U.S. patent application number 12/917085 was filed with the patent office on 2011-02-24 for new compounds i.
This patent application is currently assigned to ASTRAZENECA AB (PUBL). Invention is credited to Joseph W. Boyd, Giles A. Brown, Michael Higginbottom.
Application Number | 20110046135 12/917085 |
Document ID | / |
Family ID | 40409928 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110046135 |
Kind Code |
A1 |
Boyd; Joseph W. ; et
al. |
February 24, 2011 |
NEW COMPOUNDS I
Abstract
The present application relates to new compounds of formula (I),
##STR00001## to pharmaceutical compositions comprising the
compounds, to processes for their preparation, and to the use of
the compounds as leptin receptor modulator mimetics in the
preparation of medicaments against conditions associated with
weight gain, type 2 diabetes and dyslipidemias.
Inventors: |
Boyd; Joseph W.;
(Cambridgeshire, GB) ; Brown; Giles A.;
(Cambridge, GB) ; Higginbottom; Michael;
(Cambridgeshire, GB) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
ASTRAZENECA AB (PUBL)
Sodertalje
SE
|
Family ID: |
40409928 |
Appl. No.: |
12/917085 |
Filed: |
November 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12315698 |
Dec 5, 2008 |
7851471 |
|
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12917085 |
|
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61022983 |
Jan 23, 2008 |
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Current U.S.
Class: |
514/235.8 ;
514/252.11; 514/253.01; 544/121; 544/357; 544/360 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 13/12 20180101; A61P 37/00 20180101; A61P 3/06 20180101; A61P
9/00 20180101; A61P 9/12 20180101; A61P 25/00 20180101; A61P 17/02
20180101; A61P 27/02 20180101; A61P 5/50 20180101; A61P 15/08
20180101; A61P 3/04 20180101; C07D 265/30 20130101; C07D 241/04
20130101; A61P 3/00 20180101; A61P 3/10 20180101; A61P 17/00
20180101; C07D 211/22 20130101; A61P 9/10 20180101; A61P 29/00
20180101 |
Class at
Publication: |
514/235.8 ;
544/360; 544/357; 544/121; 514/253.01; 514/252.11 |
International
Class: |
A61K 31/5355 20060101
A61K031/5355; C07D 401/12 20060101 C07D401/12; C07D 241/14 20060101
C07D241/14; C07D 413/12 20060101 C07D413/12; A61K 31/497 20060101
A61K031/497; A61P 17/02 20060101 A61P017/02; A61P 9/10 20060101
A61P009/10; A61P 9/00 20060101 A61P009/00; A61P 37/00 20060101
A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 2007 |
SE |
0702696-6 |
Claims
1. A compound of formula (I) ##STR00060## or a pharmaceutically
acceptable salt thereof, wherein: X is selected from O, S,
N(R.sup.1) and CH(R.sup.2a), provided that the ring containing X is
not 3-pyrrolidine; Y is CH.sub.2, O or N(R.sup.5); each R.sup.1 is
independently selected from hydrogen, C.sub.1-6-alkyl which is
unsubstituted or optionally substituted with one or more
substituents independently selected from halogen, hydroxy, cyano
and C.sub.1-6-alkoxy, and C.sub.1-6-acyl which is unsubstituted or
optionally substituted with one or more substituents independently
selected from halogen, hydroxy and C.sub.1-6-alkoxy; each R.sup.2
and each R.sup.3 is independently selected from halogen, hydroxy,
C.sub.1-6-alkyl which is unsubstituted or optionally substituted
with one or more substituents independently selected from halogen,
hydroxy and C.sub.1-6-alkoxy, and C.sub.1-6-alkoxy which is
unsubstituted or optionally substituted with one or more
substituents independently selected from halogen, hydroxy and
C.sub.1-6-alkoxy; R.sup.2a is hydrogen or R.sup.2; each R.sup.4 is
independently selected from halogen, hydroxy, cyano, nitro,
CF.sub.3, C.sub.1-6-alkyl and C.sub.1-6-alkoxy; R.sup.5 is hydrogen
or C.sub.1-4-alkyl; a, b and c are each independently 0, 1, 2 or 3;
d is 0, 1 or 2; e is 1, 2 or 3; and f and g are each independently
0, 1 or 2.
2. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein Y is O.
3. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein X is O, N(R.sup.1) or
CH(R.sup.2a).
4. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 is hydrogen, methyl, ethyl
or acetyl.
5. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein a and b are each independently 0
or 1.
6. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein c is 0, 1 or 2.
7. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein each R.sup.4 is independently
selected from fluoro, chloro, methyl and ethyl.
8. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein the compound is of formula (I')
##STR00061## wherein: X.sup.1 and X.sup.2 are each independently
selected from O, N(R.sup.1) and CH(R.sup.2a), provided that at
least one of X.sup.11 and X.sup.2 is N(R.sup.1); R.sup.1, R.sup.2,
R.sup.2a and R.sup.4 are as defined in claim 1; c is 0, 1, 2 or 3;
e is 1 or 2; and g is 0 or 1.
9. A compound according to claim 1, or a pharmaceutically
acceptable salt thereof, which is selected from:
[(3R)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate;
[(3S)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate;
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate;
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate;
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
4-phenylpiperazine-1-carboxylic acid
2-(1,4-dimethylpiperazin-2-yl)ethyl ester;
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate;
[(2S)-4-methylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate;
(1,4-dimethylpiperazin-2-yl)methyl
4-benzylpiperazine-1-carboxylate; morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; (2S)-morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; (2R)-morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; (4-methylmorpholin-2-yl)methyl
4-phenylpiperazine-1-carboxylate;
[(2S)-4-methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate;
[(2R)-4-methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate;
[(2S)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
[(2R)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
[(2S)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate;
[(2R)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate;
morpholin-2-ylmethyl 4-(4-fluorophenyl)piperazine-1-carboxylate;
(2S)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
(2R)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
(4-methylmorpholin-2-yl)methyl
4-(4-chlorophenyl)piperazine-1-carboxylate;
(4-methylmorpholin-2-yl)methyl
4-(4-fluorobenzyl)piperazine-1-carboxylate;
(4-acetylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate;
morpholin-3-ylmethyl 4-(4-fluorophenyl)piperazine-1-carboxylate;
(3S)-morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
(3R)-morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate;
(4-methylmorpholin-3-yl)methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; morpholin-3-ylmethyl
4-phenylpiperazine-1-carboxylate; morpholin-3-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate;
(4-methylmorpholin-3-yl)methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate;
(2S)-morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; and
(2R)-morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; and
pharmaceutically acceptable salts thereof.
10. A pharmaceutical formulation containing a compound according to
claim 1, or a pharmaceutically acceptable salt thereof, as an
active ingredient, in combination with a pharmaceutically
acceptable diluent or carrier.
11. A method for treatment or prevention of conditions or diseases
associated with weight gain, which comprises administering to a
mammal, including man, in need of such treatment an effective
amount of a compound according to claim 1, or a pharmaceutically
acceptable salt thereof.
12. The method according to claim 11, wherein the condition or
disease is obesity, type 2 diabetes, lipodystrophy, insulin
resistance, metabolic syndrome, hyperglycemia, hyperinsulinemia,
dyslipidemia, hepatic steatosis, hyperphagia, hypertension,
hypertriglyceridemia, infertility, a skin disorder associated with
weight gain or macular degeneration.
13. A method for treatment or prevention of severe weight loss,
dysmenorrhea, amenorrhea, female infertility or immunodeficiency,
or the treatment of wound healing, which comprises administering to
a mammal, including man, in need of such treatment an effective
amount of a compound according to claim 1, or a pharmaceutically
acceptable salt thereof.
14. method for treatment or prevention of inflammatory conditions
or diseases, low level inflammation associated with obesity and
excess plasma leptin, atherosclerosis, macro or micro vascular
complications of type 1 or 2 diabetes, retinopathy, nephropathy,
autonomic neuropathy, or blood vessel damage caused by ischaemia or
atherosclerosis, which comprises administering to a mammal,
including man, in need of such treatment an effective amount of a
compound according to claim 1, or a pharmaceutically acceptable
salt thereof.
15. A method for inhibition of angiogenesis, which comprises
administering to a mammal, including man, in need of such treatment
an effective amount of a compound according to claim 1, or a
pharmaceutically acceptable salt thereof.
16. A process for the preparation of a compound of claim 1,
comprising: (a) reacting a compound of formula (II): ##STR00062##
wherein R.sup.3, R.sup.4, b, c, f and g are as defined in claim 1,
with 4-nitrophenyl chloroformate or bis-(4-nitrophenyl)carbonate in
the presence of a suitable base (such as DIPEA or NEt.sub.3) in a
suitable solvent (such as DCM or THF), at -10 to 40.degree. C., to
form a compound of formula (III): ##STR00063## (b) reacting the
compound of formula (III) with a compound of formula (IV):
##STR00064## wherein X, R.sup.1, R.sup.2, a, d and e are as defined
in claim 1, in the presence of a suitable base, (such as NaH or
NMM), in a suitable solvent (such as THF or DCM), at -10 to
40.degree. C., to obtain a compound of formula (I); and (c)
optionally, in one or several steps transforming a compound of
formula (I) into another compound of formula (I).
17. A process for the preparation of a compound of claim 1,
comprising: (a) reacting a compound of formula (IV): ##STR00065##
Wherein X, R.sup.1, R.sup.2, a, d and e are as defined in claim 1,
with 4-nitrophenyl chloroformate or bis-(4-nitrophenyl)carbonate in
the presence of a suitable base (such as DIPEA or NMM) in a
suitable solvent (such as DCM), at -10 to 40.degree. C., to form a
compound of formula (V): ##STR00066## (b) reacting the compound of
formula (V) with a compound of formula (II): ##STR00067## wherein
R.sup.3, R.sup.4, b, c, f and g are as defined in claim 1, in the
presence of a suitable base, (such as DIPEA), in a suitable solvent
(such as DCM or DMF), at -10 to 40.degree. C., to obtain a compound
of formula (I); and (c) optionally, in one or several steps
transforming a compound of formula (I) into another compound of
formula (I).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/315,698, filed Dec. 5, 2008, which is pending, which claims
the benefit of Swedish Application No. 0702696-6, filed Dec. 5,
2007 and of U.S. Provisional Application No. 61/022,983, filed Jan.
23, 2008. The entire disclosures of the aforementioned related
applications are hereby incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present application relates to new piperazine
derivatives, to pharmaceutical compositions comprising the
compounds, to processes for their preparation, and to the use of
the compounds as leptin receptor modulator mimetics in the
preparation of medicaments against conditions associated with
weight gain, type 2 diabetes and dyslipidemias.
BACKGROUND ART
[0003] The prevalence of obesity is increasing in the
industrialized world. Typically, the first line of treatment is to
offer diet and life style advice to patients, such as reducing the
fat content of their diet and increasing their physical activity.
However, some patients may also need to undergo drug therapy to
maintain the beneficial results obtained from adapting the
aforementioned diet and lifestyle changes.
[0004] Leptin is a hormone synthesized in fat cells that is
believed to act in the hypothalamus to reduce food intake and body
weight (see, e.g., Bryson, J. M. (2000) Diabetes, Obesity and
Metabolism 2: 83-89).
[0005] It has been shown that in obese humans the ratio of leptin
in the cerebrospinal fluid to that of circulating leptin is
decreased (Koistinen et al., (1998) Eur. J. Clin. Invest. 28:
894-897). This suggests that the capacity for leptin transport into
the brain is deficient in the obese state. Indeed, in animal models
of obesity (NZO mouse and Koletsky rat), defects in leptin
transport have been shown to result in reduced brain leptin content
(Kastin, A. J. (1999) Peptides 20: 1449-1453; Banks, W. A. et al.,
(2002) Brain Res. 950: 130-136). In studies involving
dietary-induced obese rodents (a rodent model that is believed to
more closely resemble human obesity, see, e.g., Van Heek et al.
(1997) J. Clin. Invest. 99: 385-390), excess leptin administered
peripherally was shown to be ineffective in reducing food intake
and body weight, whereas leptin injected directly into the brain
was effective in reducing food intake and body weight. It has also
been shown that in obese humans with excess circulating leptin, the
signaling system became desensitized to the continual stimulation
of the leptin receptors (Mantzoros, C. S. (1999) Ann. Intern. Med.
130: 671-680).
[0006] Amgen has conducted clinical trials with recombinant
methionyl human leptin. The results from these trials were mixed,
as even in the presence of high plasma concentrations of leptin
weight loss was variable, and the average weight reduction in the
cohort of patients tested relatively small (Obesity Strategic
Perspective, Datamonitor, 2001).
[0007] Several attempts at finding active fragments have been
reported in the literature since the discovery of the leptin gene
coding sequence. An example is by Samson et al. (1996) Endocrinol.
137: 5182-5185 which describes an active fragment at the N-terminal
(22 to 56). This sequence was shown to reduce food intake when
injected ICV whereas a sequence taken at the C-terminal was shown
not to have any effect. Leptin fragments are also disclosed in
International Patent Application WO 97/46585.
[0008] Other reports looking at the C-terminus part of the sequence
reported a possible stimulation of luteinising hormone production
by a 116-130 fragment (Gonzalez et al., (1999) Neuroendocrinology
70:213-220) and an effect on GH production following GHRH
administration (fragment 126-140) (Hanew (2003) Eur. J. Endocrin.
149: 407-412).
[0009] Leptin has recently been associated with inflammation. It
has been reported that circulating leptin levels rise during
bacterial infection and in inflammation (see Otero, M et al. (2005)
FEBS Lett. 579: 295-301 and references therein). Leptin can also
act to increase inflammation by enhancing the release of
pro-inflammatory cytokines TNF and IL-6 from inflammatory cells
(Zarkesh-Esfahani, H. et al. (2001) J. Immunol. 167: 4593-4599).
These agents in turn can contribute to the insulin resistance
commonly seen in obese patients by reducing the efficacy of insulin
receptor signaling (Lyon, C. J. et al. (2003) Endocrinol. 44:
2195-2200). Continuous low grade inflammation is believed to be
associated with obesity (in the presence and absence of insulin
resistance and Type II diabetes) (Browning et al. (2004) Metabolism
53: 899-903, Inflammatory markers elevated in blood of obese women;
Mangge et al. (2004) Exp. Clin. Endocrinol. Diabetes 112: 378-382,
Juvenile obesity correlates with serum inflammatory marker
C-reactive protein; Maachi et al. (2004) Int. J. Obes. Relat.
Metab. Disord. 28: 993-997, Systemic low grade inflammation in
obese people). Leptin has also been implicated in the process of
atherogenesis, by promoting lipid uptake into macrophages and
endothelial dysfunction, thus promoting the formation of
atherosclerotic plaques (see Lyon, C. J. et al. (2003) Endocrinol.
144: 2195-2200).
[0010] Leptin has also been shown to promote the formation of new
blood vessels (angiogenesis) a process implicated in the growth of
adipose tissue (Bouloumie A, et al. (1998) Circ. Res. 83:
1059-1066). Angiogenesis has also been implicated in diabetic
retinopathy (Suganami, E. et al. (2004) Diabetes. 53:
2443-2448).
[0011] Angiogenesis is also believed to be involved with the growth
of new blood vessels that feed abnormal tumour cells. Elevated
leptin levels have been associated with a number of cancers, in
particular breast, prostate and gastrointestinal cancers in humans
(Somasundar P. et al. (2004) J. Surg. Res. 116: 337-349).
[0012] Leptin receptor agonists may also be used in the manufacture
of a medicament to promote wound healing (Gorden, P. and Gavrilova,
O. (2003) Current Opinion in Pharmacology 3: 655-659).
[0013] Further, it has been shown that elevating leptin signaling
in the brain may represent an approach for the treatment of
depressive disorders (Lu, Xin-Yun et al. (2006) PNAS 103:
1593-1598).
DISCLOSURE OF THE INVENTION
[0014] It has surprisingly been found that compounds of formula (I)
are effective in reducing body weight and food intake in rodents.
While not wishing to be bound by theory, it is proposed that the
compounds of formula I modulate the leptin receptor signaling
pathway. In some embodiments, compounds with leptin receptor
agonistic like properties can be useful for the treatment of
disorders relating to leptin signaling, as well as conditions
associated with weight gain, such as obesity. The inventors
hypothesized that small molecule CNS penetrant leptin mimetics
would be able to by-pass the limiting uptake system into the brain.
Further, assuming that this situation mirrors the human obese
condition, the inventors believe that a CNS-active leptinoid with a
relatively long duration of action would make an effective therapy
for the obese state and its attendant complications, in particular
(but not limited to) diabetes.
[0015] In other embodiments, compounds with leptin receptor
antagonistic like properties could be useful for the treatment of
inflammation, atherosclerosis, diabetic retinopathy and
nephropathy.
[0016] In a first aspect, the disclosure relates to a compound of
formula (I),
##STR00002##
and pharmaceutically acceptable salts, hydrates, geometrical
isomers, racemates, tautomers, optical isomers or N-oxides thereof,
wherein:
[0017] X is selected from O, S, N(R.sup.1) and CH(R.sup.2),
provided that the ring containing X is not 3-pyrrolidine;
[0018] Y is CH.sub.2, O or N(R.sup.5);
[0019] R.sup.1 is independently selected from hydrogen,
C.sub.1-6-alkyl (unsubstituted or optionally substituted with one
or more substituents independently selected from halogen, hydroxy,
cyano and C.sub.1-6-alkoxy) and C.sub.1-6-acyl (unsubstituted or
optionally substituted with one or more substituents independently
selected from halogen, hydroxy and C.sub.1-6-alkoxy);
[0020] R.sup.2 and R.sup.3 are independently selected from
hydrogen, halogen, hydroxy, C.sub.1-6-alkyl (unsubstituted or
optionally substituted with one or more substituents independently
selected from halogen, hydroxy and C.sub.1-6-alkoxy) and
C.sub.1-6-alkoxy (unsubstituted or optionally substituted with one
or more substituents independently selected from halogen, hydroxy
and C.sub.1-6-alkoxy);
[0021] R.sup.4 is independently selected from hydrogen, halogen,
hydroxy, cyano, nitro, CF.sub.3, C.sub.1-6-alkyl and
C.sub.1-6-alkoxy;
[0022] R.sup.5 is hydrogen or C.sub.1-4-alkyl;
[0023] a, b and c are each independently 1, 2 or 3;
[0024] d is 0, 1 or 2;
[0025] e is 1, 2 or 3; and
[0026] f and g are each independently 0, 1 or 2.
[0027] In a preferred embodiment, Y is O.
[0028] In another embodiment, X is preferably selected from O,
N(R.sup.1) and CH(R.sup.2).
[0029] R.sup.1 is preferably selected from hydrogen,
C.sub.1-4-alkyl and C.sub.1-4-acyl.
[0030] In a most preferred embodiment, R.sup.1 is hydrogen, methyl
or acetyl.
[0031] R.sup.2 and R.sup.3 are preferably independently selected
from hydrogen and C.sub.1-4-alkyl.
[0032] In a most preferred embodiment, R.sup.2 and R.sup.3 are
hydrogen.
[0033] R.sup.4 is preferably independently selected from hydrogen,
halogen, CF.sub.3 and C.sub.1-4-alkyl.
[0034] In a most preferred embodiment, R.sup.4 is independently
selected from hydrogen, fluoro, i chloro or methyl.
[0035] d and f are each preferably 1.
[0036] e is preferably 1 or 2.
[0037] g is preferably 0 or 1, and more preferably 0.
[0038] Particular preferred compounds of formula (I) are the
compounds of formula (I')
##STR00003##
wherein:
[0039] X.sup.1 and X.sup.2 are each independently selected from O,
N(R.sup.1) or CH(R.sup.2), provided that at least one of X.sup.1
and X.sup.2 is N(R.sup.1);
[0040] R.sup.1 is as defined in formula (I), and preferably
hydrogen, methyl or acetyl;
[0041] R.sup.2 is as defined in formula (I), and preferably
hydrogen;
[0042] R.sup.4 is hydrogen, fluoro, chloro or methyl;
[0043] c is 1, 2 or 3;
[0044] e is 1 or 2; and
[0045] g is 0 or 1.
[0046] Specific preferred compounds of formula (I) are those
selected from the group consisting of [0047]
[(3R)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate; [0048]
[(3S)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate; [0049]
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate; [0050]
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate; [0051]
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0052]
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0053]
4-phenylpiperazine-1-carboxylic acid
2-(1,4-dimethylpiperazin-2-yl)ethyl ester; [0054]
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; [0055]
[(2S)-4-methylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate; [0056]
(1,4-dimethylpiperazin-2-yl)methyl
4-benzylpiperazine-1-carboxylate; [0057] morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; [0058] (2S)-morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; [0059] (2R)-morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate; [0060]
(4-methylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate;
[0061] [(2S)-4-methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate; [0062]
[(2R)-4-methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate; [0063]
[(2S)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0064]
[(2R)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0065]
[(2S)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; [0066]
[(2R)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; [0067]
morpholin-2-ylmethyl 4-(4-fluorophenyl)piperazine-1-carboxylate;
[0068] (2S)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0069]
(2R)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0070]
(4-methylmorpholin-2-yl)methyl
4-(4-chlorophenyl)piperazine-1-carboxylate; [0071]
(4-methylmorpholin-2-yl)methyl
4-(4-fluorobenzyl)piperazine-1-carboxylate; [0072]
(4-acetylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate;
[0073] morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0074]
(3S)-morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0075]
(3R)-morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0076]
(4-methylmorpholin-3-yl)methyl
4-(4-fluorophenyl)piperazine-1-carboxylate; [0077]
morpholin-3-ylmethyl 4-phenylpiperazine-1-carboxylate; [0078]
morpholin-3-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; [0079]
(4-methylmorpholin-3-yl)methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; [0080]
(2S)-morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate; and [0081]
(2R)-morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate.
[0082] Another aspect of the present disclosure is a compound of
formula (I) for use in therapy.
[0083] In a further aspect, the disclosure relates to a compound of
formula (I) for use in the treatment or prevention of any of the
disorders or conditions described herein.
[0084] In yet a further aspect, the invention relates to the use of
a compound of formula (I) in the manufacture of a medicament for
the treatment or prevention of any of the disorders or conditions
described herein.
[0085] In some embodiments, said compounds may be used in the
manufacture of a medicament for the treatment or prevention of a
condition that is prevented, treated, or ameliorated by selective
action on the leptin receptor.
[0086] In some embodiments, said compounds may be used in the
manufacture of a medicament for the treatment or prevention of
conditions (in particular, metabolic conditions) that are
associated with weight gain. Conditions associated with weight gain
include diseases, disorders, or other conditions that have an
increased incidence in obese or overweight subjects. Examples
include: lipodystrophy, HIV lipodystrophy, diabetes (type 2),
insulin resistance, metabolic syndrome, hyperglycemia,
hyperinsulinemia, dyslipidemia, hepatic steatosis, hyperphagia,
hypertension, hypertriglyceridemia, infertility, a skin disorder
associated with weight gain, macular degeneration. In some
embodiments, the compounds may also be used in the manufacture of a
medicament for maintaining weight loss of a subject.
[0087] In some embodiments, compounds of formula (I) which are
leptin receptor agonist mimetics may also be used in the
manufacture of a medicament to promote wound healing.
[0088] In some embodiments, compounds of formula (I) which are
leptin receptor agonist mimetics may also be used in the
manufacture of a medicament for the treatment or prevention of
conditions that cause a decrease in circulating leptin
concentrations, and the consequent malfunction of the immune and
reproductive systems. Examples of such conditions and malfunctions
include severe weight loss, dysmenorrhea, amenorrhea, female
infertility, immunodeficiency and conditions associated with low
testosterone levels.
[0089] In some embodiments, compounds of formula (I) which are
leptin receptor agonist mimetics may also be used in the
manufacture of a medicament for the treatment or prevention of
conditions caused as a result of leptin deficiency, or a leptin or
leptin receptor mutation.
[0090] In some other embodiments, compounds of formula (I) which
are leptin receptor antagonist mimetics may be used for the
treatment or prevention of inflammatory conditions or diseases, low
level inflammation associated with obesity and excess plasma leptin
and in reducing other complications associated with obesity
including atherosclerosis, and for the correction of insulin
resistance seen in Metabolic Syndrome and diabetes.
[0091] In some embodiments, compounds of formula (I) which are
leptin receptor antagonist mimetics can be used for the treatment
or prevention of inflammation caused by or associated with: cancer
(such as leukemias, lymphomas, carcinomas, colon cancer, breast
cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma,
kidney cancer, melanoma, hepatic, lung, breast, and prostate
metastases, etc.); auto-immune disease (such as organ transplant
rejection, lupus erythematosus, graft v. host rejection, allograft
rejections, multiple sclerosis, rheumatoid arthritis, type I
diabetes mellitus including the destruction of pancreatic islets
leading to diabetes and the inflammatory consequences of diabetes);
autoimmune damage (including multiple sclerosis, Guillam Barre
Syndrome, myasthenia gravis); cardiovascular conditions associated
with poor tissue perfusion and inflammation (such as atheromas,
atherosclerosis, stroke, ischaemia-reperfusion injury,
claudication, spinal cord injury, congestive heart failure,
vasculitis, haemorrhagic shock, vasospasm following subarachnoid
haemorrhage, vasospasm following cerebrovascular accident,
pleuritis, pericarditis, the cardiovascular complications of
diabetes); ischaemia-reperfusion injury, ischaemia and associated
inflammation, restenosis following angioplasty and inflammatory
aneurysms; epilepsy, neurodegeneration (including Alzheimer's
Disease), arthritis (such as rheumatoid arthritis, osteoarthritis,
rheumatoid spondylitis, gouty arthritis), fibrosis (for example of
the lung, skin and liver), multiple sclerosis, sepsis, septic
shock, encephalitis, infectious arthritis, Jarisch-Herxheimer
reaction, shingles, toxic shock, cerebral malaria, Lyme's disease,
endotoxic shock, gram negative shock, haemorrhagic shock, hepatitis
(arising both from tissue damage or viral infection), deep vein
thrombosis, gout; conditions associated with breathing difficulties
(e.g. chronic obstructive pulmonary disease, impeded and obstructed
airways, bronchoconstriction, pulmonary vasoconstriction, impeded
respiration, chronic pulmonary inflammatory disease, silicosis,
pulmonary sarcosis, cystic fibrosis, pulmonary hypertension,
pulmonary vasoconstriction, emphysema, bronchial allergy and/or
inflammation, asthma, hay fever, rhinitis, vernal conjunctivitis
and adult respiratory distress syndrome); conditions associated
with inflammation of the skin (including psoriasis, eczema, ulcers,
contact dermatitis); conditions associated with inflammation of the
bowel (including Crohn's disease, ulcerative colitis and pyresis,
irritable bowel syndrome, inflammatory bowel disease); HIV
(particularly HIV infection), cerebral malaria, bacterial
meningitis, osteoporosis and other bone resorption diseases,
osteoarthritis, infertility from endometriosis, fever and myalgia
due to infection, and other conditions mediated by excessive
anti-inflammatory cell (including neutrophil, eosinophil,
macrophage and T-cell) activity.
[0092] In some embodiments, compounds of formula (I) which are
leptin receptor antagonists mimetics may be used for the treatment
or prevention of macro or micro vascular complications of type 1 or
2 diabetes, retinopathy, nephropathy, autonomic neuropathy, or
blood vessel damage caused by ischaemia or atherosclerosis.
[0093] In some embodiments, compounds of formula (I) which are
leptin receptor antagonist mimetics may be used to inhibit
angiogenesis. Compounds that inhibit angiogenesis may be used for
the treatment or prevention of obesity or complications associated
with obesity. Compounds that inhibit angiogenesis may be used for
the treatment or prevention of complications associated with
inflammation diabetic retinopathy, or tumour growth particularly in
breast, prostate or gastrointestinal cancer.
[0094] In a further aspect, the disclosure relates to a method for
the treatment or prevention of any of the disorders or conditions
described herein, which includes administering to a subject (e.g.,
a subject in need thereof; e.g., a mammal) an effective amount of a
compound of formula I.
[0095] Methods delineated herein include those wherein the subject
is identified as in need of a particular stated treatment.
Identifying a subject in need of such treatment can be in the
judgment of a subject or a health care professional and can be
subjective (e.g. opinion) or objective (e.g. measurable by a test
or diagnostic method).
[0096] In other aspects, the methods herein include those further
comprising monitoring subject response to the treatment
administrations. Such monitoring may include periodic sampling of
subject tissue, fluids, specimens, cells, proteins, chemical
markers, genetic materials, etc. as markers or indicators of the
treatment regimen. In other methods, the subject is prescreened or
identified as in need of such treatment by assessment for a
relevant marker or indicator of suitability for such treatment.
[0097] In one embodiment, the disclosure provides a method of
monitoring treatment progress. The method includes the step of
determining a level of diagnostic marker (Marker) (e.g., any target
or cell type delineated herein modulated by a compound herein) or
diagnostic measurement (e.g., screen, assay) in a subject suffering
from or susceptible to a disorder or symptoms thereof delineated
herein, in which the subject has been administered a therapeutic
amount of a compound herein sufficient to treat the disease or
symptoms thereof. The level of Marker determined in the method can
be compared to known levels of Marker in either healthy normal
controls or in other afflicted patients to establish the subject's
disease status. In preferred embodiments, a second level of Marker
in the subject is determined at a time point later than the
determination of the first level, and the two levels are compared
to monitor the course of disease or the efficacy of the therapy. In
certain preferred embodiments, a pre-treatment level of Marker in
the subject is determined prior to beginning treatment according to
this disclosure; this pre-treatment level of Marker can then be
compared to the level of Marker in the subject after the treatment
commences, to determine the efficacy of the treatment.
[0098] In certain method embodiments, a level of Marker or Marker
activity in a subject is determined at least once. Comparison of
Marker levels, e.g., to another measurement of Marker level
obtained previously or subsequently from the same patient, another
patient, or a normal subject, may be useful in determining whether
therapy according to the disclosure is having the desired effect,
and thereby permitting adjustment of dosage levels as appropriate.
Determination of Marker levels may be performed using any suitable
sampling/expression assay method known in the art or described
herein. Preferably, a tissue or fluid sample is first removed from
a subject. Examples of suitable samples include blood, urine,
tissue, mouth or cheek cells, and hair samples containing roots.
Other suitable samples would be known to the person skilled in the
art. Determination of protein levels and/or mRNA levels (e.g.,
Marker levels) in the sample can be performed using any suitable
technique known in the art, including, but not limited to, enzyme
immunoassay, ELISA, radiolabeling/assay techniques,
blotting/chemiluminescence methods, real-time PCR, and the
like.
[0099] In some embodiments, it may be advantageous if a compound of
formula (I) is able to penetrate the central nervous system. In
other embodiments, it may be advantageous if a compound of formula
(I) is not able to penetrate the CNS. In general, it is expected
that compounds that are leptin receptor agonist mimetics may be
particularly useful for the treatment or prevention of obesity,
insulin resistance, or diabetes (particularly glucose intolerance)
if these compounds can penetrate the CNS. A person of ordinary
skill in the art can readily determine whether a compound can
penetrate the CNS. A suitable method that may be used is described
in the Biological Methods section.
[0100] A leptin receptor response may be measured in any suitable
way. In vitro, this may be done be measuring leptin receptor
signaling. For example, phosphorylation of Akt, STAT3, STAT5, MAPK,
shp2 or the leptin receptor in response to binding of leptin or a
compound of the disclosure to the leptin receptor may be measured.
The extent of phosphorylation of Akt, STAT3, STAT5, MAPK, shp2 or
the leptin receptor may be determined for example by Western
blotting or by ELISA. Alternatively, a STAT reporter assay may be
used, for example STAT driven luciferase expression. A cell line
expressing the leptin receptor may be used for such assays. In
vivo, leptin receptor response may be measured by determining the
reduction in food intake and body weight after administration of
leptin or a compound of the disclosure.
[0101] The Biological Methods below describe assays and methods
that can be used to determine whether a compound of the disclosure
is a leptin receptor agonist mimetic or a leptin receptor
antagonist mimetic.
[0102] A compound of formula (I) may be administered with or
without other therapeutic agents. For example, where it is desired
to reduce inflammation, the compound may be administered with an
anti-inflammatory agent (for example, disease modifying
anti-rheumatic drugs such as methotrexate, sulphasalazine and
cytokine inactivating agents, steroids, NSAIDs, cannabinoids,
tachykinin modulators, or bradykinin modulators). Where it is
desired to provide an anti-tumour effect, a compound of formula (I)
may be administered with a cytotoxic agent (for example,
methotrexate, cyclophosphamide) or another anti-tumour drug.
[0103] Compounds of formula (I) may be radiolabeled (for example
with tritium or radioactive iodine) for in vitro or in vivo
applications, such as receptor displacement studies or receptor
imaging.
[0104] A further aspect of the present disclosure relates to
processes for the manufacture of compounds of formula (I) as
defined above. In one embodiment, the process comprises:
(a) reacting a compound of formula (II):
##STR00004##
wherein R.sup.3, R.sup.4, b, c, f and g are as defined in formula
(I), with 4-nitrophenyl chloroformate or
bis-(4-nitrophenyl)carbonate in the presence of a suitable base
(such as DIPEA or NEt.sub.3) in a suitable solvent (such as DCM or
THF), at -10 to 40.degree. C., to form a compound of formula
(III):
##STR00005##
(b) reacting the compound of formula (III) with a compound of
formula (IV):
##STR00006##
wherein X, R.sup.1, R.sup.2, a, d and e are as defined in formula
(I), in the presence of a suitable base, (such as NaH or NMM), in a
suitable solvent (such as THF or DCM), at -10 to 40.degree. C., to
obtain a compound of formula (I); and (c) optionally, in one or
several steps transforming a compound of formula (I) into another
compound of formula (I).
[0105] In another embodiment, the process comprises:
(a) reacting a compound of formula (IV):
##STR00007##
wherein X, R.sup.1, R.sup.2, a, d and e are as defined in formula
(I), with 4-nitrophenyl chloroformate or
bis-(4-nitrophenyl)carbonate in the presence of a suitable base
(such as DIPEA or NMM) in a suitable solvent (such as DCM), at -10
to 40.degree. C., to form a compound of formula (V):
##STR00008##
(b) reacting the compound of formula (V) with a compound of formula
(II):
##STR00009##
wherein R.sup.3, R.sup.4, b, c, f and g are as defined in formula
(I), in the presence of a suitable base, (such as DIPEA), in a
suitable solvent (such as DCM or DMF), at -10 to 40.degree. C., to
obtain a compound of formula (I); and (c) optionally, in one or
several steps transforming a compound of formula (I) into another
compound of formula (I).
DEFINITIONS
[0106] The following definitions shall apply throughout the
specification and the appended claims.
[0107] Unless otherwise stated or indicated, the term
"C.sub.1-6-alkyl" denotes a straight or branched alkyl group having
from 1 to 6 carbon atoms. Examples of said C.sub.1-6-alkyl include
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,
t-butyl, and straight- and branched-chain pentyl and hexyl. For
parts of the range "C.sub.1-6-alkyl" all subgroups thereof are
contemplated such as C.sub.1-5-alkyl, C.sub.1-4-alkyl,
C.sub.1-3-alkyl, C.sub.1-2-alkyl, C.sub.2-6-alkyl, C.sub.2-5-alkyl,
C.sub.2-4-alkyl, C.sub.2-3-alkyl, C.sub.3-6-alkyl, C.sub.4-5-alkyl,
etc.
[0108] Unless otherwise stated or indicated, the term
"C.sub.1-6-acyl" denotes a carbonyl group that is attached through
its carbon atom to a hydrogen atom (i.e., a formyl group) or to a
straight or branched C.sub.1-5-alkyl group, where alkyl is defined
as above. Examples of said C.sub.1-6-acyl include formyl, acetyl,
propionyl, n-butyryl, 2-methylpropionyl and n-pentoyl. For parts of
the range "C.sub.1-6-acyl" all subgroups thereof are contemplated
such as C.sub.1-5-acyl, C.sub.1-4-acyl, C.sub.1-3-acyl,
C.sub.1-2-acyl, C.sub.2-6-acyl, C.sub.2-5-acyl, C.sub.2-4-acyl,
C.sub.2-3-acyl, C.sub.3-6-acyl, C.sub.4-5-acyl, etc. If a
C.sub.1-6-acyl group is optionally substituted with one or more
substituents independently selected from halogen, hydroxy and
C.sub.1-6-alkoxy, said substituent can not be attached to the
carbonyl carbon atom.
[0109] Unless otherwise stated or indicated, the term
"C.sub.1-6-alkoxy" denotes a straight or branched alkoxy group
having from 1 to 6 carbon atoms. Examples of said C.sub.1-6-alkoxy
include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,
iso-butoxy, sec-butoxy, t-butoxy, and straight- and branched-chain
pentoxy and hexoxy. For parts of the range "C.sub.1-6-alkoxy" all
subgroups thereof are contemplated such as C.sub.1-5-alkoxy,
C.sub.1-4-alkoxy, C.sub.1-3-alkoxy, C.sub.1-2-alkoxy,
C.sub.2-6-alkoxy, C.sub.2-5-alkoxy, C.sub.2-4-alkoxy,
C.sub.2-3-alkoxy, C.sub.3-6-alkoxy, C.sub.4-5-alkoxy, etc.
[0110] "Halogen" refers to fluorine, chlorine, bromine or
iodine.
[0111] "Hydroxy" refers to the --OH radical.
[0112] "Nitro" refers to the --NO.sub.2 radical.
[0113] "Cyano" refers to the --CN radical.
[0114] "Optional" or "optionally" means that the subsequently
described event or circumstance may but need not occur, and that
the description includes instances where the event or circumstance
occurs and instances in which it does not.
[0115] The term "mammal" includes organisms, which include mice,
rats, cows, sheep, pigs, rabbits, goats, and horses, monkeys, dogs,
cats, and preferably humans. The subject may be a human subject or
a non human animal, particularly a domesticated animal, such as a
dog. "Pharmaceutically acceptable" means being useful in preparing
a pharmaceutical composition that is generally safe, non-toxic and
neither biologically nor otherwise undesirable and includes being
useful for veterinary use as well as human pharmaceutical use.
[0116] "Treatment" as used herein includes prophylaxis of the named
disorder or condition, or amelioration or elimination of the
disorder once it has been established.
[0117] "An effective amount" refers to an amount of a compound that
confers a therapeutic effect (e.g., treats, controls, ameliorates,
prevents, delays the onset of, or reduces the risk of developing a
disease, disorder, or condition or symptoms thereof) on the treated
subject. The therapeutic effect may be objective (i.e., measurable
by some test or marker) or subjective (i.e., subject gives an
indication of or feels an effect).
[0118] "Prodrugs" refers to compounds that may be converted under
physiological conditions or by solvolysis to a biologically active
compound of formula (I). A prodrug may be inactive when
administered to a subject in need thereof, but is converted in vivo
to an active compound of formula (I). Prodrugs are typically
rapidly transformed in vivo to yield the parent compound, e.g. by
hydrolysis in the blood. The prodrug compound usually offers
advantages of solubility, tissue compatibility or delayed release
in a mammalian organism (see Silverman, R. B., The Organic
Chemistry of Drug Design and Drug Action, 2.sup.nd Ed., Elsevier
Academic Press (2004), pp. 498-549). Prodrugs may be prepared by
modifying functional groups, such as a hydroxy, amino or mercapto
groups, present in a compound of formula (I) in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compound. Examples of prodrugs include, but are
not limited to, acetate, formate and succinate derivatives of
hydroxy functional groups or phenyl carbamate derivatives of amino
functional groups.
[0119] Throughout the specification and the appended claims, a
given chemical formula or name shall also encompass all hydrates
and solvates thereof. Further, a given chemical formula or name
shall encompass all tautomeric and stereoisomeric forms thereof.
Stereoisomers include enantiomers and diastereomers. Enantiomers
can be present in their pure forms, or as racemic (equal) or
unequal mixtures of two enantiomers. Diastereomers can be present
in their pure forms, or as mixtures of diastereomers. Diastereomers
also include geometrical isomers, which can be present in their
pure cis or trans forms or as mixtures of those.
[0120] The compounds of formula (I) may be used as such or, where
appropriate, as pharmacologically acceptable salts (acid or base
addition salts) thereof. The pharmacologically acceptable addition
salts mentioned below are meant to comprise the therapeutically
active non-toxic acid and base addition salt forms that the
compounds are able to form. Compounds that have basic properties
can be converted to their pharmaceutically acceptable acid addition
salts by treating the base form with an appropriate acid. Exemplary
acids include inorganic acids, such as hydrogen chloride, hydrogen
bromide, hydrogen iodide, sulphuric acid, phosphoric acid; and
organic acids such as formic acid, acetic acid, propanoic acid,
hydroxyacetic acid, lactic acid, pyruvic acid, glycolic acid,
maleic acid, malonic acid, oxalic acid, benzenesulphonic acid,
toluenesulphonic acid, methanesulphonic acid, trifluoroacetic acid,
fumaric acid, succinic acid, malic acid, tartaric acid, citric
acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic
acid, ascorbic acid and the like. Exemplary base addition salt
forms are the sodium, potassium, calcium salts, and salts with
pharmaceutically acceptable amines such as, for example, ammonia,
alkylamines, benzathine, and amino acids, such as, e.g. arginine
and lysine. The term addition salt as used herein also comprises
solvates which the compounds and salts thereof are able to form,
such as, for example, hydrates, alcoholates and the like.
Compositions
[0121] For clinical use, the compounds of the disclosure are
formulated into pharmaceutical formulations for various modes of
administration. It will be appreciated that the compounds may be
administered together with a physiologically acceptable carrier,
excipient, or diluent. The pharmaceutical compositions may be
administered by any suitable route, preferably by oral, rectal,
nasal, topical (including buccal and sublingual), sublingual,
transdermal, intrathecal, transmucosal or parenteral (including
subcutaneous, intramuscular, intravenous and intradermal)
administration.
[0122] Other formulations may conveniently be presented in unit
dosage form, e.g., tablets and sustained release capsules, and in
liposomes, and may be prepared by any methods well known in the art
of pharmacy. Pharmaceutical formulations are usually prepared by
mixing the active substance, or a pharmaceutically acceptable salt
thereof, with conventional pharmaceutically acceptable carriers,
diluents or excipients. Examples of excipients are water, gelatin,
gum arabicum, lactose, microcrystalline cellulose, starch, sodium
starch glycolate, calcium hydrogen phosphate, magnesium stearate,
talcum, colloidal silicon dioxide, and the like. Such formulations
may also contain other pharmacologically active agents, and
conventional additives, such as stabilizers, wetting agents,
emulsifiers, flavouring agents, buffers, and the like. Usually, the
amount of active compounds is between 0.1-95% by weight of the
preparation, preferably between 0.2-20% by weight in preparations
for parenteral use and more preferably between 1-50% by weight in
preparations for oral administration.
[0123] The formulations can be further prepared by known methods
such as granulation, compression, microencapsulation, spray
coating, etc. The formulations may be prepared by conventional
methods in the dosage form of tablets, capsules, granules, powders,
syrups, suspensions, suppositories or injections. Liquid
formulations may be prepared by dissolving or suspending the active
substance in water or other suitable vehicles. Tablets and granules
may be coated in a conventional manner. To maintain therapeutically
effective plasma concentrations for extended periods of time,
compounds of the disclosure may be incorporated into slow release
formulations.
[0124] The dose level and frequency of dosage of the specific
compound will vary depending on a variety of factors including the
potency of the specific compound employed, the metabolic stability
and length of action of that compound, the patient's age, body
weight, general health, sex, diet, mode and time of administration,
rate of excretion, drug combination, the severity of the condition
to be treated, and the patient undergoing therapy. The daily dosage
may, for example, range from about 0.001 mg to about 100 mg per
kilo of body weight, administered singly or multiply in doses, e.g.
from about 0.01 mg to about 25 mg each. Normally, such a dosage is
given orally but parenteral administration may also be chosen.
Preparation of Compounds of the Invention
[0125] The compounds of formula (I) above may be prepared by, or in
analogy with, conventional methods. Formation of the central
urethane or urea linker is the key synthetic step in preparing the
compounds formula (I). A large number of activating reagents can be
used for the formation of a urethane or urea linker e.g. phosgene
to form chloroformate of alcohols, or carbonyldiimidazole (CDI) to
form imidazole carboxylates. Typically the urethane linkers
incorporated into compounds of formula (I) have been synthesized
utilizing 4-nitrophenyl chloroformate or
bis-(4-nitrophenyl)carbonate as the activating agent. The
preparation of intermediates and compounds according to the
examples of the present disclosure may in particular be illuminated
by the following Schemes 1 and 2. Definitions of variables in the
structures in the schemes herein are commensurate with those of
corresponding positions in the formulae delineated herein.
##STR00010##
wherein X, R.sup.1-R.sup.4 and a-g are as defined in formula 1.
[0126] In one procedure, as generally represented in Scheme 1, the
piperazine moiety is activated by treating piperazine derivative
(II) with 4-nitrophenyl chloroformate or
bis-(4-nitrophenyl)carbonate in the presence of a base (such as
DIPEA) to form the corresponding carbamate derivative (III).
Treatment of this activated intermediate with the appropriate
alcohol moiety (IV) in the presence of a base (such as NaH) results
in the formation of the desired compound of formula (I).
##STR00011##
wherein X, R.sup.1-R.sup.4 and a-g are as defined in formula 1.
[0127] In an alternative procedure, the alcohol moiety is activated
by treating alcohol (IV) with 4-nitrophenyl chloroformate or
bis-(4-nitrophenyl)carbonate in the presence of a base (such as
DIPEA) to form the corresponding 4-nitrophenyl carbonate derivative
(V). In the next step, the activated carbonate (V) is treated with
the appropriate piperazine moiety (II) in the presence of a base
(such as DIPEA), resulting in the formation of the desired compound
of formula (I). This is generally represented in Scheme 2.
[0128] The formation of the urethane is typically a two step
process but this may also be performed in a one-pot reaction by
formation of the activated intermediate in situ. The experimental
section below gives examples of all of these synthetic
alternatives.
[0129] The necessary starting materials for preparing the compounds
of formula (I) are either commercially available, or may be
prepared by methods known in the art.
[0130] The processes described below in the experimental section
may be carried out to give a compound in the form of a free base or
as an acid addition salt. A pharmaceutically acceptable acid
addition salt may be obtained by dissolving the free base in a
suitable organic solvent and treating the solution with an acid, in
accordance with conventional procedures for preparing acid addition
salts from base compounds. Examples of addition salt forming acids
are mentioned above.
[0131] The compounds of formula (I) may possess one or more chiral
carbon atoms, and they may therefore be obtained in the form of
optical isomers, e.g., as a pure enantiomer, or as a mixture of
enantiomers (racemate) or as a mixture containing diastereomers.
The separation of mixtures of optical isomers to obtain pure
enantiomers is well known in the art and may, for example, be
achieved by fractional crystallization of salts with optically
active (chiral) acids or by chromatographic separation on chiral
columns.
[0132] The chemicals used in the synthetic routes delineated herein
may include, for example, solvents, reagents, catalysts, and
protecting group and deprotecting group reagents. Examples of
protecting groups are t-butoxycarbonyl (Boc), benzyl and trityl
(triphenylmethyl). The methods described above may also
additionally include steps, either before or after the steps
described specifically herein, to add or remove suitable protecting
groups in order to ultimately allow synthesis of the compounds. In
addition, various synthetic steps may be performed in an alternate
sequence or order to give the desired compounds. Synthetic
chemistry transformations and protecting group methodologies
(protection and deprotection) useful in synthesizing applicable
compounds are known in the art and include, for example, those
described in R. Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective
Groups in Organic Synthesis, 3.sup.rd Ed., John Wiley and Sons
(1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for
Organic Synthesis, John Wiley and Sons (1994); and L. Paquette,
ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and
Sons (1995) and subsequent editions thereof.
[0133] The following abbreviations have been used:
t-AmOH tert-Amylalcohol Boc tert-Butoxycarbonyl t-Bu tert-Butyl
DCM Dichloromethane
DIPEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
ES.sup.+Electrospray
[0134] Et.sub.2O Diethyl ether EtOAc Ethyl acetate HIV Human
immunodeficiency virus HPLC High performance liquid
chromatography
ICV Intracerebroventricular
LCMS Liquid Chromatography Mass Spectrometry
M Molar
[0135] [MH].sup.+Protonated molecular ion
NEt.sub.3 Triethylamine
[0136] NMM N-methyl morpholine
RP Reverse Phase
[0137] tert Tertiary TFA Trifluoroacetic acid
THF Tetrahydrofuran
[0138] Embodiments of the disclosure are described in the following
examples with reference to the accompanying drawings, in which:
[0139] FIG. 1 shows an example of body weight separation between
animals fed on a high carbohydrate diet. The error bars represent
mean+/-SEM.
[0140] FIG. 2 shows the cumulative body weight change (%) observed
in a 4 day study in DIO rats for Example 7.
[0141] FIG. 3 shows the cumulative body weight change (%) observed
in a 4 day study in DIO rats for Example 20.
[0142] FIG. 4 shows the cumulative body weight change (%) observed
in a 4 day study in DIO rats for Example 30.
[0143] FIG. 5 shows the concentration-dependent increase in
[.sup.3H]-thymidine incorporation by JEG-3 cells for leptin.
[0144] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment herein includes that embodiment as any
single embodiment or in combination with any other embodiments or
portions thereof.
[0145] The disclosure will now be further illustrated by the
following non-limiting examples. The specific examples below are to
be construed as merely illustrative, and not limitative of the
remainder of the disclosure in any way whatsoever. Without further
elaboration, it is believed that one skilled in the art can, based
on the description herein, utilize the present disclosure to its
fullest extent. All references and publications cited herein are
hereby incorporated by reference in their entirety.
EXAMPLES AND INTERMEDIATE COMPOUNDS
Experimental Methods
[0146] All reagents were commercial grade and were used as received
without further purification, unless otherwise specified.
Commercially available anhydrous solvents were used for reactions
conducted under inert atmosphere. Reagent grade solvents were used
in all other cases, unless otherwise specified. The methyl
isocyanate resin was supplied by NovaBiochem (Cat. No. 01-64-0169).
Analytical LCMS was performed on a Waters ZQ mass spectrometer
connected to an Agilent 1100 HPLC system. Analytical HPLC was
performed on an Agilent 1100 system. High-resolution mass spectra
(HRMS) were obtained on an Agilent MSD-TOF connected to an Agilent
1100 HPLC system. During the analyses the calibration was checked
by two masses and automatically corrected when needed. Spectra are
acquired in positive electrospray mode. The acquired mass range was
m/z 100-1100. Profile detection of the mass peaks was used. Flash
chromatography was performed on a Flash Master Personal system
equipped with Strata SI-1 silica gigatubes. Reverse phase
chromatography was performed on a Gilson system equipped with Merck
LiChoprep.RTM. RP-18 (40-63 .mu.m) 460.times.26 mm column, 30
mL/min, gradient of methanol in water. Preparative HPLC was
performed on a Gilson system equipped with Phenomenex Hydro RP
150.times.20 mm, 20 mL/min, gradient of acetonitrile in water. The
compounds were automatically named using ACD 6.0 or 8.0.
[0147] Analytical HPLC and LCMS data were obtained with:
[0148] System A: Phenomenex Synergi Hydro RP, (150.times.4.6 mm, 4
.mu.m), gradient 5-100% CH.sub.3CN (+0.085% TFA) in H.sub.2O (+0.1%
TFA), 1.5 mL/min, with a gradient time of 7 min, 200-300 nm,
30.degree. C.; or
[0149] System B: Phenomenex Synergi Hydro RP, (150.times.4.6 mm, 4
.mu.m), gradient 5-95% CH.sub.3CN (+0.085% TFA) in H.sub.2O (+0.1%
TFA), 1 mL/min, with a gradient time of 15.5 min, 200-300 nm,
40.degree. C.
Analytical LCMS data were also obtained with:
[0150] System C: Phenomenex Synergi Hydro RP (30.times.4.6 mm, 4
.mu.m), gradient 5-100% CH.sub.3CN (+0.085% TFA) in H.sub.2O (+0.1%
TFA), 1.5 mL/min, with a gradient time of 1.75 min, 30.degree.
C.
Intermediate 1
4-Nitrophenyl 4-(4-methyl)phenylpiperazine-1-carboxylate
##STR00012##
[0152] To a solution of 1-(4-methylphenyl)piperazine
dihydrochloride (10.65 g, 42.7 mmol) and DIPEA (22 mL, 133 mmol) in
DCM (100 mL) at 0.degree. C. was added 4-nitrophenyl chloroformate
(9.5 g, 47.1 mmol). The reaction mixture was stirred for 30 minutes
at 0.degree. C. and then washed with saturated sodium hydrogen
carbonate solution (3.times.200 mL), dried (MgSO.sub.4) and
concentrated in vacuo to give 4-nitrophenyl
4-(4-methyl)phenylpiperazine-1-carboxylate (17.84 g, quantitative)
as a yellow solid which was used without further purification.
[0153] Analytical LCMS: purity .about.90% (System C, R.sub.T=2.20
min), ES.sup.+: 342.1 [MH].sup.+.
Intermediate 2
4-Nitrophenyl 4-phenylpiperazine-1-carboxylate
##STR00013##
[0155] To a solution of phenylpiperazine (12.0 g, 74.0 mmol) and
DIPEA (13.5 mL, 81.6 mmol) in DCM (70 mL) at 0.degree. C. add
4-nitrophenyl chloroformate (16.5 g, 81.71 mmol). The reaction
mixture was stirred for 30 minutes at 0.degree. C. and then washed
with saturated sodium hydrogen carbonate solution (3.times.200 mL),
dried (MgSO.sub.4) and concentrated in vacuo to give a
4-nitrophenyl 4-phenylpiperazine-1-carboxylate (24.8 g, 102%) as a
yellow solid which was used without further purification.
[0156] Analytical LCMS: purity .about.90% (System C, R.sub.T=2.40
min), ES.sup.+: 328.1 [MH].sup.+.
Intermediate 3
1,4-Dimethyl-(S)-2-hydroxymethyl piperazine
##STR00014##
[0158] (S)-2-piperazine carboxylic acid dihydrochloride (5.10 g,
25.1 mmol) was dissolved in water (10 mL) and dioxane (40 mL) and
cooled to 0.degree. C. Sodium hydroxide (3.10 g, 77.5 mmol) in
water (6 mL) and di-tert-butyl dicarbonate (11.5 g, 52.7 mmol) were
added. The reaction mixture was stirred at room temperature for 16
hours and then concentrated in vacuo. The residue was suspended in
DCM (400 mL), stirred for 30 minutes at room temperature and then
filtered. The filtrate was concentrated in vacuo to give
(S)-2-piperazine-1,2,4-tricarboxylic acid 1,4-di-tert-butyl ester
(9.90 g, 119%) as a white solid which was used without further
purification.
[0159] Analytical LCMS: purity 100% (System C, R.sub.T=2.28 min),
ES.sup.+: 331.1 [MH].sup.+.
[0160] (S)-2-piperazine-1,2,4-tricarboxylic acid 1,4-di-tert-butyl
ester (9.90 g, 30.0 mmol) was dissolved in THF (14 mL). 1M borane
in THF (90 mL, 90.0 mmol) was added at room temperature. The
reaction mixture was stirred at reflux for 4 hours and then cooled
to 0.degree. C. and quenched with the drop-wise addition of
methanol (100 mL) and then concentrated in vacuo to give
(S)-2-hydroxymethyl-piperazine-1,4-dicarboxylic acid di-tert-butyl
ester (12.07 g, 127%) as a white solid which was used without
further purification.
[0161] Analytical LCMS: purity 100% (System C, R.sub.T=2.08 min),
ES.sup.+: 339.4 [MH].sup.+.
[0162] (S)-2-hydroxymethyl-piperazine-1,4-dicarboxylic acid
di-tert-butyl ester (5.66 g, 17.9 mmol) was dissolved in THF (10
mL) and cooled to 0.degree. C. 2M LiAlH.sub.4 in THF (28.5 mL, 57.0
mmol) was added. The reaction mixture was stirred at reflux for 4
hours and then cooled to 0.degree. C. and quenched with the
drop-wise addition of 1M sodium hydroxide solution (20 mL). The
reaction mixture was diluted with THF (50 mL), filtered and the
filtrate was concentrated in vacuo to give
1,4-dimethyl-(S)-2-hydroxymethyl piperazine (1.44 g, 56%) as a
colourless oil which was used without further purification.
[0163] Analytical LCMS: purity .about.90% (System C, R.sub.T=0.38
min), ES.sup.+: 145.2 [MH].sup.+.
Intermediate 4
1,4-Dimethyl-(R)-2-hydroxymethyl piperazine
##STR00015##
[0165] (R)-piperazine-1,3-dicarboxylic acid 1-tert-butyl ester
(4.94 g, 21.5 mmol) was dissolved in water (10 mL) and dioxane (20
mL) and cooled to 0.degree. C. Sodium hydroxide (1.72 g, 43.0 mmol)
in water (4 mL) and di-tent-butyl dicarbonate (5.20 g, 23.8 mmol)
were added. The reaction mixture was stirred at room temperature
for 16 hours and then concentrated in vacuo. The residue was
suspended in DCM (400 mL) and stirred for 30 minutes at room
temperature, filtered and the filtrate was concentrated in vacuo to
give (R)-2-piperazine-1,2,4-tricarboxylic acid 1,4-di-tert-butyl
ester (9.78 g, 138%) as a pale yellow oil which was used without
further purification.
[0166] Analytical LCMS: purity .about.90% (System C, R.sub.T=1.56
min), ES.sup.+: 131.7 [M+H-2Boc].sup.+.
[0167] (R)-2-piperazine-1,2,4-tricarboxylic acid 1,4-di-tert-butyl
ester (2.16 g, 6.55 mmol) was dissolved in THF (10 mL). 1M borane
in THF (20 mL, 20.0 mmol) was added at room temperature. The
reaction mixture was stirred at reflux for 4 hours and then cooled
to 0.degree. C. and quenched with the drop-wise addition of
methanol (60 mL) and then concentrated in vacuo to give
(R)-2-hydroxymethyl-piperazine-1,4-dicarboxylic acid di-tert-butyl
ester (2.60 g, 125%) as an off white solid which was used without
further purification.
[0168] Analytical LCMS: purity .about.90% (System C, R.sub.T=2.06
min), ES.sup.+: 116.9 [M+H-2Boc].sup.+.
[0169] (R)-2-hydroxymethyl-piperazine-1,4-dicarboxylic acid
di-tert-butyl ester (6.24 g, 19.7 mmol) was dissolved in THF (10
mL) and cooled to 0.degree. C. 2M LiAlH.sub.4 in THF (30.0 mL, 60.0
mmol) was added. The reaction mixture was stirred at reflux for 4
hours and then cooled to 0.degree. C. and quenched with the
drop-wise addition of 1M sodium hydroxide solution (20 mL). The
reaction mixture was diluted with THF (50 mL), filtered and the
filtrate was concentrated in vacuo to give
1,4-dimethyl-(R)-2-hydroxymethyl piperazine (1.77 g, 62%) as a
colourless oil which was used without further purification.
[0170] Analytical LCMS: purity 100% (System C, R.sub.T=0.44 min),
ES.sup.+: 145.2 [MH].sup.+.
Intermediate 5
(S)-(4-Methyl-piperazin-2-yl)-methanol
##STR00016##
[0172] To a stirred suspension of (S)-piperazine-1,3-dicarboxylic
acid 1-tert-butyl ester (5.00 g, 21.7 mmol) in THF (40 mL) was
slowly added 1.0 M borane.THF complex solution (32.6 mL, 32.6
mmol). The reaction was heated to 90.degree. C. and stirred under
reflux for 2 hours. The reaction mixture was removed from the heat
before a further 1.5 equivalents of 1.0 M borane.THF complex
solution (32.6 mL, 32.6 mmol) was added. The reaction was re-heated
to 90.degree. C. and stirred under reflux for a further 2 hours.
The reaction was cooled to 0.degree. C. and quenched by the slow
addition of MeOH. The reaction mixture was then concentrated in
vacuo. The white solid obtained was dissolved in THF (30 mL),
cooled to 0.degree. C. and slowly added a 2.0M solution of
LiAlH.sub.4 in THF (27 mL, 54.0 mmol). The reaction was heated to
90.degree. C. and stirred under reflux for 2 h. A further portion
of 2.0M solution of LiAlH.sub.4 in THF (27 mL, 54.0 mmol) was added
and the reaction stirred under reflux for 4 h and then at room
temperature overnight. The reaction mixture was cooled to 0.degree.
C. and quenched by the slow addition of 1.0M aq NaOH solution until
the exothermic reaction subsided. The resulting gel was diluted
with THF and the solids filtered off. The filtrate was then
concentrated in vacuo to afford
(S)-(4-methyl-piperazin-2-yl)-methanol (2.84 g, 101% crude yield)
as a colourless oil.
Intermediate 6
4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester
##STR00017##
[0174] 1-(4-Fluoro-phenyl)-piperazine (12.2 g, 67.7 mmol) and
triethylamine (4.63 mL, 67.7 mmol) were dissolved in anhydrous THF
(150 mL) under nitrogen and the reaction mixture was cooled to
0.degree. C. 4-Nitrophenyl chloroformate (13.4 g, 67.7 mmol)
dissolved in anhydrous THF (150 mL) at 0.degree. C. was added and
the reaction mixture was stirred at room temperature overnight. The
solvent was removed in vacuo and the residue suspended between
water and EtOAc. The aqueous layer was extracted with two further
portions of EtOAc, the organic layers were combined, dried
(MgSO.sub.4), dried in vacuo and then recrystallised from toluene
(50 mL)/hexane (60 mL) to give
4-(4-fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl ester
(15.8 g, 67%) as fine yellow needles.
Intermediate 7
3-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
##STR00018##
[0176] Morpholine-3,4-dicarboxylic acid 4-tert-butyl ester (9.50 g,
41 mmol) was dissolved in anhydrous THF (50 mL) under nitrogen and
cooled to -10.degree. C. A 1M solution of borane (82 mL, 82 mmol)
was added dropwise whilst maintaining the temperature below
0.degree. C. The reaction mixture was allowed to warm to room
temperature and stirred overnight. The reaction mixture was cooled
to -5.degree. C. and water (10 mL) was added cautiously followed by
Na.sub.2CO.sub.3 (9.5 g) in water (20 mL). After stirring for 30
min at room temperature the THF was removed in vacuo, water was
added and the reaction mixture was extracted with diethylether
(.times.3). The combined organic extracts were dried (MgSO.sub.4)
and the solvent was evaporated in vacuo to give
3-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester (8.9
g, 100%) as a colourless oil.
Intermediate 8
(S)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester
##STR00019##
[0178] (S)-3-Amino-1,2-propanediol (16.8 g, 184 mmol) was dissolved
in MeOH (90 mL) at room temperature and the solution was diluted
with MeCN (550 mL). NEt.sub.3 (30.5 mL, 219 mmol) was added and the
reaction mixture was cooled to -10.degree. C. Chloroacetyl chloride
(22.2 g, 15.6 mL, 197 mmol) was added dropwise at -10.degree. C.
during 1.5 h under nitrogen. The temperature was maintained at
-10.degree. C. for an additional hour and then the reaction mixture
was allowed to reach room temperature and stirred overnight (16 h).
The solvents were removed in vacuo. The residue was purified by
column chromatography (normal phase, Apollo Scientific silica,
40-60.mu., 60 .ANG., gradient 1% to 10% MeOH in EtOAc). The
combined fractions were evaporated to afford
2-chloro-N--[(S)-2,3-dihydroxypropyl]-acetamide (28.5 g, 92%) as a
colourless oil which gave a white solid on standing.
[0179] To a solution of t-BuOK (13.7 g, 122 mmol) in t-AmOH (175
mL) was added a solution of
2-chloro-N--[(S)-2,3-dihydroxypropyl]acetamide (8.1 g, 48.3 mmol)
in t-AmOH (325 mL) at room temperature over 2 h. The resulting
suspension was stirred for 1 h at room temperature. Methanol (50
mL) and water (5 mL) were added and the resulting solution was
stirred for 0.5 h at room temperature before adjusting the pH to 5
by the addition of conc. HCl. The suspension was filtered and the
solid washed with methanol. The filtrate was evaporated in vacuo,
the resulting solid was slurried in methanol (50 mL) and filtered
off to afford (S)-6-(hydroxymethyl)morpholin-3-one (1.3 g) as a
white solid. A second crop was obtained by adding EtOAc (200 mL) to
the filtrate and filtering the resulting solution through a silica
plug (1.times.10 cm). The solvent was evaporated in vacuo to afford
(S)-6-(hydroxymethyl)morpholin-3-one (3.9 g, overall yield 5.2 g,
82%) as a white solid.
[0180] To a solution of (S)-6-(hydroxymethyl)morpholin-3-one (7.50
g, 57.2 mmol) in anhydrous THF (100 mL) at 0.degree. C. was added
BH.sub.3 in THF (110 mL, 85.8 mmol). The reaction mixture was
allowed to reach room temperature and then heated at reflux
overnight. The reaction was cooled to room temperature and then
quenched with a mixture of water (5 mL) in THF (16 mL) followed by
7M methanolic ammonia (30 mL). The reaction mixture was heated to
reflux for 30 min, cooled and the solvents removed in vacuo The
residue was dissolved in methanol (15 mL), loaded onto an Iso lute
SCX-2 20 g column, washed with MeOH (100 mL) and eluted with
methanolic ammonia. The solvent was removed in vacuo to give
(S)-morpholin-2-yl-methanol (1.67 g, 24%) as a brown liquid.
[0181] To a solution of (S)-morpholin-2-yl-methanol (1.63 g, 13.9
mmol) in DCM (75 mL) was added a solution of NaOH (0.61 g, 15.3
mmol) in water (2 mL). Di-tent-butyldicarbonate (3.04 g, 13.9 mmol)
was added and the reaction mixture stirred overnight at room
temperature. The aqueous phase was separated and further extracted
with DCM (2.times.15 mL). The combined organic phases were dried
(Na.sub.2SO.sub.4) and the solvent removed in vacuo. The residue
was purified by column chromatography (normal phase silica, 20 g
Isolute-Si column, gradient 1% to 10% MeOH in DCM) and dried in
vacuo to give (S)-2-hydroxymethyl-morpholine-4-carboxylic acid
tert-butyl ester (2.3 g, 71%) as a white solid.
Intermediate 9
(R)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester
##STR00020##
[0183] (R)-2-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester was prepared similar to the procedure described for
Intermediate 8, but using (R)-3-amino-1,2-propanediol instead of
(S)-3-amino-1,2-propanediol. The title compound was obtained as a
white solid (10% overall yield, 4 steps)
Intermediate 10
(S)-(4-Methylmorpholin-2-yl)-methanol
##STR00021##
[0185] (S)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 8; 1.20 g, 5.52 mmol) in THF (5 mL) was added
to a suspension of LiAlH.sub.4 (1.30 g, 34.3 mmol) in anhydrous THF
(15 mL) at -10.degree. C. and stirred under nitrogen for 15 min.
The cooling bath was removed and the reaction mixture was gently
heated to reflux for 4 h. The reaction mixture was quenched by
careful addition of a mixture of water (4 mL) in THF (25 mL) with
ice bath cooling. THF (50 mL) was added to the reaction mixture,
stirred for 15 min, filtered and the solid washed with THF (50 mL).
The combined filtrates were evaporated in vacuo. The residue was
dissolved in DCM (50 mL), dried (MgSO.sub.4) and the solvent
removed in vacuo to give (S)-(4-methylmorpholin-2-yl)-methanol (490
mg, 67%) as a colourless oil.
Intermediate 11
(R)-(4-Methylmorpholin-2-yl)-methanol
##STR00022##
[0187] (R)-(4-Methylmorpholin-2-yl)-methanol was prepared similar
to the procedure described for Intermediate 10, but using
(R)-2-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
(Intermediate 9) instead of
(S)-2-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
(Intermediate 8). The title compound was obtained as a colourless
oil (560 mg, 77%).
Intermediate 12
4-(2,4-Difluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester
##STR00023##
[0189] 1-(2,4-Difluoro-phenyl)-piperazine (3.70 g, 18.9 mmol) and
triethylamine (2.8 mL, 19.8 mmol) were dissolved in anhydrous THF
(30 mL) under nitrogen and the reaction mixture was cooled to
0.degree. C. 4-Nitrophenyl chloroformate (3.8 g, 18.9 mmol),
dissolved in anhydrous THF (25 mL), was added dropwise and the
reaction mixture was stirred at room temperature overnight. The
solvent was removed in vacuo and the residue triturated with water,
and then filtered. The yellow solid was recrystallised from a
mixture of hexane and toluene (3:7) using charcoal to give
4-(2,4-difluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester (4.3 g, 63%) as a yellow crystalline solid.
Intermediate 13
2-(1,4-Dimethylpiperazin-2-yl)ethanol
##STR00024##
[0191] To a solution of methyl-2-piperazineacetate dihydrochloride
(500 mg, 2.16 mmol) in dioxane (12 mL) and water (6.0 mL) at
0.degree. C. was slowly added a solution of NaOH (182 g, 4.55 mmol)
in water (0.4 mL) followed by di-tert-butyl dicarbonate (992 mg,
4.55 mmol). The reaction mixture was stirred at room temperature
for 4 hours, and then concentrated in vacuo. To the resulting salts
was added DCM (75 mL) and the suspension stirred vigorously for 30
minutes. The salts were filtered off and washed with DCM (50 mL).
The filtrate was concentrated in vacuo to afford
2-methoxycarbonylmethylpiperazine-1,4-dicarboxylic acid
di-tert-butyl ester (662 mg, 85%) as a pale yellow oil.
[0192] To a stirred solution of
2-methoxycarbonylmethylpiperazine-1,4-dicarboxylic acid
di-tert-butyl ester (662 mg, 1.85 mmol) in anhydrous THF (10 mL) at
0.degree. C. was slowly added a 1M solution of LiAlH.sub.4 in THF
(5.7 mL, 5.70 mmol). The stirred reaction mixture was allowed to
warm to room temperature over a period of 15 minutes before being
refluxed for 2 hours. After this time the reaction mixture was
cooled to 0.degree. C. and cautiously quenched by the dropwise
addition of 1.0M aqueous NaOH solution until the effervescing
ceased. The resulting gel was diluted with THF (50 mL) and the
solids filtered off. The filtrate was concentrated in vacuo to
afford 2-(1,4-dimethylpiperazin-2-yl)ethanol (191 mg, 65%) as a
colourless oil.
Example 1
[(3R)-1-Methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate
##STR00025##
[0194] A 2M solution of LiAlH.sub.4 in THF (14.0 mL, 28.0 mmol) was
added drop-wise to a solution of (R)-tert-butyl
3-(hydroxymethyl)piperidine-1-carboxylate (5.00 g, 23.2 mmol) in
THF (30 mL) under argon at 0.degree. C. The reaction mixture was
allowed to warm to room temperature over 2 hours and stirred at
room temperature for 17 hours. The reaction mixture was cooled to
0.degree. C. and a 1M aq NaOH solution (4.0 mL) was added
drop-wise. Water (2 mL) was added and the resulting mixture stirred
at room temperature for 2 hours. The white solid was removed by
filtration. The filtrate was concentrated in vacuo to give
(R)-(1-methylpiperidin-3-yl)methanol (3.19 g, 106%) as a colourless
oil which was used without further purification.
[0195] Analytical LCMS: purity .about.90% (System C, R.sub.T=0.46
min), ES.sup.+: 130.1 [MH].sup.+.
[0196] Sodium hydride (0.70 g, 60% dispersion in mineral oil, 17.5
mmol) was suspended in heptane (10 mL) under an argon atmosphere.
The heptane was decanted off, and the flask was charged with THF
(20 mL) and cooled to 0.degree. C. A solution of
(R)-(1-methylpiperidin-3-yl)methanol (0.75 g, 5.83 mmol) in THF (20
mL) was added drop-wise, followed by a solution of 4-nitrophenyl
4-(4-methylphenyl)piperazine-1-carboxylate (Intermediate 1; 2.19 g,
6.42 mmol) in THF (20 mL). The reaction mixture was allowed to warm
to room temperature and stirred for 18 hours. The reaction mixture
was then cooled to 0.degree. C. and quenched with the drop-wise
addition of sat aq NaHCO.sub.3 solution and concentrated in vacuo.
The residue was dissolved in ethyl acetate (200 mL), washed with
sat aq NaHCO.sub.3 solution (4.times.50 mL), dried (MgSO.sub.4) and
concentrated in vacuo. The residue was purified by reverse phase
chromatography (gradient eluting with methanol in water, with 1%
formic acid in each solvent, 0-25%). The resulting residue was
dissolved in DCM (50 mL) and stirred with solid K.sub.2CO.sub.3 for
20 min, filtered and concentrated in vacuo. The residue was further
purified by normal phase column chromatography (eluting with DCM,
followed by a 90:10 mixture of DCM:MeOH) to give
[(3R)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate (619 mg, 32%) as a pale
brown solid.
[0197] Analytical HPLC: purity 99.9% (System A, R.sub.T=4.07 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.46 min),
ES.sup.+: 332.5 [MH].sup.+; HRMS calcd for
C.sub.19H.sub.29N.sub.3O.sub.2: 331.2260, found 331.2275.
Example 2
[(3S)-1-Methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate
##STR00026##
[0199] (S)-(1-Methylpiperidin-3-yl)methanol (1.50 g, 11.6 mmol;
prepared according to Example 1 but starting from (S)-tent-butyl
3-(hydroxymethyl)piperidine-1-carboxylate) was dissolved in DCM (20
mL) and cooled to 0.degree. C. NMM (1.30 mL, 12.2 mmol) and
nitrophenyl chloroformate (2.46 g, 12.2 mmol) were added. The
reaction mixture was stirred at 0.degree. C. for 2 hours and then a
solution of 4-(4-methylphenyl)piperazine dihydrochloride (1.88 g,
7.5 mmol) and DIPEA (3.70 mL, 22.1 mmol) in DMF (40 mL) was added.
The reaction mixture was stirred at room temperature for 2 hours
and then concentrated in vacuo. The residue was dissolved in EtOAc
(300 mL) and then washed sequentially with 1M aq Na.sub.2CO.sub.3
solution (5.times.200 mL), dried (MgSO.sub.4) and concentrated in
vacuo. The residue was purified by normal phase column
chromatography (eluting with DCM, followed by a 85:15 mixture of
DCM:MeOH) followed by reverse phase HPLC (Advanced Chromatography
Technologies ACE-122-1030 RP silica 100.times.30 mm column, packed
with Ace 5 C8 (5 .mu.m), Pore Size 100 .ANG., 30 mL/min, gradient
of CH.sub.3CN in water, with 0.1% TFA in each solvent, 8-38%). The
residue was dissolved in DCM (70 mL) and stirred with solid
K.sub.2CO.sub.3 for 20 min, filtered and concentrated in vacuo to
give a yellow oil which was recrystallised from heptane/EtOAc to
give [(3S)-1-methylpiperidin-3-yl]methyl
4-(4-methylphenyl)piperazine-1-carboxylate (521 mg, 13.5%) as a
white solid.
[0200] Analytical HPLC: purity 100% (System A, R.sub.T=3.88 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.39 min),
ES.sup.+: 332.2 [MH].sup.+; HRMS calcd for
C.sub.19H.sub.29N.sub.3O.sub.2: 331.2260, found 331.2270.
Example 3
[(2S)-1,4-Dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate
##STR00027##
[0202] To a solution of 1,4-dimethyl-(S)-2-hydroxymethyl piperazine
(Intermediate 3; 1.49 g, 10.3 mmol) in THF (20 mL) at 0.degree. C.
was added sodium hydride (1.24 g, 60% dispersion in mineral oil,
31.0 mmol). The reaction mixture was stirred for several minutes at
0.degree. C. and then 4-nitrophenyl
4-phenylpiperazine-1-carboxylate (Intermediate 2; 3.72 g, 11.4
mmol) in THF (20 mL). The reaction mixture was allowed to warm to
room temperature and stirred for 7 hours. The reaction mixture was
then cooled to 0.degree. C. and quenched with the drop-wise
addition of sat aq NaHCO.sub.3 solution. The THF was removed in
vacuo, the aqueous phase extracted with EtOAc (.times.3), combined
organic phases washed with sat aq NaHCO.sub.3 solution (.times.6),
dried (MgSO.sub.4) and concentrated in vacuo. The residue was
suspended in water:formic acid solution [1:1] (20 mL), filtered and
the filtrate was purified by reverse phase column chromatography
(gradient eluting with methanol in water, with 1% formic acid in
each solvent, 0-15%). The resulting residue was dissolved in DCM
(50 mL) and stirred with solid K.sub.2CO.sub.3 for 20 minutes,
filtered and concentrated in vacuo to give
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate (1.25 g, 36%) as a pale yellow
solid.
[0203] Analytical HPLC: purity 99.8% (System A, R.sub.T=3.50 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.86 min),
ES.sup.+: 333.6 [MH].sup.+; HRMS calcd for
C.sub.15H.sub.28N.sub.4O.sub.2: 332.2212, found 332.2227.
Example 4
[(2R)-1,4-Dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate
##STR00028##
[0205] Sodium hydride (1.14 g, 60% dispersion in mineral oil, 28.6
mmol) was suspended in heptane (10 mL) under an argon atmosphere.
The heptane was decanted off, and the flask was charged with THF
(20 mL) and cooled to 0.degree. C. A solution of
1,4-dimethyl-(R)-2-hydroxymethyl piperazine (Intermediate 4; 1.38
g, 9.5 mmol) in THF (20 mL) was added drop-wise, followed by a
solution of the 4-nitrophenyl 4-phenylpiperazine-1-carboxylate
(Intermediate 2; 4.06 g, 12.4 mmol) in THF (20 mL). The reaction
mixture was allowed to warm to room temperature, stirred for 16
hours, the reaction mixture was cooled to 0.degree. C. then
quenched with the drop-wise addition of sat aq NaHCO.sub.3 solution
and concentrated in vacuo. The residue was dissolved in EtOAc (200
mL), washed with sat aq NaHCO.sub.3 solution (4.times.50 mL), dried
(MgSO.sub.4) and concentrated in vacuo. The residue was suspended
in water:formic acid solution [1:1] (20 mL), filtered and the
filtrate was purified by reverse phase column chromatography
(gradient eluting with methanol in water, with 1% formic acid in
each solvent, 0-15%). The resulting residue was dissolved in DCM
(50 mL) and stirred with solid K.sub.2CO.sub.3 for 20 minutes,
filtered and concentrated in vacuo to give
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate (1.93 g, 61%) as a pale yellow
solid.
[0206] Analytical HPLC: purity 100% (System A, R.sub.T=3.55 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.90 min),
ES.sup.+: 333.5 [MH].sup.+; HRMS calcd for
C.sub.18H.sub.28N.sub.4O.sub.2: 332.2212, found 332.2225.
Example 5
[(2S)-1,4-Dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00029##
[0208] 4-Nitrophenyl chloroformate (5.17 g, 25.7 mmol) was
dissolved in DCM (200 mL) at room temperature and the reaction
mixture was cooled to 0.degree. C. and DIPEA (6.94 g, 9.38 mL, 53.9
mmol) and 1,4-dimethyl-(S)-2-hydroxymethyl piperazine (Intermediate
3; 3.70 g, 25.7 mmol) were added. The reaction mixture was stirred
at room temperature for 2 h, and then split into three equal
volumes. To one portion was added 1-(4-fluoro-phenyl)-piperazine
(1.53 g, 8.5 mmol) and the mixture was stirred for 48 h. The
solvent was removed in vacuo and the residue partitioned between
EtOAc (500 mL) and 1.0M NaOH solution (200 mL). The organic layer
was washed with 1.0M aq NaOH solution (5.times.125 mL), brine (100
mL), dried (MgSO.sub.4) and the solvent removed in vacuo. The
residue was dissolved in DCM (100 mL) and isocyanate resin (3 g)
was added, the reaction mixture was shaken for 14 h, filtered and
the solvent was removed in vacuo. The residue was purified by
reverse phase column chromatography (LiChroprep RP-18, 40-63 .mu.m,
460.times.26 mm (100 g), 30 mL/min, gradient 0% to 30% (over 75
min) to 100% (over 13 min) MeOH in water with 1% formic acid). The
residue was de-salted using K.sub.2CO.sub.3 in DCM to give
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate (1.78 g, 60.0%) as a
light yellow gum.
[0209] Analytical HPLC: purity 99.1% (System A, R.sub.T=3.69 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.10 min),
ES.sup.+: 351.1 [MH].sup.+.
Example 6
4-Phenylpiperazine-1-carboxylic acid
2-(1,4-dimethylpiperazin-2-yl)ethyl ester
##STR00030##
[0211] 2-(1,4-Dimethylpiperazin-2-yl)ethanol (703 mg, 4.44 mmol)
was dissolved in anhydrous THF (20 mL) and the stirred solution
cooled to 0.degree. C. before NaH (60% dispersion in mineral oil;
550 mg, 13.8 mmol) was added. The suspension was left to stir for
10 minutes. 4-Nitrophenyl 4-phenylpiperazine-1-carboxylate
(Intermediate 2; 1.89 g, 5.77 mmol) was added to the reaction
mixture, which was left to stir at room temperature for 3 hours.
After this time the reaction was terminated by cooling the mixture
to 0.degree. C. and cautiously quenching with saturated aqueous
NaHCO.sub.3 solution (50 mL). The THF was removed in vacuo and
transferred to a separating funnel where the organic product was
extracted with EtOAc (3.times.75 mL). The combined organic extracts
were collected together and washed with saturated aqueous
NaHCO.sub.3 solution (3.times.75 mL). The organic layer was dried
(MgSO.sub.4), filtered and concentrated in vacuo to give a crude
yellow slurry. The crude residue was purified by reverse phase
column chromatography (LiChroprep RP-18, 40-63 .mu.m, 460.times.26
mm (100 g), 30 mL/min, gradient 0% to 15% (over 70 min) to 100%
(over 5 min) MeOH in water with 1% formic acid). De-salting (using
K.sub.2CO.sub.3 in DCM), filtration and concentration in vacuo
afforded 4-phenylpiperazine-1-carboxylic acid
2-(1,4-dimethylpiperazin-2-yl)ethyl ester (236 mg, 13%) as a dark
yellow oil.
[0212] Analytical HPLC: purity 100% (System A, R.sub.T=3.50 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.92 min),
ES.sup.+: 347.6 [MH].sup.+; HRMS calcd for
C.sub.19H.sub.30N.sub.4O.sub.2: 346.2369, found 346.2378.
Example 7
[(2S)-1,4-Dimethylpiperazin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00031##
[0214] 4-Nitrophenyl chloroformate (5.17 g, 25.7 mmol) was
dissolved in DCM (200 mL) at room temperature and the reaction
mixture was cooled to 0.degree. C. and DIPEA (6.94 g, 9.38 mL, 53.9
mmol) and 1,4-dimethyl-(S)-2-hydroxymethyl piperazine (Intermediate
3; 3.70 g, 25.7 mmol) were added. The reaction mixture was stirred
at room temperature for 2 h and then split into three equal
volumes. To one portion was added
1-(2,4-difluoro-phenyl)-piperazine (1.68 g, 8.5 mmol) and stirred
for 48 h. The solvent was removed in vacuo and the residue was
partitioned between EtOAc (500 mL) and 1.0M aq NaOH solution (200
mL). The organic layer was washed with 1.0M aq NaOH solution
(5.times.125 mL), brine (100 mL), dried (MgSO.sub.4) and the
solvent removed in vacuo. The residue was dissolved in DCM (100 mL)
and isocyanate resin (3 g) was added, the reaction mixture was
shaken for 14 h, filtered and the solvents were removed in vacuo.
The residue was purified by reverse phase column chromatography
(LiChroprep RP-18, 40-63 .mu.m, 460.times.26 mm (100 g), 30 mL/min,
gradient 0% to 30% (over 75 min) to 100% (over 13 min) MeOH in
water with 1% formic acid). The residue was de-salted using
K.sub.2CO.sub.3 in DCM to give
[(2S)-1,4-dimethylpiperazin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate (1.23 g, 39.4%) as a
light yellow gum.
[0215] Analytical HPLC: purity 100% (System A, R.sub.T=4.12 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.53 min),
ES.sup.+: 369.1 [MH].sup.+; HRMS calcd for
C.sub.18H.sub.26F.sub.2N.sub.4O.sub.2: 368.2024, found
368.2038.
Example 8
[(2S)-4-Methylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate trihydrochloride
##STR00032##
[0217] To a suspension of (S)-(4-methyl-piperazin-2-yl)-methanol
(Intermediate 5; 2.84 g, 21.7 mmol) in a mixture of H.sub.2O (20
mL) and dioxane (40 mL) at 0.degree. C. was added 50% w/w aq NaOH
solution (0.96 g, 24.0 mmol). Di-tert-butyl dicarbonate (5.01 g,
22.9 mmol) was added and the reaction mixture stirred at room
temperature overnight. The solvents were removed in vacuo. The
residue was dissolved in anhydrous THF (25 mL) and the solution
cooled to 0.degree. C. NaH (60% wt dispersion in oil; 4.05 g, 101
mmol) was added and the grey suspension stirred for 10 minutes
before 4-nitro-phenyl 4-phenyl-piperazine-1-carboxylate
(Intermediate 2; 11.2 g, 34.1 mmol) was added. The reaction was
stirred at room temperature over the weekend. The reaction mixture
was cooled to 0.degree. C. and quenched with sat aq NaHCO.sub.3
solution (20 mL) before the THF was removed in vacuo. The organic
product was extracted with EtOAc (3.times.50 mL) and the organic
layers combined and washed with sat aq NaHCO.sub.3 solution
(3.times.50 mL), dried (MgSO.sub.4), and the solvent removed in
vacuo. MeOH was added to the crude slurry and the solids filtered
off and washed with MeOH. The filtrate was concentrated and
purified in two batches by reverse phase column chromatography
(LiChroprep RP-18, 40-63 nm, 460.times.26 mm (100 g), 30 mL/min,
gradient 0% to 50% (over 75 min) MeOH in water with 1% formic
acid). The product was de-salted using K.sub.2CO.sub.3 in DCM to
give
(S)-4-methyl-2-(4-phenyl-piperazine-1-carbonyloxymethyl)-piperazine--
1-carboxylic acid tert-butyl ester (1.65 g, 18%) as a brown
oil.
[0218] To a solution of
(S)-4-methyl-2-(4-phenyl-piperazine-1-carbonyloxymethyl)-piperazine-1-car-
boxylic acid tert-butyl ester (1.65 g, 3.9 mmol) in MeOH (10 mL)
was added 2M HCl in Et.sub.2O solution (11.8 mL, 23.6 mmol). The
reaction was stirred at room temperature for 3 days. The reaction
mixture was concentrated in vacuo. The residue was purified by
reverse phase column chromatography (LiChroprep RP-18, 40-63 .mu.m,
460.times.26 mm (100 g), 30 mL/min, gradient 0% to 15% (over 75
min) MeOH in water with 0.1% TFA. The material was then purified
further by reverse phase HPLC (YMC ODS-A 100.times.20 mm, 5 .mu.m,
25 mL/min, gradient 50% to 100% (over 7 min) then 100% (3 min) MeOH
in 10% MeOH/water). The pure product was stirred in 4M aq HCl
solution (10 mL) for 4 hours before being concentrated in vacuo.
The resulting oil was washed with heptane, concentrated in vacuo
and then dried in a vacuum oven overnight to afford the
[(2S)-4-methylpiperazin-2-yl]methyl
4-phenylpiperazine-1-carboxylate trihydrochloride salt (1.63 g,
96%) as a pale brown solid.
[0219] Analytical HPLC: purity 100% (System A, R.sub.T=3.27 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.71 min),
ES.sup.+: 319.2 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.26N.sub.4O.sub.2: 318.2056, found 318.2071.
Example 9
(1,4-Dimethylpiperazin-2-yl)methyl
4-benzylpiperazine-1-carboxylate
##STR00033##
[0221] 1,4-dimethyl-2-hydroxymethyl piperazine (1.00 g, 6.94 mmol)
was dissolved in DCM (50 mL) at room temperature and NMM (0.74 g,
7.29 mmol) was added. The reaction mixture was cooled to 0.degree.
C. and 4-nitrophenylchloroformate (1.4 g, 6.94 mmol) was added. The
reaction mixture was stirred at room temperature for 4 h and then
divided into two equal volumes. To one portion was added DIPEA
(1.35 g, 10.4 mmol), 1-benzyl piperazine (0.61 g, 3.47 mmol) and
the reaction mixture was stirred for 4 h. The solvents were removed
in vacuo and the residue was partitioned between EtOAc (300 mL) and
1.0M aq NaOH solution (100 mL). The organic layer was washed with
1.0M aq NaOH solution (5.times.100 mL), brine (100 mL), dried
(MgSO.sub.4) and the solvents were removed in vacuo. The residue
was dissolved in DCM (100 mL) and isocyanate resin was added, the
reaction mixture was shaken for 16 h, filtered and the solvents
were removed in vacuo. The residue was purified by reverse phase
column chromatography (LiChroprep RP-18, 40-63 .mu.m, 460.times.26
mm (100 g), 30 mL/min, gradient 0% to 30% (over 88 min) MeOH in
water with 1% formic acid). The residue was de-salted using
K.sub.2CO.sub.3 in DCM to give (1,4-dimethylpiperazin-2-yl)methyl
4-benzylpiperazine-1-carboxylate (0.32 g, 26.3%) as a light yellow
oil.
[0222] Analytical HPLC: purity 100% (System A, R.sub.T=2.94 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.40 min),
ES.sup.+: 347.2 [MH].sup.+; HRMS calcd for
C.sub.19H.sub.30N.sub.4O.sub.2: 346.2369, found 346.2382.
Example 10
[0223] Morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate
dihydrochloride
##STR00034##
[0224] 2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (4.13 g, 19.0 mmol) was dissolved in anhydrous THF (20 mL)
and the solution cooled to 0.degree. C. NaH (60% wt dispersion in
oil; 2.28 g, 57.0 mmol) was added and the grey suspension stirred
for 10 minutes before 4-nitrophenyl
4-phenylpiperazine-1-carboxylate (Intermediate 2; 7.46 g, 22.8
mmol) was added. The reaction was stirred at room temperature over
the weekend. A further 1.5 equivalents of NaH (60% wt dispersion in
oil) (1.14 g, 28.5 mmol) was added and the reaction stirred at room
temperature overnight. The reaction mixture was cooled to 0.degree.
C. and quenched with sat aq NaHCO.sub.3 solution (50 mL) before the
THF was removed in vacuo. The organic product was extracted with
EtOAc (3.times.50 mL) and the organic layers combined, washed with
sat aq NaHCO.sub.3 solution (3.times.50 mL), dried (MgSO.sub.4) and
the solvent removed in vacuo. The residue was purified by reverse
phase column chromatography (LiChroprep RP-18, 40-63 .mu.m,
460.times.36 mm (200 g), 30 mL/min, gradient 0% to 80% (over 120
mins) MeOH in water with 1% formic acid) to afford
2-(4-phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxylic
acid tert-butyl ester (6.67 g, 87% yield) as a dark brown oil.
[0225] To a solution of
2-(4-phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxylic
acid tert-butyl ester (6.67 g, 16.5 mmol) in MeOH (10 mL) was added
2M HCl in Et.sub.2O solution (16.5 mL, 32.9 mmol). The brown
solution was stirred at room temperature overnight. The reaction
mixture was concentrated in vacuo. The residue purified by reverse
phase column chromatography (LiChroprep RP-18, 40-63 .mu.m,
460.times.26 mm (100 g), 30 mL/min, gradient 0% to 15% (over 75
min) MeOH in water with 0.1% TFA). The pure sample was stirred in a
4M aq HCl solution (20 mL) overnight before being concentrated in
vacuo. The resulting yellow oil was washed with heptane,
concentrated in vacuo and dried in a vacuum oven overnight to give
the morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate
dihydrochloride salt (4.72 g, 76%) as a pale yellow solid.
[0226] Analytical HPLC: purity 99.6% (System A, R.sub.T=3.57 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.90 min),
ES.sup.+: 306.5 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.23N.sub.3O.sub.3: 305.1739, found 305.1749.
Example 11
(2S)-Morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate
##STR00035##
[0228] (S)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 8; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(5 mL) and stirred under nitrogen for 30 min. 4-Nitrophenyl
4-phenylpiperazine-1-carboxylate (Intermediate 2, 180 mg, 0.55
mmol) and THF (2 mL) were added and the reaction mixture was
stirred at room temperature for 48 h. The reaction mixture was
filtered through celite and the solid washed with TI-IF (10 mL).
The filtrates were combined and the solvent removed in vacuo. The
residue was purified by column chromatography (normal phase silica,
5 g Isolute-Si column, gradient 10% to 30% EtOAc in hexane) to give
(2S)-(4-phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxylic
acid tert-butyl ester (180 mg, 96%) as a colourless oil.
[0229]
(2S)-(4-Phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxyl-
ic acid tert-butyl ester (180 mg, 0.44 mmol) was dissolved in DCM
(2 mL) and TFA (3 mL, 10% in DCM) was added. The reaction mixture
was stirred at room temperature overnight. The solvents were
removed in vacuo. Heptane was added and the solvent was removed in
vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol) was added to a solution
of the residue in DCM (2 mL) and stirred for 30 min before adding
water (0.3 mL) and stirred for 1 h. The mixture was filtered and
the filtrate dried (Na.sub.2SO.sub.4) and solvents removed in
vacuo. The residue was purified by column chromatography (normal
phase, 5 g Isolute-Si column, EtOAc in Hexane (1:1) followed by
1-10% MeOH in EtOAc). The residue was dried in vacuo to give
(2S)-morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate (101 mg,
75%) as a white solid.
[0230] Analytical HPLC: purity 98.9% (System B, R.sub.T=6.63 min);
Analytical LCMS: purity 100% (System B, R.sub.T=6.57 min),
ES.sup.+: 306.7 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.23N.sub.3O.sub.3: 305.1739, found 305.1753.
Example 12
(2R)-Morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate
##STR00036##
[0232] (R)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 9; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(5 mL) and stirred under nitrogen for 30 min. 4-Nitrophenyl
4-phenylpiperazine-1-carboxylate (Intermediate 2; 180 mg, 0.55
mmol) was added and the reaction mixture was stirred at room
temperature for 48 h. The reaction mixture was filtered through
celite and the solid washed with THF (10 mL). The filtrates were
combined and the solvent removed in vacuo. The residue was purified
by column chromatography (normal phase, silica, 5 g Isolute-Si
column, gradient 10% to 50% EtOAc in hexane) to give
(2R)-(4-phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxyli-
c acid tert-butyl ester (170 mg, 91%) as a light yellow oil.
[0233]
(2R)-(4-Phenylpiperazine-1-carbonyloxymethyl)-morpholine-4-carboxyl-
ic acid tert-butyl ester (130 mg, 0.32 mmol) was dissolved in DCM
(2 mL) and TFA (3 mL, 10% in DCM) was added. The reaction mixture
was stirred at room temperature overnight. The solvents were
removed in vacuo. Heptane was added and the solvent removed in
vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol) was added to a solution
of the residue in DCM (2 mL) and stirred for 30 min before adding
water (0.3 mL) and stirred for 1 h. The mixture was filtered and
the filtrate dried (Na.sub.2SO.sub.4) and solvents removed in
vacuo. The residue was purified by column chromatography (normal
phase, 5 g Isolute-Si column, EtOAc in Hexane (1:1) followed by
1-10% MeOH in EtOAc). The residue was dried in vacuo to give
(2R)-morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate (57 mg,
58%) as a white solid.
[0234] Analytical HPLC: purity 99.8% (System B, R.sub.T=6.63 min);
Analytical LCMS: purity 100% (System B, R.sub.T=6.54 min),
ES.sup.+: 306.7 [MH].sup.+; FIRMS calcd for
C.sub.16H.sub.23N.sub.3O.sub.3: 305.1739, found 305.1753.
Example 13
(4-Methylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate
##STR00037##
[0236] Morpholin-2-ylmethyl 4-phenylpiperazine-1-carboxylate
(non-HCl salt of Example 10; 1.00 g, 3.27 mmol) and 37%
formaldehyde in water (1.97 g, 1.82 mL, 65.5 mmol) were dissolved
in MeOH (20 mL) at room temperature followed by addition of sodium
triacetoxyborohydride (2.78 g, 13.1 mmol) portionwise over 5
minutes. The reaction mixture was stirred for 2 h. The reaction
mixture was quenched by the addition of saturated aq
Na.sub.2CO.sub.3 solution. The solvents were removed in vacuo and
the residual aqueous phase was loaded onto an Isolute HM-N 20 mL
cartridge. The desired product was eluted with DCM (200 mL) and the
solvent removed in vacuo to give (4-methylmorpholin-2-yl)methyl
4-phenylpiperazine-1-carboxylate (0.93 g, 88.9%) as a colourless
gum.
[0237] Analytical HPLC: purity 99.5% (System A, R.sub.T=3.64 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.01 min),
ES.sup.+: 320.1 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.25N.sub.3O.sub.3: 319.1896, found 319.1899.
Example 14
[(2S)-4-Methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate
##STR00038##
[0239] (S)-(4-Methyl-morpholin-2-yl)-methanol (Intermediate 10; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol,) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-Nitrophenyl 4-phenylpiperazine-1-carboxylate
(Intermediate 2; 300 mg, 0.92 mmol) was added and the reaction
mixture was stirred at room temperature for 48 h. The reaction
mixture was filtered through celite and the solid washed with THF
(10 mL). The filtrates were combined and the solvent removed in
vacuo. The residue was purified by column chromatography (normal
phase silica, 5 g Isolute-Si column, gradient 1-5% MeOH in DCM)
then by reverse phase HPLC (YMC ODS-A 100.times.20 mm, 5 .mu.m, 25
mL/min, gradient 20% to 100% (over 7 min) then 100% (3 min) MeOH in
10% MeOH/water). The pure product was taken up in DCM (5 mL),
filtered, and dried in vacuo to give
[(2S)-4-methylmorpholin-2-yl]methyl
4-phenyl-piperazine-1-carboxylate (50 mg, 20%) as a colourless
gum.
[0240] Analytical HPLC: purity 99.4% (System A, R.sub.T=3.65 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.66 min),
ES.sup.+: 320.3 [MH].sup.+; FIRMS calcd for
C.sub.17H.sub.25N.sub.3O.sub.3: 319.1896, found 319.1909.
Example 15
[(2R)-4-Methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate
##STR00039##
[0242] (R)-(4-Methyl-morpholin-2-yl)-methanol (Intermediate 11; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-Nitrophenyl 4-phenylpiperazine-1-carboxylate
(Intermediate 2; 300 mg, 0.92 mmol) was added and the reaction
mixture was stirred at room temperature for 48 h. The reaction
mixture was filtered through celite and the solid washed with THF
(10 mL). The filtrates were combined and the solvent removed in
vacuo. The residue was purified by column chromatography (normal
phase silica, 5 g Isolute-Si column, gradient 1-5% MeOH in DCM).
The solvents were removed in vacuo to give
[(2R)-4-methylmorpholin-2-yl]methyl
4-phenylpiperazine-1-carboxylate (175 mg, 72%) as a light yellow
gum
[0243] Analytical HPLC: purity 99.8% (System B, R.sub.T=6.84 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.67 min),
ES.sup.+: 320.5 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.25N.sub.3O.sub.3: 319.1896, found 319.1895.
Example 16
[(2S)-4-Methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00040##
[0245] (S)-(4-Methyl-morpholin-2-yl)-methanol (Intermediate 10; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-(4-Fluorophenyl)-piperazine-1-carboxylic
acid 4-nitrophenyl ester (Intermediate 6; 314 mg, 0.91 mmol) was
added and the reaction mixture was stirred at room temperature for
48 h. The reaction mixture was filtered through celite and the
solid washed with THF (10 mL). The filtrates were combined and the
solvent removed in vacuo. The residue was purified by column
chromatography (normal phase silica, 5 g Isolute-Si column,
gradient 1-5% MeOH in DCM) and the solvents were removed in vacuo
to give [(2S)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)-piperazine-1-carboxylate (130 mg, 51%) as a
light brown solid.
[0246] Analytical HPLC: purity 96.0% (System B, R.sub.T=7.46 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.97 min),
ES.sup.+: 338.6 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.24FN.sub.3O.sub.3: 337.1802, found 337.1814.
Example 17
[(2R)-4-Methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00041##
[0248] (R)-(4-Methyl-morpholin-2-yl)-methanol (Intermediate 11; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-(4-Fluorophenyl)-piperazine-1-carboxylic
acid 4-nitrophenyl ester (Intermediate 6; 314 mg, 0.91 mmol) was
added and the reaction mixture was stirred at room temperature for
48 h. The reaction mixture was filtered through celite and the
solid washed with THF (10 mL). The filtrates were combined and the
solvent removed in vacuo. The residue was purified by column
chromatography (normal phase silica, 5 g Isolute-Si column,
gradient 1-5% MeOH in DCM) and then reverse phase HPLC (YMC ODS-A
100.times.20 mm, 5 .mu.m, 25 mL/min, gradient 20% to 100% (over 7
min) then 100% (3 min) MeOH in 10% MeOH/water). The pure product
was taken up in DCM (5 mL), filtered, and dried in vacuo to give
[(2R)-4-methylmorpholin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate (100 mg, 28%) as a
colourless gum.
[0249] Analytical HPLC: purity 99.7% (System A, R.sub.T=3.97 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.94 min),
ES.sup.+: 338.3 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.24FN.sub.3O.sub.3: 337.1802, found 337.1815.
Example 18
[(2S)-4-Methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00042##
[0251] (S)-(4-Methylmorpholin-2-yl)-methanol (Intermediate 10; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-(2,4-Difluorophenyl)-piperazine-1-carboxylic
acid 4-nitrophenyl ester (Intermediate 12, 330 mg, 0.91 mmol) was
added and the reaction mixture was stirred at room temperature for
48 h. The reaction mixture was filtered through celite and the
solid washed with THF (10 mL). The filtrates were combined and the
solvent removed in vacuo. The residue was purified by column
chromatography (normal phase silica, 5 g Isolute-Si column,
gradient 1-5% MeOH in DCM) and the solvents were removed in vacuo
to give [(2S)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)-piperazine-1-carboxylate (104 mg, 38%) as a
colourless gum.
[0252] Analytical HPLC: purity 99.3% (System B, R.sub.T=8.55 min);
Analytical LCMS: purity 100% (System B, R.sub.T=8.32 min),
ES.sup.+: 356.7 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.23F.sub.2N.sub.3O.sub.3: 355.1707, found
355.1724.
Example 19
[(2R)-4-Methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00043##
[0254] (R)-(4-Methylmorpholin-2-yl)-methanol (Intermediate 11; 100
mg, 0.76 mmol) was added to a suspension of NaH (60% dispersion in
oil; 90.0 mg, 2.28 mmol) in anhydrous THF (7 mL) and stirred under
nitrogen for 30 min. 4-(2,4-Difluorophenyl)-piperazine-1-carboxylic
acid 4-nitrophenyl ester (Intermediate 12; 330 mg, 0.91 mmol) was
added and the reaction mixture was stirred at room temperature for
48 h. The reaction mixture was filtered through celite and the
solid washed with THF (10 mL). The filtrates were combined and the
solvent removed in vacuo. The residue was purified by column
chromatography (normal phase silica, 5 g Isolute-Si column,
gradient 1-5% MeOH in DCM) and the solvents were removed in vacuo
to give [(2R)-4-methylmorpholin-2-yl]methyl
4-(2,4-difluorophenyl)-piperazine-1-carboxylate (87 mg, 32%) as a
colourless gum.
[0255] Analytical HPLC: purity 100% (System B, R.sub.T=8.57 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.62 min),
ES.sup.+: 356.4 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.23F.sub.2N.sub.3O.sub.3: 355.1707, found
355.1722.
Example 20
Morpholin-2-ylmethyl 4-(4-fluorophenyl)piperazine-1-carboxylate
dihydrochloride
##STR00044##
[0257] 2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (2.17 g, 10 mmol) was dissolved in anhydrous THF (50 mL) and
the reaction mixture was cooled to 0.degree. C. NaH (60% dispersion
in oil; 0.40 g, 10 mmol) was added and stirred for 10 minutes
before 4-(4-fluorophenyl)-piperazine-1-carboxylic acid
4-nitrophenyl ester (Intermediate 6; 3.45 g, 10 mmol) was added.
The reaction mixture was stirred at room temperature overnight and
then cautiously quenched by dropwise addition of water (1 mL)/THF
(10 mL) mixture before the THF was removed in vacuo. The residue
was suspended between sat aq Na.sub.2CO.sub.3 (50 mL) and EtOAc
(200 mL). The organic layer was washed with sat aq Na.sub.2CO.sub.3
(5.times.50 mL), dried (MgSO.sub.4) and dried in vacuo. The residue
was purified by reverse phase column chromatography (LiChroprep
RP-18, 40-63 .mu.m, 460.times.26 mm (100 g), 30 mL/min, gradient 0%
to 60% (over 60 min) MeOH in water) to give .about.80% pure
material by HPLC. The crude intermediate was dissolved in DCM (100
mL) and TFA (10 mL, excess) and was stirred at room temperature
overnight. The solvent was removed in vacuo and the residue was
repurified by reverse phase column chromatography (LiChroprep
RP-18, 40-63 .mu.m, 460.times.26 mm (100 g), 30 mL/min, gradient 0%
to 20% (over 70 min) then held at 20% (over 120 min) MeOH in water
with 1% formic acid. The solvent was removed in vacuo and the
residue was de-salted using K.sub.2CO.sub.3 in DCM. The residue was
dissolved in DCM and HCl (2M in Et.sub.2O, 3.2 mL, 6.4 mmol), dried
in a vacuum oven overnight to give the morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate dihydrochloride salt
(2.30 g, 58%) as a colourless gum.
[0258] Analytical HPLC: purity 97.5% (System A, R.sub.T=3.90 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.27 min),
ES.sup.+: 324.1 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1660.
Example 21
(2S)-Morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00045##
[0260] (S)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 8; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(2 mL) and stirred under nitrogen for 30 min.
4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl ester
(Intermediate 6; 191 mg, 0.55 mmol) was added and the reaction
mixture was stirred at room temperature for 48 h. The reaction
mixture was filtered through celite and the solid washed with THF
(10 mL). The filtrates were combined and the solvent removed in
vacuo. The residue was purified by column chromatography (normal
phase silica, 5 g Isolute-Si column, gradient 10% to 50% EtOAc in
hexane) to give
(2S)-[4-(4-fluoro-phenyl)-piperazine-1-carbonyloxy-methyl]-morpholine-4-c-
arboxylic acid tent-butyl ester (200 mg, quantitative) as a light
yellow oil.
[0261]
(2S)-[4-(4-Fluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morpholin-
e-4-carboxylic acid tert-butyl ester (200 mg, 0.46 mmol) was
dissolved in DCM (2 mL) and TFA (3 mL, 10% in DCM) was added. The
reaction mixture was stirred at room temperature overnight. The
solvents were removed in vacuo. Heptane was added and then the
solvent was removed in vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol)
was added to a solution of the residue in DCM (2 mL) and stirred
for 30 min before adding water (0.3 mL) and stirred for 1 h. The
mixture was filtered and the filtrate dried (Na.sub.2SO.sub.4) and
solvents removed in vacuo. The residue was purified by column
chromatography (normal phase, 5 g Isolute-Si column, EtOAc in
hexane (1:1) followed by 1-10% MeOH in EtOAc). The residue was
dried in vacuo to give (2S)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate (135 mg, 89%) as a white
solid.
[0262] Analytical HPLC: purity 98.2% (System B, R.sub.T=7.23 min);
Analytical LCMS: purity 100% (System B, R.sub.T=7.12 min),
ES.sup.+: 324.7 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1659.
Example 22
(2R)-Morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00046##
[0264] (R)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 9; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(2 mL) and stirred under nitrogen for 30 min.
4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl ester
(Intermediate 6; 191 mg, 0.55 mmol) was added and the reaction
mixture was stirred at room temperature for 48 h. The reaction
mixture was filtered through celite and the solid washed with THF
(10 mL). The filtrates were combined and the solvent removed in
vacuo. The residue was purified by column chromatography (normal
phase silica, 5 g Isolute-Si column, gradient 10% to 50% EtOAc in
hexane) to give
(2R)-[4-(4-fluoro-phenyl)-piperazine-1-carbonyloxy-methyl]-morpholine-4-c-
arboxylic acid tert-butyl ester (130 mg, 67%) as a colourless
oil.
[0265]
(2R)-[4-(4-Fluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morpholin-
e-4-carboxylic acid tert-butyl ester (120 mg, 0.40 mmol) was
dissolved in DCM (2 mL) and TFA (3 mL, 10% in DCM) was added. The
reaction mixture was stirred at room temperature overnight. The
solvents were removed in vacuo. Heptane was added and then the
solvents were removed in vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol)
was added to a solution of the residue in DCM (2 mL) and stirred
for 30 min before adding water (0.3 mL) and stirred for 1 h. The
mixture was filtered and the filtrate dried (Na.sub.2SO.sub.4) and
solvents removed in vacuo. The residue was purified by column
chromatography (normal phase, 5 g Isolute-Si column, EtOAc in
hexane (1:1) followed by 1-10% MeOH in EtOAc). The residue was
dried in vacuo to give (2R)-morpholin-2-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate (117 mg, 91%) as a white
solid.
[0266] Analytical HPLC: purity 99.3% (System B, R.sub.T=7.25 min);
Analytical LCMS: purity 97.9% (System B, R.sub.T=7.13 min),
ES.sup.+: 324.6 [MH].sup.+; FIRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1659.
Example 23
(4-Methylmorpholin-2-yl)methyl
4-(4-chlorophenyl)piperazine-1-carboxylate dihydrochloride
##STR00047##
[0268] (4-Methyl-morpholin-2-yl)-methanol (1.05 g, 8.0 mmol) and
DIPEA (2.79 ml, 16.0 mmol were dissolved in DCM (80 mL) and the
reaction mixture was cooled to 0.degree. C. and p-nitrophenyl
chloroformate (3.23 g, 16.0 mmol) was added. The reaction mixture
was allowed to warm to room temperature and stirred for 2 h. The
reaction mixture was divided into 4 batches and concentrated in
vacuo. One portion was dissolved in DMF (20 mL) and
1-(4-chlorophenyl)-piperazine dihydrochloride (539 mg, 2 mmol) was
added, and the resulting reaction mixture was stirred at room
temperature for 24 h. The solvent was removed in vacuo and the
residue was dissolved in DCM (30 mL). The organic layer was washed
with 1.0M Na.sub.2CO.sub.3 solution (3.times.30 mL), dried
(MgSO.sub.4) and the solvents were removed in vacuo. The residue
was purified by column chromatography (normal phase, 20 g, Strata
SI-1, silica gigatube, 20 mL/min, gradient 0% to 5% MeOH in DCM,
residue dry loaded). The solvents were removed in vacuo and the
residue was dissolved in DCM (50 mL), filtered and 2M HCl in
Et.sub.2O (2 mL, 4 mmol) added. The solvents were removed in vacuo
and the resulting solid was dried in a vacuum oven overnight to
give the (4-methylmorpholin-2-yl)methyl
4-(4-chlorophenyl)piperazine-1-carboxylate dihydrochloride salt
(0.510 g, 60%) as a white solid.
[0269] Analytical HPLC: purity 100% (System A, R.sub.T=4.69 min);
Analytical LCMS: purity 100% (System A, R.sub.T=5.12 min),
ES.sup.+: 354.4 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.24ClN.sub.3O.sub.3: 353.1506, found 353.1516.
Example 24
(4-Methylmorpholin-2-yl)methyl
4-(4-fluorobenzyl)piperazine-1-carboxylate dihydrochloride
##STR00048##
[0271] (4-Methyl-morpholin-2-yl)-methanol (1.05 g, 8.0 mmol) and
DIPEA (2.79 ml, 16.0 mmol were dissolved in DCM (80 mL) and the
reaction mixture was cooled to 0.degree. C. and p-nitrophenyl
chloroformate (3.23 g, 16.0 mmol) was added. The reaction mixture
was allowed to warm to room temperature and stirred for 2 h. The
reaction mixture was divided into 4 batches and concentrated in
vacuo. One portion was dissolved in DMF (20 mL) and
1-(4-fluoro-benzyl)-piperazine (0.39 g, 2 mmol) added and the
resulting reaction mixture stirred at room temperature for 24 h.
The solvent was removed in vacuo and the residue was taken up in
DCM (30 mL). The organic layer was washed with 1.0M
Na.sub.2CO.sub.3 solution (3.times.30 mL), dried (MgSO.sub.4) and
the solvents were removed in vacuo. The residue was purified by
column chromatography (normal phase, 20 g, Strata SI-1, silica
gigatube, 20 mL/min, gradient 0% to 5% MeOH in DCM, residue dry
loaded). The solvents were removed in vacuo and the residue was
dissolved in DCM (50 mL), filtered and 2M HCl in Et.sub.2O (2 mL, 4
mmol) added. The solvent was removed in vacuo and the residue was
dried in a vacuum oven overnight to give the
(4-methylmorpholin-2-yl)methyl
4-(4-fluorobenzyl)piperazine-1-carboxylate dihydrochloride salt
(0.375 g, 44%) as a white solid.
[0272] Analytical HPLC: purity 99.2% (System A, R.sub.T=3.13 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.60 min),
ES.sup.+: 352.5 [MH].sup.+.
Example 25
(4-Acetylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate
##STR00049##
[0274] To a stirred solution of morpholin-2-ylmethyl
4-phenylpiperazine-1-carboxylate dihydrochloride (Example 10; 0.52
g, 1.40 mmol) in pyridine (2.25 mL, 27.7 mmol) was slowly added
acetyl chloride (0.15 mL, 2.10 mmol). The reaction mixture was
stirred at room temperature for 1 h before the pyridine was removed
in vacuo and the resulting residue diluted with sat aq NaHCO.sub.3
solution (50 mL). The organic product was extracted with EtOAc
(3.times.50 mL). The combined organic layers were washed with sat
aq NaHCO.sub.3 solution (3.times.50 mL), dried (MgSO.sub.4) and the
solvent removed in vacuo. The residue was purified by column
chromatography (normal phase, 20 g, Strata SI-1, silica gigatube,
20 mL/min, gradient 0% to 5% MeOH in EtOAc). The solvents were
removed in vacuo and dried in a vacuum oven overnight to afford
(4-acetylmorpholin-2-yl)methyl 4-phenylpiperazine-1-carboxylate
(0.38 g, 78%) as a pale brown oil.
[0275] Analytical HPLC: purity 99.0% (System A, R.sub.T=4.34 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.60 min),
ES.sup.+: 348.5 [MH].sup.+; HRMS calcd for
C.sub.18H.sub.25N.sub.3O.sub.4: 347.1845, found 347.1859.
Example 26
[0276] Morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00050##
[0277] NaH (60% in oil, prewashed with hexane; 1.30 g, 31.7 mmol)
was suspended in anhydrous THF (50 mL) under nitrogen and the
reaction mixture was cooled to -10.degree. C. with stirring. A
solution of 3-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 7; 2.30 g, 10.6 mmol) in THF (50 mL) was added
dropwise. The reaction mixture was stirred for 20 min at 0.degree.
C. 4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester (Intermediate 6; 4.38 g, 12.7 mmol) was added and the
reaction mixture was stirred overnight at room temperature. Sat aq
NaHCO.sub.3 solution (10 mL) was added and the solvents were
removed in vacuo. The residue was partitioned between water and
EtOAc and the aqueous phase extracted with EtOAc. The combined
organic extracts were washed with sat aq NaHCO.sub.3 (3.times.100
mL), dried (MgSO.sub.4) and the solvent removed in vacuo. The
residue was purified by flash normal column chromatography (Apollo
silica, 40-63 .mu.m, 60A) eluting with EtOAc/hexane. Impure
material was further purified by flash normal phase chromatography
using 1% MeOH in DCM to give
3-[4-(4-fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4-carbox-
ylic acid tert-butyl ester (3.05 g, 68%).
[0278]
3-[4-(4-fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4--
carboxylic acid tert-butyl ester (2.98 g, 7.04 mmol) was stirred in
DCM (30 mL) at 0.degree. C. TFA (6 mL) dissolved in DCM (24 mL) was
added dropwise. The reaction mixture was stirred overnight at room
temperature. The solvent was removed in vacuo and the residue was
partitioned between DCM (30 mL) and aqueous NaHCO.sub.3 (5 g in 30
mL). The aqueous layer was extracted with DCM (3.times.30 mL),
dried (MgSO.sub.4) and the solvent removed in vacuo to give
morpholin-3-ylmethyl 4-(4-fluorophenyl)piperazine-1-carboxylate
(2.18 g, 96%) as a white solid.
[0279] Analytical HPLC: purity 98.3% (System A, R.sub.T=3.88 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.27 min),
ES.sup.+: 324.0 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1661.
Example 27
(3S)-Morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00051##
[0281] NaH (60% in oil, prewashed with hexane; 2.76 g, 69.0 mmol)
was suspended in anhydrous THF (100 mL) under nitrogen and the
reaction mixture was cooled to 0.degree. C. with stirring. A
solution of (S)-3-hydroxymethyl-morpholine-4-carboxylic acid
tert-butyl ester (5.00 g, 23.0 mmol) in THF (100 mL) was added
dropwise. The reaction mixture was stirred for 30 minutes at
0.degree. C. 4-(4-Fluorophenyl)-piperazine-1-carboxylic acid
4-nitrophenyl ester (Intermediate 6; 10.0 g, 29.0 mmol) was added
and the reaction mixture was stirred overnight at room temperature
before standing for 24 h. The reaction mixture was cooled to
5-10.degree. C. A 10% aqueous solution of NaHCO.sub.3 (22 mL) was
added and the solvents were removed in vacuo. The residue was
partitioned between EtOAc (700 mL) and 10% aqueous solution of
NaHCO.sub.3 (200 mL)/water (400 mL). The EtOAc layer was dried
(MgSO.sub.4) and the solvent removed in vacuo. The residue was
purified by column chromatography (normal phase silica, 100 g
Isolute-Si column, gradient of EtOAc in hexane). The residue was
dissolved in DCM and evaporated in vacuo and repeated before drying
in vacuo to give
(3S)-3-[4-(4-fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4-c-
arboxylic acid tert-butyl ester (8.19 g, 84%) as an orange gum.
[0282]
(3S)-3-[4-(4-Fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholi-
ne-4-carboxylic acid tert-butyl ester (8.19 g, 19.3 mmol) was
stirred in DCM (96 mL) at -5 to 0.degree. C. TFA (28.9 mL)
dissolved in DCM (67 mL) was added dropwise and the cooling
maintained for 30 minutes. The reaction mixture was stirred for 20
h at room temperature. The reaction mixture was diluted with
heptane (200 mL) and a red-brown gum separated. The gum was dried
in vacuo and then washed with heptane (200 mL). The residue was
partitioned between DCM (450 mL) and 1.2M aqueous NaHCO.sub.3 (135
mL). The aqueous layer was extracted with DCM (2.times.225 mL). The
combined DCM extracts were dried (Na.sub.2SO.sub.4) and the solvent
removed in vacuo to give (3S)-morpholin-3-ylmethyl
4-(4-fluorophenyl)-piperazine-1-carboxylate (5.83 g, 93%) as a
beige solid.
[0283] Analytical HPLC: purity 99.5% (System A, R.sub.T=3.86 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.82 min),
ES.sup.+: 324.7 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1652.
Example 28
(3R)-Morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00052##
[0285] NaH (60% in oil, prewashed with hexane; 1.38 g, 34.5 mmol)
was suspended in anhydrous THF (50 mL) under nitrogen, the reaction
mixture cooled to -5.degree. C. with stirring and a solution of
(R)-3-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
(2.50 g, 11.5 mmol) in THF (50 mL) was added dropwise. The reaction
mixture was stirred for 30 min at 0 to 3.degree. C.
4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl ester
(Intermediate 6; 5.01 g, 14.5 mmol) was added and the reaction
mixture was stirred overnight at room temperature. The reaction
mixture was cooled with an ice bath and a 10% aq NaHCO.sub.3
solution (11 mL) was added. The solvents were removed in vacuo and
the residue was partitioned between EtOAc (350 mL) and 10% aq
NaHCO.sub.3 solution (100 mL)/water (200 mL). The EtOAc layer was
dried (MgSO.sub.4) and the solvent removed in vacuo. The residue
was purified by column chromatography (normal phase silica, 70 g
Isolute-Si column, gradient of EtOAc in hexane). The residue was
dissolved in DCM and evaporated in vacuo and repeated once before
drying in vacuo to give
(3R)-3-[4-(4-fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4-c-
arboxylic acid tert-butyl ester (2.80 g) as a pale yellow gum.
[0286] This procedure was repeated on a further batch of
(R)-3-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
(2.50 g, 11.5 mmol). After repurification of the impure fractions
from both batches, 8.17 g (83.9%) of
(3R)-3-[4-(4-fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4-c-
arboxylic acid tert-butyl ester was obtained.
[0287]
(3R)-3-[4-(4-Fluorophenyl)-piperazine-1-carbonyloxymethyl]-morpholi-
ne-4-carboxylic acid tert-butyl ester (5.07 g, 12.0 mmol) was
stirred in dry dioxane (35 mL) at room temperature and HCl in
dioxane (4M; 15 mL, 60 mmol) was added dropwise. The reaction
mixture was stirred for 24 h at room temperature. A further portion
of HCl in dioxane (4M; 10 mL, 40 mmol) was added and stirred
overnight. The dioxane was decanted and the residual gum was washed
with dioxane (.times.3) and Et.sub.2O (.times.2). The residue was
partitioned between DCM (300 mL) and 1.2M aqueous NaHCO.sub.3. The
aqueous layer was extracted with DCM (2.times.200 mL). The combined
DCM extracts were dried (Na.sub.2SO.sub.4) and the solvent removed
in vacuo. The residue was dissolved in DCM and dried in vacuo to
give (3R)-morpholin-3-ylmethyl
4-(4-fluorophenyl)piperazine-1-carboxylate (3.87 g, quant.) as an
off white solid.
[0288] Analytical HPLC: purity 100% (System A, R.sub.T=3.87 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.82 min),
ES.sup.+: 324.7 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.22FN.sub.3O.sub.3: 323.1645, found 323.1657.
Example 29
(4-Methylmorpholin-3-yl)methyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00053##
[0290] LiAlH.sub.4 (4.2 g, 110 mmol) was suspended in anhydrous THF
(60 mL) under nitrogen, cooled to -10.degree. C. with stirring and
a solution of 3-hydroxymethyl-morpholine-4-carboxylic acid
tert-butyl ester (Intermediate 7; 4.1 g, 18.4 mmol) in THF (50 mL)
was added dropwise. The reaction mixture was stirred for 20 minutes
at 0.degree. C., heated to reflux for 3 h and then stirred at room
temperature overnight. The reaction mixture was cooled to
-10.degree. C. and quenched with the dropwise addition of 10% water
in THF. The reaction mixture was diluted with THF (50 mL), stirred
for 1 h at room temperature, filtered and the filtrate was
concentrated in vacuo. The residue was dissolved in DCM, dried
(MgSO.sub.4) and the solvent removed in vacuo to give
(4-methyl-morpholin-3-yl)-methanol (2.05 g, 85%) as a colourless
liquid.
[0291] NaH (60% in oil, prewashed with hexane; 0.91 g, 22.9 mmol)
was suspended in anhydrous THF (25 mL) under nitrogen and cooled to
0.degree. C. with stirring. A solution of
(4-methyl-morpholin-3-yl)-methanol (1.00 g, 2.62 mmol) in THF (25
mL) was added dropwise. 4-(4-Fluorophenyl)-piperazine-1-carboxylic
acid 4-nitrophenyl ester (Intermediate 6; 3.16 g, 9.15 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. A saturated aqueous solution of NaHCO.sub.3 (10 mL)
was added and the solvents were removed in vacuo. The residue was
partitioned between water and EtOAc and the aqueous phase was
extracted with EtOAc. The combined organic extracts were washed
with sat aq NaHCO.sub.3 (3.times.100 mL), dried (MgSO.sub.4) and
the solvents were removed in vacuo. The residue was purified by
flash normal column chromatography (Apollo silica, 40-63 .mu.m,
60A) eluting with 1% MeOH in DCM to give
(4-methylmorpholin-3-yl)methyl
4-(4-fluorophenyl)piperazine-1-carboxylate (2.15 g, 83%) as a white
solid.
[0292] Analytical HPLC: purity 100% (System A, R.sub.T=3.99 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.38 min),
ES.sup.+: 338.0 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.24FN.sub.3O.sub.3: 337.1802, found 337.1811.
Example 30
[0293] Morpholin-3-ylmethyl 4-phenylpiperazine-1-carboxylate
##STR00054##
[0294] NaH (60% in oil, prewashed with hexane; 1.23 g, 30.1 mmol)
was suspended in anhydrous THF (50 mL) under nitrogen and cooled to
0.degree. C. with stirring. A solution of
3-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
(Intermediate 7; 2.23 g, 10.3 mmol) in THF (50 mL) was added
dropwise. The reaction mixture was stirred for 20 min at 0.degree.
C. 4-Nitrophenyl 4-phenylpiperazine-1-carboxylate (Intermediate 2;
4.03 g, 12.3 mmol) was added and the reaction mixture was stirred
for 48 h at room temperature. A sat aq NaHCO.sub.3 solution (10 mL)
was added and the solvents were removed in vacuo. The residue was
partitioned between water and EtOAc, the aqueous phase was
extracted with EtOAc (2.times.100 mL). The combined organic
extracts were washed with sat aq NaHCO.sub.3 (3.times.100 mL),
dried (MgSO.sub.4) and the solvents were removed in vacuo. The
residue was purified by flash normal column chromatography (Apollo
silica, 40-63 .mu.m, 60A) eluting with 1% MeOH in DCM to give
3-(4-phenyl-piperazine-1-carbonyloxymethyl)-morpholine-4-carboxylic
acid tert-butyl ester (3.35 g, 80%).
[0295]
3-(4-Phenyl-piperazine-1-carbonyloxymethyl)-morpholine-4-carboxylic
acid tert-butyl ester (3.31 g, 8.16 mmol) was stirred in DCM (30
mL) at 0.degree. C. TFA (6 mL) dissolved in DCM (24 mL) was added
dropwise. The reaction mixture was stirred overnight at room
temperature. The solvents were removed in vacuo and the residue was
partitioned between DCM (30 mL) and aq NaHCO.sub.3 (5 g in 30 mL).
The aqueous layer was extracted with DCM (3.times.30 mL), dried
(MgSO.sub.4) and the solvents were removed in vacuo to give
morpholin-3-ylmethyl 4-phenylpiperazine-1-carboxylate (2.55 g,
quant.) as a white solid.
[0296] Analytical HPLC: purity 100% (System A, R.sub.T=3.60 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.97 min),
ES.sup.+: 306.0 [MH] HRMS calcd for C.sub.16H.sub.23N.sub.3O.sub.3:
305.1739, found 305.1741.
Example 31
[0297] Morpholin-3-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00055##
[0298] 3-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 7; 250 mg, 0.76 mmol) in THF (5 mL) was added
dropwise to a suspension of NaH (60% dispersion in oil, prewashed
with hexane; 138 mg, 3.45 mmol) in anhydrous THF (5 mL) at
0.degree. C. and stirred under nitrogen for 35 minutes.
4-(2,4-Difluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester (Intermediate 12; 501 mg, 1.38 mmol) was added and the
reaction mixture was stirred at room temperature overnight. The
reaction mixture was quenched with the addition of aqueous
NaHCO.sub.3 (0.5 mL) at 0.degree. C. and the solvents removed in
vacuo. The residue was suspended between aq Na.sub.2CO.sub.3 (20
mL) and EtOAc (20 mL). The aqueous phase was extracted with EtOAc
(2.times.20 mL). The combined organic layers were washed with aq
Na.sub.2CO.sub.3 (20 mL), dried (MgSO.sub.4) and the solvent
removed in vacuo. The residue was purified by column chromatography
(normal phase, Apollo silica, 1% MeOH in DCM) and the solvents were
removed in vacuo to give
3-[4-(2,4-difluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morpholine-4-c-
arboxylic acid tent-butyl ester (170 mg, 34%).
[0299] To
3-[4-(2,4-difluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morph-
oline-4-carboxylic acid tert-butyl ester (170 mg, 0.39 mmol) in DCM
(5 mL) at 0.degree. C. was added TFA (1 mL in 5 mL DCM). The
reaction mixture was allowed to warm to room temperature and
stirred overnight. The solvent was removed in vacuo and the residue
suspended between sat aq NaHCO.sub.3 (10 mL) and DCM (10 mL). The
aqueous phase was extracted with DCM (2.times.10 mL). The combined
organic layers were dried (MgSO.sub.4) and the solvent removed in
vacuo to give morpholin-3-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate (104 mg, 79%) as a
colourless oil.
[0300] Analytical HPLC: purity 98.7% (System B, R.sub.T=8.29 min);
Analytical LCMS: purity 100% (System B, R.sub.T=8.09 min),
ES.sup.+: 342.6 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.21F.sub.2N.sub.3O.sub.3: 341.1551, found
341.1561.
Example 32
(4-Methylmorpholin-3-yl)methyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00056##
[0302] (4-Methyl-morpholin-3-yl)-methanol (387 mg, 2.95 mmol) in
THF (7.5 mL) was added dropwise to a suspension of NaH (60%
dispersion in oil, prewashed with hexane; 354 mg, 8.85 mmol) in
anhydrous THF (7.5 mL) at 0.degree. C. and stirred under nitrogen
for 40 minutes. 4-(2,4-Difluorophenyl)-piperazine-1-carboxylic acid
4-nitrophenyl ester (Intermediate 12; 1.28 g, 3.54 mmol) was added
and the reaction mixture was stirred at room temperature overnight.
The reaction mixture was quenched with the addition of aqueous
NaHCO.sub.3 (0.5 mL) at 0.degree. C. The reaction mixture was
stirred for 1 h and then the solvents were removed in vacuo. The
residue was suspended between water and EtOAc. The aqueous phase
was extracted with EtOAc (.times.3). The combined organic layers
were washed with aqueous Na.sub.2CO.sub.3, dried (MgSO.sub.4) and
the solvent removed in vacuo. The residue was purified by column
chromatography (normal phase, Apollo silica, 1% MeOH in DCM) and
the solvents were removed in vacuo to give
(4-methylmorpholin-3-yl)methyl
difluorophenyl)piperazine-1-carboxylate (197 mg, 19%) as a
colourless oil.
[0303] Analytical HPLC: purity 99.3% (System B, R.sub.T=8.45 min);
Analytical LCMS: purity 100% (System A, R.sub.T=4.61 min),
ES.sup.+: 356.5 [MH].sup.+; HRMS calcd for
C.sub.17H.sub.23F.sub.2N.sub.3O.sub.3: 355.1707, found
355.1724.
Example 33
(2S)-Morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00057##
[0305] (S)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 8; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(2 mL) and stirred under nitrogen for 30 min.
4-(2,4-Difluoro-phenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester (Intermediate 12; 191 mg, 0.55 mmol) was added and the
reaction mixture was stirred at room temperature for 24 h. The
reaction mixture was filtered through celite and the solid washed
with THF (10 mL). The filtrates were combined and the solvent
removed in vacuo. The residue was purified by column chromatography
(normal phase silica, 5 g Isolute-Si column, gradient 10% to 50%
EtOAc in hexane) to give
(2S)-[4-(2,4-difluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morpholine--
4-carboxylic acid tert-butyl ester (210 mg, quant.) as an oil.
[0306]
(2S)-[4-(2,4-Difluoro-phenyl)-piperazine-1-carbonyloxymethyl]morpho-
line-4-carboxylic acid tert-butyl ester (205 mg, 0.46 mmol) was
dissolved in DCM (2 mL) and TFA (3 mL, 10% in DCM) was added. The
reaction mixture was stirred at room temperature overnight. The
solvents were removed in vacuo. Heptane was added and the solvent
was then removed in vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol) was
added to a solution of the residue in DCM (2 mL) and stirred for 1
h. The mixture was filtered and the filtrate dried
(Na.sub.2SO.sub.4) and solvents removed in vacuo. The residue was
purified by column chromatography (normal phase, 5 g, Isolute-Si,
EtOAc in hexane (1:1) followed by 1-10% MeOH in EtOAc). The residue
was dried in vacuo to give (2S)-morpholin-2-ylmethyl
difluorophenyl)piperazine-1-carboxylate (104 mg, 69%) as a white
solid.
[0307] Analytical HPLC: purity 99.8% (System B, R.sub.T=8.40 min);
Analytical LCMS: purity 100% (System B, R.sub.T=8.15 min),
ES.sup.+: 342.6 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.21F.sub.2N.sub.3O.sub.3: 341.1551, found
341.1559.
Example 34
(2R)-Morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate
##STR00058##
[0309] (R)-2-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl
ester (Intermediate 9; 100 mg, 0.46 mmol) was added to a suspension
of NaH (60% dispersion in oil; 55.0 mg, 1.38 mmol) in anhydrous THF
(2 mL) and stirred under nitrogen for 30 min.
4-(2,4-difluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl
ester (Intermediate 12; 191 mg, 0.55 mmol) was added and the
reaction mixture was stirred at room temperature for 24 h. The
reaction mixture was filtered through celite and the solid washed
with THF (10 mL). The filtrates were combined and the solvent
removed in vacuo. The residue was purified by column chromatography
(normal phase silica, 5 g Isolute-Si column, gradient 10% to 50%
EtOAc in hexane) to give
(2R)-[4-(2,4-difluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morpholine--
4-carboxylic acid tert-butyl ester (176 mg, 86%).
[0310]
(2R)-[4-(2,4-difluoro-phenyl)-piperazine-1-carbonyloxymethyl]-morph-
oline-4-carboxylic acid tert-butyl ester (176 mg, 0.40 mmol) was
dissolved in DCM (2 mL) and TFA (3 mL, 10% in DCM) was added. The
reaction mixture was stirred at room temperature overnight. The
solvents were removed in vacuo. Heptane was added and then the
solvent removed in vacuo. K.sub.2CO.sub.3 (500 mg, 3.6 mmol) was
added to a solution of the residue in DCM (2 mL) and stirred for 30
min before adding water (0.3 mL) and stirred for 1 h. The mixture
was filtered and the filtrate dried (Na.sub.2SO.sub.4) and solvents
removed in vacuo. The residue was purified by column chromatography
(normal phase, 5 g Isolute-Si column, EtOAc in hexane (1:1)
followed by 1-10% MeOH in EtOAc). The residue was dried in vacuo to
give (2R)-morpholin-2-ylmethyl
4-(2,4-difluorophenyl)piperazine-1-carboxylate (89 mg, 65%) as a
white solid.
[0311] Analytical HPLC: purity 99.8% (System B, R.sub.T=8.38 min);
Analytical LCMS: purity 100% (System B, R.sub.T=8.23 min),
ES.sup.+: 342.6 [MH].sup.+; HRMS calcd for
C.sub.16H.sub.21F.sub.2N.sub.3O.sub.3: 341.1551, found
341.1561.
Example 35
[(2R)-1,4-Dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate
##STR00059##
[0313] NaH (60% dispersion in mineral oil; 0.45 g, 11.3 mmol) was
suspended in anhydrous THF (20 mL) under nitrogen and the reaction
mixture was cooled to 0.degree. C. with stirring. A solution of
1,4-dimethyl-(R)-2-hydroxymethyl piperazine (Intermediate 4; 0.50
g, 3.47 mmol) in THF (20 mL) was added dropwise. The reaction
mixture was stirred for 10 minutes at 0.degree. C.
4-(4-Fluorophenyl)-piperazine-1-carboxylic acid 4-nitrophenyl ester
(Intermediate 6; 1.50 g, 4.34 mmol) was added and the reaction
mixture was stirred overnight at room temperature. The reaction
mixture was quenched with sat. aq. NaHCO.sub.3 (40 mL) and the THF
was removed in vacuo. The aqueous phase was extracted with EtOAc
(100 mL). The organic layer was washed with sat. aq. NaHCO.sub.3
(5.times.100 mL), dried (MgSO.sub.4) and the solvent removed in
vacuo. The residue was purified by reverse phase column
chromatography (LiChroprep RP-18, 40-63 .mu.m, 460.times.26 mm (100
g), 30 mL/min, gradient 0% to 30% (over 75 min) to 100% (over 13
min) MeOH in water with 1% formic acid). The residue was de-salted
using K.sub.2CO.sub.3 in DCM to give
[(2R)-1,4-dimethylpiperazin-2-yl]methyl
4-(4-fluorophenyl)piperazine-1-carboxylate (0.42 g, 35%) as a
colourless gum.
[0314] Analytical HPLC: purity 100% (System A, R.sub.T=3.88 min);
Analytical LCMS: purity 100% (System A, R.sub.T=3.58 min),
ES.sup.+: 351.2 [MH].sup.+; HRMS calcd for
C.sub.18H.sub.27FN.sub.4O.sub.2: 350.2118, found 350.2125.
Biological Methods
Animal Model of Human Obesity (Dietary-Induced Obese Rat)
[0315] Rodent models of obesity are valuable tools for studying the
underlying factors that contribute to the initiation and
maintenance of the obese state in humans. The model of diet-induced
obesity (DIO) in rodents is particularly suited to this task as DIO
rats share a number of traits with human obesity.
[0316] These include polygenic inheritance, insulin resistance,
hyperleptinemia, lowered growth hormone secretion, proclivity to
preferentially oxidize carbohydrate over fat, and the ability to
decrease metabolic rate when calorie-restricted, leading to weight
regain after restriction. In outbred rats fed a high energy diet,
about one-half develop DIO, while the rest are resistant to obesity
and gain no more weight than chow-fed controls (diet resistant,
DR). The model of diet-induced obesity (DIO) is of special interest
with regard to regulation of energy homeostasis. When fed a diet
moderately high in fat, sucrose, and energy content (HE diet),
about one-half of the rats will put on substantially more weight
than the others (DIO vs. DR).
[0317] Rats predisposed to develop DIO will gain weight at rates
comparable to rats fed a low-energy (chow) diet and will not become
obese unless fed an HE diet. However, once the DIO and DR
phenotypes are established on a HE diet, the resulting weight gains
and body composition changes persist, even when animals are
switched back to a normal chow diet. Changes in body weight and
composition, which occur during the development and perpetuation of
the DIO and DR phenotypes, are associated with several alterations
in brain function that may underlie these adjustments
DIO Protocol
[0318] The diet-induced obesity protocol as described by Widdowson,
P. S. et al. (Diabetes (1997) 46:1782-1785) was followed for
selection of obese-prone animals. Wistar male rats (.about.200-250
g at start of modified dietary intervention) are put on a
high-carbohydrate (HE) diet for 8-10 weeks. The composition of the
diet is 33% (w/v) powdered chow (RM1), 33% (w/v) condensed milk
(Nestle), 7% (w/v) Castor sugar (Tate & Lyle), and 27% (w/v)
water. Body weights are recorded and following an 8-week period,
animals are separated in 2 groups according to their weight. As in
any outbred strain of animals (rodents, primates) a population will
naturally separate in two groups: individuals prone to obesity
(putting on more weight) or obesity-resistant (putting on less
weight). The obese animals weigh up to 60 g more after 6 weeks.
Obese-prone animals are kept to perform studies on the effect on
body weight and food intake of compounds of formula (I). FIG. 1
shows an example of body weight separation between animals fed on
the highly palatable diet (high carbohydrate).
In Vivo Experiments on the Effect of the Compounds on Body
Weight
[0319] Obese-prone animals are treated with a compound of formula
(I) and the effect on their body weight is measured. The compounds
are dosed bid at 10 mg/kg PO, with a dose-volume of 1 mL/kg or an
equivalent vehicle dose (saline) for comparison. The doses are
administered AM (09:00) and PM (16:00) and the body weight is
measured in the morning before dosing. There are typically 8
animals per group. FIGS. 2 to 4 show the cumulative body weight
change (%) observed in a 4 day study in DIO rats for Examples 7, 20
and 30, respectively.
Leptin Assay in Non-Recombinant System
[0320] Although well-characterised in recombinant systems (e.g.
ObRb-transfected HEK293 cells), where leptin elicits a very marked
increase in STAT3 phosphorylation, these systems have often failed
to provide an accurate measure of activity of a test compound
towards the leptin receptor. It seems that overexpression of the
receptor (as well as the possibility for different drugs to act on
different parts of the signaling pathway triggered by leptin
association with its receptor) results in most cases in the absence
of activity of the drugs tested.
[0321] The leptin receptor expression in non-recombinant system is
often fluctuating and care must be given to identify a system where
signal stability remains within experiments. Using such a system,
leptin receptor antagonist mimetics could be identified by
evaluating their action vs. leptin (see below).
[0322] Leptin is produced chiefly in adipose cells, but in humans,
mRNA encoding leptin is also present in the placenta. Here, leptin
might play an important proliferative role in the microvasculature.
The possibility to use this hypothesis in a native cell line was
evaluated.
JEG-3 Protocol
[0323] In JEG-3 cells (choriocarcinoma cell line) leptin is able to
stimulate proliferation up to 3 fold (Biol. Reprod. (2007) 76:
203-10). Leptin also causes a concentration-dependent increase in
[.sup.3H]-thymidine incorporation in JEG-3 cells (FIG. 5, maximal
effect at 100 nM (EC.sub.50=2.1 nM)). The radioactivity
incorporated by the cells is an index of their proliferative
activity and is measured in counts per minute (CPM) with a liquid
scintillation beta counter.
[0324] This finding can be applied to test whether a compound is
able to either reproduce the effect of leptin on cell proliferation
(leptin receptor agonist mimetic) (i.e., a given compound will
cause an increase in incorporated [.sup.3H]-Thymidine by the cells)
or to inhibit the effect of leptin (antagonistic effect) by
preventing the leptin-mediated increase in [.sup.3H]-thymidine
incorporation.
[0325] This approach has the advantage of using a non-recombinant
system and has reasonable reproducibility and robustness.
Measurement of Brain Penetration
[0326] The test species (rodent) is given a bolus dose of the
substrate under investigation, usually via intravenous (IV) or oral
(PO) routes. At appropriate time points, blood samples are taken
and the resultant plasma extracted and analysed for substrate
concentration and, where appropriate, metabolite concentration. At
similar time points, animals from another group are sacrificed,
brains isolated and the brain surface cleaned. Brain samples are
then homogenised, extracted and analysed for substrate
concentration and, where appropriate, metabolite concentration.
Alternatively, microdialysis probes are implanted into one or more
brain regions of the test species and samples collected at
appropriate time points for subsequent analysis. This method has
the advantage of measuring only extra-cellular substrate
concentration. Plasma and brain concentrations are then compared
and ratios calculated, either by comparison of averaged
concentrations at individual time points, or by calculation of the
area-under-the-curve (AUC) of the concentration-time plots.
* * * * *