U.S. patent application number 17/488137 was filed with the patent office on 2022-01-13 for pth prodrugs.
This patent application is currently assigned to ASCENDIS PHARMA BONE DISEASES A/S. The applicant listed for this patent is ASCENDIS PHARMA BONE DISEASES A/S. Invention is credited to Felix Cleemann, Mathias Krusch, Guillaume Maitro, Kennett Sprogoe, Thomas Wegge, Joachim Zettler.
Application Number | 20220008516 17/488137 |
Document ID | / |
Family ID | 1000005869361 |
Filed Date | 2022-01-13 |
United States Patent
Application |
20220008516 |
Kind Code |
A1 |
Sprogoe; Kennett ; et
al. |
January 13, 2022 |
PTH Prodrugs
Abstract
The present invention relates to PTH prodrugs, pharmaceutical
compositions comprising such PTH prodrugs and their uses.
Inventors: |
Sprogoe; Kennett; (Holte,
DK) ; Cleemann; Felix; (Mainz, DE) ; Maitro;
Guillaume; (Mannheim, DE) ; Krusch; Mathias;
(Hirschhorn, DE) ; Wegge; Thomas; (Heidelberg,
DE) ; Zettler; Joachim; (Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASCENDIS PHARMA BONE DISEASES A/S |
HELLERUP |
|
DK |
|
|
Assignee: |
ASCENDIS PHARMA BONE DISEASES
A/S
HELLERUP
DK
|
Family ID: |
1000005869361 |
Appl. No.: |
17/488137 |
Filed: |
September 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16118155 |
Aug 30, 2018 |
|
|
|
PCT/EP2017/054550 |
Feb 28, 2017 |
|
|
|
17488137 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/545 20170801;
A61K 47/60 20170801; A61K 47/54 20170801; A61K 38/29 20130101; A61K
9/0019 20130101 |
International
Class: |
A61K 38/29 20060101
A61K038/29; A61K 47/54 20060101 A61K047/54; A61K 47/60 20060101
A61K047/60; A61K 9/00 20060101 A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2016 |
EP |
16158048.5 |
Jul 13, 2016 |
EP |
16179294.0 |
Sep 29, 2016 |
EP |
16191484.1 |
Feb 13, 2017 |
EP |
17155839.8 |
Claims
1-18. (canceled)
19. A parathyroid hormone (PTH) conjugate of formula (IIf-i) or a
pharmaceutically acceptable salt thereof: ##STR00074## wherein the
unmarked dashed line indicates the attachment to the N-terminal
nitrogen of a PTH moiety having the sequence of SEQ ID NO:51; and
the dashed line marked with the asterisk indicates attachment to a
moiety ##STR00075## wherein m and p are independently an integer
ranging from and including 400 to 500.
20. The PTH conjugate or a pharmacological salt thereof of claim
19, wherein the moiety ##STR00076## has a molecular weight of about
40 kDa.
21. The PTH conjugate or a pharmacological salt thereof of claim
19, wherein m and p are the same integer.
22. The PTH conjugate or a pharmacological salt thereof of claim
19, wherein m and p are about 450.
23. A pharmaceutical composition comprising at least one PTH
conjugate or a pharmaceutically acceptable salt thereof of claim 19
and at least one excipient.
24. The pharmaceutical composition of claim 19, wherein the
pharmaceutical composition has a pH ranging from and including pH 4
to pH 6.
25. A method of treating a patient having hypoparathyroidism
comprising administering an effective amount of the PTH conjugate
or pharmaceutically acceptable salt thereof of claim 1 or a
pharmaceutical composition comprising such PTH conjugate or
pharmaceutically acceptable salt thereof to the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/118,155 filed Aug. 30, 2018, the US national stage entry of
International Application No. PCT/EP2017/054550 filed Feb. 28,
2017, which is incorporated by reference in its entirety for all
purposes, and which claims the benefit of EP Application No.
16158048.5 filed Mar. 1, 2016; EP application Ser. No., 16179294.0
filed Jul. 13, 2016; EP Application No. 16191484.1 filed Sep. 29,
2016; and EP Application No. 17155839.8 filed Feb. 13, 2017.
REFERENCE TO SEQUENCE LISTING
[0002] This application includes an electronic sequence listing in
a file named 565511SEQLST.TXT, created on Sep. 24, 2021 and
containing 75,897 bytes, which is incorporated by reference in its
entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to PTH prodrugs,
pharmaceutical compositions comprising such PTH prodrugs and their
uses.
BACKGROUND
[0004] Hypoparathyroidism is a rare endocrine disease with low
serum calcium and inappropriately low (insufficient) circulating
parathyroid hormone levels, most often in adults secondary to
thyroid surgery. Standard treatment includes activated vitamin D
analogues and calcium supplementation, which increases calcium and
phosphorus absorption and serum levels at the expense of abnormally
increased urinary calcium excretion. Hypoparathyroidism is the only
major endocrine condition today, where the hormonal insufficiency
in general is not treated by substitution of the missing hormone
(PTH).
[0005] The prevalence of hypoparathyroidism has recently been
systematically studied in Denmark, where a total of more than 2000
patients were identified giving a prevalence of 24/100 000
inhabitants, among whom only a minority (2/100 000) had
hypoparathyroidism due to non-surgical causes. These estimates are
in agreement with recent data from the USA, showing a prevalence of
the same magnitude for patients with chronic
hypoparathyroidism.
[0006] Endogenous PTH is synthesized and secreted by the
parathyroid glands and is the principal endocrine hormone
regulating systemic calcium and phosphorus homeostasis.
Physiological actions of PTH include releasing calcium and
phosphorus from bone, retaining calcium but not phosphorus in the
kidney by increasing renal tubular reabsorption of calcium but
decreasing renal tubular reabsorption of phosphate, and stimulating
the renal production of active vitamin D (1,25(OH).sub.2vitamin D3)
which in turn enhances intestinal calcium and phosphorus
absorption. Without the renal actions of PTH to conserve calcium
and excrete phosphorus, conventional therapy with vitamin D analogs
and calcium supplementation may lead to renal insufficiency or
failure due to progressive nephrocalcinosis, as well as ectopic
calcifications (in the basal ganglia, the lens of the eye, and the
vascular system) due to a chronically increased calcium x
phosphorus product, which precipitates out as calcium phosphate
crystals when this product is maintained elevated for long
periods.
[0007] Recently Natpara.RTM., PTH(1-84) was approved by the FDA for
the treatment of hypoparathyroidism. Historically Forteo.RTM.
PTH(1-34) has also been used as once, twice or thrice daily
injections for hypoparathyroidism, despite not being approved for
this indication.
[0008] When PTH is delivered intermittently, such as by current
daily or multiple daily injections of PTH(1-84) or PTH(1-34) it
acts on bone as an anabolic agent by preferentially activating
osteoblasts over osteoclasts. This anabolic effect of intermittent
PTH exposure contrasts with the net bone catabolism that can occur
with continuous exposure to PTH. The anabolic potential of
intermittent administration of PTH agonists has successfully been
utilized for the treatment of osteoporosis, where bone turnover is
usually high and bone mineral density (BMD) is low, whereas the
converse is the case for hypoparathyroidism.
[0009] A major complication of hypoparathyroidism is
hypercalciuria, due to the lack of PTH dependent calcium
reabsorption in the distal renal tubules. Hypercalciuria is
associated with an increased risk of nephrocalcinosis,
nephrolithiasis and kidney failure. According to the FDAs review of
Natpara, daily injections of PTH failed to provide adequate control
of urinary calcium excretion, due to the short half-life of this
PTH agonist in the body.
[0010] Furthermore, unphysiological levels of PTH may be associated
with hypercalcemia and hypocalcemia. Treatment with Natpara did not
improve the incidence of these complications compared to placebo.
This can in part be explained by the unfavorable PK of Natpara. For
example, administration of the currently approved doses of Natpara
results in greatly supraphysiological levels of PTH with a
C.sub.max of 300 pg/ml, which returns to baseline at 12 hours. As a
result patients are over treated in the initial phase following
administration and under treated in the phase leading up to
subsequent dosing.
[0011] Hypocalcemia is associated with numerous symptoms, some of
which can be life threatening, including: tetany; paresthesias;
impaired cognition; loss of consciousness with convulsions (grand
mal seizures); impaired kidney function; heart arrhythmias and
fainting, and even heart failure.
[0012] As such, there is a high unmet need for a more physiological
PTH therapy, providing a sustained exposure to PTH that enables
alleviation of symptoms relating to hypocalcemia, hypercalciuria,
and hyperphosphatemia, without causing hypercalcemia.
[0013] PTH replacement therapy would be more physiologic if
delivered by continuous infusion, such as using an insulin pump.
This has for example been demonstrated by Winer et al. (J Pediatr,
2014, 165(3), 556-563), where pump delivery simultaneously
normalized bone turnover markers and urine and serum mineral
levels, whereas intermittent injection delivery did not.
[0014] The normal PTH range is 15-50 pg/ml, and it is important to
appreciate that intermittent PTH agonist administration does not
constitute physiological replacement therapy. PTH polypeptides have
inherently short circulating half-lives because of rapid hepatic
metabolism. The rapid clearance of the drug from the body prevents
sufficient drug coverage throughout the dosing interval, despite
initial supraphysiological drug levels.
[0015] Several approaches have been applied to create longer acting
version of PTH, including encapsulation of PTH in PLGA
microparticles and permanent conjugation of the PTH molecule to
either synthetic or peptidic polymers. Kostenuik et al. (J Bone
Miner Res, 2007, 22(10), 1534-1547), described a PTH-Fc fusion
protein with a longer half-life than PTH(1-34) and studies were
conducted in osteopenic ovariectomized rats and mice to determine
whether intermittent (one to two per week) injections of PTH-Fc
would increase bone mass, density, and strength despite the
prolonged duration of exposure to PTH. It was demonstrated that a
PTH-derived molecule with a sustained circulating half-life
provided comparable anabolic effects on cortical and cancellous
bone to daily PTH, but with a reduced dosing frequency. Another
approach has been suggested by Ponnapakkam et al. (Drug Discov
Today, 2014, 19(3), 204.208), in which a hybrid polypeptide of PTH
and a collagen binding domain caused long-term (up to 12 months)
increases in bone mineral density in normal female mice after a
single dose.
[0016] Patients with hypoparathyroidism typically demonstrate an
abnormally low rate of bone turnover resulting in increased bone
mineral density, and as such the anabolic effects of PTH should be
avoided when treating Hypoparathyroidism, and optimally treatment
should normalize their rate of bone turnover, but not increase it
to above the normal range, as has been demonstrated with daily
treatment with PTH(1-34) and PTH(1-84).
[0017] In summary, there is a need for a more efficacious PTH
treatment.
SUMMARY OF THE INVENTION
[0018] It is therefore an object of the present invention to at
least partially overcome the shortcomings described above.
[0019] This object is achieved with a PTH prodrug or a
pharmaceutically acceptable salt thereof, wherein the prodrug is of
formula (Ia) or (Ib)
##STR00001## [0020] wherein [0021] -D is a PTH moiety; [0022]
-L.sup.1- is a reversible prodrug linker moiety connected to the
PTH moiety -D through a functional group of PTH; [0023] -L.sup.2-
is a single chemical bond or a spacer moiety; [0024] --Z is a
water-soluble carrier moiety; [0025] x is an integer selected from
the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15 or 16; and [0026] y is an integer selected from the group
consisting of 1, 2, 3, 4 and 5.
[0027] It was surprisingly found that the PTH prodrugs of the
present invention exhibit a low residual activity of the prodrug
and provide a sustained release of PTH. As a result, administration
of the PTH prodrugs of the present invention leads to a concurrent
normalization of serum calcium and a reduction in serum phosphate
and thus to an increased serum calcium to serum phosphate ratio
compared to treatment with PTH1-84, the current standard of care.
At the same time no advese effects on bone resorption and formation
markers and overall bone health in the relevant animal model for
the human condition were observed upon administration of
physiological doses.
[0028] It was also surprisingly found that such PTH prodrugs are
capable of achieving a stable plasma profile of PTH which ensures
physiological serum and urinary calcium levels or even lower than
normal urinary calcium levels.
DETAILED DESCRIPTION
[0029] Within the present invention the terms are used having the
meaning as follows.
[0030] As used herein the term "PTH" refers to all PTH
polypeptides, preferably from mammalian species, more preferably
from human and mammalian species, more preferably from human and
murine species, as well as their variants, analogs, orthologs,
homologs, and derivatives and fragments thereof, that are
characterized by raising serum calcium and renal phosphorus
excretion, and lowering serum phosphorus and renal calcium
excretion. The term "PTH" also refers to all PTHrP polypeptides,
such as the polypeptide of SEQ ID NO:121, that bind to and activate
the common PTH/PTHrP1 receptor. Preferably, the term "PTH" refers
to the PTH polypeptide of SEQ ID NO:51 as well as its variants,
homologs and derivatives exhibiting essentially the same biological
activity, i.e. raising serum calcium and renal phosphorus
excretion, and lowering serum phosphorus and renal calcium
excretion.
[0031] Preferably, the term "PTH" refers to the following
polypeptide sequences:
TABLE-US-00001 (PTH 1-84) SEQ ID NO: 1
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (PTH 1-83) SEQ ID NO: 2
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAKS (PTH 1-82) SEQ ID NO: 3
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAK (PTH 1-81) SEQ ID NO: 4
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKA (PTH 1-80) SEQ ID NO: 5
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTK (PTH 1-79) SEQ ID NO: 6
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLT (PTH 1-78) SEQ ID NO: 7
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVL (PTH 1-77) SEQ ID NO: 8
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNV (PTH 1-76) SEQ ID NO: 9
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVN (PTH 1-75) SEQ ID NO: 10
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADV (PTH 1-74) SEQ ID NO: 11
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKAD (PTH 1-73) SEQ ID NO: 12
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKA (PTH 1-72) SEQ ID NO: 13
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADK (PTH 1-71) SEQ ID NO: 14
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEAD (PTH 1-70) SEQ ID NO: 15
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEA (PTH 1-69) SEQ ID NO: 16
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGE (PTH 1-68) SEQ ID NO: 17
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLG (PTH 1-67) SEQ ID NO: 18
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSL (PTH 1-66) SEQ ID NO: 19
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKS
(PTH 1-65) SEQ ID NO: 20
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEK
(PTH 1-64) SEQ ID NO: 21
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHE
(PTH 1-63) SEQ ID NO: 22
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESH
(PTH 1-62) SEQ ID NO: 23
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVES
(PTH 1-61) SEQ ID NO: 24
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVE (PTH
1-60) SEQ ID NO: 25
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLV (PTH
1-59) SEQ ID NO: 26
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVL (PTH
1-58) SEQ ID NO: 27
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNV (PTH
1-57) SEQ ID NO: 28
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDN (PTH
1-56) SEQ ID NO: 29
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKED (PTH
1-55) SEQ ID NO: 30
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKE (PTH 1-54)
SEQ ID NO: 31 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKK (PTH 1-53) SEQ ID NO: 32
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRK (PTH 1-52)
SEQ ID NO: 33 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PR
(PTH 1-51) SEQ ID NO: 34
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ RP (PTH 1-50) SEQ
ID NO: 35 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR (PTH
1-49) SEQ ID NO: 36
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ (PTH 1-48) SEQ ID
NO: 37 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS (PTH 1-47)
SEQ ID NO: 38 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG (PTH
1-46) SEQ ID NO: 39 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA
(PTH 1-45) SEQ ID NO: 40
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD (PTH 1-44) SEQ ID NO:
41 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR (PTH 1-43) SEQ ID
NO: 42 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP (PTH 1-42) SEQ
ID NO: 43 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA (PTH 1-41) SEQ
ID NO: 44 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL (PTH 1-40) SEQ
ID NO: 45 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP (PTH 1-39) SEQ
ID NO: 46
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA (PTH 1-38) SEQ ID NO: 47
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG (PTH 1-37) SEQ ID NO: 48
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL (PTH 1-36) SEQ ID NO: 49
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA (PTH 1-35) SEQ ID NO: 50
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV (PTH 1-34) SEQ ID NO: 51
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (PTH 1-33) SEQ ID NO: 52
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN (PTH 1-32) SEQ ID NO: 53
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH (PTH 1-31) SEQ ID NO: 54
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV (PTH 1-30) SEQ ID NO: 55
SVSEIQLMHNLGKHLNSMERVEWLRKKLQD (PTH 1-29) SEQ ID NO: 56
SVSEIQLMHNLGKHLNSMERVEWLRKKLQ (PTH 1-28) SEQ ID NO: 57
SVSEIQLMHNLGKHLNSMERVEWLRKKL (PTH 1-27) SEQ ID NO: 58
SVSEIQLMHNLGKHLNSMERVEWLRKK (PTH 1-26) SEQ ID NO: 59
SVSEIQLMHNLGKHLNSMERVEWLRK (PTH 1-25) SEQ ID NO: 60
SVSEIQLMHNLGKHLNSMERVEWLR (amidated PTH 1-84) SEQ ID NO: 61
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ; wherein the C-terminus is
amidated (amidated PTH 1-83) SEQ ID NO: 62
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAKS; wherein the C-terminus is
amidated (amidated PTH 1-82) SEQ ID NO: 63
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKAK; wherein the C-terminus is
amidated (amidated PTH 1-81) SEQ ID NO: 64
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTKA; wherein the C-terminus is amidated
(amidated PTH 1-80) SEQ ID NO: 65
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLTK; wherein the C-terminus is amidated
(amidated PTH 1-79) SEQ ID NO: 66
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVLT; wherein the C-terminus is amidated
(amidated PTH 1-78) SEQ ID NO: 67
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNVL; wherein the C-terminus is amidated
(amidated PTH 1-77) SEQ ID NO: 68
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVNV; wherein the C-terminus is amidated
(amidated PTH 1-76) SEQ ID NO: 69
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADVN; wherein the C-terminus is amidated
(amidated PTH 1-75) SEQ ID NO: 70
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKADV; wherein the C-terminus is amidated
(amidated PTH 1-74) SEQ ID NO: 71
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKAD; wherein the C-terminus is amidated
(amidated PTH 1-73) SEQ ID NO: 72
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADKA; wherein the C-terminus is amidated
(amidated PTH 1-72) SEQ ID NO: 73
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADK; wherein the C-terminus is amidated
(amidated PTH 1-71) SEQ ID NO: 74
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEAD; wherein the C-terminus is amidated (amidated
PTH 1-70) SEQ ID NO: 75
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEA; wherein the C-terminus is amidated (amidated
PTH 1-69) SEQ ID NO: 76
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGE; wherein the C-terminus is amidated (amidated
PTH 1-68) SEQ ID NO: 77
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLG; wherein the C-terminus is amidated (amidated
PTH 1-67) SEQ ID NO: 78
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSL; wherein the C-terminus is amidated (amidated PTH
1-66) SEQ ID NO: 79
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKS; wherein the C-terminus is amidated (amidated PTH
1-65) SEQ ID NO: 80
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEK;
wherein the C-terminus is amidated (amidated PTH 1-64) SEQ ID NO:
81 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHE; wherein the C-terminus is amidated (amidated PTH
1-63) SEQ ID NO: 82
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESH;
wherein the C-terminus is amidated (amidated PTH 1-62) SEQ ID NO:
83 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVES;
wherein the C-terminus is amidated (amidated PTH 1-61) SEQ ID NO:
84 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVE;
wherein the C-terminus is amidated (amidated PTH 1-60) SEQ ID NO:
85 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLV;
wherein the C-terminus is amidated (amidated PTH 1-59) SEQ ID NO:
86 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVL;
wherein the C-terminus is amidated (amidated PTH 1-58) SEQ ID NO:
87 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNV;
wherein the C-terminus is amidated (amidated PTH 1-57) SEQ ID NO:
88 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKEDN; wherein the C-terminus is amidated (amidated PTH 1-56) SEQ
ID NO: 89 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR
PRKKED; wherein the C-terminus is amidated (amidated PTH 1-55) SEQ
ID NO: 90 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKE;
wherein the C-terminus is amidated (amidated PTH 1-54) SEQ ID NO:
91 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKK; wherein
the C-terminus is amidated (amidated PTH 1-53) SEQ ID NO: 92
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRK; wherein the
C-terminus is amidated (amidated PTH 1-52) SEQ ID NO: 93
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PR; wherein the
C-terminus is amidated (amidated PTH 1-51) SEQ ID NO: 94
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR P; wherein the
C-terminus is amidated (amidated PTH 1-50) SEQ ID NO: 95
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ R; wherein the
C-terminus is amidated (amidated PTH 1-49) SEQ ID NO: 96
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ; wherein the
C-terminus is amidated (amidated PTH 1-48) SEQ ID NO: 97
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS; wherein the
C-terminus is amidated (amidated PTH 1-47) SEQ ID NO: 98
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG; wherein the
C-terminus is amidated (amidated PTH 1-46) SEQ ID NO: 99
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA; wherein the
C-terminus is amidated (amidated PTH 1-45) SEQ ID NO: 100
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD; wherein the
C-terminus is amidated (amidated PTH 1-44) SEQ ID NO: 101
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR; wherein the
C-terminus is amidated (amidated PTH 1-43) SEQ ID NO: 102
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP; wherein the C-terminus
is amidated (amidated PTH 1-42) SEQ ID NO: 103
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA; wherein the C-terminus
is amidated (amidated PTH 1-41) SEQ ID NO: 104
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL; wherein the C-terminus
is amidated (amidated PTH 1-40) SEQ ID NO: 105
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP; wherein the C-terminus is
amidated (amidated PTH 1-39) SEQ ID NO: 106
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA; wherein the C-terminus is
amidated (amidated PTH 1-38) SEQ ID NO: 107
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG; wherein the C-terminus is
amidated (amidated PTH 1-37) SEQ ID NO: 108
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL; wherein the C-terminus is
amidated (amidated PTH 1-36) SEQ ID NO: 109
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA; wherein the C-terminus is
amidated (amidated PTH 1-35) SEQ ID NO: 110
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV; wherein the C-terminus is
amidated (amidated PTH 1-34) SEQ ID NO: 111
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; wherein the C-terminus is
amidated (amidated PTH 1-33) SEQ ID NO: 112
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN; wherein the C-terminus is
amidated (amidated PTH 1-32) SEQ ID NO: 113
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH; wherein the C-terminus is
amidated (amidated PTH 1-31) SEQ ID NO: 114
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV; wherein the C-terminus is amidated
(amidated PTH 1-30) SEQ ID NO: 115 SVSEIQLMHNLGKHLNSMERVEWLRKKLQD;
wherein the C-terminus is amidated (amidated PTH 1-29) SEQ ID NO:
116 SVSEIQLMHNLGKHLNSMERVEWLRKKLQ; wherein the C-terminus is
amidated (amidated PTH 1-28) SEQ ID NO: 117
SVSEIQLMHNLGKHLNSMERVEWLRKKL; wherein the C-terminus is amidated
(amidated PTH 1-27) SEQ ID NO: 118 SVSEIQLMHNLGKHLNSMERVEWLRKK;
wherein the C-terminus is amidated (amidated PTH 1-26) SEQ ID NO:
119 SVSEIQLMHNLGKHLNSMERVEWLRK; wherein the C-terminus is amidated
(amidated PTH 1-25) SEQ ID NO: 120 SVSEIQLMHNLGKHLNSMERVEWLR;
wherein the C-terminus is amidated (PTHrP) SEQ ID NO: 121
AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSP
NTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQ
EKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH
[0032] More preferably, the term "PTH" refers to the sequence of
SEQ ID:NOs 47, 48, 49, 50, 51, 52, 53, 54, 55, 107, 108, 109, 110,
111, 112, 113, 114 and 115. Even more preferably, the term "PTH"
refers to the sequence of SEQ ID:NOs 50, 51, 52, 110, 111 and 112.
In a particularly preferred embodiment the term "PTH" refers to the
sequence of SEQ ID NO:51.
[0033] As used herein, the term "PTH polypeptide variant" refers to
a polypeptide from the same species that differs from a reference
PTH or PTHrP polypeptide. Preferably, such reference is a PTH
polypeptide sequence and has the sequence of SEQ ID NO:51.
Generally, differences are limited so that the amino acid sequence
of the reference and the variant are closely similar overall and,
in many regions, identical. Preferably, PTH polypeptide variants
are at least 70%, 80%, 90%, or 95% identical to a reference PTH or
PTHrP polypeptide, preferably to the PTH polypeptide of SEQ ID
NO:51. By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence, it is
intended that the amino acid sequence of the subject polypeptide is
identical to the query sequence except that the subject polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the query amino acid sequence. These alterations of
the reference sequence may occur at the amino (N-terminal) or
carboxy terminal (C-terminal) positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence. The
query sequence may be an entire amino acid sequence of the
reference sequence or any fragment specified as described herein.
Preferably, the query sequence is the sequence of SEQ ID NO:51.
[0034] Such PTH polypeptide variants may be naturally occurring
variants, such as naturally occurring allelic variants encoded by
one of several alternate forms of a PTH or PTHrP occupying a given
locus on a chromosome or an organism, or isoforms encoded by
naturally occurring splice variants originating from a single
primary transcript. Alternatively, a PTH polypeptide variant may be
a variant that is not known to occur naturally and that can be made
by mutagenesis techniques known in the art.
[0035] It is known in the art that one or more amino acids may be
deleted from the N-terminus or C-terminus of a bioactive
polypeptide without substantial loss of biological function. Such
N- and/or C-terminal deletions are also encompassed by the term PTH
polypeptide variant.
[0036] It is also recognized by one of ordinary skill in the art
that some amino acid sequences of PTH or PTHrP polypeptides can be
varied without significant effect of the structure or function of
the polypeptide. Such mutants include deletions, insertions,
inversions, repeats, and substitutions selected according to
general rules known in the art so as to have little effect on
activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided in Bowie
et al. (1990), Science 247:1306-1310, which is hereby incorporated
by reference in its entirety, wherein the authors indicate that
there are two main approaches for studying the tolerance of the
amino acid sequence to change.
[0037] The term PTH polypeptide also encompasses all PTH and PTHrP
polypeptides encoded by PTH and PTHrP analogs, orthologs, and/or
species homologs. It is also recognized by one of ordinary skill in
the art that PTHrP and PTHrP analogs bind to activate the common
PTH/PTHrP1 receptor, so the term PTH polypeptide also encompasses
all PTHrP analogs. As used herein, the term "PTH analog" refers to
PTH and PTHrP of different and unrelated organisms which perform
the same functions in each organism but which did not originate
from an ancestral structure that the organisms' ancestors had in
common. Instead, analogous PTH and PTHrP arose separately and then
later evolved to perform the same or similar functions. In other
words, analogous PTH and PTHrP polypeptides are polypeptides with
quite different amino acid sequences but that perform the same
biological activity, namely raising serum calcium and renal
phosphorus excretion, and lowering serum phosphorus and renal
calcium excretion.
[0038] As used herein the term "PTH ortholog" refers to PTH and
PTHrP within two different species which sequences are related to
each other via a common homologous PTH or PTHrP in an ancestral
species, but which have evolved to become different from each
other.
[0039] As used herein, the term "PTH homolog" refers to PTH and
PTHrP of different organisms which perform the same functions in
each organism and which originate from an ancestral structure that
the organisms' ancestors had in common. In other words, homologous
PTH polypeptides are polypeptides with quite similar amino acid
sequences that perform the same biological activity, namely raising
serum calcium and renal phosphorus excretion, and lowering serum
phosphorus and renal calcium excretion. Preferably, PTH polypeptide
homologs may be defined as polypeptides exhibiting at least 40%,
50%, 60%, 70%, 80%, 90% or 95% identity to a reference PTH or PTHrP
polypeptide, preferably the PTH polypeptide of SEQ ID NO:51.
[0040] Thus, a PTH polypeptide according to the invention may be,
for example: (i) one in which at least one of the amino acids
residues is substituted with a conserved or non-conserved amino
acid residue, preferably a conserved amino acid residue, and such
substituted amino acid residue may or may not be one encoded by the
genetic code; and/or (ii) one in which at least one of the amino
acid residues includes a substituent group; and/or (iii) one in
which the PTH polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol); and/or (iv) one in which additional amino
acids are fused to the PTH polypeptide, such as an IgG Fc fusion
region polypeptide or leader or secretory sequence or a sequence
which is employed for purification of the above form of the
polypeptide or a pre-protein sequence.
[0041] As used herein, the term "PTH polypeptide fragment" refers
to any polypeptide comprising a contiguous span of a part of the
amino acid sequence of a PTH or PTHrP polypeptide, preferably the
polypeptide of SEQ ID NO:51.
[0042] More specifically, a PTH polypeptide fragment comprises at
least 6, such as at least 8, at least 10 or at least 17 consecutive
amino acids of a PTH or PTHrP polypeptide, more preferably of the
polypeptide of SEQ ID NO:51. A PTH polypeptide fragment may
additionally be described as sub-genuses of PTH or PTHrP
polypeptides comprising at least 6 amino acids, wherein "at least
6" is defined as any integer between 6 and the integer representing
the C-terminal amino acid of a PTH or PTHrP polypeptide, preferably
of the polypeptide of SEQ ID No:51. Further included are species of
PTH or PTHrP polypeptide fragments at least 6 amino acids in
length, as described above, that are further specified in terms of
their N-terminal and C-terminal positions. Also encompassed by the
term "PTH polypeptide fragment" as individual species are all PTH
or PTHrP polypeptide fragments, at least 6 amino acids in length,
as described above, that may be particularly specified by a
N-terminal and C-terminal position. That is, every combination of a
N-terminal and C-terminal position that a fragment at least 6
contiguous amino acid residues in length could occupy, on any given
amino acid sequence of a PTH or PTHrP polypeptide, preferably the
PTH polypeptide of SEQ ID:NO51, is included in the present
invention.
[0043] The term "PTH" also includes poly(amino acid) conjugates
which have a sequence as described above, but having a backbone
that comprises both amide and non-amide linkages, such as ester
linkages, like for example depsipeptides. Depsipeptides are chains
of amino acid residues in which the backbone comprises both amide
(peptide) and ester bonds. Accordingly, the term "side chain" as
used herein refers either to the moiety attached to the
alpha-carbon of an amino acid moiety, if the amino acid moiety is
connected through amine bonds such as in polypeptides, or to any
carbon atom-comprising moiety attached to the backbone of a
poly(amino acid) conjugate, such as for example in the case of
depsipeptides. Preferably, the term "PTH" refers to polypeptides
having a backbone formed through amide (peptide) bonds.
[0044] As the term PTH includes the above-described variants,
analogs, orthologs, homologs, derivatives and fragments of PTH and
PTHrP, all references to specific positions within a reference
sequence also include the equivalent positions in variants,
analogs, orthologs, homologs, derivatives and fragments of a PTH or
PTHrP moiety, even if not specifically mentioned.
[0045] As used herein, the term "random coil" refers to a peptide
or protein adopting/having/forming, preferably having, a
conformation which substantially lacks a defined secondary and
tertiary structure as determined by circular dichroism spectroscopy
performed in aqueous buffer at ambient temperature, and pH 7.4.
Preferably, ambient temperature is about 20.degree. C., i.e.
between 18.degree. C. and 22.degree. C., most preferably ambient
temperature is 20.degree. C.
[0046] As used herein the term "pharmaceutical composition" refers
to a composition containing one or more active ingredients, for
example a drug or a prodrug, here specifically the PTH prodrugs of
the present invention, and optionally one or more excipients, as
well as any product which results, directly or indirectly, from
combination, complexation or aggregation of any two or more of the
ingredients of the composition, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions of the present invention encompass any
composition made by admixing one or more PTH prodrugs of the
present invention and optionally a pharmaceutically acceptable
excipient.
[0047] As used herein the term "liquid composition" refers to a
mixture comprising water-soluble PTH prodrug and one or more
solvents, such as water.
[0048] The term "suspension composition" relates to a mixture
comprising water-insoluble PTH prodrug, where for example the
carrier Z' is a hydrogel, and one or more solvents, such as water.
Due to the water-insoluble polymer, the polymeric prodrug cannot
dissolve and renders the prodrug in a particulate state.
[0049] As used herein, the term "dry composition" means that a
pharmaceutical composition is provided in a dry form. Suitable
methods for drying are spray-drying and lyophilization, i.e.
freeze-drying. Such dry composition of prodrug has a residual water
content of a maximum of 10%, preferably less than 5% and more
preferably less than 2%, determined according to Karl Fischer.
Preferably, the pharmaceutical composition of the present invention
is dried by lyophilization.
[0050] The term "drug" as used herein refers to a substance used in
the treatment, cure, prevention, or diagnosis of a disease or used
to otherwise enhance physical or mental well-being. If a drug is
conjugated to another moiety, the moiety of the resulting product
that originated from the drug is referred to as "biologically
active moiety". The PTH prodrug of the present invention comprise a
PTH moiety which is released from the PTH prodrug in the form of
the drug PTH.
[0051] As used herein the term "prodrug" refers to a conjugate
comprising a biologically active moiety reversibly and covalently
connected to a specialized protective group through a reversible
prodrug linker moiety which is a linker moiety comprising a
reversible linkage with the biologically active moiety and wherein
the specialized protective group alters or eliminates undesirable
properties in the parent molecule. This also includes the
enhancement of desirable properties in the drug and the suppression
of undesirable properties. The specialized non-toxic protective
group is referred to as "carrier". A prodrug releases the
reversibly and covalently bound biologically active moiety in the
form of its corresponding drug. In other words, a prodrug is a
conjugate comprising a biologically active moiety which is
covalently and reversibly conjugated to a carrier moiety via a
reversible prodrug linker moiety, which covalent and reversible
conjugation of the carrier to the reversible prodrug linker moiety
is either directly or through a spacer. Such conjugate releases the
formerly conjugated biologically active moiety in the form of a
free drug.
[0052] A "biodegradable linkage" or a "reversible linkage" is a
linkage that is hydrolytically degradable, i.e. cleavable, in the
absence of enzymes under physiological conditions (aqueous buffer
at pH 7.4, 37.degree. C.) with a half-life ranging from one hour to
two months, preferably from one hour to one month. Accordingly, a
stable linkage is a linkage having a half-life under physiological
conditions (aqueous buffer at pH 7.4, 37.degree. C.) of more than
two months.
[0053] Accordingly, a "reversible prodrug linker moiety" is a
moiety which is covalently conjugated to a biologically active
moiety, such as PTH, through a reversible linkage and is also
covalently conjugated to a carrier moiety, such as --Z or --Z',
wherein the covalent conjugation to said carrier moiety is either
directly or through a spacer moiety, such as -L.sup.2-. Preferably
the linkage between --Z or --Z' and -L.sup.2- is a stable
linkage.
[0054] As used herein, the term "traceless prodrug linker" means a
reversible prodrug linker which upon cleavage releases the drug in
its free form. As used herein, the term "free form" of a drug means
the drug in its unmodified, pharmacologically active form.
[0055] As used herein, the term "excipient" refers to a diluent,
adjuvant, or vehicle with which the therapeutic, such as a drug or
prodrug, is administered. Such pharmaceutical excipient can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, including but not
limited to peanut oil, soybean oil, mineral oil, sesame oil and the
like. Water is a preferred excipient when the pharmaceutical
composition is administered orally. Saline and aqueous dextrose are
preferred excipients when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions are preferably employed as liquid excipients
for injectable solutions. Suitable pharmaceutical excipients
include starch, glucose, lactose, sucrose, mannitol, trehalose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like. The
pharmaceutical composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, pH buffering agents,
like, for example, acetate, succinate, tris, carbonate, phosphate,
HEPES (4-(2-hydroxy ethyl)-1-piperazineethanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid), or can contain detergents,
like Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids
like, for example, glycine, lysine, or histidine. These
pharmaceutical compositions can take the form of solutions,
suspensions, emulsions, tablets, pills, capsules, powders,
sustained-release formulations and the like. The pharmaceutical
composition can be formulated as a suppository, with traditional
binders and excipients such as triglycerides. Oral formulation can
include standard excipients such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Such compositions will contain
a therapeutically effective amount of the drug or biologically
active moiety, together with a suitable amount of excipient so as
to provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0056] As used herein, the term "reagent" means a chemical compound
which comprises at least one functional group for reaction with the
functional group of another chemical compound or drug. It is
understood that a drug comprising a functional group (such as a
primary or secondary amine or hydroxyl functional group) is also a
reagent.
[0057] As used herein, the term "moiety" means a part of a
molecule, which lacks one or more atom(s) compared to the
corresponding reagent. If, for example, a reagent of the formula
"H--X--H" reacts with another reagent and becomes part of the
reaction product, the corresponding moiety of the reaction product
has the structure "H--X--" or "--X--", whereas each "-" indicates
attachment to another moiety. Accordingly, a biologically active
moiety is released from a prodrug as a drug.
[0058] It is understood that if the sequence or chemical structure
of a group of atoms is provided which group of atoms is attached to
two moieties or is interrupting a moiety, said sequence or chemical
structure can be attached to the two moieties in either
orientation, unless explicitly stated otherwise. For example, a
moiety "--C(O)N(R)--" can be attached to two moieties or
interrupting a moiety either as "--C(O)N(R)--" or as
"--N(R)C(O)--". Similarly, a moiety
##STR00002##
[0059] can be attached to two moieties or can interrupt a moiety
either as
##STR00003##
[0060] As used herein, the term "functional group" means a group of
atoms which can react with other groups of atoms. Functional groups
include but are not limited to the following groups:
[0061] carboxylic acid (--(C.dbd.O)OH), primary or secondary amine
(--NH.sub.2, --NH--), maleimide, thiol (--SH), sulfonic acid
(--(O.dbd.S=O)OH), carbonate, carbamate (--O(C.dbd.O)N<),
hydroxyl (--OH), aldehyde (--(C.dbd.O)H), ketone (--(C.dbd.O)--),
hydrazine (>N--N<), isocyanate, isothiocyanate, phosphoric
acid (--O(P.dbd.O)OHOH), phosphonic acid (--O(P.dbd.O)OHH),
haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine,
disulfide, sulfonamides, sulfuric acid, vinyl sulfone, vinyl
ketone, diazoalkane, oxirane, and aziridine.
[0062] In case the prodrugs of the present invention comprise one
or more acidic or basic groups, the invention also comprises their
corresponding pharmaceutically or toxicologically acceptable salts,
in particular their pharmaceutically utilizable salts. Thus, the
prodrugs of the present invention comprising acidic groups can be
used according to the invention, for example, as alkali metal
salts, alkaline earth metal salts or as ammonium salts. More
precise examples of such salts include sodium salts, potassium
salts, calcium salts, magnesium salts or salts with ammonia or
organic amines such as, for example, ethylamine, ethanolamine,
triethanolamine or amino acids. Prodrugs of the present invention
comprising one or more basic groups, i.e. groups which can be
protonated, can be present and can be used according to the
invention in the form of their addition salts with inorganic or
organic acids. Examples for suitable acids include hydrogen
chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric
acid, lactic acid, salicylic acid, benzoic acid, formic acid,
propionic acid, pivalic acid, diethylacetic acid, malonic acid,
succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,
sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic
acid, isonicotinic acid, citric acid, adipic acid, and other acids
known to the person skilled in the art. For the person skilled in
the art further methods are known for converting the basic group
into a cation like the alkylation of an amine group resulting in a
positively-charge ammonium group and an appropriate counterion of
the salt. If the prodrugs of the present invention simultaneously
comprise acidic and basic groups, the invention also includes, in
addition to the salt forms mentioned, inner salts or betaines
(zwitterions). The respective salts can be obtained by customary
methods which are known to the person skilled in the art like, for
example by contacting these prodrugs with an organic or inorganic
acid or base in a solvent or dispersant, or by anion exchange or
cation exchange with other salts. The present invention also
includes all salts of the prodrugs of the present invention which,
owing to low physiological compatibility, are not directly suitable
for use in pharmaceuticals but which can be used, for example, as
intermediates for chemical reactions or for the preparation of
pharmaceutically acceptable salts.
[0063] The term "pharmaceutically acceptable" means a substance
that does cause harm when administered to a patient and preferably
means approved by a regulatory agency, such as the EMA (Europe)
and/or the FDA (US) and/or any other national regulatory agency for
use in animals, preferably for use in humans.
[0064] As used herein the term "about" in combination with a
numerical value is used to indicate a range ranging from and
including the numerical value plus and minus no more than 10% of
said numerical value, more preferably no more than 8% of said
numerical value, even more preferably no more than 5% of said
numerical value and most preferably no more than 2% of said
numerical value. For example, the phrase "about 200" is used to
mean a range ranging from and including 200+/-10%, i.e. ranging
from and including 180 to 220; preferably 200+/-8%, i.e. ranging
from and including 184 to 216; even more preferably ranging from
and including 200+/-5%, i.e. ranging from and including 190 to 210;
and most preferably 200+/-2%, i.e. ranging from and including 196
to 204. It is understood that a percentage given as "about 20%"
does not mean "20%+/-10%", i.e. ranging from and including 10 to
30%, but "about 20%" means ranging from and including 18 to 22%,
i.e. plus and minus 10% of the numerical value which is 20.
[0065] As used herein, the term "polymer" means a molecule
comprising repeating structural units, i.e. the monomers, connected
by chemical bonds in a linear, circular, branched, crosslinked or
dendrimeric way or a combination thereof, which may be of synthetic
or biological origin or a combination of both. It is understood
that a polymer may also comprise one or more other chemical
group(s) and/or moiety/moieties, such as, for example, one or more
functional group(s). Preferably, a soluble polymer has a molecular
weight of at least 0.5 kDa, e.g. a molecular weight of at least 1
kDa, a molecular weight of at least 2 kDa, a molecular weight of at
least 3 kDa or a molecular weight of at least 5 kDa. If the polymer
is soluble, it preferable has a molecular weight of at most 1000
kDa, such as at most 750 kDa, such as at most 500 kDa, such as at
most 300 kDa, such as at most 200 kDa, such as at most 100 kDa. It
is understood that for insoluble polymers, such as hydrogels, no
meaningful molecular weight ranges can be provided. It is
understood that also a protein is a polymer in which the amino
acids are the repeating structural units, even though the side
chains of each amino acid may be different.
[0066] As used herein, the term "polymeric" means a reagent or a
moiety comprising one or more polymer(s) or polymer
moiety/moieties. A polymeric reagent or moiety may optionally also
comprise one or more other moiety/moieties, which are preferably
selected from the group consisting of: [0067] C.sub.1-50 alkyl,
C.sub.2-50 alkenyl, C.sub.2-50 alkynyl, C.sub.3-10 cycloalkyl, 3-
to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,
phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and [0068]
linkages selected from the group comprising
[0068] ##STR00004## wherein dashed lines indicate attachment to the
remainder of the moiety or reagent, and --R and --Ra are
independently of each other selected from the group consisting of
--H, methyl, ethyl, propyl, butyl, pentyl and hexyl.
[0069] The person skilled in the art understands that the
polymerization products obtained from a polymerization reaction do
not all have the same molecular weight, but rather exhibit a
molecular weight distribution. Consequently, the molecular weight
ranges, molecular weights, ranges of numbers of monomers in a
polymer and numbers of monomers in a polymer as used herein, refer
to the number average molecular weight and number average of
monomers, i.e. to the arithmetic mean of the molecular weight of
the polymer or polymeric moiety and the arithmetic mean of the
number of monomers of the polymer or polymeric moiety.
[0070] Accordingly, in a polymeric moiety comprising "x" monomer
units any integer given for "x" therefore corresponds to the
arithmetic mean number of monomers. Any range of integers given for
"x" provides the range of integers in which the arithmetic mean
numbers of monomers lies. An integer for "x" given as "about x"
means that the arithmetic mean numbers of monomers lies in a range
of integers of x+/-10%, preferably x+/-8%, more preferably x+/-5%
and most preferably x+/-2%.
[0071] As used herein, the term "number average molecular weight"
means the ordinary arithmetic mean of the molecular weights of the
individual polymers.
[0072] As used herein the term "water-soluble" with reference to a
carrier means that when such carrier is part of the PTH prodrug of
the present invention at least 1 g of the PTH prodrug comprising
such water-soluble carrier can be dissolved in one liter of water
at 20.degree. C. to form a homogeneous solution. Accordingly, the
term "water-insoluble" with reference to a carrier means that when
such carrier is part of the PTH prodrug of the present invention
less than 1 g of the PTH prodrug comprising such water-insoluble
carrier can be dissolved in one liter of water at 20.degree. C. to
form a homogeneous solution.
[0073] As used herein, the term "hydrogel" means a hydrophilic or
amphiphilic polymeric network composed of homopolymers or
copolymers, which is insoluble due to the presence of hydrogen
bonds, ionic interactions and/or covalent chemical crosslinks. The
crosslinks provide the network structure and physical
integrity.
[0074] As used herein the term "thermogelling" means a compound
that is a liquid or a low viscosity solution having a viscosity of
less than 500 cps at 25.degree. C. at a shear rate of about
0.1/second at a low temperature, which low temperature ranges
between about 0.degree. C. to about 10.degree. C., but which is a
higher viscosity compound of less than 10000 cps at 25.degree. C.
at a shear rate of about 0.1/second at a higher temperature, which
higher temperature ranges between about 30.degree. C. to about
40.degree. C., such as at about 37.degree. C.
[0075] As used herein, the term "PEG-based" in relation to a moiety
or reagent means that said moiety or reagent comprises PEG.
Preferably, a PEG-based moiety or reagent comprises at least 10%
(w/w) PEG, such as at least 20% (w/w) PEG, such as at least 30%
(w/w) PEG, such as at least 40% (w/w) PEG, such as at least 50%
(w/w), such as at least 60 (w/w) PEG, such as at least 70% (w/w)
PEG, such as at least 80% (w/w) PEG, such as at least 90% (w/w)
PEG, such as at least 95%. The remaining weight percentage of the
PEG-based moiety or reagent are other moieties preferably selected
from the following moieties and linkages: [0076] C.sub.1-50 alkyl,
C.sub.2-50 alkenyl, C.sub.2-50 alkynyl, C.sub.3-10 cycloalkyl, 3-
to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,
phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and [0077]
linkages selected from the group comprising
[0077] ##STR00005## wherein dashed lines indicate attachment to the
remainder of the moiety or reagent, and --R and --Ra are
independently of each other selected from the group consisting of
--H, methyl, ethyl, propyl, butyl, pentyl and hexyl.
[0078] As used herein, the term "PEG-based comprising at least X %
PEG" in relation to a moiety or reagent means that said moiety or
reagent comprises at least X % (w/w) ethylene glycol units
(--CH.sub.2CH.sub.2O--), wherein the ethylene glycol units may be
arranged blockwise, alternating or may be randomly distributed
within the moiety or reagent and preferably all ethylene glycol
units of said moiety or reagent are present in one block; the
remaining weight percentage of the PEG-based moiety or reagent are
other moieties preferably selected from the following moieties and
linkages: [0079] C.sub.1-50 alkyl, C.sub.2-50 alkenyl, C.sub.2-50
alkynyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8-
to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl,
and tetralinyl; and [0080] linkages selected from the group
comprising
[0080] ##STR00006## wherein dashed lines indicate attachment to the
remainder of the moiety or reagent, and --R and --Ra are
independently of each other selected from the group consisting of
--H, methyl, ethyl, propyl, butyl, pentyl and hexyl.
[0081] The term "hyaluronic acid-based comprising at least X %
hyaluronic acid" is used accordingly.
[0082] The term "substituted" as used herein means that one or more
--H atom(s) of a molecule or moiety are replaced by a different
atom or a group of atoms, which are referred to as
"substituent".
[0083] Preferably, the one or more further optional substituents
are independently of each other selected from the group consisting
of halogen, --CN, --COOR.sup.x1, --OR.sup.x1, --C(O)R.sup.x1,
--C(O)N(R.sup.x1R.sup.x1a), --S(O).sub.2N(R.sup.x1R.sup.x1a),
--S(O)N(R.sup.x1R.sup.x1a), --S(O).sub.2R.sup.x1, --S(O)R.sup.x1,
--N(R.sup.x1)S(O).sub.2N(R.sup.x1aR.sup.x1b), --SR.sup.x1,
--N(R.sup.x1R.sup.x1a), --NO.sub.2, --OC(O)R.sup.x1,
--N(R.sup.x1)C(O)R.sup.x1a, --N(R.sup.x1)S(O).sub.2R.sup.x1a,
--N(R.sup.x1)S(O)R.sup.x1a, --N(R.sup.x1)C(O)OR.sup.x1a,
--N(R.sup.x1)C(O)N(R.sup.x1aR.sup.x1b),
--OC(O)N(R.sup.x1R.sup.x1a), -T.sup.0, C.sub.1-50 alkyl, C.sub.2-50
alkenyl, and C.sub.2-50 alkynyl; wherein -T.sup.0, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
substituted with one or more --R.sup.x2, which are the same or
different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T.sup.0-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.x3)--, --S(O).sub.2N(R.sup.x3)--,
--S(O)N(R.sup.x3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.x3)S(O).sub.2N(R.sup.x3a)--, --S--, --N(R.sup.x3)--,
--OC(OR.sup.x3)(R.sup.x3a)--, --N(R.sup.x3)C(O)N(R.sup.x3a)--, and
--OC(O)N(R.sup.x3)--; --R.sup.x1, --R.sup.x1a, --R.sup.x1b are
independently of each other selected from the group consisting of
--H, -T.sup.0, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50
alkynyl; wherein -T.sup.0, C.sub.1-50 alkyl, C.sub.2-50 alkenyl,
and C.sub.2-50 alkynyl are optionally substituted with one or more
--R.sup.x2, which are the same or different and wherein C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
interrupted by one or more groups selected from the group
consisting of -T.sup.0-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.x3)--, --S(O).sub.2N(R.sup.x3)--,
--S(O)N(R.sup.x3)--; --S(O).sub.2--, --S(O)--,
--N(R.sup.x3)S(O).sub.2N(R.sup.x3a)--, --S--, --N(R.sup.x3)--,
--OC(OR.sup.x3)(R.sup.x3a)--, --N(R.sup.x3)C(O)N(R.sup.x3a)--, and
--OC(O)N(R.sup.x3)--;
[0084] each T.sup.0 is independently selected from the group
consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to
11-membered heterobicyclyl; wherein each T.sup.0 is independently
optionally substituted with one or more --R.sup.x2, which are the
same or different;
[0085] each --R.sup.x2 is independently selected from the group
consisting of halogen, --CN, oxo (.dbd.O), --COOR.sup.x4,
--OR.sup.x4, --C(O)R.sup.x4, --C(O)N(R.sup.x4R.sup.x4a),
--S(O).sub.2N(R.sup.x4R.sup.x4a), --S(O)N(R.sup.x4R.sup.x4a),
--S(O).sub.2R.sup.x4, --S(O)R.sup.x4,
--N(R.sup.x4)S(O).sub.2N(R.sup.x4aR.sup.x4b), --SR.sup.x4,
--N(R.sup.x4R.sup.x4a), --NO.sub.2, --OC(O)R.sup.x4,
--N(R.sup.x4)C(O)R.sup.x4a, --N(R.sup.x4)S(O).sub.2R.sup.x4a,
--N(R.sup.x4)S(O)R.sup.x4a, --N(R.sup.x4)C(O)OR.sup.x4a,
--N(R.sup.x4)C(O)N(R.sup.x4a R.sup.x4b),
[0086] --OC(O)N(R.sup.x4R.sup.x4a), and C.sub.1-6 alkyl; wherein
C.sub.1-6 alkyl is optionally substituted with one or more halogen,
which are the same or different;
[0087] each --R.sup.x3, --R.sup.x3a, --R.sup.x4, --R.sup.x4a,
--R.sup.x4b is independently selected from the group consisting of
--H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally
substituted with one or more halogen, which are the same or
different.
[0088] More preferably, the one or more further optional
substituents are independently of each other selected from the
group consisting of halogen, --CN, --COOR.sup.x1, --OR.sup.x1,
--C(O)R.sup.x1, --C(O)N(R.sup.x1R.sup.x1a),
--S(O).sub.2N(R.sup.x1R.sup.x1a), --S(O)N(R.sup.x1R.sup.x1a),
--S(O).sub.2R.sup.x1, --S(O)R.sup.x1,
--N(R.sup.x1)S(O).sub.2N(R.sup.x1aR.sup.x1b), --SR.sup.x1,
--N(R.sup.x1R.sup.x1a), --NO.sub.2, --OC(O)R.sup.x1,
--N(R.sup.x1)C(O)R.sup.x1a, --N(R.sup.x1)S(O).sub.2R.sup.x1a,
--N(R.sup.x1)S(O)R.sup.x1a, --N(R.sup.x1)C(O)OR.sup.x1a,
--N(R.sup.x1)C(O)N(R.sup.x1aR.sup.x1b),
--OC(O)N(R.sup.x1R.sup.x1a), -T.sup.0, C.sub.1-10 alkyl, C.sub.2-10
alkenyl, and C.sub.2-10 alkynyl; wherein -T.sup.0, C.sub.1-10
alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally
substituted with one or more --R.sup.x2, which are the same or
different and wherein C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and
C.sub.2-10 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T.sup.0-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.x3)--, --S(O).sub.2N(R.sup.x3)--,
--S(O)N(R.sup.x3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.x3)S(O).sub.2N(R.sup.x3a)--, --S--, --N(R.sup.x3)--,
--OC(OR.sup.x3)(R.sup.x3a)--, --N(R.sup.x3)C(O)N(R.sup.x3a)--, and
--OC(O)N(R.sup.x3)--;
[0089] each --R.sup.x1, --R.sup.x1a, --R.sup.x1b, --R.sup.x3,
--R.sup.x3a is independently selected from the group consisting of
--H, halogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6
alkynyl;
[0090] each T.sup.0 is independently selected from the group
consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to
11-membered heterobicyclyl; wherein each T.sup.0 is independently
optionally substituted with one or more --R.sup.x2, which are the
same or different;
[0091] each --R.sup.x2 is independently selected from the group
consisting of halogen, --CN, oxo (.dbd.O), --COOR.sup.x4,
--OR.sup.x4, --C(O)R.sup.x4, --C(O)N(R.sup.x4R.sup.x4a),
--S(O).sub.2N(R.sup.x4R.sup.x4a), --S(O)N(R.sup.x4R.sup.x4a),
--S(O).sub.2R.sup.x4, --S(O)R.sup.x4,
--N(R.sup.x4)S(O).sub.2N(R.sup.x4aR.sup.x4b), --SR.sup.x4,
--N(R.sup.x4R.sup.x4a), --NO.sub.2, --OC(O)R.sup.x4,
--N(R.sup.x4)C(O)R.sup.x4a, --N(R.sup.x4)S(O).sub.2R.sup.x4a,
--N(R.sup.x4)S(O)R.sup.x4a, --N(R.sup.x4)C(O)OR.sup.x4a,
--N(R.sup.x4)C(O)N(R.sup.x4aR.sup.x4b),
--OC(O)N(R.sup.x4R.sup.x4a), and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different;
[0092] each --R.sup.x4, --R.sup.x4a, --R.sup.x4b is independently
selected from the group consisting of --H, halogen, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; Even more
preferably, the one or more further optional substituents are
independently of each other selected from the group consisting of
halogen, --CN, --COOR.sup.x1, --OR.sup.x1, --C(O)R.sup.x1,
--C(O)N(R.sup.x1R.sup.x1a), --S(O).sub.2N(R.sup.x1R.sup.x1a),
--S(O)N(R.sup.x1R.sup.x1a), --S(O).sub.2R.sup.x1, --S(O)R.sup.x1,
--N(R.sup.x1)S(O).sub.2N(R.sup.x1aR.sup.x1b), --SR.sup.x1,
--N(R.sup.x1R.sup.x1a), --NO.sub.2, --OC(O)R.sup.x1,
--N(R.sup.x1)C(O)R.sup.x1a, --N(R.sup.x1)S(O).sub.2R.sup.x1a,
--N(R.sup.x1)S(O)R.sup.x1a, --N(R.sup.x1)C(O)OR.sup.x1a,
--N(R.sup.x1)C(O)N(R.sup.x1aR.sup.x1b),
--OC(O)N(R.sup.x1R.sup.x1a), -T.sup.0, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, and C.sub.2-6 alkynyl; wherein -T.sup.0, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl are optionally substituted
with one or more --R.sup.x2, which are the same or different and
wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl
are optionally interrupted by one or more groups selected from the
group consisting of -T.sup.0-, --C(O)O--, --O--C(O)--,
--C(O)N(R.sup.x3)--, --S(O).sub.2N(R.sup.x3)--,
--S(O)N(R.sup.x3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.x3)S(O).sub.2N(R.sup.x3a)--, --S--, --N(R.sup.x3)--,
--OC(OR.sup.x3)(R.sup.x3a)--, --N(R.sup.x3)C(O)N(R.sup.x3a)--, and
--OC(O)N(R.sup.x3)--;
[0093] each --R.sup.x1, --R.sup.x1a, --R.sup.x1b, --R.sup.x2,
--R.sup.x3, --R.sup.x3a is independently selected from the group
consisting of --H, halogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and
C.sub.2-6 alkynyl;
[0094] each T.sup.0 is independently selected from the group
consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to
11-membered heterobicyclyl; wherein each T.sup.0 is independently
optionally substituted with one or more --R.sup.x2, which are the
same or different.
[0095] Preferably, a maximum of 6 --H atoms of an optionally
substituted molecule are independently replaced by a substituent,
e.g. 5 --H atoms are independently replaced by a substituent, 4 --H
atoms are independently replaced by a substituent, 3 --H atoms are
independently replaced by a substituent, 2 --H atoms are
independently replaced by a substituent, or 1 --H atom is replaced
by a substituent.
[0096] The term "interrupted" means that a moiety is inserted
between two carbon atoms or--if the insertion is at one of the
moiety's ends--between a carbon or heteroatom and a hydrogen atom,
preferably between a carbon and a hydrogen atom.
[0097] The term "spacer" refers to any moiety that is suitable to
connect two moieties. Preferably, a spacer is selected from the
group consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.y1)--, --S(O).sub.2N(R.sup.y1)--,
--S(O)N(R.sup.y1)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y1)S(O).sub.2N(R.sup.y1a)--, --S--, --N(R.sup.y1)--,
--OC(OR.sup.y1)(R.sup.y1a)--, --N(R.sup.y1)C(O)N(R.sup.y1a)--,
--OC(O)N(R.sup.y1)--, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50
alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one
or more --R.sup.y2, which are the same or different and wherein
C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.y3)--, --S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--,
[0098] --S(O)--, --N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--, --S--,
--N(R.sup.y3)--, --OC(OR.sup.y3)(R.sup.y3a)--,
--N(R.sup.y3)C(O)N(R.sup.y3a)--, and --OC(O)N(R.sup.y3)--;
[0099] --R.sup.y1 and --R.sup.y1a are independently of each other
selected from the group consisting of --H, -T, C.sub.1-50 alkyl,
C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
substituted with one or more --R.sup.y2, which are the same or
different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y4)--, --S(O).sub.2N(R.sup.y4)--,
--S(O)N(R.sup.y4)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y4)S(O).sub.2N(R.sup.y4a)--, --S--, --N(R.sup.y4)--,
--OC(OR.sup.y4)(R.sup.y4a)--, --N(R.sup.y4)C(O)N(R.sup.y4a)--, and
--OC(O)N(R.sup.y4)--;
[0100] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl; wherein each T is independently
optionally substituted with one or more --R.sup.y2, which are the
same or different;
[0101] each --R.sup.y2 is independently selected from the group
consisting of halogen, --CN, oxo (.dbd.O), --COOR.sup.y5,
--OR.sup.y5, --C(O)R.sup.y5, --C(O)N(R.sup.y5R.sup.y5a),
--S(O).sub.2N(R.sup.y5R.sup.y5a), --S(O)N(R.sup.y5R.sup.y5a),
--S(O).sub.2R.sup.y5, --S(O)R.sup.y5,
--N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), --SR.sup.y5,
--N(R.sup.y5R.sup.y5a), --NO.sub.2, --OC(O)R.sup.y5,
--N(R.sup.y5)C(O)R.sup.y5a, --N(R.sup.y5)S(O).sub.2R.sup.y5a,
--N(R.sup.y5)S(O)R.sup.y5a, --N(R.sup.y5)C(O)OR.sup.y5a,
--N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b),
--OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different; and
[0102] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently selected
from the group consisting of --H, and C.sub.1-6 alkyl, wherein
C.sub.1-6 alkyl is optionally substituted with one or more halogen,
which are the same or different.
[0103] Even more preferably the spacer is selected from -T-,
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.y1)--,
--S(O).sub.2N(R.sup.y1)--, --S(O)N(R.sup.y1)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.y1)S(O).sub.2N(R.sup.y1a), --S--,
--N(R.sup.y1)--, --OC(OR.sup.y1)(R.sup.y1a)--,
--N(R.sup.y1)C(O)N(R.sup.y1a)--, --OC(O)N(R.sup.y1)--, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-,
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are
optionally substituted with one or more --R.sup.y2, which are the
same or different and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
and C.sub.2-20 alkynyl are optionally interrupted by one or more
groups selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y3)--S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--, --S--, --N(R.sup.y3)--,
--OC(OR.sup.y3)(R.sup.y3a)--, --N(R.sup.y3)C(O)N(R.sup.y3a)--, and
--OC(O)N(R.sup.y3)--; --R.sup.y1 and --R.sup.y1a are independently
of each other selected from the group consisting of --H, -T,
C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl;
wherein -T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10
alkynyl are optionally substituted with one or more --R.sup.y2,
which are the same or different, and wherein C.sub.1-10 alkyl,
C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally
interrupted by one or more groups selected from the group
consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.y4)--S(O).sub.2N(R.sup.y4)--, --S(O)N(R.sup.y4)--,
--S(O).sub.2--, --S(O)--, --N(R.sup.y4)S(O).sub.2N(R.sup.y4a)--,
--S--, --N(R.sup.y4)--, --OC(OR.sup.y4)(R.sup.y4a)--,
--N(R.sup.y4)C(O)N(R.sup.y4a)--, and --OC(O)N(R.sup.y4)--;
[0104] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl; wherein each T is independently
optionally substituted with one or more --R.sup.y2, which are the
same or different;
[0105] --R.sup.y2 is selected from the group consisting of halogen,
--CN, oxo (.dbd.O), --COOR.sup.y5, --OR.sup.y5, --C(O)R.sup.y5,
--C(O)N(R.sup.y5R.sup.y5a), --S(O).sub.2N(R.sup.y5R.sup.y5a),
--S(O)N(R.sup.y5R.sup.y5a), --S(O).sub.2R.sup.y5, --S(O)R.sup.y5,
--N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), --SR.sup.y5,
--N(R.sup.y5R.sup.y5a), --NO.sub.2, --OC(O)R.sup.y5,
--N(R.sup.y5)C(O)R.sup.y5a, --N(R.sup.y5)S(O).sub.2R.sup.y5a,
--N(R.sup.y5)S(O)R.sup.y5a, --N(R.sup.y5)C(O)OR.sup.y5a,
--N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b),
--OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different; and
[0106] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently of each
other selected from the group consisting of --H, and C.sub.1-6
alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0107] Even more preferably the spacer is selected from the group
consisting of -T-, --C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.y1)--,
--S(O).sub.2N(R.sup.y1)--, --S(O)N(R.sup.y1)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.y1)S(O).sub.2N(R.sup.y1a)--, --S--,
--N(R.sup.y1)--, --OC(OR.sup.y1)(R.sup.y1a)--,
--N(R.sup.y1)C(O)N(R.sup.y1a)--, --OC(O)N(R.sup.y1)--, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-,
C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are
optionally substituted with one or more --R.sup.y2, which are the
same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl,
and C.sub.2-50 alkynyl are optionally interrupted by one or more
groups selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y3)--, --S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--, --S--, --N(R.sup.y3)--,
--OC(OR.sup.y3)(R.sup.y3a)--, --N(R.sup.y3)C(O)N(R.sup.y3a)--, and
--OC(O)N(R.sup.y3)--;
[0108] --R.sup.y1 and --R.sup.y1a are independently selected from
the group consisting of --H, -T, C.sub.1-10 alkyl, C.sub.2-10
alkenyl, and C.sub.2-10 alkynyl;
[0109] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl;
[0110] each --R.sup.y2 is independently selected from the group
consisting of halogen, and C.sub.1-6 alkyl; and
[0111] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently of each
other selected from the group consisting of --H, and C.sub.1-6
alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0112] As used herein, the term "C.sub.1-4 alkyl" alone or in
combination means a straight-chain or branched alkyl moiety having
1 to 4 carbon atoms. If present at the end of a molecule, examples
of straight-chain or branched C.sub.1-4 alkyl are methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
When two moieties of a molecule are linked by the C.sub.1-4 alkyl,
then examples for such C.sub.1-4 alkyl groups are --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH(C.sub.2H.sub.5)--,
--C(CH.sub.3).sub.2--. Each hydrogen of a C.sub.1-4 alkyl carbon
may optionally be replaced by a substituent as defined above.
Optionally, a C.sub.1-4 alkyl may be interrupted by one or more
moieties as defined below.
[0113] As used herein, the term "C.sub.1-6 alkyl" alone or in
combination means a straight-chain or branched alkyl moiety having
1 to 6 carbon atoms. If present at the end of a molecule, examples
of straight-chain and branched C.sub.1-6 alkyl groups are methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl,
2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl,
2,3-dimethylbutyl and 3,3-dimethylpropyl. When two moieties of a
molecule are linked by the C.sub.1-6 alkyl group, then examples for
such C.sub.1-6 alkyl groups are --CH.sub.2--,
--CH.sub.2--CH.sub.2--,
--CH(CH.sub.3)--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH(C.sub.2H.sub.5)-- and --C(CH.sub.3).sub.2--. Each hydrogen
atom of a C.sub.1-6 carbon may optionally be replaced by a
substituent as defined above. Optionally, a C.sub.1-6 alkyl may be
interrupted by one or more moieties as defined below.
[0114] Accordingly, "C.sub.1-10 alkyl", "C.sub.1-20 alkyl" or
"C.sub.1-50 alkyl" means an alkyl chain having 1 to 10, 1 to 20 or
1 to 50 carbon atoms, respectively, wherein each hydrogen atom of
the C.sub.1-10, C.sub.1-20 or C.sub.1-50 carbon may optionally be
replaced by a substituent as defined above.
[0115] Optionally, a C.sub.1-10 or C.sub.1-50 alkyl may be
interrupted by one or more moieties as defined below.
[0116] As used herein, the term "C.sub.2-6 alkenyl" alone or in
combination means a straight-chain or branched hydrocarbon moiety
comprising at least one carbon-carbon double bond having 2 to 6
carbon atoms. If present at the end of a molecule, examples are
--CH.dbd.CH.sub.2, --CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH.sub.2, --CH.dbd.CHCH.sub.2--CH.sub.3 and
--CH.dbd.CH--CH.dbd.CH.sub.2. When two moieties of a molecule are
linked by the C.sub.2-6 alkenyl group, then an example for such
C.sub.2-6 alkenyl is --CH.dbd.CH--. Each hydrogen atom of a
C.sub.2-6 alkenyl moiety may optionally be replaced by a
substituent as defined above. Optionally, a C.sub.2-6 alkenyl may
be interrupted by one or more moieties as defined below.
[0117] Accordingly, the term "C.sub.2-10 alkenyl", "C.sub.2-20
alkenyl" or "C.sub.2-50 alkenyl" alone or in combination means a
straight-chain or branched hydrocarbon moiety comprising at least
one carbon-carbon double bond having 2 to 10, 2 to 20 or 2 to 50
carbon atoms. Each hydrogen atom of a C.sub.2-10 alkenyl,
C.sub.2-20 alkenyl or C.sub.2-50 alkenyl group may optionally be
replaced by a substituent as defined above. Optionally, a
C.sub.2-10 alkenyl, C.sub.2-20 alkenyl or C.sub.2-50 alkenyl may be
interrupted by one or more moieties as defined below.
[0118] As used herein, the term "C.sub.2-6 alkynyl" alone or in
combination means straight-chain or branched hydrocarbon moiety
comprising at least one carbon-carbon triple bond having 2 to 6
carbon atoms. If present at the end of a molecule, examples are
--C.ident.CH, --CH.sub.2--C.ident.CH,
CH.sub.2--CH.sub.2--C.ident.CH and CH.sub.2--C.ident.C--CH.sub.3.
When two moieties of a molecule are linked by the alkynyl group,
then an example is --C.ident.C--. Each hydrogen atom of a C.sub.2-6
alkynyl group may optionally be replaced by a substituent as
defined above. Optionally, one or more double bond(s) may occur.
Optionally, a C.sub.2-6 alkynyl may be interrupted by one or more
moieties as defined below.
[0119] Accordingly, as used herein, the term "C.sub.2-10 alkynyl",
"C.sub.2-20 alkynyl" and "C.sub.2-50 alkynyl" alone or in
combination means a straight-chain or branched hydrocarbon moiety
comprising at least one carbon-carbon triple bond having 2 to 10, 2
to 20 or 2 to 50 carbon atoms, respectively. Each hydrogen atom of
a C.sub.2-10 alkynyl, C.sub.2-20 alkynyl or C.sub.2-50 alkynyl
group may optionally be replaced by a substituent as defined above.
Optionally, one or more double bond(s) may occur. Optionally, a
C.sub.2-10 alkynyl, C.sub.2-20 alkynyl or C.sub.2-50 alkynyl may be
interrupted by one or more moieties as defined below.
[0120] As mentioned above, a C.sub.1-4 alkyl, C.sub.1-6 alkyl,
C.sub.1-10 alkyl, C.sub.1-20 alkyl, C.sub.1-50 alkyl, C.sub.2-6
alkenyl, C.sub.2-10 alkenyl, C.sub.2-20 alkenyl, C.sub.2-50
alkenyl, C.sub.2-6 alkynyl, C.sub.2-10 alkynyl, C.sub.2-20 alkenyl
or C.sub.2-50 alkynyl may optionally be interrupted by one or more
moieties which are preferably selected from the group consisting
of
##STR00007## [0121] wherein [0122] dashed lines indicate attachment
to the remainder of the moiety or reagent; and [0123] --R and --Ra
are independently of each other selected from the group consisting
of --H, methyl, ethyl, propyl, butyl, pentyl and hexyl.
[0124] As used herein, the term "C.sub.3-10 cycloalkyl" means a
cyclic alkyl chain having 3 to 10 carbon atoms, which may be
saturated or unsaturated, e.g. cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,
cyclononyl or cyclodecyl. Each hydrogen atom of a C.sub.3-10
cycloalkyl carbon may be replaced by a substituent as defined
above. The term "C.sub.3-10 cycloalkyl" also includes bridged
bicycles like norbornane or norbornene.
[0125] The term "8- to 30-membered carbopolycyclyl" or "8- to
30-membered carbopolycycle" means a cyclic moiety of two or more
rings with 8 to 30 ring atoms, where two neighboring rings share at
least one ring atom and that may contain up to the maximum number
of double bonds (aromatic or non-aromatic ring which is fully,
partially or un-saturated). Preferably a 8- to 30-membered
carbopolycyclyl means a cyclic moiety of two, three, four or five
rings, more preferably of two, three or four rings.
[0126] As used herein, the term "3- to 10-membered heterocyclyl" or
"3- to 10-membered heterocycle" means a ring with 3, 4, 5, 6, 7, 8,
9 or 10 ring atoms that may contain up to the maximum number of
double bonds (aromatic or non-aromatic ring which is fully,
partially or un-saturated) wherein at least one ring atom up to 4
ring atoms are replaced by a heteroatom selected from the group
consisting of sulfur (including --S(O)--, --S(O).sub.2--), oxygen
and nitrogen (including=N(O)--) and wherein the ring is linked to
the rest of the molecule via a carbon or nitrogen atom. Examples
for 3- to 10-membered heterocycles include but are not limited to
aziridine, oxirane, thiirane, azirine, oxirene, thiirene,
azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,
imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,
isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,
isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,
tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,
oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,
thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,
imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine,
piperazine, piperidine, morpholine, tetrazole, triazole,
triazolidine, tetrazolidine, diazepane, azepine and homopiperazine.
Each hydrogen atom of a 3- to 10-membered heterocyclyl or 3- to
10-membered heterocyclic group may be replaced by a substituent as
defined below.
[0127] As used herein, the term "8- to 11-membered heterobicyclyl"
or "8- to 11-membered heterobicycle" means a heterocyclic moiety of
two rings with 8 to 11 ring atoms, where at least one ring atom is
shared by both rings and that may contain up to the maximum number
of double bonds (aromatic or non-aromatic ring which is fully,
partially or un-saturated) wherein at least one ring atom up to 6
ring atoms are replaced by a heteroatom selected from the group
consisting of sulfur (including --S(O)--, --S(O).sub.2--), oxygen
and nitrogen (including .dbd.N(O)--) and wherein the ring is linked
to the rest of the molecule via a carbon or nitrogen atom. Examples
for an 8- to 11-membered heterobicycle are indole, indoline,
benzofuran, benzothiophene, benzoxazole, benzisoxazole,
benzothiazole, benzisothiazole, benzimidazole, benzimidazoline,
quinoline, quinazoline, dihydroquinazoline, quinoline,
dihydroquinoline, tetrahydroquinoline, decahydroquinoline,
isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline,
dihydroisoquinoline, benzazepine, purine and pteridine. The term 8-
to 11-membered heterobicycle also includes spiro structures of two
rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles
like 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an 8- to
11-membered heterobicyclyl or 8- to 11-membered heterobicycle
carbon may be replaced by a substituent as defined below.
[0128] Similarly, the term "8- to 30-membered heteropolycyclyl" or
"8- to 30-membered heteropolycycle" means a heterocyclic moiety of
more than two rings with 8 to 30 ring atoms, preferably of three,
four or five rings, where two neighboring rings share at least one
ring atom and that may contain up to the maximum number of double
bonds (aromatic or non-aromatic ring which is fully, partially or
unsaturated), wherein at least one ring atom up to 10 ring atoms
are replaced by a heteroatom selected from the group of sulfur
(including --S(O)--, --S(O).sub.2--), oxygen and nitrogen
(including=N(O)--) and wherein the ring is linked to the rest of a
molecule via a carbon or nitrogen atom.
[0129] It is understood that the phrase "the pair R.sup.x/R.sup.y
is joined together with the atom to which they are attached to form
a C.sub.3-10 cycloalkyl or a 3- to 10-membered heterocyclyl" in
relation with a moiety of the structure
##STR00008##
means that Rx and Ry form the following structure:
##STR00009##
wherein R is C.sub.3-10 cycloalkyl or 3- to 10-membered
heterocyclyl.
[0130] It is also understood that the phrase "the pair
R.sup.x/R.sup.y is joint together with the atoms to which they are
attached to form a ring A" in relation with a moiety of the
structure
##STR00010##
[0131] means that R.sup.x and R.sup.y form the following
structure:
##STR00011##
[0132] As used herein, the term "terminal alkyne" means a
moiety
##STR00012##
[0133] As used herein, "halogen" means fluoro, chloro, bromo or
iodo. It is generally preferred that halogen is fluoro or
chloro.
[0134] In general, the term "comprise" or "comprising" also
encompasses "consist of" or "consisting of".
[0135] It is understood that in formula (Ia) and (Ib) -D is
connected to -L.sup.1- via a covalent and reversible linkage.
[0136] In another aspect the present invention relates to a PTH
prodrug or a pharmaceutically acceptable salt thereof comprising a
conjugate D-L, wherein [0137] -D is a PTH moiety; and [0138] -L
comprises a reversible prodrug linker moiety -L.sup.1-, which
moiety -L.sup.1- is connected to the PTH moiety -D through a
functional group of PTH; [0139] wherein -L.sup.1- is substituted
with -L.sup.2-Z' and is optionally further substituted; wherein
[0140] -L.sup.2- is a single chemical bond or a spacer moiety; and
[0141] --Z' is a water-insoluble carrier moiety.
[0142] It is understood that a multitude of moieties
-L.sup.2-L.sup.1-D is connected to a water-insoluble carrier --Z'
and that the linkage between -D and -L.sup.1- is covalent and
reversible.
[0143] Preferably, -D has the sequence of SEQ ID NO:47, SEQ ID
NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ
ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:107, SEQ ID NO:108,
SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114 or SEQ ID NO:115. More preferably -D has the
sequence of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID
NO:110, SEQ ID NO:111 or SEQ ID NO: 112.
[0144] In one embodiment -D has the sequence of SEQ ID NO:50.
[0145] In another embodiment -D has the sequence of SEQ ID NO:
52.
[0146] In another embodiment -D has the sequence of SEQ ID
NO:110.
[0147] In another embodiment -D has the sequence of SEQ ID
NO:111.
[0148] In another embodiment -D has the sequence of SEQ ID NO:
112.
[0149] Most preferably -D has the sequence of SEQ ID NO:51.
[0150] The moiety -L.sup.1- is either conjugated to a functional
group of the side chain of an amino acid residue of -D, to the
N-terminal amine functional group or to the C-terminal carboxyl
functional group of -D or to a nitrogen atom in the backbone
polypeptide chain of -D.
[0151] Attachment to either the N-terminus or C-terminus can either
be directly through the corresponding amine or carboxyl functional
group, respectively, or indirectly wherein a spacer moiety is first
conjugated to the amine or carboxyl functional group to which
spacer moiety -L.sup.1- is conjugated.
[0152] Preferably, the amino acid residue of PTH to which -L.sup.1-
is conjugated comprises a functional group selected from the group
consisting carboxylic acid, primary and secondary amine, maleimide,
thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde,
ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid,
phosphonic acid, haloacetyl, alkyl halide, acryloyl, aryl fluoride,
hydroxylamine, sulfate, disulfide, vinyl sulfone, vinyl ketone,
diazoalkane, oxirane, guanidine and aziridine. Even more preferably
the amino acid residue of PTH to which -L.sup.1- is conjugated
comprises a functional group selected from the group consisting
hydroxyl, primary and secondary amine and guanidine. Even more
preferably the amino acid residue of PTH to which -L.sup.1- is
conjugated comprises a primary or secondary amine functional group.
Most preferably the amino acid residue of PTH to which -L.sup.1- is
conjugated comprises a primary amine functional group.
[0153] If the moiety -L.sup.1- is conjugated to a functional group
of the side chain of an amino acid residue of PTH said amino acid
residue is selected from the group consisting of proteinogenic
amino acid residues and non-proteinogenic amino acid residues.
[0154] In one embodiment -L.sup.1- is conjugated to a functional
group of the side chain of a non-proteinogenic amino acid residue
of PTH. It is understood that such non-proteinogenic amino acid is
not found in the sequence of native PTH or fragments thereof and
that it may only be present in variants, analogs, orthologs,
homologs and derivatives of PTH.
[0155] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a proteinogenic amino acid
residue of PTH. Preferably, said amino acid is selected from the
group consisting of histidine, lysine, tryptophan, serine,
threonine, tyrosine, aspartic acid, glutamic acid and arginine.
Even more preferably said amino acid is selected from the group
consisting of lysine, aspartic acid, arginine and serine. Even more
preferably said amino acid is selected from the group consisting of
lysine, arginine and serine.
[0156] In one embodiment -L.sup.1- is conjugated to a functional
group of the side chain of a histidine of PTH.
[0157] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a lysine of PTH.
[0158] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a tryptophan of PTH.
[0159] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a serine of PTH.
[0160] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a threonine of PTH.
[0161] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a tyrosine of PTH.
[0162] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a aspartic acid of PTH.
[0163] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of a glutamic acid of PTH.
[0164] In another embodiment -L.sup.1- is conjugated to a
functional group of the side chain of an arginine of PTH.
[0165] It is understood that not every PTH moiety may comprise all
of these amino acid residues.
[0166] In a preferred embodiment -L.sup.1- is conjugated to the
N-terminal amine functional group of PTH, either directly through
the corresponding amine functional group or indirectly wherein a
spacer moiety is first conjugated to the amine functional group to
which spacer moiety -L.sup.1- is conjugated. Even more preferably,
-L.sup.1- is directly conjugated to the N-terminal amine functional
group of PTH, preferably PTH 1-34, i.e. PTH having the sequence of
SEQ ID NO:51.
[0167] It was surprisingly found that N-terminal attachment of
-L.sup.1- is advantageous, i.e. attachment of -L.sup.1- to the
N-terminus of PTH, because it was found that such attachment site
protects the N-terminus which is crucial for PTH activity.
Furthermore, it was surprisingly found that the main metabolite
formed from a PTH prodrug with N-terminal attachment of -L.sup.1-
is PTH 1-33, i.e. the 33 N-terminal amino acids of PTH, which
metabolite is known to be active.
[0168] In another embodiment -L.sup.1- is conjugated to the
C-terminal functional group of PTH, either directly through the
corresponding carboxyl functional group or indirectly wherein a
spacer moiety is first conjugated to the carboxyl functional group
to which spacer moiety -L.sup.1- is conjugated.
[0169] Most preferably L.sup.1- is directly conjugated to the
N-terminal amine functional group of PTH.
[0170] The moiety -L.sup.1- can be connected to -D through any type
of linkage, provided that it is reversible. Preferably, -L.sup.1-
is connected to -D through a linkage selected from the group
consisting of amide, ester, carbamate, acetal, aminal, imine,
oxime, hydrazone, disulfide and acylguanidine. Even more preferably
-L.sup.1- is connected to -D through a linkage selected from the
group consisting of amide, ester, carbamate and acylguanidin. It is
understood that some of these linkages per se are not reversible,
but that in the present invention neighboring groups comprised in
-L.sup.1- render these linkage reversible.
[0171] In one embodiment -L.sup.1- is connected to -D through an
ester linkage.
[0172] In another embodiment -L.sup.1- is connected to -D through a
carbamate linkage.
[0173] In another embodiment -L.sup.1- is connected to -D through
an acylguanidine.
[0174] In a preferred embodiment -L.sup.1- is connected to -D
through an amide linkage.
[0175] The moiety -L.sup.1- is a reversible prodrug linker from
which the drug, i.e. PTH, is released in its free form, i.e. it is
a traceless prodrug linker. Suitable prodrug linkers are known in
the art, such as for example the reversible prodrug linker moieties
disclosed in WO 2005/099768 A2, WO 2006/136586 A2, WO 2011/089216
A1 and WO 2013/024053 A1, which are incorporated by reference
herewith.
[0176] In another embodiment -L.sup.1- is a reversible prodrug
linker as described in WO 2011/012722 A1, WO 2011/089214 A1, WO
2011/089215 A1, WO 2013/024052 A1 and WO 2013/160340 A1 which are
incorporated by reference herewith.
[0177] A particularly preferred moiety -L.sup.1- is disclosed in WO
2009/095479 A2. Accordingly, in a preferred embodiment the moiety
-L.sup.1- is of formula (II):
##STR00013## [0178] wherein the dashed line indicates the
attachment to a nitrogen, hydroxyl or thiol of -D which is a PTH
moiety; [0179] --X-- is --C(R.sup.4R.sup.4a)--; --N(R.sup.4)--;
--O--; --C(R.sup.4R.sup.4a)--C(R.sup.5R.sup.5a)--;
--C(R.sup.5R.sup.5a)--C(R.sup.4R.sup.4a)--;
--C(R.sup.4R.sup.4a)--N(R.sup.6)--;
--N(R.sup.6)--C(R.sup.4R.sup.4a)--; --C(R.sup.4R.sup.4a)--O--;
--O--C(R.sup.4R.sup.4a)--; or --C(R.sup.7R.sup.7a)--; [0180]
X.sup.1 is C; or S(O); [0181] --X.sup.2-- is
--C(R.sup.8R.sup.8a)--; or
--C(R.sup.8R.sup.8a)--C(R.sup.9R.sup.9a)--; [0182] .dbd.X.sup.3 is
.dbd.O; .dbd.S; or .dbd.N--CN; [0183] --R.sup.1, --R.sup.1a,
--R.sup.2, --R.sup.2a, --R.sup.4, --R.sup.4a, --R.sup.5,
--R.sup.5a, --R.sup.6, --R.sup.8, --R.sup.8a, --R.sup.9, --R.sup.9a
are independently selected from the group consisting of --H; and
C.sub.1-6 alkyl; [0184] --R.sup.3, --R.sup.3a are independently
selected from the group consisting of --H; and C.sub.1-6 alkyl,
provided that in case one of --R.sup.3, --R.sup.3a or both are
other than --H they are connected to N to which they are attached
through an SP.sup.3-hybridized carbon atom; [0185] --R.sup.7 is
--N(R.sup.10R.sup.10a); or --NR.sup.10--(C.dbd.O)--R.sup.11; [0186]
--R.sup.7a, --R.sup.10, --R.sup.10a, --R.sup.11 are independently
of each other --H; or C.sub.1-6 alkyl; [0187] optionally, one or
more of the pairs --R.sup.1a/--R.sup.4a, --R.sup.1a/--R.sup.5a,
--R.sup.1a/--R.sup.7a, --R.sup.4a/--R.sup.5a, --R.sup.8a/--R.sup.9a
form a chemical bond; [0188] optionally, one or more of the pairs
--R.sup.1/--R.sup.1a, --R.sup.2/--R.sup.2a, --R.sup.4/--R.sup.4a,
--R.sup.5/--R.sup.5a, --R.sup.8/--R.sup.8a, --R.sup.9/--R.sup.9a
are joined together with the atom to which they are attached to
form a C.sub.3-10 cycloalkyl; or 3- to 10-membered heterocyclyl;
[0189] optionally, one or more of the pairs --R.sup.1/--R.sup.4,
--R.sup.1/--R.sup.5, --R.sup.1/--R.sup.6, --R.sup.1/--R.sup.7a,
--R.sup.4/--R.sup.5, --R.sup.4/--R.sup.6, --R.sup.8/--R.sup.9,
--R.sup.2/--R.sup.3 are joined together with the atoms to which
they are attached to form a ring A; [0190] optionally,
R.sup.3/R.sup.3a are joined together with the nitrogen atom to
which they are attached to form a 3- to 10-membered heterocycle;
[0191] A is selected from the group consisting of phenyl; naphthyl;
indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 3- to
10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and
[0192] wherein -L.sup.1- is substituted with -L.sup.2-Z or
-L.sup.2-Z' and wherein -L.sup.1- is optionally further
substituted, provided that the hydrogen marked with the asterisk in
formula (II) is not replaced by -L.sup.2-Z or -L.sup.2-Z' or a
substituent; [0193] wherein [0194] -L 2- is a single chemical bond
or a spacer; [0195] --Z is a water-soluble carrier; and [0196] --Z'
is a water-insoluble carrier.
[0197] Preferably -L.sup.1- of formula (II) is substituted with one
moiety -L.sup.2-Z or -L.sup.2-Z'.
[0198] In one embodiment -L.sup.1- of formula (II) is not further
substituted.
[0199] It is understood that if --R.sup.3/--R.sup.3a of formula
(II) are joined together with the nitrogen atom to which they are
attached to form a 3- to 10-membered heterocycle, only such 3- to
10-membered heterocycles may be formed in which the atoms directly
attached to the nitrogen are SP.sup.3-hybridized carbon atoms. In
other words, such 3- to 10-membered heterocycle formed by
--R.sup.3/--R.sup.3a together with the nitrogen atom to which they
are attached has the following structure
##STR00014## [0200] wherein [0201] the dashed line indicates
attachment to the rest of -L.sup.1-; [0202] the ring comprises 3 to
10 atoms comprising at least one nitrogen; and [0203] R.sup.# and
R.sup.## represent an SP.sup.3-hybridized carbon atom.
[0204] It is also understood that the 3- to 10-membered heterocycle
may be further substituted.
[0205] Exemplary embodiments of suitable 3- to 10-membered
heterocycles formed by --R.sup.3/--R.sup.3a of formula (II)
together with the nitrogen atom to which they are attached are the
following:
##STR00015## [0206] wherein [0207] dashed lines indicate attachment
to the rest of the molecule; and [0208] --R is selected from the
group consisting of --H and C.sub.1-6 alkyl.
[0209] -L.sup.1- of formula (II) may optionally be further
substituted. In general, any substituent may be used as far as the
cleavage principle is not affected, i.e. the hydrogen marked with
the asterisk in formula (II) is not replaced and the nitrogen of
the moiety
##STR00016##
of formula (II) remains part of a primary, secondary or tertiary
amine, i.e. --R.sup.3 and --R.sup.3a are independently of each
other --H or are connected to --N< through an
SP.sup.3-hybridized carbon atom.
[0210] In one embodiment --R.sup.1 or --R.sup.1a of formula (II) is
substituted with -L.sup.2-Z or -L.sup.2-Z'. In another embodiment
--R.sup.2 or --R.sup.2a of formula (II) is substituted with
-L.sup.2-Z or -L.sup.2-Z'. In another embodiment --R.sup.3 or
--R.sup.3a of formula (II) is substituted with -L.sup.2-Z or
-L.sup.2-Z'. In another embodiment --R.sup.4 of formula (II) is
substituted with -L.sup.2-Z or -L.sup.2-Z'. In another embodiment
--R.sup.5 or --R.sup.5a of formula (II) is substituted with
-L.sup.2-Z or -L.sup.2-Z'. In another embodiment --R.sup.6 of
formula (II) is substituted with -L.sup.2-Z or -L.sup.2-Z'. In
another embodiment --R.sup.7 or --R.sup.7a of formula (II) is
substituted with -L.sup.2-Z or -L.sup.2-Z'. In another embodiment
--R.sup.8 or --R.sup.8a of formula (II) is substituted with
-L.sup.2-Z or -L.sup.2-Z'. In another embodiment --R.sup.9 or
--R.sup.9a of formula (II) is substituted with -L.sup.2-Z or
-L.sup.2-Z'. In another embodiment --R.sup.10 is substituted with
-L.sup.2-Z or -L.sup.2-Z'. In another embodiment --R.sup.11 is
substituted with -L.sup.2-Z or -L.sup.2-Z'.
[0211] Preferably, --X-- of formula (II) is selected from the group
consisting of --C(R.sup.4R.sup.4a)--, --N(R.sup.4)-- and
--C(R.sup.7R.sup.7a)--.
[0212] In one embodiment --X-- of formula (II) is
--C(R.sup.4R.sup.4a)--In one preferred embodiment --X-- of formula
(II) is --C(R.sup.7R.sup.7a)--.
[0213] Preferably, --R.sup.7 of formula (II) is
--NR.sup.10--(C.dbd.O)--R.sup.11.
[0214] Preferably, --R.sup.7a of formula (II) is selected from --H,
methyl and ethyl. Most preferably --R.sup.7a of formula (II) is
--H.
[0215] Preferably, --R.sup.10 is selected from --H, methyl and
ethyl. Most preferably --R.sup.10 is methyl.
[0216] Preferably, --R.sup.11 is selected from --H, methyl and
ethyl. Most preferably --R.sup.11 is --H.
[0217] Preferably, --R.sup.11 is substituted with -L.sup.2-Z or
-L.sup.2-Z'.
[0218] In another preferred embodiment --X-- of formula (II) is
--N(R.sup.4)--.
[0219] Preferably, --R.sup.4 is selected from the group consisting
of --H, methyl and ethyl. Preferably, --R.sup.4 is --H.
[0220] Preferably, X.sup.1 of formula (II) is C.
[0221] Preferably, .dbd.X.sup.3 of formula (II) is .dbd.O.
[0222] Preferably, --X.sup.2-- of formula (II) is
--C(R.sup.8R.sup.8a)--.
[0223] Preferably --R.sup.8 and --R.sup.8a of formula (II) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (II) is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (II) are --H.
[0224] Preferably, --R.sup.1 and --R.sup.1a of formula (II) are
independently selected from the group consisting of --H, methyl and
ethyl.
[0225] In one preferred embodiment at least one of --R.sup.1 and
--R.sup.1a of formula (II) is --H, more preferably both --R.sup.1
and --R.sup.1a of formula (II) are --H.
[0226] In another preferred embodiment at least one of --R.sup.1
and --R.sup.1a of formula (II) is methyl, more preferably both
--R.sup.1 and --R.sup.1a of formula (II) are methyl.
[0227] Preferably, --R.sup.2 and --R.sup.2a of formula (II) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.2 and --R.sup.2a of
formula (II) is --H. Even more preferably both --R.sup.2 and
--R.sup.2a of formula (II) are H.
[0228] Preferably, --R.sup.3 and --R.sup.3a of formula (II) are
independently selected from the group consisting of --H, methyl,
ethyl, propyl and butyl.
[0229] In one preferred embodiment at least one of --R.sup.3 and
--R.sup.3a of formula (II) is methyl, more preferably --R.sup.3 of
formula (II) is methyl and --R.sup.3a of formula (II) is --H.
[0230] In another preferred embodiment --R.sup.3 and --R.sup.3a of
formula (II) are both --H.
[0231] Preferably, -D is connected to -L.sup.1- through a nitrogen
by forming an amide bond.
[0232] In one preferred embodiment the moiety -L.sup.1- is of
formula (IIa-i):
##STR00017## [0233] wherein [0234] the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0235] --R.sup.1, --R.sup.1a, --R.sup.2, --R.sup.2a,
--R.sup.3, --R.sup.3a, --R.sup.7, --R.sup.7a and --X.sup.2-- are
used as defined in formula (II); [0236] and [0237] wherein
-L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein
-L.sup.1- is optionally further substituted, provided that the
hydrogen marked with the asterisk in formula (IIa-i) is not
replaced by -L.sup.2-Z or -L.sup.2-Z' or a substituent.
[0238] Preferably -L.sup.1- of formula (IIa-i) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0239] Preferably the moiety -L.sup.1- of formula (IIa-i) is not
further substituted.
[0240] Preferably, --R.sup.1 and --R.sup.1a of formula (IIa-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.1 and --R.sup.1a of
formula (IIa-i) is --H. Even more preferably both --R.sup.1 and
--R.sup.1a of formula (IIa-i) are --H.
[0241] Preferably, --R.sup.7 of formula (IIa-i) is
--NR.sup.10--(C.dbd.O)--R.sup.11.
[0242] Preferably, --R.sup.7a of formula (II-i) is selected from
--H, methyl and ethyl. Most preferably --R.sup.7a of formula (II-i)
is --H.
[0243] Preferably, --R.sup.10 of formula (IIa-i) is selected from
--H, methyl and ethyl. Most preferably --R.sup.10 of formula
(IIa-i) is methyl.
[0244] Preferably, --R.sup.11 of formula (IIa-i) is selected from
--H, methyl and ethyl. Most preferably --R.sup.11 of formula
(IIa-i) is --H.
[0245] Preferably, --R.sup.11 of formula (IIa-i) is substituted
with -L.sup.2-Z or -L.sup.2-Z'.
[0246] Preferably, --X.sup.2-- of formula (IIa-i) is
--C(R.sup.8R.sup.8a)--.
[0247] Preferably --R.sup.8 and --R.sup.8a of formula (IIa-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIa-i) is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIa-i) are --H.
[0248] Preferably, --R.sup.2 and --R.sup.2a of formula (IIa-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.2 and --R.sup.2a of
formula (IIa-i) is --H. Even more preferably both --R.sup.2 and
--R.sup.2a of formula (IIa-i) are H.
[0249] Preferably, --R.sup.3 and --R.sup.3a of formula (IIa-i) are
independently selected from the group consisting of --H, methyl,
ethyl, propyl and butyl. Even more preferably at least one of
--R.sup.3 and --R.sup.3a of formula (IIa-i) is methyl.
[0250] Preferably, --R.sup.3 of formula (IIa-i) is --H and
--R.sup.3a of formula (IIa-i) is methyl.
[0251] More preferably the moiety -L.sup.1- is of formula
(IIa-ii)
##STR00018## [0252] wherein the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0253] --R.sup.2, --R.sup.2a, --R.sup.10, --R.sup.11
and --X.sup.2-- are used as defined in formula (II); and [0254]
wherein -L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and
wherein -L.sup.1- is optionally further substituted, provided that
the hydrogen marked with the asterisk in formula (IIa-ii) is not
replaced by -L.sup.2-Z or -L.sup.2-Z' or a substituent.
[0255] Preferably -L.sup.1- of formula (IIa-ii) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0256] Preferably the moiety -L.sup.1- of formula (IIa-ii) is not
further substituted.
[0257] Preferably, --X.sup.2-- of formula (IIa-ii) is
--C(R.sup.8R.sup.8a)--.
[0258] Preferably --R.sup.8 and --R.sup.8a of formula (IIa-ii) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIa-ii) is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIa-ii) are --H.
[0259] Preferably, --R.sup.3 and --R.sup.3a of formula (IIa-ii) are
independently selected from the group consisting of --H, methyl,
ethyl, propyl and butyl. Even more preferably at least one of
--R.sup.3 and --R.sup.3a of formula (IIa-ii) is methyl.
[0260] Preferably, --R.sup.3 of formula (IIa-ii) is --H and
--R.sup.3a of formula (IIa-ii) is methyl.
[0261] Preferably, --R.sup.10 of formula (IIa-ii) is selected from
--H, methyl and ethyl. Most preferably --R.sup.10 of formula
(IIa-ii) is methyl.
[0262] Preferably, --R.sup.11 of formula (IIa-ii) is selected from
--H, methyl and ethyl. Most preferably --R.sup.11 of formula
(IIa-ii) is --H.
[0263] Preferably, --R.sup.11 of formula (IIa-ii) is substituted
with -L.sup.2-Z or -L.sup.2-Z'.
[0264] In an even more preferred embodiment the moiety -L.sup.1- is
of formula (IIa-ii'):
##STR00019## [0265] wherein [0266] wherein the dashed line
indicates the attachment to a nitrogen of D which is a PTH moiety
by forming an amide bond; [0267] the dashed line marked with the
asterisk indicates attachment to -L.sup.2-; [0268] --R.sup.3,
--R.sup.3a --R.sup.10 and --X.sup.2-- are used as defined in
formula (II); and [0269] wherein -L.sup.1- is optionally further
substituted, provided that the hydrogen marked with the asterisk in
formula (IIa-ii') is not replaced by a substituent.
[0270] Preferably the moiety -L.sup.1- of formula (IIa-ii') is not
further substituted.
[0271] Preferably, --X.sup.2-- of formula (IIa-ii') is
--C(R.sup.8R.sup.8a)--.
[0272] Preferably --R.sup.8 and --R.sup.8a of formula (IIa-ii') are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIa-ii') is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIa-ii') are --H.
[0273] Preferably, --R.sup.3 and --R.sup.3a of formula (IIa-ii')
are independently selected from the group consisting of --H,
methyl, ethyl, propyl and butyl. Even more preferably at least one
of --R.sup.3 and --R.sup.3a of formula (IIa-ii') is methyl.
[0274] Preferably, --R.sup.3 of formula (IIa-ii') is --H and
--R.sup.3a of formula (IIa-ii') is methyl.
[0275] Preferably, --R.sup.10 of formula (IIa-ii') is selected from
--H, methyl and ethyl. Most preferably --R.sup.10 of formula
(IIa-ii') is methyl.
[0276] Even more preferably the moiety -L.sup.1- is of formula
(IIa-iii):
##STR00020## [0277] wherein the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; and [0278] wherein -L.sup.1- is substituted with
-L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is optionally
further substituted, provided that the hydrogen marked with the
asterisk in formula (IIa-iii) is not replaced by -L.sup.2-Z or
-L2-Z' or a substituent.
[0279] Preferably -L.sup.1- of formula (IIa-iii) is substituted
with one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0280] Preferably the moiety -L.sup.1- of formula (IIa-iii) is not
further substituted.
[0281] Most preferably the moiety -L.sup.1- is of formula
(IIa-iii'):
##STR00021## [0282] wherein [0283] wherein the dashed line
indicates the attachment to a nitrogen of D which is a PTH moiety
by forming an amide bond; [0284] the dashed line marked with the
asterisk indicates attachment to -L.sup.2-; [0285] --R.sup.2,
--R.sup.2a, --R.sup.3, --R.sup.3a and --X.sup.2-- are used as
defined in formula (II); and [0286] wherein -L.sup.1- is optionally
further substituted, provided that the hydrogen marked with the
asterisk in formula (IIa-iii') is not replaced by a
substituent.
[0287] Preferably the moiety -L.sup.1- of formula (IIa-iii') is not
further substituted.
[0288] In another preferred embodiment the moiety -L.sup.1- is of
formula (IIb-i)
##STR00022## [0289] wherein [0290] the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0291] --R.sup.1, --R.sup.1a, --R.sup.2, --R.sup.2a,
--R.sup.3, --R.sup.3a, --R.sup.4 and --X.sup.2-- are used as
defined in formula (II); and [0292] wherein -L.sup.1- is
substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is
optionally further substituted, provided that the hydrogen marked
with the asterisk in formula (IIb-i) is not replaced by -L.sup.2-Z
or -L.sup.2-Z' or a substituent.
[0293] Preferably -L.sup.1- of formula (IIb-i) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0294] Preferably the moiety -L.sup.1- of formula (IIb-i) is not
further substituted.
[0295] Preferably, --R.sup.1 and --R.sup.1a of formula (IIb-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.1 and --R.sup.1a of
formula (IIb-i) is methyl. Even more preferably both --R.sup.1 and
--R.sup.1a of formula (IIb-i) are methyl.
[0296] Preferably, --R.sup.4 of formula (IIb-i) is selected from
the group consisting of --H, methyl and ethyl. More preferably,
--R.sup.4 of formula (IIb-i) is --H.
[0297] Preferably, --X.sup.2-- of formula (IIb-i) is
--C(R.sup.8R.sup.8a)--.
[0298] Preferably --R.sup.8 and --R.sup.8a of formula (IIb-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIb-i) is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIb-i) are --H.
[0299] Preferably, --R.sup.2 and --R.sup.2a of formula (IIb-i) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.2 and --R.sup.2a of
formula (IIb-i) is --H. Even more preferably both --R.sup.2 and
--R.sup.2a of formula (IIb-i) are H.
[0300] Preferably, --R.sup.3 and --R.sup.3a of formula (IIb-i) are
independently selected from the group consisting of --H, methyl,
ethyl, propyl and butyl. Even more preferably at least one of
--R.sup.3 and --R.sup.3a of formula (IIb-i) is --H. Even more
preferably both --R.sup.3 and --R.sup.3a of formula (IIb-i) are
--H.
[0301] More preferably the moiety -L.sup.1- is of formula
(IIb-ii):
##STR00023## [0302] wherein the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0303] --R.sup.2, --R.sup.2a, --R.sup.3, --R.sup.3a and
--X.sup.2-- are used as defined in formula (II); and [0304] wherein
-L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein
-L.sup.1- is optionally further substituted, provided that the
hydrogen marked with the asterisk in formula (IIb-ii) is not
replaced by -L.sup.2-Z or -L.sup.2-Z' or a substituent.
[0305] Preferably -L.sup.1- of formula (IIb-ii) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0306] Preferably the moiety -L.sup.1- of formula (IIb-ii) is not
further substituted.
[0307] Preferably, --X.sup.2-- of formula (IIb-ii) is
--C(R.sup.8R.sup.8a)--.
[0308] Preferably --R.sup.8 and --R.sup.8a of formula (IIb-ii) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIb-ii) is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIb-ii) are --H.
[0309] Preferably, --R.sup.2 and --R.sup.2a of formula (IIb-ii) are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably, at least one of --R.sup.2 and --R.sup.2a of
formula (IIb-ii) is --H. Even more preferably both --R.sup.2 and
--R.sup.2a of formula (IIb-ii) are H.
[0310] Preferably, --R.sup.3 and --R.sup.3a of formula (IIb-ii) are
independently selected from the group consisting of --H, methyl,
ethyl, propyl and butyl. Even more preferably at least one of
--R.sup.3 and --R.sup.3a of formula (IIb-ii) is --H. Even more
preferably both --R.sup.3 and --R.sup.3a of formula (IIb-ii) are
--H.
[0311] Even more preferably the moiety -L.sup.1- is of formula
(IIb-ii'):
##STR00024## [0312] wherein [0313] the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0314] --R.sup.2, --R.sup.2a, --R.sup.3, --R.sup.3a and
--X.sup.2-- are used as defined in formula (II); and [0315] wherein
-L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein
-L.sup.1- is optionally further substituted, provided that the
hydrogen marked with the asterisk in formula (IIb-ii') is not
replaced by -L.sup.2-Z or -L.sup.2-Z' or a substituent.
[0316] Preferably the moiety -L.sup.1- of formula (IIb-ii') is not
further substituted.
[0317] Preferably, --X.sup.2-- of formula (IIb-ii') is
--C(R.sup.8R.sup.8a)--.
[0318] Preferably --R.sup.8 and --R.sup.8a of formula (IIb-ii') are
independently selected from the group consisting of --H, methyl and
ethyl. More preferably at least one of --R.sup.8 and --R.sup.8a of
formula (IIb-ii') is --H. Even more preferably both --R.sup.8 and
--R.sup.8a of formula (IIb-ii') are --H.
[0319] Preferably, --R.sup.2 and --R.sup.2a of formula (IIb-ii')
are independently selected from the group consisting of --H, methyl
and ethyl. More preferably, at least one of --R.sup.2 and
--R.sup.2a of formula (IIb-ii') is --H. Even more preferably both
--R.sup.2 and --R.sup.2a of formula (IIb-ii') are H.
[0320] Preferably, --R.sup.3 and --R.sup.3a of formula (IIb-ii')
are independently selected from the group consisting of --H,
methyl, ethyl, propyl and butyl. Even more preferably at least one
of --R.sup.3 and --R.sup.3a of formula (IIb-ii') is --H. Even more
preferably both --R.sup.3 and --R.sup.3a of formula (IIb-ii') are
--H.
[0321] Even more preferably the moiety -L.sup.1- is of formula
(IIb-iii):
##STR00025## [0322] wherein [0323] the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; and [0324] wherein -L.sup.1- is substituted with
-L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is optionally
further substituted, provided that the hydrogen marked with the
asterisk in formula (IIb-iii) is not replaced by -L.sup.2-Z or
-L2-Z' or a substituent.
[0325] Preferably -L.sup.1- of formula (IIb-iii) is substituted
with one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0326] Preferably the moiety -L.sup.1- of formula (IIb-iii) is not
further substituted.
[0327] Most preferably the moiety -L.sup.1- is of formula
(IIb-iii'):
##STR00026## [0328] wherein [0329] the dashed line indicates the
attachment to a nitrogen of -D which is a PTH moiety by forming an
amide bond; [0330] --R.sup.2, --R.sup.2a, --R.sup.3, --R.sup.3a and
--X.sup.2-- are used as defined in formula (II); and [0331] wherein
-L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein
-L.sup.1- is optionally further substituted, provided that the
hydrogen marked with the asterisk in formula (IIb-iii') is not
replaced by -L.sup.2-Z or -L.sup.2-Z' or a substituent.
[0332] Preferably the moiety -L.sup.1- of formula (IIb-iii') is not
further substituted.
[0333] Another preferred moiety -L.sup.1- is disclosed in
unpublished European patent application 14180004, which corresponds
to the international application with the application number
PCT/EP2015/067929. Accordingly, in another preferred embodiment the
moiety -L.sup.1- is of formula (III):
##STR00027## [0334] wherein [0335] the dashed line indicates
attachment to a primary or secondary amine or hydroxyl of -D which
is a PTH moiety by forming an amide or ester linkage, respectively;
[0336] --R.sup.1, --R.sup.1a, --R.sup.2, --R.sup.2a, --R.sup.3 and
--R.sup.3a are independently of each other selected from the group
consisting of --H, --C(R.sup.8R.sup.8aR.sup.8b),
--C(.dbd.O)R.sup.8, --C.dbd.N, --C(.dbd.NR.sup.8)R.sup.8a,
--CR.sup.8(.dbd.CR.sup.8aR.sup.8b), --C.ident.CR.sup.8 and -T;
[0337] --R.sup.4, --R.sup.5 and --R.sup.5a are independently of
each other selected from the group consisting of --H,
--C(R.sup.9R.sup.9aR.sup.9b) and -T; [0338] a1 and a2 are
independently of each other 0 or 1; [0339] each --R.sup.6,
--R.sup.6a, --R.sup.7, --R.sup.7a, --R.sup.8, --R.sup.8a,
--R.sup.8b, --R.sup.9, --R.sup.9a, --R.sup.9b are independently of
each other selected from the group consisting of --H, halogen,
--CN, --COOR.sup.10, --OR.sup.10, --C(O)R.sup.10,
--C(O)N(R.sup.10R.sup.10a), --S(O).sub.2N(R.sup.10R.sup.10a),
--S(O)N(R.sup.10R.sup.10a), --S(O).sub.2R.sup.10, --S(O)R.sup.10,
--N(R.sup.10)S(O).sub.2N(R.sup.10aR.sup.10b), --SR.sup.10,
--N(R.sup.10R.sup.10a), --NO.sub.2, --OC(O)R.sup.10,
--N(R.sup.10)C(O)R.sup.10a, --N(R.sup.10)S(O).sub.2R.sup.10a,
--N(R.sup.10)S(O)R.sup.10a, --N(R.sup.10)C(O)OR.sup.10a,
--N(R.sup.10)C(O)N(R.sup.10aR.sup.10b),
--OC(O)N(R.sup.10R.sup.10a), -T, C.sub.1-20 alkyl, C.sub.2-20
alkenyl, and C.sub.2-20 alkynyl; wherein -T, C.sub.1-20 alkyl,
C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally
substituted with one or more --R.sup.11, which are the same or
different and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and
C.sub.2-20 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.12), --S(O).sub.2N(R.sup.12)--,
--S(O)N(R.sup.12)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.12)S(O).sub.2N(R.sup.12a)--S--, --N(R.sup.12)--,
--OC(OR.sup.12)(R.sup.12a)--, --N(R.sup.12)C(O)N(R.sup.12a)--, and
--OC(O)N(R.sup.12)--; each --R.sup.10, --R.sup.10a, --R.sup.10b is
independently selected from the group consisting of --H, -T,
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl;
wherein -T, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20
alkynyl are optionally substituted with one or more --R.sup.11,
which are the same or different and wherein C.sub.1-20 alkyl,
C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally
interrupted by one or more groups selected from the group
consisting of -T-, --C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.12)--,
--S(O).sub.2N(R.sup.12)--, --S(O)N(R.sup.12)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.12)S(O).sub.2N(R.sup.12a)--, --S--,
--N(R.sup.12)--, --OC(OR.sup.12)(R.sup.12a)--,
--N(R.sup.12)C(O)N(R.sup.12a)--, and --OC(O)N(R.sup.12)--; [0340]
each T is independently of each other selected from the group
consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to
11-membered heterobicyclyl; wherein each T is independently
optionally substituted with one or more --R.sup.11, which are the
same or different; [0341] each --R.sup.11 is independently of each
other selected from halogen, --CN, oxo (.dbd.O), --COOR.sup.13,
--OR.sup.13, --C(O)R.sup.13, --C(O)N(R.sup.13R.sup.13a),
--S(O).sub.2N(R.sup.13R.sup.13a), --S(O)N(R.sup.13R.sup.13a),
--S(O).sub.2R.sup.13, --S(O)R.sup.13,
--N(R.sup.13)S(O).sub.2N(R.sup.13aR.sup.13b), --SR.sup.13,
--N(R.sup.13R.sup.13a), --NO.sub.2, --OC(O)R.sup.13,
--N(R.sup.13)C(O)R.sup.13a, --N(R.sup.13)S(O).sub.2R.sup.13a,
--N(R.sup.13)S(O)R.sup.13a, --N(R.sup.13)C(O)OR.sup.13a,
--N(R.sup.13)C(O)N(R.sup.13aR.sup.13b),
--OC(O)N(R.sup.13R.sup.13a), and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different; [0342] each --R.sup.12, --R.sup.12a,
--R.sup.13, --R.sup.13a, --R.sup.13b is independently selected from
the group consisting of --H, and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different; [0343] optionally, one or more of the pairs
--R/--R.sup.1a, --R.sup.2/--R.sup.2a, --R.sup.3/--R.sup.3a,
--R.sup.6/--R.sup.6a, --R.sup.7/--R.sup.7a are joined together with
the atom to which they are attached to form a C.sub.3-10 cycloalkyl
or a 3- to 10-membered heterocyclyl; [0344] optionally, one or more
of the pairs --R.sup.1/--R.sup.2, --R.sup.1/--R.sup.3,
--R.sup.1/--R.sup.4, --R.sup.1/--R.sup.5, --R.sup.1/--R.sup.6,
--R.sup.1/--R.sup.7, --R.sup.2/--R.sup.3, --R.sup.2/--R.sup.4,
--R.sup.2/--R.sup.5, --R.sup.2/--R.sup.6, --R.sup.2/--R.sup.7,
--R.sup.3/--R.sup.4, --R.sup.3/--R.sup.5, --R.sup.3/--R.sup.6,
--R.sup.3/--R.sup.7, --R.sup.4/--R.sup.5, --R.sup.4/--R.sup.6,
--R.sup.4/--R.sup.7, --R.sup.5/--R.sup.6, --R.sup.5/--R.sup.7,
--R.sup.6/--R.sup.7 are joint together with the atoms to which they
are attached to form a ring A; [0345] A is selected from the group
consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl;
C.sub.3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to
11-membered heterobicyclyl; [0346] wherein -L.sup.1- is substituted
with -L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is optionally
further substituted; [0347] wherein [0348] -L2- is a single
chemical bond or a spacer; [0349] --Z is a water-soluble carrier;
and [0350] --Z' is a water-insoluble carrier.
[0351] The optional further substituents of -L.sup.1- of formula
(III) are preferably as described above.
[0352] Preferably -L.sup.1- of formula (III) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0353] In one embodiment -L.sup.1- of formula (III) is not further
substituted.
[0354] Additional preferred embodiments for -L.sup.1- are disclosed
in EP1536334B1, WO2009/009712A1, WO2008/034122A1, WO2009/143412A2,
WO2011/082368A2, and U.S. Pat. No. 8,618,124B2, which are herewith
incorporated by reference in their entirety.
[0355] Additional preferred embodiments for -L.sup.1- are disclosed
in U.S. Pat. No. 8,946,405B2 and U.S. Pat. No. 8,754,190B2, which
are herewith incorporated by reference in their entirety.
Accordingly, a preferred moiety -L.sup.1- is of formula (IV):
##STR00028## [0356] wherein [0357] the dashed line indicates
attachment to -D which is a PTH moiety and wherein attachment is
through a functional group of -D selected from the group consisting
of --OH, --SH and --NH.sub.2; [0358] m is 0 or 1; [0359] at least
one or both of --R.sup.1 and --R.sup.2 is/are independently of each
other selected from the group consisting of --CN, --NO.sub.2,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted alkenyl, optionally substituted alkynyl,
--C(O)R.sup.3, --S(O)R.sup.3, --S(O).sub.2R.sup.3, and --SR.sup.4,
[0360] one and only one of --R.sup.1 and --R.sup.2 is selected from
the group consisting of --H, optionally substituted alkyl,
optionally substituted arylalkyl, and optionally substituted
heteroarylalkyl; [0361] --R.sup.3 is selected from the group
consisting of --H, optionally substituted alkyl, optionally
substituted aryl, optionally substituted arylalkyl, optionally
substituted heteroaryl, optionally substituted heteroarylalkyl,
--OR.sup.9 and --N(R.sup.9).sub.2; [0362] --R.sup.4 is selected
from the group consisting of optionally substituted alkyl,
optionally substituted aryl, optionally substituted arylalkyl,
optionally substituted heteroaryl, and optionally substituted
heteroarylalkyl; [0363] each --R.sup.5 is independently selected
from the group consisting of --H, optionally substituted alkyl,
optionally substituted alkenylalkyl, optionally substituted
alkynylalkyl, optionally substituted aryl, optionally substituted
arylalkyl, optionally substituted heteroaryl and optionally
substituted heteroarylalkyl; [0364] --R.sup.9 is selected from the
group consisting of --H and optionally substituted alkyl; [0365]
--Y-- is absent and --X-- is --O-- or --S--; or [0366] --Y-- is
--N(Q)CH.sub.2-- and --X-- is --O--; [0367] Q is selected from the
group consisting of optionally substituted alkyl, optionally
substituted aryl, optionally substituted arylalkyl, optionally
substituted heteroaryl and optionally substituted heteroarylalkyl;
[0368] optionally, --R.sup.1 and --R.sup.2 may be joined to form a
3 to 8-membered ring; and [0369] optionally, both --R.sup.9
together with the nitrogen to which they are attached form a
heterocyclic ring; [0370] wherein -L.sup.1- is substituted with
-L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is optionally
further substituted; [0371] wherein [0372] -L.sup.2- is a single
chemical bond or a spacer; [0373] --Z is a water-soluble carrier;
and [0374] --Z' is a water-insoluble carrier.
[0375] Only in the context of formula (IV) the terms used have the
following meaning:
[0376] The term "alkyl" as used herein includes linear, branched or
cyclic saturated hydrocarbon groups of 1 to 8 carbons, or in some
embodiments 1 to 6 or 1 to 4 carbon atoms.
[0377] The term "alkoxy" includes alkyl groups bonded to oxygen,
including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy,
and similar.
[0378] The term "alkenyl" includes non-aromatic unsaturated
hydrocarbons with carbon-carbon double bonds.
[0379] The term "alkynyl" includes non-aromatic unsaturated
hydrocarbons with carbon-carbon triple bonds.
[0380] The term "aryl" includes aromatic hydrocarbon groups of 6 to
18 carbons, preferably 6 to carbons, including groups such as
phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes
aromatic rings comprising 3 to 15 carbons containing at least one
N, O or S atom, preferably 3 to 7 carbons containing at least one
N, O or S atom, including groups such as pyrrolyl, pyridyl,
pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,
isothiazolyl, quinolyl, indolyl, indenyl, and similar.
[0381] In some instance, alkenyl, alkynyl, aryl or heteroaryl
moieties may be coupled to the remainder of the molecule through an
alkylene linkage. Under those circumstances, the substituent will
be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or
heteroarylalkyl, indicating that an alkylene moiety is between the
alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to
which the alkenyl, alkynyl, aryl or heteroaryl is coupled.
[0382] The term "halogen" includes bromo, fluoro, chloro and
iodo.
[0383] The term "heterocyclic ring" refers to a 4 to 8 membered
aromatic or non-aromatic ring comprising 3 to 7 carbon atoms and at
least one N, O, or S atom. Examples are piperidinyl, piperazinyl,
tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as
the exemplary groups provided for the term "heteroaryl" above.
[0384] When a ring system is optionally substituted, suitable
substituents are selected from the group consisting of alkyl,
alkenyl, alkynyl, or an additional ring, each optionally further
substituted. Optional substituents on any group, including the
above, include halo, nitro, cyano, --OR, --SR, --NR.sub.2, --OCOR,
--NRCOR, --COOR, --CONR.sub.2, --SOR, --SO.sub.2R, --SONR.sub.2,
--SO.sub.2NR.sub.2, wherein each R is independently alkyl, alkenyl,
alkynyl, aryl or heteroaryl, or two R groups taken together with
the atoms to which they are attached form a ring.
[0385] Preferably -L.sup.1- of formula (IV) is substituted with one
moiety -L.sup.2-Z or -L.sup.2-Z'.
[0386] An additional preferred embodiment for -L.sup.1- is
disclosed in WO2013/036857A1, which is herewith incorporated by
reference in its entirety. Accordingly, a preferred moiety
-L.sup.1- is of formula (V):
##STR00029## [0387] wherein [0388] the dashed line indicates
attachment to -D which is a PTH moiety and wherein attachment is
through an amine functional group of -D; [0389] --R.sup.1 is
selected from the group consisting of optionally substituted
C.sub.1-C.sub.6 linear, branched, or cyclic alkyl; optionally
substituted aryl; optionally substituted heteroaryl; alkoxy; and
--NR %; [0390] --R.sup.2 is selected from the group consisting of
--H; optionally substituted C.sub.1-C.sub.6 alkyl; optionally
substituted aryl; and optionally substituted heteroaryl; [0391]
--R.sup.3 is selected from the group consisting of --H; optionally
substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and
optionally substituted heteroaryl; [0392] --R.sup.4 is selected
from the group consisting of --H; optionally substituted
C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and optionally
substituted heteroaryl; [0393] each --R.sup.5 is independently of
each other selected from the group consisting of --H; optionally
substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and
optionally substituted heteroaryl; or when taken together two
--R.sup.5 can be cycloalkyl or cycloheteroalkyl; [0394] wherein
-L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and wherein
-L.sup.1- is optionally further substituted; [0395] wherein [0396]
-L2- is a single chemical bond or a spacer; [0397] --Z is a
water-soluble carrier; and [0398] --Z' is a water-insoluble
carrier.
[0399] Only in the context of formula (V) the terms used have the
following meaning:
[0400] "Alkyl", "alkenyl", and "alkynyl" include linear, branched
or cyclic hydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4
carbons wherein alkyl is a saturated hydrocarbon, alkenyl includes
one or more carbon-carbon double bonds and alkynyl includes one or
more carbon-carbon triple bonds. Unless otherwise specified these
contain 1-6 C.
[0401] "Aryl" includes aromatic hydrocarbon groups of 6-18 carbons,
preferably 6-10 carbons, including groups such as phenyl, naphthyl,
and anthracene "Heteroaryl" includes aromatic rings comprising 3-15
carbons containing at least one N, O or S atom, preferably 3-7
carbons containing at least one N, O or S atom, including groups
such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl,
isoxazolyl, thiszolyl, isothiazolyl, quinolyl, indolyl, indenyl,
and similar.
[0402] The term "substituted" means an alkyl, alkenyl, alkynyl,
aryl, or heteroaryl group comprising one or more substituent groups
in place of one or more hydrogen atoms. Substituents may generally
be selected from halogen including F, Cl, Br, and I; lower alkyl
including linear, branched, and cyclic; lower haloalkyl including
fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower
alkoxy including linear, branched, and cyclic; SH; lower alkylthio
including linear, branched and cyclic; amino, alkylamino,
dialkylamino, silyl including alkylsilyl, alkoxysilyl, and
arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic
ester, carboxylic amide, aminocarbonyl; aminoacyl; carbamate; urea;
thiocarbamate; thiourea; ketne; sulfone; sulfonamide; aryl
including phenyl, naphthyl, and anthracenyl; heteroaryl including
5-member heteroaryls including as pyrrole, imidazole, furan,
thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole,
triazole, oxadiazole, and tetrazole, 6-member heteroaryls including
pyridine, pyrimidine, pyrazine, and fused heteroaryls including
benzofuran, benzothiophene, benzoxazole, benzimidazole, indole,
benzothiazole, benzisoxazole, and benzisothiazole.
[0403] Preferably -L.sup.1- of formula (V) is substituted with one
moiety -L.sup.2-Z or -L.sup.2-Z'.
[0404] A further preferred embodiment for -L.sup.1- is disclosed in
U.S. Pat. No. 7,585,837B2, which is herewith incorporated by
reference in its entirety. Accordingly, a preferred moiety
-L.sup.1- is of formula (VI):
##STR00030## [0405] wherein [0406] the dashed line indicates
attachment to -D which is a PTH moiety and wherein attachment is
through an amine functional group of -D; [0407] R.sup.1 and R.sup.2
are independently selected from the group consisting of hydrogen,
alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro,
--SO.sub.3H, --SO.sub.2NHR.sup.5, amino, ammonium, carboxyl,
PO.sub.3H.sub.2, and OPO.sub.3H.sub.2; R.sup.3, R.sup.4, and
R.sup.5 are independently selected from the group consisting of
hydrogen, alkyl, and aryl; [0408] wherein -L.sup.1- is substituted
with -L.sup.2-Z or -L.sup.2-Z' and wherein -L.sup.1- is optionally
further substituted; [0409] wherein [0410] -L2- is a single
chemical bond or a spacer; [0411] --Z is a water-soluble carrier;
and [0412] --Z' is a water-insoluble carrier.
[0413] Suitable substituents for formulas (VI) are alkyl (such as
C.sub.1-6 alkyl), alkenyl (such as C.sub.2-6 alkenyl), alkynyl
(such as C.sub.2-6 alkynyl), aryl (such as phenyl), heteroalkyl,
heteroalkenyl, heteroalkynyl, heteroaryl (such as aromatic 4 to 7
membered heterocycle) or halogen moieties.
[0414] Only in the context of formula (VI) the terms used have the
following meaning:
[0415] The terms "alkyl", "alkoxy", "alkoxyalkyl", "aryl",
"alkaryl" and "aralkyl" mean alkyl radicals of 1-8, preferably 1-4
carbon atoms, e.g. methyl, ethyl, propyl, isopropyl and butyl, and
aryl radicals of 6-10 carbon atoms, e.g. phenyl and naphthyl. The
term "halogen" includes bromo, fluoro, chloro and iodo.
[0416] Preferably -L.sup.1- of formula (VI) is substituted with one
moiety -L.sup.2-Z or -L.sup.2-Z'.
[0417] A further preferred embodiment for -L.sup.1- is disclosed in
WO2002/089789A1, which is herewith incorporated by reference in its
entirety. Accordingly, a preferred moiety -L.sup.1- is of formula
(VII):
##STR00031## [0418] wherein [0419] the dashed line indicates
attachment to -D which is a PTH moiety and wherein attachment is
through an amine functional group of -D; [0420] L.sub.1 is a
bifunctional linking group, [0421] Y.sub.1 and Y.sub.2 are
independently 0, S or NR.sup.7; [0422] R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6 and R.sup.7 are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyls, C.sub.3-12 branched
alkyls, C.sub.3-8 cycloalkyls, C.sub.1-6 substituted alkyls,
C.sub.3-8 substituted cycloalkyls, aryls, substituted aryls,
aralkyls, C.sub.1-6 heteroalkyls, substituted C.sub.1-6
heteroalkyls, C.sub.1-6 alkoxy, phenoxy, and C.sub.1-6
heteroalkoxy; [0423] Ar is a moiety which when included in formula
(VII) forms a multisubstituted aromatic hydrocarbon or a
multi-substituted heterocyclic group; [0424] X is a chemical bond
or a moiety that is actively transported into a target cell, a
hydrophobic moiety, or a combination thereof, [0425] y is 0 or 1;
[0426] wherein -L.sup.1- is substituted with -L.sup.2-Z or
-L.sup.2-Z' and wherein -L.sup.1- is optionally further
substituted; [0427] wherein [0428] -L.sup.2- is a single chemical
bond or a spacer; [0429] --Z is a water-soluble carrier; and [0430]
--Z' is a water-insoluble carrier.
[0431] Only in the context of formula (VII) the terms used have the
following meaning:
[0432] The term "alkyl" shall be understood to include, e.g.
straight, branched, substituted C.sub.1-12 alkyls, including
alkoxy, C.sub.3-8 cycloalkyls or substituted cycloalkyls, etc.
[0433] The term "substituted" shall be understood to include adding
or replacing one or more atoms contained within a functional group
or compounds with one or more different atoms.
[0434] Substituted alkyls include carboxyalkyls, aminoalkyls,
dialkylaminos, hydroxyalkyls and mercaptoalkyls; substituted
cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls
include moieties such as napthyl; substituted aryls include
moieties such as 3-bromo-phenyl; aralkyls include moieties such as
toluyl; heteroalkyls include moieties such as ethylthiophene;
substituted heteroalkyls include moieties such as
3-methoxythiophone; alkoxy includes moeities such as methoxy; and
phenoxy includes moieties such as 3-nitrophenoxy. Halo- shall be
understood to include fluoro, chloro, iodo and bromo.
[0435] Preferably -L.sup.1- of formula (VII) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0436] In another preferred embodiment -L.sup.1- comprises a
substructure of formula (VIII)
##STR00032## [0437] wherein [0438] the dashed line marked with the
asterisk indicates attachment to a nitrogen of -D which is a PTH
moiety by forming an amide bond; [0439] the unmarked dashed lines
indicate attachment to the remainder of -L.sup.1-; and [0440]
wherein -L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and
wherein -L.sup.1- is optionally further substituted; [0441] wherein
[0442] -L.sup.2- is a single chemical bond or a spacer; [0443] --Z
is a water-soluble carrier; and [0444] --Z' is a water-insoluble
carrier.
[0445] Preferably -L.sup.1- of formula (VIII) is substituted with
one moiety -L.sup.2-Z or -L.sup.2-Z'.
[0446] In one embodiment -L.sup.1- of formula (VIII) is not further
substituted.
[0447] In another preferred embodiment -L.sup.1- comprises a
substructure of formula (IX)
##STR00033## [0448] wherein [0449] the dashed line marked with the
asterisk indicates attachment to a nitrogen of -D which is a PTH
moiety by forming a carbamate bond; [0450] the unmarked dashed
lines indicate attachment to the remainder of -L.sup.1-; and [0451]
wherein -L.sup.1- is substituted with -L.sup.2-Z or -L.sup.2-Z' and
wherein -L.sup.1- is optionally further substituted; [0452] wherein
[0453] -L2- is a single chemical bond or a spacer; [0454] --Z is a
water-soluble carrier; and [0455] --Z' is a water-insoluble
carrier.
[0456] Preferably -L.sup.1- of formula (IX) is substituted with one
moiety -L.sup.2-Z or -L.sup.2-Z'.
[0457] In one embodiment -L.sup.1- of formula (IX) is not further
substituted.
[0458] In the prodrugs of the present invention -L.sup.2- is a
chemical bond or a spacer moiety.
[0459] In one embodiment -L.sup.2- is a chemical bond.
[0460] In another embodiment -L.sup.2- is a spacer moiety.
[0461] When -L.sup.2- is other than a single chemical bond,
-L.sup.2- is preferably selected from the group consisting of -T-,
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.y1)--,
--S(O).sub.2N(R.sup.y1)--, --S(O)N(R.sup.y1)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.y1)S(O).sub.2N(R.sup.y1a)--, --S--,
--N(R.sup.y1)--,
--OC(OR.sup.y1)(R.sup.y1a)--N(R.sup.y1)C(O)N(R.sup.y1a)--,
--OC(O)N(R.sup.y1)--, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50
alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one
or more --R.sup.y2, which are the same or different and wherein
C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.y3)--, --S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--,
[0462] --S(O)--, --N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--, --S--,
--N(R.sup.y3)--, --OC(OR.sup.y3)(R.sup.y3a)--,
--N(R.sup.y3)C(O)N(R.sup.y3a)--, and --OC(O)N(R.sup.y3)--;
[0463] --R.sup.y1 and --R.sup.y1a are independently of each other
selected from the group consisting of --H, -T, C.sub.1-50 alkyl,
C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
substituted with one or more --R.sup.y2, which are the same or
different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y4)--, --S(O).sub.2N(R.sup.y4)--,
--S(O)N(R.sup.y4)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y4)S(O).sub.2N(R.sup.y4a)--, --S--, --N(R.sup.y4)--,
--OC(OR.sup.y4)(R.sup.y4a)--, --N(R.sup.y4)C(O)N(R.sup.y4a)--, and
--OC(O)N(R.sup.y4)--;
[0464] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl; wherein each T is independently
optionally substituted with one or more --R.sup.y2, which are the
same or different;
[0465] each --R.sup.y2 is independently selected from the group
consisting of halogen, --CN, oxo (.dbd.O), --COOR.sup.y5,
--OR.sup.y5, --C(O)R.sup.y5, --C(O)N(R.sup.y5R.sup.y5a),
--S(O).sub.2N(R.sup.y5R.sup.y5a), --S(O)N(R.sup.y5R.sup.y5a),
--S(O).sub.2R.sup.y5, --S(O)R.sup.y5,
--N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), --SR.sup.y5,
--N(R.sup.y5R.sup.y5a), --NO.sub.2, --OC(O)R.sup.y5,
--N(R.sup.y5)C(O)R.sup.y5a, --N(R.sup.y5)S(O).sub.2R.sup.y5a,
--N(R.sup.y5)S(O)R.sup.y5a, --N(R.sup.y5)C(O)OR.sup.y5a,
--N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b),
--OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6
alkyl is optionally substituted with one or more halogen, which are
the same or different; and
[0466] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently selected
from the group consisting of --H, and C.sub.1-6 alkyl, wherein
C.sub.1-6 alkyl is optionally substituted with one or more halogen,
which are the same or different.
[0467] When -L.sup.2- is other than a single chemical bond,
-L.sup.2- is even more preferably selected from -T-, --C(O)O--,
--O--, --C(O)--, --C(O)N(R.sup.y1)--, --S(O).sub.2N(R.sup.y1)--,
--S(O)N(R.sup.y1)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y1)S(O).sub.2N(R.sup.y1a)--, --S--, --N(R.sup.y1)--,
--OC(OR.sup.y1)(R.sup.y1a)--,
--N(R.sup.y1)C(O)N(R.sup.y1a)--OC(O)N(R.sup.y1)--, C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-,
C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are
optionally substituted with one or more --R.sup.y2, which are the
same or different and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl,
and C.sub.2-20 alkynyl are optionally interrupted by one or more
groups selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y3)--, --S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--,
[0468] --S--, --N(R.sup.y3)--, --OC(OR.sup.y3)(R.sup.y3a)--,
--N(R.sup.y3)C(O)N(R.sup.y3a)--, and --OC(O)N(R.sup.y3)--;
[0469] --R.sup.y1 and --R.sup.y1a are independently of each other
selected from the group consisting of --H, -T, C.sub.1-10 alkyl,
C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl; wherein -T, C.sub.1-10
alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally
substituted with one or more --R.sup.y2, which are the same or
different, and wherein C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and
C.sub.2-10 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of -T-, --C(O)O--, --O--,
--C(O)--, --C(O)N(R.sup.y4)--, --S(O).sub.2N(R.sup.y4)--,
--S(O)N(R.sup.y4)--, --S(O).sub.2--, --S(O)--,
--N(R.sup.y4)S(O).sub.2N(R.sup.y4a)--, --, --N(R.sup.y4)--,
--OC(OR.sup.y4)(R.sup.y4a)--, --N(R.sup.y4)C(O)N(R.sup.y4a)--, and
--OC(O)N(R.sup.y4)--;
[0470] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl; wherein each T is independently
optionally substituted with one or more --R.sup.y2, which are the
same or different;
[0471] --R.sup.y2 is selected from the group consisting of halogen,
--CN, oxo (.dbd.O), --COOR.sup.y5, --OR.sup.y5, --C(O)R.sup.y5,
--C(O)N(R.sup.y5R.sup.y5a), --S(O).sub.2N(R.sup.y5R.sup.y5a),
--S(O)N(R.sup.y5R.sup.y5a), --S(O).sub.2R.sup.y5, --S(O)R.sup.y5,
--N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), --SR.sup.y5,
--N(R.sup.y5R.sup.y5a), --NO.sub.2, --OC(O)R.sup.y5,
--N(R.sup.y5)C(O)R.sup.y5a, --N(R.sup.y5)S(O).sub.2R.sup.y5a,
--N(R.sup.y5)S(O)R.sup.y5a, --N(R.sup.y5)C(O)OR.sup.y5a,
--N(R.sup.y5)C(O)N(R.sup.y5a R.sup.y5b),
[0472] --OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein
C.sub.1-6 alkyl is optionally substituted with one or more halogen,
which are the same or different; and
[0473] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently of each
other selected from the group consisting of --H, and C.sub.1-6
alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0474] When -L.sup.2- is other than a single chemical bond,
-L.sup.2- is even more preferably selected from the group
consisting of -T-, --C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.y1)--,
--S(O).sub.2N(R.sup.y1)--, --S(O)N(R.sup.y1)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.y1)S(O).sub.2N(R.sup.y1a)-, --S--,
--N(R.sup.y1)--,
--OC(OR.sup.y1)(R.sup.y1a)--N(R.sup.y1)C(O)N(R.sup.y1a)-,
--OC(O)N(R.sup.y1)--, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50
alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one
or more --R.sup.y2, which are the same or different and wherein
C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of -T-, --C(O)O--, --O--, --C(O)--,
--C(O)N(R.sup.y3)--, --S(O).sub.2N(R.sup.y3)--,
--S(O)N(R.sup.y3)--, --S(O).sub.2--,
[0475] --S(O)--, --N(R.sup.y3)S(O).sub.2N(R.sup.y3a)--, --S--,
--N(R.sup.y3)--, --OC(OR.sup.y3)(R.sup.y3a)--,
--N(R.sup.y3)C(O)N(R.sup.y3a)--, and --OC(O)N(R.sup.y3)--;
[0476] --R.sup.y1 and --R.sup.y1a are independently selected from
the group consisting of --H, -T, C.sub.1-10 alkyl, C.sub.2-10
alkenyl, and C.sub.2-10 alkynyl;
[0477] each T is independently selected from the group consisting
of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10
cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered
heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to
30-membered heteropolycyclyl;
[0478] each --R.sup.y2 is independently selected from the group
consisting of halogen, and C.sub.1-6 alkyl; and
[0479] each --R.sup.y3, --R.sup.y3a, --R.sup.y4, --R.sup.y4a,
--R.sup.y5, --R.sup.y5a and --R.sup.y5b is independently of each
other selected from the group consisting of --H, and C.sub.1-6
alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0480] Even more preferably, -L.sup.2- is a C.sub.1-20 alkyl chain,
which is optionally interrupted by one or more groups independently
selected from --O--, -T- and --C(O)N(R.sup.y1)--; and which
C.sub.1-20 alkyl chain is optionally substituted with one or more
groups independently selected from --OH, -T and
--C(O)N(R.sup.y6R.sup.y6a); wherein --R.sup.y1, --R.sup.y6,
--R.sup.y6a are independently selected from the group consisting of
H and C.sub.1-4 alkyl and wherein T is selected from the group
consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,
C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to
11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and
8- to 30-membered heteropolycyclyl.
[0481] Preferably, -L.sup.Z- has a molecular weight in the range of
from 14 g/mol to 750 g/mol.
[0482] Preferably, -L.sup.Z- comprises a moiety selected from
##STR00034## ##STR00035##
[0483] wherein
[0484] dashed lines indicate attachment to the rest of -L.sup.2-,
-L.sup.1-, --Z and/or --Z', respectively; and
[0485] --R and --Ra are independently of each other selected from
the group consisting of --H, methyl, ethyl, propyl, butyl, pentyl
and hexyl.
[0486] In one preferred embodiment -L.sup.2- has a chain lengths of
1 to 20 atoms.
[0487] As used herein the term "chain length" with regard to the
moiety -L.sup.2- refers to the number of atoms of -L.sup.2- present
in the shortest connection between -L.sup.1- and --Z.
[0488] Preferably, -L.sup.2- is of formula (i)
##STR00036## [0489] wherein [0490] the dashed line marked with the
asterisk indicates attachment to -L.sup.1-; [0491] the unmarked
dashed line indicates attachment to --Z or --Z'; [0492] n is
selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17 and 18; and [0493] wherein the
moiety of formula (i) is optionally further substituted.
[0494] Preferably, n of formula (i) is selected from the group
consisting of 3, 4, 5, 6, 7, 8, and 9. Even more preferably n of
formula (i) is 4, 5, 6, or 7. In one embodiment n of formula (i) is
4. In another embodiment n of formula (i) is 5. In another
embodiment n of formula (i) is 6.
[0495] In one preferred embodiment the moiety -L.sup.1-L.sup.2- is
selected from the group consisting of
##STR00037## [0496] wherein [0497] the unmarked dashed line
indicates the attachment to a nitrogen of -D which is a PTH moiety
by forming an amide bond; and [0498] the dashed line marked with
the asterisk indicates attachment to --Z or --Z'.
[0499] In one preferred embodiment the moiety -L.sup.1-L.sup.2- is
selected from the group consisting of
##STR00038## [0500] wherein [0501] the unmarked dashed line
indicates the attachment to a nitrogen of -D which is a PTH moiety
by forming an amide bond; and [0502] the dashed line marked with
the asterisk indicates attachment to --Z or --Z'.
[0503] In a preferred embodiment the moiety -L.sup.1-L.sup.2- is of
formula (IIca-ii).
[0504] In another preferred embodiment the moiety -L.sup.1-L.sup.2-
is of formula (IIcb-iii).
[0505] Preferably, the PTH prodrug of the present invention is of
formula (Ia) with x=1.
[0506] The carrier --Z comprises a C.sub.5-24 alkyl or a polymer.
Preferably, --Z comprises a polymer, preferably a polymer selected
from the group consisting of 2-methacryloyl-oxyethyl phosphoyl
cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides),
poly(alkyloxy) polymers, poly(amides), poly(amidoamines),
poly(amino acids), poly(anhydrides), poly(aspartamides),
poly(butyric acids), poly(glycolic acids), polybutylene
terephthalates, poly(caprolactones), poly(carbonates),
poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),
poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),
poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic
acids), poly(hydroxyethyl acrylates),
poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates),
poly(hydroxypropylmethacrylamides), poly(hydroxypropyl
methacrylates), poly(hydroxypropyloxazolines),
poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic
acids), poly(methacrylamides), poly(methacrylates),
poly(methyloxazolines), poly(organophosphazenes), poly(ortho
esters), poly(oxazolines), poly(propylene glycols),
poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl
amines), poly(vinylmethylethers), poly(vinylpyrrolidones),
silicones, celluloses, carbomethyl celluloses, hydroxypropyl
methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins,
hyaluronic acids and derivatives, functionalized hyaluronic acids,
mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl
starches, hydroxyethyl starches and other carbohydrate-based
polymers, xylans, and copolymers thereof.
[0507] Preferably, --Z has a molecular weight ranging from 5 to 200
kDa. Even more preferably, --Z has a molecular weight ranging from
8 to 100 kDa, even more preferably ranging from 10 to 80 kDa, even
more preferably from 12 to 60, even more preferably from 15 to 40
and most preferably --Z has a molecular weight of about 20 kDa. In
another equally preferred embodiment --Z has a molecular weight of
about 40 kDa.
[0508] In one embodiment such water-soluble carrier --Z comprises a
protein. Preferred proteins are selected from the group consisting
of carboxyl-terminal polypeptide of the chorionic gonadotropin as
described in US 2012/0035101 A1 which are herewith incorporated by
reference; albumin; XTEN sequences as described in WO 2011123813 A2
which are herewith incorporated by reference; proline/alanine
random coil sequences as described in WO 2011/144756 A1 which are
herewith incorporated by reference; proline/alanine/serine random
coil sequences as described in WO 2008/155134 A1 and WO 2013/024049
A1 which are herewith incorporated by reference; and Fc fusion
proteins.
[0509] In one embodiment --Z is a polysarcosine.
[0510] In another preferred embodiment --Z comprises a
poly(N-methylglycine).
[0511] In a particularly preferred embodiment --Z comprises a
random coil protein moiety.
[0512] In one preferred embodiment --Z comprises one random coil
protein moiety.
[0513] In another preferred embodiment --Z comprises two random
coil proteins moieties.
[0514] In another preferred embodiment --Z comprises three random
coil proteins moieties.
[0515] In another preferred embodiment --Z comprises four random
coil proteins moieties.
[0516] In another preferred embodiment --Z comprises five random
coil proteins moieties.
[0517] In another preferred embodiment --Z comprises six random
coil proteins moieties.
[0518] In another preferred embodiment --Z comprises seven random
coil proteins moieties.
[0519] In another preferred embodiment --Z comprises eight random
coil proteins moieties.
[0520] Preferably such random coil protein moiety comprises at
least 25 amino acid residues and at most 2000 amino acids. Even
more preferably such random coil protein moiety comprises at least
30 amino acid residues and at most 1500 amino acid residues. Even
more preferably such random coil protein moiety comprises at least
50 amino acid residues and at most 500 amino acid residues.
[0521] In a preferred embodiment, --Z comprises a random coil
protein moiety of which at least 80%, preferably at least 85%, even
more preferably at least 90%, even more preferably at least 95%,
even more preferably at least 98% and most preferably at least 99%
of the total number of amino acids forming said random coil protein
moiety are selected from alanine and proline. Even more preferably,
at least 10%, but less than 75%, preferably less than 65%, of the
total number of amino acid residues of such random coil protein
moiety are proline residues. Preferably, such random coil protein
moiety is as described in WO 2011/144756 A1 which is hereby
incorporated by reference in its entirety. Even more preferably --Z
comprises at least one moiety selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:51 and SEQ ID NO:61 as
disclosed in WO2011/144756 which are hereby incorporated by
reference. A moiety comprising such random coil protein comprising
alanine and proline will be referred to as "PA" or "PA moiety".
[0522] Accordingly, --Z comprises a PA moiety.
[0523] In an equally preferred embodiment, --Z comprises a random
coil protein moiety of which at least 80%, preferably at least 85%,
even more preferably at least 90%, even more preferably at least
95%, even more preferably at least 98% and most preferably at least
99% of the total number of amino acids forming said random coil
protein moiety are selected from alanine, serine and proline. Even
more preferably, at least 4%, but less than 40% of the total number
of amino acid residues of such random coil protein moiety are
proline residues. Preferably, such random coil protein moiety is as
described in WO 2008/155134 A1 which is hereby incorporated by
reference in its entirety. Even more preferably --Z comprises at
least one moiety selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42,
SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54 and SEQ ID NO:56 as disclosed in WO 2008/155134 A1, which are
hereby incorporated by reference. A moiety comprising such random
coil protein moiety comprising alanine, serine and proline will be
referred to as "PAS" or "PAS moiety".
[0524] Accordingly, --Z comprises a PAS moiety.
[0525] In an equally preferred embodiment, --Z comprises a random
coil protein moiety of which at least 80%, preferably at least 85%,
even more preferably at least 90%, even more preferably at least
95%, even more preferably at least 98% and most preferably at least
99% of the total number of amino acids forming said random coil
protein moiety are selected from alanine, glycine and proline. A
moiety comprising such random coil protein moiety comprising
alanine, glycine and proline will be referred to as "PAG" or "PAG
moiety".
[0526] Accordingly, --Z comprises a PAG moiety.
[0527] In an equally preferred embodiment, --Z comprises a random
coil protein moiety of which at least 80%, preferably at least 85%,
even more preferably at least 90%, even more preferably at least
95%, even more preferably at least 98% and most preferably at least
99% of the total number of amino acids forming said random coil
protein moiety are selected from proline and glycine. A moiety
comprising such random coil protein moiety comprising proline and
glycine will be referred to as "PG" or "PG moiety".
[0528] Preferably, such PG moiety comprises a moiety of formula
(a-0)
[(Gly).sub.p-Pro-(Gly).sub.q]r (a-0); [0529] wherein [0530] p is
selected from the group consisting of 0, 1, 2, 3, 4 and 5; [0531] q
is selected from the group consisting of 0, 1, 2, 3, 4 and 5;
[0532] r is an integer ranging from and including 10 to 1000;
[0533] provided that at least one of p and q is at least 1;
[0534] Preferably, p of formula (a-0) is selected from the group
consisting of 1, 2 and 3.
[0535] Preferably, q of formula (a-0) is selected from 0, 1 and
2.
[0536] Even more preferably the PG moiety comprises the sequence of
SEQ ID:NO 122: GGPGGPGPGGPGGPGPGGPG
[0537] Even more preferably, the PG moiety comprises the sequence
of SEQ ID:NO 122 of formula (a-0-a)
(GGPGGPGPGGPGGPGPGGPG), (a-0-a), [0538] wherein [0539] v is an
integer ranging from and including 1 to 50.
[0540] Accordingly, --Z comprises a PG moiety.
[0541] In an equally preferred embodiment, --Z comprises a random
coil protein moiety of which at least 80%, preferably at least 85%,
even more preferably at least 90%, even more preferably at least
95%, even more preferably at least 98% and most preferably at least
99% of the total number of amino acids forming said random coil
protein moiety are selected from alanine, glycine, serine,
threonine, glutamate and proline. Preferably, such random coil
protein moiety is as described in WO 2010/091122 A1 which is hereby
incorporated by reference. Even more preferably --Z comprises at
least one moiety selected from the group consisting of SEQ ID
NO:182, SEQ ID NO: 183, SEQ ID NO:184; SEQ ID NO:185, SEQ ID NO:
186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190,
SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID
NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199,
SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID
NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,
SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID
NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217,
SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID
NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763,
SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID
NO:768, SEQ ID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO:772,
SEQ ID NO:773, SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQ ID
NO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID
NO:1716, SEQ ID NO:1718, SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID
NO:1721 and SEQ ID NO:1722 as disclosed in WO2010/091122A1, which
are hereby incorporated by reference. A moiety comprising such
random coil protein moiety comprising alanine, glycine, serine,
threonine, glutamate and proline will be referred to as "XTEN" or
"XTEN moiety" in line with its designation in WO 2010/091122
A1.
[0542] Accordingly, --Z comprises an XTEN moiety.
[0543] In another preferred embodiment, --Z comprises a fatty acid
derivate. Preferred fatty acid derivatives are those disclosed in
WO 2005/027978 A2 and WO 2014/060512 A1 which are herewith
incorporated by reference.
[0544] In another preferred embodiment --Z is a hyaluronic
acid-based polymer.
[0545] In one embodiment --Z is a carrier as disclosed in WO
2012/02047 A1 which is herewith incorporated by reference.
[0546] In another embodiment --Z is a carrier as disclosed in WO
2013/024048 A1 which is herewith incorporated by reference.
[0547] In another preferred embodiment --Z is a PEG-based polymer,
such as a linear, branched or multi-arm PEG-based polymer.
[0548] In one embodiment --Z is a linear PEG-based polymer.
[0549] In another embodiment --Z is a multi-arm PEG-based polymer.
Preferably, --Z is a multi-arm PEG-based polymer having at least 4
PEG-based arms.
[0550] Preferably, such multi-arm PEG-based polymer --Z is
connected to a multitude of moieties -L.sup.2-L.sup.1-D, wherein
each moiety -L.sup.2-L.sup.1-D is preferably connected to the end
of an arm, preferably to the end of an arm. Preferably such
multi-arm PEG-based polymer --Z is connected to 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15 or 16 moieties -L.sup.2-L.sup.1-D.
Even more preferably such multi-arm PEG-based polymer --Z is
connected to 2, 3, 4, 6 or 8 moieties -L.sup.2-L.sup.1-D. Even more
preferably such multi-arm PEG-based polymer --Z is connected to 2,
4 or 6 moieties -L.sup.2-L.sup.1-D, even more preferably such
multi-arm PEG-based polymer --Z is connected to 4 or 6 moieties
-L.sup.2-L.sup.1-D, and most preferably such multi-arm PEG-based
polymer --Z is connected to 4 moieties -L.sup.2-L.sup.1-D.
[0551] Preferably, such multi-arm PEG-based polymer --Z is a
multi-arm PEG derivative as, for instance, detailed in the products
list of JenKem Technology, USA (accessed by download from
http://www.jenkemusa.com/Pages/PEGProducts.aspx on Dec. 18, 2014),
such as a 4-arm-PEG derivative, in particular a 4-arm-PEG
comprising a pentaerythritol core, an 8-arm-PEG derivative
comprising a hexaglycerin core, and an 8-arm-PEG derivative
comprising a tripentaerythritol core. More preferably, the
water-soluble PEG-based carrier --Z comprises a moiety selected
from:
[0552] a 4-arm PEG Amine comprising a pentaerythritol core:
##STR00039##
[0553] with n ranging from 20 to 500;
[0554] an 8-arm PEG Amine comprising a hexaglycerin core:
##STR00040##
[0555] with n ranging from 20 to 500; and
[0556] R=hexaglycerin or tripentaerythritol core structure; and
[0557] a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol
core:
##STR00041##
[0558] with n ranging from 20 to 500; and
[0559] R=comprising a sorbitol or dipentaerythritol core;
[0560] and wherein dashed lines indicate attachment to the rest of
the PTH prodrug.
[0561] In a preferred embodiment --Z is a branched PEG-based
polymer. In one embodiment --Z is a branched PEG-based polymer
having one, two, three, four, five or six branching points.
Preferably, --Z is a branched PEG-based polymer having one, two or
three branching points. In one embodiment --Z is a branched
PEG-based polymer having one branching point. In another embodiment
--Z is a branched PEG-based polymer having two branching points. In
another embodiment --Z is a branched PEG-based polymer having three
branching points. A branching point is preferably selected from the
group consisting of --N<, --CH< and >C<.
[0562] Preferably, such branched PEG-based moiety --Z has a
molecular weight of at least 10 kDa.
[0563] In one embodiment such branched moiety --Z has a molecular
weight ranging from and including 10 kDa to 500 kDa, more
preferably ranging from and including 10 kDa to 250 Da, even more
preferably ranging from and including 10 kDa to 150 kDa, even more
preferably ranging from and including 12 kDa to 100 kDa and most
preferably ranging from and including 15 kDa to 80 kDa.
[0564] Preferably, such branched moiety --Z has a molecular weight
ranging from and including 10 kDa to 80 kDa. In one embodiment the
molecular weight is about 10 kDa. In another embodiment the
molecular weight of such branched moiety --Z is about 20 kDa. In
another embodiment the molecular weight of such branched moiety --Z
is about 30 kDa. In another embodiment the molecular weight of such
a branched moiety --Z is about 40 kDa. In another embodiment the
molecular weight of such a branched moiety --Z is about 50 kDa. In
another embodiment the molecular weight of such a branched moiety
--Z is about 60 kDa. In another embodiment the molecular weight of
such a branched moiety --Z is about 70 kDa. In another embodiment
the molecular weight of such a branched moiety --Z is about 80 kDa.
Most preferably, such branched moiety --Z has a molecular weight of
about 40 kDa.
[0565] Preferably, --Z or --Z' comprises a moiety
##STR00042##
[0566] In an equally preferred embodiment --Z comprises an amide
bond.
[0567] Preferably --Z comprises a moiety of formula (a)
##STR00043## [0568] wherein [0569] the dashed line indicates
attachment to -L.sup.2- or to the remainder of --Z; [0570] BP.sup.a
is a branching point selected from the group consisting of --N<,
--CR< and >C<; [0571] --R is selected from the group
consisting of --H and C.sub.1-6 alkyl; [0572] a is 0 if BP.sup.a is
--N< or --CR< and n is 1 if BP.sup.a is >C<; [0573]
--S.sup.a--, --S.sup.a'--, --S.sup.a''-- and --S.sup.a'''-- are
independently of each other a chemical bond or are selected from
the group consisting of C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and
C.sub.2-50 alkynyl; wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl,
and C.sub.2-50 alkynyl are optionally substituted with one or more
--R.sup.1, which are the same or different and wherein C.sub.1-50
alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally
interrupted by one or more groups selected from the group
consisting of -T-, --C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.2)--,
--S(O).sub.2N(R.sup.2)--, --S(O)N(R.sup.2)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.2)S(O).sub.2N(R.sup.2a)--, --S--,
--N(R.sup.2)--, --OC(OR.sup.2)(R.sup.2a)--,
--N(R.sup.2)C(O)N(R.sup.2a)--, and --OC(O)N(R.sup.2)--; [0574] each
-T- is independently selected from the group consisting of phenyl,
naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3-
to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-
to 30-membered carbopolycyclyl, and 8- to 30-membered
heteropolycyclyl; wherein each -T- is independently optionally
substituted with one or more --R.sup.1, which are the same or
different; [0575] each --R.sup.1 is independently selected from the
group consisting of halogen, --CN, oxo (.dbd.O), --COOR.sup.3,
--OR.sup.3, --C(O)R.sup.3, --C(O)N(R.sup.3R.sup.3a),
--S(O).sub.2N(R.sup.3R.sup.3a), --S(O)N(R.sup.3R.sup.3a),
--S(O).sub.2R.sup.3, --S(O)R.sup.3,
--N(R.sup.3)S(O).sub.2N(R.sup.3aR.sup.3b), --SR.sup.3,
--N(R.sup.3R.sup.3a), --NO.sub.2, --OC(O)R.sup.3,
--N(R.sup.3)C(O)R.sup.3a, --N(R.sup.3)S(O).sub.2R.sup.3a,
--N(R.sup.3)S(O)R.sup.3a, --N(R.sup.3)C(O)OR.sup.3a,
--N(R.sup.3)C(O)N(R.sup.3aR.sup.3b), --OC(O)N(R.sup.3R.sup.3a), and
C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different; [0576]
each --R.sup.2, --R.sup.2a, --R.sup.3, --R.sup.3a and --R.sup.3b is
independently selected from the group consisting of --H, and
C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different; and
[0577] --P.sup.a', --P.sup.a'' and --P.sup.a''' are independently a
polymeric moiety.
[0578] In one embodiment BP.sup.a of formula (a) is --N<.
[0579] In another embodiment BP.sup.a of formula (a) is
>C<.
[0580] In a preferred embodiment BP.sup.a of formula (a) is
--CR<. Preferably, --R is --H. Accordingly, a of formula (a) is
0.
[0581] In one embodiment --S.sup.a-- of formula (a) is a chemical
bond.
[0582] In another embodiment --S.sup.a-- of formula (a) is selected
from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl
and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10 alkenyl
and C.sub.2-10 alkynyl are optionally interrupted by one or more
chemical groups selected from the group consisting of -T-,
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.4)--,
--S(O).sub.2N(R.sup.4)--, --S(O)N(R.sup.4)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.4)S(O).sub.2N(R.sup.4a)--, --S--,
--N(R.sup.4)--, --OC(OR.sup.4)(R.sup.4a)--,
--N(R.sup.4)C(O)N(R.sup.4a)--, and --OC(O)N(R.sup.4)--; wherein -T-
is a 3- to 10-membered heterocyclyl; and --R.sup.4 and --R.sup.4a
are independently selected from the group consisting of --H,
methyl, ethyl, propyl and butyl.
[0583] Preferably --S.sup.a-- of formula (a) is selected from the
group consisting of C.sub.1-10 alkyl which is interrupted by one or
more chemical groups selected from the group consisting of -T-,
--C(O)N(R.sup.4)-- and --O--.
[0584] In one embodiment --S.sup.a'-- of formula (a) is a chemical
bond.
[0585] In another embodiment --S.sup.a'-- of formula (a) is
selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or
more chemical groups selected from the group consisting of
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.4)--,
--S(O).sub.2N(R.sup.4)--, --S(O)N(R.sup.4)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.4)S(O).sub.2N(R.sup.4a)--, --S--,
--N(R.sup.4)--, --OC(OR.sup.4)(R.sup.4a)--,
--N(R.sup.4)C(O)N(R.sup.4a)--, and --OC(O)N(R.sup.4)--; wherein
--R.sup.4 and --R.sup.4a are independently selected from the group
consisting of --H, methyl, ethyl, propyl and butyl. Preferably
--S.sup.a'-- of formula (a) is selected from the group consisting
of methyl, ethyl, propyl, butyl, which are optionally interrupted
by one or more chemical groups selected from the group consisting
of --O--, --C(O)-- and --C(O)N(R.sup.4)--.
[0586] In one embodiment --S.sup.a''-- of formula (a) is a chemical
bond.
[0587] In another embodiment --S.sup.a''-- of formula (a) is
selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or
more chemical groups selected from the group consisting of
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.4)--,
--S(O).sub.2N(R.sup.4)--, --S(O)N(R.sup.4)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.4)S(O).sub.2N(R.sup.4a)--, --S--,
--N(R.sup.4)--, --OC(OR.sup.4)(R.sup.4a)--,
--N(R.sup.4)C(O)N(R.sup.4a)--, and --OC(O)N(R.sup.4)--; wherein
--R.sup.4 and --R.sup.4a are independently selected from the group
consisting of --H, methyl, ethyl, propyl and butyl. Preferably
--S.sup.a''-- of formula (a) is selected from the group consisting
of methyl, ethyl, propyl, butyl, which are optionally interrupted
by one or more chemical groups selected from the group consisting
of --O--, --C(O)-- and --C(O)N(R.sup.4)--.
[0588] In one embodiment --S.sup.a'''-- of formula (a) is a
chemical bond.
[0589] In another embodiment --S.sup.a'''-- of formula (a) is
selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10
alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or
more chemical groups selected from the group consisting of
--C(O)O--, --O--, --C(O)--, --C(O)N(R.sup.4)--,
--S(O).sub.2N(R.sup.4)--, --S(O)N(R.sup.4)--, --S(O).sub.2--,
--S(O)--, --N(R.sup.4)S(O).sub.2N(R.sup.4a)--, --S--,
--N(R.sup.4)--, --OC(OR.sup.4)(R.sup.4a)--,
--N(R.sup.4)C(O)N(R.sup.4a)--, and --OC(O)N(R.sup.4)--; wherein
--R.sup.4 and --R.sup.4a are independently selected from the group
consisting of --H, methyl, ethyl, propyl and butyl. Preferably
--S.sup.a'''-- of formula (a) is selected from the group consisting
of methyl, ethyl, propyl, butyl, which are optionally interrupted
by one or more chemical groups selected from the group consisting
of --O--, --C(O)-- and --C(O)N(R.sup.4)--.
[0590] Preferably, --P.sup.a', --P.sup.a'' and --P.sup.a''' of
formula (a) independently comprise a polymer selected from the
group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins,
poly(acrylic acids), poly(acrylates), poly(acrylamides),
poly(alkyloxy) polymers, poly(amides), poly(amidoamines),
poly(amino acids), poly(anhydrides), poly(aspartamides),
poly(butyric acids), poly(glycolic acids), polybutylene
terephthalates, poly(caprolactones), poly(carbonates),
poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),
poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),
poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic
acids), poly(hydroxyethyl acrylates),
poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates),
poly(hydroxypropylmethacrylamides), poly(hydroxypropyl
methacrylates), poly(hydroxypropyloxazolines),
poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic
acids), poly(methacrylamides), poly(methacrylates),
poly(methyloxazolines), poly(organophosphazenes), poly(ortho
esters), poly(oxazolines), poly(propylene glycols),
poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl
amines), poly(vinylmethylethers), poly(vinylpyrrolidones),
silicones, celluloses, carbomethyl celluloses, hydroxypropyl
methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins,
hyaluronic acids and derivatives, functionalized hyaluronic acids,
mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl
starches, hydroxyethyl starches and other carbohydrate-based
polymers, xylans, and copolymers thereof.
[0591] More preferably, --P.sup.a', --P.sup.a'' and --P.sup.a''' of
formula (a) independently comprise a PEG-based moiety. Even more
preferably, --P.sup.a', --P.sup.a'' and --P.sup.a''' of formula (a)
independently comprise a PEG-based moiety comprising at least 20%
PEG, even more preferably at least 30%, even more preferably at
least 40% PEG, even more preferably at least 50% PEG, even more
preferably at least 60% PEG, even more preferably at least 70% PEG,
even more preferably at least 80% PEG and most preferably at least
90% PEG.
[0592] Preferably, --P.sup.a', --P.sup.a'' and --P.sup.a''' of
formula (a) independently have a molecular weight ranging from and
including 5 kDa to 50 kDa, more preferably have a molecular weight
ranging from and including 5 kDa to 40 kDa, even more preferably
ranging from and including 7.5 kDa to 35 kDa, even more preferably
ranging from and 7.5 to 30 kDa, even more preferably ranging from
and including 10 to 30 kDa.
[0593] In one embodiment --P.sup.a', --P.sup.a'' and --P.sup.a'''
of formula (a) have a molecular weight of about 5 kDa.
[0594] In another embodiment --P.sup.a', --P.sup.a'' and
--P.sup.a''' of formula (a) have a molecular weight of about 7.5
kDa.
[0595] In another embodiment --P.sup.a', --P.sup.a'' and
--P.sup.a''' of formula (a) have a molecular weight of about
kDa.
[0596] In another embodiment --P.sup.a', --P.sup.a'' and
--P.sup.a''' of formula (a) have a molecular weight of about 12.5
kDa.
[0597] In another embodiment --P.sup.a', --P.sup.a'' and
--P.sup.a''' of formula (a) have a molecular weight of about
kDa.
[0598] In another embodiment --P.sup.a', --P.sup.a'' and
--P.sup.a''' of formula (a) have a molecular weight of about
kDa.
[0599] In one embodiment --Z comprises one moiety of formula
(a).
[0600] In another embodiment --Z comprises two moieties of formula
(a).
[0601] In another embodiment --Z comprises three moieties of
formula (a).
[0602] Preferably, --Z is a moiety of formula (a).
[0603] More preferably, --Z comprises a moiety of formula (b)
##STR00044## [0604] wherein [0605] the dashed line indicates
attachment to -L.sup.2- or to the remainder of --Z; and [0606] m
and p are independently of each other an integer ranging from and
including 150 to 1000; preferably an integer ranging from and
including 150 to 500; more preferably an integer ranging from and
including 200 to 500; and most preferably an integer ranging from
and including 400 to 500.
[0607] Preferably, m and p of formula (b) are the same integer.
[0608] Most preferably m and p of formula (b) are about 450.
[0609] Preferably, --Z is a moiety of formula (b).
[0610] The carrier --Z' is a water-insoluble polymer, even more
preferably a hydrogel. Preferably, such hydrogel comprises a
polymer selected from the group consisting of
2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),
poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,
poly(amides), poly(amidoamines), poly(amino acids),
poly(anhydrides), poly(aspartamides), poly(butyric acids),
poly(glycolic acids), polybutylene terephthalates,
poly(caprolactones), poly(carbonates), poly(cyanoacrylates),
poly(dimethylacrylamides), poly(esters), poly(ethylenes),
poly(ethyleneglycols), poly(ethylene oxides), poly(ethyl
phosphates), poly(ethyloxazolines), poly(glycolic acids),
poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),
poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),
poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),
poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic
acids), poly(methacrylamides), poly(methacrylates),
poly(methyloxazolines), poly(organophosphazenes), poly(ortho
esters), poly(oxazolines), poly(propylene glycols),
poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl
amines), poly(vinylmethylethers), poly(vinylpyrrolidones),
silicones, celluloses, carbomethyl celluloses, hydroxypropyl
methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins,
hyaluronic acids and derivatives, functionalized hyaluronic acids,
mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl
starches, hydroxyethyl starches and other carbohydrate-based
polymers, xylans, and copolymers thereof.
[0611] If the carrier --Z' is a hydrogel, it is preferably a
hydrogel comprising PEG or hyaluronic acid. Most preferably such
hydrogel comprises PEG.
[0612] Even more preferably, the carrier --Z' is a hydrogel as
described in WO 2006/003014 A2, WO 2011/012715 A1 or WO 2014/056926
A1, which are herewith incorporated by reference in their
entirety.
[0613] In another embodiment --Z' is a polymer network formed
through the physical aggregation of polymer chains, which physical
aggregation is preferably caused by hydrogen bonds,
crystallization, helix formation or complexation. In one embodiment
such polymer network is a thermogelling polymer.
[0614] Preferably, the total mass of the PTH prodrug of the present
invention is at least 10 kDa, such as at least 12 kDa, such as at
least 15 kDa, such as at least 20 kDa or such as at least kDa. It
is preferred that the total mass of the PTH prodrug of the present
invention is at most 250 kDa, such as at most 200 kDa, 180 kDa, 150
kDa or 100 kDa.
[0615] In a preferred embodiment the PTH prodrug of the present
invention is of formula (IIe-i):
##STR00045## [0616] wherein [0617] the unmarked dashed line
indicates the attachment to a nitrogen of -D which is a PTH moiety
by forming an amide bond; and [0618] the dashed line marked with
the asterisk indicates attachment to a moiety
[0618] ##STR00046## [0619] wherein [0620] m and p are independently
an integer ranging from and including 400 to 500.
[0621] Preferably, -D is attached to the PTH prodrug of formula
(IIe-i) through the N-terminal amine functional group of the PTH
moiety.
[0622] In another preferred embodiment the PTH prodrug of the
present invention is of formula (IIf-i).
##STR00047## [0623] wherein [0624] the unmarked dashed line
indicates the attachment to a nitrogen of -D which is a PTH moiety
by forming an amide bond; and [0625] the dashed line marked with
the asterisk indicates attachment to a moiety
[0625] ##STR00048## [0626] wherein [0627] m and p are independently
an integer ranging from and including 400 to 500.
[0628] Preferably, -D is attached to the PTH prodrug of formula
(IIf-i) through the N-terminal amine functional group of the PTH
moiety.
[0629] In a preferred embodiment the residual activity of the PTH
prodrug of the present invention is less than 10%, more preferably
less than 1%, even more preferably less than 0.1%, even more
preferably less than 0.01%, even more preferably less than 0.001%
and most preferably less than 0.0001%.
[0630] As used herein the term "residual activity" refers to the
activity exhibited by the PTH prodrug of the present invention with
the PTH moiety bound to a carrier in relation to the activity
exhibited by the corresponding free PTH. In this context the term
"activity" refers to binding to an activation of the PTH/PTHrP1
receptor resulting in activation of adenylate cyclase to generate
cAMP, phospholipase C to generate intracellular calcium, or
osteoblastic expression of RANKL (which binds to RANK (Receptor
Activator of Nuclear Factor kB) on osteoclasts. It is understood
that measuring the residual activity of the PTH prodrug of the
present invention takes time during which a certain amount of PTH
will be released from the PTH prodrug of the present invention and
that such released PTH will distort the results measured for the
PTH prodrug. It is thus accepted practice to test the residual
activity of a prodrug with a conjugate in which the drug moiety, in
this case PTH, is non-reversibly, i.e. stably, bound to a carrier,
which as closely as possible resembles the structure of the PTH
prodrug for which residual activity is to be measured.
[0631] A suitable assay for measuring PTH activity and the residual
activity of the PTH prodrug of the present invention, preferably in
the form of a stable analog, is for example measuring cAMP
production from HEK293 cells over-expressing the PTH/PTHrP1
receptor (Hohenstein et al., Journal of Pharmaceutical and
Biomedical Analysis, September 2014, 98: 345-350), or a cell-based
assay to detect cyclicAMP release, detected by homogenous
time-resolved fluorescence (HTRF) or ELISA, that has been validated
according to ICHQ2(R1)
(http://www.criver.com/files/pdfs/bps/bp_r_in_vitro_bioassays.aspx).
[0632] It was surprisingly found that using N-terminal attachment
of -L.sup.1- and using a branched PEG carrier for --Z, i.e. a
2.times.20 kDa PEG, results in a particularly low residual
activity. Reduced residual activity is desirable as it reduces
side-effects.
[0633] It was also surprisingly found that PTH produgs of the
present invention are capable of achieving a stable plasma profile
of PTH which ensures physiological serum and urinary calcium levels
or even reduced urinary calcium levels.
[0634] Preferably, after subcutaneous administration the
pharmacokinetic profile of a PTH prodrug of the present invention
exhibits a peak to trough ratio of less than 4 within one injection
interval.
[0635] As used herein the term "injection interval" refers to the
time between two consecutive administrations of the pharmaceutical
composition of the present invention.
[0636] As used herein the term "peak to trough ratio" refers to the
ratio between the highest plasma concentration and the lowest
plasma concentration of PTH released from the PTH prodrug of the
present invention within the time period between two consecutive
administrations to a non-human primate, preferably to a cynomolgus
monkey.
[0637] The time period between two consecutive subcutaneous
administrations, i.e. the administration interval, is preferably at
least 24 hours, such as 24 hours, 36 hours, 48 hours, 60 hours, 72
hours, every 84 hours, 96 hours, 108 hours, 120 hours, 132 hours,
144 hours, 156 hours, one week, two weeks, three weeks or four
weeks.
[0638] In one embodiment the time period between two consecutive
subcutaneous administrations is 24 hours.
[0639] In another embodiment the time period between two
consecutive subcutaneous administrations is 48 hours.
[0640] In another embodiment the time period between two
consecutive subcutaneous administrations is 72 hours.
[0641] In another embodiment the time period between two
consecutive subcutaneous administrations is 96 hours.
[0642] In another embodiment the time period between two
consecutive subcutaneous administrations is 120 hours.
[0643] In another embodiment the time period between two
consecutive subcutaneous administrations is 144 hours.
[0644] In another embodiment the time period between two
consecutive subcutaneous administrations is one week.
[0645] The peak to trough ratio measured in each administration
interval is less than 4, preferably less than 3.8, more preferably
less than 3.6, even more preferably less than 3.4, even more
preferably less than 3.2, even more preferably less than 3, even
more preferably less than 2.8, even more preferably less than 2.6,
even more preferably less than 2.4, even more preferably less than
2.2 and most preferably less than 2.
[0646] Another aspect of the present invention is a pharmaceutical
composition comprising at least one PTH prodrug of the present
invention and at least one excipient.
[0647] Preferably, the pharmaceutical composition comprising at
least one PTH prodrug of the present invention has a pH ranging
from and including pH 3 to pH 8. More preferably, the
pharmaceutical composition has a pH ranging from and including pH 4
to pH 6. Most preferably, the pharmaceutical composition has a pH
ranging from and including pH 4 to pH 5.
[0648] In one embodiment the pharmaceutical composition comprising
at least one PTH prodrug of the present invention and at least one
excipient is a liquid or suspension formulation. It is understood
that the pharmaceutical composition is a suspension formulation if
the PTH prodrug of the present invention comprises a
water-insoluble carrier --Z'.
[0649] In another embodiment the pharmaceutical composition
comprising at least one PTH prodrug of the present invention and at
least one excipient is a dry formulation.
[0650] Such liquid, suspension or dry pharmaceutical composition
comprises at least one excipient. Excipients used in parenteral
formulations may be categorized as, for example, buffering agents,
isotonicity modifiers, preservatives, stabilizers, anti-adsorption
agents, oxidation protection agents, viscosifiers/viscosity
enhancing agents, or other auxiliary agents. However, in some
cases, one excipient may have dual or triple functions. Preferably,
the at least one excipient comprised in the pharmaceutical
composition of the present invention is selected from the group
consisting of [0651] (i) Buffering agents: physiologically
tolerated buffers to maintain pH in a desired range, such as sodium
phosphate, bicarbonate, succinate, histidine, citrate and acetate,
sulphate, nitrate, chloride, pyruvate; antacids such as
Mg(OH).sub.2 or ZnCO.sub.3 may be also used; [0652] (ii)
Isotonicity modifiers: to minimize pain that can result from cell
damage due to osmotic pressure differences at the injection depot;
glycerin and sodium chloride are examples; effective concentrations
can be determined by osmometry using an assumed osmolality of
285-315 mOsmol/kg for serum; [0653] (iii) Preservatives and/or
antimicrobials: multidose parenteral formulations require the
addition of preservatives at a sufficient concentration to minimize
risk of patients becoming infected upon injection and corresponding
regulatory requirements have been established; typical
preservatives include m-cresol, phenol, methylparaben,
ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl
alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium
sorbate, benzoic acid, chlorocresol, and benzalkonium chloride;
[0654] (iv) Stabilizers: Stabilisation is achieved by strengthening
of the protein-stabilising forces, by destabilisation of the
denatured state, or by direct binding of excipients to the protein;
stabilizers may be amino acids such as alanine, arginine, aspartic
acid, glycine, histidine, lysine, proline, sugars such as glucose,
sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,
salts such as potassium phosphate, sodium sulphate, chelating
agents such as EDTA, hexaphosphate, ligands such as divalent metal
ions (zinc, calcium, etc.), other salts or organic molecules such
as phenolic derivatives; in addition, oligomers or polymers such as
cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA
may be used; [0655] (v) Anti-adsorption agents: Mainly ionic or
non-ionic surfactants or other proteins or soluble polymers are
used to coat or adsorb competitively to the inner surface of the
formulation's container; e.g., poloxamer (Pluronic F-68), PEG
dodecyl ether (Brij 35), polysorbate 20 and 80, dextran,
polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatins;
chosen concentration and type of excipient depends on the effect to
be avoided but typically a monolayer of surfactant is formed at the
interface just above the CMC value; [0656] (vi) Oxidation
protection agents: antioxidants such as ascorbic acid, ectoine,
methionine, glutathione, monothioglycerol, morin, polyethylenimine
(PEI), propyl gallate, and vitamin E; chelating agents such as
citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be
used; [0657] (vii) Viscosifiers or viscosity enhancers: in case of
a suspension retard settling of the particles in the vial and
syringe and are used in order to facilitate mixing and resuspension
of the particles and to make the suspension easier to inject (i.e.,
low force on the syringe plunger); suitable viscosifiers or
viscosity enhancers are, for example, carbomer viscosifiers like
Carbopol 940, Carbopol Ultrez 10, cellulose derivatives like
hydroxypropylmethylcellulose (hypromellose, HPMC) or
diethylaminoethyl cellulose (DEAE or DEAE-C), colloidal magnesium
silicate (Veegum) or sodium silicate, hydroxyapatite gel,
tricalcium phosphate gel, xanthans, carrageenans like Satia gum UTC
30, aliphatic poly(hydroxy acids), such as poly(D,L- or L-lactic
acid) (PLA) and poly(glycolic acid) (PGA) and their copolymers
(PLGA), terpolymers of D,L-lactide, glycolide and caprolactone,
poloxamers, hydrophilic poly(oxyethylene) blocks and hydrophobic
poly(oxypropylene) blocks to make up a triblock of
poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g.
Pluronic.RTM.), polyetherester copolymer, such as a polyethylene
glycol terephthalate/polybutylene terephthalate copolymer, sucrose
acetate isobutyrate (SAIB), dextran or derivatives thereof,
combinations of dextrans and PEG, polydimethylsiloxane, collagen,
chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides,
poly (acrylamide-co-diallyldimethyl ammonium (DADMA)),
polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as
dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin,
heparan sulfate, hyaluronan, ABA triblock or AB block copolymers
composed of hydrophobic A-blocks, such as polylactide (PLA) or
poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such
as polyethylene glycol (PEG) or polyvinyl pyrrolidone; such block
copolymers as well as the abovementioned poloxamers may exhibit
reverse thermal gelation behavior (fluid state at room temperature
to facilitate administration and gel state above sol-gel transition
temperature at body temperature after injection); [0658] (viii)
Spreading or diffusing agent: modifies the permeability of
connective tissue through the hydrolysis of components of the
extracellular matrix in the intrastitial space such as but not
limited to hyaluronic acid, a polysaccharide found in the
intercellular space of connective tissue; a spreading agent such as
but not limited to hyaluronidase temporarily decreases the
viscosity of the extracellular matrix and promotes diffusion of
injected drugs; and [0659] (ix) Other auxiliary agents: such as
wetting agents, viscosity modifiers, antibiotics, hyaluronidase;
acids and bases such as hydrochloric acid and sodium hydroxide are
auxiliary agents necessary for pH adjustment during
manufacture.
[0660] The pharmaceutical composition comprising at least one PTH
prodrug may be administered to a patient by various modes of
administration, such as via topical, enteral or parenteral
administration and by methods of external application, injection or
infusion, including intraarticular, periarticular, intradermal,
subcutaneous, intramuscular, intravenous, intraosseous,
intraperitoneal, intrathecal, intracapsular, intraorbital,
intravitreal, intratympanic, intravesical, intracardiac,
transtracheal, subcuticular, subcapsular, subarachnoid,
intraspinal, intraventricular, intrasternal injection and infusion,
direct delivery to the brain via implanted device allowing delivery
of the invention or the like to brain tissue or brain fluids (e.g.,
Ommaya Reservoir), direct intracerebroventricular injection or
infusion, injection or infusion into brain or brain associated
regions, injection into the subchoroidal space, retro-orbital
injection and ocular instillation. Preferably the pharmaceutical
composition comprising at least one PTH prodrug is administered via
subcutaneous injection.
[0661] Subcutaneous injection is preferably done with a syringe and
needle or with a pen injector, even more preferably with a pen
injector.
[0662] Another aspect of the present invention is the use of the
PTH prodrug or a pharmaceutically acceptable salt thereof or a
pharmaceutical composition comprising at least one PTH prodrug of
the present invention as a medicament.
[0663] Another aspect of the present invention is the PTH prodrug
or a pharmaceutically acceptable salt thereof or the pharmaceutical
composition comprising at least one PTH prodrug of the present
invention for use in the treatment of a disease which can be
treated with PTH.
[0664] Preferably, said disease is selected from the group
consisting of hypoparathyroidism, hyperphosphatemia, osteoporosis,
fracture repair, osteomalacia, osteomalacia and osteoporosis in
patients with hypophosphatasia, steroid-induced osteoporosis, male
osteoporosis, arthritis, osteoarthritis, osteogenesis imperfect,
fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral
hypercalcemia associated with malignancy, osteopenia, periodontal
disease, bone fracture, alopecia, chemotherapy-induced alopecia,
and thrombocytopenia. Most preferably said disease is
hypoparathyroidism.
[0665] In one embodiment the patient undergoing the method of
treatment of the present invention is a mammalian patient,
preferably a human patient.
[0666] Another aspect of the present invention is the use of the
PTH prodrug or a pharmaceutically acceptable salt thereof or the
pharmaceutical composition comprising at least one PTH prodrug of
the present invention for the manufacture of a medicament for
treating a disease which can be treated with PTH.
[0667] Preferably, said disease is selected from the group
consisting of hypoparathyroidism, hyperphosphatemia, osteoporosis,
fracture repair, osteomalacia, osteomalacia and osteoporosis in
patients with hypophosphatasia, steroid-induced osteoporosis, male
osteoporosis, arthritis, osteoarthritis, osteogenesis imperfect,
fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral
hypercalcemia associated with malignancy, osteopenia, periodontal
disease, bone fracture, alopecia, chemotherapy-induced alopecia,
and thrombocytopenia. Most preferably said disease is
hypoparathyroidism.
[0668] In one embodiment the disease to be treated with the PTH
prodrug or a pharmaceutically acceptable salt thereof or the
pharmaceutical composition comprising at least one PTH prodrug of
the present invention occurs in a mammalian patient, preferably in
a human patient.
[0669] A further aspect of the present invention is a method of
treating, controlling, delaying or preventing in a mammalian
patient, preferably a human patient, in need of the treatment of
one or more diseases which can be treated with PTH, comprising the
step of administering to said patient in need thereof a
therapeutically effective amount of PTH prodrug or a
pharmaceutically acceptable salt thereof or a pharmaceutical
composition comprising PTH prodrug of the present invention.
[0670] Preferably, the one or more diseases which can be treated
with PTH is selected from the group consisting of
hypoparathyroidism, hyperphosphatemia, osteoporosis, fracture
repair, osteomalacia, osteomalacia and osteoporosis in patients
with hypophosphatasia, steroid-induced osteoporosis, male
osteoporosis, arthritis, osteoarthritis, osteogenesis imperfect,
fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral
hypercalcemia associated with malignancy, osteopenia, periodontal
disease, bone fracture, alopecia, chemotherapy-induced alopecia,
and thrombocytopenia. Most preferably said disease is
hypoparathyroidism.
[0671] An additional aspect of the present invention is a method of
administering the PTH prodrug, a pharmaceutically acceptable salt
thereof or the pharmaceutical composition of the present invention,
wherein the method comprises the step of administering the PTH
prodrug, a pharmaceutically acceptable salt thereof or the
pharmaceutical composition of the present invention via topical,
enteral or parenteral administration and by methods of external
application, injection or infusion, including intraarticular,
periarticular, intradermal, subcutaneous, intramuscular,
intravenous, intraosseous, intraperitoneal, intrathecal,
intracapsular, intraorbital, intravitreal, intratympanic,
intravesical, intracardiac, transtracheal, subcuticular,
subcapsular, subarachnoid, intraspinal, intraventricular,
intrasternal injection and infusion, intranasal, oral,
transpulmonary and transdermal administration, direct delivery to
the brain via implanted device allowing delivery of the invention
or the like to brain tissue or brain fluids (e.g., Ommaya
Reservoir), direct intracerebroventricular injection or infusion,
injection or infusion into brain or brain associated regions,
injection into the subchoroidal space, retro-orbital injection and
ocular instillation, preferably via subcutaneous injection.
[0672] In a preferred embodiment, the present invention relates to
a PTH prodrug or pharmaceutically acceptable salt thereof or a
pharmaceutical composition of the present invention, for use in the
treatment of hypoparathyroidism via subcutaneous injection.
EXAMPLES
[0673] Materials and Methods
[0674] Side chain protected PTH(1-34) (SEQ ID NO:51) on TCP resin
having Boc protected N-terminus and ivDde protected side chain of
Lys26 (synthesized by Fmoc-strategy) was obtained from CASLO ApS,
Kongens Lyngby, Denmark and Peptide Specialty Laboratories GmbH,
Heidelberg, Germany.
[0675] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus (synthesized by Fmoc-strategy) was obtained
from CASLO ApS, Kongens Lyngby, Denmark and Peptide Specialty
Laboratories GmbH, Heidelberg, Germany.
[0676] PEG 2.times.20 kDa maleimide, Sunbright GL2-400MA and PEG
2.times.10 kDa maleimide, Sunbright GL2-200MA were purchased from
NOF Europe N.V., Grobbendonk, Belgium. S-Trityl-6-mercaptohexanoic
acid was purchased from Polypeptide, Strasbourg, France. HATU was
obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany.
Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc.,
Louisville, Ky., USA. Fmoc-Aib-OH was purchased from Iris Biotech
GmbH, Marktredwitz, Germany. All other chemicals and reagents were
purchased from Sigma Aldrich GmbH, Taufkirchen, Germany, unless a
different supplier is mentioned.
[0677] Compound 11a (examples 11-15) was synthesized following the
procedure described in patent WO 2009/05479 A2, example 1.
[0678] Syringes equipped with polyethylenene frits (MultiSynTech
GmbH, Witten, Germany) were used as reaction vessels or for washing
steps of peptide resins.
[0679] General procedure for the removal of ivDde protecting group
from side chain protected PTH on resin: The resin was pre-swollen
in DMF for 30 min and the solvent was discarded. The ivDde group
was removed by incubating the resin with DMF/hydrazine hydrate 4/1
(v/v, 2.5 mL/g resin) for 8.times.15 min. For each step fresh
DMF/hydrazine hydrate solution was used. Finally, the resin was
washed with DMF (10.times.), DCM (10.times.) and dried in
vacuo.
[0680] General procedure for the removal of Fmoc protecting group
from protected PTH on resin: The resin was pre-swollen in DMF for
30 min and the solvent was discarded. The Fmoc group was removed by
incubating the resin with DMF/piperidine/DBU 96/2/2 (v/v/v, 2.5
mL/g resin) for 3.times.10 min. For each step fresh
DMF/piperidine/DBU solution was used. Finally, the resin was washed
with DMF (10.times.), DCM (10.times.) and dried in vacuo.
[0681] RP-HPLC purification:
[0682] For preparative RP-HPLC a Waters 600 controller and a 2487
Dual Absorbance Detector was used, equipped with the following
columns: Waters XBridge.TM. BEH300 Prep C18 5 .mu.m, 150.times.10
mm, flow rate 6 mL/min, or Waters XBridge.TM. BEH300 Prep C18 10
.mu.m, 150.times.30 mm, flow rate 40 mL/min. Linear gradients of
solvent system A (water containing 0.1% TFA v/v) and solvent system
B (acetonitrile containing 0.1% TFA v/v) were used. HPLC fractions
containing product were pooled and lyophilized if not stated
otherwise.
[0683] Flash Chromatography:
[0684] Flash chromatography purifications were performed on an
Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil
silica cartridges and n-heptane and ethyl acetate as eluents.
Products were detected at 254 nm.
[0685] Ion Exchange Chromatography:
[0686] Ion exchange chromatography (IEX) was performed using an
Amersham Bioscience AEKTAbasic system equipped with a MacroCap SP
cation exchanger column (Amersham Bioscience/GE Healthcare). 17 mM
acetic acid pH 4.5 (solvent A) and 17 mM acetic acid, 1 M NaCl, pH
4.5 (solvent B) were used as mobile phases.
[0687] Size Exclusion Chromatography:
[0688] Size exclusion chromatography (SEC) was performed using an
Amersham Bioscience AEKTAbasic system equipped with HiPrep 26/10
desalting columns (Amersham Bioscience/GE Healthcare). 0.1% (v/v)
acetic acid was used as mobile phase.
[0689] For in vitro release kinetics studies of compound 31, a pH
7.40 buffer (100 mM phosphate, 10 mM L-methionine, 3 mM EDTA, 0.05%
Tween-20) was used instead of 0.1% AcOH as mobile phase.
[0690] Analytical Methods
[0691] Analytical ultra-performance LC (UPLC)-MS was performed on a
Waters Acquity system equipped with a Waters BEH300 C18 column
(2.1.times.50 mm, 1.7 .mu.m particle size, flow: 0.25 mL/min;
solvent A: water containing 0.04% TFA (v/v), solvent B:
acetonitrile containing 0.05% TFA (v/v)) coupled to a LTQ Orbitrap
Discovery mass spectrometer from Thermo Scientific or coupled to a
Waters Micromass ZQ.
[0692] Quantitative measurements of serum calcium (sCa), urinary
calcium and serum phosporous (sP) were performed on a Roche-Hitachi
P800 modular biochemistry instrument.
[0693] Quantification of plasma total PTH(1-34) concentrations:
[0694] Plasma total PTH(1-34) concentrations were determined by
quantification of a signature peptide close to the N-terminus
(sequence: IQLMHNLGK (SEQ ID NO: 123)) and a C-terminal signature
peptide (sequence: LQDVHNF (SEQ ID NO: 124)) after plasma protein
precipitation, followed by sequential digestion with Endoproteinase
Lys-C(origin: Lysobacter enzymogenes) and Endoproteinase
Glu-C(origin: Staphylococcus aureus V8) of the supernatant.
Subsequently, analysis by reversed phase liquid chromatography and
detection by mass spectrometry (RP-HPLC-MS) was performed.
[0695] Calibration standards of PTH(1-34) conjugate in blank plasma
were prepared as follows: The PTH(1-34) conjugate formulation was
pre-diluted with formulation buffer to aqueous standard solutions
ranging from 5 to 300 pg/mL PTH(1-34) eq (concentration range 1)
and 0.5 pg/mL to 100 pg/mL PTH(1-34) eq (concentration range 2),
respectively. Each aqueous standard solution was then diluted 1:100
with thawed heparinized plasma to obtain concentration ranges from
50 to 3000 ng/mL PTH(1-34) eq (dilution with rat plasma of
concentration range 1) and 5 to 1000 ng/mL PTH(1-34) eq (dilution
with monkey plasma of concentration range 2).
[0696] These solutions were used for the generation of a
calibration curve. Calibration curves were weighted 1/x2 for both
signature peptides. For quality control, three samples independent
from the calibration standard solutions were prepared accordingly.
Concentrations at the lower end (3-5 fold concentration of the
respective LLOQ), the middle range (0.05-0.1 fold concentration of
the respective ULOQ) and the upper end (0.5-0.8 fold concentration
of the respective ULOQ).
[0697] Sample preparation volumes can be altered depending on the
targeted signal response after sample preparation. Processing
procedure of the protein precipitation is described here for the
analysis of plasma samples originated in monkey species. Protein
precipitation was carried out by addition of 200 pL of precooled
(5-10.degree. C.) methanol to 100 pL of the plasma sample. 180 pL
of the supernatant were transferred into a new well-plate and
evaporated to dryness (under a gentle nitrogen stream at 45.degree.
C.). 50 pL of reconstitution solvent (50 mM Tris 0.5 mM CaCl.sub.2)
buffer, adjusted to pH 8.0) were used to dissolve the residue.
Proteolytic digestion was performed as follows:
[0698] 20 .mu.g of Lys-C(order number 125-05061, Wako Chemicals
GmbH, Neuss, Germany) were dissolved in 80 pL of 10 mM acetic acid.
3 pL of the Lys-C solution were added to each cavity and samples
incubated for 15 hours at 37.degree. C. Afterwards 10 pg of
Glu-C(order number V1651, Promega GmbH, Mannheim, Germany) were
dissolved in 25 pL water, added to each cavity and incubation
continued for 1.5 hours at 37.degree. C. After incubation samples
were acidified with 2 pL water/formic acid 4:6 (v/v) and 10 pL were
injected into the UPLC-MS system.
[0699] LC-MS analysis was carried out by using an Agilent 1290 UPLC
coupled to an Agilent 6460 TripleQuad mass spectrometer via an ESI
probe. Chromatography was performed on a Waters Acquity BEH300 C18
analytical column (1.7 .mu.m particle size; column dimensions used
are 50.times.2.1 mm for analysis of samples originated from rat
species or 100.times.2.1 mm for analysis of samples originated from
monkey species) with pre-filter at a flow rate of 0.30 mL/min
(T=60.degree. C.). Water (UPLC grade) containing 0.1% formic acid
(v/v) was used as mobile phase A and acetonitrile (UPLC grade) with
0.1% formic acid as mobile phase B.
[0700] The gradient system for the analysis of samples originated
from rat plasma comprised a linear increase from 0.1% B to 40% B in
7 min. The gradient system for the analysis of samples originated
from monkey plasma comprised a linear increase from 8.0% B to 11.0%
B in 6 min, followed by a linear increase to 26% B in 4 minutes.
Mass analysis was performed in multiple reaction monitoring (MRM)
mode, monitoring the transitions m/z 437.2 to 131.0 and m/z 352.3
to 463.0.
[0701] Alternatively, quantification of plasma total PTH(1-34)
concentrations was performed according to the following
procedure:
[0702] Plasma total PTH(1-34) concentrations were determined by
quantification of a signature peptide close to the N-terminus
(sequence: IQLMHNLGK (SEQ ID NO: 123)) and a C-terminal signature
peptide (sequence: LQDVHNF (SEQ ID NO: 124)) after plasma protein
precipitation, followed by sequential digestion with Endoproteinase
Lys-C(origin: Lysobacter enzymogenes) and Endoproteinase
Glu-C(origin: Staphylococcus aureus V8) of the supernatant.
Subsequently, analysis by reversed phase liquid chromatography and
detection by mass spectrometry (RP-HPLC-MS) was performed.
[0703] Calibration standards of PTH(1-34) conjugate in blank
heparinized plasma were prepared in concentration ranges from 50 to
3000 ng/mL PTH(1-34) eq (dilution with rat plasma) and 1 to 1000
ng/mL PTH(1-34) eq (dilution with monkey plasma).
[0704] These solutions were used for the generation of a
calibration curve. For quality control, three samples independent
from the calibration standard solutions were prepared accordingly.
Concentrations at the lower end (3-5 fold concentration of the
respective LLOQ), the middle range (0.05-0.1 fold concentration of
the respective ULOQ) and the upper end (0.5-0.8 fold concentration
of the respective ULOQ).
[0705] Sample preparation volumes can be altered depending on the
targeted signal response after sample preparation. Processing
procedure of the protein precipitation is described here for the
analysis of plasma samples originated in rat species. Protein
precipitation was carried out by addition of 100 pL of precooled
(5-10.degree. C.) methanol to 50 pL of the plasma sample. 60 .mu.L
of the supernatant were transferred into a new well-plate and
evaporated to dryness (under a gentle nitrogen stream at 45.degree.
C.). 60 .mu.L of reconstitution solvent (50 mM Tris 0.5 mM
CaCl.sub.2 buffer, adjusted to pH 8.0) were used to dissolve the
residue. Proteolytic digestion was performed as follows:
[0706] 20 .mu.g of Lys-C(order number 125-05061, Wako Chemicals
GmbH, Neuss, Germany) were dissolved in 80 .mu.L of 10 mM acetic
acid. 3 .mu.L of the Lys-C solution were added to each cavity and
samples incubated for 15 hours at 37.degree. C. Afterwards 10 pg of
Glu-C(order number V1651, Promega GmbH, Mannheim, Germany) were
dissolved in 25 .mu.L water, and 1.5 .mu.L of the Glu-C solution
added to each cavity and incubation continued for 1.5 hours at
37.degree. C. After incubation samples were acidified with 2 .mu.L
water/formic acid 4:6 (v/v) and 10 .mu.L were injected into the
UPLC-MS system.
[0707] Chromatography was performed on a Waters Acquity BEH300
C.sub.18 analytical column (1.7 .mu.m particle size; column
dimensions 50.times.2.1 mm). Water (UPLC grade) containing 0.1%
formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC
grade) with 0.1% formic acid as mobile phase B.
[0708] Quantification of Plasma PEG Concentrations:
[0709] Plasma total PEG concentrations were determined by
quantification of the polymeric part of PTH(1-34) conjugates after
plasma protein precipitation and enzymatic digestion of the
supernatant. Analysis by size exclusion chromatography and
detection by mass spectrometry (SEC-MS) followed.
[0710] Calibration standards of PTH(1-34) conjugate in blank
heparinized monkey plasma were prepared in concentration ranges
from 50 to 1200 ng/mL PEG equivalents.
[0711] These solutions were used for the generation of a quadratic
calibration curve. Calibration curves were weighted 1/x. For
quality control, three samples independent from the calibration
standard solutions were prepared accordingly. Concentrations at the
lower end (2-4 fold concentration of the LLOQ), the middle range
(0.1-0.2 fold concentration of the ULOQ) and the upper end (0.8
fold concentration of the ULOQ). Protein precipitation was carried
out by addition of 200 .mu.L of precooled (5-10.degree. C.)
methanol to 100 .mu.L of the plasma sample. 180 .mu.L of the
supernatant were transferred into a new well-plate and evaporated
to dryness (under a gentle nitrogen stream at 45.degree. C.). 50
.mu.L of reconstitution solvent (50 mM Tris 0.5 mM CaCl.sub.2
buffer, adjusted to pH 8.0) were used to dissolve the residue.
Proteolytic digestion was performed as follows: 20 pg of
Lys-C(order number 125-05061, Wako Chemicals GmbH, Neuss, Germany)
were dissolved in 80 .mu.L of 10 mM acetic acid. 3 .mu.L of the
Lys-C solution were added to each cavity and samples incubated for
15 hours at 37.degree. C. Afterwards 10 pg of Glu-C(order number
V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 .mu.L
water, and 1.5 .mu.L of the Glu-C solution added to each cavity and
incubation continued for 1.5 hours at 37.degree. C. After
incubation samples were acidified with 2 .mu.L water/formic acid
4:6 (v/v) and 5 .mu.L were injected into the SEC-MS system.
[0712] SEC-MS analysis was carried out by using an Agilent 1290
UPLC coupled to an Agilent 6460 TripleQuad mass spectrometer via an
ESI probe. Acquisition of a distinct precursor ion of the polymer
was achieved by applying high voltage in-source fragmentation
(200-300V) at the MS interface. Chromatography was performed on a
TOSOH TSK Gel SuperAW3000 analytical column (4.0 .mu.m particle
size; column dimensions 150.times.6.0 mm) at a flow rate of 0.50
mL/min (T=65.degree. C.). Water (UPLC grade) containing 0.1% formic
acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade)
with 0.1% formic acid as mobile phase B. The chromatographic setup
for sample analysis comprises an isocratic elution of 50% B over 8
minutes.
[0713] Mass analysis was performed in single reaction monitoring
(SRM) mode, monitoring the transition m/z 133.1 to 45.1.
[0714] Due to the reversible nature of the attachment of -L.sup.1-
to -D, measurements for PTH receptor activity were made using
stable analogs of the PTH prodrugs of the present invention, i.e.
they were made using similar structures to those of the PTH
prodrugs of the present invention, which instead of a reversible
attachment of --Z to -D have a stable attachment.
[0715] This was necessary, because the PTH prodrugs of the present
invention would release PTH in the course of the experiment and
said released PTH would have influenced the result.
Example 1
[0716] Synthesis of Linker Reagent 1f
[0717] Linker reagent if was synthesized according to the following
scheme:
##STR00049##
[0718] To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48
mmol) and NaCNBH.sub.3 (819 mg, 12.63 mmol) in MeOH (20 mL) was
added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion
wise. The mixture was stirred at rt for 90 min, acidified with 3 M
HCl (4 mL) and stirred further 15 min. The reaction mixture was
added to saturated NaHCO.sub.3 solution (200 mL) and extracted
5.times. with DCM. The combined organic phases were dried over
Na.sub.2SO.sub.4 and the solvents were evaporated in vacuo. The
resulting N-methyl-N-Boc-N'-Tmob-ethylenediamine 1a was dried in
high vacuum and used in the next reaction step without further
purification.
[0719] Yield: 3.76 g (11.48 mmol, 89% purity, la: double Tmob
protected product=8:1)
[0720] MS: m/z 355.22=[M+H].sup.+, (calculated monoisotopic
mass=354.21).
[0721] To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU
(4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol)
and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction
mixture was stirred for 3 h at rt, diluted with DCM (250 mL) and
washed 3.times. with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3.times.
with brine (100 mL). The aqueous phases were re-extracted with DCM
(100 mL). The combined organic phases were dried over
Na.sub.2SO.sub.4, filtrated and the residue concentrated to a
volume of 24 mL. 1b was purified using flash chromatography.
[0722] Yield: 5.31 g (148%, 6.66 mmol)
[0723] MS: m/z 796.38=[M+H].sup.+, (calculated monoisotopic
mass=795.37).
[0724] To a solution of 1b (5.31 g, max. 4.52 mmol ref. to
N-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3% v/v) was
added. The solution was stirred for 12 min at rt, diluted with DCM
(400 mL) and washed 3.times. with 0.1 M H.sub.2SO.sub.4 (150 mL)
and 3.times. with brine (150 mL). The aqueous phases were
re-extracted with DCM (100 mL). The combined organic phases were
dried over Na.sub.2SO.sub.4 and filtrated. 1c was isolated upon
evaporation of the solvent and used in the next reaction without
further purification.
[0725] MS: m/z 574.31=[M+H].sup.+, (calculated monoisotopic
mass=573.30).
[0726] 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile
(26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid
(2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were
added. The reaction mixture was stirred for 4 h at rt, diluted with
DCM (400 mL) and washed 3.times. with 0.1 M H.sub.2SO.sub.4 (100
mL) and 3.times. with brine (100 mL).
[0727] The aqueous phases were re-extracted with DCM (100 mL). The
combined organic phases were dried over Na2SO.sub.4, filtered and
1d was isolated upon evaporation of the solvent.
[0728] Product 1d was purified using flash chromatography.
[0729] Yield: 2.63 g (62%, 94% purity) MS: m/z 856.41=[M+H].sup.+,
(calculated monoisotopic mass=855.41).
[0730] To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL)
and H.sub.2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the
reaction mixture was stirred for 70 min at rt. The mixture was
diluted with DCM (200 mL) and washed 3.times. with 0.1 M
H.sub.2SO.sub.4 (50 mL) and 3.times. with brine (50 mL). The
aqueous phases were re-extracted with DCM (100 mL). The combined
organic phases were dried over Na2SO.sub.4, filtered and 1e was
isolated upon evaporation of the solvent. 1e was purified using
flash chromatography.
[0731] Yield: 2.1 g (88%)
[0732] MS: m/z 878.4=[M+Na].sup.+, (calculated monoisotopic
mass=837.40).
[0733] To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4
mL) were added DCC (123 mg, 0.59 mmol), and a catalytic amount of
DMAP. After 5 min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was
added and the reaction mixture was stirred at rt for 1 h. The
reaction mixture was filtered, the solvent was removed in vacuo and
the residue was taken up in 90% acetonitrile plus 0.1% TFA (3.4
mL). The crude mixture was purified by RP-HPLC. Product fractions
were neutralized with 0.5 M pH 7.4 phosphate buffer and
concentrated. The remaining aqueous phase was extracted with DCM
and if was isolated upon evaporation of the solvent.
[0734] Yield: 154 mg (81%)
[0735] MS: m/z 953.4=[M+H].sup.+, (calculated monoisotopic
mass=952.43).
Example 2
[0736] Synthesis of Linker Reagent 2g
##STR00050##
[0737] 4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was
dissolved in DCM (20 mL) and added dropwise under stirring to a
solution of ethylenediamine 2a (6.5 mL, 97.3 mmol) in DCM (20 mL).
The reaction mixture was stirred for 2 h at rt after which it was
diluted with diethyl ether (300 mL), washed 3.times. with brine/0.1
M NaOH 30/1 (v/v) and once with brine. The organic phase was dried
over Na.sub.2SO.sub.4 and 2b was isolated upon evaporation of the
solvent.
[0738] Yield: 3.18 g (98%)
[0739] Mint protected intermediate 2b (3.18 g, 9.56 mmol) was
dissolved in DCM (30 mL). 6-(Tritylthio)-hexanoic acid (4.48 g,
11.5 mmol), PyBOP (5.67 g, 10.9 mmol) and DIPEA (5.0 mL, 28.6 mmol)
were added and the mixture was stirred for 30 min at rt. The
solution was diluted with diethyl ether (250 mL), washed 3.times.
with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic
phase was dried over Na.sub.2SO.sub.4 and the solvent was removed
in vacuo. 2c was purified using flash chromatography.
[0740] Yield: 5.69 g (85%)
[0741] MS: m/z 705.4=[M+H].sup.+, (calculated monoisotopic
mass=704.34).
[0742] Compound 2c (3.19 g, 4.53 mmol) was dissolved in abhydrous
THF (50 mL), 1 M BH.sub.3.THF solution in THF (8.5 mL, 8.5 mmol)
was added and the mixture was stirred for 16 h at rt. More 1 M
BH.sub.3 THF solution in THF (14 mL, 14.0 mmol) was added and the
mixture was stirred for further 16 h at rt. Methanol (8.5 mL) and
N,N'-dimethyl-ethylendiamine (3.00 mL, 27.9 mmol) were added and
the mixture was heated under reflux for 3 h. The mixture was
allowed to cool down and ethyl acetate (300 mL) was added. The
solution was washed 2.times. with aqueous Na.sub.2CO.sub.3 and
2.times. with aqueous NaHCO.sub.3. The organic phase was dried over
Na2SO.sub.4 and the solvent was removed in vacuo to obtain 2d.
[0743] Yield: 3.22 g (103%)
[0744] MS: m/z 691.4=[M+H].sup.+, (calculated monoisotopic
mass=690.36).
[0745] Di-tert-butyl dicarbonate (2.32 g, 10.6 mmol) and DIPEA
(3.09 mL, 17.7 mmol) were dissolved in DCM (5 mL) and added to a
solution of 2d (2.45 g, 3.55 mmol) in DCM (5 mL). The mixture was
stirred for 30 min at rt. The solution was concentrated in vacuo
and purified by flash chromatography to obtain product 2e.
[0746] Yield: 2.09 g (74%)
[0747] MS: m/z 791.4=[M+H].sup.+, (calculated monoisotopic
mass=790.42).
[0748] Compound 2e (5.01 g, 6.34 mmol) was dissolved in
acetonitrile (80 mL). 0.4 M aqueous HCl (80 mL) followed by
acetonitrile (20 mL) was added and the mixture was stirred for 1 h
at rt. The pH was adjusted to pH 5.5 by addition of aqueous 5 M
NaOH. The organic solvent was removed in vacuo and the remaining
aqueous solution was extracted 4.times. with DCM. The combined
organic phases were dried over Na.sub.2SO.sub.4 and the solvent was
removed in vacuo to obtain product 2f.
[0749] Yield: 4.77 g (95%)
[0750] MS: m/z 519.3=[M+H].sup.+, (calculated monoisotopic
mass=518.30).
[0751] Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL)
and added to a solution of p-nitrophenyl chloroformate (2.01 g,
9.98 mmol) in DCM (25 mL). 2,4,6-trimethylpyridine (4.38 mL, 33.3
mmol) was added and the solution was stirred for 45 min at rt. The
solution was concentrated in vacu and purified by flash
chromatography to obtain product 2g.
[0752] Yield: 4.04 g (89%)
[0753] MS: m/z 706.32=[M+Na].sup.+, (calculated monoisotopic
mass=683.30).
Example 3
[0754] Synthesis of Permanent S1 PTH(1-34) Conjugate 3
##STR00051##
[0755] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of
6-tritylmercaptohexanoic acid (62.5 mg, 160 .mu.mol), PyBOP (80.1
mg, 154 .mu.mol) and DIPEA (53 .mu.L, 306 .mu.mol) in DMF (2 mL)
was added to 0.21 g (51 .mu.mol) of the resin. The suspension was
agitated for 80 min at rt. The resin was washed 10.times. with DMF,
10.times. with DCM and dried in vacuo. Cleavage of the peptide from
the resin and removal of protecting groups was achieved by adding
10 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)
TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h
at rt. Crude 3 was precipitated in pre-cooled diethyl ether
(-18.degree. C.). The precipitate was dissolved in ACN/water and
purified by RP-HPLC. The product fractions were freeze-dried.
[0756] Yield: 36 mg (14%), 3*8 TFA
[0757] MS: m/z 1062.31=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1062.30).
Example 4
[0758] Synthesis of Permanent K26 PTH(1-34) Conjugate 4
##STR00052##
[0759] Side chain protected PTH(1-34) on TCP resin having Boc
protected N-terminus and ivDde protected side chain of Lys26 was
ivDde deprotected according to the procedure given in Materials and
Methods. A solution of 6-tritylmercaptohexanoic acid (107 mg, 273
.mu.mol), PyBOP (141 mg, 273 .mu.mol) and DIPEA (95 .mu.L, 545
.mu.mol) in DMF (3 mL) was added to 0.80 g (90.9 .mu.mol) of the
resin. The suspension was agitated for 1 h at rt. The resin was
washed 10.times. with DMF, 10.times. with DCM and dried in vacuo.
Cleavage of the peptide from the resin and removal of protecting
groups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1
(v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the
suspension for 1 h at rt. Crude 4 was precipitated in pre-cooled
diethyl ether (-18.degree. C.). The precipitate was dissolved in
ACN/water and purified by RP-HPLC. The product fractions were
freeze-dried.
[0760] Yield: 40 mg (8%), 4*8 TFA
[0761] MS: m/z 1062.30=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1062.30).
Example 5
[0762] Synthesis of Transient S1 PTH(1-34) Conjugate
##STR00053##
[0763] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of Fmoc-Aib-OH
(79 mg, 244 .mu.mol), PyBOP (127 mg, 244 .mu.mol) and DIPEA (64
.mu.L, 365 .mu.mol) in DMF (1.5 mL) was added to 0.60 g (61
.mu.mol) of the resin. The suspension was agitated for 16 h at rt.
The resin was washed 10.times. with DMF and Fmoc-deprotected as
described above. A solution of 2g (167 mg, 244 .mu.mol) and DIPEA
(64 .mu.L, 365 .mu.mol) in DMF (1.5 mL) was added to the resin. The
suspension was agitated for 24 h at rt. The resin was washed
10.times. with DMF, 10.times. with DCM and dried in vacuo. Cleavage
of the peptide from the resin and removal of protecting groups was
achieved by adding 7 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)
TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h
at rt. Crude 5 was precipitated in pre-cooled diethyl ether
(-18.degree. C.). The precipitate was dissolved in ACN/water and
purified by RP-HPLC. The product fractions were freeze-dried.
[0764] Yield: 78 mg (24%), 5*9 TFA
[0765] MS: m/z 1101.59=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1101.57).
Example 6
[0766] Synthesis of Transient S1 PTH(1-34) Conjugate 6
##STR00054##
[0767] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of Fmoc-Ala-OH
(32 mg, 102 .mu.mol), PyBOP (53 mg, 102 .mu.mol) and DIPEA (27
.mu.L, 152 .mu.mol) in DMF (3 mL) was added to 0.25 g (25 .mu.mol)
of the resin. The suspension was shaken for 1 h at rt. The resin
was washed 10.times. with DMF, 10.times. with DCM and dried under
vacuum. Fmoc-deprotection was performed as described above. A
solution of 2g (69 mg, 102 .mu.mol) and DIPEA (27 .mu.L, 152
.mu.mol) in DMF (3 mL) was added to the resin. The suspension was
agitated for 1.5 h at rt. The resin was washed 10.times. with DMF,
10.times. with DCM and dried in vacuo. Cleavage of the peptide from
the resin and removal of protecting groups was achieved by adding 3
mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)
TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h
at rt. Crude 6 was precipitated in pre-cooled diethyl ether
(-18.degree. C.). The precipitate was dissolved in ACN/water and
purified by RP-HPLC. The product fractions were freeze-dried.
[0768] Yield: 25 mg (18%), 6*9 TFA
[0769] MS: m/z 1098.75=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1098.07).
Example 7
[0770] Synthesis of Transient S1 PTH(1-34) Conjugate 7
##STR00055##
[0771] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of
Fmoc-Ser(Trt)-OH (117 mg, 205 .mu.mol), PyBOP (108 mg, 207 .mu.mol)
and DIPEA (53 .mu.L, 305 .mu.mol) in DMF (2 mL) was added to 0.50 g
(51 .mu.mol) of the resin. The suspension was agitated for 1 h at
rt. The resin was washed 10.times. with DMF, 10.times. with DCM and
dried under vacuum. Fmoc-deprotection was performed as described
above. A solution of 2g (144 mg, 211 .mu.mol) and DIPEA (53 .mu.L,
305 .mu.mol) in DMF (1.8 mL) was added to the resin. The suspension
was shaken for 7 h at rt. The resin was washed 10.times. with DMF,
10.times. with DCM and dried in vacuo. Cleavage of the peptide from
the resin and removal of protecting groups was achieved by adding 6
mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)
TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h
at rt. Crude 7 was precipitated in pre-cooled diethyl ether
(-18.degree. C.). The precipitate was dissolved in ACN/water and
purified by RP-HPLC. The product fractions were freeze-dried.
[0772] Yield: 54 mg (20%), 7*9 TFA
[0773] MS: m/z 1102.08=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1102.07).
Example 8
[0774] Synthesis of Transient S1 PTH(1-34) Conjugate 8
##STR00056##
[0775] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of Fmoc-Leu-OH
(36 mg, 102 .mu.mol), PyBOP (53 mg, 102 .mu.mol) and DIPEA (27
.mu.L, 152 .mu.mol) in DMF (3 mL) was added to 0.25 g (25 .mu.mol)
of the resin. The suspension was agitated for 1 h at rt. The resin
was washed 10.times. with DMF, 10.times. with DCM and dried under
vacuum. Fmoc-deprotection was performed as described above. A
solution of 2g (69 mg, 102 .mu.mol) and DIPEA (27 .mu.L, 152
.mu.mol) in DMF (3 mL) was added to the resin. The suspension was
agitated for 1.5 h at rt. The resin was washed 10.times. with DMF,
10.times. with DCM and dried in vacuo. Cleavage of the peptide from
the resin and removal of protecting groups was achieved by adding 3
mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)
TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h
at rt. Crude 8 was precipitated in pre-cooled diethyl ether
(-18.degree. C.). The precipitate was dissolved in ACN/water and
purified by RP-HPLC. The product fractions were freeze-dried.
[0776] Yield: 31 mg (22%), 8*9 TFA
[0777] MS: m/z 1109.32=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1108.58).
Example 9
[0778] Synthesis of Transient S1 PTH(1-34) Conjugate 9
##STR00057##
[0779] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of 1e (182 mg,
213 .mu.mol), PyBOP (111 mg, 213 .mu.mol) and DIPEA (93 .mu.L, 532
.mu.mol) in DMF (5 mL) was added to 2.00 g (107 .mu.mol) of the
resin. The suspension was agitated for 16 h at rt. The resin was
washed 10.times. with DMF, 10.times. with DCM and dried under
vacuum. Cleavage of the peptide from the resin and removal of
protecting groups was achieved by adding 20 mL cleavage cocktail
100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating
the suspension for 1 h at rt. Crude 9 was precipitated in
pre-cooled diethyl ether (-18.degree. C.). The precipitate was
dissolved in ACN/water and purified by RP-HPLC. The product
fractions were freeze-dried.
[0780] Yield: 47 mg (8%), 9*9 TFA
[0781] MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1108.57).
Example 10
[0782] Synthesis of Transient K26 PTH(1-34) Conjugate 10
##STR00058##
[0783] Side chain protected PTH(1-34) on TCP resin having Boc
protected N-terminus and ivDde protected side chain of Lys26 was
ivDde deprotected according to the procedure given in Materials and
Methods. A solution of if (867 mg, 910 .mu.mol) and DIPEA (0.24 mL,
1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 .mu.mol) of the
resin. The suspension was agitated for 1 h at rt. The resin was
washed 10.times. with DMF, 10.times. with DCM and dried under
vacuum. Cleavage of the peptide from the resin and removal of
protecting groups was achieved by adding 20 mL cleavage cocktail
100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and shaking
the suspension for 1 h at rt. Crude 10 was precipitated in
pre-cooled diethyl ether (-18.degree. C.). The precipitate was
dissolved in ACN/water and purified by RP-HPLC. The product
fractions were freeze-dried.
[0784] Yield: 92 mg (7%), 10*9 TFA
[0785] MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1108.57).
Example 11
[0786] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate
11b
##STR00059##
[0787] 0.15 mL of a 0.5 M NaH.sub.2PO.sub.4 buffer (pH 7.4) was
added to 0.5 mL of a 20 mg/mL solution of thiol 5 (10 mg, 1.84
.mu.mol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v).
The solution was incubated at rt for 10 min after which 238 .mu.L
of a 10 mg/mL solution of maleimide 11a (2.4 mg, 2.21 .mu.mol) in
1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added.
The solution was incubated for 20 min at rt. 10 .mu.L TFA was added
and the mixture was purified by RP-HPLC. The product fractions were
freeze-dried to obtain 11b.
[0788] Yield: 3.1 mg (26%), 11b*9 TFA
[0789] MS: m/z 1097.00=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+5H].sup.5+=1096.99).
Example 12
[0790] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate
12
##STR00060##
[0791] Conjugate 12 was synthesized as described for 11b by using
thiol 6 (10 mg, 1.85 .mu.mol) and maleimide 11a (2.4 mg, 2.21
.mu.mol).
[0792] Yield: 10 mg (83%), 12*9 TFA
[0793] MS: m/z 1094.20=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1094.19).
Example 13
[0794] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate
13
##STR00061##
[0795] Conjugate 13 was synthesized as described for 11b by using
thiol 7 (10 mg, 1.84 .mu.mol) and maleimide 11a (2.4 mg, 2.21
.mu.mol).
[0796] Yield: 8 mg (67%), 13*9 TFA
[0797] MS: m/z 1097.40=[M+5H].sup.5+, (calculated monoisotopic mass
for [M+5H].sup.5+=1097.39).
Example 14
[0798] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate
14
##STR00062##
[0799] Conjugate 14 was synthesized as described for 11b by using
thiol 8 (10 mg, 1.83 .mu.mol) and maleimide 11a (2.4 mg, 2.21
.mu.mol).
[0800] Yield: 4 mg (33%), 14*9 TFA
[0801] MS: m/z 1378.01=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1378.00).
Example 15
[0802] Synthesis of Low Molecular Weight Transient K26 PEG
Conjugate 15
##STR00063##
[0803] Conjugate 15 was synthesized as described for 11b by using
thiol 10 (5.2 mg, 0.95 .mu.mol) and maleimide 11a (1.23 mg, 1.14
.mu.mol).
[0804] Yield: 2.1 mg (33%), 15*9 TFA
[0805] MS: m/z 1102.60=[M+5H].sup.5+, (calculated monoisotopic mass
for [M+5H].sup.5+=1102.59).
Example 16
[0806] Synthesis of Permanent 2.times.20 kDa S1 PEG Conjugate
16
##STR00064##
[0807] 772 .mu.L of a solution containing thiol 3 (19.4 mg/mL, 15
mg, 3.54 .mu.mol) and 2.5 mg/mL Boc-L-Met in 1/1 (v/v)
acetonitrile/water containing 0.1% TFA (v/v) were added to 1.87 mL
of a solution containing PEG 2.times.20 kDa maleimide (Sunbright
GL2-400MA, 187 mg, 4.32 .mu.mol) and 2.5 mg/mL Boc-L-Met in water
containing 0.1% TFA (v/v). 0.5 M NaH.sub.2PO.sub.4 buffer (0.66 mL,
pH 7.0) was added and the mixture was stirred for 30 min at rt. 10
.mu.L of a 270 mg/mL solution of 2-mercaptoethanol in water was
added. The mixture was stirred for 5 min at rt and 0.33 mL 1 M HCl
were added. Conjugate 16 was purified by IEX followed by RP-HPLC
using a linear gradient of solvent system A (water containing 0.1%
AcOH v/v) and solvent system B (acetonitrile containing 0.1% AcOH
v/v). The product containing fractions were freeze-dried.
[0808] Yield: 97 mg (2.01 .mu.mol, 57%) conjugate 16*8 AcOH
Example 17
[0809] Synthesis of Permanent 2.times.20 kDa K26 PEG Conjugate
17
##STR00065##
[0810] Conjugate 17 was prepared as described for 16 by reaction of
thiol 4 (15 mg, 3.53 .mu.mol) and PEG 2.times.20 kDa maleimide
(Sunbright GL2-400MA, 187 mg, 4.32 .mu.mol).
[0811] Yield: 80 mg (1.79 .mu.mol, 51%) conjugate 17*8 AcOH
Example 18
[0812] Synthesis of Transient 2.times.20 kDa S1 PEG Conjugate
18
##STR00066##
[0813] Conjugate 18 was prepared as described for 16 by reaction of
thiol 5 (37 mg, 8.40 .mu.mol) and PEG 2.times.20 kDa maleimide
(Sunbright GL2-400MA, 445 mg, 9.24 .mu.mol). The reaction was
quenched by addition of 50 .mu.L TFA without prior addition of
2-mercaptoethanol. Conjugate 18 was purified by IEX followed by SEC
for desalting. The product containing fractions were
freeze-dried.
[0814] Yield: 161 mg (3.33 .mu.mol, 40%) conjugate 18*9 AcOH
Example 19
[0815] Synthesis of Transient 2.times.20 kDa S1 PEG Conjugate
19
##STR00067##
[0816] Conjugate 19 was prepared as described for 16 by reaction of
thiol 7 (27 mg, 6.14 .mu.mol) and PEG 2.times.20 kDa maleimide
(Sunbright GL2-400MA, 325 mg, 7.50 .mu.mol).
[0817] Yield: 249 mg (5.16 .mu.mol, 84%) conjugate 19*9 AcOH
Example 20
[0818] Synthesis of Transient 2.times.20 kDa S1 PEG Conjugate
20
##STR00068##
[0819] Conjugate 20 was prepared as described for 16 by reaction of
thiol 9 (38 mg, 8.59 .mu.mol) and PEG 2.times.20 kDa maleimide
(Sunbright GL2-400MA, 455 mg, 9.45 .mu.mol). The reaction was
quenched by addition of 50 .mu.L TFA without prior addition of
2-mercaptoethanol. Conjugate 20 was purified by IEX followed by SEC
for desalting. The product containing fractions were
freeze-dried.
[0820] Yield: 194 mg (4.01 .mu.mol, 47%) conjugate 20*9 AcOH
Example 21
[0821] Synthesis of Transient 2.times.20 kDa K26 PEG Conjugate
21
##STR00069##
[0822] Conjugate 21 was prepared as described for 16 by reaction of
thiol 10 (34 mg, 7.58 .mu.mol) and PEG 2.times.20 kDa maleimide
(Sunbright GL2-400MA, 401 mg, 9.26 .mu.mol).
[0823] Yield: 256 mg (5.30 .mu.mol, 70%) conjugate 21*9 AcOH
Example 22
[0824] In Vitro Release Kinetics of Transient Low Molecular Weight
PEG Conjugates
[0825] Conjugates 11b, 12, 13, 14, and 15 were dissolved in pH 7.4
phosphate buffer (60 mM NaH.sub.2PO.sub.4, 3 mM EDTA, 0.01%
Tween-20, adjusted to pH 7.4 by NaOH) containing 0.05 mg/mL
pentafluorophenol as internal standard at a concentration of
approximately 1 mg conjugate/mL. The solutions were filtered
sterile and incubated at 37.degree. C. At time points, aliquots
were withdrawn and analysed by RP-HPLC and ESI-MS. The fraction of
released PTH at a particular time point was calculated from the
ratio of UV peak areas of liberated PTH and PEG conjugate. The %
released PTH was plotted against incubation time. Curve-fitting
software was applied to calculate the corresponding half times of
release.
[0826] Results:
[0827] For conjugate 11b a release half life time of 3.2 d was
obtained.
[0828] For conjugate 12 a release half life time of 8.7 d was
obtained.
[0829] For conjugate 13 a release half life time of 10.8 d was
obtained.
[0830] For conjugate 14 a release half life time of 25.3 d was
obtained.
[0831] For conjugate 15 a release half life time of 6.9 d was
obtained.
Example 23
[0832] In vitro release kinetics of transient 2.times.20 kDa PEG
conjugates Conjugates 18, 19, 20, and 21 were dissolved in pH 7.4
phosphate buffer (60 mM NaH.sub.2PO.sub.4, 3 mM EDTA, 0.01%
Tween-20, adjusted to pH 7.4 by NaOH) containing 0.08 mg/mL
pentafluorophenol as internal standard at a concentration of
approximately 5 mg conjugate/mL. The solutions were filtered
sterile and incubated at 37.degree. C. At time points, aliquots
were withdrawn and analysed by RP-HPLC. The fraction of released
PTH at a particular time point was calculated from the ratio of UV
peak areas of liberated PTH and PEG conjugate. The % released PTH
was plotted against incubation time. Curve-fitting software was
applied to calculate the corresponding half times of release.
[0833] Results:
[0834] For conjugate 18 a release half life time of 2.8 d was
obtained.
[0835] For conjugate 19 a release half life time of 13.4 d was
obtained.
[0836] For conjugate 20 a release half life time of 1.3 d was
obtained.
[0837] For conjugate 21 a release half life time of 7.1 d was
obtained.
Example 24
[0838] PTH Receptor Activity of Permanent 2.times.20 kDa PEG
Conjugates 16 and 17 in Cell Based Assay
[0839] The residual PTH activity of permanently PEGylated
conjugates 16 and 17 was quantified by measuring cAMP production
from HEK293 cells over-expressing the PTH/PTHrP1 receptor
(Hohenstein A, Hebell M, Zikry H, El Ghazaly M, Mueller F, Rohde,
J. Development and validation of a novel cell-based assay for
potency determination of human parathyroid hormone (PTH), Journal
of Pharmaceutical and Biomedical Analysis September 2014, 98:
345-350). PTH(1-34) from NIBSC (National Institute for Biological
Standards and Control, UK) was used as reference standard.
[0840] Results:
[0841] For conjugate 16 a receptor activity of 0.12% was found
relative to PTH(1-34) reference.
[0842] For conjugate 17 a receptor activity of 0.11% was found
relative to PTH(1-34) reference.
[0843] The results indicate an effective lowering of receptor
activity in the permanent 2.times.20 kDa PEG conjugates 16 and 17.
It can be concluded that similar conjugates with transiently Ser1
or Lys26 linked PTH (like e.g. 18 and 21) are suitable PTH prodrugs
providing low residual receptor activity. Direct analysis of
transient conjugates in the cell assay is not possible due to
linker cleavage under the assay conditions. The released PTH would
influence the assay result.
Example 25
[0844] Pharmacokinetic Study of Permanent 2.times.20 kDa PEG
Conjugates 16 and 17 in Rats
[0845] Male Wistar rats (6 weeks, 230-260 g) received either a
single intravenous (2 groups, n=3 animals each) or a single
subcutaneous (2 groups, n=3 animals each) administration of 16 or
17 at doses of 29 pg/rat PTH.sub.eq and 31 pg/rat PTH.sub.eq
respectively. Blood samples were collected up to 168 h post dose,
and plasma was generated. Plasma PTH(1-34) concentrations were
determined by quantification of the N-terminal signature peptide
(sequence: IQLMHNLGK (SEQ ID NO: 123)) and the C-terminal signature
peptide (sequence: LQDVHNF (SEQ ID NO: 124)) after LysC and GluC
digestion as described in Materials and Methods.
[0846] Results: Dose administrations were well tolerated with no
visible signs of discomfort during administration and following
administration. No dose site reactions were observed any time
throughout the study. After intraveneous injection of 16 and 17 the
total PTH(1-34) t.sub.max was observed at 15 min (earliest time
point analyzed), followed by a slow decay in total PTH(1-34)
content with a half life time of approx. 13 h and 11 h
respectively. After subcutaneous injection the total PTH(1-34)
concentration peaked at a t.sub.max of 24 h for both 16 and 17,
followed by a slow decay in total PTH(1-34) content with half life
times of approx. 1.5 days for both conjugates. The bioavailability
was approx. 40% and 60% respectively. Similar PK curves were
obtained for the N- and the C-terminal signature peptide up to 168
h post dose, indicating the presence of intact PTH(1-34) in the
conjugate.
[0847] The favourable long lasting PK and the stability of PTH in
the conjugates indicate the suitability of the permanent 2.times.20
kDa PEG model compounds as slow releasing PTH prodrugs after
subcutaneous injection. It can be concluded that similar conjugates
with transiently Ser1 (like e.g. 18) or Lys26 linked PTH are
suitable PTH prodrugs providing long lasting levels of released
bioactive PTH.
Example 26
[0848] Pharmacokinetic Study of Transient 2.times.20 kDa S1 PEG
Conjugate 19 in Cynomolgus Monkeys
[0849] Male non naive cynomolgus monkeys (2-4 years, 3.7-5.4 kg)
received a single subcutaneous (n=3 animals) administration of 19
at a dose of 70 .mu.g/kg PTH.sub.eq. Blood samples were collected
up to 504 h post dose, and plasma was generated. Total plasma
PTH(1-34) concentrations were determined by quantification of the
N-terminal signature peptide (sequence: IQLMHNLGK (SEQ ID NO: 123))
and the C-terminal signature peptide (sequence: LQDVHNF (SEQ ID NO:
124)) after LysC and GluC digestion as described in Materials and
Methods. The PEG concentrations were determined using the method
described in Materials and Methods.
[0850] Results: Dose administrations were well tolerated with no
visible signs of discomfort during administration. One animal
showed showed visible signs of discomfort 72 h post dose, but
recovered the days after. No dose site reactions were observed any
time throughout the study. The total PTH(1-34) concentration peaked
at a t.sub.max of 24 h, followed by a slow decay in total PTH(1-34)
content with a half life time of approx. 2.5 d for the N-terminal
signature peptide and 0.9 d for the C-terminal signature peptide.
The PEG concentration peaked at t.sub.max of 24 h, followed by a
slow decay in PEG concentration with a half life time of 3.5 d.
[0851] It can be concluded that conjugate 19 is a suitable prodrug
for sustained delivery of PTH.
Example 27
[0852] Pharmacokinetic Study of Transient 2.times.20 kDa S1 PEG
Conjugate 18 in Cynomolgus Monkeys
[0853] Non naive cynomolgus monkeys (2-3 years, 2.5-4 kg) received
daily subcutaneous (n=2 animals--1 male/1 female) administration of
18 at dose levels of 0.2, 0.5, and 1 .mu.g/kg PTH.sub.eq for 28
days. Blood samples were collected up to 28 days (at days 1, 13,
and, 27 samples were collected at pre-dose, 2 h, 4 h, 8 h, 12 h,
and 24 h post-dose) and plasma was generated. Plasma PTH(1-34)
concentrations were determined by quantification of the N-terminal
signature peptide (sequence: IQLMHNLGK (SEQ ID NO: 123)) and the
C-terminal signature peptide (sequence: LQDVHNF (SEQ ID NO: 124))
after LysC and GluC digestion as described in Materials and
Methods.
[0854] Results: All dose administrations were performed without
incident. No dose site reactions were observed any time throughout
the study. Dose linearity was observed in the three groups. Dose
stacking was observed from day 1 compared with day 13 and day 27.
Total PTH(1-34) concentrations were quantified via the N-terminal
signature peptide (sequence: IQLMHNLGK (SEQ ID NO: 123)) at steady
state (during day 27).
[0855] A low peak-to-trough ratio of total PTH(1-34) for all dose
groups of below 3 was observed after daily subcutaneous application
at steady state in cynomolgus monkeys. As free peptide
concentrations at steady state are correlated to total PTH(1-34)
concentration, the peak-to-trough ratio for the free peptide is
below 4 in cynomolgus monkeys.
Example 28
[0856] Pharmacodynamic Actions in Thyroparathyroidectomised (TPTx)
Rats During a 28-Days Study with Daily Subcutaneous Injections with
Conjugate 18 or PTH(1-84)
[0857] This study was performed in order to test and compare the
effect of daily subcutaneous injection of compound 18 and
PTH(1-84), the current standard of care, in an animal disease model
relevant for investigating treatment of hypoparathyroidism (HP).
Rats subjected to thyroparathyroidectomy (TPTx) by blunt dissection
are unable to produce parathyroid hormone, PTH, the major regulator
of calcium homeostasis. Hence, TPTx rats develop hypocalcemia and
hyperphosphatemia characteristic of HP. 17 weeks old female SD TPTx
rats (n=9/group) were dosed subcutaneously for 28 days with
compound 18 (5 .mu.g PTH eq/kg/d; 1.2 nmol/kg/d, in 10 mM succinic
acid, 46 g/L mannitol, pH 4.0), PTH(1-84) (70 .mu.g PTH eq/kg/d;
7.3 nmol/kg/d; in 10 mM citrate, mannitol 39.0 g/L, pH 5.0) or
vehicle.
[0858] Additionally, one group of sham operated rats (n=9)
representing normophysiological background control were also given
vehicle. Serum calcium (sCa) and phosporous (sP) levels in the
animals were measured pre- and post-dose on days 1, 6, 12 and 27.
Moreover, bone turnover markers (P1NP and CTx) were measured and
bone quality assessed by ex vivo pQCT.
[0859] Results: The average sCa in the TPTx rats pre-dosing at day
1 was 8.3 mg/dL compared to 10.9 mg/dL in the sham operated control
rats. The sP values were 8.7 mg/dL and 5.9 mg/dL, respectively.
Compound 18 given daily at 1.2 nmol/kg elevated sCa to near-normal
levels while lowering sP within a few days of administration. At
day 12 (day 5 at steady state with compound 18) sCa had stabilised
at normal level (10.7 mg/dL) in this group of animals (compound
18/sham-control ratio=1.01) as opposed to the hypocalceamic level
(8.1 mg/dL) measured in the PTH(1-84) treated rats
(PTH(1-84)/sham-control ratio=0.76). Additionally, the 24-hour
urinary Ca excretion at day 12 was comparable between the animals
treated with compound 18 and sham-control. Bone mineral density
(BMD) and bone mineral content (BMC) were increased in TPTx
controls as seen in HP patients. Treatment with Compound 18
decreased BMD, BMC and area in parallel with an increase in CTx
compared to sham and vehicle-treated TPTx animals. A significant
increase in trabecular BMD was observed in animals dosed with
PTH(1-84) compared to both control groups.
[0860] It was concluded that compound 18 at a dosage even as low as
less than 20% of the molar equivalent of the here tested dose of
PTH(1-84) was able to maintain sCa at a level comparable to the sCa
level in sham-control animals (here representing normal level) over
a 24 hour period. In contrast, PTH(1-84) at a dose of 7.3 nmol/kg/d
did not lead to increase in sCa as compared to the levels in the
vehicle-injected TPTx rats. However, a minimal decrease in sP was
observed in the PTH(1-84) dosed animals confirming exposure and
response to PTH(1-84) in the rats. Following the 28-days of
treatment with compound 18, trabecular and cortical BMD in
vertebrae were within normal range, whereas an anabolic effect was
observed for PTH(1-84) on trabecular and cortical bone in
vertebrae.
Example 29
[0861] Synthesis of Linker Reagent 29h
##STR00070## ##STR00071##
[0862] To a solution of compound 29a (250 g, 294 mmol, 1 eq) in
dichloromethane (1 L) was added a solution of Na.sub.2CO.sub.3 (187
g, 1.8 mol, 6 eq) in H.sub.2O (1 L). The reaction solution was
stirred at 15-30.degree. C. for 0.5 hour. TLC (DCM/MeOH=10:1,
R.sub.f=0.5) showed the starting material was consumed completely.
The organic layer was separated and the aqueous phase was extracted
with dichloromethane (1 L). The organic layers were combined and
washed with brine (800 mL), then dried with anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuum to give
compound 29b as a yellow oil.
[0863] Yield: 200 g, 272 mmol, 93%
[0864] Four reactions were carried out in parallel.
[0865] To a solution of compound 29b (50 g, 68.1 mmol, 1 eq) and
Fmoc-5-aminovaleric acid (25.4 g, 74.9 mmol, 1.1 eq), DIPEA (61.6
g, 477 mmol, 83.3 mL, 7 eq) in acetonitrile (500 mL) was added
drop-wise T3P 50% [EtOAc] (130 g, 204 mmol, 122 mL, 3 eq) at
15-30.degree. C. for 1 hour. After addition, the reaction mixture
was stirred at 15-30.degree. C. for 18 hours. TLC (Petroleum
ether/Ethyl acetate=1:1, R.sub.f=0.5) showed the starting material
was consumed completely. The four reactions were combined for
workup. The mixture was diluted with water (3 L), then adjusted to
pH=3-4 with 0.5 N HCl solution. The mixture was extracted with
EtOAc (3 L), then the aqueous phase was extracted with EtOAc (2 L).
The organic layers were combined and washed with brine (1 L), then
dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in
vacuum to give the crude product as yellow oil. The crude product
was purified by column chromatography on silica gel with petroleum
ether/ethyl acetate to give compound 29c as a yellow solid.
[0866] Yield: 220 g, 199 mmol, 73%
[0867] Four reactions were carried out in parallel.
[0868] To a solution of compound 29c (55 g, 52 mmol, 1 eq) in
dichloromethane (275 mL) was added piperidine (47.3 g, 555 mmol, 55
mL, 10.7 eq). The reaction solution was stirred for 3 hours at
15-30.degree. C. TLC (Petroleum ether/Ethyl acetate=1:1, R.sub.f=0)
showed the starting material was consumed completely. The four
reactions were combined and the mixture was diluted with water (800
mL) and dichloromethane (800 L), then adjusted to pH=3-4 with 0.5 N
HCl solution. The organic layer was separated and the aqueous phase
was extracted with dichloromethane (800 mL). The organic layers
were combined and washed with brine (1 L), then dried with
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum to
give the crude product. The crude product was purified by column
chromatography on silica gel with DCM/MeOH to give compound 29d as
a white solid.
[0869] Yield: 140 g, 168 mmol, 81%
[0870] Four reactions were carried out in parallel.
[0871] To a solution of compound 29d (30 g, 36 mmol, 1 eq) in THF
(300 mL) was added
(1,3-dioxo-1,3-dihydro-2H-isoindol-2-y1)acetaldehyde (6.8 g, 36
mmol, 1 eq) and NaBH(OAc).sub.3 (15.3 g, 72 mmol, 2 eq) in one
portion. After addition, the reaction mixture was stirred at
15-30.degree. C. for 18 hours. TLC (DCM/MeOH=10:1, R.sub.f=0.4)
showed the starting material was consumed completely. The four
reactions were combined and the mixture was diluted with water (2
L) and EtOAc (1.5 L). The organic layer was separated and the
aqueous phase was extracted with EtOAc (1 L). The organic layers
were combined and washed with brine (1 L), then dried with
anhydrous Na2SO.sub.4, filtered and concentrated in vacuum to give
compound 29e as yellow oil.
[0872] Yield: 164 g, crude
[0873] Three reactions were carried out in parallel.
[0874] To a solution of compound 29e (50 g, 49.7 mmol, 1 eq) in DCM
(150 mL) was added Et.sub.3N (25.1 g, 248 mmol, 34.4 mL, 5 eq) and
Boc.sub.2O (21.7 g, 99.4 mmol, 22.8 mL, 2 eq). After addition, the
reaction mixture was stirred at 15-30.degree. C. for 12 hours. TLC
(Petroleum ether/Ethyl acetate=1:1, R.sub.f=0.4) showed the
starting material was consumed completely. The three reactions were
combined and the mixture was diluted with water (800 mL), then
adjusted to pH=3-4 with 0.5 N HCl solution. The organic layer was
separated and the aqueous phase was extracted with dichloromethane
(800 mL). The organic layers were combined and washed with brine
(800 mL), then dried with anhydrous Na2SO.sub.4, filtered and
concentrated in vacuum to give the crude product. The crude product
was purified by column chromatography on silica gel with petroleum
ether/ethyl acetate to give compound 29f as yellow solid.
[0875] Yield: 80 g, 72.3 mmol, 48.5%
[0876] Three reactions were carried out in parallel.
[0877] To a solution of compound 29f (25 g, 22.6 mmol, 1 eq) in DCM
(125 mL) and EtOH (300 mL) was added NH.sub.2NH.sub.2.H.sub.2O
(28.9 g, 565 mmol, 28 mL, 98% purity, 25 eq) in one portion. After
addition, the reaction mixture was stirred at 15-30.degree. C. for
18 hours. TLC (Petroleum ether/Ethyl acetate=1:1, R.sub.f=0.03)
showed the starting material was consumed completely. The three
reactions were combined and the mixture was diluted with water (1
L) and dichloromethane (800 mL), then adjusted to pH=3-4 with 0.5 N
HCl solution. The organic layer was separated and the aqueous phase
was extracted with dichloromethane (500 mL). The organic layers
were combined and washed with brine (800 mL), then dried with
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum to
give the crude product. The crude product was purified by column
chromatography on silica gel with DCM/MeOH to give compound 29g as
yellow oil.
[0878] Yield: 45 g, 46.1 mmol, 68%
[0879] Four reactions were carried out in parallel.
[0880] To a solution of compound 29g (11 g, 11.3 mmol, 1.0 eq) in
THF (100 mL) was added Et.sub.3N (3.4 g, 33.8 mmol, 4.7 mL, 3.0 eq)
and 4-nitrophenyl carbonochloridate (2.5 g, 12.4 mmol, 1.1 eq).
After addition, the reaction mixture was stirred at 15-30.degree.
C. for 18 hours. TLC (Petroleum ether/Ethyl acetate=1:1,
R.sub.f=0.4) showed the starting material was consumed completely.
The four reactions were combined and the mixture was diluted with
water (800 mL) and EtOAc (800 mL), then adjusted to pH=3-4 with 0.5
N HCl solution. The organic layer was separated and the aqueous
phase was extracted with EtOAc (500 mL). The organic layers were
combined and washed with brine (800 mL), then dried with anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuum to give the
crude product. The crude product was purified by column
chromatography on silica gel with petroleum ether: ethyl acetate to
give 29h as pale yellow sticky oil.
[0881] Yield: 29 g, 25.4 mmol, 56%
Example 30
[0882] Synthesis of Transient Si PTH(1-34) Conjugate 30
##STR00072##
[0883] Side chain protected PTH(1-34) on TCP resin having Fmoc
protected N-terminus was Fmoc deprotected according to the
procedure given in Materials and Methods. A solution of
Fmoc-Ser(Trt)-OH (997 mg, 1.75 mmol), PyBOP (911 mg, 1.75 mmol) and
DIPEA (305 .mu.L, 1.75 mmol) in DMF (5 mL) was added to 5.0 g (0.58
mmol) of the resin. The suspension was agitated overnight at rt.
The resin was washed 10.times. with DMF and Fmoc-deprotection was
performed as described above. A solution of 29h (2.66 g, 2.33 mmol)
and DIPEA (611 .mu.L, 3.50 mmol) in DMF (5 mL) was added to the
resin. The suspension was agitated overnight at rt. The resin was
washed 10.times. with DMF, 10.times. with DCM and dried in vacuo.
Cleavage of the peptide from the resin and removal of protecting
groups was achieved by adding 30 mL cleavage cocktail 100/3/3/2/1
(v/w/v/v/v) TFA/EDT/TES/water/thioanisole and agitating the
suspension for 1 h at rt. Crude 30 was precipitated in pre-cooled
diethyl ether (-18.degree. C.). The precipitate was dissolved in
ACN/water and purified by RP-HPLC. The product fractions were
freeze-dried.
[0884] Yield: 168 mg (5%), 30*9 TFA
[0885] MS: m/z 1155.92=[M+4H].sup.4+, (calculated monoisotopic mass
for [M+4H].sup.4+=1155.85).
Example 31
[0886] Synthesis of Transient 4.times.10 kDa S1 PEG Conjugate
31
##STR00073##
[0887] 2.3 mL of a solution containing 30 (13 mg/mL, 30 mg, 5.31
.mu.mol) in 8/2 (v/v) water/ethanol containing 0.1% TFA (v/v) and
10 mM methionine were added to 3.4 mL of a solution containing PEG
2.times.10 kDa maleimide (Sunbright GL2-200MA, 342 mg, 15.9
.mu.mol) in the same solvent. 0.5 M NaH.sub.2PO.sub.4 buffer (0.8
mL, pH 7.0) was added and the mixture was stirred for 30 min at rt.
20 .mu.L of TFA was added and the mixture was stored at 4.degree.
C. overnight. Conjugate 31 was purified by IEX followed by RP-HPLC
using a linear gradient of solvent system A (water containing 0.2%
AcOH v/v) and solvent system B (acetonitrile containing 0.2% AcOH
v/v). The product containing fractions were freeze-dried.
[0888] Yield: 161 mg (3.55 .mu.mol, 67%) conjugate 31*9 AcOH
Example 32
[0889] In Vitro Release Kinetics of Transient 4.times.10 kDa PEG
Conjugate 31 Conjugate 31 (11 mg) was dissolved in 1 vol % acetic
acid in water (1.8 mL) at 0.5 mg PTHeq/mL. Buffer exchange to pH
7.4 phosphate buffer (100 mM NaH.sub.2PO.sub.4, 10 mM L-methionine,
3 mM EDTA, 0.05% Tween-20, adjusted to pH 7.4 by NaOH) was
performed by SEC chromatography. The eluate was further diluted
with phosphate buffer to reach a concentration of 0.1 mg PTHeq/mL.
The resulting solution was sterile filtered and incubated at
37.degree. C. At time points, aliquots were withdrawn and analysed
by RP-HPLC. The fraction of released PTH at a particular time point
was calculated from the ratio of UV peak areas of liberated PTH and
PEG conjugate. The % released PTH was plotted against incubation
time. Curve-fitting software was applied to calculate the
corresponding half times of release.
[0890] Results:
[0891] For conjugate 31 a release half-life time of 14.5 d was
obtained.
Example 33
[0892] Pharmacodynamic Actions in Cynomolgus Monkeys During a
Single Dose PK/PD Study with Compound 18
[0893] Compound 18 was administered at 1 .mu.g/kg to male
cynomolgus monkeys (N=3) in a single subcutaneous PK/PD study
assessing serum calcium (sCa) levels and urinary calcium excretion
for 96 hours post-dose.
[0894] Results: Following compound 18 administration at 1 .mu.g/kg
to cynomolgus monkeys, sCa levels remained within the normal range
for 96 hours post-dose and a clear trend for decreased urinary
calcium levels over the first 24 hours was observed.
[0895] Conclusion: At a dose maintaining sCa in the normocalcemic
range, a concurrent decrease in urinary Ca excretion was observed
and compound 18 hereby addresses a key unmet medical need in
patients with HP.
Example 34
[0896] Pharmacokinetic Study of Transient 2.times.20 kDa S1 PEG
Conjugate 18 in Cynomolgus Monkeys
[0897] Naive cynomolgus monkeys (2-3.5 years, 2-5 kg) (3-5
males/3-5 females) received daily subcutaneous administrations of
18 at dose levels of 0.2, 0.5 and 1.5 .mu.g PTH/kg. Blood samples
were collected at; Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h
post-dose, at Day 8: pre-dose, at Day 14: predose, 8h, and 12 h and
at Day 28:3 h, 6 h, 8 h, 12 h, 18 h, 24 h, 72 h, 168 h, and 336 h)
and plasma was generated. Total PTH plasma concentrations were
determined by quantification of the N-terminal signature peptide
(sequence: IQLMHNLGK (SEQ ID NO: 123)) after LysC and GluC
digestion as presented earlier in Materials and Methods.
[0898] Results: Systemic exposure expressed as C.sub.max and AUC
increased in an approximately dose proportional manner. Systemic
exposure of Total PTH expressed as AUC accumulated approximately
3-fold from Day 1 to Day 28.
[0899] A low mean peak-to-trough ratio of Total PTH for all dose
groups was observed after daily subcutaneous administration in
cynomolgus monkeys at Day 28 (steady state observed from Day
8).
Example 35
[0900] Pharmacokinetic Study of Transient 2.times.20 kDa S1 PEG
Conjugate 18 in Sprague-Dawley Rats
[0901] Sprague-Dawley Crl:CD(SD) rats (initiation of dosing at 8
weeks of age) received daily subcutaneous administrations of 18 at
dose levels of 10, 30 and 60 .mu.g PTH/kg for 28 days. A TK group
containing of 9 males and 9 females per dose group was divided into
3 subgroups with 3 rats per subgroup. Blood samples were collected
up to 28 days with 3 rats per sex, per sampling time point. Samples
were collected at Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h
post-dose, and at Day 28:3 h, 6 h, 8 h, 12 h, 18 h, 24 h, and 336 h
and plasma was generated. The total PTH plasma concentrations were
determined by quantification of the N-terminal signature peptide
(sequence: IQLMHNLGK (SEQ ID NO: 123)) after LysC and GluC
digestion as presented earlier in Materials and Methods.
[0902] Results: Systemic exposure expressed as mean C.sub.max and
AUC increased in an approximately dose proportional manner.
Systemic exposure of Total PTH expressed as mean AUC accumulated
3-6 fold from Day 1 to Day 28. Systemic exposure in the female rat
was approximately 2-fold higher than in males.
[0903] A low mean peak-to-trough ratio of Total PTH for all dose
groups was observed after daily subcutaneous administration in
Sprague-Dawley rats at Day 28 (steady state observed from Day
8).
ABBREVIATIONS
[0904] ACN acetonitrile [0905] AcOH acetic acid [0906] Aib
2-aminoisobutyric acid [0907] BMD bone mineral density [0908] Bn
benzyl [0909] Boc tert-butyloxycarbonyl [0910] COMU
(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeni-
um hexafluorophosphate [0911] cAMP cyclic adenosine monophosphate
[0912] d day [0913] DBU 1,3-diazabicyclo[5.4.0]undecene [0914] DCC
N,N'-dicyclohexylcarbodiimide [0915] DCM dichloromethane [0916]
DIPEA N,N-diisopropylethylamine [0917] DMAP dimethylamino-pyridine
[0918] DMF N,N-dimethylformamide [0919] DMSO dimethylsulfoxide
[0920] DTT dithiothreitol [0921] EDTA ethylenediaminetetraacetic
acid [0922] eq stoichiometric equivalent [0923] ESI-MS electrospray
ionization mass spectrometry [0924] Et ethyl [0925] Fmoc
9-fluorenylmethyloxycarbonyl [0926] Glu-C endoproteinase Glu-C
[0927] h hour [0928] HATU
O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0929] HP hypoparathyroidism [0930] HPLC high
performance liquid chromatography [0931] ivDde
4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl [0932] LC
liquid chromatography [0933] LTQ linear trap quadrupole [0934]
Lys-C endoproteinase Lys-C [0935] LLOQ lower limit of
quantification [0936] Mal 3-maleimido propyl [0937] Me methyl
[0938] MeOH methanol [0939] min minutes [0940] Mint
monomethoxytrityl [0941] MS mass spectrum/mass spectrometry [0942]
m z mass-to-charge ratio [0943] OtBu tert-butyloxy [0944] PEG
poly(ethylene glycol) [0945] pH potentia Hydrogenii [0946] PK
pharmacokinetics [0947] Pr propyl [0948] PTH parathyroid hormone
[0949] PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate [0950] Q-TOF quadrupole time-of-flight [0951]
RP-HPLC reversed-phase high performance liquid chromatography
[0952] rt room temperature [0953] sCa serum calcium [0954] SIM
single ion monitoring [0955] SEC size exclusion chromatography
[0956] sc subcutaneous [0957] sP serum phosphate [0958] t.sub.1/2
half life [0959] TCP tritylchloride polystyrol [0960] TES
triethylsilane [0961] TFA trifluoroacetic acid [0962] THF
tetrahydrofuran [0963] TK toxicokinetic [0964] Tmob
2,4,6-trimethoxybenzyl [0965] TPTx thyroparathyroidectomy [0966]
Trt triphenylmethyl, trityl [0967] ULOQ upper limit of
quantification [0968] UPLC ultra performance liquid chromatography
[0969] UV ultraviolet [0970] ZQ single quadrupole
Sequence CWU 1
1
124184PRTHomo sapiens 1Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu
Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp
Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp
Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75 80Ala Lys Ser
Gln283PRTArtificial SequenceHuman PTH 1-83 2Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75
80Ala Lys Ser382PRTArtificial Sequencehuman PTH 1-82 3Ser Val Ser
Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn
Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu
50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr
Lys65 70 75 80Ala Lys481PRTArtificial Sequencehuman PTH 1-81 4Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly
Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser
His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val
Leu Thr Lys65 70 75 80Ala580PRTArtificial Sequencehuman PTH 1-80
5Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5
10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala
Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu
Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn
Val Leu Thr Lys65 70 75 80679PRTArtificial Sequencehuman PTH 1-79
6Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5
10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala
Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu
Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn
Val Leu Thr65 70 75778PRTArtificial Sequencehuman PTH 1-78 7Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His
Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val
Leu65 70 75877PRTArtificial Sequencehuman PTH 1-77 8Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe
Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln
Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55
60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val65 70
75976PRTArtificial Sequencehuman PTH 1-76 9Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu Gly Glu Ala Asp Lys Ala Asp Val Asn65 70 751075PRTArtificial
Sequencehuman PTH 1-75 10Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro
Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu
Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala
Asp Lys Ala Asp Val65 70 751174PRTArtificial Sequencehuman PTH 1-74
11Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1
5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala
Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu
Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp65
701273PRTArtificial Sequencehuman PTH 1-73 12Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys
Ser Leu Gly Glu Ala Asp Lys Ala65 701372PRTArtificial Sequencehuman
PTH 1-72 13Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg
Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu
Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys65
701471PRTArtificial Sequencehuman PTH 1-71 14Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys
Ser Leu Gly Glu Ala Asp65 701570PRTArtificial Sequencehuman PTH
1-70 15Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp
Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val
Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala65
701669PRTArtificial Sequencehuman PTH 1-69 16Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys
Ser Leu Gly Glu651768PRTArtificial Sequencehuman PTH 1-68 17Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His
Glu 50 55 60Lys Ser Leu Gly651867PRTArtificial Sequencehuman PTH
1-67 18Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp
Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val
Glu Ser His Glu 50 55 60Lys Ser Leu651966PRTArtificial
Sequencehuman PTH 1-66 19Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro
Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu
Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys
Ser652065PRTArtificial Sequencehuman PTH 1-65 20Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55
60Lys652164PRTArtificial Sequencehuman PTH 1-64 21Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe
Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln
Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55
602263PRTArtificial Sequencehuman PTH 1-63 22Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His 50 55
602362PRTArtificial Sequencehuman PTH 1-62 23Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser 50 55
602461PRTArtificial Sequencehuman PTH 1-61 24Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu 50 55
602560PRTArtificial Sequencehuman PTH 1-60 25Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn Val Leu Val 50 55 602659PRTArtificial
Sequencehuman PTH 1-59 26Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro
Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu
Asp Asn Val Leu 50 552758PRTArtificial Sequencehuman PTH 1-58 27Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly
Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val 50
552857PRTArtificial Sequencehuman PTH 1-57 28Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu Asp Asn 50 552956PRTArtificial Sequencehuman
PTH 1-56 29Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg
Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp 50
553055PRTArtificial Sequencehuman PTH 1-55 30Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg Lys Lys Glu 50 553154PRTArtificial Sequencehuman PTH 1-54
31Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1
5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala
Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys 503253PRTArtificial
Sequencehuman PTH 1-53 32Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro
Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
503352PRTArtificial Sequencehuman PTH 1-52 33Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
Pro Arg 503451PRTArtificial Sequencehuman PTH 1-51 34Ser Val Ser
Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn
Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
45Gln Arg Pro 503550PRTArtificial Sequencehuman PTH 1-50 35Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn
Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
45Gln Arg 503649PRTArtificial Sequencehuman PTH 1-49 36Ser Val Ser
Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn
Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
45Gln3748PRTArtificial Sequencehuman PTH 1-48 37Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
453847PRTArtificial Sequencehuman PTH 1-47 38Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly 35 40
453946PRTArtificial Sequencehuman PTH 1-46 39Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala 35 40
454045PRTArtificial Sequencehuman PTH 1-45 40Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp 35 40 454144PRTArtificial
Sequencehuman PTH 1-44 41Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro
Leu Ala Pro Arg 35 404243PRTArtificial Sequencehuman PTH 1-43 42Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro 35
404342PRTArtificial Sequencehuman PTH 1-42 43Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro Leu Ala 35 404441PRTArtificial Sequencehuman
PTH-41 44Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu 35
404540PRTArtificial Sequencehuman PTH 1-40 45Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu Gly Ala Pro 35 404639PRTArtificial Sequencehuman PTH 1-39
46Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1
5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val
His 20 25 30Asn Phe Val Ala Leu Gly Ala 354738PRTArtificial
Sequencehuman PTH 1-38 47Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
354837PRTArtificial Sequencehuman PTH 1-37 48Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
Ala Leu 354936PRTArtificial Sequencehuman PTH 1-36 49Ser Val Ser
Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn
Phe Val Ala 355035PRTArtificial Sequencehuman PTH 1-35 50Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val 355134PRTArtificial Sequencehuman PTH 1-34 51Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe5233PRTArtificial Sequencehuman PTH 1-33 52Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn5332PRTArtificial Sequencehuman PTH 1-32 53Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
305431PRTArtificial Sequencehuman PTH 1-31 54Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val 20 25
305530PRTArtificial Sequencehuman PTH 1-30 55Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 305629PRTArtificial
Sequencehuman PTH 1-29 56Ser Val Ser Glu Ile Gln Leu Met His Asn
Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln 20 255728PRTArtificial Sequencehuman PTH 1-28 57Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 20
255827PRTArtificial Sequencehuman PTH 1-27 58Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys 20 255926PRTArtificial Sequencehuman
PTH 1-26 59Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys 20
256025PRTArtificial Sequencehuman PTH 1-25 60Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg
Val Glu Trp Leu Arg 20 256184PRTArtificial Sequenceamidated human
PTH 1-84MOD_RES(84)..(84)AMIDATION 61Ser Val Ser Glu Ile Gln Leu
Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu
Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu
Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg
Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu
Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75 80Ala
Lys Ser Gln6283PRTArtificial Sequenceamidated human PTH
1-83MOD_RES(83)..(83)AMIDATION 62Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75 80Ala Lys
Ser6382PRTArtificial Sequenceamidated human PTH
1-82MOD_RES(82)..(82)AMIDATION 63Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75 80Ala
Lys6481PRTArtificial Sequenceamidated human PTH
1-81MOD_RES(81)..(81)AMIDATION 64Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75
80Ala6580PRTArtificial Sequenceamidated human PTH
1-80MOD_RES(80)..(80)AMIDATION 65Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75
806679PRTArtificial Sequenceamidated human PTH
1-79MOD_RES(79)..(79)AMIDATION 66Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr65 70
756778PRTArtificial Sequenceamidated human PTH
1-78MOD_RES(78)..(78)AMIDATION 67Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn Val Leu65 70 756877PRTArtificial
Sequenceamidated human PTH 1-77MOD_RES(77)..(77)AMIDATION 68Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His
Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val65
70 756976PRTArtificial Sequenceamidated human PTH
1-76MOD_RES(76)..(76)AMIDATION 69Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp Val Asn65 70 757075PRTArtificial
Sequenceamidated human PTH 1-75MOD_RES(75)..(75)AMIDATION 70Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His
Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val65 70
757174PRTArtificial Sequenceamidated human PTH
1-74MOD_RES(74)..(74)AMIDATION 71Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu Ala Asp Lys Ala Asp65 707273PRTArtificial Sequenceamidated
human PTH 1-73MOD_RES(73)..(73)AMIDATION 72Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu Gly Glu Ala Asp Lys Ala65 707372PRTArtificial Sequenceamidated
human PTH 1-72MOD_RES(72)..(72)AMIDATION 73Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu Gly Glu Ala Asp Lys65 707471PRTArtificial Sequenceamidated
human PTH 1-71MOD_RES(71)..(71)AMIDATION 74Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu Gly Glu Ala Asp65 707570PRTArtificial Sequenceamidated human
PTH 1-70MOD_RES(70)..(70)AMIDATION 75Ser Val Ser Glu Ile Gln Leu
Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu
Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu
Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg
Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu
Gly Glu Ala65 707669PRTArtificial Sequenceamidated human PTH
1-69MOD_RES(69)..(69)AMIDATION 76Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu
Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly
Glu657768PRTArtificial Sequenceamidated human PTH
1-68MOD_RES(68)..(68)AMIDATION 77Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu
Gly657867PRTArtificial Sequenceamidated human PTH
1-67MOD_RES(67)..(67)AMIDATION 78Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser
Leu657966PRTArtificial Sequenceamidated human PTH
1-66MOD_RES(66)..(66)AMIDATION 79Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys
Ser658065PRTArtificial Sequenceamidated human PTH
1-65MOD_RES(65)..(65)AMIDATION 80Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55
60Lys658164PRTArtificial Sequenceamidated human PTH
1-64MOD_RES(64)..(64)AMIDATION 81Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55
608263PRTArtificial Sequenceamidated human PTH
1-63MOD_RES(63)..(63)AMIDATION 82Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu Ser His 50 55 608362PRTArtificial
Sequenceamidated human PTH 1-62MOD_RES(62)..(62)AMIDATION 83Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser 50
55 608461PRTArtificial Sequenceamidated human PTH
1-61MOD_RES(61)..(61)AMIDATION 84Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu Val Glu 50 55 608560PRTArtificial
Sequenceamidated human PTH 1-60MOD_RES(60)..(60)ACETYLATION 85Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly
Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val 50 55
608659PRTArtificial Sequenceamidated human PTH
1-59MOD_RES(59)..(59)AMIDATION 86Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys Glu Asp Asn Val Leu 50 558758PRTArtificial Sequenceamidated
human PTH 1-58MOD_RES(58)..(58)AMIDATION 87Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn Val 50 558857PRTArtificial Sequenceamidated
human PTH 1-57MOD_RES(57)..(57)AMIDATION 88Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp Asn 50 558956PRTArtificial Sequenceamidated
human PTH 1-56MOD_RES(56)..(56)AMIDATION 89Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
Arg Lys Lys Glu Asp 50 559055PRTArtificial Sequenceamidated human
PTH 1-55MOD_RES(55)..(55)AMIDATION 90Ser Val Ser Glu Ile Gln Leu
Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu
Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu
Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg
Lys Lys Glu 50 559154PRTArtificial Sequenceamidated human PTH
1-54MOD_RES(54)..(54)AMIDATION 91Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
Lys 509253PRTArtificial Sequenceamidated human PTH
1-53MOD_RES(53)..(53)AMIDATION 92Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys
509352PRTArtificial Sequenceamidated human PTH
1-52MOD_RES(52)..(52)AMIDATION 93Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg
509451PRTArtificial Sequenceamidated human PTH
1-51MOD_RES(51)..(51)AMIDATION 94Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro
509550PRTArtificial Sequenceamidated human PTH
1-50MOD_RES(50)..(50)AMIDATION 95Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg
509649PRTArtificial Sequenceamidated human PTH
1-49MOD_RES(49)..(49)AMIDATION 96Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40
45Gln9748PRTArtificial Sequenceamidated human PTH
1-48MOD_RES(48)..(48)AMIDATION 97Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 459847PRTArtificial
Sequenceamidated human PTH 1-47MOD_RES(47)..(47)AMIDATION 98Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly 35 40
459946PRTArtificial Sequenceamidated human PTH
1-46MOD_RES(46)..(46)AMIDATION 99Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg Asp Ala 35 40 4510045PRTArtificial
Sequenceamidated human PTH 1-45MOD_RES(45)..(45)AMIDATION 100Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp 35 40
4510144PRTArtificial Sequenceamidated human PTH
1-44MOD_RES(44)..(44)AMIDATION 101Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu Ala Pro Arg 35 4010243PRTArtificial Sequenceamidated
human PTH 1-43MOD_RES(43)..(43)AMIDATION 102Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
Leu Gly Ala Pro Leu Ala Pro 35 4010342PRTArtificial
Sequenceamidated human PTH 1-42MOD_RES(42)..(42)AMIDATION 103Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu Ala 35
4010441PRTArtificial Sequenceamidated human PTH
1-41MOD_RES(41)..(41)AMIDATION 104Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro Leu 35 4010540PRTArtificial Sequenceamidated human PTH
1-40MOD_RES(40)..(40)AMIDATION 105Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala Pro 35 4010639PRTArtificial Sequenceamidated human PTH
1-39MOD_RES(39)..(39)AMIDATION 106Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
Ala 3510738PRTArtificial Sequenceamidated human PTH
1-38MOD_RES(38)..(38)AMIDATION 107Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly
3510837PRTArtificial Sequenceamidated human PTH
1-37MOD_RES(37)..(37)AMIDATION 108Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu
3510936PRTArtificial Sequenceamidated human PTH
1-36MOD_RES(36)..(36)AMIDATION 109Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala
3511035PRTArtificial Sequenceamidated human PTH
1-35MOD_RES(35)..(35)AMIDATION 110Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val
3511134PRTArtificial Sequenceamidated human PTH
1-34MOD_RES(34)..(34)AMIDATION 111Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn
Phe11233PRTArtificial Sequenceamidated human PTH
1-33MOD_RES(33)..(33)AMIDATION 112Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn11332PRTArtificial
Sequenceamidated human PTH 1-32MOD_RES(32)..(32)AMIDATION 113Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 3011431PRTArtificial Sequenceamidated human PTH
1-31MOD_RES(31)..(31)AMIDATION 114Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val 20 25 3011530PRTArtificial
Sequenceamidated human PTH 1-30MOD_RES(30)..(30)AMIDATION 115Ser
Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10
15Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25
3011629PRTArtificial Sequenceamidated human PTH
1-29MOD_RES(29)..(29)AMIDATION 116Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln 20 2511728PRTArtificial Sequenceamidated
human PTH 1-28MOD_RES(28)..(28)AMIDATION 117Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu 20 2511827PRTArtificial
Sequenceamidated human PTH
1-27MOD_RES(27)..(27)AMIDATION 118Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys 20 2511926PRTArtificial Sequenceamidated human PTH
1-26MOD_RES(26)..(26)AMIDATION 119Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys 20 2512025PRTArtificial Sequenceamidated human PTH
1-25MOD_RES(25)..(25)AMIDATION 120Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg 20 25121141PRTHomo sapiens 121Ala Val Ser Glu His Gln Leu
Leu His Asp Lys Gly Lys Ser Ile Gln1 5 10 15Asp Leu Arg Arg Arg Phe
Phe Leu His His Leu Ile Ala Glu Ile His 20 25 30Thr Ala Glu Ile Arg
Ala Thr Ser Glu Val Ser Pro Asn Ser Lys Pro 35 40 45Ser Pro Asn Thr
Lys Asn His Pro Val Arg Phe Gly Ser Asp Asp Glu 50 55 60Gly Arg Tyr
Leu Thr Gln Glu Thr Asn Lys Val Glu Thr Tyr Lys Glu65 70 75 80Gln
Pro Leu Lys Thr Pro Gly Lys Lys Lys Lys Gly Lys Pro Gly Lys 85 90
95Arg Lys Glu Gln Glu Lys Lys Lys Arg Arg Thr Arg Ser Ala Trp Leu
100 105 110Asp Ser Gly Val Thr Gly Ser Gly Leu Glu Gly Asp His Leu
Ser Asp 115 120 125Thr Ser Thr Thr Ser Leu Glu Leu Asp Ser Arg Arg
His 130 135 14012220PRTArtificial SequenceArtificial random coil
122Gly Gly Pro Gly Gly Pro Gly Pro Gly Gly Pro Gly Gly Pro Gly Pro1
5 10 15Gly Gly Pro Gly 201239PRTArtificial SequenceSignature
peptide used in analysis 123Ile Gln Leu Met His Asn Leu Gly Lys1
51247PRTArtificial SequenceSignature peptide used in analysis
124Leu Gln Asp Val His Asn Phe1 5
* * * * *
References