [M – C(CH)3 + 2 H]+ 216.0984,
found 216.0957; calcd. for C7H14N3O2 [M – CO2C(CH)3 + 2 H]+ 172.1086, found
172.1068. IR (neat): (cmꢀ1) = 3379/3194 (w, NꢀH), 2882 (w, CꢀH), 1686 (s, C=O),
1643 (s, C=O).
5.5.8.
tertꢀButyl (4aRS,8RS,8aRS)ꢀ2ꢀoxoꢀ8ꢀ(pyrrolidinꢀ1ꢀyl)ꢀ2,3,4a,5,6,7,8,8aꢀ
octahydroꢀ1Hꢀpyrido[3,4ꢀb][1,4]oxazineꢀ6ꢀcarboxylate (14)
A mixture of primary amine 13 (951 mg, 3.5 mmol), 1,4ꢀdiiodobutane (1.8 mL,
14.0 mmol), NaHCO3 (2.0 g, 23.8 mmol) and CH3CN (100 mL) was stirred under
reflux for 20 h. After evaporation of the solvent in vacuum the residue was purified by
fc (∅ = 8 cm, h = 14 cm, CH2Cl2/CH3OH = 9.75/0.35 → CH2Cl2/CH3OH = 9.5/0.5,
V = 65 mL, Rf = 0.21 (CH2Cl2/CH3OH = 9.5/0.5)). Colorless solid, mp 135 °C
(decomposition), yield 626 mg (55 %). C16H27N3O4 (M = 325.4 g/mol). 1H NMR
(CDCl3): δ (ppm) = 1.46 (s, 9 H, C(CH3)3), 1.66 – 1.73 (m, 4 H, N(CH2CH2)2), 2.55 –
2.84 (m, 7 H, N(CH2CH2)2, 5ꢀCH2, 7ꢀCH2, 8ꢀCH), 3.50 (dd, 3J = 10.3 Hz, 3J = 2.8 Hz,
1 H, 8aꢀCH), 3.86 – 3.99 (m, 1 H, 4aꢀCH), 4.13 – 4.31 (m, 2 H, 3ꢀCH2), 4.31 – 4.43
(m, 1 H, 5ꢀCH2 or 7ꢀCH2), 4.46 – 4.62 (m, 1 H, 7ꢀCH2 or 5ꢀCH2), 6.69 – 7.40 (m, 1 H,
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13
NH). C NMR (CDCl3): δ (ppm) = 23.4 (2 C, N(CH2CH2)2), 28.5 (3 C, C(CH3)3), 46.0
(Cꢀ5 or Cꢀ7), 47.0 (Cꢀ7 or Cꢀ5), 52.9 (2 C, N(CH2CH2)2), 59.9 (Cꢀ8a), 60.8 (Cꢀ8), 67.8
(Cꢀ3), 69.4 (Cꢀ4a), 80.5 (C(CH3)3), 154.5 (C=Ocarbamate), 168.9 (C=Oamide). MS (EM,
ESI): m/z = calcd. for C16H28N3O4 [M + H]+ 326.2080, found 326.2106; calcd. for
C12H20N3O4 [M – C(CH)3 + 2 H]+ 270.1454, found 270.1460. IR (neat):
(cmꢀ1) = 2967/2940/2897 (w, CꢀH), 1674 (s, C=O). Elemental analysis: calcd. C
59.06, H 8.36, N 12.91, found 57.73, H 8.18, N 12.58.
5.5.9.
(4aRS,8RS,8aRS)ꢀ8ꢀ(Pyrrolidinꢀ1ꢀyl)ꢀ4a,5,6,7,8,8aꢀhexahydroꢀ1Hꢀ
pyrido[3,4ꢀb][1,4]oxazinꢀ2(3H)ꢀone (15a)
Trifluoroacetic acid (0.23 mL, 3.0 mmol) was added dropwise to a solution of Bocꢀ
protected pyridooxazine 14 (43.1 mg, 0.13 mmol) in CH2Cl2 (3 mL). After stirring for
2 h, the solvent was removed under reduced pressure. The residue was dissolved in
CH2Cl2 (5 mL), washed with a saturated solution of NaHCO3 (5 mL) and the aqueous
layer was extracted with CH2Cl2 (3 x 5 mL). The organic layers were combined and
the solvent was removed in vacuum. Rf = 0.13 (CH2Cl2/CH3OH = 8/2). Colorless
1
crystals, yield 5.5 mg (18 %). C11H19N3O2 (M = 225.3 g/mol). H NMR (CDCl3): δ
(ppm) = 1.71 – 1.74 (m, 4 H, N(CH2CH2)2), 1.79 – 1.99 (m, 2 H, 5ꢀCH2, 7ꢀCH2),
2.44 – 2.49 (m, 1 H, NHamine), 2.65 – 2.72 (m, 5 H, N(CH2CH2)2, 8ꢀCH), 3.19 – 3.27
3
3
(m, 1 H, 5ꢀCH2 or 7ꢀCH2), 3.30 (dd, J = 13.0 Hz, J = 4.9 Hz, 1 H, 7ꢀCH2 or 5ꢀCH2),
3
3.57 (dd, 3J = 9.7 Hz, J = 2.4 Hz, 1 H, 8aꢀCH), 3.83 – 3.91 (m, 1 H, 4aꢀCH), 4.18 (d,
3J = 16.7 Hz 1 H, 3ꢀCH2), 4.27 (d, 2J = 16.8 Hz, 1 H, 3ꢀCH2), 6.90 (s, 1 H, NHamide).
MS (APCI): m/z = 226 [M + H]+.
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5.5.10. (4aRS,8RS,8aRS)ꢀ6ꢀEthylꢀ8ꢀ(pyrrolidinꢀ1ꢀyl)ꢀ2,3,4a,5,6,7,8,8aꢀoctahydroꢀ
1Hꢀpyrido[3,4ꢀb][1,4]oxazinꢀ2ꢀone (15c)
According to General Procedure A Bocꢀprotected pyridooxazine 14 (70.9 mg,
0.22 mmol) was reacted with trifluoroacetic acid (0.50 mL, 6.5 mmol) in CH2Cl2
(5 mL). The subsequent alkylation was performed with acetaldehyde (25 ꢃL,
0.44 mmol), NaBH(OAc)3 (65.3 mg, 0.31 mmol) and THF (4 mL). After 40 min the
transformation was complete and the product was purified by fc (∅ = 2 cm,
h = 18 cm, CH2Cl2/CH3OH/NH3 = 9.5/0.47/0.03, V = 10 mL, Rf = 0.20 (CH2Cl2/CH3OH
= 9/1)). Colorless solid, mp 164 – 166 °C, yield 46.6 mg (84 %). C13H23N3O2
(M = 253.3 g/mol). 1H NMR (CDCl3): δ (ppm) = 1.08 (t, 3J = 7.2 Hz, 3 H, CH3), 1.75 –
1.84 (m, 4 H, N(CH2CH2)2), 2.00 – 2.10 (m, 2 H, 5ꢀCH2ꢀax, 7ꢀCH2ꢀax), 2.37 – 2.47 (m,
1 H, CH2CH3), 2.50 – 2.60 (m, 1 H, CH2CH3), 2.75 – 2.84 (m, 2 H, N(CH2CH2)2),
2.85 – 2.98 (m, 3 H, 8ꢀCH, N(CH2CH2)2), 3.14 – 3.20 (m, 1 H, 7ꢀCH2ꢀeq), 3.23 (dd,
3
3
3
2J = 10.3 Hz, J = 4.6 Hz, 1 H, 5ꢀCH2ꢀeq), 3.42 (dd, J = 9.8 Hz, J = 3.5 Hz, 1 H, 8aꢀ
2
CH), 4.02 – 4.10 (m, 1 H, 4aꢀCH), 4.20 (d, J = 16.8 Hz, 1 H, 3ꢀCH2), 4.25 (d,
2J = 16.7 Hz, 1 H, 3ꢀCH2), 7.76 (s broad, 1 H, NH). 13C NMR (CDCl3):
δ (ppm) = 11.94 (CH3), 23.4 (2 C, N(CH2CH2)2), 51.9 (CH2CH3), 53.5 (2 C,
N(CH2CH2)2), 53.9 (Cꢀ7), 56.2 (Cꢀ5), 59.4 (Cꢀ8a), 61.2 (Cꢀ8), 67.7 (Cꢀ3), 70.5 (Cꢀ4a),
169.9 (C=O). MS (EM, APCI): m/z = calcd. for C13H24N3O2 [M + H]+ 254.1869, found
254.1845. IR (neat): (cmꢀ1) = 2962/2936/2870 (m, CꢀH), 1670 (s, C=O).
5.5.11. (4aRS,8RS,8aRS)ꢀ6ꢀMethylꢀ8ꢀ(pyrrolidinꢀ1ꢀyl)ꢀ2,3,4a,5,6,7,8,8aꢀoctahydroꢀ
1Hꢀpyrido[3,4ꢀb][1,4]oxazine (16b)
A solution of AlH3 (1.33 mmol) was freshly prepared by General Procedure B. Then
Bocꢀprotected pyridooxazine 14 (60 g, 0.18 mmol) dissolved in THF (3 mL) was
added dropwise to the alane solution at 0 °C. The mixture was stirred at 0 °C for
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45 min and at rt for 20 min. 2 M NaOH (5 mL) was carefully added at 0 °C. CH2Cl2
(5 mL) was added, the layers were separated and the aqueous layer was extracted
with CH2Cl2 (4 x 5 mL). The combined organic layer was washed with brine, dried
(Na2SO4) and filtered. The solvent was removed under reduced pressure. Colorless
1
crystals, yield 45 mg (> 99 %). C12H23N3O (M = 225.3 g/mol). H NMR (CD3OD): δ
(ppm) = 1.67 – 1.79 (m, 4 H, 2 x N(CH2CH2)2), 1.80 – 1.87 (m, 1 H, 5ꢀCH2 or 7ꢀCH2),
2.03 – 2.10 (m, 1 H, 7ꢀCH2 or 5ꢀCH2), 2.22 (s, 3 H, CH3), 2.39 – 2.47 (m, 1 H, 8ꢀCH
or 8aꢀCH), 2.67 – 2.78 (m, 3 H, N(CH2CH2)2, 8aꢀCH or 8ꢀCH), 2.80 – 2.95 (m, 5 H,
N(CH2CH2)2, 2ꢀCH2, 5ꢀCH2 or 7ꢀCH2), 3.66 (d, 1 H, 7ꢀCH2 or 5ꢀCH2), 3.57 (ddd,
3
3
2J = 11.4 Hz, J = 10.3 Hz, J = 4.3 Hz, 1 H, 3ꢀCH2ꢀax), 3.66 (m, 1 H, 4aꢀCH), 3.76
3
3
3
(ddd, J = 11.6 Hz, J = 3.0 Hz, J = 1.5 Hz, 1 H, 3ꢀCH2ꢀeq). A signal for NH is not
observed in the 1H NMR spectrum.
5.5.12.
2ꢀ(3,4ꢀDichlorophenyl)ꢀ1ꢀ[(4aRS,8RS,8aRS)ꢀ8ꢀ(pyrrolidinꢀ1ꢀyl)ꢀ
2,3,4a,5,6,7,8,8aꢀoctahydropyrido[3,4ꢀb][1,4]oxazinꢀ1ꢀyl]ethanone (17a) WMSꢀ
29ꢀ08
Pd/C (40 % (m/m), 12 mg) and conc. HCl (0.5 mL) were added to a solution of amide
17f (29.9 mg, 0.06 mmol) in THF/water (1/1, 5 mL) and the suspension was stirred
under H2 atmosphere (1 bar, balloon) at rt for 30 min. The mixture was filtered and
THF was removed under reduced pressure. The pH value of the aqueous solution
was adjusted to pH 8 with 2 M NaOH. The solution was extracted with CH2Cl2
(5 x 3 mL), the organic layer was dried (Na2SO4), filtered and concentrated in
vacuum.
The
product
was
purified
by
fc
(∅ = 2 cm,
h = 16 cm,
CH2Cl2/CH3OH/NH3 = 8.9/1.09/0.01, V = 10 mL, Rf = 0.46). Pale yellow solid,
1
mp 87 – 90 °C, yield 21.9 mg (90 %). C19H25Cl2N3O2 (M = 398.3 g/mol). H NMR
(CD3OD): δ (ppm) = 1.62 – 1.77 (m, 4 H, N(CH2CH2)2), 2.40 – 2.59 (m, 5 H,
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2
N(CH2CH2)2, 5ꢀCH2ꢀax), 2.71 (d, J = 14.1 Hz, 1 H, 7ꢀCH2ꢀax), 3.07 – 3.23 (m, 3 H, 5ꢀ
2
CH2ꢀeq, 7ꢀCH2ꢀeq, 8ꢀCH), 3.74 (d, J = 15.4 Hz, 1 H, ArCH2), 3.79 – 4.02 (m, 6 H, 2ꢀ
3
CH2, 3ꢀCH2, 8aꢀCH, ArCH2), 4.28 – 4.39 (m, 1 H, 4aꢀCH), 7.21 (dd, J = 8.2 Hz,
4J = 2.0 Hz, 1 H, 6ꢀCHar), 7.44 – 7.50 (m, 2 H, 2ꢀCHar, 5ꢀCHar). 13C NMR (CD3OD):
δ (ppm) = 24.1 (2 C, N(CH2CH2)2), 40.7 (ArCH2), 42.5 (Cꢀ8a), 49.8 (Cꢀ7), 51.4 (Cꢀ5),
54.1 (2 C, N(CH2CH2)2), 62.6 (Cꢀ8), 65.7 (Cꢀ2), 66.8 (Cꢀ3), 67.4 (Cꢀ4a), 130.4 (Carꢀ6),
131.6 (Carꢀ2), 131.9 (Cq), 132.4 (Carꢀ5), 133.3 (Cq), 136.9 (Cq), 173.1 (C=O). MS (EM,
APCI): m/z = calcd. for C19H26Cl2N3O2 [M + H]+ 398.1402, found 398.1401. IR (neat):
(cmꢀ1) = 2955/2924/2770 (m, CꢀH), 1636 (s, C=O). Purity (HPLC): 99.3 %,
tR = 15.83 min.
5.5.13. 2ꢀ(3,4ꢀDichlorophenyl)ꢀ1ꢀ[(4aRS,8RS,8aRS)ꢀ6ꢀmethylꢀ8ꢀ(pyrrolidinꢀ1ꢀyl)ꢀ
2,3,4a,5,6,7,8,8aꢀoctahydropyrido[3,4ꢀb][1,4]oxazinꢀ1ꢀyl]ethanone (17b)
(WMSꢀ29ꢀ01)
2ꢀ(3,4ꢀDichlorophenyl)acetyl chloride (33.5 ꢃL, 0.22 mmol) was added to a solution of
amine 16b (40.6 mg, 0.18 mmol) in CH2Cl2 (5.5 mL) and the mixture was stirred for
30 min at rt. Then 2 M NaOH (5.5 mL) was added and the mixture was stirred for
additional 12 h at rt. The layers were separated and the aqueous layer was extracted
with CH2Cl2 (2 x 3 mL). The combined organic layer was dried (Na2SO4) and filtered.
The filtrate was concentrated under reduced pressure and the product was purified
by fc (∅ = 2 cm, h = 17 cm, CH2Cl2/CH3OH = 9.8/0.2 → CH2Cl2/CH3OH = 9.5/0.5,
V = 10 mL, Rf = 0.57 (CH2Cl2/CH3OH = 9/1)). Pale yellow solid, mp 111 – 112 °C,
yield 62.2 mg (84 %). C20H27Cl2N3O2 (M = 412.35 g/mol). 1H NMR (CDCl3): δ
2
3
(ppm) = 1.62 – 1.83 (m, 4 H, N(CH2CH2)2), 1.90 (t, J = 9.9 Hz, J = 9.9 Hz, 1 H, 5ꢀ
CH2ꢀax), 2.18 (d, 2J = 13.0 Hz, 1 H, 7ꢀCH2ꢀax), 2.26 (s, 3 H, CH3), 2.45 – 2.77 (m, 4 H,
2
2
N(CH2CH2)2), 2.90 – 2.98 (d, J = 13.0 Hz, 1 H, 7ꢀCH2ꢀeq), 3.12 (dd, J = 10.1 Hz,
29