Synthesis and characterization of a series of chiral alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists

Article abstract of DOI:10.1016/j.bmcl.2016.03.102

Herein, we report the synthesis and structure-activity relationship of a series of chiral alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel potent and selective dopamine D4 receptor antagonist with selectivity against the other dopamine receptors tested (<10% inhibition at 1 μM against D₁, D_2L, D_2S, D₃, and D₅).

Full text of DOI:10.1016/j.bmcl.2016.03.102

Accepted Manuscript
Synthesis and characterization of a series of chiral alkoxymethyl morpholine
analogs as dopamine receptor 4 (D4R) antagonists
Jonathan O. Witt, Andrea L. McCollum, Miguel A. Hurtado, Eric D. Huseman,
Daniel E. Jeffries, Kayla J. Temple, Hyekyung C. Plumley, Anna L. Blobaum,
Craig W. Lindsley, Corey R. Hopkins
PII:
S0960-894X(16)30334-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.102
BMCL 23745
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 February 2016
25 March 2016
28 March 2016
Please cite this article as: Witt, J.O., McCollum, A.L., Hurtado, M.A., Huseman, E.D., Jeffries, D.E., Temple, K.J.,
Plumley, H.C., Blobaum, A.L., Lindsley, C.W., Hopkins, C.R., Synthesis and characterization of a series of chiral
alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.102
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Synthesis and characterization of a series of
chiral alkoxymethyl morpholine analogs as
dopamine receptor 4 (D4R) antagonists
Jonathan O. Witt, Andrea L. McCollum, Miguel A. Hurtado, Eric D. Huseman, Daniel E. Jeffries, Kayla J.
Temple, Hyekyung C. Plumley, Anna L. Blobaum, Craig W. Lindsley, and Corey R. Hopkins

Bioorganic & Medicinal Chemistry Letters
Synthesis and characterization of a series of chiral alkoxymethyl morpholine
analogs as dopamine receptor 4 (D4R) antagonists
Jonathan O. Witta, Andrea L. McColluma, Miguel A. Hurtadob, Eric D. Husemana, Daniel E. Jeffriesa,
Kayla J. Templea, Hyekyung C. Plumleyb,c, Anna L. Blobaumb,c, Craig W. Lindsleya,b,c, and Corey R.
Hopkinsa,b,c,
aDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232, United States
bDepartment of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, United States
cVanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Herein, we report the synthesis and structure-activity relationship of a series of chiral
alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-
(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel
potent and selective dopamine D4 receptor antagonist with selectivity against the other
dopamine receptors tested (<10% inhibition at 1 M against D1, D2L, D2S, D3, and D5)
Keywords:
Dopamine 4 receptor
Antagonist
2009 Elsevier Ltd. All rights reserved.
Morpholine
L-DOPA-induced dyskinesia
Dopamine Selectivity
Dopamine (DA) is a major neurotransmitter and is the primary
endogenous ligand for the dopamine receptors. Dopamine
receptors are members of the Class A G-protein coupled
receptors. There are five dopamine receptor subtypes which are
subdivided into two families, the D1-like family and the D2-like
family. The D1-like family consists of the D1 and D5 receptor
subtypes which are coupled to Gs and mediate excitatory
neurotransmission. The D2-family consists of three receptor
subtypes (D2, D3, and D4) which are coupled to Gi/Go and
mediate inhibitory neurotransmission. Of the subtypes, the
dopamine D4 receptor (D4R) has received considerable attention
as a potential target for pharmacological intervention due to
Figure 1. Structure of previously disclosed chiral morpholine D4 antagonist,
ML398.
To this end, we set out to replace the ethyl linker with a
hydroxymethyl group as this would allow for significant
diversification of this portion of the molecule.11 In addition, as
we have shown previously, the activity of the chiral morpholine
scaffold resides in the (S)-enantiomer and the starting Boc-
protected (S)-2-(hydroxymethyl)morpholine is commercially
available. The synthetic procedure to access these compounds is
disorders linked to dysfunction of this receptor (schizophrenia1-3
,
Parkinson’s disease4,5, and substance abuse6-8).
Recently, we reported on the identification of a chiral
morpholine scaffold as a potent and selective D4R antagonist,
ML398 (Figure 1).9,10 ML398 was active in vivo; however, the
SAR analysis was limited due to the synthetic feasibility of
modification of the upper right-hand phenethyl group. Thus, we
wanted to evaluate alternative linker groups in order to more
fully explore the SAR around both the N-linked groups as well as
moieties adjacent to the oxygen group of the morpholine.
shown
in
Scheme
1.
The
tert-butyl
(S)-2-
hydroxymethyl)morpholine-4-carboxylate, 1, was coupled with
the appropriate aryl bromide, 2, under copper mediated
conditions to afford 3.12 Alternatively, the aryl ethers could be
formed under Mitsunobu conditions13 (ArOH, PPh3, DIAD, W,
o
180 C) in good yield. Next, the Boc group was removed under
acidic conditions and reductive amination with polymer bound
CNBH3 provided the final compounds in modest overall yields.14
———
 Corresponding author. Tel.: +1-615-936-6892; fax: +1-615-778-1414; e-mail: corey.r.hopkins@vanderbilt.edu

Structure and D4 activity of the N-linked analogs.
Compound
R
cLogPa
5.10
IC50 (nM)b
130
Ki (nM)b
36
ML398
3.73
3.68
3.08
4.40
3.15
3.84
150
45
42
4a
4b
4c
4d
4e
4f
12.3
44
160
64
17.8
310
170
1,110
62
Scheme 1. Reagents and Conditions: (a) CuI, Me4Phen, Cs2CO3, Toluene
(14-30%); (b) ArOH, PPh3, DIAD, benzene, rt (51%); (c) ArOH, PS-PPh3,
o
3.84
3.54
4.35
4.21
4.21
3.18
3.18
69
19.1
150
27
4g
4h
4i
DIAD, THF, rt (21–25%), (d) ArOH, PPh3, DIAD, THF, W, 180 C, 5 min
(62%) (e) 4M HCl in dioxanes (f) Polymer bound CNBH3, R1CHO, Acetic
Acid, DCM (16-42%)
530
97
The first set of analogs that we synthesized and evaluated kept
the upper right-hand portion constant as the unsubstituted
phenoxy moiety and modified the southern nitrogen substituents.
A key component to design of the molecules was to lower the
cLogP of the compounds since ML398 was rather lipophilic
(cLogP = 5.10), with a design on potency and pharmacokinetic
parameters. The 4-chlorobenzyl, 4a, direct comparator to ML398
(Ki = 36 nM) was equipotent to its predecessor compound (Ki =
42 nM) and the introduction of an ether linker led to a significant
improvement (lowering) of the cLogP (5.10 vs. 3.73).10 Further
substitution around the benzyl group led to a more active
compound (3,4-dimethyl, 4b, Ki = 12.3 nM). Additional steric
bulk was well tolerated as the naphthyl group was active as well
(4d, Ki = 17.8 nM). Interestingly, the 2-substituted quinolone,
4e, was significantly less potent (Ki = 310 nM) compared to the
naphthyl group. Multiple substitution patterns around the phenyl
group are well tolerated (4f – 4s) with a few notable exceptions.
Namely, the 3-chloro-4-fluorophenyl (4f, Ki = 170 nM) and 3,4-
difluorophenyl (4h, Ki = 150 nM) were less potent than the other
analogs tested. The transposed 4-chloro-3-fluorophenyl (4g, Ki =
19.1 nM) and the 3,4-dichlorophenyl (4i, Ki = 27 nM) were more
active by ~10-fold, suggesting the 4-fluoro substitution is not as
well tolerated. However, this is not a fully general phenomenon
as the 3-methoxy-4-fluoro analog, 4m, is one of the most potent
compounds in this series (Ki = 11.6 nM), along with the 3-
methoxy-4-chloro compound (4p, Ki = 11.6 nM) and 4-methoxy-
3-chloro (4n, Ki = 10.4 nM). As an additional confirmation of
the (S)-enantiomer activity, the (R)-enantiomer of 4n was made
and evaluated, and it was not active (4o, 35% inhibition at 10
M). In addition to substituted benzyl groups in the southern
portion of the molecule, heteroaryl moieties were also well
tolerated. The imidazo[1,5-a]pyridine, 4u (Ki = 35 nM), was
equipotent with ML398; however, the imidazo[1,2-a]pyridine, 4t
(Ki = 160 nM) was less potent. Moving to the 3-substituted
indole compounds yielded the most active compounds in this set
of analogs. The 6-chloro (4aa, Ki = 2.2 nM), 6-methoxy (4dd,
Ki = 5.4 nM) and 6-fluoro (4ee, Ki = 5.2 nM) were all productive
changes. The corresponding 5-substituted indole compounds
were not as active (4bb and 4cc), nor was a 4-substituted analog
(4ff).
58
16.2
89
4j
320
56
4k
4l
14.3
11.6
10.4
42
4m
3.70
3.70
3.70
38
4n
4o
4p
(R)-4n
35%c
42
11.6
51
4.48
3.04
3.75
180
150
58
4q
4r
4s
43
16.1
2.89
3.51
3.73
590
130
65
160
35
4t
4u
4v
18.1
3.77
3.83
3.20
130
260
170
37
72
47
4w
4x
4y
3.77
4.39
58
15.9
2.2
4z
8.0
4aa
3.73
460
130
4bb
4.39
3.73
130
37
4cc
19.5
5.4
4dd
Table 1.

3.84
3.18
3.80
3.80
3.18
3.70
3.04
3.70
3.41
3.41
38
10.4
13.1
10.8
10.1
26
5k
5l
3.87
3.87
18.9
5.2
4ee
47
3,830
1,060
4ff
39
5m
5n
5o
5p
5q
5r
5s
aCalculated
using
Dotmatics
Elemental
(www.dotmatics.com/products/elemental). bIC50 and Ki values were
run in duplicate in a radioligand binding assay using Spiperone at
EuroFins (www.EuroFins.com). c% inhibition at 10 M.
37
92
150
380
130
100
130
42
Next, we turned our attention to the alkoxy substituents (R2,
Table 2) in conjunction with the southern fragments (R1, Table
110
36
1).
Initial evaluation utilized the 4-chlorobenzyl and 4-
methoxybenzyl groups as these were shown to be potent
antagonists of the D4R. The first analogs tested were 5-
pyrimidine and 2-pyrimidine replacements for the phenyl group.
Neither of these replacements led to active compounds; although
5a did show weak activity (76% inhibition at 10 M), although
introduction of these polar groups led to an significant lowering
of the cLogP, as expected. However, removal of one of the
nitrogen atoms in the 5-pyrimidine analog led to the 3-pyridine
analog and resulted in significant recovery of the potency (5e, Ki
= 47 nM; 5f, Ki = 59 nM). Similar removal of a nitrogen atom in
the 2-pyrimdine series leaving the 2-pyridine analogs only led to
modest recovery of the potency in one of the analogs (5g, Ki =
730 nM). Substituted 3- or 4-methoxy groups on the phenyl
ether were comparable in activity to the unsubstitued phenethyl
derivatives (5p – 5r). The 3-fluoro and 4-fluoro substituted
compounds were well tolerated resulting in very potent
compounds (5k, Ki = 10.4 nM; 5l, Ki = 13.1 nM; 5m, Ki = 10.8
nM; 5n, Ki = 10.1 nM). Unexpectedly, the 2-halogen-6-
alkoxypyridine compounds were active; unlike the 2-
alkoxypyridine analogs (vide supra). In fact, combining the 6-
fluoro-3-indole analog (4ee) with the 2-chloro-6-alkoxypyridine
led to one of the most potent compounds from this series (5y, Ki
= 3.3 nM). Lastly, two compounds in which the sulfide linker
replaced the alkoxy linker were synthesized; this proved to be a
fruitful change as well (5aa, Ki = 9.4 nM; 5bb, Ki = 7.4 nM).
28
36
5t
3.47
76
21
5u
3.33
3.30
3.30
460
260
-3%c
130
73
5v
5w
5x
3.99
3.85
11.9
100
3.3
29
5y
5z
4.41
4.35
34
27
9.4
7.4
5aa
5bb
aCalculated using Dotmatics Elemental
(www.dotmatics.com/products/elemental). bIC50 and Ki values were run
in duplicate in a radioligand binding assay using Spiperone at EuroFins
(www.EuroFins.com). c% inhibition at 10 M.
Table 2.
Having identified a number of active D4R antagonists, we next
wanted to profile these compounds against the other dopamine
receptors (D1, D2L, D2S, D3, and D5) (Table 3). Generally
speaking, the compounds are selective against the D1-like family
of receptors (D1 and D5), both the phenoxy (4) and substituted
phenoxy or heteroarylalkoxy compounds (5) are selective against
the D1-like family of receptors. The compounds are less selective
for the D2-like family, specifically the D2S and D2L receptors.
That being said, a number of compounds prove completely
selective against all of the dopamine receptors (Table 3), despite
Structure and D4 activity of the O- and N-linked analogs.
IC50
Ki
Cmpd
ML398
5a
R1
R2
cLogPa
5.10
(nM)b
(nM)b
130
36
1.83
76%c
high sequence homology.
Notably, the 6-fluoro-3-indole
-4%c
31%c
8%c
compound (4ee) showed activity against both D2L and D2S (78%
and 76%, respectively). However, the comparator 2-halogen-6-
alkoxypyridine compounds (5u and 5y) were fully selective
against all of the dopamine receptors tested. Gratifyingly, 5y is
one of the most potent analogs that was made and tested. In
addition, the sulfide analogs were also selective (5aa and 5bb).
1.17
2.33
1.67
2.42
1.77
2.83
2.17
2.79
2.79
5b
5c
5d
5e
5f
103
47
Table 3.
Dopamine receptor selectivity of select compounds.
210
59
% Inhibition at 10 Ma
D4
Compound
4b
4l
4n
4p
4u
4v
4z
(nM)
12.3
14.3
10.4
11.6
35
D1
D2L
D2S
<50%
76%
<50%
60%
<50%
<50%
64%
D3
D5
<50%
<50%
2,630
57%c
34%c
37%c
730
5g
5h
5i
<50%
<50%
78%
52%
64%
18.1
15.9
5j
<50%
<50%

2.2
5.4
5.2
10.4
13.1
10.8
10.1
21
<50%
<50%
<50%
<50%
<50%
94%
87%
78%
83%
88%
93%
82%
76%
79%
82%
<50%
<50%
<50%
<50%
<50%
<50%
70% <50%
70% <50%
<50%
51% <50%
76% <50%
23.1
132
5.73
4aa
4dd
4ee
5k
5l
5m
5n
5u
5y
5aa
5bb
5bb
In order to better understand the nature of the instability in
liver microsomes in both human and rat, we analyzed 5y in a
metabolic
soft-spot
experiment
(Q2
Solutions,
www.q2labsolutions.com). 5y was highly metabolized in both
rat and human liver microsomal samples in the presence of
NADPH. Compound B was the major metabolite in the rat
3.3
9.4
7.4
microsomes (N-dealkylation
+ oxidation), and the major
a % inhibition values were run in duplicate in a radioligand binding
assay at EuroFins (www.EuroFins.com).
metabolite in human microsomes was Compound A (N-
dealkylation). The parent compound, 5y, was observed in the rat
and human samples in the absence of NADPH. Thus, further
analog work will concentrate on blocking the N-dealkylation
mechanism of metabolism.
Having identified
a number of potent and selective
compounds, we further profiled selected compounds in a battery
of Tier 1 in vitro DMPK assays (Table 4). The intrinsic
clearance (CLINT) was assessed in liver microsomes (rat and
human), and many of the compounds proved to be unstable to
oxidative metabolism and were predicted to display high
clearance in both species.15 However, a few compounds were
shown to have moderate predicted clearance, such as 4t
(imidazo[1,2-a]pyridine) and 4g (3-fluoro-4-chlorophenyl),
which presumably can block oxidation of the phenyl group.
Utilizing an equilibrium dialysis approach, the protein binding of
the compounds was evaluated in both human and rat plasma.
The fraction unbound (Fu) ranged from low to moderate, and
these values loosely correlated with the calculated logP of the
compounds. Although it is understood that fraction unbound is a
difficult parameter to SAR around, lowering cLogP within a
series can tend to produce better values. As such, 4t, 4l, and 4y
had the highest fraction unbound and the lowest cLogP values.
Lastly, we assessed the ability of these compounds to cross the
Figure 2. Metabolic soft-spot analysis of 5y in liver microsomes.
In conclusion, we have further elaborated our initial D4R
antagonist, ML398, by changing the ethyl linker to
hydroxymethyl linker on the chiral morpholine scaffold.
a
A
number of compounds are very potent (D4 Ki < 20 nM) with
excellent selectivity against the other dopamine receptors.
Notably, compounds 5y, 5aa, and 5bb were shown to have D4
Kis < 10 nM and be completely selective against the other
dopamine receptors (Kis >10 M, ie., >1000-fold selectivity).
Compounds 4ee and 5y are intriguing molecules as they contain
molecular handles and possess desirable physicochemical
properties (cLogP) for potential radioligand development. Many
of the compounds identified were highly cleared in both human
and rat liver microsomes, and we have shown that N-dealkylation
is a major contributor to the instability. Lastly, compounds from
this scaffold class are highly brain penetrant as assessed in a
rodent IV cassette experiment to determine brain-to-plasma ratios
(Kp values >2). Further optimization and in vivo behavioral
efficacy experiments will be disclosed in due course.
blood-brain barrier in a rodent IV cassette experiment to
17
determine brain-to-plasma ratios (Kp).16,
A selection of
compounds is shown in Table 3, and, although the compounds
show high clearance in rat, the compounds are able to cross the
BBB with Kp values >2.
Table 4.
In vitro and in vivo DMPK results of select compounds.
Microsome intrinsic
clearance
Plasma unbound
fraction (Fu)
(mL/min/kg)
Compound
4a
4g
4i
4j
4l
4n
4p
4t
D4 (nM)
12.3
19.1
27
16.2
14.3
10.4
11.6
160
47
hCLINT
147
rCLINT
4518
251
621
247
2128
1597
1505
154
433
3137
614
Human
0.007
0.006
0.002
0.002
0.031
0.010
0.010
0.057
0.039
0.035
0.006
0.037
0.017
0.006
0.011
0.009
0.015
0.004
0.008
0.001
Rat
0.007
0.012
0.006
0.007
0.037
0.019
0.025
0.215
0.074
0.069
0.010
0.075
0.047
0.013
0.014
0.015
0.016
0.008
0.005
0.004
65.3
50.5
50.5
71.9
78.7
79.7
17.3
60.5
135
46.7
31.6
71.1
93.0
68.9
101
Acknowledgments
4y
4z
The authors would like to thank the Michael J. Fox
Foundation for Parkinson’s Research for research support for
CRH (MJFF Grant ID: 10000) and Jarrett Foster, Sichen Chang,
and Xiaoyan Zhan for their contributions to the DMPK screening
tier.
15.9
2.2
5.4
4aa
4dd
4ee
5m
5n
5u
5y
5z
5aa
5bb
184
5.2
1686
1499
1774
4010
4195
5313
3217
6436
10.8
10.1
21
3.3
29
230
98.2
122
References and Notes
9.4
7.4
366
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C. I.; Seabrook, G. R., Trends Pharm. Sci. 1997, 18, 186.
Rodent IV Cassette (0.25 mg/kg, 0.25 h)
Plasma (ng/mL)
Brain (ng/g)
197
108
83.1
246
Kp
42.5
14.7
37.3
74.8
26.1
4.62
7.38
2.23
3.29
5.03
4a
4l
4y
4aa
5aa
131

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13. Mitsunobu, O., Synthesis 1981, 1.
14. Experimental Procedure for the synthesis of 5y: tert-butyl (S)-2-
(hydroxymethyl)morpholine-4-carboxylate, 1, (110.0 mg; 0.507
mmol) and DMF (2.5 mL) were added to a vial equipped with a
stir bar. The mixture was cooled to 0 oC and then NaH (24.3 mg;
0.608 mmol) was added and the mixture was stirred until fully
dissolved. Next, 2-chloro-6-fluoropyridine (133.3 mg; 1.013
mmol) was added and then the reaction mixture was warmed to
o
60 C. After 90 mins, the reaction was cooled to rt and CH2Cl2
was added along with 5% LiCl in H2O. The layers were
separated and the organic layer was dried (MgSO4) and collected
and concentrated. The crude product was purified via flash
column chromatography (5-30% EtOAc/hexanes) to provide the
N-Boc product. 1H NMR (400 MHz, CDCl3) δ 7.45 (1 H, t, J =
8.0 Hz), 6.84 (1 H, d, J = 7.5 Hz), 6.64 (1 H, d, J = 8.0 Hz), 4.28
(2 H, d, J = 4.9 Hz), 3.87 (2 H, d, J = 10.6 Hz), 3.72 – 3.69 (2 H,
m), 3.51 (1 H, dt, J = 11.6, 2.7 Hz), 2.92 (1 H, t, J = 22.3 Hz),
2.76 (1 H, s), 1.40 (9 H, s).
The product was treated with 2.0 mL of 4 M HCl/dioxanes and
stirred at rt for 30 mins. At this time, the mixture was
concentrated and the material was converted to the free base via
an SCX column. The material was added to a microwave vial
along with MP-CNBH3 (88.2 mg; 2.28 mmol/g polymer; 0.201
mmol), acetic acid (23.0 μL; 0.402 mmol) and CH2Cl2 (1.0 mL)
and then heated in a microwas at 110 oC for 7 mins. The reaction
mixture was cooled to rt, filtered and washed with CH2Cl2 and
purified by flash column chromatography (5-30%
EtOAc:hexanes) leaving the desired compound (12.0 mg; 0.0320
mmol; 42%). [α]D +18.84 (c 1.0, CH3OH); LCMS: RT = 0.786
min, >98% @ 254 nm, m/z = 376.2 [M + H]+. 1H NMR (400
MHz, CDCl3) δ 8.01 (1 H, br. s.), 7.60 (1 H, dd, J = 8.7, 5.5 Hz),
7.42 (1 H, t, J = 7.7 Hz), 7.03 (1 H, d, J = 1.4 Hz), 6.97 (1 H, dd,
J = 9.6, 2.0 Hz), 6.84 – 6.79 (1 H, m), 6.63 (1 H, d, J = 8.2 Hz),
4.24 – 4.22 (2 H, m), 3.86 (2 H, br. d., J = 10.9 Hz), 3.68 – 3.65
(1 H, m), 2.80 (1 H, d, J = 11.1 Hz), 2.68 (1 H, d, J = 11.4 Hz),
2.17 (1 H, dt, J = 11.4, 3.2 Hz), 2.00 (1 H, t, J = 10.6 Hz); 13C
NMR (400 MHz, CDCl3) δ 163.2, 161.3, 158.9, 148.1, 140.6,
136.3, 124.3, 123.8, 120.5, 116.5, 109.5, 108.5, 97.5, 74.1, 67.6,
66.9, 54.8, 54.0, 52.9.
15. Obach, R. S., Drug Metab. Disp. 1999, 27, 1350.