Synthesis and SAR study of a novel series of dopamine receptor agonists

Article abstract of DOI:10.1016/j.bmc.2013.11.012

The synthesis of a novel series of dopamine receptor agonists are described as well as their in vitro potency and efficacy on dopamine D₁ and D₂ receptors. This series was designed from pergolide and (4aR,10aR)-1-propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinolin-6-ol (PHBQ) and resulted in the synthesis of (2R,4aR,10aR)-2-methylsulfanylmethyl-4-propyl- 3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol (compound 27), which has a D₁ and D₂ receptor profile similar to that of the most recently approved drug for Parkinson's disease, rotigotine.

Full text of DOI:10.1016/j.bmc.2013.11.012

Bioorganic & Medicinal Chemistry 22 (2014) 381–392
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR study of a novel series of dopamine receptor
agonists
Rune Risgaard a,c, Martin Jensen a, Morten Jørgensen a, Benny Bang-Andersen a, Claus T. Christoffersen b,
Klaus G. Jensen b, Jesper L. Kristensen c, Ask Püschl a,
⇑
a Neuroscience Drug Discovery, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Valby, Denmark
b Drug ADME Research, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Valby, Denmark
c Department of Drug Design and Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel series of dopamine receptor agonists are described as well as their in vitro potency
and efficacy on dopamine D1 and D2 receptors. This series was designed from pergolide and (4aR,10aR)-1-
propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinolin-6-ol (PHBQ) and resulted in the synthesis of
(2R,4aR,10aR)-2-methylsulfanylmethyl-4-propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxa-
zin-9-ol (compound 27), which has a D1 and D2 receptor profile similar to that of the most recently
approved drug for Parkinson’s disease, rotigotine.
Received 20 August 2013
Revised 1 November 2013
Accepted 7 November 2013
Available online 15 November 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Dopamine receptor agonist
Parkinson’s disease
Rotigotine
1. Introduction
Furthermore, rotigotine is a phenolic amine which suffer from
the same poor oral bioavailability and rapid clearance from the cir-
Parkinson’s disease (PD) is a disorder in which the neurons that
produce dopamine (Fig. 1) are degrading.1 Dopamine receptor ago-
nists (DRA) are used to treat the symptoms of this disease by
substituting for dopamine, either as a mono-therapy or, as the
disease is progressing to more severe stages, in combination with
culation as other phenolic amines.6 Consequently, rotigotine has
been formulated as a transdermal patch4,5 to avoid first pass
metabolism and secure bioavailability. Rotigotine also contains a
thiophene which is a structural alert.7,8
The two octahydro-benzo[g]quinolines MHBQ and PHBQ (Fig. 1)
from Sandoz,9 have earlier been published as dopamine D2-like
receptor agonists, whereas pergolide has been published as a
mixed D1 and D2 receptor agonist. We used these scaffolds as inspi-
ration for a novel series of putative DRA with the aim of finding
compounds with more balanced, apomorphine-like receptor pro-
files as compared to rotigotine and without the thiophene struc-
tural alert. Thus, we set out to synthesize an improved version of
rotigotine by the synthesis and evaluation of a novel set of ligands
designed as outlined above.
L-DOPA or apomorphine. Five subtypes of the dopamine receptors
are known and they can be divided into two subclasses, ‘D1 like’
(D1 and D5) and ‘D2 like’ (D2, D3 and D4).1 Common for all DRA indi-
cated for PD is their high selectivity for D2 and D3 receptors, and
their low-to-neglectible interaction with D1 receptors. Dopamine,
coming from the precursor drug L-DOPA, and apomorphine dis-
plays a more balanced D1/D2 ratio when compared to the DRA
and are both considered gold standard PD pharmacotherapies.2
Recently, rotigotine (Fig. 1) was approved for the treatment of
PD as well.3–5
In vitro, rotigotine mimic the profiles of dopamine and apomor-
phine to some extent, displaying activity as an agonist at both D2
and D3 receptors as well as D1 receptors. However, rotigotine still
displays a preference for D2 and D3 receptors,4 and when compared
to apomorphine and dopamine itself, a relative more potent D1
receptor component would be required in a putative improved
version of rotigotine.
2. Results and discussion
2.1. Chemistry
The novel DRAs 6a–10a (Table 1) were prepared in the follow-
ing manner. Racemic azido alcohol 1 was prepared from naphtha-
len-1-ol as described in the literature (Scheme 1).10 The procedure
was slightly modified, and lithium was used instead of sodium in
the Birch reduction of the naphthalen-1-ol. The hydroxy group
was methylated using dimethyl sulfate, and the epoxide was pre-
pared using m-CPBA as described in the literature.10 It was possible
⇑
Corresponding author. Tel.: +45 36433286.
E-mail address: askp@lundbeck.com (A. Püschl).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.012

382
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
NH2
N
N
HN
S
HO
OH
SMe
OH
Dopamine
Rotigotine
Pergolide
N
N
N
O
SMe
OH
OH
OH
MHBQ
Figure 1. Structures of dopamine and the dopamine receptor agonists (DRA) rotigotine, Pergolide, MHBQ, PHBQ and 27.
PHBQ
27
to use this procedure on a 0.3 mol scale in 66% isolated yield. Com-
pound 1 was then prepared by reacting this epoxide with sodium
azide under acidic conditions and then separating the two regioiso-
meric azido alcohols by crystalization.10 Compound 1 was reduced
to 2 by catalytic hydrogenation and then reacted with chloro-acetyl
chloride to give 3. The lactam 3 was reduced with lithium alumi-
num hydride (LAH), and the resulting morpholine was protected
with a Boc group to yield 4. Compound 4 was separated into its
two enantiomers 4a and 4b by chiral supercritical fluid chromatog-
raphy (SFC on a chiral phase). The Boc group was removed to pro-
duce 5a (Scheme 2), which was converted into 8a–10a by
alkylation followed by deprotection with L-selectride under micro-
wave conditions. The microwave conditions were convenient be-
cause deprotection of methyl aryl ethers with L-selectride,
without electron withdrawing substituents, usually takes two days
in refluxing THF.11 It was not possible to prepare 8a–10a with BBr3
because using this method, the morpholine ring was also cleaved.
Compound 6a was prepared from 4a by reducing the Boc group
with LAH under microwave conditions and then cleaving the
methyl group as described above. Compound 7a was prepared from
4a by reductive amination after deprotection. Compounds 6a–10a
were precipitated as their HCl salts. The HCl salt of the enantiomer
10b was prepared in a similar manner from 4b and a single crystal
for X-ray was obtained (see Supplementary data). The configuration
of 10b was determined to be the (4aS, 10aS), whereas 6a–10a have
the opposite configuration.
The novel DRA 19–27 (Table 2) were prepared in the following
manner. Compounds 11a and 11b were prepared from racemic 2
Table 1
Data for reference compounds and 6a–10a, 10b
R
N
R
N
O
O
OH
OH
6a, 7a, 8a, 9a, 10a
10b
R
D1 EC50
(%eff)
D2 EC50
(%eff)
D1/D2
H.Mic
(L/min)
H.Hep
(L/min)
cLogD
PSA
MPO
Dopamine
Rotigotine
MHBQ
Figure 1
Figure 1
Figure 1
Figure 1
39 (100)
28 (110)
>10000
83 (95)
36 (100)
0.7 (110)
25 (94)
1
40
—
nd
3.7
1.2
nd
nd
16
6.9
nd
nd
nd
nd
2.8
1.0
1.9
23.5
23.5
23.5
3.3
4.5
4.0
PHBQ
0.17 (96)
488
>10000
4 (94)
—
0.2
0.7
3.4
20
1.2
1.6
32.7
32.7
5.5
5.5
6a
7a
120 (85)
0.8 (120)
150
150 (89)
330 (87)
0.14 (110)
1.1 (87)
1071
66
0.8
0.9
6.9
11
2.2
2.0
32.7
32.7
5.4
5.5
8a
9a
>10000
>10000
18 (99)
—
—
0.9
nd
9.8
nd
2.1
2.1
32.7
32.7
5.4
5.4
10a
1400 (90)
10b
Values from efficacy assays (EC50) is in nM and % efficacy is relative to dopamine. Calculated polar surface area (PSA) is topological PSA and calculated lipophilicity (cLogD) is
at pH 7.4. In vitro CLint are determined in human microsomes (H.Mic) and human hepatocytes (H.Hep).

R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
383
N3
NH2
OH
Ref 9
Ref 9
a
b,c
O
OH
OH
OMe
OMe
OMe
2
1
boc
N
boc
N
boc
H
N
O
N
d,e
f
+
O
O
O
O
OMe
OMe
OMe
OMe
3
4b
4
4a
Scheme 1. Reagents and conditions: (a) Pd/C, H2 (45 psi), EtOH; (b) Chloroacetyl chloride, Et3N, THF; (c) NaH, TBAI, THF; (d) LiAlH4, THF; (e) Boc2O, Et3N, THF; (f) SFC
separation.
boc
N
R
N
HCl
H
N
HCl
8a: R = Propyl
9a
b-d
a
: R = Allyl
10a
: R = Cyclopropylmethyl
O
O
O
OMe
OMe
OH
5a
8a-10a
4a
Me
N
boc
Et
HCl
HCl
N
N
O
f
e
O
O
OH
OMe
OH
4a
7a
6a
Scheme 2. Reagents and conditions: (a) HCl, Ether, MeOH; (b) 8a: (i) Propyliodide, K2CO3, DMF, 55 °C, (ii) L-selectride, THF, (iii) HCl/ether/MeOH; (c) 9a: (i) allylbromide,
K2CO3, DMF, 55 °C, (ii) L-selectride/THF, (iii) HCl/ether/MeOH; (d) 10a: (i) cyclopropylmethylbromide, K2CO3, DMF, 55 °C, 5 h, (ii) L-selectride/THF, (iii) HCl/Ether/MeOH; (e)
6a: (i) LiAlH4/THF, (ii) L-selectride, THF, (iii) HCl/Ether/MeOH; (f) 7a: (i) L-selectride, THF, microwave, 100 °C, (ii) HCl, ether, MeOH, (iii) acetaldehyde, NaBH3CN, MeOH; (iv)
HCl, ether, MeOH.
by Boc protection of the amine and separation of the enantiomers
using SFC on a chiral phase (Scheme 3). The Boc group was re-
moved after the separation using HCl in diethyl ether and MeOH
as solvent to afford the pure enantiomers as the HCl salts. 11a
was alkylated with propionyl chloride to yield 12. Compound 12
was alkylated with either (S)- or (R)-epichlorohydrine, and the
intermediate was treated with base to yield a mixture of 13 and
14, which were separated by chromatography.12 Surprisingly, upon
treatment of 11a with either (S)- or (R)-epichlorhydrin the same
diastereoisomers were observed in both cases depending on the
site of attack of the nucleophile. Compounds 13 and 14 could inde-
pendently be tosylated to give intermediates 15 and 16 (Scheme 4).
The tosyl group was then displaced with different nucleophiles to
afford compounds 19 to 27. Finally, the methoxy group was
cleaved with either L-selectride under microwave conditions (as
described above) or with thiophenol and KF in DMA.
Compounds 25 and 27 were precipitated as their HCl salts
whereas 19–24 and 26 were prepared and tested as the free bases.
The absolute stereochemistry was determined by X-ray crystallog-
raphy of a single crystal from the HCl salt of 27 (see Supplementary
data), and the configuration was found to be (2R,4aR,10aR).
The references compounds MHBQ and PHBQ were prepared as
described in the Supplementary data.
measuring compound induced changes in cAMP in CHO cells
expressing either the human D1 or D2 receptor (Tables 1 and 2).
We first prepared the close analogues 6a of MHBQ and 8a of
PHBQ. Compound 6a was 6 times more potent on D2 than MHBQ,
whereas the EC50’s on D1 were >1000 nM for both compounds.
On the contrary, 8a was equipotent to PHBQ on both D1 and D2.
In general, the N-alkyl structure–activity relationship (SAR) on
the D2 receptor within our series is similar to the SAR described
for the known benzo[g]quinoline series, exemplified by MHBQ
and PHBQ.9 In short, the potency increases from methyl to propyl
but the cyclopropylmethyl is apparently too large since the EC50
of 10a is approximately 20 times weaker than 7a–9a, and this con-
firms that the N-alkyl binding pocket probably is small.9
Hence, a relative small number of N-alkyl analogues were pre-
pared and no real optimization of the D1/D2 ratio could be obtained
by this strategy. The synthesis of analogues with a substituent at
the 2 position was then pursued (Table 2), and we incorporated
small lipophilic substituents at the 2 position. This is in agreement
with a D2 homology model recently published, which shows lipo-
philic residues Trp 386 and Phe 389 to be close to this position.13
Compounds 19–27 were prepared in low to moderate yields
and the absolute configuration of a crystal of the HCl salt of 27
were determined by X-ray crystallography (Supplementary data).
Compounds 19, 21, 23, 25, and 27 have the (2R,4aR,10aR) configu-
ration, while 20, 22, 24 and 26 are the corresponding-2-epimers,
having the (2S, 4aR,10aR) configuration.
2.2. In vitro functional potencies at dopamine receptors and
SAR considerations
The compounds 20, 22, 24 and 26 with the 2S configuration are
more selective D2 receptor agonists, when compared to their 2R
counterparts, because of their relative weaker D1 receptor agonisms.
In vitro functional potencies and efficacies of the novel
compounds and key reference compounds were determined by

384
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
Table 2
Data for 19–27
N
O
N
O
R
R
OH
OH
19, 21, 23, 25, 27
20, 22, 24, 26
R =
D1 EC50
(%eff)
D2 EC50
(%eff)
D1/D2
H.Mic.
(L/min)
H.Hep.
(L/min)
cLogD
PSA
MPO
N
89 (90)
1.5 (99)
1.0 (95)
59
2.5
9.8
nd
2.1
2.1
50.5
50.5
5.8
5.8
19
N
N
210 (88)
210
nd
nd
20
N
N
N
N
21
22
360 (90)
13 (98)
33 (99)
28
nd
nd
4.2
4.2
50.5
50.5
3.7
3.7
Ph
N
3900 (110)
118
nd
Ph
N
23
24
150 (96)
230 (71)
1.7 (96)
88
2.5
nd
8.4
nd
1.4
1.4
63.4
63.4
5.8
5.8
N
N
N
0.5 (110)
460
N
N
N
N
25
74 (85)
2.1 (98)
35
16
14
2.9
50.5
5.2
Cl
N
N
26
27
400 (94)
19 (93)
3.5 (99)
0.6 (96)
114
32
nd
13
nd
14
2.9
3.2
50.5
32.7
5.2
4.8
Cl
S
Values from efficacy assays (EC50) is in nM and % efficacy is relative to dopamine. Calculated polar surface area (PSA) is topological PSA and calculated lipophilicity (cLogD) is
at pH 7.4. In vitro CLint are determined in human microsomes and human hepatocytes.
NH2
OH
NH2
OH
NH2
OH
HCl
HCl
a,b,c
+
OMe
OMe
OMe
OMe
11b
2
11a
Pr
Pr
N
Pr
N
NH2
OH
HCl
NH
e
d
+
OH
O
O
OH
OMe
OMe
OH
OMe
11a
14
12
13
Scheme 3. Reagents and conditios: (a) Boc2O; (b) SFC separation; (c) HCl; (d) (i) propionyl chloride, (ii) LiAlH4; (e) S(ꢀ)-epichlorohydrin or R(+)-epichlorohydrin.
Compound 27 incorporates a small methyl sulfide group in
the 2 position (as in pergolide) and is the strongest D1 receptor
agonists among the novel DRAs. Compound 27 shows similar
potency and efficacy on D1 (19 nM, 93%) and D2 (0.6 nM, 96%)
receptors as rotigotine (D1 (28 nM, 110%) and D2 (0.7 nM,
110%)). However, compound 27 is predicted to be a better CNS
drug than rotigotine based on MPO scores8 and is devoid of
any structural alerts.

R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
385
Pr
N
Pr
N
Pr
N
Pr
N
a
c
b
O
O
O
O
OH
OMe
OTs
OMe
N
N
OMe
OH
13
15
19
17
N
N
Pr
N
Pr
N
Pr
N
Pr
N
a
c
b
O
O
O
O
OH
OMe
OTs
OMe
N
N
OMe
OH
14
16
20
18
N
N
Scheme 4. Reagents and conditions: (a) tosylchloride, pyridine, 4 h rt; (b) pyrazole, NaH, DMF, 130 °C, 0.5 h; (c) thiophenol, KF, DMA, microwave, 100 °C; compounds 21–27
(Table 1) were prepared in a similar manner.
Table 3
N-ethyl (7a) and the N-propyl (8a) analogues also displayed some
potency at the D1 receptor. Therefore, the N-propyl group was cho-
In vivo rat metabolism for rotigotine, 6a and 7a, N = 5
Clp (L/h/kg)
Vss (L/kg)
T1/2 (h)
F (%)
sen as the N-substituent in the corresponding 2-substituted napt-
hooxazine analogues 19–27. In general, these compounds were
synthesized in low to moderate yield. The absolute configuration
of 27 was determined by X-ray crystallography to be the
(2R,4aR,10aR) configuration.
In short, compound 27 shows similar in vitro potency and effi-
cacy on both D1 and D2 receptors as does rotigotine, and the hepa-
tic clearance of the two compounds are also similar. However,
compound 27 has no thiophene structural alert and may also have
better CNS-drug like properties if this can be reliably predicted in
this series via calculated MPO scores (4.8 vs 3.3, Table 2).8
Rotigotine
6a
7a
5.5
5.5
2.3
9.7
1.9
1.0
2
1
0.3
<5
<5
<5
Recently a D1 receptor homology model was published,14 and
our data could help to refine this model further.
2.3. Metabolism
As the clinical use of most of the DRA’s are hampered by fre-
quent daily dosing due to a high metabolic clearance, the meta-
bolic stability of novel DRA ligands is an important selection
criteria. Thus, the intrinsic clearance (Clint) in human microsomes
(H.Mic) and human hepatocytes (H.Hep) was determined for the
new ligands and the reference compounds as well (see Tables 1
and 2). We found a high microsomal Clint, well over the liver blood
flow of 1.4 L/min for all the tested compounds. An even higher
Clint were seen using hepatocytes, which in addition to micro-
somes harbors conjugation enzymes catalyzing, for example, glu-
curonidation of the free hydroxyl group in the molecules. Except
for a modest improvement of Clint for 6a, which did not meet
the desired pharmacological profile, all compounds showed high
Clint values in excess of the liver blood flow, indicating similar high
metabolic clearances and thus no apparent improvement of meta-
bolic stability.
The in vivo PK parameters in rats dosed po and iv at 0.5 and
1.0 mg/kg for Rotigotine, 6a and 7a are shown in Table 3. The iv
clearance and volume of distribution for 6a (Clp = 5.5, Vss = 1.9)
and 7a (Clp = 2.3, Vss = 1.0) resulted in short half-life’s and low bio-
availabilities for both 6a (T1/2 = 1 h, F <5%) and 7a (T1/2 = 0.3 h, F
<5%). Rotigotine showed higher Vss (9.7), but similar high clea-
rence (5.5), resulting in short half-life’s and low bioavailability
for Rotigotine (T1/2 = 2 h, F <5%). Because of these low bioavailabil-
ities and short half-life’s for 6a and 7a, and the high in vitro clea-
rence for these compounds (Tables 1 and 2) no further in vivo PK
parameters were measured for compound 8a–10a and 19–27. In
case one of these compounds should be progressed into humans,
alternative routes of administration, such as, for example, trans-
dermal or subcutaneous administration, should be invistigated.
4. Experimental
4.1. Chemistry
Reactions were generally performed under either argon or
nitrogen gas in dry solvents. Organic extracts were dried over
anhydrous sodium or magnesium sulfate and filtered before the
solvents were removed under reduced pressure (described as
‘dried and concentrated’). Microwave-assisted reactions were per-
formed with Emrys Optimizer from Personal Chemistry operating
at 2450 MHz, in sealed vials (described as ‘using microwave heat-
ing’) with internal pressure below 20 bar. An increasing amount of
etylacetate in heptane was used for silica gel chromatography,
unless otherwise noted. Hydrogenations were performed on a
standard Parr shaker at rt under a hydrogen pressure of 1–3 bar.
Tetrahydrofuran
(THF),
N,N0-dimethylformamide
(DMF),
N,N0-dimethylacetamide (DMA) dimethylsulphoxide (DMSO),
N-methylpyrrolidone (NMP), 1,4-dioxane (dioxane), toluene,
pyridine, acetonitrile (MeCN), dichloromethane (DCM) and 1,2-
dichloroethane (DCE) were dried over 4 Å MS and were HPLC
grade. Methanol (MeOH) and cyclohexane were also HPLC grade.
96% Ethanol (EtOH), 99.8% ethanol (abs EtOH), ethyl acetate
(EtOAc), diethylether (ether) and heptane were technical grade.
Thin layer chromatography was performed on Merck 60 F254 silica
gel plates. The spots were visualized using a UV lamp operating at
254 nm. Melting points were measured on a Buchi 535 apparatus.
NMR spectra were recorded either on a Bruker 600-Avance-III
spectrometer equipped with a 5 mm TCI cryoprobe operating at
600 MHz for 1H and 151 MHz for 13C, or using a Bruker DRX-500
spectrometer equipped with a 5 mm QNP probe operating at
511 MHz for 1H and 126 MHz for 13C. Chemical shifts are reported
in ppm. TMS was used as internal references for H NMR in CDCl3,
and residual CHCl3 (d = 77.1) for C NMR. As internal references
3. Conclusion
The N-alkyl DRAs 6a–10a were prepared in good yields and all
displayed relative potent D2 receptor agonism, whereas the

386
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
for NMR in DMSO-d6, were residual DMSO used (d = 2.50 for H
NMR, and d = 39.5 for C NMR). Coupling constants are in hertz.
The following abbreviations are used for multiplicity of NMR sig-
nals: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
LC–MS data were obtained on a PE Sciex API150EX instrument
equipped with an ion spray source and Shimadzu LC-8A/SLC-10A
LC system: column, 30 mm ꢁ 4.6 mm Waters Symmmetry C18 col-
MH+: 176.9. 1H NMR (CDCl3) d 2.80 (d, 1H), 3.15 (d, 1H), 3.30 (d,
1H), 3.45 (m, 3H), 3.80 (s, 3H), 6.65 (t, 2H) 7.10 (t, 1H). 13C NMR
(CDCl3) d 157.7, 132.9, 127.2, 121.9, 120.6, 108.2, 55.7, 52.2, 51.9,
29.9, 23.5. Calcd for C11H12O2: C, 74.98; H, 6.86. Found: C, 74.65;
H, 6.94. This epoxide (13.99 g, 79.39 mmol) was dissolved in DMSO
(300 mL). NaN3 (41.29 g, 635.15 mmol) was suspended in DMSO
(300 mL), and concentrated H2SO4 (11.67 g, 119.1 mmol) was dis-
solved in DMSO (133 mL). The solutions and suspensions were
combined and stirred for 23 h at 60 °C. To the mixture was added
EtOAc (1.0 L) and water (1.5 L). The phases were separated, and
the aqueous phase was extracted with EtOAc (2 ꢁ 500 mL). The
combined organic phases were washed with brine, dried and con-
centrated to give 19.66 g of the two isomers as a red solid. The
regioisomers were separated by fractional crystallization in tolu-
ene (100 mL) where 1 precipitated. Yield: 4.61 g (27%) of 1 as col-
orless crystals, mp 143–144 °C (lit.8 mp 145–147 °C); 1H NMR
(500 MHz, CDCl3) d 2.30 (b, 1H), 2.55 (dd, 1H), 2.85 (dd, 1H), 3.20
(dd, 1H), 3.35 (dd, 1H), 3.70 (m, 1H), 3.80 (s, 3H), 3.85 (m, 1H),
6.70 (m, 2H), 7.15 (t, 1H). Anal. Calcd for C11H13N3O2: C, 60.26;
H, 5.98; N, 19.17. Found: C, 60.27; H, 6.14; N, 18.94.
umn with 3.5 lm particle size; solvent system, A = water/TFA
(100:0.05) and B = water/acetonitrile/TFA (5:95:0.03) (TFA = triflu-
oroacetic acid); method, linear gradient elution with 90% A to 100%
B in 2.4 min and then 10% B in 0.4 min, with a flow rate of 3.3 mL/
min. Total time including equilibration was 2.8 min. Injection vol-
ume was 10 lL from a Gilson 215 autosampler. The purity of com-
pounds submitted for biological testing were in all cases P95% as
determined using evaporative light scattering detection (ELSD) and
P95% using UV detection (254 nm) unless noted otherwise. Pre-
parative LC–MS were performed on a PE Sciex API150EX instru-
ment equipped with an ion spray source and Shimadzu LC-8A/
SLC-10A LC system: column, 50 mm ꢁ 10 mm Waters Symmetry
C18 column with 5 lm particle size; solvent system, A = water/
TFA (100:0.05) and B = water/acetonitrile/TFA (5:95:0.03); meth-
od, linear gradient elution with 90% A to 100% B in 7 min and then
10% B in 1 min, with a flow rate of 5.7 mL/min. Injection volume
4.1.2. Racemic-trans-9-methoxy-4a,5,10,10a-hexahydro-4H-
naphtho[2,3-b][1,4]oxazine-3-one (3)
was 0–300
l
L from a Gilson 233XL autosampler. HRMS spectra
Compound 1 (4.61 g, 21 mmol) was dissolved in abs EtOH
(350 mL) and 10% Pd/C (490 mg) was added under an argon atmo-
sphere. The mixture was hydrogenated at 2.5 bar overnight. The
crude mixture was filtered through Celite, concentrated, and dried
in vacuo to give racemic trans-3-amino-8-methoxy-1,2,3,4-tetra-
hydro-naphthalen-2-ol (2) as a white solid. Yield: 4.15 g (96%).
This material was dissolved in THF (200 mL) and cooled in an ice-
bath. Et3 N (5.04 mL, 36 mmol) and then chloroacetyl chloride
(2.32 mL, 29.2 mmol) in THF (11 mL) was added over 2 min. The
mixture was stirred for 2 h at rt. EtOAc (400 mL) and 1 M HCl
(400 mL) were added and the layers were separated. The organic
layer was washed with brine, dried (MgSO4), filtered and concen-
trated to give 6.01 g of a brown solid. This intermediate was dis-
solved in THF (200 mL) and 60% NaH dispersion (1.25 g,
31 mmol) was added slowly at rt and the solution was stirred 3 h
at rt. The solution was subsequently quenched by adding 6 M
HCl (5.2 mL) and 3 precipitated. The product 3 was filtered off
and washed several times with ether, then water and finally ether
again, and then dried in vacuo. Yield: 3.14 g (60%) of 3 as white so-
lid. 1H NMR (500 MHz, DMSO-d6) d 2.41 (dd, 1H), 2.64 (t, 1H), 2.99
(dd, 1H), 3.10 (dd, 1H), 3.43 (m, 1H), 3.66 (m, 1H), 3.77 (s, 3H), 4.10
(m, 1H), 6.72 (d, 1H), 6.81 (d, 1H), 7.15 (t, 1H), 8.35 (s, 1H).
were obtained on a Bruker Daltonic MicroTOF with internal cali-
bration using ESI in positive mode. Chiral compounds were re-
solved by chiral super critical fluid chromatography (SFC) on a
Berger SFC multigram II instrument equipped with a Chiralpak
AD 21.2 mm ꢁ 250 mm column. Solvent system: CO2/EtOH/Et2NH
(70:29.9:0.1) or as specified below. The method consisted of a con-
stant gradient with a flow rate of 50 mL/min. The fraction collec-
tion was performed by UV 230 nm detection.
4.1.1. Racemic trans-3-azido-8-methoxy-1,2,3,4-
tetrahydronaphthalen-2-ol (1)
To a solution of naphthalen-1-ol (8.65 g, 60.0 mmol) in abs.
EtOH (10 mL) under nitrogen, was added NH3(l) (60 mL) at
ꢀ78 °C. Li (2.07 g, 300 mmol) was added in small portions over
5 min. After 65 min abs. EtOH (50 mL) was added after which the
mixture was quenched with ice-cold water (100 mL), acidified
with 12 M HCl, and extracted with ether (2 ꢁ 150 mL). The com-
bined organic phases were washed with brine, dried and concen-
trated to give 9.38 g (>100%) of the intermediate alkene as a light
red solid. GS/MS: m/z 146 (79%). Dimethylsulfate (28.4 g,
225.4 mmol) was added to a stirred suspension of this alkene
(9.38 g, 64.2 mmol), K2CO3 (32.1 g, 232 mmol) and acetone
(108 mL). The mixture was refluxed for 2.5 h, cooled to rt, dried
and concentrated. Ether (100 mL) and half satd NaHCO3 (100 mL)
was added. The phases were separated, and the aqueous phase
was extracted with more ether (2 ꢁ 100 mL). The combined organ-
ic phases were washed with brine, dried and concentrated to give
18 g of a light brown oil. The remaining dimethylsulfate was distil-
lated off at 30–33 °C (0.3 mbar). Yield: 7.01 g (68%) of the methyl-
ated intermediate as a light red oil. GS/MS: m/z 160 (78%). mCPBA
(11.89 g, 68.89 mmol) was added in small portions to a stirred
solution of the intermediate (7.01 g, 43.76 mmol) in DCM
(110 mL) at 0 °C. The mixture was stirred for 5 h at rt. The reaction
was quenched by adding 2 M NaOH (140 mL) at 0 °C, and the
phases were separated. The organic phase was filtered through sil-
ica gel using EtOAc/heptane (0–50%) as eluent. Yield: 4.99 g (65%)
of 3-methoxy-1a,2,7,7a-tetrahydronaphtho[2,3-b]oxirene as a dark
yellow solid. LC/MS: UV: 87.4%; MH+: 177.0. The product was
recrystallized from heptane/EtOH (4/1). Yield: 2.64 g (34%) as col-
orless crystals. Mp 48–9 °C. Rf: 0.49 (EtOAc); LC/MS: UV: 97.4%;
4.1.3. Racemic trans-9-methoxy-2,3,4a,5,10,10a-hexahydro-
naphtho[2,3-b][1,4]oxazine-4-carboxylic acid tert-butyl ester
(4)
1 M LAH in THF (15 mL, 15 mmol) was added dropwise to a stir-
red solution of compound 4 (1.61 g, 6.90 mmol) in THF (83 mL).
The mixture was stirred for 3 h at rt. The solution was quenched
with ice-cold water and ether was added. The aqueous layer was
extracted with ether. The combined organic layers were washed
with brine, dried and concentrated to give an yellow oil. This inter-
mediate was dissolved in THF (70 mL), and Et3N (1.45 mL,
10.4 mmol) and Boc2O (1.52 g, 7.0 mmol) were added, before the
mixture was stirred for 3 days at rt. The solution was concentrated
and the crude product was purified by silica gel chromatography
(eluent: increasing amount of EtOAc in heptanes). Yield: 1.45 g
(66%) of 4 as an oil. 1H NMR (500 MHz, CDCl3) d 1.41 (s, 9H),
2.42 (dd, 1H), 2.72 (dd, 1H), 3.17 (dd, 1H), 3.52–3.65 (m, 4H),
3.72 (m, 1H), 3.76 (s, 3H), 3.82 (m, 1H), 3.99 (m, 1H), 6.59 (d,
1H), 6.64 (d, 1H), 7.04 (t, 1H).

R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
387
4.1.4. Resolution of racemic trans-9-methoxy-2,3,4a,5,10,10a-
4.1.8. (4aR,10aR)-4-Ethyl-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazin-9-ol hydrochloride (7a)
hexahydro-naphtho[2,3-b][1,4]oxazine-4-carboxylic acid tert-
butyl ester (resolution of compound 4 into compounds 4a and
4b)
L-selectride (1 M in THF, 18 mL, 18 mmol) was added dropwise
at rt to a solution of compound 4a (577 mg, 1.8 mmol) in THF
(5 mL). The solution was heated to 100 °C for 6 h using microwave
heating. After cooling to 0 °C, ice-cold water (125 mL) and satd
NaHCO3 (50 mL) were slowly added, and the product was extracted
with ether (3 ꢁ 75 mL). The combined organic phases were washed
with brine, dried, and concentrated. The residue was purified by
silicagel chromatography to give (4aR,10aR)-9-hydroxy-2,3,4a,5,
10,10a-hexahydro-naphtho[2,3-b][1,4]oxazine-4-carboxylic acid
tert-butyl ester as a white solid. Yield: 297 mg (54%). Mp: 203–
205 °C. 141 mg, (0.46 mmol) of this material was dissolved in MeOH
(4 mL) and HCl (5 M in Et2O) was added. The solution was stirred for
45 min at rt and concentrated. The residue was dissolved in abs EtOH
(2.3 mL). NaCNBH3 (129 mg, 1.9 mmol), acetic acid (0.3 mL) and
acetaldehyde (0.16 mL, 2.8 mmol) were added and the solution
was heated to 90 °C for 15 min using microwave heating. After cool-
ing to 0 °C, ice-cold water (25 mL) and satd NaHCO3 (10 mL) were
slowly added, and the product was extracted with Et2O
(3 ꢁ 15 mL). The combined organic phases were washed with brine,
dried and concentrated. The crude product was purified by chroma-
tography to give 49 mg. The product was dissolved in MeOH (1 mL)
and HCl (1.5 mL, 5 M in ether) was added. The generated HCl salt was
isolated and dried in vacuo. Yield of 7a: 38 mg (31%) as a white solid.
Mp dec >280 °C; LC/MS: ELSD 99%, UV 100%. MH+: 234.1. 1H NMR
(500 MHz, DMSO-d6) d 1.25 (t, 3H), 2.40 (dd, 1H , 3.05–3.20 (br m,
4H), 3.35–3.50 (m, 3H), 4.00–4.10 (br m, 3H), 6.60 (d, 1H), 6.65 (d,
1H), 7.00 (t, 1H), 9.55 (s, 1H), 11.45–11.55 (b, 1H). Anal. Calcd for C14-
H19NO2ꢂHCl: C, 61.31; H, 7.53; N, 5.11. (1/4H2O) Found: C, 61.69; H,
Compound 4 (1.38 g, 43 mmol) was resolved by chiral SFC on a
Berger SFC multigram II instrument equipped with a Chiralpak AD
21.2 ꢁ 250 mm column. Solvent system: CO2/EtOH/Et2NH
(70:29.9:0.1) Method: constant gradient with a flow rate of
50 mL/min. Fraction collection was performed by UV at 230 nm
detection. Fast eluting enantiomer (4aS, 10aS enantiomer; com-
pound 4b): 0.622 g (45%) as a white solid. Mp = 89–90 °C. aD
+190.1 (c 0.25, MeOH). Slow eluting enantiomer (4aR,10aR enan-
tiomer; compound 4a): 0.633 g (46%) as a white solid. Mp = 89–
90 °C. aD ꢀ184.8 (c 0.25, MeOH).
4.1.5. (4aR,10aR)-9-Methoxy-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazine hydrochloride (5A)
To a stirred solution of compound 4a (545 mg, 1.71 mmol) in
MeOH (11 mL) was added HCl (14 mL, 5 M in ether). After stirring
for 45 min, the mixture was concentrated. Yield: 418 mg (96%) of
intermediate 5a as a white solid. Mp dec >280 °C; LC/MS: ELSD:
99.6%; UV: 99.2%; MH+: 220.2. NMR data identical to the data re-
ported for intermediate 5b. Anal. Calcd for C13H17NO2 HCl: C,
61.05; H, 7.09; N, 5.48. Found: C, 60.93; H, 7.26; N, 5.42.
4.1.6. (4aS,10aS)-9-Methoxy-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazine hydrochloride (5b)
To a stirred solution of compound 4b (543 mg, 1.71 mmol) in
MeOH (11 mL) was added HCl (14 mL, 5 M in ether). After stirring
for 45 min, the mixture was concentrated. Yield: 436 mg (100%) of
intermediate 5b as a white solid. Mp dec >280 °C; LC/MS: ELSD:
98.7%; UV: 93.7%; MH+: 220.2. 1H NMR (500 MHz, DMSO-d6) d
2.40 (dd, 1H), 3.00–3.20 (br m, 4H), 3.30 (m, 1H), 3.75 (s, 3H),
3.90 (m, 2H), 4.00 (dd, 1H), 6.75 (d, 1H), 6.80 (d, 1H), 7.15 (t,
1H), 9.75 (b, 1H). 13C NMR (DMSO-d6) d: 157.0, 133.3, 127.6,
121.6, 120.8, 108.6, 73.2, 63.1, 55.7, 54.3, 43.2, 31.4, 29.1. Anal.
Calcd for C13H17NO2ꢂHCl: C, 61.05; H, 7.09; N, 5.48. Found: C,
61.11; H, 7.25; N, 5.41.
7.57; N, 5.12.
aD: ꢀ116.6° (c 0.5, DMSO).
4.1.9. (4aR,10aR)-4-n-Propyl-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazin-9-ol hydrochloride (8a)
K2CO3 (176 mg, 1.50 mmol) and n-propyl iodide (375 mg,
2.2 mmol) were added to
a stirred solution of 5a (100 mg,
0.39 mmol) in DMF (9 mL). The mixture was stirred at 55 °C for
3 h. Water (20 mL) was added and the product was extracted into
ether (3 ꢁ 10 mL). The combined organic phases were washed with
brine and satd NH4Cl, dried and concentrated to give (4aR,10aR)-9-
methoxy-4-n-propyl-3,4,4a,5,10,10a-hexahydro-2H-naph-
tho[2,3b][1,4]oxazine as a white solid. Yield: 93 mg. LC/MS (method
25): RT 0.58 min, ELSD 100%, UV 92%. TLC: Rf = 0.51 (EtOAc). The L-
selectride de-protection procedure described for 6a was followed
to give example 8a. Yield: 61 mg (55%) as a white solid. Mp dec
>300 °C; LC/MS: ELSD 100%, UV 98%. MH+: 248.2. 1H NMR
(500 MHz, DMSO-d6) d 0.95 (t, 3H), 1.73 (m, 2H), 2.40 (dd, 1H),
3.00 (m, 1H), 3.10–3.20 (br m, 4H), 3.35–3.50 (br m, 2H), 4.05 (m,
3H), 6.60 (d, 1H), 6.65 (d, 1H), 7.00 (t, 1H), 9.55 (s, 1H), 11.30–
11.40 (b, 1H); Anal. Calcd for C15H21NO2ꢂHClꢂ0.25H2O: C, 62.48; H,
7.88; N, 4.86. Found: C, 62.71; H, 7.99; N, 5.05. aD ꢀ109.3 (c 0.5,
DMSO).
4.1.7. (4aR,10aR)-4-Methyl-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazin-9-ol hydrochloride (6a)
Compound 4a was dissolved in dry THF (5 mL). LAH (1 M in THF,
1.5 mL, 1.5 mmol) was added dropwise at rt and the solution was
heated to 90 °C for 15 min using microwave heating. After cooling
to rt, MeOH (0.3 mL) and ice-cold water (10 mL) was slowly added,
and the product was extracted with Et2O (3 ꢁ 20 mL). The com-
bined organic phases were washed with brine, dried and concen-
trated to give 107 mg intermediate (TLC: Rf = 0.19 (EtOAc)). L-
selectride (1 M in THF, 4.6 mL, 4.6 mmol) was added at rt to this
intermediate (107 mg). The solution was heated to 100 °C for 6 h
using microwave heating. After cooling to 0 °C, ice-cold water
(25 mL) and satd NaHCO3 (10 mL) were slowly added, and the prod-
uct was extracted with Et2O (3 ꢁ 15 mL). The combined organic
phases were washed with brine, dried, and concentrated. The crude
product was purified by silica gel chromatography. The free base
was dissolved in MeOH (1 mL) and precipitated as the HCl salt using
5 M HCl in ether (1.5 mL). Yield: 49 mg of 6a as a white solid. Mp
dec >280 °C; LC/MS: ELSD: 99.0%, UV: 100.0%, MH+: 220.3. 1H
NMR (500 MHz, DMSO-d6) d 2.40 (dd, 1H), 2.85 (s, 3H), 3.05–3.20
(br m, 2H), 3.25 (m, 1H), 3.35–3.50 (br m, 2H), 3.95–4.05 (br m,
3H), 6.60 (d, 1H), 6.65 (d, 1H), 7.00 (t, 1H), 9.55 (s, 1H), 11.30–
11.40 (b, 1H). Anal. Calcd for C13H17NO2ꢂHCl: C, 61.05; H, 7.09; N,
5.48. Found: C, 60.46; H, 7.18; N, 5.28. aD ꢀ122.9° (c 0.5, DMSO).
4.1.10. (4aR,10aR)-4-Allyl-3,4,4a,5,10,10a-hexahydro-2H-
naphtho[2,3-b][1,4]oxazin-9-ol hydrochloride (9a)
K2CO3 (202 mg, 1.46 mmol) and allyl bromide (0.21 mL,
300 mg, 2.48 mmol) were added to a stirred solution of 5a
(115 mg, 0.45 mmol) in DMF (10 mL). The mixture was stirred at
55 °C for 5 h. Water (20 mL) and satd NaHCO3 (10 mL) were added
and the product was extracted into ether (3 ꢁ 20 mL). The com-
bined organic phases were washed with brine, dried and concen-
trated to give (4aR,10aR)-4-allyl-9-methoxy-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3b][1,4]oxazine as a white solid. Yield:

388
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
117 mg. LC/MS (method 25): RT 0.97 min, ELSD 97%, UV 48%. MH+:
260.3. The L-selectride de-protecting procedure described for com-
pound 6a was followed to give 9a. Yield: 28 mg (21%) as a white
solid. Mp dec >280 °C; LC/MS: ELSD 99%, UV 96%. MH+: 246.2. 1H
NMR (500 MHz, DMSO-d6) d 2.40 (dd, 1H), 3.10–3.20 (br m, 3H),
3.40 (m, 1H), 3.45 (dd, 1H), 3.75 (m, 1H), 4.00–4.10 (br m, 4H),
5.55 (d, 1H), 5.65 (d, 1H), 6.05 (m, 1H), 6.60 (d, 1H), 6.65 (d, 1H),
7.00 (t, 1H), 9.55 (s, 1H), 11.65–11.75 (b, 1H). Anal. Calcd for C15-
H19NO2ꢂHClꢂ0.25H2O: C, 62.93; H, 7.22; N, 4.89. Found: C, 62.53;
H, 7.48; N, 4.71. aD ꢀ115.4° (c 0.5, DMSO).
(2R,3R enantiomer; Boc-2A): 5.17 g as a white solid. Mp = 161–
162 °C.
4.1.15. (2R,3R)-3-Amino-8-methoxy-1,2,3,4-tetrahydro-
naphthalen-2-ol (compound 11A)
To a stirred solution of Boc-2A (7.19 g, 24.5 mmol) in MeOH
(100 mL) was added HCl (99 mL, 2 M in ether). After stirring for
two hours the solution was filtered to afford compound 11A as a
white solid. Yield: 5.05 g (90%). LC/MS: ELSD 97.0%, UV 100%.
MH+: 194.1. ꢀ105.5° (c 0.25, MeOH) 1H NMR (500 MHz, DMSO-
D
d6) d: 2.41 (dd, 1H), 2.95 (dd, 1H), 3.09–3,17 (m, 3H), 3.77 (s,
3H), 3.87 (m, 1H), 5.75 (1, 1H), 6.71 (d, 1H), 6.79 (d, 1H), 7.13 (t,
1H), 8.32 (s, 3H). 13C NMR (DMSO-d6) d: 157.0, 133.8, 127.3,
122.7, 120.7, 108.3, 67.2, 55.7, 52.3, 32.6, 31.9.
4.1.11. (4aR,10aR)-4-Cyclo-propylmethyl-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3b][1,4]oxazin-9-ol hydrochloride
(10a)
K2CO3 (176 mg, 1.27 mmol) and cyclopropylmethyl bromide
(0.21 mL, 292 mg, 2.16 mmol) were added to a stirred solution of
intermediate 5a (100 mg, 0.39 mmol) in DMF (9 mL). The mixture
was stirred at 55 °C for 3 h. Water (20 mL) and satd NaHCO3
(10 mL) was added and the product was extracted into ether
(3 ꢁ 20 mL). The combined organic phases were washed with brine,
dried and concentrated to give (4aR,10aR)-4-cyclopropylmethyl-9-
methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3b][1,4]oxa-
zine as a white solid. Yield: 107 mg. LC/MS: ELSD 100%, UV 92%.
MH+: 274.3. The L-selectride deprotection procedure described for
example 6a was followed to give 10a. Yield: 79 mg (72%) as a white
solid. Mp dec >280 °C; LC/MS: ELSD 99%, UV 98%. MH+: 260.2. 1H
NMR (500 MHz, DMSO-d6) d 0.45 (m, 2H), 0.65 (m, 2H), 1.15 (m,
1H), 2.40 (dd, 1H), 3.05–3.20 (br m, 3H), 3.25 (m, 1H), 3.35–3.45
(br m, 2H), 3.70 (d, 1H), 4.00–4.10 (br m, 3H), 6.60 (d, 1H), 6.65 (d,
1H), 7.00 (t, 1H), 9.55 (s, 1H), 11.10–11.20 (b, 1H). aD ꢀ109.5° (c
0.5, DMSO).
4.1.16. (2S,3S)-3-Amino-8-methoxy-1,2,3,4-tetrahydro-
naphthalen-2-ol (compound 11B)
This material was synthesized from Boc-2B (7.44 g, 25.4 mmol)
in a similar manner as described for compound 11A. Yield: 5.34 g
(91%) of compound 11B as a white solid. LC/MS: ELSD 100%, UV
100%. MH+: 194.1. aD +104,6 (c 0.25, MeOH). NMR data identical
to the data reported for compound 11A.
4.1.17. (2R,3R)-8-Methoxy-3-propylamino-1,2,3,4-tetrahydro-
naphthalen-2-ol (12)
Propionylchlorid (0.60 mL, 6.90 mmol) and Et3N (1.80 mL,
12.9 mmol) were added to a stirred solution of compound 11a
(1.50 g, 6.5 mmol) suspended in THF (15.0 mL, 185 mmol). The
solution was stirred for 20 min. LAH (1 M in THF, 10 mL) was
added dropwise under stirring at rt. and the reaction mixture were
heated to 90 °C for 20 min using microwave heating. The solution
was quenched by pouring it into wet Na2SO4. The mixture was di-
luted with THF (50 mL), filtered over dry Na2SO4 and concentrated.
Yield: 1.21 g (81%) of 12 as an oil. LC/MS: ELSD: 100%; UV: 100%;
MH+ 235.9. 1H NMR (500 MHz, CDCl3) d 0.89 (t, J = 7,6 Hz, 3H),
1.43–1.52 (m, 2H), 2.37–2.51 (m, 3H), 2.61–2.68 (m, 1H), 2.74–
2.80 (m, 1H), 3.14 (dd, J = 4.93, J = 16.0, 1H), 3.22–3.29 (dd,
J = 16.9, J = 5.9, 1H), 3.56–3.63 (m, 1H), 3.73 (s, 1H), 6.60 (d,
J = 8.05, 1H), 6.62 (d, J = 7.89, 1H), 7.03 (t, J = 7.86, 1H). 13C NMR
(500 MHz, CDCl3) d: 157.7 136.4, 127.0, 123.8, 121.2, 107.7, 71.0,
59.8, 55.7, 49.5, 35,7, 32.1, 24.1, 12.2.
4.1.12. (4aS,10aS)-4-Cyclo-propylmethyl-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol hydrochloride
(10b)
The procedure described for example 10a was followed starting
from intermediate 5b (100 mg, 0.39 mmol) to give (4aS,10aS)-4-
cyclopropylmethyl-9-methoxy-3,4,4a,5,10,10a-hexahydro-2H-
naphtho [2,3b] [1,4]oxazine. Yield: 107 mg. LC/MS: ELSD 99%, UV
90%. MH+: 274.1.
The L-selectride deprotection procedure described for 6a was
followed to give 10b. Yield: 73 mg as a white solid. Mp dec
>280 °C; LC/MS: ELSD 99%, UV 96%. MH+: 260.2. 1H NMR data iden-
tical to the data reported for example 10a. Anal. Calcd for C16H21-
NO2ꢂHCl: C, 64,48; H, 7.52; N, 4.70. Found: C, 64.32; H, 7.66; N,
4.64. aD +114.9° (c 0.5, DMSO-d6).
4.1.18. ((2S,4aR,10aR)-9-Methoxy-4-propyl-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3-b][1,4]oxazin-2-yl)-methanol (14)
and ((2R,4aR,10aR)-9-methoxy-4-propyl-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3-b][1,4]oxazin-2-yl)-methanol (13)
S-(+)-Epichlorohydrin (1.22 mL, 15.5 mmol) was added to a stir-
red solution of compound 12 (751 mg, 3.20 mmol) dissolved in 1,2-
DCE (3.2 mL). The reaction mixture was refluxed at 120 °C over-
night, and concentrated. NaOH (8.7 mL, 2 M) was added to this
intermediate (392 mg) and the mixture was refluxed at 130 °C
overnight. The solution was allowed to cool, and THF (17.5 mL)
was added. The phases were separated and the organic phase
washed with brine (5 mL) and concentrated. The crude product
was purified by silica gel chromatography to afford 116 mg (13%)
of 14 and 76 mg (8%) of 13.
4.1.13. Racemic trans-3-hydroxy-5-methoxy-1,2,3,4-tetrahydro-
naphthalen-2-yl)-carbamic acid tert-butyl ester (Boc-2)
Compound
2 (7.36 g, 38.1 mmol) was dissolved in THF
(175 mL), and Et3N (7.7 mL, 5.58 g, 55 mmol) and Boc2O (7.74 g,
35.5 mmol) were added. The mixture was stirred at rt overnight
and concentrated. The crude product was purified by silica gel
chromatography to afford 6.90 g (62%) of Boc-2 as a white solid.
4.1.14. Resolution of racemic trans-3-hydroxy-5-methoxy-
1,2,3,4-tetrahydro-naphthalen-2-yl)-carbamic acid tert-butyl
ester (resolution of Boc-2 into Boc-2A and 2B)
Compound 14 LC/MS: ELSD: 100.0%; UV: 90.4%; MH+ 291.8. 1H
NMR (500 MHz, CDCl3) d 0.87 (t, J = 7.48, 3H), 1.38–1.59 (m, 2H),
2.09–2.15 (m, 1H), 2.30–2.40 (m, 1H), 2.59 (dd, J = 15.86,
J = 11.69, 1H), 2.65 (dd, J = 11.87, J = 3.99, 1H), 2.99 (d, J = 11.81,
1H), 3.11 (t, J = 5.59, 1H), 3.14 (t, J = 5.14, 1H), 3.74 (s, 3H), 3.83–
3.92 (m, 2H), 4.07–4.14 (m, 2H), 6.60 (d, J = 8.21, 1H), 6.63 (d,
J = 7.86, 1H), 7.04 (t, J = 8.03, 1H). 13C NMR (500 MHz, CDCl3) d:
157.5 135.5, 127.1, 123.4, 121.1, 107.7, 72.4, 72.3, 67.0, 60.9,
55,7, 55.1, 53.2, 33.9, 30.7, 19.1, 12.3.
Boc-2 (13 g, 44 mmol) was resolved by chiral SFC on a Berger SFC
multigram II instrument equipped with
a
Chiralpak AD
21.2 ꢁ 250 mm column. Solvent system: CO2/EtOH/Et2NH
(70:29.9:0.1) Method: constant gradient with a flow rate of
50 mL/min. Fraction collection was performed by UV 230 nm detec-
tion. Fast eluting enantiomer (2S, 3S enantiomer; Boc-2B): 5.14 g
(40%) as a white solid. Mp = 161–162 °C. Slow eluting enantiomer

R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
389
Compound 13 LC/MS: ELSD: 100.0%; UV: 100.0%; MH+ 291.8. 1H
4.1.22. (2S,4aR,10aR)-9-Methoxy-4-propyl-2-pyrazol-1-
ylmethyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazine (18)
NMR (500 MHz, CDCl3) d 0.85 (t, J = 7,3 Hz, 3H), 1.45–1.59 (m, 2H),
2.24–2.32 (m, 3H), 2.43 (dd, J = 16.9, J = 10.6, 1H), 2.60 (dd, J = 11.8,
J = 15.4, 1H), 2.73 (m, 1H), 2.79 (d, J = 11.18, 1H), 3.10–3.19 (m, 2H),
3,55 (dd, J = 6.35, J = 11.64, 1H), (3.58–3.66 (m, 2H), 3.74 (s, 3H),
3.78 (br s, 1H), 6.61 (d, J = 8.21, 1H), 6.66 (d, J = 7.92, 1H), 7.05 (t,
J = 7.88, 1H). 13C NMR (500 MHz, CDCl3) d: 157.5 135.8, 127.1,
123.4, 121.2, 107.7, 76.9, 76.0, 64.7, 60.9, 55,7, 55.6, 53.4, 33.9,
30.1, 18.7, 12.4.
NaH (5.0 mg, 0.18 mmol) was added to a solution of 16 (42 mg,
0.09 mmol) and pyrazol (18.4 mg, 0.27 mmol) in DMF (1.0 mL). The
solution was heated at 130 °C for 30 min using microwave condi-
tions. The reaction was quenched with brine (10 mL) and EtOAc
(10 mL) was added. The phases were separated and the organic
phase washed with additional brine and concentrated. The product
was purified by silica gel chromatography. Yield: 33 mg (95%) of
compound 18 as an oil. LC/MS: ELSD: 100.0%; UV: 100.0%; MH+
328.0. Anal. Calcd for C20H28N3O2Clꢂ3.5H2O: C, 54.42; H, 7.90; N,
9.52. Found: C, 54.35; H, 7.57; N, 9.25. 1H NMR (500 MHz,
DMSO-d6) d 0.95 (t, J = 7,34 Hz, 3H), 1.67–1.82 (m, 2H), 2.33 (dd,
J = 16.80, J = 10.21, 1H), 2.99–3.08 (mm, 2H), 3.32–3.52 (m, 7H),
3.77 (s, 3H), 4.35–4.42 (m, 1H), 4.44–4.51 (m, 1H), 4.69 (dd,
J = 14.29, J = 6.06, 1H), 5.13 (dd, J = 14.33, J = 9.29, 1H), 6.27 (t,
J = 1.87, 1H), 6.78 (d, J = 7.60, 1H), 6.82 (d, J = 7.98, 1H), 7.18 (t,
J = 7.78, 1H), 7.50 (d, J = 1.70, 1H), 7.92 (d, J = 2.08, 1H).
4.1.19. Toluene-4-sulfonic acid (2S,4aR,10aR)-9-methoxy-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naptho[2,3-b][1,4]oxazin-
2-ylmethyl ester (15)
Compound 15 was synthesized from 13 (294 mg, 1.01 mmol) as
described for 16. Yield: 330 mg (73%) of compound 15 as yellow/
brown oil. LC/MS: ELSD: 100.0%; UV: 29.6%; MH+ 446.0. 1H NMR
(500 MHz, CDCl3) d 0.92 (t, J = 7,34 Hz, 3H), 1.39–1.59 (m, 2H),
2.16 (t, J = 10.89, 1H), 2.21–2.34 (m, 2H), 2.37 (dd, J = 17.40,
J = 6.52, 1H), 2.47 (s, 3H), 2.60 (dd, J = 15.01, J = 11.89, 1H), 2.71–
2.81 (m, 1H), 2.61–2.68 (m, 1H), 2.89 (d, J = 12.13, 1H), 3.10 (dd,
J = 16.91, J = 5.81, 1H), 3.17 (dd, J = 11.25, J = 4.90, 1H), 3.55 (dd,
J = 15.76 J = 9.46, 1H), 3.82 (s, 3H), 3.87–3.94 (m, 1H), 4.04 (dd,
J = 10.30, J = 4.92, 1H), 4.10 (dd, J = 10.30, J = 5.35, 1H), 6.68 (d,
J = 8.17, 1H), 6.72 (d, J = 7.56, 1H), 7.12 (t, J = 7.91, 1H), 7.37 (d,
J = 7.84, 2H), 7.83 (d, J = 8.12, 2H).
4.1.23. (2R,4aR,10aR)-4-Propyl-2-pyrazol-1-ylmethyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol
(19)
Compound 19 was synthesized from 17 as described for 20 from
18. Yield: 7.5 mg (29%) of compound 19 as an oil. 1H NMR
(500 MHz, DMSO-d6)
d 0.96 (t, J = 7,27 Hz, 3H), 1.54–1.67
4.1.20. Toluene-4-sulfonic acid (2R,4aR,10aR)-9-methoxy-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naptho[2,3-b][1,4]oxazin-
2-ylmethyl ester (16)
(m, 1H), 1.70–1.82 (m, 1H) 2.41 (dd, J = 16.92, J = 10.69, 1H), 2.51
(br s, 1H), 2.85 (t, J = 13.90, 2H), 2.97–3.14 (m, 2H), 3.34–3.71 (m,
3H), 3.92–3.99 (m, 1H), 4.25–4.42 (m, 3H), 6.29 (s, 1H), 6.60 (dd,
J = 7.45, 1H), 6.66 (d, J = 7.66, 1H), 6.99 (t, J = 7.89, 1H), 7.51 (s,
1H), 7.75 (d, J = 1.53, 1H); HRMS C19H26N3O2 [M+H+] calcd
328.2020, found 328.2011.
TsCl (343 mg, 1.80 mmol) was added to a solution of 14 (116 mg,
0.40 mmol) in pyridine (5.5 mL, 68 mmol). The solution was stirred
at rt for 4 h and quenched by addition of water (0.4 mL). EtOAc
(18.0 mL) and satd NaHCO3 (7.4 mL) were added. The phases were
separated and the organic phase extracted with satd NaHCO3
(7.4 mL) and washed with brine, and concentrated. The crude prod-
uct was purified by silica gel chromatography to afford 118 mg
(quantitative) of 16. LC/MS: ELSD: 100.0%; UV: 100.0%; MH+
446.0. Mp: 139.3–141.6. Anal. Calcd for C23H29NO5S: C, 64.01; H,
6.77; N, 3.25. Found: C, 64.67; H, 7.18; N, 3.02. 1H NMR
(500 MHz, CDCl3) d 0.86 (t, J = 7,44 Hz, 3H), 1.30–1.48 (m, 2H),
2.11–2.17 (m, 1H), 2.21–2.27 (mm, 1H), 2.26 (s, 3H), 2.31 (dd,
J = 16.65, J = 5.98, 1H), 2.45 (dd, J = 16.15, J = 4.57, 1H), 2.51 (dd,
J = 12.15, J = 8.08, 1H), 2.61–2.68 (m, 1H), 2.78 (d, J = 12.15, 1H),
2.92 (dd, J = 16.77, J = 11.22, 1H), 3.09 (dd, J = 16.05, J = 10.88, 1H),
3.18–3.24 (m, 1H), 3.81 (s, 3H), 4.03–4.09 (m, 1H), 4.31 (dd,
J = 10.34, J = 6.08, 1H), 4.52 (dd, J = 10.34, J = 7.76, 1H), 6.66 (d,
J = 8.15, 1H), 6.67 (d, J = 7.87, 1H), 7.09 (t, J = 7.90, 1H), 7.28 (d,
J = 7.98, 2H), 7.82 (d, J = 7.98, 2H). 13C NMR (500 MHz, CDCl3) d:
157.4, 145.3, 135.6, 133.2, 130.2, 128.5, 127.1, 123.2, 121.2, 107.6,
71.3, 70.8, 68.4, 61.6, 55.7, 54.6, 51.5, 33.8, 30.1, 21.8, 19.1, 12.2.
4.1.24. (2S,4aR,10aR)-4-Propyl-2-pyrazol-1-ylmethyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol
(20)
KF and thiophenol were added to 18 (21 mg, 0.06 mmol) dis-
solved in DMA. The reaction mixture was heated to 210 °C for
1 h using microwave heating, and then concentrated. The resulting
mixture was purified with silica gel chromatography (eluent: 0–5%
MeOH in EtOAc) and 20 was further purified by HPLC. Yield: 12 mg
(60%) of 20 as an oil. 1H NMR (500 MHz, DMSO-d6) d 0.77
(t, J = 7,27 Hz, 3H), 1.41–1.62 (m, 2H), 2.14 (dd, J = 16.67,
J = 10.24, 1H), 2.31 (br s, 1H), 2.77–2.94 (m, 3H), 3.19–3.32 (m,
4H), 3.38 (d, J = 13.28, 3H), 4.15 (dd, J = 16.23, J = 10.05, 1H),
4.22–4.34 (m, 2H), 4.70 (dd, J = 14.11, J = 9.45 1H), 6.09 (s, 1H),
6.43 (dd, J = 7.62, 1H), 6.48 (d, J = 7.99, 1H), 6.81 (t, J = 7.62, 1H),
7.31 (s, 1H), 7.63 (s, 1H); HRMS
328.2020, found 328.2016.
C
19H26N3O2 [M+H+] calcd
4.1.25. (2R,4aR,10aR)-2-(3-Phenyl-pyrazol-1-ylmethyl)-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazin-9-ol (21)
4.1.21. (2R,4aR,10aR)-9-Methoxy-4-propyl-2-pyrazol-1-
ylmethyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazine (17)
NaH (4.3 mg, 0.18 mmol) was added to a solution of 15 (42 mg,
0.09 mmol) and 3-phenyl-pyrazol (38.9 mg, 0.27 mmol) in DMF
(1 mL). The solution was heated at 130 °C for 30 min using micro-
wave heating. The reaction was quenched with saturated NaHCO3
(10 mL) and EtOAc (15 mL) was added. The phases were separated
and the organic phase extracted with NaHCO3 (10 mL) and concen-
trated. Yield: 60 mg of the crude intermediate (2R,4aR,10aR)-
9-methoxy-2-(3-phenyl-pyrazol-1-ylmethyl)-4-propyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazine. LC/MS:
ELSD: 99.9%; UV: 71.5%; MH+ 418.1. This intermediate was dis-
solved in DMA (6.3 mL), and KF (11 mg, 0.18 mmol) and thiophenol
(0.07 mL, 0.68 mmol) were added. The solution was heated at
210 °C for 1 h using microwave heating. The mixture was purified
Compound 17 was synthesized from compound 15 as described
for 18 from 16. Yield: 20 mg (59%) of compound 17 as a white solid.
LC/MS: ELSD: 100.0%; UV: 100.0%; MH+ 328.0. Anal. Calcd for C20-
H28N3O2Clꢂ2H2O: C, 58.00; H, 7.73; N, 10.14. Found: C, 58.47; H,
7.39; N, 10.04. 1H NMR (500 MHz, DMSO-d6) d 0.95 (t, J = 7,34 Hz,
3H), 1.63–1.81 (m, 2H), 2.39 (dd, J = 16.71, J = 10.83, 1H), 2.90–
3.06 (mm, 2H), 3.07–3.19 (m, 2H), 3.30–3.40 (m, 3H), 3.48 (dd,
J = 16.03, J = 5.34, 1H), 3.65 (s, 1H) 3.76 (s, 3H), 4.08–4.16 (m,
1H), 4.29 (dd, J = 14.32, J = 6.74, 1H), 4.39 (dd, J = 14.32, J = 4.29,
1H), 4.43–4.49 (m, 1H), 6.28 (t, J = 2.04, 1H), 6.76 (d, J = 7.93, 1H),
6.82 (d, J = 8.21, 1H), 7.17 (t, J = 8.00, 1H), 7.50 (d, J = 1.89, 1H),
7.74 (d, J = 2.23, 1H).

390
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
by silica gel chromatography and then HPLC. Yield of 21: 16 mg
(22%). LC/MS: ELSD: 97.5%; UV: 85.6%; MH+ 404.3. 1H NMR
(500 MHz, DMSO-d6) d 0.79 (t, J = 7,16 Hz, 3H), 1.57–1.69 (m,
1H), 1.70–1.84 (m, 1H), 2.44 (dd, J = 16.74, J = 10.37, 1H), 2.51 (br
s, 2H), 2.55 (s, 1H), 2.87 (dd, J = 14.88, J = 12.08, 1H), 3.00–3.09
(m, 1H), 3.12 (dd, J = 16.57, J = 6.15, 1H) (s, 1H) 3.73 (d, J = 12.23,
3H), 3.99 (m, 1H), 4.31–4.48 (m, 3H), 6.61 (d, J = 7.60, 1H), 6.67
(d, J = 8.00, 1H), 6.77 (d, J = 2.13, 1H), 6.99 (t, J = 7.74, 1H), 7.31 (t,
J = 7.27, 1H), 7.41 (t, J = 7.55, 1H), 7.79–7.83 (m, 3H), 9.67 (br s,
1H). HRMS C25H30N3O2 [M+H+] calcd 404.2333, found 404.2339.
MH+ 376.3; 1H NMR (500 MHz, CDCl3) d 1.08 (br s, 3H), 1.73 (br s,
1H), 1.99 (br s, 1H), 2.51 (dd, J = 9.25, J = 16.47, 1H), 2.97–3.44 (m,
7H), 3.83 (s, 3H), 3.94 (m, 1H), 4.51 (br s, 1H), 4.80 (br s, 1H), 5.57
(br s, 1H), 6.72 (d, J = 8.21, 2H), 7.17 (br s, 1H), 7.52 (s, 1H), 8.23 (s,
1H). 13C NMR (500 MHz, CDCl3) d: 157.3 138.3, 132.3, 131.2, 128.1,
121.2, 121.0, 110.9, 108.4, 69.5, 68.5, 62.9, 55.8, 55.5, 51.7, 49.2,
30.1, 29.8, 16.5, 11.8. This intermediate (8 mg, 0.02 mmol) was dis-
solved in DMA (0.7 mL) in a vial. KF (ꢃ2 equiv) and thiophenol
(0.01 mL) were added and the vial was sealed. The reaction mix-
ture was heated to 220 °C for 1 h. and the solvent was evaporated
off. The resulting mixture was purified with silica gel chromatogra-
phy using 0–100% (EtOAc in heptane) and HPLC. Yield: 2.6 mg
(26%) of 25 as an oil. LC/MS: ELSD: 100.0%; UV: 100.0%; MH+
362.4. 1H NMR (500 MHz, DMSO-d6) d 0.95 (t, J = 7,26 Hz, 3H),
1.55–1.68 (m, 1H), 1.69–1.81 (m, 1H), 2.40 (dd, J = 16.60,
J = 10.62, 1H), 2.51 (br s, 1H), 2.82–3.05 (m, 2H), 3.10 (dd,
J = 16.71, J = 5.83, 1H), 3.25–3.72 (m, 2H), 3.95 (d, J = 5.99, 1H),
(dd, J = 15.78, J = 10.27, 1H), 4.24–4.40 (m, 3H), 4.44–4.50 (m,
1H), 6.60 (d, J = 7.67, 1H), 6.66 (d, J = 7.67, 1H), 6.99 (t, J = 7.82,
1H), 7.61 (s, 1H), 7.99 (s, 1H); 9.66 (br s, 1H). HRMS C19H25Cl1N3O2
[M+H+] calcd 362.1630, found 362.1628.
4.1.26. (2S,4aR,10aR)-2-(3-Phenyl-pyrazol-1-ylmethyl)-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazin-9-ol (22)
Compound 22 was synthesized from 16 as described for 21 from
15. Yield: 66 mg of the crude intermediate (2S,4aR,10aR)-9-meth-
oxy-2-(3-phenyl-pyrazol-1-ylmethyl)-4-propyl-3,4,4a,5,10,10a-
hexahydro-2H-naphtho[2,3-b][1,4]oxazine. LC/MS: ELSD: 91.9%;
UV: 79.7%; MH+ 418.1. Yield: 5 mg (8%) of compound 22 as an
oil. LC/MS: ELSD: 100.0%; UV: 88.7%; MH+ 404.3. 1H NMR
(500 MHz, DMSO-d6) d 0.79 (t, J = 7,22 Hz, 3H), 1.39–1.65 (m,
2H), 2.16 (dd, J = 16.74, J = 10.37, 1H), 2.32 (br s, 2H), 2.36 (s,
1H), 2.80–2.98 (m, 3H), 3.11–3.47 (m, 3H), 4.10–4.30 (m, 1H),
4.31–4.40 (m, 1H), 4.76 (dd, J = 15.23, J = 11.56 3.58, 1H), 6.45 (d,
J = 7.82, 1H), 6.49 (d, J = 7.48, 1H), 6.57 (d, J = 2.25, 1H), 6.82 (t,
J = 7.80, 1H), 7.12 (t, J = 7.33, 1H), 7.22 (t, J = 7.62, 2H), 7.62 (d,
J = 7.33, 2H), 7.73 (d, J = 1.89, 1H), 9.43 (br s, 1H). HRMS
4.1.30. (2S,4aR,10aR)-2-(4-Chloro-pyrazol-1-ylmethyl)-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazin-9-ol (26)
Compound 26 was synthesized from 16 as described for 25 from
15. Yield: 43 mg of the intermediate (2R,4aR,10aR)-2-(4-chloro-
pyrazol-1-ylmethyl)-9-methoxy-4-propyl-3,4,4a,5,10,10a-hexahy-
dro-2H-naphtho[2,3-b][1,4]oxazine: 43 mg as an oil. LC/MS: ELSD:
100.0%; UV: 100.0%; MH+ 376.1; 1H NMR (500 MHz, CDCl3) d 1.05
(t, J = 7,20 Hz, 3H), 1.69–1.83 (m, 1H), 1.88–2.04 (m, 1H), 2.50 (dd,
J = 17.01, J = 10.40, 1H), 2.57 (dd, J = 18.94, J = 9.44, 1H), 2.90 (br s,
2H), 3.21 (dd, J = 15.56, J = 4.75, 1H), 3.29 (t, J = 11.30, 1H), 3.37 (dd,
J = 17.08, J = 6.31, 1H), 3.54 (d, J = 11.18, 1H), 3.67 (t, J = 13.14, 1H),
3.84 (s, 3H), 4.29–4.41 (m, 2H), 4.55–4.66 (m, 1H), 4.91 (d, J = 8.33,
1H), 6.69 (d, J = 7.74, 1H), 6.72 (d, J = 8.17, 1H), 6.82 (d, J = 8.21, 1H),
7.15 (t, J = 8.00, 1H), 7.44 (s, 1H), 7.52 (s, 1H). 13C NMR (500 MHz,
CDCl3) d: 157.3 138.6, 132.3, 129.6, 128.0, 121.4, 121.0, 111.0,
108.4, 73.7, 70.6, 62.3, 55.8, 54.9, 54.6, 52.4, 30.1, 29.9, 16.4,
11.6. Yield of the product (26): 6.4 mg (19%) as an oil. LC/MS: ELSD:
100.0%; UV: 100.0%; MH+ 362.4; 1H NMR (500 MHz, DMSO-d6) d
0.96 (t, J = 7,27 Hz, 3H), 1.57–1.81 (m, 2H), 2.32 (dd, J = 16.32,
J = 10.42, 1H), 2.51 (br s, 1H), 2.93–3.06 (m, 3H), 3.42–3.60 (br s,
4H), 4.30 (dd, J = 15.78, J = 10.27, 1H), 4.39 (dd, J = 14.50, J = 4.50,
1H), 4.44–4.50 (m, 1H), 4.89 (dd, J = 14.32, J = 10.32 1H), 6.63 (d,
J = 7.67, 1H), 6.67 (d, J = 7.67, 1H), 7.00 (t, J = 7.82, 1H), 7.61 (s,
1H), 8.10 (s, 1H); 9.67 (br s, 1H). HRMS C19H25Cl1N3O2 [M+H+] calc
362.1630, found 362.1624.
C
25H30N3O2 [M+H+] calcd 404.2333, found 404.2330.
4.1.27. (2R,4aR,10aR)-4-Propyl-2-[1,2,3]triazol-1-ylmethyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol
TFA salt (23)
Compound 15 (42 mg, 0.09 mmol) dissolved in DMF (1.0 mL)
was added to a mixture of 1H-[1,2,4]Triazole (18.7 mg, 0.27 mmol)
and NaH (4.3 mg, 0.18 mmol). The solution was heated at 130 °C
for 30 min using microwave heating. The reaction was quenched
with satd NaHCO3 (10 mL), and EtOAc (15 mL) was added. The
phases were separated and the organic phase washed with
NaHCO3 (10 mL) and concentrated. Yield: 18 mg of the crude inter-
mediate (2R,4aR,10aR)-9-methoxy-4-propyl-2-[1,2,3]triazol-1-yl-
methyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxa-
zine). The L-selectride de-protecting procedure described for 6a
was followed to give 23. Yield of 23: 1.7 mg as a white oil. LC/
MS: ELSD 100%, UV 100%. MH+ = 329.5.
4.1.28. (2S,4aR,10aR)-4-Propyl-2-[1,2,3]triazol-1-ylmethyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol
(24)
Compound 24 was synthesized from 16 as described for 23 from
15, except that the deprotection was achieved with the thiophenol
method. Yield: 2.6 mg (14.1%) of compound 24 as a white solid. LC/
MS: ELSD: 100.0%; UV: 100.0%; MH+ 342.1.
4.1.31. (2R,4aR,10aR)-2-Methylsulfanylmethyl-4-propyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol
Hydrochloride (27)
Compound 15 (300 mg, 0.67 mmol) was dissolved in DMF
(37.0 mL) under an argon atmosphere. Sodium methyl sulfide
(142 mg, 2.02 mmol) and 18-crown-6 (178 mg, 0.67 mmol) were
added to a vial and the solution was afterwards transferred to
the vial. The solution was heated in the sealed vial to 110 °C for
30 min under microwaves. The reaction was quenched with satd
NaHCO3 (50 mL), and EtOAc (80 mL) were added. The phases were
separated and the organic phase washed with additional satd
NaHCO3 (20 mL) and concentrated in vacou. The crude product
was purified by silica gel chromatography (eluent 0–100% EtOAc
in heptanes). Yield 60 mg (28%) of the intermediate
(2R,4aR,10aR)-9-methoxy-2-methylsulfanylmethyl-4-propyl-
3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazine as an
oil. LC/MS (method 350): ELSD: 100%; UV: 100%; MH+ 322.3;
4.1.29. (2R,4aR,10aR)-2-(4-Chloro-pyrazol-1-ylmethyl)-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazin-9-ol (25)
Compound 15 (42 mg, 0.09 mmol) dissolved in DMF (1.0 mL)
was added to
a mixture of 4-chloro-1H-pyrazole (27.8 mg,
0.27 mmol) and NaH (4.3 mg, 0.18 mmol). The solution was heated
at 130 °C for 30 min using microwave heating. The reaction was
quenched with satd NaHCO3 (10 mL), and EtOAc (15 mL) were
added. The phases were separated and the organic phase extracted
with NaHCO3 (10 mL) and concentrated. Yield 13 mg (38%) of the
intermediate
(2R,4aR,10aR)-2-(4-chloro-pyrazol-1-ylmethyl)-9-
methoxy-4-propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-
b][1,4]oxazine as a white solid. LC/MS: ELSD: 100.0%; UV: 100.0%;

R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
391
Rt = 0.58. 1H NMR (500 MHz, CDCl3) d 0.92 (t, J = 7.48 Hz, 3H), 1.43–
1.62 (m, 2H), 2.12–2.22 (m, 4H), 2.26–2.39 (m, 2H), 2.43–2.58 (m,
2H), 2.59–2.67 (m, 1H), 2.70 (dd, J = 12.72 Hz, J = 4.89 Hz, 1H),
2.74–2.84 (m, 1H), 3.09 (d, J = 11.25 Hz, 1H), 3.16–3.29 (m, 2H),
3.60–3.68 (m, 1H), 3.79 (s, 3H), 3.85 (br s, 1H), 6.66 (d,
J = 7.82 Hz, 1H), 6.72 (d, J = 6.85 Hz, 1H), 7.11 (t, J = 7.09 Hz, 1H).
13C NMR (500 MHz, CDCl3) d: 157.6, 135.8, 127.0, 123.6, 121.2,
107.6, 77.2, 75.3, 60.6, 56.5, 55.7, 55.6, 46.5, 37.7, 34.0, 30.1,
29.8, 20.7, 17.1, 12.4. This intermediate (50 mg, 0.16 mmol) was
dissolved in DMA (7.3 mL) in a vial. KF (ꢃ2 equiv) and benzenethiol
(0.80 mL, 0.80 mmol) were added and the vial was sealed. The
reaction mixture was heated to 230 °C for 1 h. and the solvent
was evaporated off. The resulting mixture was purified with silica
gel chromatography using (eluent 0–50% EtOAc in heptanes) and
HPLC. Yield: 26 mg (54%) of (2R,4aR,10aR)-2-mercaptomethyl-4-
propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-
9-ol as an oil. This oil was dissolved in a minimum amount of
MeOH and HCl in ether was slowly added to afford 14 mg (26%)
of precipitated 27. LC/MS (Method 111): ELSD: 100%; UV: 100%;
MH+ 308.3. 1H NMR (500 MHz, DMSO-d6) d 0.96 (t, J = 6,36 Hz,
3H), 1.63–1.84 (m, 2H), 2.17 (s, 3H), 2.34–2.44 (m, 1H), 2.71 (d,
J = 3.42 Hz, 2H), 2.96–3.10 (m, 3H), 3.11–3.19 (m, 1H), 3.42–3.49
(m, 1H), 3.64 (d, J = 11.74 Hz, 1H), 4.07 (br s, 1H), 4.21 (br s, 1H),
6.61 (d, J = 7.34 Hz, 1H), 6.67 (d, J = 7.82 Hz, 1H), 6.99 (t,
J = 7.34 Hz, 1H), 9.55 (br s, 1H), 10.93 (br s, 1H).
0.1% sodium azide, 12 mM CaCl2, 1% BSA and 0.15 micro-Ci/mL
125I-cAMP) were added. Following an 18 h incubation at 4 °C the
plates were washed once and counted in a Wallac TriLux counter.
The standard deviation of the pEC50’s were below 0.27 for all
compounds tested.
4.3. Experimental for in vitro stability determination
4.3.1. In vitro stability in human liver microsomes
The stability of compounds in liver microsomes is determined
by the T method, that is, we measure the disappearance of
½
1 lM drug over time by LCMS. Using 0.5 mg/mL of microsomal
protein (liver microsomes from several donors pooled to obtain
an average enzyme content) in a NADPH generating system
(1.3 mM NADP, 3.3 mM glucose 6-phosphate and 0.4 U/mL glucose
6-phosphate dehydrogenase), 3.3 mM MgCl2, 0.1 M Potassium
phosphate buffer (pH 7.4), in a total volume of 100 lL, and stop-
ping the incubations at time points 0, 5, 15, 30 and 60 min with
1:1 v/v acetonitrile. The T is scaled to the metabolic competence
½
of a whole liver using 45 mg microsome/g liver, 20 g liver/kg and
Std. weight 70 kg.
4.3.2. In vitro stability in human hepatocytes
The stability of compounds in hepatocytes is determined by the
T
½
method, that is, we measure the disappearance of 1 lM drug
over time by LCMS. We use pooled hepatocytes from 10 donors
4.2. Experimental for receptor pharmacology
(male and female). Cells are thawed in 37 °C water bath, live cells
counted and seeded in a total of 100 lL in Dulbecco’s modified Ea-
4.2.1. D1 cAMP assay
gle medium (high glucose) with 5 mM hepes buffer in 96 well
plates. 500.000 cells/mL are used for human hepatocytes. Incuba-
tions are started after 15 min of preincubation and stopped at time
The ability of the compounds to stimulate the D1 receptor med-
iated cAMP formation in CHO cells stably expressing the human re-
combinant D1 receptor was measured as follows. Cells were seeded
in 96-well plates at a concentration of 11,000 cells/well 3 days
prior to the experiment. On the day of the experiment the cells
were washed once in preheated G buffer (1 mM MgCl2, 0.9 mM
CaCl2, 1 mM IBMX (3-i-butyl-1-methylxanthine) in PBS (phosphate
points 0, 30, 60, 90 and 120 min for human hepatocytes. The T is
½
scaled to the metabolic competence of a whole liver using scaling
factors are 120 million cells per gram of liver, 20 g liver per kg and
standard weight of /70 kg for human,
buffered saline)) and the assay was initiated by addition of 100
lL
4.4. Experimental for in vivo pharmacokinetics in rats
of test compound diluted in G buffer. The cells were incubated for
20 min at 37 °C and the reaction was stopped by the addition of
In vivo pharmacokinetics was studied in rats following single
intravenous (0.5 mg/kg) or oral (1 mg/kg) administration of com-
pounds. Serial blood samples were collected at various time points
up to 6 h after dosing. In vivo pharmacokinetic parameters were
obtained following compartmental modeling. Bioanalysis of sam-
ples were analyzed by liquid chromatography coupled to a tandem
mass spectrometer (LCMS/MS, Waters QuattroMicro, Manchester,
U.K.).
100
placed at 4 °C for 1 h. 68
NaOAc) was added and the plates were shaken for 10 min. 60
of the reaction were transferred to cAMP FlashPlates (DuPont
NEN) containing 40 L 60 mM Sodium acetate pH 6.2 and 100 mi-
l
L S buffer (0.1 M HCl and 0.1 mM CaCl2) and the plates were
lL N buffer (0.15 M NaOH and 60 mM
lL
l
cro-L IC mix (50 mM Sodium acetate pH 6.2, 0.1% sodium azide,
12 mM CaCl2, 1% BSA (bovine serum albumin) and 0.15 micro-Ci/
mL 125I-cAMP) were added. Following an 18 h incubation at 4 °C
the plates were washed once and counted in a Wallac TriLux
counter.
4.5. Experimental for X-ray determination
X-ray crystal structure determinations were performed as fol-
lows. The crystal of the compounds was cooled to 120 K using a
Cryostream nitrogen gas cooler system. The data were collected
on a Siemens SMART Platform diffractometer with a CCD area sen-
sitive detector. The structures were solved by direct methods and
refined by full-matrix least-squares against F2 of all data. The
hydrogen atoms in the structures could be found in the electron
density difference maps. The non-hydrogen atoms were refined
anisotropically. All the hydrogen atoms were at calculated
positions using a riding model with O–H = 0.84, C–H = 0.99–1.00,
N–H = 0.92–0.93 Å. For all hydrogen atoms the thermal parameters
were fixed [U(H) = 1.2 U for attached atom]. The Flack x-parame-
ters are in the range 0.0(1)–0.05(1), indicating that the absolute
structures are correct. Programs used for data collection, data
4.2.2. D2 cAMP assay
The ability of the compounds to stimulate the D2 receptor med-
iated inhibition of cAMP formation in CHO cells transfected with
the human D2 receptor was measure as follows. Cells were seeded
in 96 well plates at a concentration of 8000 cells/well 3 days prior
to the experiment. On the day of the experiment the cells were
washed once in preheated G buffer (1 mM MgCl2, 0.9 mM CaCl2,
1 mM IBMX in PBS) and the assay was initiated by addition of
100
buffer. The cells were incubated 20 min at 37 °C and the reaction
was stopped by the addition of 100 L S buffer (0.1 M HCl and
0.1 mM CaCl2) and the plates were placed at 4 °C for 1 h. 68 L N
buffer (0.15 M NaOH and 60 mM Sodium acetate) were added
and the plates were shaken for 10 min. 60 L of the reaction were
transferred to cAMP FlashPlates (DuPont NEN) containing 40
60 mM NaOAc pH 6.2 and 100 L IC mix (50 mM NaOAc pH 6.2,
lL of a mixture of 10 lM forskolin and test compound in G
l
l
l
reduction and absorption were SMART, SAINT and SADABS [cf. ‘SMART
and SAINT, Area Detector Control and Integration Software’, Version
5.054,Bruker Analytical X-ray Instruments Inc., Madison, USA
lL
l

392
R. Risgaard et al. / Bioorg. Med. Chem. 22 (2014) 381–392
(1998), Sheldrick ‘SADABS, Program for Empirical Correction of Area
Detector Data’ Version 2.03, University of Göttingen, Germany
(2001)]. The program SHELXTL [cf. Sheldrick ‘SHELXTL, Structure
Determination Programs’, Version 6.12, Bruker Analytical X-ray
Instruments Inc., Madison, USA (2001)] was used to solve the
structures and for molecular graphics.
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The crystallographic data for 10b was obtained at The Technical
University of Denmark, Department of Chemistry (Dr. Inger Sot-
ofte), and 27 was obtained at University of southern Denmark,
Departments of Physics, Chemistry and Pharmacy (Dr. Andrew D.
Bond). Dr. Henrik Pedersen (Discovery and DMPK, H. Lundbeck A/
S) and Dr. Heidi Lopez de_Diego (Biologics and Pharmaceutical Sci-
ence, H. Lundbeck) are thanked for helpful discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.012.