Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 71
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NOTE: For additional volumes, please contact the Canadian Patent Office
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NOTE POUR LE TOME / VOLUME NOTE:
CA 02607566 2007-11-05
WO 2006/121860 PCT/US2006/017411
GLUCAGON-LIKE PEPTIDE 1 (GLP-1) RECEPTOR AGONISTS AND THEIR
PHARMACOLOGICAL METHODS OF USE
[001] This application claims benefit of U.S. Provisional Application Serial
No. 60/678,723; filed
on May 6, 2005, the contents of which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[002] This invention relates to novel modifications that provide suitable
derivatization sites to
improve the pharmacokinetic properties of the peptides. Such N-terminal
modifications at the main
chain amino group of the first amino acid residue or C-terminal modifications
at the main chain
carboxylate group of the last amino acid residue may include aliphatics, C3 to
C7 cycloalkyl, aryl,
or mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen,
and/or sulfur
heteroatoms. In addition, the N-terminal or C-terminal modifications may
provide suitable
derivatization sites (exemplified, but not limited to, amino and thiol
groups). The modified peptides
of the present invention are useful in stimulating the release of insulin from
pancreatic R-cells in a
glucose-dependent manner, thereby providing a treatment option for those
individuals afflicted with
metabolic disorders such as diabetes or impaired glucose tolerance, a
prediabetic state.
BACKGROUND OF THE INVENTION
[003] Diabetes is characterized by impaired glucose metabolism manifesting
itself, among other
things, by an elevated blood glucose level in the diabetic patient. Underlying
defects lead to a
classification of diabetes into two major groups: type 1 diabetes, or insulin
dependent diabetes
mellitus (IDDM), which arises when patients lack (3-cells producing insulin in
their pancreatic islets
of Langerhans; and type 2 diabetes, or non-insulin dependent diabetes mellitus
(NIDDM), which
occurs in patients with an impaired R-cell function and alterations in insulin
action.
[004] Type 1 diabetic patients are currently treated with insulin, while the
majority of type 2
diabetic patients are treated with agents that stimulate R-cell function or
with agents that enhance
the tissue sensitivity of the patients towards insulin. Over time almost one-
half of type 2 diabetic
subjects lose their response to these agents and then must be placed on
insulin therapy. The
drugs presently used to treat type 2 diabetes are described below.
[005] Alpha-glucosidase inhibitors (e.g., Precose @, VogliboseTM, and
Miglitol@) reduce the
excursion of postprandial glucose by delaying the absorption of glucose from
the gut. These drugs
are safe and provide treatment for mild to moderately affected diabetic
subjects. However,
gastrointestinal side effects have been reported in the literature.
[006] Insulin sensitizers are drugs that enhance the body's response to
insulin.
Thiozolidinediones such as AvandiaTM (rosiglitazone) and ActosTM
(pioglitazone) activate the
CA 02607566 2007-11-05
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peroxisome proliferator-activated receptor (PPAR) gamma subtype and modulate
the activity of a
set of genes that have not been well described. RezulinTM (troglitazone), the
first drug in this class,
was withdrawn because elevated liver enzyme levels and drug induced
hepatotoxicity. These
hepatic effects do not appear to be a significant problem in patients using
AvandiaTM and ACtOSTM.
Even so, liver enzyme testing is recommended every 2 months in the first year
of therapy and
periodically thereafter. AvandiaTM and ActosTM seem to be associated with
fluid retention and
edema. Anotherpotential side effect is weight gain. AvandiaTM is not indicated
for use with insulin
because of concern about congestive heart failure.
[007] Insulin secretagogues (e.g., sulfonylureas (SFUs) and other agents that
act by the ATP-
dependent K+ channel) are another drug type presently used to treat type 2
diabetes. SFUs are
standard therapy for type 2 diabetics that have mild to moderate fasting
hyperglycemia. The SFUs
have limitations that include a potential for inducing hypoglycemia, weight
gain, and high primary
and secondary failure rates. Ten to 20% of initially treated patients fail to
show a significant
treatment effect (primary failure). Secondary failure is demonstrated by an
additional 20-30% loss
of treatment effect after six months on an SFU. Insulin treatment is required
in 50% of the SFU
responders after 5-7 years of therapy (Scheen, et al., Diabetes Res. Clin.
Pract. 6:533-543, 1989).
[008] GlucophageTM (metformin HCI) is a biguanide that lowers blood glucose by
decreasing
hepatic glucose output and increasing peripheral glucose uptake and
utilization. The drug is
effective at lowering blood glucose in mildly and moderately affected subjects
and does not have
the side effects of weight gain or the potential to induce hypoglycemia.
However, GlucophageTM
has a number of side effects including gastrointestinal disturbances and the
potential for lactic
acidosis. GlucophageTM is contraindicated in diabetics over the age of 70 and
in subjects with
impairment in renal or liver function. Finally, GlucophageTM has primary and
secondary failure
rates similar to the SFUs.
[009] Insulin treatment is instituted after diet, exercise, and oral
medications have failed to
adequately control blood glucose. This treatment has the drawbacks that it is
an injectable, that it
can produce hypoglycemia, and that it causes weight gain.
[010] Because of the problems with current treatments, new therapies to treat
type 2 diabetes
are-needed: In-partieular, newtreatments -to retain normal (glucose-dependent)
insulin secretion
are needed. Such new drugs should have the following characteristics:
dependent on glucose for
promoting insulin secretion (i.e., produce insulin secretion only in the
presence of elevated blood
glucose); low primary and secondary failure rates; and preservation of islet
cell function. The
strategy to develop the new therapy disclosed herein is based on the cyclic
adenosine
monophosphate (cAMP) signaling mechanism and its effects on insulin secretion.
[011] Cyclic AMP is a major regulator of the insulin secretion process.
Elevation of this signaling
molecule promotes the closure of the K+ channels following the activation of
protein kinase A
pathway. Closure of the K+ channels causes cell depolarization and subsequent
opening of Ca++
channels, which in turn leads to exocytosis of insulin granules. Little if any
effects on insulin
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secretion occurs in the absence of low glucose concentrations (Weinhaus, et
al., Diabetes
47:1426-1435, 1998). Secretagogues like PACAP (pituitary adenylate cyclase
activating peptide),
VIP (vasoactive intestinal peptide), GIP (glucose-dependent insulinotropic
polypeptide), and GLP-1
(glucagon-like peptide 1) use the cAMP system to regulate insulin secretion in
a glucose-
dependent fashion (Komatsu, et al., Diabetes 46:1928-1938, 1997; Filipsson, et
al., Diabetes
50:1959-1969, 2001; Drucker, Endocrinology 142:521-527, 2001). Insulin
secretagogues working
throughthe-elevation of_ cAMP such as GLP-1, VIP, GIP, and PACAP are also.able
to enhance
insulin synthesis in addition to insulin release (Skoglund, et al., Diabetes
49:1156-1164, 2000;
Borboni, et al., Endocrinology 140:5530-5537, 1999).
[012] GLP-1 is released from the intestinal L-cell after a meal and functions
as an incretin
hormone (i.e., it potentiates glucose-induced insulin release from the
pancreatic (3-cell). It is a 37-
amino acid peptide that is differentially expressed by the glucagon gene,
depending upon tissue
type. The clinical data that support the beneficial effect of raising cAMP
levels in F3-cells have
been collected with GLP-1. Infusions of GLP-1 in poorly controlled type 2
diabetics normalized
their fasting blood glucose levels (Gutniak, et al., New Eng. J. Med. 326:1316-
1322, 1992) and
with longer infusions improved the R-cell function to those of normal subjects
(Rachman, et al.,
Diabetes 45:1524-1530, 1996). A recent report has shown that GLP-1 improves
the ability of R-
cells to respond to glucose in subjects with impaired glucose tolerance
(Byrne, et al., Diabetes
47:1259-1265, 1998). All of these effects, however, are short-lived because of
the short half-life of
the peptide.
[013] Amylin Pharmaceuticals is conducting Phase III trials with Exendin-4
(AC2993), a 39
amino acid peptide originally identified in Gila Monster. Amylin has reported
that clinical studies
demonstrated improved glycemic control in type 2 diabetic patients treated
with Exendin-4.
However, the incidence of nausea and vomiting was significant.
[014] Glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue gut
peptide involved in
the regulation of fat and glucose metabolism, with the insulinotropic function
localized to residues
1-30. GIP is degraded by dipeptidyl peptidase IV (DPPIV) proteolysis and
eliminated by renal
clearance. Limited clinical data have been collected with GIP. Intravenous
(IV) administration or
continuous in type 2 diabetics caused an aute increase in plasma insulin
levels (Kindmark, et al., J.
Clin. Endocrinol. Metab. 86:2015-2019, 2001; Meier,et al., Diabetes 53 (Suppl
3):S220-S224,
2004). These effects, however, are short-lived because of the short half-life
of the peptide.
[015] PACAP is a potent stimulator of glucose-dependent insulin secretion from
pancreatic R-
cells. Three different PACAP receptor types (PAC1, VPAC1, and VPAC2) have been
described
(Harmar, et al., Pharmacol. Reviews 50:265-270, 1998; Vaudry, et al.,
Pharmacol. Reviews
52:269-324, 2000). PACAP displays no receptor selectivity, having comparable
activities and
potencies at all three receptors. PAC1 is located predominately in the CNS,
whereas VPAC1 and
VPAC2 are more widely distributed. VPAC1 is located in the CNS as well as in
liver, lungs, and
intestine. VPAC2 is located in the CNS, pancreas, skeletal muscle, heart,
kidney, adipose tissue,
3
CA 02607566 2007-11-05
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testis, and stomach. Recent work argues that VPAC2 is responsible for the
insulin secretion from
E3-cells (Inagaki, et al., Proc. Nati. Acad. Sci. USA 91:2679-2683, 1994;
Tsutsumi, et al., Diabetes
51:1453-1460, 2002). This insulinotropic action of PACAP is mediated by the
GTP binding protein
Gs. Accumulation of intracellular cAMP in turn activates the nonselective
cation channels in R-
cells increasing [Ca++], and promotes exocytosis of insulin-containing
secretory granules.
[016] GLP-1 is a member of a family of structurally related peptide hormones,
the
glucagon/secretin'family. 'Within this family, GLP-1 (7-36) and GLP-1 (7-37)
(30 amino acids and 31
amino acids, respectively) originate from the same precursor, preproglucagon,
as does glucagon
(30 amino acids). Due to tissue-specific processing, preproglucagon is
converted into GLP-1
predominantly in the intestine and glucagon in the pancreas. The GLP-1
receptor belongs to the
family of G-protein coupled receptors.
[017] GLP-1 lowers plasma glucose concentrations mediated by glucose dependent
insulin
secretion. Given the important role of GLP-1 in maintaining normal blood
glucose concentrations,
there has been considerable interest in the identification of GLP-1 receptor
agonists. Clinical
studies have demonstrated the ability of GLP-1 infusion to promote insulin
secretion and to
normalize plasma glucose in diabetic subjects. However, GLP-1 is rapidly
degraded and has a
very short half-life in the body. Furthermore, GLP-1 causes gut motility side
effects at or near its
therapeutic doses. Therefore, GLP-1 itself has significant limitations as a
therapeutic agent, and
modified versions of the peptide with enhanced stability are being pursued.
Non-peptide agonists
of the GLP-1 receptor have not been described to date.
[018] Recent studies have demonstrated diverse biological effects of GLP-1.
GLP-1 causes
glucose-dependent insulin secretion from the pancreas (Holst, Curr. Opin.
Edocrinol. Diabetes
12:56-62, 2005). GLP-1 also enhances satiety and reduces food intake (Flint,
et al., J. Clin. Invest.
101:515-520, 1998). In addition, GLP-1 protects ischemic and reperfused
myocardium (Nikolaidis,
et al., Circ. 109:962-965, 2004), as well as improving endothelial dysfunction
(Nystrom, Am. J.
Physiol. Endocrinol. Metab., 287:E1209-E1215). GLP-1 has also been associated
with improved
learning and displayed neuroprotective effects (During, et al., Nat. Med.
9:1173-1179, 2003).
[019] Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide that was
first isolated from
hog upper small intestine (Said and Mutt, Science 169:1217-1218, 1970; U.S.
Patent No.
3,879,371). This peptide belongs to a family of structurally-related, small
polypeptides that
includes helodermin, secretin, the somatostatins, and glucagon. The biological
effects of VIP are
mediated by the activation of membrane-bound receptor proteins that are
coupled to the
intracellular cAMP signaling system. These receptors were originally known as
VIP-R1 and VIP-
R2, however, they were later found to be the same receptors as VPAC1 and
VPAC2. VIP displays
comparable activities and potencies at VPAC1 and VPAC2.
[020] To improve the stability of GLP-1, efforts have focused on modifying the
sequence of the
native peptide to improve half-life and exposure, or using non-human proteins
(Exendin-4) that are
GLP-1 receptor agonists. These products include Exenatide, a peptide from the
saliva of the Gila
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monster, that is currently in a Phase III clinical trials as a twice daily
injectable. Another GLP-1
analogue is Liraglutide (NN-2211), an acylated form of native GLP-1 currently
in Phase II as a
once daily injection. Other reported modifications include conjugation to
albumin and other
technologies to improve in vivo half-life.
[021] There exists a need for improved peptides that have the glucose-
dependent insulin
secretagogue activity of PACAP, VIP, GIP, GLP-1, or Exendin-4, but with fewer
side-effects, and
preferably which are stable in formulation and have long plasrria half-lives
in vivo. Such improved
in vivo half-life results from peptides with both decreased clearance and
decreased susceptibility to
proteolysis. Furthermore, tighter control of plasma glucose levels may prevent
long-term diabetic
complications. Thus, new diabetic drugs should provide an improved quality of
life for patients.
SUMMARY OF THE INVENTION
[022] The present invention provides novel modifications that provide suitable
derivatization
sites to improve the pharmacokinetic properties of the peptides. Such N-
terminal modifications at
the amino group of the first peptide residue may include aliphatics, C3 to C7
cycloalkyl, aryl, or
mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen,
and/or sulfur
heteroatoms. In addition, the N-terminal modifications may provide suitable
derivatization sites
(exemplified, but not limited to, amino and thiol groups). Several examples of
such N-terminal
modifications include, but are not limited to, 2-amino benzoic acid, 3-amino
benzoic acid, 4-amino
benzoic acid, 4-amino-2-chloro-benzoic acid, 4-amino-3-methoxy-benzoic acid, 4-
amino-3-methyl-
benzoic acid, 1-amino-cyclopentane-3-carboxylic acid, trans-3-aminocyclohexane
carboxylic acid,
D-pipecolinic acid, 4-amino-1 -methyl-1 H-imidazole-2-carboxylic acid, 4-
methythiobenzoic acid, 2-
methythiobenzoic acid, 2-methythionicotinic acid, proline, 6-aminohexanoic
acid, benzoic acid, (S)-
tetrahydroisoquinoline acetic acid, indoline-2-carboxylic acid, cis-3-
aminocyclohexane carboxylic
acid, L-pipecolinic acid, 9-gluorenylmethoxycarbonyl, 2-thio-polyethylene
glycol benzoic acid, 2-
thio-polyethylene glycol nicotinic acid, 4-amino-1 -methyl-1 H-imidazole-2-
carboxylic acid, 1-amino-
cyclopentane-3-carboxylic acid, 4-amino-1 -methyl-1 H-imidazole-2-carboxylic
acid, 1-amino-
cyclopentane-3-carboxylic acid, 1 -amino-cyclopentane-3-carboxylic acid, (2-
mercapto-1 H-
benzimidazol-l-yl)acetic acid, 2-(tritylthio)ethyl]amino}nicotinate, 2-{[2-
(tritylthio)ethyl]
arnino}nicotinate, 1-[2-(tritylthio)ethyl]-1-H-irnidazole-2-carboxylate, 4-{[2-
(tritylthio)ethyl]amino} -
pyrimidine-5-carboxylate, 1-[2-(tritylthio)ethyl]-1 H-benzimidazole-2-
carboxylate, 2-mercapto-1 H-
imidazol-l-yl)acetic acid, ({i-[2-(tritylthio)ethyl]-iH-imidazol-2-
yl}thio)acetate, 3-(2-
tritylsulfanylethylamino)pyrazine-2-carboxylate, 4-mercaptothiazole-5-
carboxylic acid, 2-
mercaptothiazole-5-carboxylic acid, 2-(2-tritylsulfanylethylamino)thiazole-5-
carboxylae, 2-
mercapto-6-methylpyrimidine-4-carboxylic acid, 5-mercaptonicotinic acid, 5-
isopropyl-2-
mercaptothiazole-4-carboxylic acid, 1-hexadecyl-1 H-benzoimidazol-2-
ylsulfanyl)acetic acid, and 2-
(2-tert-butoxycarbonylaminoethylamino)thiazole-5-carboxylic acid.
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[023] The invention relates to a peptide of Formula (I)
Zi -A1-A2-A3-GIy-A5-Phe-Thr-A8-Asp-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-
A20-A21-Phe-A23-A24-A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-
A38-A39-A40-Z2 (SEQ ID NO: 1)
wherein
Al is His, Phe, Tyr, d-histidine, desamino-histidine, 2-amino-histidine, R-
hydroxy-
histidine, homohistidine, Na-acetylhistidine, a-fluoromethyl-histidine, a-
methyl-
histidine, 3-pyridylalanine, 2-pyridylalanine, or 4-pyridylalanine;
A2 is Ser, Gly, Ala, Val, Leu, lie, Lys, Aib, (1-aminocyclopropyl) carboxylic
acid, (1-
aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-
aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-
aminocyclooctyl) carboxylic acid;
A3 is Glu, Gln, or Ala;
A5 is Thr or Ala;
A8 is Ser or Ala;
A10 is Val, Leu, Tyr, or Ala;
A11 is Ser, Ala, Arg, Gly, Lys, or Thr;
A12 is Lys, Ser, Arg, Ala, Asn, His, or Gin;
A13 is Tyr or Gln;
A14 is Leu, Met, or Ala;
A15 is Asp or Glu;
A16 is Gly, Glu, Ala, Ser, Phe, Trp, Thr, His, Lys, Arg, Val, Ile, Leu, Met,
Asn, Gin, or
Tyr;
A17 is GIn, Glu, Arg, Ala, LYs,_Leu,_Met, Val, Phe, Ile, Trp, Asn, Asp, His,
Ser, Thr, Gly,
or Tyr;
A18 is Ala, Arg, Lys, Phe, Ile, Leu, Tyr, Val, Met, Gly, or His;
A19 is Ala or Val;
A20 is Lys, Arg, Ala, Gln, or Cys;
A21 is Glu, Leu, Ala, or Asp;
A23 is Ile or Val;
A24 is Ala, Glu, Lys, Gin, Asn, or Lys-X;
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CA 02607566 2007-11-05
WO 2006/121860 PCT/US2006/017411
A25 is Trp or Ala;
A26 is Leu or Ala;
A27 is Val, Lys, Ala, or Met;
A28 is Lys, Asn, Arg, Ala, Gin, or Cys;
A29 is Gly, Ala, Lys, Thr, or Cys;
A30 is Arg, Gly, Ala, Cys, or deleted;
A31 is Gly Pro, Lys, Cys, Lys-X or deleted;
A32 is Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
A33 is Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
A34 is Gly, Lys, Cys, Lys-X, Cys-PEG or deleted;
A35 is Ala, Lys, Cys, Lys-X, Cys-PEG or deleted;
A36 is Pro, Lys, Cys, Lys-X, Cys-PEG or deleted;
A37 is Pro, Lys, Cys, Lys-X, Cys-PEG or deleted;
A38 is Pro, Lys, Cys, Lys-X, Cys-PEG or deleted;
A39 is Ser, Lys, Cys, Lys-X, Cys-PEG or deleted; and
A40 is Lys-X, Cys-PEG or deleted.
[024] Lys-X is Lys modified at NE with a fatty acid exemplified by
CH3(CH2)nCOOH where n
ranges from 0 to about 24.
[025] Z1 is selected from
CH3(CH2)n\C.
H n=O..to22 u
/ / H2N
t,
H2N ~ C"~' \ =~
NH2 O
H2N / H2N /
H2N
I
C?~- \p \ C \ 1-~
11 ci 0 7 O
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N I H
H N Cf \ \ ~ ~
2 IO O N C
H 11
0
NH2
H
H H aNC~'R~~,2
H cH I ''
0
H\ N.
H 01~c
xz
N ~1 ~~
, 0 0
H2N_ /"-NH
H N S
aNc'-',< N/
~~~'',,,= H
I 0 II
0 0
H2N
NH . ..
HO~ S acN
C~/ O N)/-S
/
~Co
a
8
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Cx N~ ( / C
~ v
N C'~
S Ip S ,~ H o
/
C" 'a
H2N N cI,
O H Io
HS
NH2
-~N
N C~~ HN v ~ '~t
H
0
HS
~
?~,~ l'
)--lN cLC"'~ 911
HS~ fo HS 0
SH
_ .. N ----- -
Hg__._/ NH
~
~--S ~ ~
C'
N ~C'~ HS N S ~~ II
ll ~
0
9
CA 02607566 2007-11-05
WO 2006/121860 PCT/US2006/017411
SH
HS N SH
\ I \ I i
c~ ~ II
~o ~o 0
H
HS N N N
~~
/ I \ I ~
C~ HS '~~ HS
(O C IO
/N
HS
-,yN r/
I I ~ I ~
N Cl~ N N C
c
HS OH 0 HS~~NH IO
~~NH 0
PEG-S
HS
N HN ~ ~
HS
%
oi ~o
PEG-S
PEG-S ~N
i S
N~ N/\II C!V
O PEG-S Ip II ~
O
CA 02607566 2007-11-05
WO 2006/121860 PCT/US2006/017411
/~~
C NH S-PEG
(/ II PEG S-/
i
PEG S~ 11 N i C
C/ IO
S-PEG
PEG-S N S-PEG
C~ \ I C2,~ \ C~/
~O I I' "~ IO
O
PEG-S N N
i I \ I ' PEG-S' N
Yi
\ C~' PEG-S C~ N~
IOI IOI I i k
OH O
H
PEG-5\ ~N
CN C~< PEG~
IoI ~,NH ~o S i ~
S O
PEG
PEG~
II PEG
O S ~~
O
CH3(CH2)14 O CH3(CH2)14
/j--NH
/ NH o/
~ / ! I
\ N C, ~ N SC~(
0 CH3(CH2)14 lo
O
11
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[026] Z2 may be a hydroxyl group such that the peptide has an unmodified
carboxylate C-
terminus or Z2 may be a modification of the C-terminal carboxylate group. Z2
may be a
modification such as amidation, or Z2 may also be an unnatural amino acid or
amide. Z2 may be
exemplified by, but not limited to,
f-N
~~O __ ~\s' /OH
H II / N OH
O
~ / lo lo
OH H
C N /
NH O ~~N \ C~OH I
OH
H \
O ~i
H _N
/ I ac
c"OH \ ~~OH II1_1OH
cl o O o
/-NH
N--_
OH s
/OH N O,
C /OH
C
O
[027] For the peptide of Formula (1), the N-terminal modifications may be
attached via an amide
bond to the alpha-amino group of the first amino acid of said peptide. The C-
terminal
modifications may attached via an amide bond to the main chain carboxylate
group of the last
amino acid of said peptide. Examples of peptides of Formula (I) may be found
in, but are not
limited to, the peptides described in Table 1(e.g., SEQ ID NO: 1-56).
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[028] Derivatives of the present invention may include peptides that have been
fused with
another compound, such as a compound to increase the half-life of the peptide
and/or to reduce
potential immunogenicity of the peptide (e.g., polyethylene glycol, "PEG").
For example,
PEGylated peptides typically have greater half-life in vivo (Greenwald, Adv.
Drug. Del. Rev.
55:217-250, 2003).
[029] In the case of PEGylation, the fusion of the peptide to PEG may be
accomplished by any
means known to one skilled in the art: For example, PEGylation may be
accomplished by first
introducing a cysteine mutation into the peptide to provide a linker upon
which to attach the PEG,
followed by site-specific derivatization with PEG-maleimide. Alternatively,
the N-terminal
modification may incorporate a reactive moiety for coupling to PEG, as
exemplified by the amine
group, the mercapto group, or the carboxylate group of the N-terminal
modifying compounds
disclosed above. For example, PEGylation may be accomplished by first
introducing a mercapto
moiety into the peptide via the N-terminal modifying group to provide a linker
upon which to attach
the PEG, followed by site-specific derivatization with methoxy-PEG-maleimide
reagents supplied
by, for example, either Nektar Therapeutics (San Carlos, CA, USA) and/or NOF
(Tokyo, Japan).
In addition to maleimide, numerous Cys reactive groups are known to those
skilled in the art of
protein cross-linking, such as the use of alkyl halides and vinyl sulfones
(see, e.g., Proteins,
Structure and Molecular Properties, 2nd ed., T. E. Creighton, W.H. Freeman and
Company, New
York, 1993). In addition, the PEG could be introduced by direct attachment to
the C-terminal
carboxylate group, or to an internal amino acid such as Cys, Lys, Asp, or Glu
or to unnatural amino
acids that contain similar reactive sidechain moieties.
[030] Various size PEG groups can be used, as exemplified but not limited to,
PEG polymers of
from about 5 kDa to about 43 kDa. The PEG modification may include a single,
linear PEG. For
example, linear 5, 20, or 30 kDa PEGs that are attached to maleidmide or other
cross-linking
groups are available from Nektar and/or NOF (see, e.g., Table 2). Also, the
modification may
involve branched PEGs that contain two or more PEG polymer chains that are
attached to
maleimide or other cross-linking groups are available from Nektar and NOF
(see, e.g., Table 2).
[031] It is possible that PEGylation with a smaller PEG (e.g., a linear 5 kDa
PEG) will less likely
reduce activity of the peptide, whereas a larger PEG (e.g., a branched 40 kDa
PEG) will more
likely reduce activity. However, a larger PEG-wilf-increase plasrna half-life
fLirther so that once a
week injection may be possible (Harris, et al., Clin. Pharmacokinet. 40:539-
551, 2001).
[032] The linker between the PEG and the peptide cross-linking group can be
varied. For
example, the commercially available thiol-reactive 40 kDa PEG (mPEG2-MAL) from
Nektar
(Huntsville, AI) employs a maleimide group for conjugation to Cys, and the
maleimide group is
attached to the PEG via a linker that contains a Lys (see, e.g., Table 2). As
a second example, the
commercially available thiol-reactive 43 kDa PEG (GL2-400MA) from NOF employs
a maleimide
group for conjugation to Cys, and the maleimide group is attached to the PEG
via a bi-substituted
alkane linker (see, e.g., Table 2). In addition, the PEG polymer can be
attached directly to the
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maleimide, as exemplified by PEG reagents of molecular weight 5 and 20 kDa
available form
Nektar Therapeutics (Huntsville, Al) (see, e.g., Table 2).
[033] The present invention exemplifies, but is not limited to, the use of a
mercapto group as a
cross-linking site. It is well known that other moieties present in amino
acids such as the amino
group of the N-terminal modifying compound, the C-terminal carboxylate of
either an unmodified
C-terminus or a peptide modified with Z2, and the side chains of amino acids
such as Lys, Arg,
Asp, and Glu provide reactive groups that provide moieties suitable for
covalent modification and
attachment to PEG. Numerous examples of suitable cross-linking agents are
known to those
skilled in the art (see, e.g., Proteins, Structure and Molecular Properties,
2nd ed., T. E. Creighton,
W.H. Freeman and Company, New York, 1993). Such cross-linking agents can be
linked to PEG
as exemplified by, but not limited to, commercially available PEG derivatives
containing amines,
aldehydes, acetals, maleimide, succinimides, and thiols that are marketed, for
example, by Nektar
and NOF (e.g., Harris, et al., Clin. Pharmokinet. 40:539-551, 2001).
[034] In addition to PEGylation, the peptides of the present invention may be
modified with fatty
acids that improve pharmacodynamic properties. For example, the amine
containing N-terminal
modifying compounds can be derivatized with palmitate or myristolate or other
fatty acids using
methods known to those skilled in the art or an alkyl (e.g., C6-C18) moiety
can be included directly
as part of the N-terminal modifying compound.
[035] The peptides of the present invention have improved stability to
proteolysis by DPPIV and
in plasma as compared to GLP-1. The derivatives of the present invention
demonstrate an
extended duration of action in vivo, supporting a dosing interval of less than
once per day or once
per week or greater, when derivatized.
[036] The peptides of the present invention (e.g., Table 1) provide a new
therapy for patients
with, for example, metabolic disorders such as those resulting from decreased
endogenous insulin
secretion, in particular type 2 diabetics, or for patients with impaired
glucose tolerance, a
prediabetic state that has a mild alteration in insulin secretion. In
addition, the peptides of the
present invention may be useful in the prevention and/or treatment of type 1
diabetes, gestational
diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune
diabetes adult (LADA),
and associated diabetic dyslipidemia and other diabetic complications, as well
as hyperglycemia,
hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose,
dyslipidemia,
hypertriglyceridemia, Syndrome X, and insulin resistance.
[037] The peptides of the present invention (e.g., Table 1) may also be
effective in such
disorders as obesity, and in the treatment of cardiovascular disease,
including atherosclerosis,
coronary heart disease, coronary artery disease, hyperlipidemia,
hypercholesteremia, low HDL
levels, hypertension; cerebrovascular disease; and peripheral vessel disease;
and for the
treatment of neurodegenerative diseases (including Parkinson's and
Alzheimer's).
[038] One aspect of the invention is a peptide of Formula (I), and fragments,
derivatives, and
variants thereof that demonstrate at least one biological function that is
substantially the same as
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the peptides of Formula (I) (collectively, "peptides of this invention"),
including functional
equivalents thereof(e.g., Table 1).
[039] Antibodies and antibody fragments that selectively bind the peptides of
this invention (e.g.,
Table 1) are also provided. Such antibodies are useful in detecting the
peptides of this invention,
and can be identified and made by procedures well known in the art. A
polyclonal N-terminal IgG
antibody and a monoclonal C-terminal Fab antibody have been generated which
recognize
peptides of this invention.
[040] The invention is also directed to a method of treating diabetes,
diabetes-related disorders,
and/or other diseases or conditions affected by the peptides of this
invention, for example, effected
by the GLP-1 agonist function of the peptides of this invention, in a mammal,
comprising
administering a therapeutically effective amount of any of the peptides of the
present invention or
any peptide active at GLP-1 such as a peptide of Formula (I) to said mammal
(e.g., Table 1).
[041] Also disclosed are methods of making the peptides of this invention.
DETAILED DESCRIPTION OF THE INVENTION
[042] This invention provides novel modified peptides, and fragments,
derivatives, and variants
thereof that demonstrate at least one biological function that is
substantially the same as the
peptides of Formula (I) (peptides of this invention). The peptides of this
invention function (e.g.,
Table 1) in vivo as GLP-1 agonists or otherwise in the prevention and/or
treatment of such
diseases or conditions as diabetes including both type 1 and type 2 diabetes,
gestational diabetes,
maturity-onset diabetes of the young (MODY) (Herman, et al., Diabetes 43:40,
1994); latent
autoimmune diabetes adult (LADA) (Zimmet, et al., Diabetes Med. 11:299, 1994);
and associated
diabetic dyslipidemia and other diabetic complications, as well as
hyperglycemia,
hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose,
dyslipidemia,
hypertriglyceridemia, Syndrome X, and insulin resistance.
[043] The peptides of the present invention (e.g., Table 1) may also be
effective in such
disorders as obesity, and in the treatment of cardiovascular disease,
including atherosclerosis,
coronary heart disease, coronary artery disease, hyperlipidemia,
hypercholesteremia, low HDL
levels,-hypertension; cerebrovascular disease; and penpheral vessel disease;
and or t e
treatment of neurodegenerative diseases (including Parkinson's and
Alzheimer's).
[044] The peptides of this invention (e.g., Table 1) will stimulate insulin
release from pancreatic
R-cells in a glucose-dependent fashion. Furthermore, the peptides of this
invention are stable in
both aqueous and non-aqueous formulations and exhibit a plasma half-life of
greater than one
hour, for example, demonstrating a plasma half-life greater than 6 hours.
[045] The peptides of this invention are GLP-1 agonists (e.g., Table 1). In
addition, these
peptides may possess activities at other related receptors including, but not
limited to, VPAC2
receptor agonism, GIP receptor agonism, or glucagon receptor antagonism.
Furthermore, the
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peptides of this invention stimulate insulin release into plasma in a glucose-
dependent fashion
without inducing a stasis or increase in the level of plasma glucose that is
counterproductive to the
treatment of, for example, type 2 diabetes. Additionally, the peptides of this
invention may be
selective agonists of the GLP-1 receptor, thereby causing, for example, an
increase in insulin
release into plasma, while being selective against other receptors that are
responsible for such
disagreeable or dangerous side effects as gastrointestinal water retention,
and/or unwanted
cardiovascular effects such.as increased heart_r.ate or blood pressure.
[046] The peptides of this invention are also stable in aqueous and non-
aqueous formulations.
The peptides of this invention will exhibit less than 10% degradation at 37-40
C over a period of
one week, when dissolved in water (at pH between 7-8) or non-aqueous organic
solvent, or the
peptides of this invention will exhibit less than 5% degradation at 37-40 C
over a period of one
week, when dissolved in water (at pH between 7-8) or non-aqueous organic
solvent. Furthermore,
compositions and formulations of the present invention may comprise peptides
of the present
invention and about 2% to about 30% DMSO. In another embodiment of the present
invention, the
compositions and formulations may optionally include about 0.2% to about 3%
(w/v) of additional
solvents such as propylene glycol, dimethyl formamide, propylene carbonate,
polyethylene glycol,
and triglycerides.
[047] Finally, the derivatized peptides of this invention may exhibit a plasma
half-life of, for
example, at least 1 hour in rats after IV injection, at least 3 hours, or at
least 6 hours. Furthermore,
the derivatized peptide may demonstrate a significant lowering of the plasma
glucose AUC
following subcutaneous injection in rats, for example, at least 24 hours, at
least 41 hours, or at
least 65 hours following injection.
[048] The peptides of this invention provide a new therapy for patients with
decreased
endogenous insulin secretion or impaired glucose tolerance, in particular,
type 2 diabetes. That is,
the peptides of the present invention are long-acting GLP-1 receptor agonists
that may be used to
maintain, improve, and restore glucose-stimulated insulin secretion.
Furthermore, a selective
peptide agonist of the GLP-1 receptor will enhance glucose-dependent insulin
secretion in the
pancreas without causing the side effects associated with non-selective
activation of the other
related receptors.
[049] Certain terms used throughout this specification are defined below, and
others will be
defined as introduced. The single letter abbreviation for a particular amino
acid, its corresponding
amino acid, and three letter abbreviation are as follows: A, alanine (Ala); C,
cysteine (Cys); D,
aspartic acid (Asp); E, glutamic acid (Glu); F, phenylalanine (Phe); G,
glycine (Gly); H, histidine
(His); I, isoleucine (IIe); K, lysine (Lys); L, leucine (Leu); M, methionine
(Met); N, asparagine (Asn);
P, proline (Pro); Q, glutamine (Gln); R, arginine (Arg); S, serine (Ser); T,
threonine (Thr); V, valine
(Val); W, tryptophan (Trp); and Y, tyrosine (Tyr).
[050] "Functional equivalent" and "substantially the same biological function
or activity" each
means that degree of biological activity that is within about 30% to about
100% or more of that
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biological activity demonstrated by the peptide to which it is being compared
when the biological
activity of each peptide is determined by the same procedure.
[051] "Biological activity," "activity," or "biological function," which are
used interchangeably,
herein mean an effector function that is directly or indirectly performed by a
peptide (whether in its
native or denatured conformation), or by any fragments, derivatives, and
variants thereof.
Biological activities include, for example, binding to peptides, binding to
other proteins or
molecules, activity as a DNA binding protein, as a transcriptioh regulator,
ability to bind damaged
DNA, etc.
[052] The terms "fragment," "derivative," and "variant," when referring to the
peptides of the
present invention, means fragments, derivatives, and variants of the peptides
which retain
substantially the same biological function or activity as such peptides, as
described further below.
[053] A fragment is a portion of the peptide which retains substantially
similar functional activity,
for example, as described in the in vivo models disclosed herein.
[054] A derivative includes all modifications to the peptide which
substantially preserve the
functions disclosed herein and include additional structure and attendant
function (e.g., modified
N-terminus peptides, modified C-terminus peptides, or PEGylated peptides),
fusion peptides which
confer targeting specificity or an additional activity such as toxicity to an
intended target, as
described further below.
[055] The peptides of the present invention may be synthetic peptides.
[056] The fragment, derivative, or variant of the peptides of the present
invention may be (i) one
in which one or more of the amino acid residues are substituted with a
conserved or non-
conserved amino acid residue and such substituted amino acid residue may or
may not be one
encoded by the genetic code, or (ii) one in which one or more of the amino
acid residues includes
a substituent group, or (iii) one in which the mature peptide is fused with
another compound, such
as a compound to increase the half-life of the peptide (e.g., polyethylene
glycol), or (iv) one in
which the additional amino acids are fused to the mature peptide, such as a
leader or secretory
sequence or a sequence which is employed for purification of the mature
peptide, or (v) one in
which the peptide sequence is fused with a larger peptide (e.g., human
albumin, an antibody or Fc,
- -- - - for increased duration of effect). Such fragments, derivatives,and
variants and anafogsare deemed to be within the scope of those skilled in the
art from the teachings herein.
[057] The derivatives of the present invention may contain conservative amino
acid substitutions
(defined further below) made at one or more nonessential amino acid residues.
A"nonessentiaP'
amino acid residue is a residue that can be altered from the wild-type
sequence of a protein
without altering the biological activity, whereas an "essential" amino acid
residue is required for
biological activity. A "conservative amino acid substitution" is one in which
the amino acid residue
is replaced with an amino acid residue having a similar side chain. Families
of amino acid
residues having similar side chains have been defined in the art. These
families include amino
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acids with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine).
Fragments, or biologically active portions include peptide fragments suitable
for use as a
medicament, to generate.antibodies, as a research reagent,. and the like.
Fragments include
peptides comprising amino acid sequences sufficiently similar to or derived
from the amino acid
sequences of a peptide of this invention and exhibiting at least one activity
of that peptide, but
which include fewer amino acids than the full-length peptides disclosed
herein. Typically,
biologically active portions comprise a domain or motif with at least one
activity of the peptide. A
biologically active portion of a peptide can be a peptide which is, for
example, five or more amino
acids in length. Such biologically active portions can be prepared
synthetically or by recombinant
techniques and can be evaluated for one or more of the functional activities
of a peptide of this
invention by means disclosed herein and/or well known in the art.
[058] Variants of the peptides of this invention include peptides having an
amino acid sequence
sufficiently similar to the amino acid sequence of the peptides of this
invention or a domain thereof.
The term "sufficiently similar" means a first amino acid sequence that
contains a sufficient or
minimum number of identical or equivalent amino acid residues relative to a
second amino acid
sequence such that the first and second amino acid sequences have a common
structural domain
and/or common functional activity. For example, amino acid sequences that
contain a common
structural domain that is at least about 45%, about 75% through 98%, identical
are defined herein
as sufficiently similar. Variants will be sufficiently similar to the amino
acid sequence of the
peptides of this invention. Such variants generally retain the functional
activity of the peptides of
this invention.
[059] Variants include peptides that differ in amino acid sequence due to
mutagenesis. Variants
that function as GLP-1 receptor agonists may be identified by screening
combinatorial libraries of
mutants, for example truncation mutants, of the peptides of this invention for
GLP-1 receptor
agonist activity.
_._ _
[060] The invention also provides chimeric or fusion peptides. The targeting
sequence is
designed to localize the delivery of the peptide to minimize potential side
effects. The peptides of
this invention may be composed of amino acids joined to each other by peptide
bonds or modified
peptide bonds (i.e., peptide isosteres), and may contain amino acids other
than the 20 gene-
encoded amino acids. The peptides may be modified by either natural processes,
such as
posttranslational processing, or by chemical modification techniques which are
well known in the
art. Such modifications are well described in basic texts and in more detailed
monographs, as well
as in a voluminous research literature. Modifications may occur anywhere in a
peptide, including
the peptide backbone, the amino acid side-chains, and the amino or carboxyl
termini. It will be
appreciated that the same type of modification may be present in the same or
varying degrees at
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several sites in a given peptide. Also, a given peptide may contain many types
of modifications.
Peptides may be branched, for example, as a result of ubiquitination, and they
may be cyclic, with
or without branching. Cyclic, branched, and branched cyclic peptides may
result from
posttranslation natural processes or may be made by synthetic methods.
Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent
attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide
derivative,
covalent.attachment.of.a.lipid or lipid.derivative, covalent attachment of
phosphotidylinositol, cross-
linking, cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links,
formation of cysteine, formation of pyroglutamate, formulation, gamma-
carboxylation,
glycosylation, GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation,
oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation,
racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins such as
arginylation, and ubiquitination (see, e.g., Proteins, Structure and Molecular
Properties, 2nd ed., T.
E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational
Covalent
Modification of Proteins, B. C. Johnson, ed., Academic Press, New York, pgs. 1-
12 (1983); Seifter,
et al., Meth. Enzymol. 182:626-646, 1990; Rattan, et al., Ann. N.Y. Acad. Sci.
663:48-62, 1992).
[061] The peptides of the present invention include the peptides of Formula
(I) (e.g., Table 1), as
well as those sequences having insubstantial variations in sequence from them.
An "insubstantial
variation" would include any sequence addition, substitution, or deletion
variant that maintains
substantially at least one biological function of the peptides of this
invention, for example, GLP-1
receptor agonist activity, and/or enhancement of insulin secretion or lowering
of blood glucose
demonstrated herein. These functional equivalents may include peptides which
have at least
about 90% identity to the peptides of the present invention, at least 95%
identity to the peptides of
the present invention, and at least 99% identity to the peptides of the
present invention, and also
include portions of such peptides having substantially the same biological
activity. However, any
peptide having insubstantial variation in amino acid sequence from the
peptides of the present
invention that demonstrates functional equivalency as described further herein
is included in the
description of the present invention.
[062] The peptides of this invention may be a product of chemical synthetic
procedures.
[663j The peptides of this invention may be conveniently isolated by methods
that are well
known in the art. Purity of the preparations may also be assessed by any means
known in the art,
such as SDS-polyacrylamide gel electrophoresis and mass spectroscopy and
liquid
chromatography.
[064] Also provided are related peptides within the understanding of those
with skill in the art,
such as chemical mimetics, organomimetics, or peptidomimetics. As used herein,
the terms
"mimetic," "peptide mimetic," "peptidomimetic," "organomimetic," and "chemical
mimetic" are
intended to encompass peptide derivatives, peptide analogs, and chemical
compounds having an
arrangement of atoms in a three-dimensional orientation that is equivalent to
that of a peptide of
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the present invention. It will be understood that the phrase "equivalent to"
as used herein is
intended to encompass peptides having substitution(s) of certain atoms, or
chemical moieties in
said peptide, having bond lengths, bond angles, and arrangements in the
mimetic peptide that
produce the same or sufficiently similar arrangement or orientation of said
atoms and moieties to
have the biological function of the peptides of the invention. In peptide
mimetics, the three-
dimensional arrangement of the chemical constituents may be structurally
and/or functionally
equivalent_to the three-dimensional arrangementof the peptide backbone and
component amino
acid sidechains in the peptide, resulting in such peptido-, organo-, and
chemical mimetics of the
peptides of the invention having substantial biological activity. These terms
are used according to
the understanding in the art, as illustrated, for example, by Fauchere, (Adv.
Drug Res. 15:29,
1986); Veber & Freidinger, (TINS p.392, 1985); and Evans, et al., (J. Med.
Chem. 30:1229, 1987),
incorporated herein by reference.
[065] It is understood that a pharmacophore exists for the biological activity
of each peptide of
the invention. A pharmacophore is understood in the art as comprising an
idealized, three-
dimensional definition of the structural requirements for biological activity.
Peptido-, organo-, and
chemical mimetics may be designed to fit each pharmacophore with current
computer modeling
software (computer aided drug design). Said mimetics may be produced by
structure-function
analysis, based on the positional information from the substituent atoms in
the peptides of the
invention.
[066] Peptides as provided by the invention may be advantageously synthesized
by any of the
chemical synthesis techniques known in the art, particularly solid-phase
synthesis techniques, for
example, using commercially-available automated peptide synthesizers. The
mimetics of the
present invention may be synthesized by solid phase or solution phase methods
conventionally
used for the synthesis of peptides (see, e.g., Merrifield, J. Amer. Chem. Soc.
85:2149-54, 1963;
Carpino, Acc. Chem. Res. 6:191-98, 1973; Birr, Aspects of the Merrifield
Peptide Synthesis,
Springer-Veriag: Heidelberg, 1978; The Peptides: Analysis, Synthesis, Biology,
Vols. 1, 2, 3, and
5, (Gross & Meinhofer, eds.), Academic Press: New York, 1979; Stewart, et al.,
Solid Phase
Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, III., 1984; Kent,
Ann. Rev. Biochem.
57:957-89, 1988; and Gregg, et al., Int. J. Peptide Protein Res. 55:161-214,
1990, which are
incorporated herein by reference in their entirety.)
[067] Peptides of the present invention may be prepared by solid phase
methodology. Briefly,
an N-protected C-terminal amino acid residue is linked to an insoluble support
such as
divinylbenzene cross-linked polystyrene, polyacrylamide resin,
Kieselguhr/polyamide (pepsyn K),
controlled pore glass, cellulose, polypropylene membranes, acrylic acid-coated
polyethylene rods,
or the like. Cycles of deprotection, neutralization, and coupling of
successive protected amino acid
derivatives are used to link the amino acids from the C-terminus according to
the amino acid
sequence. For some synthetic peptides, an FMOC strategy using an acid-
sensitive resin may be
used. Solid supports in this regard may be divinylbenzene cross-linked
polystyrene resins, which
are commercially available in a variety of functionalized forms, including
chloromethyl resin,
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hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine (BHA) resin, 4-
methylbenzhydrylamine (MBHA) resin, oxime resins, 4-alkoxybenzyl alcohol resin
(Wang resin), 4-
(2',4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin, 2,4-
dimethoxybenzhydryl-amine resin,
and 4-(2',4'-dim ethoxyphenyl-FMOC-amino-methyl)-ph enoxyacetamidonorleucyl-M
BHA resin
(Rink amide MBHA resin). In addition, acid-sensitive resins also provide C-
terminal acids, if
desired. A protecting group for alpha amino acids is base-labile 9-
fluorenylmethoxy-carbonyl
(FMOC). .
[068] Suitable protecting groups for the side chain functionalities of amino
acids chemically
compatible with BOC (t-butyloxycarbonyl) and FMOC groups are well known in the
art. When
using FMOC chemistry, the following protected amino acid derivatives may be
utilized: FMOC-
Cys(Trt), FMOC-Ser(But), FMOC-Asn(Trt), FMOC-Leu, FMOC-Thr(Trt), FMOC-Val,
FMOC-Gly,
FMOC-Lys(Boc), FMOC-Gln(Trt), FMOC-Glu(OBut), FMOC-His(Trt), FMOC-Tyr(But),
FMOC-
Arg(PMC (2,2,5,7,8-pentamethylchroman-6-sulfonyl)), FMOC-Arg(BOC)2, FMOC-Pro,
and FMOC-
Trp(BOC). The amino acid residues may be coupled by using a variety of
coupling agents and
chemistries known in the art, such as direct coupling with DIC (diisopropyl-
carbodiimide), DCC
(dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-
oxytrisdimethylaminophosphonium hexa-
fluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluoro-
phosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate);
via performed
symmetrical anhydrides; via active esters such as pentafluorophenyl esters; or
via performed
HOBt (1 -hydroxybenzotriazole) active esters or by using FMOC-amino acid
fluoride and chlorides
or by using FMOC-amino acid-N-carboxy anhydrides. For example, activation may
be performed
with HBTU (2-(1H-benzotriazole-1-yl),1,1,3,3-tetramethyluronium
hexafluorophosphate) or HATU
(2-(1 H-7-aza-benzotriazole-1-yl),1,1,3,3-tetramethyluronium hexafluoro-
phosphate) in the
presence of HOBt or HOAt (7-azahydroxybenztriazole).
[069] The solid phase method may be carried out manually, or by automated
synthesis on a
commercially available peptide synthesizer (e.g., Applied Biosystems 431 A or
the like; Applied
Biosystems, Foster City, CA). In a typical synthesis, the first (C-terminal)
amino acid is loaded on
the chlorotrityl resin. Successive deprotection (with 20% piperidine/NMP (N-
methylpyrrolidone))
and coupling cycles according to ABI FastMoc protocols (Applied Biosystems)
may be used to
generate the peptide sequence. Double and triple coupling, with capping.by
acetic anhydride, may
also be used.
[070] The synthetic mimetic peptide may be cleaved from the resin and
deprotected by
treatment with TFA (trifluoroacetic acid) containing appropriate scavengers.
Many such cleavage
reagents, such as Reagent K (0.75 g crystalline phenol, 0.25 mL ethanedithiol,
0.5 mL thioanisole,
0.5 mL deionized water, 10 mL TFA) and others, may be used. The peptide is
separated from the
resin by filtration and isolated by ether precipitation. Further purification
may be achieved by
conventional methods, such as gel filtration and reverse phase HPLC (high
performance liquid
chromatography). Synthetic mimetics according to the present invention may be
in the form of
pharmaceutically acceptable salts, especially base-addition salts including
salts of organic bases
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and inorganic bases. The base-addition salts of the acidic amino acid residues
are prepared by
treatment of the peptide with the appropriate base or inorganic base,
according to procedures well
known to those skilled in the art, or the desired salt may be obtained
directly by lyophilization of the
appropriate base.
[071] Generally, those skilled in the art wil{ recognize that peptides as
described herein may be
modified by a variety of chemical techniques to produce peptides having
essentially the same
activity as the'unmodified peptide, and optionally having other desirable
properties. For example,
carboxylic acid groups of the peptide may be provided in the form of a salt of
a pharmaceutically-
acceptable cation. Amino groups within the peptide may be in the form of a
pharmaceutically-
acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene
sulfonic, maleic,
tartaric, and other organic salts, or may be converted to an amide. Thiols may
be protected with
any one of a number of well-recognized protecting groups, such as acetamide
groups. Those
skilled in the art will also recognize methods for introducing cyclic
structures into the peptides of
this invention so that the native binding configuration will be more nearly
approximated. For
example, a carboxyl terminal or amino terminal cysteine residue may be added
to the peptide, so
that when oxidized the peptide will contain a disulfide bond, thereby
generating a cyclic peptide.
Other peptide cyclizing methods include the formation of thioethers and
carboxyl- and amino-
terminal amides and esters.
[072] Specifically, a variety of techniques are available for constructing
peptide derivatives and
analogs with the same or similar desired biological activity as the
corresponding peptide but with
more favorable activity than the peptide with respect to solubiiity,
stability, and susceptibility to
hydrolysis and proteolysis. Such derivatives and analogs include peptides
modified at the N-
terminal amino group, as exemplified by, but not limited to, the peptides of
Formula (I) (e.g., Table
1), the C-terminal amide group, and/or changing one or more of the amido
linkages in the peptide
to a non-amido linkage. It will be understood that two or more such
modifications may be coupled
in one peptide mimetic structure (e.g., modification at the C-terminal amide
group and inclusion of
a-CH2- carbamate linkage between two amino acids in the peptide).
[073] Peptide mimetics as understood in the art and provided by the invention
are structurally
similar to the peptides of the invention, but have one or more peptide
linkages optionally replaced
by aJinkage, for example, --CH2NH--, --CH2S--; --CH2CH2--, --CH=CH-(in both
cis and trans
conformers), --COCH2--, --CH(OH)CH2 --, and --CH2SO--, by methods known in the
art and further
described in the following references: Spatola, Chemistry and Biochemistry of
Amino Acids,
Peptides, and Proteins, (Weinstein, ed.), Marcel Dekker: New York, p. 267,
1983; Spatola, Peptide
Backbone Modifications 1:3, 1983; Morley, Trends Pharm. Sci. pp. 463-468,
1980; Hudson, et al.,
Int. J. Pept. Prot. Res. 14:177-185, 1979; Spatola, et al., Life Sci. 38:1243-
1249, 1986; Hann, J.
Chem. Soc. Perkin Trans. I 307-314, 1982; Almquist, et al., J. Med. Chem.
23:1392-1398, 1980;
Jennings-White, et al., Tetrahedron Lett. 23:2533, 1982; Szelke, et al.,
EP045665A; Holladay, et
al., Tetrahedron Lett. 24:4401-4404, 1983; and Hruby, Life Sci. 31:189-199,
1982; each of which is
incorporated herein by reference. Such peptide mimetics may have significant
advantages over
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WO 2006/121860 PCT/US2006/017411
peptide embodiments, including, for example, more economical to produce,
having greater
chemical stability or enhanced pharmacological properties (such as half-life,
absorption, potency,
efficacy, etc.), reduced antigenicity, and other properties.
[074] Mimetic analogs of the peptides of the invention may also be obtained
using the principles
of conventional or rational drug design (see, e.g., Andrews, et al., Proc.
Alfred Benzon Symp.
28:145-165, 1990; McPherson, Eur. J. Biochem. 189:1-24, 1990; Hol, et al., in
Molecular
'Recognition: Chemical'and Biochemidal Problems, (Roberts, ed.); Royal Society
of Chemistry; pp.
84-93, 1989a; Hol, Arzneim-Forsch. 39:1016-1018, 1989b; Hol, Agnew Chem. Int.
Ed. Engl.
25:767-778, 1986; the disclosures of which are herein incorporated by
reference).
[075] In accordance with the methods of conventional drug design, the desired
mimetic
molecules may be obtained by randomly testing molecules whose structures have
an attribute in
common with the structure of a "native" peptide. The quantitative contribution
that results from a
change in a particular group of a binding molecule may be determined by
measuring the biological
activity of the putative mimetic in comparison with the activity of the
peptide. In one embodiment
of rational drug design, the mimetic is designed to share an attribute of the
most stable three-
dimensional conformation of the peptide. Thus, for example, the mimetic may be
designed to
possess chemical groups that are oriented in a way sufficient to cause ionic,
hydrophobic, or van
der Waals interactions that are similar to those exhibited by the peptides of
the invention, as
disclosed herein.
[076] One method for performing rational mimetic design employs a computer
system capable
of forming a representation of the three-dimensional structure of the peptide,
such as those
exemplified by Hol, 1989a; Hol, 1989b; and Hol, 1986. Molecular structures of
the peptido-,
organo-, and chemical mimetics of the peptides of the invention may be
produced using computer-
assisted design programs commercially available in the art. Examples of such
programs include
SYBYL 6.50, HQSARTM, and ALCHEMY 2000T"' (Tripos); GALAXYT"' and AM2000T"' (AM
Technologies, Inc., San Antonio, TX); CATALYSTT"' and CERIUSTM (Molecular
Simulations, Inc.,
San Diego, CA); CACHE PRODUCTSTM, TSARTM, AMBERTM, and CHEM-XT"' (Oxford
Molecular
Products, Oxford, CA) and CHEMBUILDER3DTM (lnteractive Simulations, Inc., San
Diego, CA).
[077] The peptido-, organo-, and chemical mimetics produced using the peptides
disclosed
herein using, for example, art-recognized molecular modeling programs may be
produced using
conventional chemical synthetic techniques, for example, designed to
accommodate high
throughput screening, including combinatorial chemistry methods. Combinatorial
methods useful
in the production of the peptido-, organo-, and chemical mimetics of the
invention include phage
display arrays, solid-phase synthesis, and combinatorial chemistry arrays, as
provided, for
example, by SIDDCO (Tuscon, Arizona); Tripos, Inc.; Calbiochem/Novabiochem
(San Diego, CA);
Symyx Technologies, Inc. (Santa Clara, CA); Medichem Research, Inc. (Lemont,
IL); Pharm-Eco
Laboratories, Inc. (Bethlehem, PA); or N.V. Organon (Oss, Netherlands).
Combinatorial chemistry
production of the peptido-, organo-, and chemical mimetics of the invention
may be produced
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WO 2006/121860 PCT/US2006/017411
according to methods known in the art, including, but not limited to,
techniques disclosed in Terrett,
(Combinatorial Chemistry, Oxford University Press, London, 1998); Gallop, et
al., J. Med. Chem.
37:1233-51, 1994; Gordon, et al., J. Med. Chem. 37:1385-1401, 1994; Look, et
al., Bioorg. Med.
Chem. Lett. 6:707-12, 1996; Ruhland, et al., J. Amer. Chem. Soc. 118: 253-4,
1996; Gordon, et al.,
Acc. Chem. Res. 29:144-54, 1996; Thompson & Ellman, Chem. Rev. 96:555-600,
1996; Fruchtel &
Jung, Angew. Chem. Int. Ed. Engl. 35:17-42, 1996; Pavia, "The Chemical
Generation of Molecular
Diversity"., Network Science Center, www.netsci.org, 1995; Adnan, et al.,
"Solid Support
Combinatorial Chemistry in Lead Discovery and SAR Optimization," Id., 1995;
Davies and Briant,
"Combinatorial Chemistry Library Design using Pharmacophore Diversity," Id.,
1995; Pavia,
"Chemically Generated Screening Libraries: Present and Future," Id., 1996; and
U.S. Patents,
Nos. 5,880,972; 5,463,564; 5,331573; and 5,573,905.
[078] The newly synthesized peptides may be substantially purified by
preparative high
performance liquid chromatography (see, e.g., Creighton, Proteins: Structures
And Molecular
Principles, WH Freeman and Co., New York, N.Y., 1983). The composition of a
synthetic peptide
of the present invention may be confirmed by amino acid analysis or sequencing
by, for example,
the Edman degradation procedure (Creighton, supra). Additionally, any portion
of the amino acid
sequence of the peptide may be altered during direct synthesis and/or combined
using chemical
methods with sequences from other proteins to produce a variant peptide or a
fusion peptide.
[079] Also included in this invention are antibodies and antibody fragments
that selectively bind
the peptides of this invention. Any type of antibody known in the art may be
generated using
methods well known in the art. For example, an antibody may be generated to
bind specifically to
an epitope of a peptide of this invention. "Antibody" as used herein includes
intact immunoglobulin
molecules, as well as fragments thereof, such as Fab, F(ab')2, and Fv, which
are capable of
binding an epitope of a peptide of this invention. Typically, at least 6, 8,
10, or 12 contiguous
amino acids are required to form an epitope. However, epitopes which involve
non-contiguous
amino acids may require more amino acids, for example, at least 15, 25, or 50
amino acids.
[080] An antibody which specifically binds to an epitope of a peptide of this
invention may be
used therapeutically, as well as in immunochemical assays, such as Western
blots, ELISAs,
radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other
immunochemical assays known in the art. Various immunoassays may be used to
identify
antibodies having the desired specificity. Numerous protocols for competitive
binding or
immunoradiometric assays are well known in the art. Such immunoassays
typically involve the
measurement of complex formation between an immunogen and an antibody which
specifically
binds to the immunogen.
[081] Typically, an antibody which specifically binds to a peptide of this
invention provides a
detection signal higher than a detection signal provided with other proteins
when used in an
immunochemical assay. Antibodies which specifically bind to a peptide of this
invention do not
24
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WO 2006/121860 PCT/US2006/017411
detect other proteins in immunochemical assays and can immunoprecipitate a
peptide of this
invention from solution.
[082] Peptides of this invention may be used to immunize a mammal, such as a
mouse, rat,
rabbit, guinea pig, goat, sheep, monkey, or human, to produce polyclonal
antibodies. If desired, a
peptide of this invention may be conjugated to a carrier protein, such as
bovine serum albumin,
thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species,
various adjuvants
may be used to increase the immunological response. Such adjuvants include;
but are not limited
to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface
active substances
(e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
keyhole limpet
hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli
Calmette-Guerin)
and Corynebacterium parvum are especially useful.
[083] Monoclonal antibodies which specifically bind to a peptide of this
invention may be
prepared using any technique which provides for the production of antibody
molecules by
continuous cell lines in culture. These techniques include, but are not
limited to, the hybridoma
technique, the human B cell hybridoma technique, and the EBV hybridoma
technique (Kohler, et
al., Nature 256:495-97, 1985; Kozbor, et al., J. Immunol. Methods 81:3142,
1985; Cote, et al.,
Proc. Natl. Acad. Sci. 80:2026-30, 1983; Cole, et al., Mol. Cell Biol. 62:109-
20, 1984).
[084] In addition, techniques developed for the production of "chimeric
antibodies," the splicing
of mouse antibody genes to human antibody genes to obtain a molecule with
appropriate antigen
specificity and biological activity, may be used (Morrison, et al., Proc.
Nati. Acad. Sci. 81:6851-55,
1984; Neuberger, et al., Nature 312:604-08, 1984; Takeda, et al., Nature
314:452-54, 1985).
Monoclonal and other antibodies also can be "humanized" to prevent a patient
from mounting an
immune response against the antibody when it is used therapeutically. Such
antibodies may be
sufficiently similar in sequence to human antibodies to be used directly in
therapy or may require
alteration of a few key residues. Sequence differences between rodent
antibodies and human
sequences may be minimized by replacing residues which differ from those in
the human
sequences by site directed mutagenesis of individual residues or by grafting
of entire
complementarity determining regions. Alternatively, humanized antibodies may
be produced using
recombinant methods (see, e.g., GB2188638B). Antibodies which specifically
bind to a peptide of
this invention may_ contain antigen binding sites which are either partially
or fully humanized, as
disclosed in U.S. Patent No. 5,565,332.
[085] Alternatively, techniques described for the production of single chain
antibodies may be
adapted using methods known in the art to produce single chain antibodies
which specifically bind
to a peptide of this invention. Antibodies with related specificity, but of
distinct idiotypic
composition, can be generated by chain shuffling from random combinatorial
immunoglobin
libraries (Burton, Proc. Natl. Acad. Sci. 88:11120-23, 1991).
[086] Single-chain antibodies also may be constructed using a DNA
amplification method, such
as PCR, using hybridoma cDNA as a template (Thirion, et al., Eur. J. Cancer
Prev. 5:507-11,
CA 02607566 2007-11-05
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1996). Single-chain antibodies can be mono- or bispecific, and can be bivalent
or tetravalent.
Construction of tetravalent, bispecific single-chain antibodies is taught, for
example, in Coloma &
Morrison (Nat. Biotechnol. 15:159-63, 1997). Construction of bivalent,
bispecific single-chain
antibodies is taught in Mallender & Voss (J. Biol. Chem. 269:199-206, 1994).
[087] A nucleotide sequence encoding a single-chain antibody may be
constructed using
manual or automated nucleotide synthesis, cloned into an expression construct
using standard
recombinant DNA metliods; and introduced into a cell to express the coding
sequence, as
described below. Alternatively, single-chain antibodies can be produced
directly using, for
example, filamentous phage technology (Verhaar, et al., Int. J. Cancer 61:497-
501, 1995; Nicholls,
et al., J. Immunol. Meth. 165:81-91, 1993).
[088] Antibodies which specifically bind to a peptide of this invention may
also be produced by
inducing in vivo production in the lymphocyte population or by screening
immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in the literature
(Orlandi, et al., Proc.
Natl. Acad. Sci. 86:38333-37, 1989; Winter, et al., Nature 349:293-99, 1991).
[089] Other types of antibodies may be constructed and used therapeutically in
methods of the
invention. For example, chimeric antibodies may be constructed as disclosed in
WO 93/03151.
Binding proteins which are derived from immunoglobulins and which are
multivalent and
multispecific, such as the "diabodies" also can be prepared (see, e.g., WO
94/13804,).
[090] Human antibodies with the ability to bind to the peptides of this
invention may also be
identified from the MorphoSys HuCAL library as follows. A peptide of this
invention may be
coated on a microtiter plate and incubated with the MorphoSys HuCAL Fab phage
library. Those
phage-linked Fabs not binding to the peptide of this invention can be washed
away from the plate,
leaving only phage which tightly bind to the peptide of this invention. The
bound phage can be
eluted, for example, by a change in pH or by elution with E. coli and
amplified by infection of E. coli
hosts. This panning process can be repeated once or twice to enrich for a
population of antibodies
that tightly bind to the peptide of this invention. The Fabs from the enriched
pool are then
expressed, purified, and screened in an EL.ISA assay.
[091] Antibodies according to the invention may be purified by methods well
known in the art.
For example, antibodies may be affinity purified by passage over a column to
which a peptide of
this invention is bound. The bound antibodies can then be eluted from the
column using a buffer
with a high salt concentration.
Methods of Use
[092] As used herein, various terms are defined below.
[093] When introducing elements of the present invention or the preferred
embodiment(s)
thereof, the articles "a," "an," "the," and "said" are intended to mean that
there are one or more of
the elements. The terms "comprising," "including," and "having" are intended
to be inclusive and
mean that there may be additional elements other than the listed elements.
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[094] The term "subject" as used herein includes mammals (e.g., humans and
animals).
[095] The term "treatment" includes any process, action, application, therapy,
or the like,
wherein a subject, including a human being, is provided medical aid with the
object of improving
the subject's condition, directly or indirectly, or slowing the progression of
a condition or disorder in
the subject.
[096] The term "combination therapy" or "co-therapy" means the administration
of two or more
therapeutic agents to treat, for example, diabetes. Such administration
encompasses co-
administration of two or more therapeutic agents in a substantially
simultaneous manner, such as
in a single capsule having a fixed ratio of active ingredients or in multiple,
separate capsules for
each inhibitor agent. In addition, such administration encompasses use of each
type of
therapeutic agent in a sequential manner.
[097] The phrase "therapeutically effective" means the amount of each agent
administered that
will achieve the goal of improvement in a diabetic condition or disorder
severity, while avoiding or
minimizing adverse side effects associated with the given therapeutic
treatment.
[098] The term "pharmaceutically acceptable" means that the subject item is
appropriate for use
in a pharmaceutical product.
[099] The peptides of Formula (I) are expected to be valuable as therapeutic
agents (e.g., Table
1). Accordingly, an embodiment of this invention includes a method of treating
the various
conditions in a patient (including mammals) which comprises administering to
said patient a
composition containing an amount of the peptide of Formula (I), that is
effective in treating the
target condition.
[100] The peptides of the present invention, as a result of the ability to
stimulate insulin secretion
from pancreatic islet cells in vitro, and by causing a decrease in blood
glucose in vivo, may be
employed in treatment diabetes, including both type 1 and type 2 diabetes (non-
insulin dependent
diabetes mellitus). Such treatment may also delay the onset of diabetes and
diabetic
complications. The peptides may be used to prevent subjects with impaired
glucose tolerance
from proceeding to develop type 2 diabetes. Other diseases and conditions that
may be treated or
prevented using peptides of the invention in methods of the invention include:
Maturity-Onset
Diabetes Of the Young (MODY) (Hermah, et al:, Diabetes 43:40, 1994); Latent
Autoimmune
Diabetes Adult (LADA) (Zimmet, et al., Diabetes Med. 11:299, 1994); impaired
glucose tolerance
(IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care
22 (Supp. 1):S5,
1999); impaired fasting glucose (IFG) (Charles, et al., Diabetes 40:796,
1991); gestational diabetes
(Metzger, Diabetes, 40:197, 1991); and metabolic syndrome X.
[101] The peptides of the present invention may also be effective in such
disorders as obesity,
and in the treatment of cardiovascular disease, including atherosclerosis,
coronary heart disease,
coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels,
hypertension;
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cerebrovascular disease; and peripheral vessel disease; and for the treatment
of
neurodegenerative diseases (including Parkinson's and Alzheimer's).
[102] In addition, the peptides of the invention may be used to enhance
satiety and reduce food
intake, and in the treatment of obesity (Flint, et al., J. Clin. Invest.
101:515-520, 1998). In addition,
the peptides of the invention may be used for the treatment of myocardial
infarction (Nikolaidis, et
al., Circ. 109:962-965, 2004), as well as improving endothelial dysfunction
(Nystrom, Am. J.
Physiol. Endocrinol. Metab. 287:E1209-E1215). The peptides of the invention
may also be used
for the treatment of neurodegenerative diseases (e.g., Alzheimer's and
Parkinson's diseases) and
other diseases which would benefit from the neuroprotective effects of GLP-1
(During, et al., Nat.
Med. 9:1173-1179, 2003).
[103] The peptides of the present invention may also be useful for tYeating
physiological
disorders related to, for example, regulation of insulin sensitivity and blood
glucose levels, which
are involved in, for example, abnormal pancreatic R-cell function, reduction
in the pancreatic P-cell
mass, insulin secretion, tissue sensitivity to insulin, liposarcoma cell
growth, polycystic ovarian
disease, chronic anovulation, hyperandrogenism, progesterone production,
steroidogenesis, redox
potential and oxidative stress in cells, plasma triglycerides, HDL, and LDL
cholesterol levels, and
the like.
[104] Peptides of the invention may also be used in methods of the invention
to treat secondary
causes of diabetes (Expert Committee on Classification of Diabetes Mellitus,
Diabetes Care 22
(Supp. 1):S5, 1999). Such secondary causes include glucocorticoid excess,
growth hormone
excess, pheochromocytoma, and drug-induced diabetes. Drugs that may induce
diabetes include,
but are not limited to, pyriminil, nicotinic acid, glucocorticoids, phenytoin,
thyroid hormone, R-
adrenergic agents, a-interferon and drugs used to treat HIV infection.
[105] The peptides of the present invention may be used alone or in
combination with additional
therapies and/or compounds known to those skilled in the art in the treatment
of diabetes and
related disorders. Alternatively, the methods and peptides described herein
may be used, partially
or completely, in combination therapy.
[106] The peptides of the invention may also be administered in combination
with other known
therapies for the treatment of diabetes, including PPAR ligands (e.g.,
agonists, antagonists),
insulin secretagogues, for example, sulfonylurea drugs and non-sulfonylurea
secretagogues, a-
glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic
glucose output lowering
compounds, insulin and insulin derivatives, and anti-obesity drugs. Such
therapies may be
administered prior to, concurrently with, or following administration of the
peptides of the invention.
Insulin and insulin derivatives include both long and short acting forms and
formulations of insulin.
PPAR ligands may include agonists and/or antagonists of any of the PPAR
receptors or
combinations thereof. For example, PPAR ligands may include ligands of PPAR-a,
PPAR-7,
PPAR-S or any combination of two or three of the receptors of PPAR. PPAR
ligands include, for
example, rosiglitazone, troglitazone, and pioglitazone. Sulfonylurea drugs
include, for example,
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glyburide, glimepiride, chlorpropamide, tolbutamide, and glipizide. a-
glucosidase inhibitors that
may be useful in treating diabetes when administered with a peptide of the
invention include
acarbose, miglitol, and voglibose. Insulin sensitizers that may be useful in
treating diabetes
include PPAR-y agonists such as the glitazones (e.g., troglitazone,
pioglitazone, englitazone,
MCC-555, rosiglitazone, and the like) and other thiazolidinedione and non-
thiazolidinedione
compounds; biguanides such as metformin and phenformin; protein tyrosine
phosphatase-1 B
(PTP-1 B) inhibitors; dipeptidyl peptidase IV (DPPIV) inhibitors; and 11 beta-
HSD inhibitors.
Hepatic glucose output lowering compounds that may be useful in treating
diabetes when
administered with a peptide of the invention include, for example, glucagon
anatgonists and
metformin, such as Glucophage and Glucophage XR. Insulin secretagogues that
may be useful in
treating diabetes when administered with a peptide of the invention include
sulfonylurea and non-
sulfonylurea drugs: GIP, VIP, PACAP, secretin, and derivatives thereof;
nateglinide, meglitinide,
repaglinide, glibenclamide, glimepiride, chlorpropamide, and glipizide. In one
embodiment of the
invention, peptides of the invention are used in combination with insulin
secretagogues to increase
the sensitivity of pancreatic (i-cells to the insulin secretagogue.
[107] Peptides of the invention may also be used in methods of the invention
in combination with
anti-obesity drugs. For example, anti-obesity drugs include (3-3 adrenergic
receptor agonists such
as CL 316,243; cannabinoid (e.g., CB-1) antagonists such as Rimonabant;
neuropeptide-Y
receptor antagonists; neuropeptide Y5 inhibitors; apo-B/MTP inhibitors; 11 P-
hydroxy steroid
dehydrogenase-1 inhibitors; peptide YY3.36 or analogs thereof; MCR4 agonists;
CCK-A agonists;
monoamine reuptake inhibitors; sympathomimetic agents; dopamine agonists;
melanocyte-
stimulating hormone receptor analogs; melanin concentrating hormone
antagonists; leptin; leptin
analogs; leptin receptor agonists; galanin antagonists; lipase inhibitors;
bombesin agonists;
thyromimetic agents; dehydroepiandrosterone or analogs thereof; glucocorticoid
receptor
antagonists; orexin receptor antagonists; ciliary neurotrophic factor; ghrelin
receptor antagonists;
histamine-3 receptor antagonists; neuromedin U receptor agonists; appetite
suppressants, such
as, for example, sibutramine (Meridia); and lipase inhibitors, such as, for
example, orlistat
(Xenical). The peptides of the present invention may also be administered in
combination with a
drug compound that modulates digestion and/or metabolism such as drugs that
modulate
thermogenesis, lipolysis, gut motility, fat absorption, and satiety.
[108] Peptides of the invention may also be used in methods of the invention
in combination with
drugs commonly used to treat lipid disorders. Such drugs include, but are not
limited to, HMG-CoA
reductase inhibitors, nicotinic acid, fatty acid lowering compounds (e.g.,
acipimox); lipid lowering
drugs (e.g., stanol esters, sterol glycosides such as tiqueside, and
azetidinones such as
ezetimibe), ACAT inhibitors (such as avasimibe), bile acid sequestrants, bile
acid reuptake
inhibitors, microsomal triglyceride transport inhibitors, and fibric acid
derivatives. HMG-CoA
reductase inhibitors include, for example, lovastatin, simvastatin,
pravastatin, fluvastatin,
atorvastatin, rivastatin, itavastatin, cerivastatin, and ZD-4522. Fibric acid
derivatives include, for
example, clofibrate, fenofibrate, bezafibrate, ciprofibrate, beclofibrate,
etofibrate, and gemfibrozil.
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Sequestrants include, for example, cholestyramine, colestipol, and
dialkylaminoalkyl derivatives of
a cross-linked dextran.
[109] Furthermore, peptides of the invention may also be administered in
combination with anti-
hypertensive drugs, such as, for example, (3-blockers and ACE inhibitors.
Examples of additional
anti-hypertensive agents for use in combination with the peptides of the
present invention include
calcium channel blockers (L-type and T-type; e.g., diltiazem, verapamil,
nifedipine, amlodipine and
mybefradil), diuretics (e.g., chlorothiazide, hydrochlorothiazide,
flumethiazide, hydroflumethiazide,
bendroflumethiazide, methylchlorothiazide, trichloromethiazide, polythiazide,
benzthiazide,
ethacrynic acid tricrynafen, chlorthalidone, furosemide, musolimine,
bumetanide, triamtrenene,
amiloride, spironolactone), renin inhibitors, ACE inhibitors (e.g., captopril,
zofenopril, fosinopril,
enalapril, ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril,
lisinopril), AT-1 receptor
antagonists (e. g., losartan, irbesartan, valsartan), ET receptor antagonists
(e.g., sitaxsentan,
atrsentan, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors
(dual NEP-ACE
inhibitors) (e.g., omapatrilat and gemopatrilat), and nitrates.
[110] Such co-therapies may be administered in any combination of two or more
drugs (e.g., a
peptide of the invention in combination with an insulin sensitizer and an anti-
obesity drug). Such
co-therapies may be administered in the form of pharmaceutical compositions,
as described
above.
Pharmaceutical Compositions
[111] Based on well known assays used to determine the efficacy for treatment
of conditions
identified above in mammals, and by comparison of these results with the
results of known
medicaments that are used to treat these conditions, the effective dosage of
the peptides of this
invention can readily be determined for treatment of each desired indication.
The amount of the
active ingredient to be administered in the treatment of one of these
conditions can vary widely
according to such considerations as the particular peptide and dosage unit
employed, the mode
of administration, the period of treatment, the age and sex of the patient
treated, and the nature
and extent of the condition treated.
[112] The total amount of the active ingredient to be administered may
generally range from,
for example; about 0.0001 mg/kg to about 200 mg/kg, or from about 0.001 mg/kg
to about 200
mg/kg body weight per day. A unit dosage may contain from, for example, about
0.05 mg to
about 1500 mg of active ingredient, and may be administered one or more times
per day. The
daily dosage for administration by injection, including intravenous,
intramuscular, subcutaneous,
and parenteral injections, and use of infusion techniques may be from, for
example, about 0.001
to about 200 mg/kg. The daily rectal dosage regimen may be from, for example,
about 0.001 to
about 200 mg/kg of total body weight. The transdermal concentration may be
that required to
maintain a daily dose of from, for example, about 0.001 to about 200 mg/kg.
[113] Of course, the specific initial and continuing dosage regimen for each
patient will vary
according to the nature and severity of the condition as determined by the
attending
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diagnostician, the activity of the specific peptide employed, the age of the
patient, the diet of the
patient, time of administration, route of administration, rate of excretion of
the drug, drug
combinations, and the like. The desired mode of treatment and number of doses
of a peptide of
the present invention may be ascertained by those skilled in the art using
conventional treatment
tests.
[114] The peptides of this invention may be utilized to achieve the desired
pharmacological
effect by administration to a subject in heed thereof in an appropriately
formulated pharmaceutical
composition. A subject, for example, may be a mammal, including a human, in
need of treatment
for a particular condition or disease. Therefore, the present invention
includes pharmaceutical
compositions which are comprised of a pharmaceutically acceptable carrier and
a
pharmaceutically effective amount of a peptide of the present invention. A
pharmaceutically
acceptable carrier is any carrier which is relatively non-toxic and innocuous
to a patient at
concentrations consistent with effective activity of the active ingredient so
that any side effects
ascribable to the carrier do not vitiate the beneficial effects of the active
ingredient. A
pharmaceutically effective amount of a peptide is that amount which produces a
result or exerts
an influence on the particular condition being treated. The peptides of the
present invention may
be administered with a pharmaceutically-acceptable carrier using any effective
conventional
dosage unit forms, including, for example, immediate and timed release
preparations, orally,
parenterally, topically, or the like.
[115] For oral administration, the peptides may be formulated into solid or
liquid preparations
such as, for example, capsules, pills, tablets, troches, lozenges, melts,
powders, solutions,
suspensions, or emulsions, and may be prepared according to methods known to
the art for the
manufacture of pharmaceutical compositions. The solid unit dosage forms may be
a capsule
which can be of the ordinary hard- or soft-shelled gelatin type containing,
for example, surfactants,
lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and
corn starch.
[116] In another embodiment, the peptides of this invention may be tableted
with conventional
tablet bases such as lactose, sucrose, and cornstarch in combination with
binders such as acacia,
cornstarch, or gelatin; disintegrating agents intended to assist the break-up
and dissolution of the
tablet following administration such as potato starch, alginic acid, corn
starch, and guar gum;
lubricants intended to improve the flow_of.-tablet granulation and to prevent
the adhesion of tablet
material to the surfaces of the tablet dies and punches, for example, talc,
stearic acid, or
magnesium, calcium or zinc stearate; dyes; coloring agents; and flavoring
agents intended to
enhance the aesthetic qualities of the tablets and make them more acceptable
to the patient.
Suitable excipients for use in oral liquid dosage forms include diluents such
as water and alcohols,
for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with
or without the addition
of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying
agent. Various
other materials may be present as coatings or to otherwise modify the physical
form of the dosage
unit. For instance tablets, pills or capsules may be coated with shellac,
sugar or both.
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[117] Dispersible powders and granules are suitable for the preparation of an
aqueous
suspension. They provide the active ingredient in admixture with a dispersing
or wetting agent, a
suspending agent, and one or more preservatives. Suitable dispersing or
wetting agents and
suspending agents are exemplified by those already mentioned above. Additional
excipients, for
example, those sweetening, flavoring and coloring agents described above, may
also be present.
[118] The pharmaceutical compositions of this invention may also be in the
form of oil-in-water
emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a
mixture of vegetable
oils. Suitable emulsifying agents may be (1) naturally occurring gums such as
gum acacia and
gum tragacanth, (2) naturally occurring phosphatides such as soy bean and
lecithin, (3) esters or
partial esters derived from fatty acids and hexitol anhydrides, for example,
sorbitan monooleate,
and (4) condensation products of said partial esters with ethylene oxide, for
example,
polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening
and flavoring
agents.
[119] Syrups and elixirs may be formulated with sweetening agents such as, for
example,
glycerol, propylene glycol, sorbitol, or sucrose. Such formulations may also
contain a demulcent,
and preservative, flavoring and coloring agents.
[120] The peptides of this invention may also be administered parenterally,
that is,
subcutaneously, intravenously, intramuscularly, or interperitoneally, as
injectable dosages of the
peptide in a physiologically acceptable diluent with a pharmaceutical carrier
which may be a sterile
liquid or mixture of liquids such as water, saline, aqueous dextrose and
related sugar solutions; an
alcohol such as ethanol, isopropanol, or hexadecyl alcohol; glycols such as
propylene glycol or
polyethylene glycol; glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-
methanol, ethers such as
poly(ethyleneglycol) 400; an oil; a fatty acid; a fatty acid ester or
glyceride; or an acetylated fatty
acid glyceride with or without the addition of a pharmaceutically acceptable
surfactant such as a
soap or a detergent, suspending agent such as pectin, carbomers,
methycellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent
and other
pharmaceutical adjuvants.
[121] Illustrative of oils which can be used in the parenteral formulations of
this invention are
those of petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil,
sesame oil,-cottonseed oil, corn oil; olive oil, petrolatum, and mineral oil.
Suitable fatty acids
include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid
esters are, for example,
ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali
metal, ammonium, and
triethanolamine salts and suitable detergents include cationic detergents, for
example, dimethyl
dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates;
anionic detergents,
for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and
sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty
acid alkanolamides,
and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for
example, alkyl-
beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as
well as mixtures.
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[122] The parenteral compositions of this invention may typically contain from
about 0.5% to
about 25% by weight of the active ingredient in solution. Preservatives and
buffers may also be
used advantageously. In order to minimize or eliminate irritation at the site
of injection, such
compositions may contain a non-ionic surfactant having a hydrophile-lipophile
balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such formulation
ranges from about 5% to
about 15% by weight. The surfactant can be a single component having the above
HLB or can be
a mixture of two or more components having the desired HLB.
[123] Illustrative of surfactants used in parenteral formulations are the
class of polyethylene
sorbitan fatty acid esters, for example, sorbitan monooleate and the high
molecular weight adducts
of ethylene oxide with a hydrophobic base, formed by the condensation of
propylene oxide with
propylene glycol.
[124] The pharmaceutical compositions may be in the form of sterile injectable
aqueous
suspensions. Such suspensions may be formulated according to known methods
using suitable
dispersing or wetting agents and suspending agents such as, for example,
sodium
carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium
alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting
agents which may be
a naturally occurring phosphatide such as lecithin, a condensation product of
an alkylene oxide
with a fatty acid, for example, polyoxyethylene stearate, a condensation
product of ethylene oxide
with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol,
a condensation
product of ethylene oxide with a partial ester derived form a fatty acid and a
hexitol such as
polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene
oxide with a partial
ester derived from a fatty acid and a hexitol anhydride, for example
polyoxyethylene sorbitan
monooleate.
[125] The sterile injectable preparation may also be a sterile injectable
solution or suspension in
a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents
that may be
employed are, for example, water, Ringer's solution, and isotonic sodium
chloride solution. In
addition, sterile fixed oils are conventionally employed as solvents or
suspending media. For this
purpose, any bland, fixed oil may be employed including synthetic mono or
diglycerides. In
addition, fatty acids such as oleic acid may be used in the preparation of
injectables.
[126] A composition of the invention may also be administered in the form of
suppositories for
rectal administration of the drug. These compositions may be prepared by
mixing the drug with a
suitable non-irritating excipient which is solid at ordinary temperatures but
liquid at the rectal
temperature and will therefore melt in the rectum to release the drug. Such
materials are, for
example, cocoa butter and polyethylene glycol.
[127] Another formulation employed in the methods of the present invention
employs
transdermal delivery devices ("patches"). Such transdermal patches may be used
to provide
continuous or discontinuous infusion of the peptides of the present invention
in controlled amounts.
The construction and use of transdermal patches for the delivery of
pharmaceutical agents is well
33
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known in the art (see, e.g., U.S. Patent No. 5,023,252, incorporated herein by
reference). Such
patches may be constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical
agents.
[128] Another formulation employs the use of biodegradable microspheres that
allow controlled,
sustained release of the peptides and PEGylated peptides of this invention.
Such formulations can
be comprised of synthetic polymers or copolymers. Such formulations allow for
injection,
inhalation, riasal or oral adriministration. The cbnstruction arid use of
biodegradable microspheres
for the delivery of pharmaceutical agents is well known in the art (e.g., US
Patent No. 6, 706,289,
incorporated herein by reference).
[129] It may be desirable or necessary to introduce the pharmaceutical
composition to the
patient via a mechanical delivery device. The construction and use of
mechanical delivery devices
for the delivery of pharmaceutical agents is well known in the art. For
example, direct techniques
for administering a drug directly to the brain usually involve placement of a
drug delivery catheter
into the patient's ventricular system to bypass the blood-brain barrier. One
such implantable
delivery system, used for the transport of agents to specific anatomical
regions of the body, is
described in U.S. Patent No. 5,011,472, incorporated herein by reference.
[130] The compositions of the invention may also contain other conventional
pharmaceutically
acceptable compounding ingredients, generally referred to as carriers or
diluents, as necessary or
desired. Any of the compositions of this invention may be preserved by the
addition of an
antioxidant such as ascorbic acid or by other suitable preservatives.
Conventional procedures for
preparing such compositions in appropriate dosage forms can be utilized.
[131] Commonly used pharmaceutical ingredients which may be used as
appropriate to
formulate the composition for its intended route of administration include:
acidifying agents, for
example, but are not limited to, acetic acid, citric acid, fumaric acid,
hydrochloric acid, nitric acid;
and alkalinizing agents such as, but are not limited to, ammonia solution,
ammonium carbonate,
diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium
carbonate,
sodium hydroxide, triethanolamine, trolamine.
[132] The peptides identified by the methods described herein may be
administered as the sole
pharmaceutical agent or in combination with one or more other pharmaceutical
agents where the
combination causes no unacceptable adverse effects. For example, the peptides
of this invention
can be combined with known anti-diabetic, or with known anti-obesity,
cardiovascular or other
indication agents, and the like, as well as with admixtures and combinations
thereof.
[133] The peptides identified by the methods described herein may also be
utilized, in free base
form or in compositions, in research and diagnostics, or as analytical
reference standards, and the
like. Therefore, the present invention includes compositions which are
comprised of an inert
carrier and an effective amount of a peptide of the present invention. An
inert carrier is any
material which does not interact with the peptide to be carried and which
lends support, means of
conveyance, bulk, traceable material, and the like to the peptide to be
carried. An effective
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amount of peptide is that amount which produces a result or exerts an
influence on the particular
procedure being performed.
[134] Formulations suitable for subcutaneous, intravenous, intramuscular, and
the like; suitable
pharmaceutical carriers; and techniques for formulation and administration may
be prepared by
any of the methods well known in the art (see, e.g., Remington's
Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 20t" edition, 2000)
[135] Peptides are known to undergo hydrolysis, deamidation, oxidation,
racemization and
isomerization in aqueous and non-aqueous environment. Degradation such as
hydrolysis,
deamidation or oxidation can readily detected by capillary electrophoresis.
Enzymatic degradation
notwithstanding, peptides having a prolonged plasma half-life, or biological
resident time, should,
at minimum, be stable in aqueous solution. For example, a peptide exhibits
less than 10%
degradation over a period of one day at body temperature or less than 5%
degradation over a
period of one day at body temperature. Stability (i.e., less than a few
percent of degradation) over
a period of weeks at body temperature will allow less frequent dosing.
Stability in the magnitude of
years at refrigeration temperature will allow the manufacturer to present a
liquid formulation, thus
avoid the inconvenience of reconstitution. Additionally, stability in organic
solvent would provide
peptide be formulated into novel dosage forms such as implant.
[136] The structures, materials, compositions, and methods described herein
are intended to be
representative examples of the invention, and it will be understood that the
scope of the invention
is not limited by the scope of the examples. Those skilled in the art will
recognize that the
invention may be practiced with variations on the disclosed structures,
materials, compositions and
methods, and such variations are regarded as within the ambit of the
invention.
[137] The following examples are presented to illustrate the invention
described herein, but
should not be construed as limiting the scope of the invention in any way.
EXAMPLES
[138] In order that this invention may be better understood, the following
examples are set forth.
These examples are for the purpose of illustration only, and are not to be
construed as limiting the
scope of the invention in any manner. All publications mentioned herein are
incorporated by
reference in their entirety.
Example 1. Preparation of N-terminal Modifying Compounds
[139] Air and moisture sensitive liquids and solutions were transferred via
syringe or cannula,
and introduced into reaction vessels through rubber septa. Commercial grade
reagents and
solvents were used without further purification. The term "concentration under
reduced pressure"
refers to use of a Buchi rotary evaporator at approximately 15 mm of Hg. All
temperatures are
reported uncorrected in degrees Celsius ( C). Thin layer chromatography (TLC)
was performed on
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EM Science pre-coated glass-backed silica gel 60 A F-254 250 pm plates. Column
chromatography (flash chromatography) was performed on a Biotage system using
32-63 micron,
60 A, silica gel pre-packed cartridges. Purification using preparative
reversed-phase HPLC
chromatography were accomplished using a Gilson 215 system and a YMC Pro-C18
AS-342 (150
x 20 mm I.D.) column. Typically, the mobile phase used was a mixture of H20
(A) and MeCN (B).
The water could be mixed or not with 0.1 % TFA. A typical gradient was:
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Time Flow
[min.] A: % B: % [mUmin.]
0.50 90.0 10.0 1.0
11.00 0.0 100.0 1.0
14.00 0.0 100.0 1.0
15.02 100.0 0.0 1.0
[140] Electron impact mass spectra (EI-MS or GC-MS) were obtained with a
Hewlett Packard
5989A mass spectrometer equipped with a Hewlett Packard 5890 Gas Chromatograph
with a J &
W DB-5 column (0.25 uM coating; 30 m x 0.25 mm). The ion source was maintained
at 250 C and
spectra were scanned from 50-800 amu at 2 sec per scan. High pressure liquid
chromatography-
electrospray mass spectra (LC-MS) were obtained using a Hewlett-Packard 1100
HPLC equipped
with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC
pro C-18 column (2
x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with
electrospray ionization.
Spectra were scanned from 120-1200 amu using a variable ion time according to
the number of
ions in the source. The eluents were A: 2% acetonitrile in water with 0.02%
TFA and B: 2% water
in acetonitrile with 0.018% TFA. Gradient elution from 10% to 95% B over 3.5
minutes at a
flowrate of 1.0 mUmin was used with an initial hold of 0.5 minutes and a final
hold at 95% B of 0.5
minutes. Total run time was 6.5 minutes. For consistency in characterization
data, the retention
time (RT) is reported in minutes at the apex of the peak as detected by the UV-
Vis detector set at
254 nm.
[141] Routine one-dimensional NMR spectroscopy was performed on 300 or 400 MHz
Varian
Mercury-plus spectrometers. The samples were dissolved in deuterated solvents
obtained from
Cambridge Isotope Labs, and transferred to 5 mm ID Wilmad NMR tubes. The
spectra were
acquired at 293 K. The chemical shifts were recorded on the ppm scale and were
referenced to
the appropriate residual solvent signals, such as 2.49 ppm for DMSO-d6, 1.93
ppm for CD3CN,
3.30 ppm for CD3OD, 5.32 ppm for CD2CI2, and 7.26 ppm for CDCI3 for 1H
spectra, and 39.5 ppm
for DMSO-d6, 1.3 ppm for. CD3CN, 49.0 ppm for CD3OD, 53.8 ppm for CD2CI2, and
77.0 ppm for
CDCI3 for 13C spectra. General methods of preparation are illustrated in the
reaction schemes,
and by the specific preparative examples that follow.
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Abbreviations and Acronyms
[142] When the following abbreviations are used throughout the disclosure,
they have the
following meaning:
Ac acetyl
AcOH acetic acid
Boc t-butoxycarbonyl
Bu butyl
CDCI3 deuterochloroform
Celite registered trademark of Celite Corp. brand of diatomaceous earth
CI chemical ionization
d doublet
dd doublet of doublet
ddd doublet of doublet of doublet
DME dimethoxyethane
DMF N,N-dimethyl formamide
DMSO dimethylsulfoxide
DMSO-d6 dimethylsulfoxide-ds
dppf 1,1'-bis(diphenylphosphino)ferrocene
El electron impact ionization
El - MS electron impact - mass spectrometry
Et ethyl
EtOH ethanol
EtOAc ethyl acetate
g gram
GC-MS gas chromatography - mass spectrometry
h hour(s)
'H NMR proton nuclear magnetic resonance
Hex hexanes
HPLC high performance liquid chromatography
LC-MS liquid chromatography/mass spectroscopy
LDA lithium diisopropylamide
m multiplet
M molar
m/z mass over charge
Me methyl
MeCN acetonitrile
mg milligram
MHz megahertz
min minute(s)
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mol mole
mmol millimole
MS mass spectrometry
N normal
NMR nuclear magnetic resonance
NaOAc sodium acetate
Pd/C palladium on carbon
PdCI2(dppf).CH2CI2 [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium
(II)
complex with dichloromethane (1:1)
Ph phenyl
PPh3 triphenylphosphine
ppm parts per million
Pr propyl
q quartet
qt quintet
quant. quantitative
Rf TLC retention factor
rt room temperature
RT retention time (HPLC)
s singlet
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin layer chromatography
TMS tetramethylsilane
v/v volume per unit volume
vol volume
w/w weight per unit weight
EXAMPLE 1A
[143]_ Preparation of (2-Mercapto-iH-benzimidazol-4yl)acetic acid
N
N-SH
N
HO2~
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[144] Step 1. Preparation of Methyl (2-chloro- 1H-benzimidazol- 1-yl)acetate
cc\>-cl
MeO2C)
[145] NaH (157.3 mg of a 60% dispersion in mineral oil, 3.93 mmol) was
suspended in dry DMF
(5 mL) and hexanes (0.5 mL). 2-chlorobenzimidazole (500 mg, 3.28 mmol) was
added, and the
resulting solution was allowed to stir at rt for 1 h. Methyl bromoacetate
(0.37 mL, 3.93 mmol) was
added, and the solution was allowed to stir overnight at rt. The reaction
mixture was then diluted
with water, and the resulting tan precipitate was collected by filtration,
triturated with ether, and
dried, yielding 385 mg (52%) of crude material. 'H NMR (400 MHz, CD3CN) S 3.78
(s, 3H), 5.03
(s, 2H), 7.28-7.38 (m, 2H), 7.43 (d, 1 H), 7.65 (d, 1 H).
[146] Step 2. Preparation of Methyl (2-mercapto-1H-benzimidazo%1 yl)acetate
N
\>-SH
N
MeO2C)
[147] Methyl (2-chloro-1 H-benzimidazol-1-yl)acetate (150 mg, 0.67 mmol) and
thiourea (101.7
mg, 1.34 mmol) were heated to reflux in ethanol (30 mL) overnight. The
reaction mixture was
concentrated, and the residue was triturated with water and dried, yielding
142.5 mg (96%) of
crude product. 'H NMR (400 MHz, CD3CN) S 3.78 (s, 3H), 5.03 (s, 2H), 7.20-7.35
(m, 4H), 10.43
(bs, 1 H).
[148] Step 3. Preparation of (2-Mercapto- 1 H-benzimidazol- 1 -yl)acetic acid
ccN\3H
HO2C)
[149] Methyl (2-mercapto-lH-benzimidazol-1-yl)acetate (143 mg, 0.64 mmol) was
dissolved in
THF (1 mL), MeOH (1 mL), and water (0.5 mL). The solution was treated with
LiOH (17.0 mg,
0.71 mmol) and heated to 80 C for 4 h. The pH was then adjusted to pH 4 with 1
N HCI. The
solution was diluted with water and extracted with 5% EtOH/EtOAc. The organics
were dried over
MgSO4 and concentrated in vacuo, yielding 75.0 mg (56%) of the desired
product. LC/MS m/z
209.1 (M+H)+; RT 1.08 min. 'H NMR (400 MHz, CD3CN) $ 5.03 (s, 2H), 7.20-7.35
(m, 4H), 10.43
(bs, 1 H).
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EXAMPLE 1 B
[150] Preparation of Lithium 2-{(2-(tritylthio)ethyljaminojnicotinate
O
(N' O-Li+
NH
S
[151] Step 1. Preparation of Ethyl2-{[2-(tritylthio)ethylJamino}nicotinate
cTCO2Et
N NH
9 ~ ~ ~
S
~ /
\ ~
[152] Ethyl 2-chloronicotinate (100 mg, 0.54 mmol) was combined with 2-
(tritylthio)ethanamine
(344 mg, 1.08 mmol), cesium carbonate (438 mg, 1.35 mmol), and [1,1'-
bis(diphenylphosphino)
ferrocene]dichloropalladium(II) complex with dichloromethane (1:1) (110 mg,
0.13 mmol). The
solids were dissolved in dioxane (2 mL), water (I mL), and heated to reflux
over 48 h. The
reaction mixture was then diluted with EtOAc and washed with water and brine.
The organics
were dried over Na2SO4 and concentrated in vacuo. The crude residue was
purified by Biotage
column chromatography (10% EtOAc/hexane), yielding 215 mg (85%) of the desired
product. 1H
NMR (400 MHz, CD2CI2) & 1.40 (t, 3H), 2.45 (t, 2H), 3.38 (t, 2H), 4.37 (q,
2H), 7.03 (m, 1 H), 7.18-
7.45 (m, 16H), 8.18 (d, 1 H), 8.45 (d, 1 H).
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[153] Step 2. Preparation of Lithium 2-([2-(tritylthio)ethyl]amino}nicotinate
O
(N) O"Lf+
NH
9 ' ~
lS
~
\ ~
[154] Ethyl 2-{[2-(tritylthio)ethyl]amino}nicotinate (215 mg, 0.46 mmol) was
dissolved in THF
(1 mL), MeOH (1 mL), and water (0.5 mL). The solution was treated with LiOH
(12.1 mg,
0.50 mmol) and heated to 80 C for 4 h. The crude aqueous mixture was extracted
with ether to
remove impurities. The solution was then diluted with water and extracted with
5% EtOH/EtOAc.
The EtOH/EtOAc extracts were dried over MgSO4 and concentrated in vacuo,
yielding 55.0 mg
(27%) of the desired product. LC/MS m/z440.7 (M+H)+; RT 2.17 min. iH NMR (400
MHz, CD3CN)
S 2.38 (t, 2H), 3.32 (t, 2H), 7.08 (m, 1 H), 7.18-7.45 (m, 16H), 8.19 (d, 1
H), 8.45 (d, 1 H).
EXAMPLEIC
[155] Preparation of Lithium 1-("2-(tritylthio)ethyl]-1H-imidazole-2-
carboxylate
N O
~
ic
N O"Li+
S
- ~ \
[156] Step 1. Preparation of 1,1;1 "-([(2-
bromoethyl)thio]methanetriyl}tribenzene
__ . . _ B r_
S \ /
I ~ e \
[157] Triphenylmethylmercaptan (3.00 g, 10.9 mmol) was dissolved in THF (10
mL) and cooled
to 0 C. Lithium hexamethyldisilazide (10.85 ml of a I M solution in THF) was
added, and the
reaction mixture was allowed to stir for 30 min. The cooling bath was removed
and dibromoethane
(1.12 mL, 13.0 mmoi) was added. The reaction mixture was allowed to stir at rt
for an additional
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30 min and was concentrated in vacuo. The crude residue was dissolved in ethyl
acetate and
washed with water and brine. The organics were dried over Na2SO4 and
concentrated, yielding
3.44 g crude material (80% pure by 'H NMR integration). This material was used
without further
purification. iH NMR (400 MHz, CD2CI2) S 2.75 (t, 2H), 2.90 (t, 2H), 7.20-7.45
(m, 18.6H).
[158] Step 2. Preparation of 1-[2-(Tritylthio)ethyl]-lH-imidazole
>
N
S
[159] NaH (70.5 mg of a 60% suspension in mineral oil, 1.76 mmol) was
suspended in dry DMF
(3 mL) and hexanes (0.5 mL). Imidazole (100 mg, 1.47 mmol) was added, and the
resulting
solution was allowed to stir at rt for 1 h. 1,1',1"-{[(2-
bromoethyl)thio]methanetriyl}tribenzene
(845 mg, 1.76 mmol) was added, and the solution was allowed to stir at rt
overnight. The reaction
mixture was diluted with water and extracted with EtOAc. The organics were
washed with water
and brine and dried over Na2SO4. The crude product was purified by Biotage
column
chromatography (50% EtOAc/hexane, 1% Et3N), yielding 280 mg (51 %) of the
desired product. 1H
NMR (400 MHz, CD2CC2) S 2.63 (t, 2H), 3.48 (t, 2H), 6.70 (s, 1 H), 6.92 (s, 1
H), 7.19 (s, 1 H), 7.22-
7.45 (m, 15H).
[160] Step 3. Preparation of Lithium 1-[2-(tritylthio)ethylJ-1 H-imidazole-2-
carboxylate
l N O
N0"Li+
S
_ _ I \ [161] 1-[2-(Trityfthio)ethyl]-1H-imidazole (140 mg, 0.38 mmol) was
dissolved in dry
dichloromethane (1.5 mL) and treated with trichloroacetyl chloride (0.06 mL,
0.57 mmol) and N,N-
diisopropylethylamine (0.07 mL, 0.42 mmol). The reaction mixture was allowed
to stir overnight at
rt. The reaction mixture was then concentrated in vacuo. The crude residue was
dissolved in THF
(1 mL), MeOH (1 mL), and water (0.5 mL), treated with LiOH (18.1 mg, 0.76
mmol) and allowed to
stir at 80 C for 4 h. The crude reaction mixture was then concentrated in
vacuo. Purification by
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HPLC gave 70 mg (44%) of the desired product. LC(MS m/z414.9 (M+H)-,; RT 2.76
min. 'H NMR
(400 MHz, CD3OD) S 2.68 (t, 2H), 4.18 (t, 2H), 6.72 (s, 1 H), 6.85 (s, 1 H),
7.19-7.40 (m, 15H).
EXAMPLE 1 D
[162] Preparation of Lithium 4-((2-(tritylthio)ethyl]amino]pyrimidine-5-
carboxylate
0
N ~ O-Li+
N~ NH
S
[163] Step 1. Preparation of Ethyl 4-hydroxypyrimidine-5-carboxylate
CO2Et
N OH
'
[164] Diethyl malonate (3.14 mL, 20.7 mmol) was combined with N,N',N"-
methylidynetrisformamide (3.00 g, 20.7 mmol), and p-toluenesulfonic acid (356
mg, 2.07 mmol),
and the reaction mixture was heated to 180 C for 4 h. The resulting red oil
was allowed to cool to
rt overnight. The crude reaction mixture was dissolved in a minimum amount of
water and allowed
to crystallize overnight. The solids were collected by filtration, washed with
water, and dried,
yielding 822 mg (24%) of the desired product. 1H NMR (400 MHz, CD3OD) S 1.38
(t, 3H), 4.32 (q,
2H), 8.32 (s, 1 H), 8.60 (s, 1 H).
[165] Step 2. Preparation of ethyl 4-chloropyrimidine-5-carboxylate
CO2Et
N
N Cl
[166] Ethyl 4-hydroxypyrimidine-5-carboxylate (380 mg, 2.26 mmol) was
dissolved in THF
(5 mL) and treated with thionyl chloride (1.65 ml, 22.6 mmol). The solution
was heated to reflux for
4 h and then concentrated in vacuo, yielding 417 mg (99%) of the crude
product. This material
was used without purification or characterization.
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[167] Step 3. Preparation of ethyl 4-{[2-(tritylthio)ethyl]amino)pyrimidine-5-
carboxylate
CO2Et
N NH
S
[168] Ethyl 4-chloropyrimidine-5-carboxylate (200 mg, 1.07 mmol) was dissolved
in THF (2 mL),
and 2-(trityithio)ethanamine (514 mg, 1.61 mmol) and N,N-diisopropylethylamine
(0.56 ml,
3.22 mmol) were added. The reaction mixture was heated to reflux overnight and
then
concentrated in vacuo. The crude residue was purified by Biotage column
chromatography,
yielding 230 mg (46%) product. Product confirmed by HNMR. iH NMR (400 MHz,
CD2CI2) S 1.42
(t, 3H), 2.48 (bs, 2H), 3.40 (bs, 2H), 4.40 (q, 2H), 7.18-7.50 (m, 16H), 8.85
(s, 1 H), 8.96 {s, 1 H).
[169] Step 4. Preparation of Lithium 4-{[2-(tritylthio)ethyl]amino)pyrimidine-
5-carboxylate
0
N ~ O Li}
N: NH
S
X
[170] Ethyl 4-{[2-(tritylthio)ethyl]amino}pyrimidine-5-carboxylate (230 mg,
0.49 mmol) was
dissolved in THF (1 mL), MeOH (1 mL), and water (0.5 mL). The solution was
treated with LIOH
(23.5 mg, 0.98 mmol) and allowed to stir at rt for two days. The crude
reaction mixture was then
diluted with water and extracted with EtOAc. The organic extracts were dried
over Na2SO4 and
concentrated in vacuo, yielding 180 mg (82%) of the desired product. LC/MS m/z
441.6 (M+H)+;
RT 2.54 min. iH NMR (400 MHz, CD3OD) S 2.43 (t, 2H), 3.32 (m beneath solvent
peak), 7.10-7.42
(m, 1 H), 8.70 (d,2H).
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EXAMPLE 1 E
[171] Preparation of Lithium 1-(2-(tritylthio)ethylj-9H-benzimidazole-2-
carboxylate
C N O
N Li+
s
[172] Step 1. Preparation of ethyl 1H-benzimidazole-2-carboxylate
~N
~ \>--C02Et
~ N
H
[173] 1 H-Benzimidazole-2-carboxylic acid (500 mg, 3.08 mmol) was suspended in
EtOH (5 mL),
treated with thionyl chloride (1.12 mL, 15.4 mmol), and heated to reflux
overnight. The reaction
mixture was concentrated in vacuo, yielding 644 mg (99%) of the crude product.
1H NMR (400
MHz, CD2CI2) S 1.32 (t, 3H), 4.38 (q, 2H), 7.30 (m, 2H), 7.63 (m, 2H).
[174] Step 2. Preparation of ethyl 1-(2-bromoethyl)-1H-benzimidazole-2-
carboxytate
N
~}-CO2Et
Br
NaH (504 mg of a 60% suspension in mineral oil, 1.07 mmol) was suspended in
dry DMF (1.5 mL).
Ethyl 1 H-benzimidazole-2-carboxylate (170 mg, 0.89 mmol) was added, and the
solution was
allowed to stir at rt for 1 h. 1,2-dibromoethane (0.23 mL, 2.7 mmol) was
added, and the solution
was heated to 50 C overnight. The reaction mixture was diluted"with water and
extracted with
EtOAc. The organic extracts were dried over Na2SO4i concentrated in vacuo, and
purified by
Biotage column chromatography (15% EtOAc/hexanes), yielding 100 mg (38%) of
the desired
product. iH NMR (400 MHz, CD2CI2) S 1.48 (t, 3H), 3.80 (t, 2H), 4.50 (q, 2H),
5.02 (t, 2H), 7.39 (t,
1 H), 7.45 (t, 1 H), 7.53 (d, 1 H), 7.87 (d, 1 H).
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[175] Step 3. Preparation of ethyl 1-[2-(tritylthio)ethyl]-1H-benzimidazole-2-
carboxylate
CN
~--CO2Et
N
S
_ I \
[176] A solution of ethyl 1-(2-bromoethyl)-1 H-benzimidazole-2-carboxylate
(100 mg, 0.34 mmol),
triphenylmethylmercaptan (112 mg, 0.40 mmol), and N,N-diisopropylethylamine
(0.07 ml,
0.40 mmol) in THF (1 mL) were allowed to stir for 2 h at rt. In a separate
flask, a solution of
triphenylmethylmercaptan (112 mg, 0.40 mmol) in THF (1 mL) was treated with
lithium
hexamethyldisilazide (0.40 mL of a 1 M solution in THF) and allowed to stir
for 10 min at rt. This
solution was added to the original reaction mixture, immediately resulting in
a red solution that was
allowed to stir at rt overnight. The reaction mixture was concentrated in
vacuo, and the crude
residue was purified by Biotage column chromatography, yielding 80 mg (48%) of
the desired
product. 1H NMR (400 MHz, CD2CI2) S 1.44 (t, 3H), 2.78 (t, 2H), 4.40-4.52 (m,
4H), 7.03 (m, 1 H),
7.20-7.40 (m, 15H), 7.42 (m, 1 H), 7.82 (m, 1 H).
[177] Step 4. Preparation of Lithium 1-[2-(tritylthio)ethyl]-1H-benzimidazole-
2-carboxylate
\ N O
0 Li+
S
_ I \
[178] Ethyl 1-[2-(tritylthio)ethyl]-1H-benzimidazole-2-carboxylate (75 mg,
0.15 mmol) was
dissolved in THF (1 mL), MeOH (1 mL)L and water (0.5 mL). The solution was
treated with LiOH
(7.3 mg, 0.30 mmol) and allowed to stir at rt for 2 days. The crude reaction
mixture was then
diluted with water and extracted with EtOAc. The organic extracts were dried
over Na2SO4 and
concentrated in vacuo, yielding 74 mg (99%) of the desired product. LC/MS
m/z464.9 (M+H)+; RT
3.16 min. iH NMR (400 MHz, CD3OD) S 2.72 (t, 2H), 4.58 (t, 2H), 6.98 (m, 1 H),
7.15-7.30 (m,
16H), 7.38 (d, 1 H), 7.63 (m, 1 H).
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EXAMPLE 1F
[179] Preparation of (2-Mercapto-1H-imidazol-1 yl)acetic Acid
\
>-SH
~CN
N
HO2C
[180] Step 1. Preparation of ethyl N-(2,2-dimethoxyethyl)glycinate
hydrochloride
~O
H
0 ,J,,_~,N,_,,C02Et
HCI
[181] Bromoacetaidehyde dimethylacetal (0.85 mL, 7.2 mmol) was added to a
solution of glycine
ethyl ester hydrochloride (500 mg, 3.58 mmol) and N,N-diisopropylethylamine
(1.37 mL,
7.88 mmol) in THF (4mL) and EtOH (1 mL). The reaction mixture was heated to 70
C and allowed
to stir overnight. The solution was diluted with water and extracted with
dichloromethane. The
organic extracts were dried over Na2SO4 and concentrated in vacuo. The crude
residue was
treated with I N HCI in ether, and the resulting precipitate was collected by
filtration and dried,
yielding 371 mg (23%) of the desired product. iH NMR (400 MHz, CD3CN) b 1.30
(t, 3H), 3.18
(bs, 2H), 3.42 (s, 6H), 3.85 (bs, 2H), 4.27 (q, 2H), 4.92 (t, 1 H), 9.40 (bs,
2H).
[182] Step 2. Preparation of ethyl (2-mercapto-lH-imidazol-1-yl)acetate
>--SH
CN
N
Et02C
[183] Ethyl N-(2,2-dimethoxyethyl)glycinate hydrochloride (370 mg, 1.63 mmol)
was dissolved in
EtOH (2 mL) and treated with a solution of potassium thiocyanate (237 mg, 2.44
mmol) in EtOH
(8 mL). The pink suspension was heated to reflux overnight. Concentrated HCI
(0.136 mL,
1.63 mmol) was added, and the solution was allowed to reflux for 3 h. The
reaction mixture was
concentrated in vacuo, and the resulting solid was recrystalized from EtOAc,
yielding 130 mg
(43%) of the desired product. iH NMR (400 MHz, CD3CN) S 1.33 (t, 3H), 4.20 (q,
2H), 4.77 (s,
2H), 6.77 (s, 1 H), 6.83 (s, 1 H), 9.92 (bs, 1 H).
[184] Step 3. Preparation of (2-Mercapto- 1H-imidazol-1-yl)acetic acid
~}-SH
~CN
N
H02C
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[185] Ethyl (2-mercapto-1H-imidazol-l-yl)acetate (130 mg, 0.70 mmol) was
dissolved in THF
(1 mL), MeOH (1 mL), and water (0.5 mL). The solution was treated with LiOH
(33.4 mg,
1.40 mmol) and allowed to stir at rt for 2 days. The crude reaction mixture
was acidified to pH 2
with 2N HCI, diluted with water, and extracted with 5:1 EtOAc/EtOH. The
organic extracts were
dried over Na2SO4 and concentrated in vacuo, yielding 70 mg (63%) of the
desired product. LC/MS
m/z 159.1 (M+H)+; RT 1.05 min. 1H NMR (400 MHz, CD3OD) S 4.82 (s, 2H), 6.82
(s, 1 H), 6.99 (s,
1 H).
EXAMPLE 1G
[186] Preparation of Lithium (f1(2-(tritylthio)ethyl]-1H-imidazol-2-
yl)thio)acetate
O
S
~O"Li}
CN
[187] Step 1. Preparation of methyl (1H-imidazol-2-ylthio)acetate
N
\>-~
N C02Me
H
[188] 2-Thioimidazole (300 mg, 3.00 mmol) was dissolved in THF (3 mL), treated
with N,N-
diisopropylethylamine (0.63 mL, 3.59 mmol) and methyl bromoacetate (0.31 mL,
3.3 mmol), and
allowed to stir at rt for 1 h. The solid precipitate was filtered off, and the
filtrate was diluted with
EtOAc. The organics were washed with water, dried over Na2SO4 and concentrated
in vacuo,
yielding 385 mg (75%) of the desired product. 'H NMR (400 MHz, CD3CN) S 3.68
(s, 3H), 3.82 (s,
2H), 7.05 (s, 2H).
[189] Step R. Preparation of Methyl ((1-f2-(tritylthio)ethyl]-1H-imidazol-2
yl]thio)acetate
N\>-~
N CO2Me
s
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[190] NaH (55.7 mg of a 60% suspension in mineral oil, 1.39 mmol) was
suspended in dry DMF
(1.5 mL). Methyl (1 H-imidazol-2-ylthio)acetate (200 mg, 1.16 mmol) was added,
and the resulting
solution was allowed to stir at rt for 1 h. 1,1',1 "-{[(2-
bromoethyl)thio]methanetriyl}tribenzene
(668 mg, 1.39 mmol) was added, and the reaction mixture was heated to 50 C
overnight. The
reaction mixture was diluted with water and extracted with EtOAc. The organic
extracts were
washed with water and brine, dried over MgSO4, and concentrated in vacuo. The
crude residue
was purified by Biotage column chromatography (20% EtOAc/hexanes), yielding
180 mg (33%) of
the desired product. 'H NMR (400 MHz, CD2CI2) S 2.60 (t, 2H), 3.65 (m, 5H),
3.78 (s, 2H), 6.69 (s,
1 H), 6.96 (s, 1 H), 7.20-7.40 (m, 15H).
[191] Step 3. Preparation of Lithium ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-
yl]thio)acetate
O
CN S~O'Li+
S
[192] Methyl ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-yl}thio)acetate (180 mg,
0.38 mmol) was
dissolved in THF (1 mL), MeOH (1 mL), and water (0.5 mL). The solution was
treated with LiOH
(18.2 mg, 0.76 mmol) and allowed to stir at rt overnight. The reaction mixture
was diluted with
water and extracted with EtOAc. The organic extracts were dried over Na2SO4
and concentrated
in vacuo. Purification of the crude material by HPLC gave 61 mg (34%) of the
desired product.
LC/MS m/z460.9 (M+H)+; RT 2.81 min. 'H NMR (400 MHz, CD3OD) S 2.59 (t, 2H),
3.60 (s, 2H),
3.92 (t, 2H), 6.89 (s, 1 H), 6.92 (s, 1 H), 7.19-7.35 (m, 15H).
EXAMPLE 1 H
[193] Preparation of Lithium 3-(2-tritylsulfanylethylamino)pyrazine-2-
carboxylate
O
O-Li+
CN
N NH
S
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[194] Step 1. Preparation of 2-tritylsulfanylethylamine
\ I -
S
H2N-j-
[195] A suspension of cysteamine hydrochloride (1.14 g, 9.80 mmol) and
triethylamine (3.0 mL,
21.6 mmol) in dichloromethane (20 mL) was stirred at rt for 10 min. N,O-
Bis(trimethylsilyl)
acetamide (1.99 g, 9.80 mmol) was then added, and the reaction was allowed to
stir for 30 min
under nitrogen. The reaction mixture was cooled to 0 C and trityl chloride
(2.46 g, 8.82 mmol) was
added in one portion. The suspension was allowed to warm to rt and stir for 16
h. The reaction
mixture was quenched with water (15 mL). The mixture was washed successively
with 1 N HCI
(2 x 10 mL), water (2 x 10 mL), 15 % ammonia solution (4 mL), and water (5 x
20 mL). The
organics were dried over Na2SO4 and concentrated in vacuo to give 1.9 g(61 %)
of a light brown
oil. 'H NMR (400 MHz, CDCI3) S 7.70-7.18 (m, 15H), 2.71 (t, 2H), 2.4 (b, 2H).
[196] Step 2. Preparation of 3-bromopyrazine-2-carboxylic acid methyl ester
O
CN
O
N Br
[197] Bromine (3.91 g, 24.46 mmol) was added dropwise to a stirred mixture of
3-
aminopyrazine-2-carboxylic acid methyl ester (1.27 g, 8.29 mmol) and
hydrobromic acid (4.70 mL,
41.5 mmol) at 0 C. A solution of sodium nitrite (1.44 g, 20.9 mmol) in water
(6 mL) was then
added dropwise. The reaction mixture was stirred for 15 min, brought to pH 8
with NaHCO3
(saturated, aqueous), and extracted with ethyl acetate (80 mL) and chloroform
(50 mL). The
combined organics were dried over MgSO4 and concentrated in vacuo to give 1.13
g (63%) of an
orange oil which solidified on standing. LC-MS m/z217 (M+H+); RT 1.15 min.
[198] Step 3. Preparation of 3-(2-tritylsulfanylethylamino)pyrazine-2-
carboxylic acid methyl ester
O
N o--
NH
S
_ I
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[199] A solution of 3-bromopyrazine-2-carboxylic acid methyl ester (0.10 g,
0.45 mmol), 2-
tritylsulfanylethylamine (0.29 g, 0.90 mmol) and triethylamine (0.06 mL, 0.44
mmol) in acetonitrile
(5 mL) was heated to reflux for 18 h under argon. The mixture was concentrated
under reduced
pressure and purified by column chromatography (3:1 Hex:EtOAc), yielding 0.058
g (28%) of the
desired product. iH NMR (400 MHz, CD2CI2) S 7.95 (s, 1 H), 8.44 (s, 1 H), 8.31
(s, 1 H), 7.57.18 (m,
15H), 4.20 (s, 3H), 3.36(t, 2H), 2.50 (t, 2H).
[200] Step 4. Preparation of lithium 3-(2-tritylsulfanylethylamino)pyrazine-2-
carboxylate
O
O-Li+
(NI N
NH
S
- I \
[201] A mixture of 3-(2-tritylsulfanylethylamino)pyrazine-2-carboxylic acid
methyl ester (0.12 g,
0.27 mmol) and LiOH (0.030 g, 1.3 mmol) in THF (5 mL), methanol (5 mL), and
water (2.5 mL)
was stirred at rt for 18 h. The reaction mixture was concentrated and purified
by HPLC, yielding
0.075 g (63%) of the desired product. 'H NMR (400 MHz, CD3OD) fi 8.26 (s, 1
H), 8.15 (s, 1 H),
7.42-7 (m, 15H), 3.30 (t, 2H), 2.40 (t, 2H).
EXAMPLE 11
[202] Preparation of 4-mercaptothiazole-5-carboxylic acid
O OH
S SH
'=N
[203] Step 1. Preparation of 4-(2-methoxycarbonylethylsulfanyl)thiazole-5-
carboxylic acid ethyl
ester
O O',~
S S
\=N ~O\
O
[204] A solution of ethyl isocyanoacetate (0.92 g, 7.7 mmol) in THF (8 mL) was
added dropwise
to a suspension of potassium tert-butoxide (1.0 g, 8.5 mmol) in THF (6 mL) at -
40 C. The mixture
was cooled to -60 C, and a solution of carbon disulfide (0.59 g, 7.7 mmol) in
THF (8 mL) was
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added dropwise while keeping the temperature below -50 C. The mixture was
warmed to 10 C
and methyl 3-bromopropionate (1.33 g, 7.70 mmol) was added. The mixture was
allowed to stir for
2 h and was concentrated in vacuo. The product was recrystalized from
dichloromethane/hexanes
to give 1.28 g (60%) of the desired product as a white solid. LC-MS m/z 276
(M+H+); RT 1.65 min.
[205] Step 2. Preparation of 4-mercaptothiazole-5-carboxylic acid ethyl ester
O O,,-,,,
S SH
\=N
[206] Sodium hydroxide (0.14 g, 3.5 mmol) was added to a solution of 4-(2-
methoxycarbonylethylsulfanyl)thiazole-5-carboxylic acid ethyl ester (0.96 g,
3.5 mmol) in methanol
(13.6 mL). The mixture was refluxed for 1 h and then concentrated in vacuo.
The residue was
taken up in ethyl acetate/water and the pH adjusted to 2 with 2 N HCI. The
organic layer was
isolated and concentrated in vacuo, yielding 0.66 g (100%) of the desired
product, which was used
without further purification. LC-MS m/z 189 (M+H+); RT 1.47 min.
[207] Step 3. Preparation of 4-mercaptothiazole-5-carboxylic acid
O OH
s ~ SH
\---N
[208] Sodium hydroxide (0.25 g, 6.3 mmol) was added to a solution of 4-
mercaptothiazole-5-
carboxylic acid ethyl ester (0.60 g, 3.2 mmol) in methanol (5 mL) and water (5
mL), and the
mixture was heated to 80 C for 3 h. Upon cooling to rt, the reaction mixture
was concentrated in
vacuo. The residue was acidified with 2 N HCI and extracted with
dichloromethane. The organic
layer was dried over MgSO4, concentrated in vacuo, and purified by HPLC to
give 0.059 g (12%)
of the desired product. 'H NMR (400 MHz, CD3OD) S 8.95 (s, 1 H); LC-MS m/z 162
(M+H+); RT
1.01 min.
EXAMPLE 1J
[209] Preparation of 2-mercaptothiazole-5-carboxylic acid
0
HS NS~ OH
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[210] Step 1. Preparation of 2-mercaptothiazole-5-carboxylic acid methyl ester
0
HS NS/ O'
[211] A mixture of 2-bromothiazole-5-carboxylic acid methyl ester (0.40 g, 1.8
mmol) and
thiourea (0.16 g, 2.1 mmol) in ethanol (6 mL) was heated to reflux for 2 h.
The mixture was
allowed to cool to rt, and the resulting suspension was filtered to give 0.19
g(61 %) of the desired
product as a yellow solid. LC-MS m/z 176.1 (M+H+); RT 1.17 min.
[212] Step 2. Preparation of 2-mercaptothiazole-5-carboxylic acid
0
HS NSj OH
[213] Sodium hydroxide (0.08 g, 1.9 mmol) was added to a solution of 2-
mercaptothiazole-5-
carboxylic acid methyl ester (0.17 g, 0.97 mmol) in methanol (5 mL) and water
(5 mL). The
reaction mixture was stirred at rt for 3 h and concentrated in vacuo. The
residue was acidified with
2 N HCI and the resulting suspension was filtered to give 0.061 g (39%) of the
desired product as
an off-white solid. 'H NMR (400 MHz, CD3OD) 5 7.80 (s, 1 H); LC-MS m/z 161
(M+H+); RT 1.02
min.
EXAMPLE y K
[214] Preparation of Lithium 2-(2-tritylsulfanylethylamino)thiazole-5-
carboxylate
0 O-Li+
S \
N
S"/'-N
H
[215] Step 1. Preparation of 2-(2-tritylsulfanyl-ethylamino)-thiazole-5-
carboxylic acid methyl
ester
0
O\
S
H
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[216] A solution of 2-bromothiazole-5-carboxylic acid methyl ester (0.20 g,
0.88 mmol), 2-
tritylsulfanylethylamine (0.42 g, 1.3 mmol), and triethylamine (0.12 mL, 0.88
mmol) in acetonitrile
(5 mL) was heated to reflux for 18 h under argon. The mixture was concentrated
in vacuo and
purified by column chromatography (3:1 Hex:EtOAc) to give 0.12 g(29 /0) of the
desired product.
'H NMR (400 MHz, CD2CI2) S 8.15 (s, 1H), 7.60-7.15 (m, 15H), 4.20 (s, 3H),
3.45(t, 2H), 2.50 (br,
2H).
[2171 Step 2. Preparation of lithium 2-(2-tritylsulfanylethylamino)-thiazole-5-
carboxylate
0 O-Li+
S
- N
S.,/--N
H
[2181 A mixture of 2-(2-tritylsulfanylethylamino)-thiazole-5-carboxylic acid
methyl ester (0.12 g,
0.26 mmol) and LiOH (0.03 g, 1.3 mmol) in THF (5 mL), methanol (5 mL) and
water (2.5 mL) was
stirred at rt for 18 h. The reaction mixture was concentrated in vacuo and
purified by HPLC to give
0.087 g (75%) of the desired product. 'H NMR (400 MHz, CD2CI2) 8 7.90 (s, I
H), 7.40-7.10 (m,
15H), 3.30 (s, 3H), 3.45(t, 2H), 2.40 (br, 2H).
EXAMPLE yL
[219] Preparation of 2-mercapto-6-methylpyrimidine-4-carboxylic acid
O
HSY, N\ OH
N /
[220] Step 1. Preparation of 2-carbamimidoylsulfanyl-6-methylpyrimidine-4-
carboxylic acid
methyl ester hydrobromide
O
HzNy SY, N Oi
HBr NH N /
[221] A mixture of 2-chloro-6-methylpyrimidine-4-carboxylic acid methyl ester
(0.50 g, 2.7 mmol)
and thiourea (0.41 g, 5.4 mmol) in ethanol (8 mL) was heated to reflux for 16
h and then
concentrated in vacuo. Attempt to purify the product by recrystalization
(methanol/ether) gave
0.229 g(38 to) of an oil which was confirmed as the desired product. LC-MS mlz
226.9 (M+H+);
RT1.07min.
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[222] Step 2. Preparation of 2-mercapto-6-methylpyrimidine-4-carboxylic acid
O
HS\ /N\ OH
~N /
[223] A mixture of 1 N sodium hydroxide (6.98 mL, 6.98 mmol) and 2-
carbamimidoyisulfanyl-6-
methylpyrimidine-4-carboxylic acid methyl ester hydrobromide (0.37 g, 1.4
mmol) was heated to
reflux for 2 h. Upon cooling to rt, the reaction mixture was concentrated in
vacuo. The residue
was taken up in water and MP-TsOH (Argonaut technologies) was added. The
mixture was stirred
for 18 h until a pH of 4 was achieved. The mixture was filtered, and the
filtrate was concentrated in
vacuo. The residue was taken up in methanol, filtered, and the filtrate was
concentrated in vacuo
to give 0.139 g (59%) of the desired product as a brownish solid. 1H NMR (400
MHz, CD3OD) S
7.10 (s, 1 H), 3.40 (s, 3H); LC-MS m/z 171 (M+H+); RT 1.15 min.
EXAMPLEIM
[224] Preparation of 5-mercaptonicotinic acid
O
HS ~ OH
~ ~
N
[225] Step 1. Preparation of 5-tert-butylsulfanylnicotinonitrile
ks N
[226] tert-Butylthiol (0.43 g, 4.8 mmol) was added to a suspension of NaH
(0.19 g, 4.8 mmol) in
DMF (15 mL), and the reaction mixture was heated at 50 C for 1 h. 5-
Bromonicotinonitrile (0.60 g,
3.2 mmol) was added to the resulting suspension, and the reaction mixture was
heated at 120 C
for 5 h. Upon cooling to rt, the mixture was concentrated in vacuo and
purified by HPLC, yielding
0.329 g (54%) of the desired product as an off-white solid. LC-MS m/z 193
(fVI+H+); RT 2.90 min.
[227] Step 2. Preparation of 5-tert-butylsulfanylnicotinic acid
O
kS OH
nN_ [228] A mixture of 5-tert-butylsulfanylnicotinonitrile (0.43 g, 2.2 mmol)
and sodium hydroxide
(0.89 g, 22 mmol) in ethanol (5 mL) and water (5 mL) was heated to reflux for
1 h. Upon cooling to
rt, the reaction mixture was diluted with water and was extracted with ether.
The aqueous layer
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was acidified with 2 N HCI and was extracted with dichloromethane. The
dichloromethane extracts
were dried over MgSO4 and concentrated to give 0.433 g (79%) of the desired
product as a white
solid. LC-MS m/z 212 (M+H+); RT 2.36 min.
[229] Step 3. Preparation 5-mercaptonicotinic acid
O
HS ~ OH
~ ~
N
[230] A solution of 5-tert-butylsulfanyinicotinic acid (0.30 g, 1.2 mmol) in 2
N HCI (9 mL,
18 mmol) was heated to reflux for 32 h. Upon cooling to rt, the reaction
mixture was concentrated
in vacuo to give 0.054 g (28%) of the desired product. iH NMR (400 MHz, CD3OD)
S 9.00-8.80
(m, 2H), 8.40 (d, 1 H); LC-MS m/z 156 (M+H+); RT 2.37 min.
EXAMPLE 1 N
[231] Preparation of 5-isopropyl-2-mercaptothiazole-4-carboxyllc acid
O
HS~N
S/ OH
[232] Step 1. Preparation of 2-chloro-4-methyl-3-oxopentanoic acid ethyl ester
O O
I-T ~cl
[233] A solution of sulfuryl chloride (1.63 mL, 19.7 mmol) in toluene (5 mL)
was added dropwise
to a solution of 4-methyl-3-oxopentanoic acid ethyl ester (3.28 g, 19.7 mmol)
in toluene (25 mL)
over 10 min. The resulting mixture was stirred at rt for 18 h and then slowly
quenched with water
and NaHCO3 (saturated, aqueous). The mixture was extracted with ethyl acetate,
and the
combined organics were dried over MgSO4 and concentrated in vacuo to give 3.4
g (70%) of the
des'ired product which was used without further purification. LC-MS m/z 194.1
(M+H+); RT 2.69
min.
[234] Step 2. Preparation of 2-amino-5-isopropylthiazole-4-carboxylic acid
ethyl ester
O
N
H2N S ~
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[235] A mixture of 2-chloro-4-methyl-3-oxopentanoic acid ethyl ester (2.0 g,
7.3 mmol) and
thiourea (0.43 g, 5.6 mmol) in ethanol (8 mL) was refluxed for 18 h and then
concentrated in
vacuo. The residue was treated with aqueous ammonia, and the resultant yellow
solid was taken
up in water and extracted with dichloromethane. The combined organics were
dried over Na2SO4
and concentrated in vacuo. The solid was taken up in a small amount of
dichloromethane and
filtered to give 1.02 g (85%) of the desired product as a cream colored solid.
LC-MS m/z 215.1
(M+H+); RT 1.96 min.
[236] Step 3. Preparation of 2-6romo-5-isopropylthiazole-4-carboxylic acid
ethyl ester
O
N
Br Si ~ O~
[237] To a dark brown solution of copper (II) bromide (2.47 g, 11.1 mmol) in
acetonitrile (10 mL)
in a two necked flask equipped with a condenser was added tert-butylnitrite
(0.63 g, 5.5 mmol)
slowly at rt. The mixture was heated to 60 C, and a suspension of 2-amino-5-
isopropylthiazole-4-
carboxylic acid ethyl ester (0.79 g, 3.7 mmol) in acetonitrile (14 mL) was
added dropwise. The
mixture was then heated at 60 C for 3 h. Upon cooling to rt, the reaction
mixture was poured into
1 N NaOH (40 mL) and extracted with ethyl acetate. The combined organics were
dried over
Na2SO4, concentrated in vacuo, and purified by column chromatography (2:1
EtOAc/Hexanes) to
give 0.88 g (86%) of the desired product as a yellow oil. LC-MS m/z 280
(M+H+); RT 3.65 min.
[238] Step 4. Preparation of 5-isopropyl-2-mercaptothiazole-4-carboxylic acid
ethyl ester
O
N
HS Si ~ O~
[239] A mixture of 2-bromo-5-isopropylthiazole-4-carboxylic acid ethyl ester
(0.20 g, 0.72 mmol)
and thiourea (0.07 g, 0.86 mmol) in ethanol (6 mL) was heated to reflux for 2
h. Upon cooling to rt,
the resulting suspension was filtered to give 0.11 g(66 to) of the desired
product as a yellow solid.
LC-MS m/z 232.1 (M+H+); RT 2.72 min.
[240] Step 5. Preparation of 5-isopropyl-2-mercaptothiazole-4-carboxylic acid
O
HS--'~N
S/ OH
[241] NaOH (1 N, 0.78 mL, 0.78 mmol) was added to a solution of 5-isopropyl-2-
mercaptothiazole-4-carboxylic acid ethyl ester (0.09 g, 0.39 mmol) in methanol
(3 mL) and water
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(2 mL), and the mixture was stirred at rt for 3 h. The reaction mixture was
concentrated in vacuo,
and the residue was acidified with 2 N HCI. The resulting suspension was
filtered to give 0.045 g
(57%) of the desired product as an off-white solid. 'H NMR (400 MHz, CD3OD) S
3.85-4.10 (m,
1 H), 1.22 (d, 6H); LC-MS m/z 204.2 (M+H+); RT 1.65 min.
EXAMPLE10
[242] Preparation of (1-hexadecyl-7H-benzoimidazol-2-ylsulfanyl)acetic acid
HO
N~
[243] Step 1. Preparation of (1H-benzoimidazol-2 ylsulfanyl)acetic acid ethyl
ester
~O
~N
~ \>--S O
N
H
[244] A mixture of 2-mercaptobenzimidazole (0.3 g, 2 mmol), ethyl bromoacetate
(0.50 g,
2.9 mmol), and potassium carbonate (0.14 g, 0.98 mmol) in ethanol (3.1 mL) was
heated to reflux
for 8 h and then concentrated in vacuo. Purification by HPLC yielded 0.22 g
(46%) of the desired
product. LC-MS m/z237.2 (M+H+); RT 1.41 min.
[245] Step 2. Preparation of (1-hexadecyl- 1H-benzoimidazol-2-
ylsulfanyl)acetic acid methyl
ester
/
O
~O
S
N__~
N
-.-
[246] Sodium hydride (0.03 g, 0.8 mmol) was added to a solution of (1 H-
benzoimidazol-2-
ylsulfanyl) acetic acid ethyl ester (0.21 g, 0.79 mmol) in DMF (10 mL) at '0
C, and the mixture was
stirred at rt for 1 h. Hexadecylbromide (0.22 g, 0.71 mmol) was added, and the
mixture was stirred
at rt for 18 h. The reaction mixture was diluted with water and methanol and
concentrated in
vacuo. Purification by column chromatography (20% ethyl acetate in hexanes)
gave 0.24 g (67%)
of the desired product. LC-MS m/z447.4 (M+H+); RT 5.03 min.
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[247] Step 3. Preparation of (1-hexadecyl-lH-benzoimidazol-2 ylsulfanyl)acetic
acid
HO
"'~O
N
N
[248] A mixture of lithium hydroxide (0.060 g, 2.4 mmol) and (1-hexadecyl-1 H-
benzoimidazol-2-
yisulfanyl) acetic acid methyl ester (0.21 g, 0.47 mmol) was heated to reflux
for 2 h, cooled to rt,
and concentrated in vacuo. The residue was taken up in water, and the
suspension was acidified
with 1 N HCI and filtered. The solid was collected and dried to give 0.16 g
(79%) of the desired
product. iH NMR (400 MHz, CD30D) S 7.70-7.20 (m, 4H), 4.20 (t, 2H), 3.80 (s,
2H), 1.85 (m, 2H),
1.21-1.50 (m, 28H), 0.90 (t, 3H); LC-MS m/z433.2 (M+H+); RT 4.72 min.
EXAMPLE 1 P
[249] Preparation of Lithium 6-(2-tritylsulfanylethylamino)nicotinate
O
O Li+
&N-
S~/~N I~ /\
H
[250] Step 1. Preparation of 6-(2-tritylsulfanylethylamino)nicotinic acid
methyl ester.
O
&N-
H
OSN [251] Methyl 6-chloronicotinate (0.20 g, 1.17 mmol), 2-
tritylsulfanylethylamine (0.56 g,
1.75 mmol), cesium carbonate (0.95 g, 2.91 mmol), and [1,1'-
bis(diphenylphosphino)ferrocene]
dichloropalladium(II) complex with dichloromethane (1:1) (0.24 g, 0.29 mmol)
were heated to
120 C overnight in 1,4-dioxane (4.0 mL) and water. Upon cooling to rt, the
reaction mixture was
filtered through Celite @, concentrated in vacuo, and purified by Biotage
column chromatography
(5% EtOAc/Hexanes). This yielded 0.3442 g of a white solid. The material was
recrystallized from
10% EtOAc/Hexanes, yielding 0.272 g(51 %) of the desired product as a white
solid. Rf = 0.42
(20% EtOAc/Hexanes). 'H NMR (400 MHz, CD2CI2) i? 2.0 (bs, 1 H), 2.45 (t, 2H),
3.40 (t, 2H), 3.92
(s, 3H), 7.15-7.30 (m, 10H), 7.42-7.50 (m, 6H), 8.02 (d, 1 H), 8.90 (s, 1 H).
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[252] Step 2. Preparation of Lithium 6-(2-tritylsulfanylethylamino)nicotinate
O
O-Li+
S
&N-
H
N [253] 6-(2-Tritylsulfanylethylamino)nicotinic acid methyl ester (270 mg,
0.59 mmol) was
suspended in THF (2 mL), MeOH (2 mL), and water (1 mL). The reaction mixture
was treated with
LiOH (20 mg, 0.65 mmol) and heated to 50 C for 2 hours. Upon cooling to rt,
the reaction mixture
was concentrated in vacuo. Recrystallization from EtOAc/Hexanes (3:1) yielded
236 mg (89%) of
the desired product as a white solid. 'H NMR (400 MHz, DMSO-ds) S 2.25 (t,
2H), 3.00 (bt, 1 H),
3.28 (t, 2H), 7.00-7.40 (m, 16H), 7.90 (d, 1 H), 8.72 (s, 1 H).
Example 2. Peptide Synthesis
[254] Peptides are synthesized with an Applied Biosystems 430A peptide
synthesizer using
FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied
Biosystems
protocols are used. The peptides are cleaved with 84.6% TFA, 4.4% phenol, 4.4%
water,
4.4% thioanisol, and 2.2% ethanedithiol. Peptides are precipitated from the
cleavage cocktail
using cold tertbutylmethyl ether. The precipitate is washed with the cold
ether and dried under
argon. Peptides are purified with by reversed phase C18 HPLC with linear
water/acetonitrile
gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and
electrospray mass
spectrometry and with amino acid analysis.
Example 3. Methods for Adding N-Terminal Modifying Compound
[255] Peptides are synthesized with an Applied Biosystems 430A peptide
synthesizer using
FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied
Biosystems
protocols are used. The N-terminal modifying compounds are coupled to the
peptide as per a
natural amino acid coupling during FMOC chemistry. N-terminal modifying
compounds are either
commercially available or synthesized as described in Example 1. In the case
of amine and
mercapto containing N-terminal modifying compounds, the amine and mercapto
groups are
protected with FMOC or trityl, respectively, during coupling to the peptide.
The peptides are
cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2%
ethanedithiol.
Peptides are precipitated from the cleavage cocktail using cold
tertbutylmethyl ether. The
precipitate is washed with the cold ether and dried under argon. Peptides are
purified with by
reversed phase C18 HPLC with linear water/acetonitrile gradients containing
0.1% TFA. Peptide
identity is confirmed with MALDI and electrospray mass spectrometry and with
amino acid
analysis.
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Example 4. Methods for Adding C-terminal Modifying Compound
[256] Peptides are synthesized with an Applied Biosystems 433A peptide
synthesizer using
FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied
Biosystems
protocols are used. The HBTU-activated C-terminal modifying compounds are
coupled to the
resin (e.g., Wang resin for producing peptides with a C-terminal modifying
compound containing
the free carboxylate or Rink Amide for producing amide variants) as per a
natural amino acid
coupling during FMOC chemistry. The peptides are then synthesized by the
stepwise addition of
amino acids using standard FMOC protocols. The peptides are cleaved with 84.6%
TFA, 4.4%
phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol. Peptides are
precipitated from the
cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed
with the cold ether
and dried under argon. Peptides are purified with by reversed phase C18 HPLC
with linear
water/acetonitrile gradients containing 0.1% TFA. Peptide identity is
confirmed with MALDI and
electrospray mass spectrometry and with amino acid analysis.
Example 5. Preparation of PEGylated Peptides
[257] PEG derivatives are prepared by incubating methoxypolyethiene glycols
derivatized with
maledimide for coupling to the mercapto moiety of the N-terminal modifying
group. mPEG-MAL or
mPEG2-MAL products supplied by Nektar Therapeutics (Huntsville, Al, USA) or
GLE-200MA or
GLE-400MA products supplied by NOF (Toyko, Japan) are used. Coupling reactions
are
performed by incubating the peptide and a two-fold molar excess of maleimide-
PEG in 50 mM Tris,
pH 7 at rt for 2-12 h. The peptide concentration may be 1 mg/ml or less.
Underivatized peptides
and PEG are purified from the PEGylated peptide with cation exchange
chromatography and
dialysis or by reversed phase C18 HPLC. The purified PEG-peptide conjugate is
then freeze dried.
Example 6. Preparation of Fatty-acid Derivatized Peptides
[258] The fatty acid (paimitate) derivatives of amine containing N-terminal
modifying compounds
are prepared as N-terminal modified peptides as described in Example 3 except
that prior to
deprotection and cleavage the FMOC protecting group of the amine moiety of the
N-terminal
modifying group was selectively removed with 0.1% TFA and derivativized with
palmitic acid using
the same conditions as for a normal amino acid coupling.
[259] The fatty acid derivative can also be prepared as described in Example 3
using 1-
hexadecyl-i H-benzoimidazol-2-ylsulfanyl)acetic acid as the N-terminal
modifying group, which was
synthesized as described in Example 1.
Example 7. Pharmaceutical Composition - IV and SC Formulations
[260] A sterile IV injectable formulation is prepared with 4 mg of a peptide
of Formula (I), or a
derivatized petide having equivalent of 4 mg peptide content, and 1 L sterile
saline, using any
manufacturing process well known in the art. Higher concentrations of peptide
may be used for
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SC formulation. In the case of the peptide of Formula (I), or a derivatized
peptide, 4 mg is
dissolved in 100 mL saline or DMSO and sterile vials after aseptic filtration,
are filled with the
composition.
Example 8. Mass Spectrometric Analysis of Peptides
[261] Forty pmol/2 l aliquots of peptides are diluted up to 10 l with water.
The HEPES buffer
is removed by application of 50% of the sample (20 pmol/5 I) to a conditioned
Millipore C18
ZipTip, as per manufacturers instructions. Samples are eluted from the ZipTip
with matrix
(10 mg/mI aipha-cyanohydroxycinnamic acid in 50% ACN, 0.1 % TFA) directly onto
the MALDI
plate. Samples are analyzed on an Applied Biosystems Voyager DE-PRO MALDI
operated in the
reflector ion mode. Data is collected in the 500 - 4000 Da range and resulting
masses were
compared to those expected by manual calculation.
Example 9. Edman Analysis of Peptides
[262] Peptide samples are supplied for Edman degradation at 1 nmol/10 l in 10
mM HEPES,
pH 7.4, 5% TFA. Prior to Edman analysis, the HEPES buffer salt is removed by
using an Applied
Biosystems ProSorb sample cartridge as per manufacturers instructions.
Briefly, the sample is
applied to a PVDF membrane and washed with 0.1 % TFA, then the membrane is
removed and
inserted into the protein sequencer for Edman degradation. Edman degradation
is carried out on
an Applied Biosystems Procise 494HT protein sequencing system using the pulsed-
liquid method
according to manufacturer instructions. Sequences are read manually.
Example 10. Stability of Peptides
[263] The formulations described in Example 4 are placed in constant stability
chamber.
Peptides are also analyzed for stability to degradation in solutions of DPPIV
and in plasma.
Samples are removed periodically for analysis by capillary electrophoresis,
mass spectrometry,
Edman degradation, ELISA, and assays of peptide activity, which are sensitive
methods to detect
degradation of peptide. The area of various peaks is summed and the area for
peak of the parent
peptide is divided by the total peak area. The quotient is the % purity. Since
there are impurities
present in the fresh peptide, the purity change- is norrna(ized by dividing
the pur'ity at different time
point by the initial purity. For stability to DPPIV and plasma, peptides at 20
pmol/ l were incubated
at 37 C in the presence of 300 pM DPPIV in 100 mM HEPES, pH 7.4. At various
timepoints, the
reaction (2 l aliquot) is terminated by addition of 1 pM DPPIV inhibitor and
freezing. For MALDI
mass spectrometric analysis, T = 0 hr, 1 hr, 5 hr, and 24 hr timepoints are
evaluated. The results
are plotted as percent intact peptide or peptide derivative as compared to
degradation products.
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Example 11. Binding of Peptides to the GLP-1 Receptor
[264] RINm5F cell membranes are prepared according to the following procedure.
Flasks of
RINm5F cells are washed with PBS, scraped in 20 mM HEPES/1 mM EDTA/250 mM
sucrose
(HES) buffer containing protease inhibitors and homogenized in a dounce
homogenizer followed
by repeated resuspension through a 23 gauge needle. Unbroken cells and nuclei
are removed by
centrifugation at 500 x g for 5 min. The pellet is resuspended in HES buffer
using a 23 gauge
needle, and the centrifugation is repeated. The supernatanfs from the two
spins are combined and
centrifuged at 40,000 x g for 20 min. The resulting plasma membrane pellet is
resuspended in
HES buffer using a 23 gauge needle followed by a 25 gauge needle. Membranes
are stored at -
80 C until use.
[265] IC50 values for the competitive binding of peptides and peptide
fragments, variants, and
analogs of the invention to the GLP-1 receptor in RINm5F membranes typically
are at least about
0.01 nM up to about 20 nM (i.e., 0.01, 0.1 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11,
12, 13, 14,15, 16, 17, 18,
19, or 20 nM). IC50 values for peptide derivatives of the invention typically
are at least 0.01 nM up
to about 500 nM (i.e., 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, 300, 350,
400, 450, or 500 nM).
[266] Competitive binding of the peptides using RINm5F cell plasma membranes
is measured
as follows. Ninety-six-well GF/C filtration plates (Millipore, Bedford, MA)
are blocked with 0.3%
PEI for at least one hour and washed twice with binding buffer consisting of
20 mM Tris, 2 mM
EDTA, pH 7.5, 1 mg/ml BSA, and 1 mg/ml bacitracin. Five micrograms of RINm5F
cell plasma
membranes diluted in binding buffer are applied to each well together with
0.05 Ci1251 labeled
GLP-1 and peptide concentrations ranging from 1x10"'2 to 1x10"5 M. Following a
60-minute
incubation at rt, the plates were washed 3 times with ice-cold PBS containing
1 mg/ml BSA. The
plates are dried, scintillant is added to each well, and cpm per well is
determined using a Wallac
Microbeta counter.
[267] The number of 1251 counts bound to the membranes at each concentration
of peptide are
plotted and analyzed by nonlinear regression using Prizm software to determine
the IC50. The
peptides disclosed bound to the GLP-1 receptor present in the plasma membranes
isolated from
RINm5F cells with IC50 values of between 1.4 nM and 248 nM (determined from a
minimum of
three trials). IC50 is the concentration of a peptide at which maximal binding
of labeled GLP-1 (7-
36) is reduced by 50%.
Example 12. Elevation of cAMP in Response to Peptides
[268] Peptide signaling of the GLP-1 receptor is measured using cAMP
scintillation proximity
assay. RINm5F cells are plated in 96-well plates (Costar) at 1.5
x105cells/well and grown at 37 C
for 24 hours in RPMI 1640, 5% FBS, antibiotic/antimycotic (Gibco BRL). The
media is removed
and the cells are washed twice with PBS. The cells are incubated with peptide
concentrations
ranging from 1x10'12 to 1x10'5 M in HEPES-PBS containing 1% BSA and 100 M
IBMX for 15 min
at 37 C. For assay of peptides conjugated with fatty acid, the BSA wasi
omitted from the
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incubation buffer. The incubation buffer is removed, and the cells are lysed
in the lysis reagent
provided with the cAMP Scintillation Proximity Assay (SPA) direct screening
assay system
(Amersham Pharmacia Biotech Inc, Piscataway, NJ). The amount of cAMP (in pmol)
present in
the lysates is determined following instructions provided with this kit. The
amount of cAMP (in
pmol) produced at each concentration of peptide is plotted and analyzed by
nonlinear regression
using Prizm software to determine the EC50 for each peptide.
Example 13. Insulin Secretion from Dispersed Rat Islet Cells
[269] Insulin secretion of dispersed rat islets mediated by a number of
peptides of the present
invention is measured as follows. Islets of Langerhans, isolated from SD rats
(200-250 g), are
digested using collagenase. The dispersed islet cells are treated with
trypsin, seeded into 96 V-
bottom plates, and pelleted. The cells are then cultured overnight in media
with or without
peptides of this invention. The media is aspirated, and the cells are pre-
incubated with Krebs-
Ringer-HEPES buffer containing 3 mM glucose for 30 minutes at 37 C. The pre-
incubation buffer
is removed, and the cells are incubated at 37 C with Krebs-Ringer-HEPES buffer
containing the
appropriate glucose concentration (e.g., 8 mM) with or without peptides for an
appropriate time. A
portion of the supernatant is removed and its insulin content is measured by
SPA. The results are
expressed as "fold over control" (FOC).
Example 14. Generation of Peptide Specific Antibodies and Peptide Measurement
by ELISA
[270] Polyclonal antibodies specific to the peptides of the present invention
are generated by
synthesizing a specific fragment of a peptide of this invention using an ABI
433A peptide
synthesizer. The peptide is then cleaved from the resin, and purified on a
Beckman System Gold
Analytical and Preparative HPLC system. A Perspective MALDI mass
spectrophotometer system
is used to identify the correct product. The peptide is dried using a
lyophilizer. The peptide (2 mg)
is then conjugated to keyhole limpet hemocyanin (KLH) via the free sulphydryl
group on the Cys.
[271] Female New Zealand White rabbits are immunized on Day 0, 14, 35, 56, and
77. On Day
0, each rabbit is injected subcutaneous with 250 g peptide and complete
Freund's adjuvant.
Subsequent immunizations utilize 125 g peptide per rabbit. Bleeds are started
on Day 21 and
continued at 21-day intervals thereafter. Purification of anti-peptide
antibodies is performed by
passing the crude serum over a specific peptide affinity purification column.
The antibody titer is
determined by ELISA.
[272] A 96-well Immulon 4HBX plate is coated with a C-terminal F(ab) antibody,
specific to the
peptides of the present invention, and allowed to incubate ovenight at 4 C.
The plate is then
blocked to prevent non-specific binding. Then, peptide standards (2500 ng/mL-1
60 pg/mL) are
diluted in 33% plasma and the samples are diluted 1:3 in buffer followed by
incubation for 1.5 h at
rt. After washing, a polyclonal N-terminal antibody specific to the peptides
of this invention is
incubated on the plate for 1 h. This is followed by the addition of
horseradish peroxidase (HRP)-
donkey-anti-rabbit antibody and the samples and standards are incubated for
another hour.
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Detection is assessed following incubation with 3,3',5,5'-tetramethylbenzidine
(TMB) solution, and
the plate is read at OD4e0 -
[273] Alternatively, the 96-well Immulon 4HBX plate is coated with a
polyclonal N-terminal
antibody, specific to the peptides of the present invention, and allowed to
incubate overnight at
4 C. The plate is then blocked to prevent non-specific binding. Then, peptide
standards (2500
ng/mL-1 60 pg/mL) are diluted in 50% plasma and the samples are diluted 1:2 in
buffer followed by
incubation for 1.5 h at rt. After washing, a monoclonal anti-PEG antibody
specific to the peptides
of this invention is incubated on the plate for one hour. This was followed by
the addition of
horseradish peroxidase (HRP)- anti-mouse antibody and the samples and
standards are incubated
for another hour. Detection is assessed following incubation with 3,3',5,5'-
tetramethylbenzidine
(TMB) solution, and the plate is read at OD450 =
Example 15: Phamacokinetics of Peptides Following IV and Subcutaneous Dosing
[274] Plasma samples are transferred to a microcentrifuge tube and an equal
volume of
acetonitrile is added to the sample (a 50% final concentration). The sample is
vigorously vortexed
for about 5 min and allowed to sit on ice for 10 min. The sample is again
vortexed for about 1 min,
and then centrifuged for 30 min in a microcentrifuge (4 C) at maximum (about
15,000 x g).
[275] Following centrifugation, the aqueous phase is carefully transferred to
a clean centrifuge
tube, and the sample is centrifuged for 5 min in a microcentrifuge (4 C) at
maximum speed (about
15,000 x g). The extracted sample is dried under vacuum using a Speed Vac
SC110 (Savant) with
a medium heat setting until dry. The sample is resuspended in an appropriate
volume of sterile
water and is maintained at 4 C. The sample is then sonicated in a sonibath for
10 min at rt prior to
analysis.
Example 16. Effect of Peptides on lntraperitoneal Glucose Tolerance in Rats
[276] The in vivo activity of the peptides of this invention when administered
subcutaneously is
examined in rats. Rats fasted overnight are given a subcutaneous injection of
control or peptide
(1-100 g/kg). Three hours later, basal blood glucose is measured, and the
rats are given 2 g/kg
of glucose intraperitoneally. Blood glucose is measured again after 15, 30,
and 60 min.
[277] Demonstration of the activity of the peptides of the present invention
may be accomplished
through in vitro, ex vivo, and in vivo assays that are well known in the art.
For example, to
demonstrate the efficacy of a pharmaceutical agent for the treatment of
diabetes and related
disorders such as Syndrome X, impaired glucose tolerance, impaired fasting
glucose, and
hyperinsulinemia; atherosclerotic disease and related disorders such as
hypertriglyceridemia and
hypercholesteremia; and obesity, the following assays may be used.
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Example 17. Method for Measuring Blood Glucose Levels
[278] db/db mice (obtained from Jackson Laboratories, Bar Harbor, ME) are bled
(by either eye
or tail vein) and grouped according to equivalent mean blood glucose levels.
They are dosed with
the test peptide for 14 days. At this point, the animals are bled again by eye
or tail vein and blood
glucose levels were determined. In each case, glucose levels are measured with
a Glucometer
Elite XL (Bayer Corporation, Elkhart, IN).
Example 18. Method for Measuring an Effect on Cardiovascular Parameters
[279] Cardiovascular parameters (e.g., heart rate and blood pressure) are also
evaluated. SHR
rats are dosed with vehicle or test peptide for 2 weeks. Blood pressure and
heart rate are
determined using a tail-cuff method as described by Grinsell, et al., (Am. J.
Hypertens. 13:370-
375, 2000). In monkeys, blood pressure and heart rate are monitored as
described by Shen, et
al., (J. Pharmacol. Exp. Therap. 278:1435-1443, 1996).
Example 19. Method for Measuring Triglyceride Levels
[280] hApoAl mice (obtained from Jackson Laboratories, Bar Harbor, ME) are
bled (by either
eye or tail vein) and grouped according to equivalent mean serum triglyceride
levels. They are
dosed with the test peptide for 8 days. The animals are then bled again by eye
or tail vein, and
serum triglyceride levels are determined. In each case, triglyceride levels
are measured using a
Technicon Axon Autoanalyzer (Bayer Corporation, Tarrytown, NY).
Example 20. Method for Measuring HDL-Cholesterol Levels
[281] To determine plasma HDL-cholesterol levels, hApoAl mice are bled and
grouped with
equivalent mean plasma HDL-cholesterol levels. The mice are dosed with vehicle
or test peptide
for 7 days, and then bled again on day 8. Plasma is analyzed for HDL-
cholesterol using the
Synchron Clinical System (CX4) (Beckman Coulter, Fullerton, CA).
Example 21. Method for Measuring Total Cholesterol, HDL-Cholesterol,
Triglycerides, and
Glucose Levels
[282] In another in vivo assay, obese monkeys are bled, then dosed with
vehicle or test peptide
for 4 weeks, and then bled again. Serum is analyzed for total cholesterol, HDL-
cholesterol,
triglycerides, and glucose using the Synchron Clinical System (CX4) (Beckman
Coulter, Fullerton,
CA). Lipoprotein subclass analysis is performed by NMR spectroscopy as
described by Oliver, et
al., (Proc. Natl. Acad. Sci. USA 98:5306-5311, 2001).
[283] All publications and patents mentioned in the above specification are
incorporated herein
by reference. Various modifications and variations of the described
compositions and methods
of the invention will be apparent to those skilled in the art without
departing from the scope and
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spirit of the invention. Although the invention has been described in
connection with specific
preferred embodiments, it should be understood that the invention as claimed
should not be
unduly limited to such specific embodiments. Indeed, various modifications of
the above-
described modes for carrying out the invention which are obvious to those
skilled in the field of
biochemistry or related fields are intended to be within the scope of the
following claims. Those
skilled in the art will recognize, or be able to ascertain using no more than
routine
experimentation, many equivalents to the specific embodiments of the invention
described herein.
Such equivalents are intended to be encompassed by the following claims.
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TABLE 1
SEQID SEQUENCE
NO
2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (GLP-1(7-37))
3 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-NH2 (GLP-1(7-36))
4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-
HSQGTFTSDYSKYLDSRRAQDFVQWLVKGR-NH2
6 HSQGTFTSDYSKYLEGQAAKEFIAWLVKGR-NH2
7 HSQGTFTSDYAKYLDARRAKEFIAWLVKGR-NH2
8 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRG
9 HSQGTFTSDYARYLDARRAKEFIAWLVKGR-NH2
HSQGTFTSDYAAYLDARRAKEFIAWLVKGR-NH2
11 HSQGTFTSDYAKYLDAARAKEFIAWLVKGR-NH2
12 HSQGTFTSDYAKYLDAKKAKEFIAWLVKGRG
13 HSQGTFTSDYARYLDAKKAKEFIAWLVKGRG
14 HSQGTFTSDYAKYLDAAKAKEFIAWLVKGRG
HSQGTFTSDYARYLDAAKAKEFIAWLVKGRG
16 HSQGTFTSDYAKYLDARRACEFIAWLVKGRG
17 HSQGTFTSDYAKYLDARRAKEFIAWLVCGRG
18 HSQGTFTSDYAKYLDARRAKEFIAWLVKCRG
19 HSQGTFTSDYAKYLDARRAKEFIAWLVKGCG
HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC
21 HSQGTFTSDYARYLDARRAKEFIAWLVRGRG
22 HSQGTFTSDYARYLDARRAREFIKWLVRGRG
23 HSQGTFTSDYARYLDARRAREFIAWLVKGRG
24 HSQGTFTSDYARYLDAR RARE FIAW LVRG RG K
HSQGTFTSDYARYLDARRAREFIKWLVRGRC
26 HSQGTFTSDYSKYLDSRRAQDFVQWLVKGR-NH2
--- - - -
27 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRK
28 HSQGTFTSDYARYLDARRAREFIKWLVRGRG
29 HSQGTFTSDYARYLDARRAREFIKWLVRGRGK
HSQGTFTSDYARYLDARRAKEFIKWLVRGRG
31 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC
32 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC
33 HSQGTFTSDYSKYLDSRRAQDFVQWLVKGRC
34 HSQGTFTSDYSKYLEGQAAKEFIAWLVKGRC
HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC
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TABLE 1 (Cont'd)
SEQID SEQUENCE
NO
36 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRGC
37 HSQGTFTSDYARYLDARRAKEFIAWLVKGRC
38 HSQGTFTSDYAAYLDARRAKEFIAWLVKGRC
39 HSQGTFTSDYAKYLDAARAKEFIAWLVKGRC
40 HSQGTFTSDYAKYLDAKKAKEFIAWLVKGRGC
41 HSQGTFTSDYARYLDAKKAKEFIAWLVKGRGC
42 HSQGTFTSDYAKYLDAAKAKEFIAWLVKGRGC
43 HSQGTFTSDYARYLDAAKAKEFIAWLVKGRGC
44 HSQGTFTSDYAKYLDARRACEFIAWLVKGRGC
45 HSQGTFTSDYAKYLDARRAKEFIAWLVCGRGC
46 HSQGTFTSDYAKYLDARRAKEFIAWLVKCRGC
47 HSQGTFTSDYAKYLDARRAKEFIAWLVKGCGC
48 HSQGTFTSDYARYLDARRAKEFIAWLVRGRGC
49 HSQGTFTSDYARYLDARRAREFIKWLVRGRGC
50 HSQGTFTSDYARYLDARRAREFIAWLVKGRGC
51 HSQGTFTSDYARYLDARRAREFIAWLVRGRGKC
52 HSQGTFTSDYSKYLDSRRAQDFVQWLVKGRC
53 HSQGTFTSDYAKYLDARRAKEFIAWLVKGRKC
54 HSQGTFTSDYARYLDARRAREFIKWLVRGRGC
55 HSQGTFTSDYARYLDARRAREFIKWLVRGRGKC
56 HSQGTFTSDYARYLDARRAKEFIKWLVRGRGC
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TABLE 2
PEG reagent Structure
0
Linear PEG
(e.g., Sunbright ME- CH30(CH2CH2O)n-(CH2)3NHC0(CH2)2-N
200MA)
O
Linear PEG
0
mPEG-MAL
(e.g., Nektar 2D2MOH01 CH30(CH2CH20)õN
and 2D2MOP01)
0
CHgO(CH2CH2O)n 0
Branched PEG
HN ~-(OCH2CH2)nOCH3
mPEG2-MAL H
0 ~/N
(e.g., Nektar 2D3XOT01) 0 N O
H
Branched PEG 0 (OCH2CH2)nOCH3
0
(e.g., NOF GL2-400MA) N H (OCH2CH2)nOCH3
O
Branched PEG - - /~(OCH2CH2)nOCH3
0 ~O/~~
(e.g., NOF GL2- O~~N (OCH2CH2)nOCH3
400MA2) '-1 H
O
71
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