CZ23397A3 - Derivatives of parathormone and parathormone related peptide, process of their preparation and pharmaceutical compositions containing thereof - Google Patents

Abstract

PTH or PTHrP compounds having potent antagonistic activity at the PTH/PTHrP receptor in which at least one of the amino acid residues naturally occurring in positions 2 and 10 is replaced by tryptophane or another amino acid residue bearing an aromatic or heteroaromatic group on its side chain, and optionally at least one of the amino acid residues naturally occurring in positions 3 and 6 is further replaced by tryptophane or another amino acid residue bearing an aromatic or heteroaromatic group on its side chain, having pharmacological activity, e.g. prevention or treatment of conditions which are associated with increased plasma calcium caused by excessive release of PTH or PTHrP.

Description

Technical Field: i

The invention relates to parathyroid hormone (PTH) derivatives and parathyroid hormone related peptide (PTHrP), to a process for their preparation, to pharmaceutical compositions containing them and to their use as medicaments.

SUMMARY OF THE INVENTION

In particular, the invention provides derivatives of parathyroid hormone and a parathyroid hormone related peptide having a potent PTH / PTHrP receptor antagonist activity in which at least one of the amino acid residues naturally occurring at positions 2 and 10 is replaced by tryptophan or another amino acid residue containing a side chain or an aromatic or heteroaromatic group thereof, and optionally further comprising at least one of the amino acid residues naturally occurring at the 3- and 6-positions is replaced by tryptophan. or another amino acid residue that contains an aromatic or heteroaromatic group in its side chain. These compounds are hereinafter referred to as compounds of the invention.

According to a particular embodiment, the invention provides derivatives of parathyroid hormone and a parathyroid hormone-related peptide in which the amino acid residue naturally occurring at position 10 is replaced by tryptophan or another amino acid residue containing an aromatic or heteroaromatic group in its side chain, and further optionally, at least one of the amino acid residues naturally occurring at positions 3 and 6 is replaced by tryptophan; or

- another 2 amino acid residue containing an aromatic or heteroaromatic group in its side chain.

The term parathyroid hormone related peptide (PTHrPj refers to any naturally occurring form of a parathyroid hormone related peptide, for example, a human, bovine, chicken, rat, or murine parathyroid hormone related peptide. For convenience, the same amino acid residue numbering system will be used in the following description of a parathyroid hormone, regardless of whether any α-amino acid residue of the Parathyroid hormone related peptide sequence is replaced or deleted.

The term parathyroid hormone (PTH) refers to any naturally occurring form of parathyroid hormone, for example human, bovine, chicken, rat, or murine parathyroid hormone. For convenience, the same parathyroid amino acid residue numbering system will be used in the following description as usual, regardless of whether any α-amino acid residue of the parathyroid hormone sequence is replaced or deleted according to the invention.

The terms parathyroid related peptide derivative (PTHrP derivative) or parathyroid hormone derivative (PTH derivative) refer to a peptide comprising an amino acid sequence based on the N-terminal fragment of a parathyroid hormone peptide or an N-terminal fragment of a parathyroid hormone, preferably a parathormone-related peptide fragment, or a parathyroid hormone fragment. which begins with any of residues 2 to 7 and ends with any of residues 27 to 38, for example the N-terminal fragment of a parathyroid hormone-related peptide or a parathyroid hormone containing up to 31. 34, 35, 36, 37, or 38 amino acid residues. Accordingly, it is understood that the terms parathyroid hormone-related peptide or parathyroid hormone include peptides in which one or more amino acid residues of said N-terminal fragment are deleted, preferably at the N-terminus.

It is to be understood that these terms also include peptides in which, in addition to the substitution at position 2 and / or 10 and optionally 3 and / or 6 of the invention, one or more amino acid residues of a naturally occurring sequence is replaced by one or more other amino acid residues. (natural or unnatural). N-terminal fragments of 1-38, 1-34, and 1-31 of the human parathyroid hormone-related peptide have the sequences set forth in SEQ ID NOs: 1, 2 or 3.

The N-terminal fragments 1-38, 1-34, and 1-31 of the human parathyroid hormone have the sequences shown in SEQ ID NOs: 4, 5 or 6.

The N-terminus of the iparathormone derivative or parathyroid hormone-related peptide can. be a free or protected amino group which bears, for example. N-protecting group,. as is described. in Protective Groups in Organic Synthesis, TW Greene, by J. Wiley Sons NY (1981), 219-287, and is preferably protected by the group R-CO-, RO-CO-, RO-CH ₂ -CO- or R-. SO ₂ , or may be an amino-bearing residue. R /,. R '.' -NH-CO-. or R-NH-CS- as defined below.

The C-terminus of the compounds of the invention may be COOH, CONHj or a mono- or disubstituted amide group, for example -CO -R _c R _d , where one of the radicals R and R _d represents a hydrogen atom and the other represents an aliphatic radical, e.g. Or both R _c and R _d represent aliphatic or R _c and R _d 'together with the nitrogen atom to which they are attached form a heterocyclic radical, for example a pyrrolidinyl or piperidinyl group.

Parathyroid-related or parathyroid hormone-related peptides of the invention exhibit potent PTH / PTHrP receptor antagonist activity, e.g., bind to the PTH / PTHrP receptor, exhibit intrinsic activity (i) in a functional bioassay, i. and antagonizing a parathyroid hormone-related peptide or parathyroid hormone or a fragment thereof, for example, a parathyroid hormone-related peptide (1-34) or parathyroid hormone (1-34) in a functional bioassay with a value of ·. pA2 of at least 6.5. Preferably, the compounds of the invention exhibit an intrinsic activity value of 0.03 or less or even undetectable in some assays. An example of a functional bioassay is the osteosarcoma-based adenylate cyclase assay commonly used in the art; This assay determines the in vitro range, i

wherein the test compound stimulates adenylate cyclase activity or antagonizes the action of parathyroid hormone related peptides or. parathyroid hormone or a fragment thereof in rat osteosarcoma cells of the UMR line, for example UMR-106-06 according to the method of Marcus and AurbachTv Endocrinology, 85, 801-810 (1969). as described below *.

By amino acid is meant a naturally occurring (natural), commercially available or non-naturally occurring (unnatural) amino acid, or an optical isomer thereof. Unnatural amino acids are amino acids that are not incorporated into proteins subject to the mRNK notation, for example, β-Nal, fluoro-α-amino acids such as fluoroalanine, cyclohexylalanine or trimethylsilylaline.

Natural amino acids are well known in the art. A list of these, together with standard abbreviations, is given in U.S.P.T.O., Trademark Official Gazette, May 15, 1990, p. 33 at 46.

Natural amino acids are listed below:

AND Ala alanine D Asp aspartic acid E Glu glutamic acid F Phe phenylalanine

G Gly . glycine H His histidine AND Ile isoleucine TO Lys lysine L Leu leucine - M Met methionine N ^: Asn asparagine Q Gin glutamine fc R Arg arginine WITH Ser serine T - Thr threonine in Wall valine. w— ' Trp · - - --tryptophan.- Y Tyr tyrosine

By amino acid residue containing, in its side chain, an aromatic or heteroaromatic group is meant an amino acid residue whose side chain is, for example, an optionally substituted ring-substituted phenylmethyl group, 1- or 2-naphthylmethyl. 14 or 2-naphthylethyl, 4- or 4-pyridylmethyl, 3-indolylmethyl or 3-indazolylmethyl, preferably an amino acid residue of the formula -NH-CHR-CO-; as defined below.

According to a preferred embodiment, the invention provides a compound of the general formula Ί

R - [<X ² ) P, (X ³ ) q, (X ^e ) _a , R ¹⁰ ] -D- (yx) R, (I) wherein x is a residue number selected from the group consisting of Numbers 31, 34, 35, .36, 37, and 38, y is a residue number selected from the group consisting of 1,

2, 3, 4, 5, 6 and 7

X ² is Val or independently has one of the meanings specified for X ¹⁰ ,

X ¹ is Ser or independently has one of the meanings specified for X ¹⁰ ,

X 0 represents Gln or independently has one of the meanings specified for X ¹⁰ ,

R ¹³ is Asp or X ¹⁰ , wherein

X ¹⁰ is Trp or a radical -NH-CHR-CO-, wherein

R 'is a radical of formula (a), (b), - (c) or <d)

(d)

n Is 1 or 2 m Is 1 or 2 0 is 0 or 1, ring A is optionally substituted with one or more

substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy, wherein the two alkyl or two alkoxy substituents may also together form a ring structure fused to ring A, each of rings B and C may be independently substituted as described above for ring A, and

_And Y represents a direct bond, -CH ₂ -, oxygen, NH or N-alkyl containing 1 to 6 carbon atoms,

D represents an amino acid sequence derived from

An N-terminal fragment, a parathyroid hormone related peptide or parathyroid hormone, each of p and pass having a value of 1. with p having a value of 0 if y is greater than 2, p having a value of 0 if y is greater than 3 and having a value of 0 if y is greater than

.....- - - · - -. ...... '. ... . ..

R means hydrogen atom-, the group R-CO-, RO-CO-, R _"-O-CH2 -CO-, R-SO ₂ -, R ', R'-NH-CO-, R'-NH- or NH ₂ -alkylene-CO- having 1 to 6 carbon atoms in the alkylene moiety, wherein

R represents an alkyl group having 1 to 8 carbon atoms, a ω-carboxyalkyl group having 1 to 6 carbon atoms in the alkyl moiety, a ω- [(alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms in the alkoxy moiety and 1 to 6 carbon atoms C 2 -C 8 alkenyl, C 5 -C 7 cycloalkyl, C 5 -C 7 cycloalkylalkyl and C 1 -C 4 alkyl, or phenyl, phenylalkyl C 1 -C 4 alkyl, 1-naphthyl, 2-naphthyl, 1-naphthylalkyl of 1 to 2 carbon atoms in the alkyl moiety or 2-naphthyl alkyl of 1 to 2 carbon atoms. alkyl moieties, wherein any of these groups is optionally substituted on the ring by one or more substituents selected from the group consisting of fluorine atom, chlorine atom, nitro group, C 1 -C 4 alkyl group and C 1 -C 4 alkoxy group, or heteroaryl,

R independently has one of the meanings given for R except for a C 1 -C 6 -carboxyalkyl group of the alkyl moiety and a C 1 -C 4 (alkoxy) carbonyl] alkyl group of the alkoxy moiety; and (C 1 -C 6) alkyl;

R _a is hydroxy or amino, with the proviso that at least one of X ² and R ¹⁰ 'has the meaning of X ^xo .

If the substituents of ring A, B or C together form a ring structure, it may be, for example, -O-CH3-CH2-O-. An example of a polycyclic cycloalkyl group is, for example, an adamantyl group.

Heteroaryl in the meaning of R is meant a 5-, 6-, or 7-membered unsaturated heterocyclic radical containing at least one nitrogen atom and optionally another heteroatom such as nitrogen, oxygen or sulfur, and optionally condensed with a benzene ring. The heteroaryl group is preferably an indolyl, quinolyl or isoquinolyl group.

The compounds of the invention may exist, for example, in free form, in salt form or in the form of their complexes.

Acid addition salts may be formed, for example, with (organic acids, polymeric acids, and inorganic acids. Such acid addition salts include, for example, hydrochlorides and acetates. hydroxides such as calcium or zinc salts and / or by adding polymeric organic substances.

In the compounds of formula I, the following meanings of the individual substituents are preferred, either individually or in any combination or sub-combination:

1: - - D- - · * represents an N-terminal fragment; hPTHrP; 2. D represents the N-terminal fragment 'hPTH.;. 3. X ^{5 * 7 8 * 10} represents Trp; 4. X ¹⁰ represents an -NH-CHR-CO- group wherein R 'is a radical of formula (a), (b) or (c)', preferably

(a) or (c), more preferably a residue of formula (a);

5. X ² is Trp or -NH-CHR'-CO-. wherein R 'is a radical of formula (a), (b) or (c);

6 .. each of X ² and X ¹⁰ is Trp or -NH-CHR'-CO- wherein R 'is a radical of formula (a), (b) or (c);

7. each of X ³ and X ^1G represents Trp or a group

-NH-CHR-CO-, wherein R is a radical of formula (a), (b) or (c);

8. each of X ^s and X ¹⁰ represents Trp or a group

-NH-CHR'-CO-, wherein R is a radical of formula (a), (b). or (c);

Ninth, each of X ^2, X ^a and X ¹⁰ is Trp or -NH-CHR'-CO- wherein R 'denotes

of formula (a), (b) or (c); 10. n. is 1; 11. y is a residue number selected from numbers 3, 4, 5, 6 and 7, preferably groups Number 3 including or 5; 12. X is the residue number selected from the numbers 31 and 34; groups including 13 . R R is H, N-alkylene-CO- having 1 to 6 atoms in the moiety; •! ¹ R-SO _2, carbon R '' or in alkylene- 14.. R represents an alkyl group having 1 up to 8 carbon atoms,

C 1 -C 4 -phenylalkyl, 1- or 2-naphthyl or i

or 2-naphthylalkyl having 1 to 2 carbon atoms in the alkyl moiety;

15. R represents a C 1 -C 6 -carboxyalkyl group in the alkyl moiety.

As already mentioned, one or more amino acid residues located at positions other than 2 and / or 10 may be further replaced. a natural or unnatural amino acid residue as mentioned above or may be omitted. When the compounds of the invention are derivatives of hPTHrP, they may be subjected to an additional amino acid substitution, for example at the 3-position, e.g. Ala, at the 4-position, e.g. Trp, at the 5-position, e.g. at position 11 and / or 13, e.g. Leu, or at position 34, e.g. D-Ala; and their amino acid sequence may optionally be truncated at the N-terminus by deleting 1 to 5. 7 amino acid residues. When the compounds of the invention are derivatives of hPTH, the amino acid residues may be further replaced, for example, in at least one of positions 8, 16, 18, 33 and 34, for example Leu ⁸ , Ala ", Gin", Thr ³³ ,

Ala ³⁴ , or their D-isomers. .

l ·

The invention also provides a process for the preparation of derivatives

X of a parathyroid hormone or a parathyroid hormone related peptide of the invention. They may be prepared sequentially either in solution or by solid phase synthesis or by genetic engineering methods or combinations thereof.

The compounds of the invention can be prepared, for example, by:

a) removing at least one protecting group which is present in a peptide derivative ^-1 PTHrP or PTH according to the invention / ^ as "compound-Obecného'- formula I in protected form; or that..se ..

»Ί _

b) joining two peptide fragments in a protected, partially protected or unprotected form to each other by amide linkage, the peptide fragments being such that they are obtained. The amino acid sequence of the desired peptide derivative of a parathyroid hormone or a parathyroid hormone, for example of the formula I, optionally followed by step a). way or that

c) introducing a protecting group or substituent in a selective manner onto the amino group of the N-terminal residue of the desired sequence or N-terminal fragment thereof in protected or unprotected form, optionally carrying out process step a) and isolating the parathyroid hormone related peptide or parathyroid hormone derivatives , in free form, in salt form or in complex form.

Process steps a), b) 'and #' may be performed analogously to known methods, for example as known in the art of peptide chemistry or as described in the following examples. If desired, protecting groups suitable for use with peptides for functional groups not participating in the reaction may be used in these reactions. The term protecting group may also mean a polymeric resin containing functional groups.

If the compounds of the invention contain unnatural and natural residues, they can be prepared by combining a chemical multistage process and genetic engineering. A peptide sequence (either a complete sequence - or fragment) composed of genetically encoded amino acid residues can be produced by genetic engineering and the desired unnatural amino acids or terminal amino substituent can be incorporated by chemical synthesis and, if desired, fragments can be joined to remove the protecting group or protecting groups, if present. .

The following abbreviations are used in the following examples:,

DMF dimethylformamide,

DMA N, N-dimethylacetamide,

Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl, tBu tert-butyl,> 4

PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexa- * ¹

Τί, ΐ fluorophosphate, '.

Nal (2) L-3- (2-naphthyl) alanine,

DIEA N, N-diisopropyl-N-ethylamine a

Fmoc 9-fluorenylmethoxycarbonyl ........ Where mass spectrometry data are reported, these are M * as determined by electrospray spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 [Trp ^4-10] hPTHrP (1-34) OH

The peptide is synthesized in multiple stages on a polystyrene-based resin support, the α-amino group being protected by. The 9-fluorenylmethoxycarbonyl and side chain functional groups are protected as follows:

Asp (OtBu), Glu (OtBu), His (Trt), Lys (Boc), Asn (Trt), Gln (Trt), Arg (Pmc), Ser (tBu), Trp (Boc), and Thr (tBu).

Fmoc-Ala-oxymethyl-4-phenoxymethyl-co (polystyrene-divinylbenzene) (about 0.5 mmol / g) is used as a starting material for multi-stage solid phase peptide synthesis, which consists of repeating cycles of deprotection of α α. - amino groups, washing, condensation (ie

The addition of an additional amino acid residue to the growing peptide chain and the washing of the .alpha.-9-fluorenylmethoxycarbonyl group is cleaved using 20% piperidine in N, N-dimethylacetamide. In the condensation reaction, 4 equivalents of 9-fluorenylmethoxycarbonylamino acid and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate and 8 equivalents of N, N-diisopropyl-N-ethyl amine-N-N-dimethylacetamide Po are used per amino group. build ._ "build.

The terminal 9-fluorenylmethoxycarbonyl protecting group is removed with piperidine in N, N-dimethylacetamide. as described above. The peptide is cleaved f

from the resin used as the carrier and all side chain protecting groups are simultaneously removed using a 5% dodecylmethylsulfide reagent and 5% water in 1% trifluoroacetic acid, which is left to react for two hours at room temperature. The resin particles were filtered off, washed with trifluoroacetic acid. and the product is precipitated from the combined filtrates by adding 10 to 20 volumes of diethyl ether. The precipitate was washed with ether and dried. The product was purified by column chromatography on C-18-large pore silica gel using a gradient of acetonitrile in 2% aqueous phosphoric acid. Fractions containing. the pure compound is isolated, filtered through an anion exchange resin (Biorad, AG4-X4 acetate form) and lyophilized to give the title compound. '

Mass spectrometry: 4146.

E

In analogy to Example 1, but using the appropriate amino acids, the following compounds can be prepared:

Example 2.

[Trp ¹⁰ , Leu ¹¹ , Leu ¹³ ] hPTHrP (1-34) OH. »»

Mass Spectrometry: 4059 .., ''

Example 3 »

·. - 17 '[Trp ¹⁰ , Leu ¹¹ ] hPTHrP (1-34) OH';

Mass Spectrometry: 4073 '' '

EXAMPLE 4 · '[Trp ^3.10] hPTHrP (1-34) OH ^υ

Mass Spectrometry: 4188 ' ^w '

Example 5 ['Trp ² ' ¹⁰ ] hPTH'rP '('1-3'4) OH '' .......... '............

, - · '· *

Mass spectrometry: 4176

Example 6 [Trp ¹⁰ ] hPTHrP (2-3) OH

Mass spectrometry: 4018.3

Example 7 [Trp ¹⁰ ] hPTHrP (3-34) OH

Mass spectrometry: 3919.2

Example 8 [Trp ² ] hPTHrP (1-34) OH

Mass spectrometry: 4104.9

Example 9 [Trp ¹⁰ ] hPTHrP (1-34) OH Mass Spectrometry: 4189

-Example .10. ..... _. _____ - j [Leu ⁸ , Trp ¹⁰ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³ '] hPTH (1-34) OH Mass spectrometry: 4036

Example 11 ~ [Leu®, Trp ¹⁰ , 'Å' ^ls ', Gin ¹⁸ '/ Thr' ³³ , Ala ³ '] hPTH' (2- 34) OH · Mass Spectrometry: 3 94 9 w

Example 12 [Leu ⁸ , Trp ¹⁰ , DLeu ¹¹ , Gin ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH

Mass spectrometry: 4079

Example 13 [Phe ² , Trp ¹⁰ ] hPTHrP (1-34) OH

Mass spectrometry: 4137.1

Example 14 [Trp ² , Trp ¹⁰ ] hPTHrP (2-34) OH

Mass spectrometry: 4105.1

Example 15 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (1-3) OH Mass Spectrometry: 4146.8

Example 16 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (6 - 3 4) OH Mass spectrometry: 3623.3

Example 17 [Trp ¹⁰ ] hPTHrP (7-34) OH Mass spectrometry: 3437.6

Example 18 [Trp ³ , Trp ¹⁰ ] hPTHrP (3-4) OH Mass Spectrometry: 4018.0

Example 19 [Trp ¹⁰ ] hPTHrP (1-34) OH Mass Spectrometry: 4088.7

Example 20 [Trp ⁶ , Trp ¹⁰ ] hPTHrP (3-34) NH ₂ Mass Spectrometry: 3976.8

EXAMPLE 21 [Phe ⁶ , Trp ¹⁰ ] hPTHrP (1-34) OH Mass Spectrometry: 3546.1

Example 22 [Phe ² , Phe *, Trp ¹⁰ ] hPTHrP (1-34) OH

Mass spectrometry: 4155.1

Example 23 ¹

N '-3-Aminopropionyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂ Mass Spectrometry: 3932.0

Example 24

N '-benzyloxycarbonyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

This peptide was prepared in an analogous manner to Example 1 except that 4- (2,4-dimethoxyphenyl-9-fluorenylmethoxycarbonylaminomethyl-phenoxy) (polystyrene-1% -dinylbenzene) (about 0.4 mmol / g) was used as starting material. For example, as described in Tetrah. Letters, 28: 3787-3790 (1987), Z-Ser-OH (benzyloxycarbonyl-Ser-OH) is added to the peptide chain in the last synthesis cycle. and the peptide is cleaved and purified as described in Example 1.

Mass spectrometry: 3975.3

Example 25

N ° -2-acetyl- [Trp ¹⁰ ] hPTHrP (3-34) OH

This peptide was assembled analogously to Example 1. At the end of the synthesis, a final cycle of 9-fluorenylmethoxycarbonyl protecting group removal and acetylation was performed using a 1: 1 mixture of acetic anhydride and dimethylformamide. The peptide is cleaved from the resin and purified as above.

Mass spectrometry: 3961.1

The following compounds can be prepared in an analogous manner to that described in Examples 1, 24 or 25.

»»

Example 26 t-Acetyl- [Trp ¹⁰ ] hPTHrP (2-34) OH Mass Spectrometry: 4060.4

Example 27 '

- isocaproyl- [Leu®, Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH w

Γ.: 1 ,. _d

MS: 4091

Example 28

N ^and -2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

Mass spectrometry :. 4097.1 · / .. ', r <

Example 29

N ^e -2-naphthylacetyl- [Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂

Mass spectrometry: 4009.7

Example 30

N ^e -2-naphthylsulfonyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

Mass spectrometry: 4119.2

Example 31 t-2-naphthoxyxyacetyl- [Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂

IN

Mass spectrometry: 4111.5

Example 32

N ^e -2-naphthylacetyl- [Ala ³ , Nal (2) ¹⁰ ] hPTHrP (3-34) NH ₂ Mass spectrometry: 4080.8

Example 33

N ** - benzyloxycarbonyl- [Trp ³ , Nal (2) ¹⁰ ] hPTHrP (2-34) NH ₂ Mass spectrometry: 4248.3.

Example 34

3-naphth-2-ylproprionyl - '[Nal (2} ^Li] hPTHrP (3-34) _NH2-Mass spectrometry: 4110.8

Example 35

-naphthyl-2-ylpropions 1- [Nal (2) ¹⁰ ] hPTHrP (3 - 3 4) NH ₂ Mass spectrometry: 4110.9

Example 36

1- (2-naphthylacetyl- [Nal (2) ¹⁰ , DAla ³⁴ ] hPTHrP (3-34) NH ₂ Mass Spectrometry: 4095.9

Example 37

N®-2-naphthylacetyl- (Nal (2) ¹⁰ ] hPTHrP (3-31) NH ₂ Mass spectrometry: 3788.0

Example 38

N ^and -3-naphth-2-ylpropionyl- [Nal (2) ¹⁰ ] hPTHrP (7-34) NH ₂ Mass spectrometry: 3630.0

Example 39 ϊΓ-Acetyl- [Phe ⁶ , NaI (2) ¹⁰ ] hPTHrP (6-34) NH ₂ .

Mass spectrometry: 3637.0

Example 40 • · *,

N, N-acetyl- [Phe ⁸ , Nal (2) ¹⁰ ] hPTHrP (4-34) NH ₂ Mass Spectrometry: 3903.1

Example 41 ¹ 1- (1-Amino-1-cyclopentylcarbonyl) [Leu ³ , Trp ¹⁰ , Glh ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTHrP (1-34) OH. * *

Mass spectrometry: 4104.,.

The 9-fluorenylmethoxycarbonyl-1-aminocyclopentane-1-carboxylic acid used to prepare the intermediate peptide resin can be prepared, for example, as described by G. Valle et al., 1988, Can. J. Chem. 66: 2575 - 2582

Example 42. * /

- adamantylcarbony1 -. [Leu ³ , Trp ¹⁰ , Gln ¹³ , Thr ³³ , Al and ³⁴ ] hPTH (1-3) OH Mass. Spectrometry ·: · 4155 .......

Example 43

1- (3-indolylcarbonyl) - [Leu ⁸ , Trp ¹⁸ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH Mass Spectrometry: 4150

Example 44

- (3-quinolylcarbonyl) - [Leu (R), Trp ¹⁰ , Ala ¹⁶ , Gln ¹³ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH

Mass spectrometry: 4104

Example 45

1- (2-naphthoyl) - (Leu ⁸ , Trp ¹⁰ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ) hPTH (1-3-4) OH Mass Spectrometry: 4147

Example 46.

N - (2-naphthyl) methyl- [Trp ¹⁰ ] hPTHrP (2-34) OH

Methyl N-2-naphthoyl valinate is treated with Lawesson's reagent, S-O. Lawesson et al., Tetrahedron 37: 3635 (1981) and the resulting methyl 7 N-2-thione phthyl valinate is reduced using. the procedure described by prop-endothiopeptides. Et al., Tetrah. Letters 23-26 (1990). The methyl ester is hydrolyzed using lithium hydroxide to give the N -hydrochloride. N-α- (2-naphthylmethyl) valine as a crystalline solid melting at 205-210 ° C with decomposition. This compound is condensed with a previously prepared protected compound using benzotriazol-1-yloxytripyrrolidinophosphonium hexaf-1-fluorophosphate.

[Trp ¹⁰ ] hPTHrP- (3-34) -O-peptide resin from which the N-α-9-fluorenylmethoxycarbonyl group has been removed. The peptide is cleaved from the resin and purified as in Example 1.

Mass spectrometry: 4158.2

Example 47

N®-2-naphth-2-ylethyl- [Trp ¹⁰ ] hPTHrP (2-34) OH

Mass spectrometry: 4171.9

Example 48

N * -2-naphth-2-ylethyl- [Ala ³ , Nal (2) ¹⁰ ] hPTHrP (3-34) NH 3

Mass spectrometry: 4066.7

Example 49

N '-succinyl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂

This peptide was prepared in an analogous manner to Example 1, but using 0.63 g of 4- (2,4,4-dimethoxyphenyl-9-fluorenylmethoxycarbonylaminomethyl) phenoxy (polystyrene-1% -dinylbenzene) starting material (approximately) 0.4 mmol / g), which can be prepared, for example, as described in Tetrah. Letters, 28: 3787-3790 (1987). After attachment of the last amino acid, 9-fluorenylmethoxycarbonyl-His (Trt) -OH, the 9-fluorenylmethoxycarbonyl group is selectively cleaved by conventional means and the peptide resin is reacted with 0.5 g of succinic anhydride and 0.86 ml of N, N-diisopropyl-N -ethylamine in dimethylformamide. The peptide is cleaved from the resin, purified and lyophilized in the acetate form as in Example 1 to give the title compound.

Mass Spectrometry: 38.32.0

The following compounds can be prepared as described in Example 49:

Example 5 0

N * -sukcinyl- [Phe ^6, Nal (2) ¹⁰⁾ hPTHrP C5-31) NH ₂

Mass spectrometry: 3522.1

Example 51 β-Succinyl- [Nal (2) ⁶ , Nal (2) ¹⁰ ] hPTHrP (5 - 3 4) NH ₂ Mass Spectrometry: 3881.7

Example 52

N ° -glutaryl- [Phe ⁶ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂

Mass spectrometry: 3845.6

Example 53

N * - sukeyl - [Phe ⁵ , Na 1 (2) ¹ ', DA1 and ³⁴ ] hPTHrP (5 - 3 4) NH ₂ Mass Spectrometry: 3832.0

Example 54

N '-succinyl- [4-Cl-Phe ⁵ , Nal (2) ¹⁰ ] hPTHrP (5-34) ΝΗ ₂

Mass spectrometry: 3866.5

Example 55. _ _ ·

KT-Succinyl- [4-Cl-Phe ⁵ , 4-Cl-Phe ⁵ , Nal (2) ¹⁰ ] hPTHrP (5-34) NH ₂ Mass Spectrometry: 3910.2

The compounds of the invention, in free form or in pharmaceutically form, with other salts and complexes, exhibit valuable pharmacological properties, as confirmed in animal tests, and are therefore suitable for therapy.

The biological activity of the compounds of the invention is assessed in vitro by measuring their ability to stimulate (agonism) or inhibit parathyroid hormone (antagonism) synthesis of cyclic AMP in rat osteosarcoma UMR 106-06 cells, according to the method of Marcus and Aurbach described in Endocrinology, £ 5, 801-810 (1969). Rat osteosarcoma UMR 106 cells were grown to a continuous growth phase in a 1: 1 mixture of DMEM and HAM F12 media with 10% calf fetal calf serum (FCS) on a 12-well plate. The medium is then changed to medium with 2% calf fetal serum and 1 - 5 µg [ ³ H] -adenine is added per well. Two hours later, cells are washed twice with serum-free medium and incubated in DMEM medium with 0.1% bovine serum albumin (BSA) containing 1mM

3-isobutyl-1-methylxanthine to avoid action on phosphodiesterases. 20 minutes later test substances are added either alone or together with parathyroid hormone (antagonist test). After 15 minutes, the medium is removed, the reaction is stopped and extracted with cyclic AMP (cAMP) by adding 0.5 ml of ice-cold 5% trichloroacetic acid. Carrier solution (0.5 ml per well) containing 0.2 mM unlabeled adenine, adenosine, AMP, 'ADP, ATP and cAMP' as well as 0.4 µCi [ ¹⁴ C] -adenosine is added to determine the yield. [ ³ H] -cAMP is separated using Dowex AG 50W-X4 chromatography (pore size 0.074-0.037) and alumina chromatography and its amount is determined as described by Salomon Y. in Advances or Cyclic Nucleotide Research, Volume 10, Raven Press, 35-55, 1979. Results are expressed as a percentage of the solvent control and EC ₅₀ values are determined from the DRC curves. Antagonist potency is calculated from the right-directed shift of the DRC curves for the parathyroid hormone-related peptide or parathyroid hormone and is expressed as pA ₂ values. The compounds of the invention are effective as antagonists at concentrations from 10 ^-3 to 10 ^-5 M. The compounds of Examples 36, 37 and 49 show pA ₂ values of 10.3, 9.7 and 9.3, respectively, in UMR 106-06 cells.

'• live ·

The compounds of the invention also show binding affinity for PTH receptors, as determined, for example, as follows:

Chicken [Tyr ³⁶ ] PTHrP (1-36) amide is iodinated to a specific activity of 2,200 Ci / mmol using the lactoperoxidase method (Anawa Lab. AG, Wangen). The opossum kidney cell layers (OKI) are washed with 200 μ 200 of a 1: 1 mixture of DMEM and HAM F12 media containing 1% bovine serum albumin (BSA) and incubated at 16 ° C with .50,000 cpm ( pulses per minute) [ ¹²⁵ I-Tyr ³⁶ ] chPTHrP (1-36) amide (ch stands for chicken PTHrP) per well in the presence or absence of TyμΜ [Tyr ³⁶ ] chPTHrP- (1-36) amide . After incubation, cells are washed with 0.5 ml of 4 ° C medium, lysed with 0.5 ml of 1N sodium hydroxide and radioactivity is determined. Specific binding is defined as total binding minus non-specific binding. Competition curves are analyzed using a non-linear regression computer program SCTFIT (Feyen et al., 1992,

Biochem. ' Biophys. Res. Commun. 187: 8-13) and data are expressed as mean pK _D values (n = 2 to 3). The compounds of Examples 36/37 and 49 have a pK _{D of} 8.3, 7.9 and 8.4, respectively.

Further, the compounds of the invention antagonize the action of parathyroid hormone after intravenous infusion, for example, as determined in thyroparathyroidectomized rats. 24 hours after thyroparathyroidektomii in rats receiving anastezi isolated jugular veins infusion of PTH (1-34) and the compound to be tested / remove urine _from the bladder ^to: which is cannulated ventral side * / ^ phosphate content "'and cAMP in urine and calcium and serum phosphates. is measured by standard methods. These parameters are used to quantify the efficacy of antagonists against parathyroid hormone activity in vivo. In this assay, the compounds of the invention antagonize the action of parathyroid hormone when administered by intravenous infusion at a dose of from 1 gg / kg / h to 1 mg / kg / h. Example '49' completely suppresses parathyroid hormone-induced phosphaturia for up to 90 minutes when administered by intravenous infusion at 190 gg / kg / h, when given by PTH (1-34) by intravenous infusion at 4 µg / kg / h.

Accordingly, the compounds of the invention are useful for the prevention or treatment of all conditions associated with increased plasma calcium due to excessive release of parathyroid hormone or parathyroid hormone related peptide, e.g. hyperparathyroidism, hypercalcemia, e.g. associated with cancer, e.g. breast cancer , lung flat cell carcinoma, esophageal cancers, head and neck area, and hematologic cancers with or without bone metastases. The compounds of the invention are further suitable for the prevention or treatment of tumor growth, tumor penetration and ingrowth into bones stimulated by parat26 hormone-related peptide, and for the treatment of dermatological disorders, e.g., tissue repair therapies, e.g. hair growth.

In the case of these indications, the appropriate dosage will, of course, vary depending upon, for example, the host, the route of administration, and the severity of the conditions being treated. However, it is generally reported that satisfactory results in animals are obtained at daily dosages of from about 0.1 to about 100 mg / kg animal body weight. In larger mammals, for example humans, the indicated daily dose ranges from about 0.1 to about 500 mg of the compounds of the invention.

The compounds of the invention may be administered in free form, in the form of pharmaceutically acceptable salts or complexes. Such salts and complexes can be prepared in conventional manner and exhibit the same activity as the free compounds. The invention also provides pharmaceutical compositions comprising a compound of the invention in free base or pharmaceutically acceptable salt or complex form and in combination with a pharmaceutically acceptable diluent or carrier. Such compositions may be formulated in conventional manner. Suitably, unit dosage forms contain from about 0.025 to 250 mg of a compound of the invention, together with a pharmaceutically acceptable diluent or carrier.

The compounds of the invention may be administered by any conventional means, for example parenterally, for example in the form of injectable solutions or suspensions, or in nasal or suppository form. Alternatively, the compounds of the invention may be administered topically, for example, in the form of cream, gel, and the like to treat skin or promote hair growth as described above.

Accordingly, the invention further includes:

(a) a compound of the invention or a pharmaceutically acceptable salt or complex thereof for use as a medicament;

b) a method of preventing or treating the conditions and disorders as set forth above in a patient in need of such treatment, the method comprising administering to said patient an effective amount of a compound of the invention or a pharmaceutically acceptable salt or complex thereof; and

c) a compound of the invention or a pharmaceutically acceptable salt or complex thereof for use in the preparation of a pharmaceutical composition for use in the method described in b) above.

According to another embodiment of the invention, the compounds of the invention may be used as adjuncts or adjuvants to other therapies, for example in the case of hypercalcemia in therapy using a bone resorption inhibitor, in particular therapy using calcitonin or an analogue or derivative thereof such as salmon, eel or human calcitonin. a bisphosphonate, a diuretic, or any combination thereof, or, in the case of tumor therapy, a "cytostatic agent" or * any combination thereof.

Accordingly, the invention includes as yet another embodiment:

d) a method of preventing or treating hypercalcemia, for example the prevention or treatment of any of the specific conditions or diseases mentioned above, in a patient in need thereof, the method comprising administering to said patient an effective amount of a) a compound of the invention; the active agent is a therapeutic agent as mentioned above.

The compounds of Examples 36, 37 and 49 are useful for preventing or treating any condition that is associated with increased plasma calcium due to excessive release of parathyroid hormone or parathyroid hormone related peptide.

Sequence list

Sequence Information SEQ ID No. 1:

(i) sequence characteristics: ^ (A) length: 38 amino acids (B) type: amino acid ^ (C) number of chains: single chain (D) topology: unknown (ii) type of molecule: protein (iii) hypothetical: not.

(iii) anti-sense: not (v) fragment type: N-terminal (xi) sequence representation, SEQ ID NO: 1:

Ala Wall Ser Glu His Gin Leu Leu His Ash Lys Gly Lys Ser Ile Gin 1 5 10 15 Dec Asn Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His 20 May 25 30 Thr Ala Glu Ile. Arg Ala

Sequence Information ¹ of SEQ ID No. 2:

(i) sequence characteristics:

(A) length: 34 amino acids (B) type: amino acid ...............

(C) number of chains: single chain (D) topology: unknown (ii) type of molecule: protein (iii) hypothetical: not (iii) anti-sense: not (v) fragment type: N-terminal

(xi) know orní Eni the sequence SEQ ^; 1 · * '· ID 2: Ala Val Glu His Gin Leu Leu His Asn Lys Gly Lys Ser Ile Gin 1 5 10 15 Dec Asn Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His 20 May 25 30

Thr Ala

Sequence Information SEQ ID NO

3:

(s) characteristics-sequence:

(A) length: 31 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology; unknown (ii) molecule type: protein · (iii) hypothetical: not (iii) anti-sense: not (v) fragment type: N-terminal (xi) representation of SEQ ID NO 3:

Ala Val Ser Glu His Gin “Leu'Leu His * Asn'Lys Gly Lys'Ser-Ile-Gln1 p .10 15

Asn 'Leu Arg Arg Arg Phe Phe Leu His Hish Leu Ile Ala Glu Ile · * 20 25 30

Sequence Information SEQ ID No. 4:

(i) character i sticky sequence: .........

(A) length · 48 amino acids (B) type: amino acid (C) number of chains: single chain '(D) topology: unknown (ii) type of molecule: protein (iii) hypothetical: not (iii) anti-sense: not ( (v) fragment type: N-terminal (xi) representation of SEQ ID NO: 4:

Ser Val Ser Glu Ile Gin Leu द्वारा किया Leu Met His Asn Leu Gly Lys His Leu Asn

Ser Met Glu Arg Val Glu Trp Leu

Asn Phe Val Ala Leu Gly

Sequence information of SEQ ID

No 5:

(i) sequence characteristics:

(A) length: 34 amino acids (B) type: amino acid (C) number of chains: single chain (D) topology: ·. unknown (ii) type of molecule: protein (iii) hypothetical: not (iii) anti-sense: not in

(v) fragment type: N-terminal

. (x and) knows zor ni: í se ikENCE SEQ ID C. 5: Ser Wall Ser 1 Glu Ile Gin 'Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 and 15 Dec Ser Met Glu Arg Wall Glu Trp Leu Arg Lys Lys Leu Gin Asp Wall His 20 May 25 30 Asn Phe

Sequence Information -SEQ ID # 6:

(i) sequence characteristics:

(A) length: 31 amino acids (B) type / amino acid (C) number of chains: single chain (D) topology,: unknown. .

(ii) - type of molecule; protein

E (iii) hypothetical: not (iii) anti-sense: not (v) fragment type: N-terminal

(xi ) shown V J * iem sequence SEQ ID No. 6: Ser Wall Ser Glu Ile Gin Leu Met Ash Leu Gly Lys His Leu 1 5 10 ... 15 Dec Ser Met Glu Arg Wall Glu Leu Arg Lys Lys Leu Gin Asp Wall 20 May 25 30

Claims (9)

PATENTOVÉ Ό χι < -ς Ό ι- Λ Μ σ ο: ί ^r R ο, Κ r C4 rc ο < A derivative of a parathyroid hormone or a parathyroid hormone-related peptide in which at least one of the amino acid residues naturally occurring at positions 2 and 10 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain and optionally further optionally at least one of the amino acid residues naturally occurring at positions 3 and 6 is replaced by tryptophan or another amino acid residue having an aromatic or heteroaromatic group in its side chain, except for derivatives in which the amino acid residue is at position 10 Tyr, at in free form or in the form of a salt or complex. 2. A derivative of a parathyroid hormone or a parathyroid hormone-related peptide in which the amino acid residue naturally occurring at position 10 is replaced by tryptophan or another amino acid-containing residue having an aromatic or heteroaromatic group in its side chain, and wherein optionally further is at least one. amino acid residues naturally occurring at positions 3 and 6 are replaced by tryptophan or another. .zbytkem _τ amino acid which contains in its side chain, aromatic or heteroaromatic group, with the exception of those derivatives in which the amino acid residue at position 10 with Tyr, in free form or in salt or complex form, The parathyroid hormone or parathyroid hormone-related peptide of claim 1, wherein the amino acid residue containing an aromatic or heteroaromatic group in its side chain is an amino acid residue whose side chain is an optionally substituted 3- or 4-pyridylmethyl group on the ring, A 3-indolylmethyl group or a 3-indazolylmethyl group, or a radical of formula (a), (b), (c) or (d) wherein n has value 1 or 2, m has value 1 or 2, 0 has value 0 or 1, ring A is optionally substituted with one or more substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl and C 1 -C 4 alkoxy, wherein the two alkyl or two alkoxy substituents may also together to form a ring structure not fused to ring A, each of rings B and C may be independently substituted as described above for ring A, and Y _a represents a direct bond, -CH ₂ -, an oxygen atom, an NH group or an N-alkyl group having 1 to 6 carbon atoms. The parathyroid hormone or parathyroid hormone-related peptide of claim 1, which is a compound of formula X R - [(X ² ) p, (X ³ ) q, (X ⁸ ) e, R ¹⁰ ] -D- (yx) R a (I) wherein x is a residue number selected from the group consisting of numbers 31, 34, 35 , 36, 37 and 38, y is a residue number selected from the group consisting of 1, 2,3,4,5,6a7, X ¹ represents Val or independently has one of the meanings given for X ¹⁰ , X ³ is Ser or independently has one of the meanings given for X ¹⁰ , X ^e represents Gln or independently has "one of the meanings" given for X- ^1a , '. R ¹⁰ is Asp or X ¹⁰ , wherein X ¹⁰ represents Trp or the radical -NH-CHR'-CO-, wherein R 'is a radical of formula (a), (b) or' (d) where n has value 1 or 2, m has value 1 or 2, o is 0 or 1, ring A is optionally substituted with one or more substituents selected from the group consisting of fluorine, chlorine, nitro, C 1 -C 4 alkyl and C 1 -C 4 alkoxy, wherein two alkyl or together the two alkoxy substituents may also form a ring structure not fused to ring A, each of rings B and C being independently substituted as above for ring A, and * 7 ··. I. _And Y '.představuje direct bond, -CH ₂ -, O,; NH or an 'N-alkyl group containing from 1 to 6 carbon atoms, D represents an amino acid sequence derived from N-terminal fragment of PTHrP or PTH,, » '· ¹ each of p and g. s has a value of 1, .s, by ¹ that p has a value of 0 if y is greater than 2, g has a value of 0 if y is greater than 3 and s has a value of 0 if y is greater than 6; ..... means '''' hydrogen, '''R'-CO-''''R''-O'-CO-,' ^{1 1} RO-CH ₂ -CO-, R-SO ₂ -, r ', r''-nh-co-, r'-nh-en or NH ₂ -alkylene-CO- with 1 to 6 carbon atoms in the alkylene moiety, where R represents an alkyl group having 1 to 8 carbon atoms, a ω-carboxyalkyl group having 1 to 6 carbon atoms in the alkyl moiety, a ω - [(alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms in the alkoxy moiety and 1 to 6 carbon atoms in the alkyl moiety, a C 2 -C 8 alkenyl group, a C 5 -C 7 cycloalkyl group, a C 5 -C 7 cycloalkylalkyl group and a C 1 -C 4 cycloalkyl group, or a phenyl group, C 1 -C 4 -phenylalkyl, 1-naphthyl, 2-naphthyl, 1-naphthylalkyl of 1 to 2 carbon atoms in the alkyl moiety or 2-naphthylalkyl of 1 to 2 carbon atoms an alkyl moiety, any of which is optionally substituted on the ring by one or more substituents selected from the group consisting of a fluorine atom, a chlorine atom, or an alkyl moiety; . nitro, alkyl. (C 1 -C 4) and (C 1 -C 4) alkoxy, or heteroaryl group, R independently has one of the meanings given for R except for the ω-carboxyalkyl group having 1 to 6 carbon atoms in the> alkyl portion and the ω- (alkoxy) carbonyl] alkyl group having 1 to 4 carbon atoms. carbon atoms in the alkoxy moiety and from 1 to 6 atoms, carbon atoms in the alkyl moiety, and * R _a is hydroxy or amino, with the proviso that. at least one of X ² and R ¹⁰ has the meaning of X ¹⁰ . 5. Parathyroid hormone or peptide derivative. . of a parathyroid hormone according to claim 1 or 4, which is derived from an N-terminal fragment of human parathyroid hormone, hPTH, or a human parathyroid hormone related peptide, hPTHrP. ^{6 * *} The parathyroid hormone or parathyroid hormone-related peptide of claim 1, selected from the group consisting of N '- 2-naphthyl tyl - [Nal (2) ¹⁰ , DAla ³⁴ ] hPTHrP (3 - 3 4) NH ₂ , N ° -2-naphthylacetyl- [Nal (2) ^{8 9 10} ] hPTHrP (3-31) NH ₂ , and N ° -sukeinyl- ^[Phe, Nal (2) ^10] hPTHrP (5-34) NH _second A process for the preparation of a parathyroid hormone derivative or a parathyroid hormone-related peptide according to claim 1 in free form or in the form of a salt or complex, which comprises: a) removes at least one protecting group that is present in the parathyroid hormone-related peptide derivative or parathyroid hormone of the invention, for example a compound of formula I, in protected form, or b) joining two peptide fragments in a protected, partially protected or unprotected form to each other by amide bonding, such peptide fragments being such that the amino acid sequence of the desired parathyroid hormone or parathyroid hormone related peptide derivative, for example of formula I, is obtained; (a) the manner in which or c). introduces a protecting group or substituent in a selective manner at. amino group of the N-terminal residue of the desired sequence or N-terminal fragment thereof in protected or unprotected form, optionally followed by step a) of the method, and recovering the thus obtained parathyroid hormone-related peptide or parathyroid hormone derivatives in free, salt or form complex. The parathyroid hormone or parathyroid hormone-related peptide of claim 1 in free form or in physiologically acceptable salt form for use as a medicament. > 9. A pharmaceutical composition comprising a parathyroid hormone derivative or a parathyroid hormone related peptide of claim 1 in free form or in a pharmaceutically acceptable salt thereof.