U.S. patent application number 11/159007 was filed with the patent office on 2005-10-27 for selective linear peptides with melanocortin-4 receptor (mc4-r) agonist activity.
Invention is credited to Chen, Li, Cheung, Adrian Wai-Hing, Chu, Xin-Jie, Danho, Waleed, Swistok, Joseph, Yagaloff, Keith Alan.
Application Number | 20050239711 11/159007 |
Document ID | / |
Family ID | 22717646 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239711 |
Kind Code |
A1 |
Chen, Li ; et al. |
October 27, 2005 |
Selective linear peptides with melanocortin-4 receptor (MC4-R)
agonist activity
Abstract
Peptides of formulae I, II and III that selectively activate
melanocortin-4 (MC-4) receptor activity. 12
Inventors: |
Chen, Li; (Westfield,
NJ) ; Cheung, Adrian Wai-Hing; (Glen Rock, NJ)
; Chu, Xin-Jie; (Livingston, NJ) ; Danho,
Waleed; (Wayne, NJ) ; Swistok, Joseph;
(Nutley, NJ) ; Yagaloff, Keith Alan; (HoHokus,
NJ) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.
PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
|
Family ID: |
22717646 |
Appl. No.: |
11/159007 |
Filed: |
June 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11159007 |
Jun 22, 2005 |
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10435466 |
May 9, 2003 |
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10435466 |
May 9, 2003 |
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09811964 |
Mar 19, 2001 |
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6600015 |
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60194450 |
Apr 4, 2000 |
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Current U.S.
Class: |
530/330 ;
514/10.7; 514/21.8; 530/329 |
Current CPC
Class: |
C07K 5/1016 20130101;
A61K 38/00 20130101; C07K 14/68 20130101; A61P 5/00 20180101; A61P
43/00 20180101; A61P 3/04 20180101; C07K 7/06 20130101 |
Class at
Publication: |
514/016 ;
530/329; 530/330; 514/017 |
International
Class: |
A61K 038/08; C07K
007/06 |
Claims
What is claimed is:
1. A compound of the formula: 263wherein m is 0 or 1; n is 0 or 1;
R.sup.1 is an unsubstituted linear or branched alkyl having from 1
to 8 carbon atoms; linear or branched alkyl having from 1 to 8
carbon atoms mono-substituted by phenyl or carboxyl; unsubstituted
phenyl; or phenyl mono-substituted by fluoro, chloro or linear or
branched alkyl having from 1 to 4 carbon atoms; X is 264wherein
R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen or a linear
or branched alkoxy having from 1 to 4 carbon atoms, wherein when
R.sup.3 is alkoxy, R.sup.2 and R.sup.4 are both hydrogen; R.sup.9
is hydrogen, linear or branched alkyl having from 1 to 3 carbons,
linear or branched alkoxy having from 1 to 3 carbons, or
unsubstituted phenoxy; R.sup.11 is cyclohexyl, cycloheptyl, or a
branched alkyl having from 3 to 8 carbon atoms; R.sup.6 is hydrogen
or methyl; R.sup.7 is 265Y is 266and R.sup.8 is hydrogen or methyl;
or Y is 267and R.sup.8 is hydrogen.
Description
CONTINUITY INFORMATION
[0001] This application is a Continuation of U.S. Ser. No.
10/435,466, filed May 9, 2003, which is a Continuation of U.S. Ser.
No. 09/811,964, filed Mar. 19, 2001 which claims priority under 35
U.S.C. .sctn.119(e) of provisional application Ser. No. 60/194,450,
filed Apr. 4, 2000.
BACKGROUND OF THE INVENTION
[0002] Obesity is widely recognized as a serious health problem for
the developed countries, and has reached epidemic status in the
United States. More than 50% of the U.S. population is considered
overweight, with >25% diagnosed as clinically obese and at
considerable risk for heart disease, non-insulin dependent diabetes
mellitus (NIDDM), hypertension, and certain cancers. This epidemic
presents a significant burden on the health care system as
projected obesity treatment costs of more than $70 billion annually
are expected in the U.S. alone. Strategies for treating obesity
include reducing food intake or enhancing the expenditure of
energy.
[0003] It has been demonstrated that, when injected into the third
ventricle of the brain or intraperitoneally, a cyclic heptapeptide
analog of .alpha.-melanocyte stimulating hormone (.alpha.MSH)
having melanocortin-4 receptor (MC4-R) agonist activity caused long
lasting inhibition of food intake in mice. This effect was
reversible when co-administered with a MC4-R antagonist. (Fan, et
al., Nature (1997) 385: 165-168) Therefore, agonists of MC4-R
activity would be useful in treating or preventing obesity.
[0004] There are five known melanocortin receptors based on
sequence homology that ranges from 35-60% homology between family
members ((Cone, et al., Rec. Prog. Hormone Res. (1996) 51:
287-318), but these receptors differ in their functions. For
example, the MC1-R is a G-protein coupled receptor that regulates
pigmentation in response to the .alpha.MSH, which is a potent
agonist of MC1-R. (Cone, et al., ibid.). Agonism of the MC1-R
receptor results in stimulation of the melanocytes which causes
eumelanin and increases the risk for cancer of the skin. Agonism of
MC1-R can also have neurological effects. Stimulation of MC2-R
activity can result in carcinoma of adrenal tissue. The effects of
agonism of the MC3-R and MC5-R are not yet known. All of the
melanocortin receptors respond to the peptide hormone class of
melanocyte stimulating hormones (MSH). These peptides are derived
from pro-opiomelanocortin (POMC), a prohormone of 131 amino acids
that is processed into three classes of hormones; the melanocortins
(.alpha.,.beta. and .gamma.), adrenocorticotropin hormone (ACTH),
and various endorphins (e.g. lipotropin) (Cone, et al., ibid.).
Because of their different functions, simultaneous agonism of the
activities of multiple melanocortin receptors has the potential of
causing unwanted side effects. Therefore it is desirable that an
agonist of MC4-R be more selective for the MC4-R than for one or
more of the other melanocortin receptors.
[0005] Haskell-Luevano, et al. (Peptides (1996) 17(6): 995-1002)
disclose peptides that contain the tripeptide (D)Phe-Arg-Trp and
exhibit melanotropic (skin darkening) activity in the frog (Rana
pipiens) skin bioassay. Haskell-Luevano, et al. (ibid) do not
disclose any compound of formula I, II or III described below.
SUMMARY OF THE INVENTION
[0006] This invention provides a compound of the formula: 3
[0007] In compounds of formula I m is 0 or 1. n is 0 or 1. R.sup.1
is an unsubstituted linear or branched alkyl having from 1 to 8
carbon atoms; linear or branched alkyl having from 1 to 8 carbon
atoms mono-substituted by phenyl or carboxyl; unsubstituted phenyl;
or phenyl mono-substituted by fluoro, chloro or linear or branched
alkyl having from 1 to 4 carbon atoms. X is 4
[0008] R.sup.2, R.sup.3 and R.sup.4 are independently hydrogen or a
linear or branched alkoxy having from 1 to 4 carbon atoms, wherein
when R.sup.3 is alkoxy, R.sup.2 and R.sup.4 are both hydrogen.
R.sup.9 is hydrogen, linear or branched alkyl having from 1 to 3
carbons, linear or branched alkoxy having from 1 to 3 carbons, or
unsubstituted phenoxy. R.sup.11 is cyclohexyl, cycloheptyl, or a
branched alkyl having from 3 to 8 carbon atoms. R.sup.6 is hydrogen
or methyl. R.sup.7 is 5
[0009] Y is 6
[0010] and R.sup.8 is hydrogen or methyl; or
[0011] Y is 7
[0012] and R.sup.8 is hydrogen.
[0013] This invention provides a compound of the formula: 8
[0014] In the compounds of formula II m is 0 or 1. n is 0 or 1.
R.sup.1 is an unsubstituted linear or branched alkyl having from 4
to 8 carbon atoms; linear or branched alkyl having from 1 to 8
carbon atoms mono-substituted by phenyl or carboxyl; or
unsubstituted phenyl; or phenyl mono-substituted by fluoro, chloro
or linear or branched alkyl having from 1 to 4 carbon atoms.
R.sup.7 is 9
[0015] Y is 10
[0016] and R.sup.8 is hydrogen or methyl; or
[0017] Y is 11
[0018] and R.sup.8 is hydrogen.
[0019] R.sup.10 is hydrogen, halo, linear or branched alkyl having
from 1 to 3 carbon atoms, linear or branched alkoxy having from 1
to 3 carbon atoms, or --NR.sup.12R.sup.13 wherein R.sup.12 and
R.sup.13 are each independently a linear or branched alkyl having
from 1 to 3 carbons or together are --(CH.sub.2).sub.q-- wherein q
is 3, 4 or 5.
[0020] This invention provides a compound of the formula: 12
[0021] In the compounds of formula III, R.sup.1 is unsubstituted
linear or branched alkyl having from 4 to 8 carbon atoms. R.sup.6
is hydrogen or methyl. R.sup.8 is hydrogen or methyl. p is 2, 3 or
4 and R.sup.14 is 13
[0022] or p is 4and R.sup.14 is 14
[0023] or p is 3 and R.sup.14 is 15
[0024] The compounds of formulae I, II and III as well as
Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-Ape-(D)Phe-Arg-Trp-Gly-N- H.sub.2 are agonists of the MC4-R.
It is known that agonists of MC4-R activity cause reduction of food
intake in a mouse model of human obesity. Therefore the compounds
of formula I are useful in the treatment or prevention of
obesity.
[0025] All of the compounds of formulae I, II and III exemplified
below as well as Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-Ape-(D)Phe-Arg-Trp-Gly-NH.sub.2 were tested for MC4-R agonist
activity and MC1-R agonist activity in the in vitro assay described
below in Biological Activity Example A. All of the tested compounds
had an EC50 for MC4-R agonist activity of less than 500 nM, and all
exhibited at least 10-fold greater MC4-R agonist activity than
MC1-R agonist activity. In contrast, the compound
Bu-His-(D)Phe-Arg-Trp-Gly-NH.sub.2 (Example 30) exhibited greater
MC1-R agonist activity than MC4-R agonist activity.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Nomenclature and Abbreviations
[0027] The nomenclature used to define the peptides is that
typically used in the art wherein the amino group at the N-terminus
appears to the left and the carboxyl group at the C-terminus
appears to the right. By natural amino acids is meant one of the
naturally occurring amino acids found in proteins, i.e., Gly, Ala,
Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met,
Phe, Tyr, Pro, Trp, and His. Where the amino acid has isomeric
forms, it is the L form of the amino acid that is represented
unless otherwise explicitly indicated.
[0028] The following abbreviations or symbols are used to represent
amino acids, protecting groups, solvents, reagents and the
like.
[0029] Symbol Meaning
[0030] .beta.-Ala beta-Alanine
[0031] (2)-Nal (2)-Naphthylalanine
[0032] Atc 2-Aminotetraline-2-carboxylic acid
[0033] 5-BrAtc 5-Bromo-2-aminotetraline-2-carboxylic acid
[0034] 5-ClAtc 5-Chloro-2-aminotetraline-2-carboxylic acid
[0035] 5-MeOAtc 5-Methoxy-2-aminotetraline-2-carboxylic acid
[0036] 5-EtOAtc 5-Ethoxy-2-aminotetraline-2-carboxylic acid
[0037] 5-iPrOAtc 5-Isopropoxy-2-aminotetraline-2-carboxylic
acid
[0038] 5-MeAtc 5-Methyl-2-aminotetraline-2-carboxylic acid
[0039] 5-EtAtc 5-Ethyl-2-aminotetraline-2-carboxylic acid
[0040] 5-iPrAtc 5-Isopropyl-2-aminotetraline-2-carboxylic acid
[0041] 5-DmaAtc 5-Dimethylamino-2-aminotetraline-2-carboxylic
acid
[0042] Sar Sarcosine (N-methylglycine)
[0043] Cit Citrulline
[0044] Apc 1-Amino-4-phenylcyclohexane-1-carboxylic acid
[0045] 4-HOApc 1-Amino-4-(4-hydroxyphenyl)cyclohexane-1-carboxylic
acid
[0046] 4-MeOApc 1-Amino-4-(4-methoxyphenyl)cyclohexane-1-carboxylic
acid
[0047] 3-MeOApc 1-Amino-4-(4-methoxyphenyl)cyclohexane-1-carboxylic
acid
[0048] 4-EtOApc 1-Amino-4-(4-ethoxyphenyl)cyclohexane-1-carboxylic
acid
[0049] 4-iPrOApc
1-Amino-4-(4-isopropoxyphenyl)cyclohexane-1-carboxylic acid
[0050] 4-MeApc 1-Amino-4-(4-methylphenyl)cyclohexane-1-carboxylic
acid
[0051] 4-ClApc 1-Amino-4-(4-chlorophenyl)cyclohexane-1-carboxylic
acid
[0052] Appc 4-Amino-1-phenylpiperidine-4-carboxylic acid
[0053] 2-MeAppc 4-Amino-1-(2-methylphenyl)piperidine-4-carboxylic
acid
[0054] 2-iProAppc
4-Amino-1-(2-isopropoxyphenyl)piperidine-4-carboxylic acid
[0055] 3-MeAppc 4-Amino-1-(3-methylphenyl)piperidine-4-carboxylic
acid
[0056] 3-MeOAppc 4-Amino-1-(3-methoxyphenyl)piperidine-4-carboxylic
acid
[0057] 4-MeAppc 4-Amino-1-(4-methylphenyl)piperidine-4-carboxylic
acid
[0058] 4-ClAppc 4-Amino-1-(4-chlorophenyl)piperidine-4-carboxylic
acid
[0059] 4-PhOAppc 4-Amino-1-(4-phenoxyphenyl)piperidine-4-carboxylic
acid
[0060] Achc 1-Amino-4-cyclohexylcyclohexane-1-carboxylic acid
[0061] Adpc 1-Amino-4-diphenylcyclohexane-1-carboxylic acid
[0062] Ape 1-Amino-4-phenylcyclohex-3-ene-1-carboxylic acid
[0063] Abc 1-Amino-4-tert-butylcyclohexane-1-carboxylic acid
[0064] 3-Amb 3-Aminomethyl benzoic acid
[0065] 4-Amb 4-Aminomethyl benzoic acid
[0066] 2-Aba 2-Aminobenzoic acid
[0067] Bu Butyl
[0068] Penta Pentyl
[0069] Fmoc 9-Fluorenylmethoxycarbonyl
[0070] Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl
[0071] CH.sub.2Cl.sub.2 Methylene chloride
[0072] CH.sub.3CN Acetonitrile
[0073] DMF Dimethylformamide
[0074] DIPEA N,N-Diisopropylethylamine
[0075] TFA Trifluoroacetic acid
[0076] HOBT N-Hydroxybenzotriazole
[0077] DIC N,N'-Diisopropylcarbodiimide
[0078] BOP Benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium
Hexafluorophosphate
[0079] PyBroP Bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate
[0080] HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
Hexafluorophosphate
[0081] FAB-MS Fast atom bombardment mass spectrometry
[0082] ES-MS Electrospray mass spectrometry
[0083] NBSC 2-Nitrobenzenesulfonyl chloride
[0084] DEAD N,N-diethylazodicarboxylate
[0085] Ph Phenyl
[0086] Setting forth the substituted amino acid, in parentheses
indicates analogs of the peptide sequence. Derivatization of the
N-terminal amino group, is indicated to the left of the N-terminal
substitution, separated by a hyphen. That is, for example,
Ac-His-(D)Phe-Arg-Trp-Gly-NH.sub.2 indicates a peptide having an
amino acid sequence in which an acetyl group has been substituted
for hydrogen at the N-terminus. The suffixes "--OH" and
"--NH.sub.2" following the hyphen or the parentheses refer to the
free acid and amide forms of the polypeptide, respectively.
DETAILED DESCRIPTION OF COMPOUNDS
[0087] In compounds of formula I, it is generally preferred that
R.sup.6 and R.sup.8 are both hydrogen, n is 1 and R.sup.7 is either
the first or the second of the substructures shown above. Also
preferred are compounds of formuae IA, IB or IC as shown below.
[0088] Compounds of formula IA, are represented as follows: 16
[0089] In the compound of formula IA, m is 0 or 1. n is 0 or 1.
R.sup.1 is an unsubstituted linear or branched alkyl having from 1
to 8 carbon atoms; linear or branched alkyl having from 1 to 8
carbon atoms mono-substituted by phenyl or carboxyl; unsubstituted
phenyl; or phenyl mono-substituted by fluoro, chloro or linear or
branched alkyl having from 1 to 4 carbon atoms. R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen; a linear or branched alkyl
having from 1 to 4 carbon atoms; hydroxy, a linear or branched
alkoxy having from 1 to 4 carbon atoms; or chloro, wherein when
R.sup.3 is alkyl, hydroxy, alkoxy or chloro, R.sup.2 and R.sup.4
are both hydrogen. R.sup.6 is hydrogen or methyl. R.sup.7 is 17
[0090] Y is 18
[0091] and R.sup.8 is hydrogen or methyl; or
[0092] Y is 19
[0093] R.sup.8 is hydrogen.
[0094] In the compounds of formula IA, R.sup.7 can be either a
tryptophan side chain or a 1- or 2-naphthyl group. In compounds of
formula IA in which R.sup.7 is a tryptophan side chain, i.e. 20
[0095] n can be either 0 or 1. Examples of such compounds in which
n is 0 include Penta-Apc-(D)Phe-Arg-Trp-NH.sub.2 and
Penta-Apc-(D)Phe-Arg-N-meth- ylTrp-NH.sub.2. In compounds of
formula IA in which R.sup.7 is a tryptophan side chain and n is 1,
Y can be a linear or branched alkyl group selected from methylene,
ethylene or methyl-substituted methylene, i.e. 21
[0096] or one of the aryl-containing moieties shown above. In
compounds of formula IA in which R.sup.7 is a tryptophan side chain
and n is 1, Y is methylene, ethylene or methyl-substituted
methylene, m can be 0 or 1. Examples of such compounds in which m
is 1 include Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Bu-carbamoyl-Apc-(D)Phe-Arg- -Trp-Ala-NH.sub.2, and
Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-.beta.-Ala-NH.sub.2- . In compounds
of formula IA in which R.sup.7 is a tryptophan side chain, n is 1,
Y is methylene, ethylene or methyl-substituted methylene and m is
0, the phenyl ring of the Apc group can be either unsubstituted
(i.e. R.sup.2, R.sup.3 and R.sup.4 are hydrogen) or substituted. In
such compounds in which the phenyl ring of the Apc group is
unsubstituted, R.sup.1 can be, for example, an unsubstituted linear
alkyl such as in the compounds
Penta-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2, Penta-Apc-(D)Phe-Arg-Trp-
-Sar-NH.sub.2, Penta-Apc-(D)Phe-Arg-N-methylTrp-Gly-NH.sub.2,
Bu-Apc-(D)Phe-Arg-Trp-Ala-NH.sub.2, or
Bu-Apc-(D)Phe-Arg-Trp-.beta.-Ala-N- H.sub.2; or unsubstituted
phenyl such as in the compounds
Phenylacetyl-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Phenylacetyl-Apc-(D)Phe-Arg- -Trp-Ala-NH2, or
Phenylacetyl-Apc-(D)Phe-Arg-Trp-Ala-NH.sub.2. In such compounds in
which the phenyl ring of the Apc group is substituted, one
preferred substitution pattern is wherein R.sup.3 is alkyl,
hydroxy, alkoxy or chloro (more preferably R.sup.3 is hydroxy or
alkoxy) and R.sup.2 and R.sup.4 are hydrogen. Examples include
Penta-4-ClApc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-4-MeApc-(D)Phe-Arg-Trp-G- ly-NH.sub.2,
Penta-4-HOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-4-MeOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-4-EtOApc-(D)Phe-Arg-Trp- -Gly-NH.sub.2, and
Penta-4-iPrOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2. Another preferred
substitution pattern is wherein R.sup.2 is alkoxy, R.sup.3 is
hydrogen and R.sup.4 is hydrogen, for example in the compound
Penta-3-MeOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2. In compounds of formula
IA in which R.sup.7 is a tryptophan side chain and n is 1, and Y is
22
[0097] m can be 0 or 1. Examples of such compounds in which m is 1
include Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2 and
Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH.sub.2. Examples of such
compounds in which m is 0 include
Bu-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2,
Phenylacetyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2,
Bu-Apc-(D)Phe-Arg-Trp-3-A- mb-NH.sub.2,
Phenylacetyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH.sub.2,
Bu-Apc-(D)Phe-Arg-Trp-4-Amb-NH.sub.2, and
Phenylacetyl-Apc-(D)Phe-Arg-Trp- -4-Amb-NH.sub.2.
[0098] In compounds of formula IA in which R.sup.7 is 2-naphthyl,
i.e. 23
[0099] it is preferred that R.sup.2, R.sup.3 and R.sup.4 are
hydrogen. Examples of such compounds include
Penta-Apc-(D)Phe-Arg-N-methyl(2)Nal-NH- .sub.2 and
Bu-Carbamoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2. In compounds of
formula IA in which R.sup.7 is 2-naphthyl, it is preferred that n
is 1 and m is 0. In compounds of formula IA in which R.sup.7 is
2-naphthyl, n is 1 and m is 0, and Y is methylene, ethylene or
methyl-substituted methylene, R.sup.1 can be, for example an
unsubstituted linear alkyl. Examples of such compounds include,
Penta-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.su- b.2,
Bu-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2,
Ac-Apc-(D)Phe-Arg-(2)Nal-Gly-N- H.sub.2,
Penta-Apc-(D)Phe-Arg-N-methyl (2)Nal-Gly-NH.sub.2,
Bu-Apc-(D)Phe-Arg-(2)Nal-Ala-NH.sub.2, and
Bu-Apc-(D)Phe-Arg-(2)Nal-beta-- Ala-NH.sub.2. Alternatively R.sup.1
can be, for example, unsubstituted phenyl, or alkyl substituted by
phenyl or carboxyl. Examples of such compounds include
Benzoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2,
3-carboxylpropanoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2, and
3-carboxylpropanoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2. In
compounds of formula IA in which R.sup.7 is 2-naphthyl, n is 1 and
m is 0, and Y is 24
[0100] It is preferred that R.sup.1 is unsubstituted lower alkyl.
Examples of such compounds include
Bu-Apc-(D)Phe-Arg-(2)Nal-3-Amb-NH.sub.2,
Bu-Apc-(D)Phe-Arg-(2)Nal-2-Aba-NH.sub.2, and
Bu-Apc-(D)Phe-Arg-(2)Nal-4-A- mb-NH.sub.2.
[0101] Compounds of formula IB are represented as follows: 25
[0102] In the compound of formula IB, R.sup.1 is an unsubstituted
linear or branched alkyl having from 1 to 8 carbon atoms. R.sup.7
is 26
[0103] R.sup.11 is cyclohexyl, or a branched alkyl having from 3 to
8 carbon atoms. Y is methylene, i.e. --CH.sub.2--. Examples of
compounds of formula IB include Penta-Abc-(D)Phe-Arg-Trp-Gly-NH2
and Penta-Achc-(D)Phe-Arg-Trp-Gly-NH.sub.2.
[0104] Compounds of formula IC are represented as follows: 27
[0105] In the compound of formula IC, R.sup.1 is an unsubstituted
linear or branched alkyl having from 1 to 8 carbon atoms. R.sup.7
is 28
[0106] Y is 29
[0107] R.sup.9 is hydrogen, a linear or branched alkyl having from
1 to 3 carbon atoms, a linear or branched alkoxy having from 1 to 3
carbon atoms, fluoro, chloro, or unsubstituted phenoxy. Examples of
compounds of formula IC in which R.sup.9 is hydrogen include
Penta-Appc-(D)Phe-Arg-Trp- -Gly-NH.sub.2 and
Penta-Appc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2. Examples of compounds of
formula IC in which R.sup.9 is a linear or branched alkyl having
from 1 to 3 carbon atoms include Penta-2-MeAppc-(D)Phe-Arg-Trp-Gly-
-NH.sub.2, Penta-2-iPrAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-3-MeAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-4-MeAppc-(D)Phe-Arg-- Trp-Gly-NH.sub.2. Examples of compounds
of formula IC in which R.sup.9 is a linear or branched alkoxy
having from 1 to 3 carbon atoms or unsubstituted phenoxy include
Penta-3-MeOAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-4-PhOAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2. Examples of compounds
of formula IC in which R.sup.9 is chloro include
Penta-4-ClAppc-(D)Phe-Arg-T- rp-Gly-NH.sub.2.
[0108] In the compound of formula II it is generally preferred that
R.sup.6 and R.sup.8 are hydrogen. R.sup.7 can be, for example a
tryptophan side chain, i.e. 30
[0109] or 2-naphthyl. When R.sup.7 is a tryptophan side chain it is
generally preferred that n is 1. Among the compounds of formula II
in which R.sup.6 and R.sup.8 are hydrogen; R.sup.7 is a tryptophan
side chain, and n is 1, are included compounds in which Y is
--CH.sub.2-- and m is 0. Examples of such compounds in which
R.sup.10 is hydrogen or a linear or branched alkyl having from 1 to
3 carbon atoms are included Bu-Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-5-Me-(D,L)Atc-(D)Phe-Arg-Trp-Gl- y-NH.sub.2,
Penta-5-Et-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-5-iPr-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2. Examples of such
compounds in which R.sup.10 is halo include
Penta-5-Br-(D,L)Atc-(D)Phe-Ar- g-Trp-Gly-NH.sub.2,
Penta-5-Br-Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-5-Cl-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2. Examples of such
compounds in which R.sup.10 is linear or branched alkoxy having
from 1 to 3 carbon atoms include
Penta-5-MeO-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2,
Penta-5-EtO-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-5-iPrO-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2. Examples of such
compounds in which R.sup.10 is --NR .sup.2R.sup.13 wherein R.sup.12
and R.sup.13 are each methyl include
Penta-5-DmaAtc-(D)Phe-Arg-Trp-Gly-NH.sub- .2.
[0110] Among the compounds of formula II in which R.sup.6 and
R.sup.8 are hydrogen; R.sup.7 is a tryptophan side chain, and n is
1, are included compounds in which Y is 31
[0111] and R.sup.10 is halo. Examples of such compounds include
Bu-(D,L)5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2,
Bu-carbamoyl-(D,L)-5-BrAtc- -(D)Phe-Arg-Trp-2-Aba-NH.sub.2 and
Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-T- rp-2-Aba-NH.sub.2.
[0112] In compounds of formula II in which wherein R.sup.6 and
R.sup.8 are hydrogen; R.sup.7 is 2-naphthyl i.e. 32
[0113] it is generally preferred that R.sup.10 is halo. Examples of
such compounds include
Penta-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2,
3-carboxylpropanoyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2,
Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2 and
Bu-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-2-Aba-NH.sub.2.
[0114] Examples of compounds of formula III include
Bu-Apc-(D)Phe-PhenylhomoArg-Trp-Gly-NH.sub.2,
Penta-Apc-(D)Phe-Cit-Trp-Gl- y-NH.sub.2,
Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH.sub.2 and
Penta-Ape-(D)Phe-Arg-Trp-Gly-NH.sub.2.
[0115] Chemical Synthesis
[0116] The compounds of this invention can be readily synthesized
by any known conventional procedure for the formation of a peptide
linkage between amino acids. Such conventional procedures include,
for example, any solution phase procedure permitting a condensation
between the free alpha amino group of an amino acid or residue
thereof having its carboxyl group or other reactive groups
protected and the free primary carboxyl group of another amino acid
or residue thereof having its amino group or other reactive groups
protected.
[0117] The synthesis of these compounds may be carried out by a
procedure whereby each amino acid in the desired sequence is added
one at a time in succession to another amino acid or residue
thereof or by a procedure whereby peptide fragments with the
desired amino acid sequence are first synthesized conventionally
and then condensed to provide the desired peptide.
[0118] Such conventional procedures for synthesizing the novel
compounds of the present invention include for example any solid
phase peptide synthesis method. In such a method the synthesis of
the novel compounds can be carried out by sequentially
incorporating the desired amino acid residues one at a time into
the growing peptide chain according to the general principles of
solid phase methods [Merrifield, R. B., J. Amer. Chem. Soc. 1963,
85, 2149-2154; Barany et al., The Peptides, Analysis, Synthesis and
Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press
1-284 (1980)].
[0119] Common to chemical syntheses of peptides is the protection
of reactive side chain groups of the various amino acid moieties
with suitable protecting groups, which will prevent a chemical
reaction from occurring at that site until the protecting group is
ultimately removed. Usually also common is the protection of the
alpha amino group of an amino acid or fragment while that entity
reacts at the carboxyl group, followed by the selective removal of
the alpha amino protecting group and allow a subsequent reaction to
take place at that site. While specific protecting groups are
mentioned below in regard to the solid phase synthesis method, it
should be noted that each amino acid can be protected by any
protective group conventionally used for the respective amino acid
in solution phase synthesis.
[0120] For example, alpha amino groups may be protected by a
suitable protecting group selected from aromatic urethane-type
protecting groups, such as benzyloxycarbonyl (Z) and substituted
benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc)
and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type
protecting groups, such as t-butyloxycarbonyl (Boc),
diisopropylmethoxycarbonyl, isopropoxycarbonyl, and
allyloxycarbonyl. Herein, Fmoc is the most preferred for alpha
amino protection.
[0121] Guanidino groups may be protected by a suitable protecting
group selected from nitro, p-toluenesulfonyl (Tos), Z,
pentamethylchromanesulfo- nyl (Pmc), adamantyloxycarbonyl, and Boc.
Pmc is the most preferred for arginine (Arg).
[0122] In the examples all solvents, isopropanol (iPrOH), methylene
chloride (CH.sub.2Cl.sub.2), dimethylformamide (DMF) and
N-methylpyrrolidinone (NMP) were purchased from Fisher or Burdick
and Jackson and were used without additional distillation.
Trifluoroacetic acid was purchased from Halocarbon or Fluka and
used without further purification. Diisopropylcarbodiimide (DIC)
and diisopropylethylamine (DIPEA) was purchased from Fluka or
Aldrich and used without further purification. Hydroxybenzotriazole
(HOBT) dimethylsulfide (DMS) and 1,2-ethanedithiol (EDT) were
purchased from Sigma Chemical Co. and used without further
purification. Protected amino acids were generally of the L
configuration and were obtained commercially from Bachem, Advanced
ChemTech, or Neosystem. Purity of these reagents was confirmed by
thin layer chromatography, NMR and melting point prior to use.
Benzhydrylamine resin (BHA) was a copolymer of styrene--1%
divinylbenzene (100-200 or 200-400 mesh) obtained from Bachem or
Advanced Chemtech. Total nitrogen content of these resins were
generally between 0.3-1.2 meq/g.
[0123] High performance liquid chromatography (HPLC) was conducted
on a LDC apparatus consisting of Constametric I and III pumps, a
Gradient Master solvent programmer and mixer, and a Spectromonitor
III variable wavelength UV detector. Analytical HPLC was performed
in reversed phase mode using Vydac C.sub.18 columns (0.4.times.30
cm). Preparative HPLC separations were run on Vydac columns
(2.times.25 cm).
[0124] Peptides were prepared using solid phase synthesis following
the principles and general method described by Merrifield, [J.
Amer. Chem. Soc., 1963, 85, 2149], although other equivalent
chemical synthesis known in the art could be used as previously
mentioned. Solid phase synthesis is commenced from the C-terminal
end of the peptide by coupling a protected alpha-amino acid to a
suitable resin. Such a starting material can be prepared by
attaching an alpha-amino-protected amino acid by an ester linkage
to a p-benzyloxybenzyl alcohol (Wang) resin, or by an amide bond
between an Fmoc-Linker, such as p-[(R,
S)-.alpha.-[1-(9H-fluoren-9-y-
l)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid
(Rink linker) to a benzhydrylamine (BHA) resin. Preparation of the
hydroxymethyl resin is well known in the art. Fmoc-Linker-BHA resin
supports are commercially available and generally used when the
desired peptide being synthesized has an unsubstituted amide at the
C-terminus.
[0125] In general, the amino acids or mimetics are coupled onto the
Fmoc-Linker-BHA resin using the Fmoc protected form of amino acid
or mimetic, with 2-5 equivalents of amino acid and a suitable
coupling reagent. After couplings, the resin may be washed and
dried under vacuum. Loading of the amino acid onto the resin may be
determined by amino acid analysis of an aliquot of Fmoc-amino acid
resin or by determination of Fmoc groups by UV analysis. Any
unreacted amino groups may be capped by reacting the resin with
acetic anhydride and diisopropylethylamine in methylene
chloride.
[0126] The resins are carried through several repetitive cycles to
add amino acids sequentially. The alpha amino Fmoc protecting
groups are removed under basic conditions. Piperidine, piperazine
or morpholine (20-40% v/v) in DMF may be used for this purpose.
Preferably 40% piperidine in DMF is utilized
[0127] Following the removal of the alpha amino protecting group,
the subsequent protected amino acids are coupled stepwise in the
desired order to obtain an intermediate, protected peptide-resin.
The activating reagents used for coupling of the amino acids in the
solid phase synthesis of the peptides are well known in the art.
For example, appropriate reagents for such syntheses are
benzotriazol-1-yloxy-tri-(dim- ethylamino)phosphonium
hexafluorophosphate (BOP), Bromo-tris-pyrrolidino-p- hosphonium
hexafluorophosphate (PyBroP), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-
-tetramethyluronium hexafluorophosphate (HBTU), and
diisopropylcarbodiimide (DIC). Preferred here are HBTU and DIC.
Other activating agents as described by Barany and Merrifield [The
Peptides, Vol. 2, J. Meienhofer, ed., Academic Press, 1979, pp
1-284] may be utilized. Various reagents such as
1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (HOSu) and
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotr- iazine (HOOBT) may be
added to the coupling mixtures in order to optimize the synthetic
cycles. Preferred here is HOBT.
[0128] The protocol for a typical synthetic cycle is as
follows:
1 Protocol 1 Step Reagent Time 1 DMF 2 .times. 30 sec 2 40%
piperidine/DMF 1 min 3 40% piperidine/DMF 15 min 4 DMF 2 .times. 30
sec 5 iPrOH 2 .times. 30 sec 6 DMF 3 .times. 30 sec 7 coupling 60
min- 18 hours 8 DMF 2 .times. 30 sec 9 iPrOH 1 .times. 30 sec 10
DMF 1 .times. 30 sec 11 CH.sub.2Cl.sub.2 2 .times. 30 sec
[0129] Solvents for all washings and couplings were measured to
volumes of 10-20 ml/g resins. Coupling reactions throughout the
synthesis were monitored by the Kaiser ninhydrin test to determine
extent of completion [Kaiser et at. Anal. Biochem. 1970, 34,
595-598]. Slow reaction kinetics was observed for Fmoc-Arg (Pmc)
and for couplings to secondary amines by sterically hindered acids.
Any incomplete coupling reactions were either recoupled with
freshly prepared activated amino acid or capped by treating the
peptide resin with acetic anhydride as described above. The fully
assembled peptide-resins were dried in vacuum for several
hours.
[0130] For each compound, the blocking groups were removed and the
peptide cleaved from the resin by the following procedure.
Generally, the peptide-resins were treated with 100 .mu.L
ethanedithiol, 100 .mu.L dimethylsulfide, 300 .mu.L anisole, and
9.5 mL trifluoroacetic acid, per gram of resin, at room temperature
for 120 min. The resin is filtered off and the filtrates are
precipitated in chilled ethyl ether. The precipitates are
centrifuged and the ether layer is decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged. The crude
products are dried under vacuum.
[0131] Purification of Crude Peptide Preparations
[0132] Purification of the crude peptides was carried out by
preparative HPLC. The peptides were applied to the columns in a
minimum volume of either AcOH/H.sub.2O or 0. 1% TFA/H.sub.2O.
Gradient elution was generally started at 10% B buffer, 10% -60% B
in 90 minutes, (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) at a flow rate of 8 mL/min. UV detection was made
at 280 nm. Fractions were collected at 1.0-2.5 minute intervals and
inspected by analytical HPLC. Fractions judged to be of high purity
were pooled and lyophilized.
[0133] Purity of the final products was checked by analytical HPLC
on a reversed phase column as stated above. Purity of all products
was judged to be approximately 95-99%. All final products were also
subjected to fast atom bombardment mass spectrometry (FAB-MS) or
electrospray mass spectrometry (ES-MS). All products yielded the
expected parent M+H ions within acceptable limits.
[0134] Utilizing the techniques described above, the compounds of
this invention can be synthesized in accordance with the following
reaction schemes. 33 34 35 36 37 38 39404142 43 44 45 46 47 48
[0135] The synthetic peptides of the current invention are prepared
by using conventional solid phase peptide synthesis methodology
discussed in the previous section. Each cycle consists of two
procedures; the initial cleavage of the Fmoc protecting group from
the terminal nitrogen in the resin bound chain followed by
acylation of the amine function with an Fmoc protected amino acid.
The cycle is generally carried out in accordance with the stepwsize
procedures outlined in Protocol 1. The deprotection is accomplished
by using an organic base, for example piperazine, morpholine or
piperidine, preferably piperidine in a suitable inert solvent, for
example N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP).
The coupling reaction can be carried out by one of the many
conditions developed for amide bond formation, for example
O-benzotriazol-1-yl N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU) in the presence of an organic base, for
example diisopropylethylamine (DIPEA) in an inert solvent, for
example DMF. Alternatively in the present instance, the amide group
can be formed using a carbodiimide, for example,
diisopropylcarbodiimide (DIC) along with an activating agent such
as 1-hydroxybenzotriazole (HOBT) in a suitable inert solvent such
as DMF.
[0136] In Scheme A, in the first cycle, the Fmoc-Linker-BHA Resin
represented by structure 1 is deprotected and condensed with
Fmoc-amino acids of structure 2 to give the resin bound compounds
of structure 3. A second cycle incorporates the Fmoc-amino acids 4
to give the compounds of structure 5 (n=1). Compounds of structure
5 in which n=0 are prepared by eliminating the first cycle, and by
coupling Fmoc-amino acids of structure 4 directly to the
deprotected Fmoc-Linker-BHA Resin. In the third cycle, treatment of
the resin linked peptide furnishes the intermediates of structure
6a where R.sup.6 represents hydrogen. The intermediates of
structure 6b where R.sup.6 represents methyl are synthesized as
shown in Scheme C. Compounds of structure 6a, prepared by treating
compounds of structure 5 as prescribed in steps 1-5 of Protocol 1,
are reacted with an arylsulfonyl chloride, preferably
2-nitrobenzenesulfonyl chloride. The reaction is carried out in the
presence of a proton acceptor, for example pyridine, triethylamine
(TEA) or DIPEA, preferably DIPEA in a suitable inert solvent,
preferably DMF. N-methylation of the formed sulfonamide group in
the washed resin bound compounds of structure 19 is accomplished
under Mitsunobu conditions. Thus the sulfonamides of structure 19
are reacted with methanol in the presence of diethyl
azodicarboxylate (DEAD) and triphenylphosphine using methanol as
solvent. After the reaction is complete, the resin bound
N-methylsulfonamide of structure 20 is washed free of residual
reagents and byproducts. The 2-nitrobenzenesulfonyl residue is
removed by reacting 20 with 2-mercaptoethanol and the strong
organic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in a suitable
solvent, preferably DMF to give the resin bound intermediate of
structure 6b. The third cycle is completed by coupling compounds of
either structures 6a and 6b with Fmoc-Arg(Pmc)-OH (7) to give the
resin bound compounds of structure 8. Two additional cycles (
Scheme B) are carried out on peptides of structure 8 where the
amino acid Fmoc-(D)-Phe-OH (2) followed by either one of the amino
acid derivatives of structure 10 or 11 are sequentially
incorporated into the resin bound peptide to give the resin bound
polypeptides of structures 12 and 13.
[0137] Removal of the Fmoc from the resin bound polypeptides 12 is
carried out by treatment of 12 with piperidine in DMF to give the
compounds of structure 14 using the reaction conditions outlined in
Steps 1-5 of Protocol 1. The polypeptide is then N-capped by
reaction with an acylating agent to form the resin bound amides of
structure 15 or by reaction with an isocyanate to form the ureas of
structure 16 (Scheme D). The acylation is carried out under a
variety of methods well known to one skilled in the art. Among the
methods used are:
[0138] (i) reaction of the compounds of structure 14 with a
carboxylic acid R.sup.1--CO.sub.2H in a suitable solvent, such as
DMF in the presence of HBTU, and an organic base, preferably DIPEA
and
[0139] (ii) reaction of the compounds of structure 14 with a
carboxylic acid chloride R.sup.1--COCl in a suitable solvent, such
as dichloromethane in the presence an organic base, such as
pyridine, TEA and DIPEA, preferably DIPEA and
[0140] (iii) reaction of the compounds of structure 14 with a
carboxylic acid anhydride (R.sup.1--CO.sub.2CO--R.sup.1 in a
suitable solvent, such as dichloromethane or DMF in the presence an
organic base, preferably DIPEA.
[0141] The reaction of the compounds of structure 14 with an
isocyanate R.sup.1--NCO is carried out in a suitable solvent, such
as dichloromethane or DMF in the presence an organic base,
preferably DIPEA.
[0142] When the acylation and urea forming reactions are complete,
the resin bound products 15 and 16 are washed free of residual
reagents and byproducts.
[0143] Using the same conditions, the resin bound polypeptides of
structure 13 are converted to the N-acylated compounds of structure
17 and the ureas of structure 18 (Scheme E).
[0144] In Scheme F, the sequencing is carried out as in Scheme A
except that Fmoc-Glu(allyl)-OH (21) is incorporated into the resin
bound polypeptide instead of Fmoc-Arg(Pmc)-OH (7) to give the resin
bound N-capped polypeptides of structure 22. The allyl group is
removed by treatment of 22 with tributyltin hydride, palladium
chloride and triphenylphosphine in an inert solvent, for example
DMF to give the resin bound polypeptide of structure 23. Coupling
of 23 with Boc-guanidine gave the acylguanidine resin bound
compounds of structure 24. The reaction can be carried out by using
standard amide forming reaction methods, for example in the
presence of HBTU and an organic base, preferably DIPEA in a
suitable solvent, such as DMF.
[0145] In Scheme G, the sequencing is carried out as in Scheme A
except that either FmocPhenylhomoArg-OH (25) or Fmoc-citrulline
(26) is incorporated into the resin bound polypeptide in the place
of Fmoc-Arg(Pmc)-OH (7) to give the resin bound N-capped
polypeptides of structures 27 and 28 respectively.
[0146] As shown in Scheme H, the cleavage of remaining protecting
groups in the N-capped polypeptides 15-18, 24, 27 and 28 and the
concomitant cleavage of the peptides from the solid support is
carried out by using a strong organic acid, preferably
trifluoroacetic acid, optionally in the presence of an inert
solvent such as dichloromethane and a trace (1%) of water. The
reaction is conveniently carried out with or without the presence
of one or more carbocation scavengers, for example ethanedithiol,
dimethyl sulfide, triethylsilane and anisole. The polypeptide
cleavage solution is filtered free from the solid support, then is
diluted with a suitable solvent, preferably diethyl ether. The
solid polypeptides of structures 29-35 produced in this manner is
purified by reversed phase chromatography over a preparative C18
column. If convenient, in those cases where a racemic Fmoc-amino
acid 11 is sequenced into the polypeptide, the individual
stereoisomers are separated during the purification procedure. The
Fmoc-amino acids 2, 4, 7, 9, 21, 25 and 26 as well as the acylating
agents and isocyanates used to N-cap the polypeptides are known
compounds that are commercially available.
[0147] The Fmoc-amino acids 10 and 11 are prepared as described
herein by methods that are well known to those of ordinary skill in
the practice of organic chemistry. In Scheme I, the preparation of
Fmoc-amino acids from cyclic ketones is outlined. The
4-phenylcyclohexanones of formula 36 are converted to the
hydantoins of formula 37 by treatment with ammonium carbonate and
potassium cyanide. The reaction is conveniently carried out an
aqueous ethanol mixture at a temperature of from 50.degree. C. to
90.degree. C., preferably between 80.degree. C. and 90.degree. C.
Direct hydrolysis of the hydantoins to the amino acids of structure
38 require a prolonged treatment with strong base, for example with
6N sodium hydroxide solution or with barium hydroxide at reflux
temperature. Alternatively, compounds of structure 37 can be
converted to the bis-Boc derivatives of structure 39. The reaction
is carried out using tert-butyl dicarbonate [(Boc).sub.2O] in an
inert solvent, preferably tetrahydrofuran (THF), in the presence of
an organic amine base, preferably TEA and a catalyst,
4-dimethylaminopyridine (DMAP) at a temperature of from zero
degrees to room temperature, preferably at room temperature. The
bis-Boc hydantoins of structure 39 are readily converted to the
amino acids of structure 38. The reaction is accomplished using 1N
sodium hydroxide in an inert solvent, preferably dimethoxyethane
(DME) at from zero degrees to 50.degree. C., preferably at about
room temperature. Protection of the amino functionality with an
Fmoc group in a compound of structure 38 is carried out under a
variety of reaction conditions to give 40. The reaction may
conveniently be performed by treatment of a solution of the amino
acid 38 in a mixture of THF or dioxane, preferably dioxane and
aqueous sodium carbonate with 9-fluorenylmethoxychloroformate
(FmocCl) at a temperature of from zero degrees to room temperature,
preferably at room temperature. Alternatively,
N-(9-fluorenylmethoxycarbo- nyloxy)succinimide (FmocOSu) is added
to a solution of the amino acid 38 in aqueous acetonitrile
containing an organic tertiary amine base, preferably TEA. The
reaction is run at from zero degrees to room temperature,
preferably at room temperature. In another variation of the
procedure, DME is evaporated from the hydrolysis mixture in the
conversion of 39 to 38 and the reaction is adjusted to .about.pH
11. The resulting solution of the sodium salt of 38 is then treated
in situ with FmocOSu or FmocCl in dioxane at a temperature of from
zero degrees to room temperature, preferably at room
temperature.
[0148] In the same manner, the tetralones 41, the
N-aryl-4-ketopiperidines 42, and the cyclohexanone derivatives 43
and 44 are converted to the corresponding Fmoc-amino acids of
structures 11 and 45-47.
[0149] Compounds of structure 40 where R.sup.3 represents a linear
or branched lower alkoxy and R.sup.2 and R.sup.4 is hydrogen, as in
the sub genus structure 49, may be prepared by 0-alkylation of the
compound of structure 48 (Scheme J). Where R.sup.16 represents an
unbranched lower alkyl moiety, the alkylation is carried out by
using a primary alkyl halide of structure R.sup.16X in the presence
of an alkali metal carbonate, for example, sodium or potassium
carbonate. The alkyl halide may be a chloro, bromo or iodo
derivative, preferably an alkyl iodide (.times..dbd.I). The
reaction may be conveniently carried out in an inert solvent that
promotes Sn2 displacement reactions, for example acetone,
2-butanone or N,N-dimethylformamide, preferably acetone, at a
temperature of from room temperature to the reflux temperature of
the solution, preferably the reflux temperature. When R.sup.16
represents a branched lower alkyl group, e.g., 2-propyl, the
alkylation is carried out by using a secondary alkyl halide of
structure R.sup.16X in the presence of an alkali metal carbonate,
e.g., potassium carbonate. The secondary alkyl halide is preferably
a secondary alkyl iodide, for example, 2-iodopropane
(.times..dbd.I). The reaction may be conveniently carried out in an
inert solvent, preferably N,N-dimethylformamide, at a temperature
of from room temperature to the reflux temperature of the solution,
preferably at about 100.degree. C.
[0150] Compounds of structure 40 can be prepared by methods that
are well known to one of ordinary skill in the practice of organic
chemistry. As outlined in Scheme K), treatment of the aryl halides
of structure 50 (X' represents bromo or iodo) with an alkyl metal
reagent, preferably t-butyl lithium, results in a transmetalation
reaction to give the corresponding aryl lithium of structure 51.
The reaction is conveniently carried out at -78.degree. C. by the
addition of a solution of the alkyl lithium in to a solution of
compounds of structure 50 an inert anhydrous solvent, such as
diethyl ether or tetrahydrofuran, preferably tetrahydrofuran. The
aryl lithium of structure 51, is then reacted in situ with a
solution of the monoketal of cyclohexane-1,4-dione (2) in an
suitable inert solvent, for example tetrahydrofuran, while the
reaction temperature is maintained below -60.degree. C., preferably
at about -78.degree. C. to give the carbinols of structure 53. The
compounds of structure 54 are obtained by the dehydration of the
carbinols of structure 53. The reaction is conveniently carried out
using a strong organic acid catalyst, preferably p-toluenesulfonic
acid in an inert solvent, for example benzene or toluene,
preferably benzene, at the reflux temperature of the solvent. The
formed water is removed from the reaction mixture by means of a
Dean Stark apparatus to enable the reaction to go to completion.
Compounds of structure 55 are produced by hydrogenation of the
olefms of structure 54. The reaction is conveniently carried out
using a noble metal catalyst, for example palladium on carbon, in a
hydrogen atmosphere in an inert solvent, for example ethanol or
ethyl acetate. The hydrogenation is usually carried out at room
temperature and 40 psi of hydrogen, however if the aryl ring in
structure 54 contains a group prone to hydrogenolysis, e.g., if
R.sup.2, R.sup.3 or R.sup.4 represents chloro, the reaction
pressure is kept at about 5 psi. Compounds of structure 55 may be
also obtained directly from carbinols of structure 53 by reductive
elimination of the hydroxyl group. In this reaction a solution of
the compound of structure 53 (R.sup.2.dbd.R.sup.3.dbd.H and
R.sup.4.dbd.OMe) in an inert solvent, for example dichloromethane,
is treated with a Lewis acid, such as boron trifluoride etherate,
and a reducing agent, for example triethylsilane, at a temperature
of from zero degrees to room temperature. Removal of the ketal
protectimg group in compounds of structure 55 gives the ketone of
formula 40. The reaction is conveniently carried out in acetone or
2-butanone, preferably acetone under acid catalysis, for example 4N
hydrochloric acid or p-toluenesulfonic acid at from room
temperature to the reflux temperature of the reaction mixture,
preferably at the reflux temperature.
[0151] 5-Substituted-beta-tetralones of structure 41 are generally
known compounds, or if they are not known they can be prepared by
methods that are well known to one of ordinary skill in the field
of organic chemistry. In the present instance, compounds of
structure 41 are prepared by two methods outlined in Schemes L and
M.
[0152] As shown in Scheme L, a 2-substituted hydrocinnamic acid of
structure 56 (R.sup.10=bromo, chloro or a linear or branched alkyl
group of from 1 to 3 carbons) is converted to the corresponding
carboxylic acid chloride of structure 57. This conversion can be
carried out by several methods, for example by treatment of the
hydrocinnamic acid with oxalyl chloride, optionally in the presence
of a catalytic amount of N,N-dimethylformamide, in an inert
solvent, such as benzene or dichloromethane, preferably
dichloromethane. The reaction may be conveniently carried out at a
temperature of from zero degrees to room temperature, preferably at
room temperature. Alternatively the compound of structure 56 is
reacted with an acyl chloride forming reagent such as sulfuryl
chloride in an inert solvent, for example benzene or toluene,
preferably toluene at a temperature between room temperature to the
reflux temperature of the solution, preferably at the reflux
temperature.
[0153] The diazoketone of structure 58 is prepared by treatment of
the thus formed acyl halide of structure 57 in an inert solvent,
e.g., dichloromethane with an excess of a freshly prepared ethereal
solution of diazomethane. The combination of reagents is
conveniently carried out at ice bath temperature and the reaction
is then allowed to proceed at a temperature of from zero degrees to
room temperature, preferably at room temperature. Cyclization of
the diazoketone of structure 58 to furnish the tetralone of
structure 41 is promoted by rhodium (II) acetate dimer in an inert
solvent, e.g., dichloromethane. The reaction is normally carried
out at from room temperature to the reflux temperature of the
solution, preferably at the reflux temperature.
[0154] Compounds of structure 41, wherein R.sup.10 represents a
linear or branched lower alkoxy group or a dialkylamino
substituent, are prepared as shown in Scheme M. The compounds of
structure 60 (R.sup.15'=an unbranched lower alkyl moiety) are
prepared by per-O-alkylation of the naphthalenediol of structure 59
with a primary alkyl iodide or bromide, preferably an iodide, in
the presence of a base such as an alkali metal carbonate, for
example, sodium or potassium carbonate. The reaction may be carried
out in an inert solvent, preferably N,N-dimethylformamide at a
temperature of from room temperature to 100.degree. C., preferably
at 35.degree. C. The compounds of structure 63 (R.sup.15"
represents a branched lower alkyl) are prepared in two steps from
the 2-tetralone of structure 61. The tetralone of structure 61 is
subjected to dehydrogenation in the presence of a noble metal
catalyst, such as palladium metal (10% on carbon) in a suitable
high boiling solvent such as p-cymene to give the aromatized
compound of structure 62. The naphthol of structure 62 is then
0-alkylated with a secondary alkyl iodide in the presence of a base
such as an alkali metal carbonate, preferably cesium carbonate to
furnish the compound of structure 63. The reaction may be
conveniently carried out in an inert solvent, preferably
N,N-dimethylformamide at a temperature of from room temperature to
100.degree. C., preferably at about 40.degree. C. The compound of
structure 65 is prepared by alkylation of 5-amino-2-naphthol (4)
with methyl iodide in the presence of a base such as an alkali
metal carbonate, preferably potassium carbonate. The reaction may
be carried out in an inert solvent, for example acetone or
2-butanone, preferably acetone, at a temperature between room
temperature and the reflux temperature of the solution, preferably
at the reflux temperature.
[0155] The tetralones of structures 41 are produced by reduction of
the compounds of structures 60, 63 and 65 under dissolving metal
conditions, followed by the acid catalyzed hydrolysis of the
intermediate enol ethers. The transformation is conveniently
carried out by the portionwise addition of a large excess of an
alkali metal, such as sodium or potassium, preferably sodium, to a
boiling solution of the substrate in an lower alcohol, preferably
alcohol until the starting material is consumed. The tetralones of
structures 41 are obtained by treatment of a solution of the
isolated intermediate enol ethers with a strong acid catalyst,
preferably p-toluenesulfonic acid. The hydrolysis may conveniently
carried out in a mixture of a lower alcohol, preferably ethanol,
and water at a temperature of between room temperature and the
reflux temperature of the solution, preferably at the reflux
temperature.
[0156] Compounds of structure 68 are can be prepared by reactions
that are known per se. For example, they can be prepared by
coupling a secondary amine of structure 66 with an aryl bromide or
iodide, preferably an aryl iodide of structure 67 (Scheme N). The
coupling reaction is catalyzed by a noble metal catalyst,
preferably tri(dibenzylideneacetone)-dipalladium, in the presence
of a chelating phosphine ligand, preferably tri-o-tolylphosphine,
and a hindered alkoxide base such as sodium tert-butoxide. The
reaction is conveniently carried out in an inert atmosphere using
an anhydrous solvent such as dioxane or toluene, preferably
dioxane, at a temperature of from 60.degree. C. to the reflux
temperature, preferably at 90.degree. C. Compounds of structure 56
and 66 are generally known compounds and are can be obtained from
commercial sources. Removal of the carbonyl protecting group in
compound 67 to give compounds of structure 42 can be carried out by
a variety of methods well known in the field of organic chemistry.
For example, the deprotection can be achieved by treatment of a
solution of compound 68 in a low boiling ketone such as acetone or
2-butanone with an aqueous mineral acid solution, for example 6N
hydrochloric acid. The reaction can be run at a temperature of from
room temperature to the reflux temperature of the mixture,
preferably at the reflux temperature.
[0157] The cyclohexanone derivatives of structures 63 are
commercially available compounds and the 4,4-diphenylcyclohexanone
(64) is prepared by published procedures.
[0158] This invention will be better understood by reference to the
following examples, which illustrate but do not limit the invention
described herein.
EXAMPLE 1
Preparation of Fmoc-1-amino-4-phenylcyclohexane-1-carboxylic acid
(Fmoc-Apc-OH)
[0159] Step 1: 49
[0160] To a solution of 4-phenylcyclohexanone (10.0 g, 57.5 mmol)
in ethanol (100 mL) and water (33 mL) in a glass pressure bottle,
were added ammonium carbonate (33 g, 344 mmol, 6 equiv.) and
potassium cyanide (5.6 g, 86.2 mmol, 1.5 equiv.). The mixture was
heated at 80-90.degree. C. for 24 hrs. The cooled reaction mixture
was added to icy water (400 ml) and stirred vigorously for 30 min.
The resulting precipitate was suction filtered, washed thoroughly
with water and dried to yield the hydantoin as a white solid (14.0
g, 100% yield). .sup.1H NMR (DMSO-d.sub.6): 8.63 (s, 1H), 7.23-7.36
(m, 4), 7.15 (m, 1), 2.50 (m, 1H), 2.10 (m, 1H), 1.85 (d, 1H) and
1.55-1.80 (m, 6H).
[0161] Step 2: 50
[0162] The hydantoin (10.0 g) was suspended in aqueous NaOH (6N,
350 mL) and heated at 130.degree. C. for 2-3 days. Upon the
completion of the hydrolysis, the reaction mixture was neutralized
with conc. HCl to slightly acidic (pH .about.6). The resulting
slurry was filtered, washed with water and dried to give
1-amino-4-phenylcyclohexane carboxylic acid (APC) as a white solid
(25 g, >100% yield. contaminated with inorganic salt) which was
used directly for next step. Small portion of the crude product was
purified on HPLC. .sup.1H NMR (DMSO-d.sub.6): 7.23.about.7.7.35
(m,2), 7.10-7.19 (m, 3H), 2.45 (m, 1H), 1.92-2.18 (m, 3H),
1.56-1.78 (m, 4H) and 1.20 (m, 1H); LRMS (electrospray) m/e 220
(M+1).sup.+, Calcd for C.sub.13H.sub.17NO.sub.2, 219.
[0163] Step 3: 51
[0164] The crude APC from the last step (25 g) was treated with
Fmoc-Cl (13.2 g., 1.25 equiv) in dioxane (300 mL) and aqueous 10%
Na.sub.2CO.sub.3 (150 ml) and stirred vigorously overnight. The
reaction mixture was concentrated to remove dioxane, neutralized
with 6N HCl to slightly acidic (pH 5-6) and extracted with EtOAc.
The combined organic extracts were washed with brine and dried over
Na.sub.2SO.sub.4. Removal of the solvent gave the crude product
which was then purified on flash chromatography (hexane/EtOAc to
CH.sub.2Cl.sub.2/MeOH) to give pure Fmoc-cis-APC (18.2 g, 72%
overall yield for two steps) and Fmoc-trans-APC (2.1 g, 8%). The
structure of cis Fmoc-APC was confirmed by single crystal X-ray
analysis of its derivative. Fmoc-cis-APC, .sup.1H NMR(CD.sub.3OD),
7.79 (d, 2H), 7.72 (d, 2H), 7.37 (t, 2), 7.24-7.32 (m, 4),
7.14-7.23 (m, 3), 4.37 (d, 2H), 4.24 (t, 1H), 2.55 (m, 1H), 2.28
(m, 2H), 1.84-1.96 (m, 2H) and 1.64-1.73 (m, 4H).
EXAMPLE 2
Preparation of
Fmoc-1-amino-4-(4-methoxyphenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-MeOApc-OH)
[0165] Step 1: 52
[0166] A solution of 4-(4-hydroxyphenyl)cyclohexanone (5.0 g, 26.3
mmol) in acetone (100 mL) was treated with K.sub.2CO.sub.3 (14.5 g,
105 mmol, 4 equiv) and iodomethane (4.9 mL, 11.2 g, 78.6 mmol, 3
equiv.). The reaction was heated at 65.degree. C. overnight. After
the solvent was removed, the residue was treated with H20 and
extracted with EtOAc. The organic extracts were combined and washed
with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuum
to give the spectroscopically pure 4-(4-methoxyphenyl)cyclohexanone
(5.34 g, 100%). .sup.1HNMR(CDCl.sub.3) 7.16 (dt, 2H), 6.87 (dt,
2H), 3.78 (s, 3H), 2.99 (tt, 1H), 2.47-2.53 (m, 4H), 2.20 (m, 2H)
and 1.83-1.98 (m, 2H); MS (electrospray) m/e, 205 (M+1).sup.+,
Calcd for C.sub.13H.sub.16O.sub.2, 204.
[0167] Step 2: 53
[0168] To a solution of the ketone (3.86 g, 18.9 mmol) in ethanol
(50 mL) and water (15 mL) in a glass pressure bottle, were added
ammonium carbonate (14.5 g, 151 mmol, 8 equiv.) and potassium
cyanide (2.0 g, 30.7 mmol, 1.6 equiv.). The mixture was heated at
80-90.degree. C. for 24 hrs. The cooled reaction mixture was added
to icy water (300 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (4.75 g,
91% yield). MS (electrospray) m/e 273 (M-H), Calcd for
C.sub.15H.sub.18N.sub.2O.sub.3, 274.
[0169] Step 3: 54
[0170] To a suspension of the hydantoin (18.7 g, 68.25 mmol) in dry
THF (450 mL) were added di-tert-butyl dicarbonate (37.2 g, 170.5
mmol, 2.5 equiv), triethylamine (10.5 mL, 7.59 g, 75.0 mmol, 1.1
equiv) and DMAP (460 mg, 3.65 mmol) in succession. About 15 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (800 mL), washed with 1N HCl
(3.times.50 mL), saturated aqueous Na.sub.2CO.sub.3 (2.times.50 mL)
and brine (2.times.50 mL), dried over anhydrous Na.sub.2SO.sub.4
and concentrated under reduced pressure. The crude light yellow
product was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.70/30) to give the pure bis-Boc hydantoin as a white
solid (27.6 g, 87%). .sup.1H NMR (CDCl.sub.3): 7.28 (dt, 2H), 6.88
(dt, 2H), 3.79 (s, 3H), 2.14-2.24 (m, 2H), 1.59 (s, 9H) and 1.38
(s, 9H); MS (electrospray) m/e 538 (M+MeCN+Na).sup.+, Calcd for
C.sub.25H.sub.34N.sub.2O.sub.7, 474.
[0171] Step 4: 55
[0172] The bis-Boc hydantoin (15.08 g, 31.78 mmol) was dissolved in
DME (500 mL) to give a clear solution. To this solution was added
1N NaOH (290 mL, 290 mmol) and the reaction was stirred overnight
at room temperature, giving a slightly cloudy mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(4-methoxyphenyl)cyclohexane carboxylic acid (4-MeOAPC)
was treated with 6N HCl to adjust the pH to 11-12. To this solution
(.about.300 mL) were added DME (300 mL) and a solution of Fmoc-OSu
(16.7 g, 49.42 mmol) in DME (200 mL) and the reaction was stirred
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove DME, acidified with
3N HCl, extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 98/2.fwdarw.90/10) to give
the pure product Fmoc-4-MeOAPC as a white solid (12.4 g, 83% yield
from the bis-Boc hydantoin). .sup.1H NMR (DMSO-d.sub.6), 7.88 (d,
2H), 7.76 (d, 2H), 7.40 (t, 2H), 7.30 (t, 2H), 7.11 (d, 2H), 6.85
(d, 2H), 3.71 (s, 3H); MS (electrospray) m/e 470 (M-H), Calcd for
C.sub.29H.sub.29NO.sub.5, 471.
EXAMPLE 3
Preparation of
Fmoc-1-amino-4-(4-ethoxyphenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-EtOApc-OH)
[0173] Step 1: 56
[0174] A solution of 4-(4-hydroxyphenyl)cyclohexanone (5.0 g, 26.3
mmol) in acetone (100 mL) was treated with K.sub.2CO.sub.3 (14.5 g,
105 mmol, 4 equiv) and iodoethane (10.5 mL, 20.5 g, 131 mmol, 5
equiv.). The reaction was heated at 65.degree. C. overnight. After
the solvent was removed, the residue was treated with H.sub.2O and
extracted with EtOAc. The organic extracts were combined and washed
with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuum
to give the spectroscopically pure 4-(4-ethoxyphenyl)cyclohexanone
(5.74 g, 100%). .sup.1H NMR (CDCl.sub.3) 7.15 (dt, 2H), 6.86 (dt,
2H), 4.02 (q, 2H), 2.99 (tt, 1H), 2.46-2.54 (m, 4H), 2.16-2.24 (m,
2H), 1.83-2.00 (m, 2H) and 1.41 (t, 3H); MS (electrospray) m/e, 219
(M+1).sup.+, Calcd for C.sub.14H.sub.18O.sub.2, 218.
[0175] Step 2: 57
[0176] To a solution of the ketone (4.15 g, 19.01 mmol) in ethanol
(50 mL) and water (15 mL) in a glass pressure bottle, were added
ammonium carbonate (14.5 g, 151 mmol, 8 equiv.) and potassium
cyanide (2.05 g, 31.42 mmol, 1.6 equiv.). The mixture was heated at
80-90.degree. C. for 19 hrs. The cooled reaction mixture was added
to icy water (300 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (5.17 g,
94% yield). MS (electrospray) m/e 287 (M-H), Calcd for
C.sub.16H.sub.20N.sub.2O.sub.3, 288.
[0177] Step 3: 58
[0178] To a suspension of the hydantoin (4.22 g, 14.65 mmol) in dry
THF (100 mL) were added di-tert-butyl dicarbonate (7.98 g, 36.60
mmol, 2.5 equiv), triethylamine (2.3 mL, 1.63 g, 16.11 mmol, 1.1
equiv) and DMAP (89.4 mg, 0.73 mmol) in succession. About 15
minutes after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (300 mL), washed with 1N HCl
(3.times.20 mL), saturated aqueous Na2CO3 (2.times.20 mL) and brine
(2.times.20 mL), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The crude light yellow product
was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.70/30) to give the pure bis-Boc hydantoin as a white
solid (7.01 g, 98%). .sup.1H NMR (CDCl.sub.3): 7.27 (dt, 2H), 6.87
(dt, 2H), 4.02 (q, 2H), 1.59 (s, 9H), 1.43 (t, 3H) and 1.38 (s,
9H); MS (electrospray) m/e 999 (2M+Na).sup.+, Calcd for
C.sub.26H.sub.36N.sub.2O.- sub.7, 488.
[0179] Step 4: 59
[0180] The bis-Boc hydantoin (6.58 g, 13.46 mmol) was dissolved in
DME (200 mL) to give a clear solution. To this solution was added
1N NaOH (121 mL, 121 mmol) and the reaction was stirred overnight
at room temperature, giving a slightly cloudy mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(4-ethoxyphenyl)cyclohexane carboxylic acid (4-EtOAPC)
was treated with 6N HCl to adjust the pH to 11-12. To this solution
(.about.130 mL) were added DME (100 mL) and a solution of Fmoc-OSu
(6.83 g, 20.24 mmol) in DME (30 mL) and the reaction was stirred
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove DME, acidified with
3N HCl, extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 98/2.fwdarw.90/10) to give
the pure product as a white solid (5.56 g, 85% yield from the
bis-Boc hydantoin). .sup.1H NMR (DMSO-d.sub.6), 7.88 (d, 2H), 7.74
(d, 2H), 7.40 (td, 2H), 7.30 (td, 2H), 7.11 (d, 2H), 6.84 (d, 2H),
3.97 (q, 2H) and 1.29 (t, 3H); MS (electrospray) m/e 484 (M-H),
Calcd for C.sub.30H.sub.31NO.sub.5, 485.
EXAMPLE 4
Preparation of
Fmoc-1-amino-4-(4-hydroxyphenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-HOApc-OH)
[0181] Step 1: 60
[0182] To a solution of 4-(4-hydroxyphenyl)cyclohexanone (2.00 g,
10.52 mmol) in ethanol (30 mL) and water (10 mL) in a glass
pressure bottle, were added ammonium carbonate (6.17 g, 64.2 mmol,
6 equiv.) and potassium cyanide (1.07 g, 15.8 mmol, 1.5 equiv.).
The mixture was heated at 80-90.degree. C. overnight. The cooled
reaction mixture was added to icy water (200 ml) and stirred
vigorously for 30 min. The resulting precipitate was suction
filtered, washed thoroughly with water and dried to yield the
hydantoin as a white solid (2.56 g, 94% yield). MS (electrospray)
m/e 261 (M+H).sup.+, Calcd for C.sub.14H.sub.16N.sub.2O.su- b.3,
260.
[0183] Step 2: 61
[0184] The hydantoin (2.10 g, 8.06 mmol) was suspended in aqueous
NaOH (6N, 100 mL) and heated at 130.degree. C. for 2-3 days. Upon
the completion of the hydrolysis, the reaction mixture was
neutralized with conc. HCl to slightly acidic (pH.about.6). The
resulting slurry was filtered, washed with water and dried to give
1-amino-4-(4-hydroxyphenyl)- cyclohexane carboxylic acid (4-HOAPC)
as a white solid (3.1 g, >100% yield contaminated with inorganic
salt). MS (electrospray) m/e 236 (M+H).sup.+, Calcd for
C.sub.13H.sub.17NO.sub.3, 235.
[0185] Step 3: 62
[0186] The crude APC from the last step (3.1 g) was treated with
Fmoc-Cl (2.6g, 1.25 equiv) in dioxane (100 mL) and aqueous 10%
Na.sub.2CO.sub.3 (50 ml) and stirred vigorously overnight. The
reaction mixture was concentrated to remove dioxane, neutralized
with 6N HCl to slightly acidic (pH 5-6) and extracted with EtOAc.
The combined organic extracts were washed with brine and dried over
Na.sub.2SO.sub.4. Removal of the solvent gave the crude product
which was purified on flash chromatography (hexane/EtOAc to
CH.sub.2Cl.sub.2/MeOH) to give pure Fmoc-4-HOAPC (2.76 g, 75%
overall yield for two steps). .sup.1H NMR(CD.sub.3OD), 7.78 (d,
2H), 7.72 (d, 2H), 7.38 (t, 2H), 7.30 (td, 2H), 7.04 (d, 2H), 6.72
(dt, 2H), 4.38 (d, 2H), 4.25 (t, 1H), 2.46 (m, 1H), 2.24-2.34 (m,
2H) and 1.81-1.92 (m, 6H); MS (electrospray) m/e 456 (M-H), Calcd
for C.sub.28H.sub.27NO.sub.5, 457.
EXAMPLE 5
Preparation of
Fmoc-1-amino-4-(4-isopropoxyphenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-iPrOApc-OH)
[0187] Step 1: 63
[0188] A solution of 4-(4-hydroxyphenyl)cyclohexanone (6.0 g, 31.6
mmol) in DMF (90 mL) was treated with K.sub.2CO.sub.3 (21 g, 158
mmol, 5 equiv) and 2-iodopropane (15 mL, 26.8 g, 158 mmol, 5
equiv.). The reaction was heated at 100.degree. C. overnight. After
the solvent was removed, the residue was treated with H.sub.2O and
extracted with EtOAc. The organic extracts were combined and washed
with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuum
to give the spectroscopically pure
4-(4-isopropoxyphenyl)cyclohexanone (7.02 g, 95%). .sup.1H NMR
(CDCl.sub.3): 7.14 (dt, 2H), 6.84 (dt, 2H), 4.3 (septet, 1H), 2.97
(tt, 1H), 2.46-2.52 (m, 4H), 2.16-2.24 (m, 2H), 1.83-1.98 (m, 2H)
and 1.33 (d, 6H).
[0189] Step 2: 64
[0190] To a solution of the ketone (5.1 g, 21.98 mmol) in ethanol
(90 mL) and water (30 mL) in a glass pressure bottle, were added
ammonium carbonate (12.6 g, 131 mmol, 6 equiv.) and potassium
cyanide (2.14 g, 32.9 mmol, 1.5 equiv.). The mixture was heated at
80-90.degree. C. for 24 hrs. The cooled reaction mixture was added
to icy water (400 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield hydantoin as a white solid (6.60 g, 99%
yield). .sup.1H NMR (DMSO-d.sub.6): 10.60 (s, 1H), 8.65 (s, 1H),
7.18 (d, 2H), 6.80 (d, 2H), 4.52 (septet, 1H), 2.43 (m, 1H),
1.85-2.15 (m, 2H), 1.56-1.80 (m, 6H) and 1.22 (d, 6H); MS
(electrospray) m/e 301 (M-H), Calcd for
C.sub.17H.sub.22N.sub.2O.sub.3, 302.
[0191] Step 3: 65
[0192] To a suspension of the hydantoin (5.8 g, 19.20 mmol) in dry
THF (180 mL) were added di-tert-butyl dicarbonate (10.46 g, 48.0
mmol, 2.5 equiv), triethylamine (2.9 mL, 2.13 g, 21.12 mmol, 1.1
equiv) and DMAP (140 mg, 1.15 mmol) in succession. About 15 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (600 mL), washed with 1N HCl
(3.times.40 mL), saturated aqueous Na.sub.2CO.sub.3 (2.times.40 mL)
and brine (2.times.40 mL), dried over anhydrous Na.sub.2SO.sub.4
and concentrated under reduced pressure. The crude light yellow
product was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.80/20) to give the pure bis-Boc hydantoin as a white
solid (9.4 g, 98%). .sup.1H NMR (CDCl.sub.3): 7.27 (dt, 2H), 6.87
(dt, 2H), 4.02 (q, 2H), 2.98 (t, 1H), 2.26-2.56 (m, 4H), 2.14-2.24
(m, 2H), 1.76-1.86 (m, 2H), 1.59 (s, 9H), 1.43 (t, 3H) and 1.38 (s,
9H); MS (electrospray) m/e 999 (2M+Na).sup.+, Calcd for
C.sub.26H.sub.36N.sub.2O.- sub.7, 488.
[0193] Step 4: 66
[0194] The bis-Boc hydantoin (4.34 g, 8.64 mmol) was dissolved in
DME (100 mL) to give a clear solution. To this solution was added
1N NaOH (78 mL, 78 mmol) and the reaction was stirred overnight at
room temperature, giving a fairly clear mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(4-isopropoxyphenyl)cyclohexane carboxylic acid
(4-iPrOAPC) was treated with 6N HCl to adjust the pH to 11-12. To
this solution (90 mL) were added DME (120 mL) and a solution of
Fmoc-OSu (3.49 g, 10.34 mmol, 1.2 equiv) in DME (20 mL) and the
reaction was stirred overnight at room temperature. The reaction
mixture was concentrated under reduced pressure to remove DME,
acidified with 3N HCl, extracted with EtOAc. The combined organic
extracts were washed with brine, dried over anhydrous
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
through flash chromatography (hexane/EtOAc.fwdarw.CH-
.sub.2Cl.sub.2/MeOH) to give the pure product as a white solid
(3.23, 75% yield from bis-Boc hydantoin). .sup.1H
NMR(DMSO-d.sub.6), 7.76 (d, 2H), 7.60 (d, 2H), 7.39 (t, 2H), 7.31
(t, 2H), 7.08 (d, 2H), 6.84 (d, 2H), 4.24 (m, 1H) and 1.34 (d, 6H);
MS (electrospray) m/e 498 (M-H), Calcd for
C.sub.31H.sub.33NO.sub.5, 499.
EXAMPLE 6
Preparation of
Fmoc-1-amino-4-(4-methylphenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-MeApc-OH)
[0195] Step 1: 67
[0196] To a solution of 4-iodotoluene (10.9 g, 50.0 mmol) in dry
THF (180 mL) at -78.degree. C. was added a solution of n-BuLi (1.6
M, 31.0 mL, 50 mmol) in hexane over 20 min. The reaction was
stirred for another 20 min before a solution of
1,4-cyclohexanedione mono-ethylene ketal (6.0 g, 38.46 mmol) in dry
THF (100 mL) was added dropwise. After stirred for 2 h at
-78.degree. C., the reaction was quenched with aqueous NH.sub.4Cl
and extracted with EtOAc. The combined organic extracts were washed
with brine, dried over Na.sub.2SO.sub.4, concentrated in vacuo to
give the spectroscopically pure product as a white solid (9.34 g,
98% yield). .sup.1H NMR (CDCl.sub.3): 7.41 (m, 2H), 7.16 (d, 2H),
3.98 (m, 4H), 2.34 (s, 3H); MS (EI) m/e 248 (M.sup.+), Calcd for
C.sub.15H.sub.20O.sub.3, 248.
[0197] Step 2: 68
[0198] To a solution of the alcohol (9.10 g, 36.65 mmol) in dry
benzene (200 mL) in a flask equipped with a Dean-Stark trap, was
added p-toluenesulfonic acid monohydrate (650 mg) and the reaction
was heated at 100.degree. C. for 3 hrs. The reaction was cooled to
rt, diluted with EtOAc (500 mL) and washed with aqueous
Na.sub.2CO.sub.3 (50 mL), brine (3.times.50 mL), dried over
Na.sub.2SO.sub.4 and concentrated under reduced pressure to give
the spectroscopically pure product (8.36 g, 100 yield), which was
used for next step without purification. MS (EI) m/e 230 (M.sup.+),
190 (M-OCH.sub.2CH.sub.2O), Calcd for C.sub.15H.sub.18O.sub.2,
230.
[0199] Step 3: 69
[0200] To a solution of the olefin (7.49 g) in EtOAc (180 mL) was
added Pd/C (5 wt % on carbon, 800 mg) and the reaction was run
under 40 psi of hydrogen for 3 hrs at room temperature. The
catalyst was filtered off and the filtrate was concentrated to give
the spectroscopically pure product as a colorless oil (7.40 g, 100%
yield). MS (EI) m/e 232 (M.sup.+), 188 (M-OCH.sub.2CH.sub.2), Calcd
for C.sub.15H.sub.20O.sub.2, 232.
[0201] Step 4: 70
[0202] A solution of the ketal (6.90 g) in acetone (140 mL) was
treated with 4N HCl (60 mL) and heated at 65.degree. C. for 4 hrs.
Solvent was removed and the residue was diluted with EtOAc and
neutralized with 4N HCl. The aqueous was extracted with EtOAc. The
combined organic extracts were washed with brine, dried and
concentrated. The resulting crude product was used for next step
without without purification (5.57 g, quantitative yield). MS (EI)
m/e 188 (M.sup.+), Calcd for C.sub.13H.sub.16O, 188.
[0203] Step 5: 71
[0204] To a solution of 4-(4-methylphenyl)cyclohexanone (5.32 g,
28.3 mmol) in ethanol (90 mL) and water (30 mL) in a glass pressure
bottle, were added ammonium carbonate (16.3 g, 169.8 mmol, 6
equiv.) and potassium cyanide (3.68 g, 56.5 mmol, 2 equiv.). The
mixture was heated at 80-90.degree. C. overnight. The cooled
reaction mixture was added to icy water (400 ml) and stirred
vigorously for 30 min. The resulting precipitate was suction
filtered, washed thoroughly with water and dried to yield the
hydantoin as a white solid (6.3 g, 86% yield). MS (electrospray)
m/e 517 (2M+H), Calcd for C.sub.15H.sub.18ClN.sub.2O.sub.2- ,
258
[0205] Step 6,: 72
[0206] To a suspension of the hydantoin (5.82 g, 22.55 mmol) in dry
THF (250 mL) were added di-tert-butyl dicarbonate (12.3 g, 56.4
mmol, 2.5 equiv), triethylamine (3.5 mL, 2.5 g, 24.7 mmol, 1.1
equiv) and DMAP (275 mg, 2.25 mmol) in succession. The reaction
turned into a clear yellow solution and was stirred overnight at
room temperature. The reaction mixture was concentrated under
reduced pressure to yield a solid that was then taken up in EtOAc
(500 mL), washed with 1N HCl (3.times.50 mL), saturated aqueous
Na.sub.2CO.sub.3 (2.times.50 mL) and brine (2.times.50 mL), dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced
pressure. The crude light yellow product was purified through flash
chromatography (hexane/EtOAc, 90/10.fwdarw.70/30) to give the pure
bis-Boc hydantoin as a white solid (10.03 g, 100% yield). .sup.1H
NMR (CDCl.sub.3): 7.26 (d, 2H), 6.87 (d, 2H), 3.00 (m, 1H), 2.32
(s, 3H), 1.59 (s, 9H) and 1.37 (s, 9H).
[0207] Step 7: 73
[0208] The bis-Boc hydantoin (6.40 g, 13.97 mmol) was dissolved in
DME (200 mL) to give a clear solution. To this solution was added
1N NaOH (120 mL, 120 mmol) and the reaction was stirred overnight
at room temperature, giving a slightly cloudy mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(4-methylphenyl)cyclohexane carboxylic acid (4-MeAPC) was
treated with 6N HCl to adjust the pH to 11-12. To this solution
(.about.140 mL) were added DME (240 mL) and a solution of Fmoc-OSu
(5.10 g, 15.13 mmol, 1.1 equiv) in DME (40 mL) and the reaction was
stirred overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove DME, acidified with
3N HCl, extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 98/2.fwdarw.90/10) to give
the pure product as a white solid (4.35 g, 69% yield from bis-Boc
hydantoin). .sup.1H NMR (DMSO-d.sub.6): 7.88 (d, 2H), 7.75 (d, 2H),
7.24-7.43 (m, 4H), 7.02-7.14 (m, 4H), 4.25 (m, 3H), 2.24 (s,
3H).
EXAMPLE 7
Preparation of
Fmoc-1-amino-4-(4-chlorophenyl)cyclohexane-1-carboxylic acid
(Fmoc-4-ClApc-OH)
[0209] Step 1: 74
[0210] A solution of 4-chlorophenylbromide (7.5 g, 39.2 mmol) in
dry THF (180 mL) was cooled to -78.degree. C. and treated dropwise
with a solution of n-BuLi (1.6 M, 25 mL, 40 mmol) in hexane over 20
min. The reaction was stirred for a further 30 min before a
solution of 1,4-cyclohexanedione mono-ethylene ketal (6.0 g, 38.46
mmol) in dry THF (100 mL) was added dropwise. After stirred for 1
hr at -78.degree. C., the reaction was quenched with aqueous
NH.sub.4Cl and extracted with EtOAc. The combined organic extracts
were washed with brine, dried over Na.sub.2SO.sub.4, concentrated
in vacuo to give the spectroscopically pure product as a white
solid (9.40 g, 91% yield). .sup.1H NMR (CDCl.sub.3): 7.45 (m 2H),
7.31 (m, 2H), 3.99 (m, 4H), 2.02-2.20 (m, 4H), 1.75-1.82 (m, 2H),
1.66-1.73 (m, 2H), 1.54 (s, 1H); MS (EI) m/e 268 (M.sup.+), 251
(M-OH), 250 (M-H.sub.2O), Calcd for C.sub.14H.sub.17ClO.sub.3,
268.
[0211] Step 2: 75
[0212] To a solution of the alcohol (6.78 g, 25.30 mmol) in dry
benzene (120 mL) in a flask equipped with a Dean-Stark trap, was
added p-toluenesulfonic acid monohydrate (960 mg) and the reaction
was heated at reflux for 3 hrs. The reaction was cooled to rt,
diluted with EtOAc (500 mL) and washed with aqueous
Na.sub.2CO.sub.3 (50 mL), brine (3.times.50 mL), dried over
Na.sub.2SO.sub.4 and concentrated under reduced pressure to give
the spectroscopically pure product (6.30 g, 100 yield), which was
used for next step without purification. MS (EI) m/e 250 (M.sup.+),
190 (M-OCH.sub.2CH.sub.2O), Calcd for C.sub.14H.sub.15ClO.sub.2,
250.
[0213] Step 3: 76
[0214] To a solution of the olefin (6.11 g) in EtOAc (120 mL) was
added Pd/C (5 wt % on carbon, 600 mg) and the reaction was run
under 5 psi of hydrogen for 3 hrs at room temperature. The catalyst
was filtered off and the filtrate was concentrated to give the
spectroscopically pure product as a colorless oil (6.10 g, 100%
yield). MS (EI) m/e 252(M.sup.+), Calcd for
C.sub.14H.sub.17ClO.sub.2, 252.
[0215] Step 4: 77
[0216] A solution of the ketal (5.81 g, 23.06 mmol) in acetone (200
mL) was treated with p-toluenesulfonic acid monohydrate (876 mg)
and heated at 60.degree. C. overnight. Solvent was removed and the
residue was taken up in EtOAc, washed with aqueous Na.sub.2CO.sub.3
solution, brine, dried and concentrated to give the crude product
as a yellow oil (5.38 g, >100% yield). Purification through
flash chromatography (heaxane/EtOAc, 80/20.fwdarw.60/40) provided
the ketone as a light yellow oil (4.54 g, 95% yield). MS (EI) m/e
208 (M.sup.+), Calcd for C.sub.12H.sub.13ClO.sub.2, 208.
[0217] Step 5: 78
[0218] To a solution of 4-(4-chlorophenyl)cyclohexanone (4.26 g,
20.48 mmol) in ethanol (90 mL) and water (30 mL) in a glass
pressure bottle, were added ammonium carbonate (13.8 g, 144 mmol, 7
equiv) and potassium cyanide (3.56 g, 54.77 mmol, 2.5 equiv). The
mixture was heated at 80-90.degree. C. overnight. The cooled
reaction mixture was added to icy water (400 ml) and stirred
vigorously for 30 min. The resulting precipitate was suction
filtered, washed thoroughly with water and dried to yield the
hydantoin as a white solid (5.58 g, 98% yield). MS (electrospray)
m/e 277 (M-H), Calcd for C.sub.14H.sub.15ClN.sub.2O.sub.2, 278
[0219] Step 6: 79
[0220] To a suspension of the hydantoin (5.15 g, 18.5 mmol) in dry
THF (250 mL) were added di-tert-butyl dicarbonate (10.1 g, 46.3
mmol, 2.5 equiv), triethylamine (2.8 mL, 2.07 g, 20.45 mmol, 1.1
equiv) and DMAP (226 mg, 1.85 mmol) in succession. The reaction
turned into a clear yellow solution and was stirred overnight at
room temperature. The reaction mixture was concentrated under
reduced pressure to yield a solid that was then taken up in EtOAc
(500 mL), washed with 1N HCl (3.times.50 mL), saturated aqueous
Na.sub.2CO.sub.3 (2.times.50 mL) and brine (2.times.50 mL), dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced
pressure. The crude light yellow product was purified through flash
chromatography (hexane/EtOAc, 90/10.fwdarw.70/30) to give the pure
bis-Boc hydantoin as a white solid (8.05 g, 91% yield). MS
(electrospray) m/e 542 (M+Ma+MeCN), Calcd for
C.sub.24H.sub.31ClN.sub.2O.- sub.6, 478
[0221] Step 7: 80
[0222] The bis-Boc hydantoin (6.41 g, 13.97 mmol) was dissolved in
DME (200 mL) to give a clear solution. To this solution was added
1N NaOH (120 mL, 120 mmol) and the reaction was stirred overnight
at room temperature, giving a slightly cloudy mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(4-chlorophenyl)cyclohexane carboxylic acid (4-ClAPC) was
treated with 6N HCl to adjust the pH to 11-12. To this solution
(.about.180 mL) were added DME (240 mL) and a solution of Fmoc-OSu
(5.31 g, 15.74 mmol, 1.1 equiv) in DME (30 mL) and the reaction was
stirred overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove DME, acidified with
3N HCl, extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 98/2.fwdarw.90/10) to give
the pure product as a white solid (5.04 g, 76% yield from the
bis-Boc hydantoin). .sup.1H NMR (DMSO-d.sub.6), 7.88 (d, 2H), 7.74
(d, 2H), 7.19-7.42 (m, 8H), 4.20-4.31 (m, 3H); MS (electrospray)
m/e 474 (M-H), Calcd for C.sub.28H.sub.26ClNO.sub.4, 475.
EXAMPLE 8
Preparation of
Fmoc-1-amino-4-(3-methoxyphenyl)cyclohexane-1-carboxylic acid
(Fmoc-3-MeOApc-OH)
[0223] Step 1: 81
[0224] To a solution of 3-iodoanisole (11.7, 50.0 mmol, 1.3 equiv)
in dry THF (180 mL) at -78.degree. C. was added a solution of
n-BuLi (1.6 M, 31.0 mL, 50 mmol, 1.3 equiv) in hexane over 25 min.
The reaction was stirred for another 30 min before a solution of
1,4-cyclohexanedione mono-ethylene ketal (6.0 g, 38.46 mmol) in dry
THF (100 mL) was added dropwise. After stirred for 2 h at
-78.degree. C., the reaction was quenched with aqueous NH.sub.4Cl
and extracted with EtOAc. The combined organic extracts were washed
with brine, dried over Na.sub.2SO.sub.4, concentrated in vacuo to
give the spectroscopically pure product as a white solid (9.34 g,
98% yield). .sup.1H NMR (CDCl.sub.3): 7.26 (dd, 1H), 7.06-7.11 (m,
2H), 6.79 (dd, 1H), 3.98 (m, 4H), 3.81 (s, 3H).
[0225] Step 2: 82
[0226] To a stirred solution of the alcohol (5.6 g, 21.21 mmol) in
dry CH.sub.2Cl.sub.2 (200 mL) under a nitrogen atmosphere at
salt-ice bath temperature, were added in succession triethylsilane
(10.2 mL,7.4 g, 63.63 mmol, 3 equiv) and boron trifluoride etherate
(21.5 mL, 24.1 g, 169.7 mmol, 8 equiv). The reaction mixture was
then allowed to warm to room temperature and stirred for 3 hrs
before washed with 10% aqueous K.sub.2CO.sub.3 solution and
H.sub.2O, dried over Na.sub.2SO.sub.4, and concentrated in vacuo to
give the deoxygenation compound as an oil (4.91 g), which was
sufficiently pure for direct use.
[0227] This crude intermediate was dissolved in acetone (130 mL)
and treated with 4N HCl (60 mL) and heated at 65.degree. C. for 4
hrs. Solvent was removed under reduced pressure and the residue was
diluted with EtOAc and neutralized with 4N NaOH solution. The
aqueous layer was extracted with EtOAc and the combined organic
extracts were washed with brine, dried and concentrated. The
resulting residue was purified by flash chromatography on silica
gel (80/20.fwdarw.60/40) to give the ketone (3.67 g, 85% overall
yield) as a yellow oil. .sup.1H NMR (CDCl.sub.3): 7.25 (dt, 1H),
6.75-6.86 (m, 3H), 3.81 (s, 3H), 3.00 (tt, 1H); MS (EI) m/e 204
(M+), Calcd for C.sub.13H.sub.16O.sub.2, 204.
[0228] Step 3: 83
[0229] To a solution of 4-(3-methoxyphenyl)cyclohexanone (3.10 g,
15.20 mmol) in ethanol (60 mL) and water (20 mL) in a glass
pressure bottle, were added ammonium carbonate (8.75 g, 91.20 mmol,
6 equiv.) and potassium cyanide (1.98 g, 30.40 mmol, 2 equiv.). The
mixture was heated at 80-90.degree. C. overnight. The cooled
reaction mixture was added to icy water (300 ml) and stirred
vigorously for 30 min. The resulting precipitate was suction
filtered, washed thoroughly with water and dried to yield the
hydantoin as a white solid (4.08 g, 98% yield). .sup.1H NMR
(DMSO-d.sub.6): 7.11 (d, 1H), 6.70-6.94 (m, 3H), 3.72 (s, 3H); MS
(electrospray) m/e 316 (M+MeCN+H), Calcd for
C.sub.15H.sub.18N.sub.2O.sub- .3, 274.
[0230] Step 4: 84
[0231] To a suspension of the hydantoin (5.29 g, 19.30 mmol) in dry
THF (250 mL) were added di-tert-butyl dicarbonate (10.5 g, 48.16
mmol, 2.5 equiv), triethylamine (3.0 mL, 2.17 g, 21.52 mmol, 1.1
equiv) and DMAP (235 mg, 1.92 mmol) in succession. The reaction
turned into a clear yellow solution and was stirred overnight at
room temperature. The reaction mixture was concentrated under
reduced pressure to yield a solid that was then taken up in EtOAc
(500 mL), washed with 1N HCl (3.times.50 mL), saturated aqueous
Na.sub.2CO.sub.3 (2.times.50 mL) and brine (2.times.50 mL), dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced
pressure. The crude light yellow product was purified through flash
chromatography (hexane/EtOAc, 80/20.fwdarw.60/40) to give the pure
bis-Boc hydantoin as a white solid (8.70 g, 95% yield). MS
(electrospray) m/e 538 (M+MeCN+Na), Calcd for
C.sub.25H.sub.34N.sub.2O.su- b.7, 474.
[0232] Step 5: 85
[0233] The bis-Boc hydantoin (2.30 g, 4.84 mmol) was dissolved in
DME (80 mL) to give a clear solution. To this solution was added 1N
NaOH (44 mL, 44 mmol) and the reaction was stirred overnight at
room temperature, giving a slightly cloudy mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
1-amino-4-(3-methoxyphenyl)cyclohexane carboxylic acid (3-MeOAPC)
was treated with 6N HCl to adjust the pH to 11-12. To this solution
(.about.40 mL) were added dioxane (80 mL) and Fmoc-Cl (1.73 g, 6.76
mmol, 1.4 equiv) and the reaction was stirred overnight at room
temperature. The reaction mixture was then concentrated under
reduced pressure to remove DME, neutralized with 3N HCl and
extracted with EtOAc. The combined organic extracts were washed
with brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated.
The crude product was purified by flash chromatography on silica
gel (CH.sub.2Cl.sub.2/MeOH, 98/2.fwdarw.90/10) to give the pure
product as a white solid (1.98 g, 87% yield from bis-Boc
hydantoin). .sup.1H NMR (DMSO-d.sub.6), 7.88 (d, 2H), 7.75 (d, 2H),
7.40 (td, 2H), 7.30 (td, 2H), 7.21 (m, 1H), 6.71-6.80 (m, 3H), 3.72
(s, 3H); MS (electrospray) m/e 494 (M+Na), Calcd for
C.sub.29H.sub.29NO.sub.5, 471.
EXAMPLE 9
Preparation of Fmoc-(D,L)-5-bromo-2 aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-Br-Atc-OH)
[0234] Step 1: 86
[0235] A mixture of 3-(2-bromophenyl)propanoic acid (prepared in 2
steps from 2-bromobenzyl bromide, 2.0 g, 8.73 mmole), oxalyl
chloride (1.14 ml, 13.1 mmole) and methylene chloride (20 ml) was
cooled in an ice bath and N,N-dimethylformamide (34 .mu.L, 0.44
mmole) was added dropwise. The mixture was stirred at room
temperature for 3 hours. Concentration in vacuo gave
3-(2-bromophenyl)propanoyl chloride which was taken up in methylene
chloride and used in the next step as a crude.
[0236] Step 2: 87
[0237] A solution of the above acid chloride (crude, 8.73 mmole) in
methylene chloride was slowly added to a solution of diazomethane
(generated from 5.70 g of 1-methyl-3-nitro-1-nitrosoguanidine) in
ether (40 ml) cooled in an ice bath. The mixture was then warmed up
to room temperature and stirred overnight. The mixture was
concentrated in vacuo and purified by column chromatography
(10.fwdarw.20% ethyl acetate/hexanes) to give
1-diazo-4-(2-bromophenyl)butan-2-one (1.88 g, 85% over 2 steps).
.sup.1H NMR (CDCl.sub.3) .delta. 7.50 (1H, d, phenyl), 7.24 (2H, m,
phenyl), 7.06 (1H, m, phenyl), 5.21 (1H, broad s, diazo), 3.05 (2H,
t, benzylic), 2.62 (2H, m).
[0238] Step 3: 88
[0239] To a mixture of rhodium (II) acetate dimer (15 mg, 0.068
mmole) in methylene chloride (120 ml) under reflux was slowly added
a solution of 1-diazo-4-(2-bromophenyl)butan-2-one (1.74 g, 6.85
mmole) in methylene chloride (30 ml). After the addition was
complete, the mixture was refluxed for an extra twenty minutes. The
mixture was cooled to room temperature, trifluoroacetic acid (1.5
ml) was added and the mixture was stirred at room temperature for
an hour. The reaction was quenched with saturated sodium
bicarbonate solution. The layers were separated and the methylene
chloride layer was washed once more with saturated sodium
bicarbonate solution. The combined aqueous layers were
back-extracted with methylene chloride. The combined organic layers
were dried over magnesium sulfate, filtered and concentrated in
vacuo to give a brown oil. Purification by column chromatography
(10.fwdarw.15% ethyl acetate/hexanes) gave 5-bromo-.beta.-tetralone
(1.18 g, 77% yield) as a colorless oil. .sup.1H NMR (CDCl.sub.3)
.delta. 7.46 (1H, t, phenyl), 7.05-7.09 (2H, m, phenyl), 3.58 (2H,
s, benzylic), 3.22 (2H, t, benzylic), 2.54 (2H, t).
[0240] Step 4: 89
[0241] A mixture of 5-bromo-.beta.-tetralone (1.18 g, 5.24 mmole),
potassium cyanide (512 mg, 7.86 mmole), ammonium carbonate (3.0 g,
31.22 mmole), ethanol (25 ml) and water (5 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 4
days. After cooling to room temperature, the white slurry was
poured into ice-water and stirred at room temperature for a couple
of hours. Filtration followed by air-drying gave hydantoin (1.31 g,
85%). .sup.1H NMR (DMSO-d.sub.6) .delta. 10.71 (1H, broad, NH),
8.28 (1H, broad s, NH), 7.0-7.5 (3H, m, phenyl). LRMS
(Electrospray): C.sub.12H.sub.11BrN.sub.2O.- sub.2, calc. 294;
observed: 293 (M-H), 295 (M-H).
[0242] Step 5: 90
[0243] A mixture of hydantoin (1.287 g, 4.36 mmole), Ba(OH).sub.2.
H.sub.2O (4.20 g, 22.2 mmole) in water (25 ml) in a sealed, thick
walled pressure flask was heated in a 125.degree. C. oil bath for 4
days. The reaction mixture was cooled to room temperature,
acidified to .about.pH 3 using 4N sulfuric acid while being stirred
vigorously. The suspension was stirred in a boiling water bath for
one hour and cooled to room temperature. The white suspension was
filtered and the precipitates rinsed with water. The combined
filtrate and washings were concentrated in vacuo to .about.20 ml.
Neutralization with concentrated ammonium hydroxide solution gave
white precipitate which were filtered, washed with water and dried
in vacuo overnight to give racemic 5-bromo-2
aminotetraline-2-carboxylic acid (893 mg, 76% yield). LRMS
(Electrospray): C.sub.11H.sub.12BrNO.sub.2, calc. 269; observed:
270 (M+H), 272 (M+H), 268 (M-H), 270 (M-H).
[0244] Step 6: 91
[0245] A mixture of racemic 5-bromo-2 aminotetraline-2-carboxylic
acid (882 mg, 3.27 mmole), triethylamine (0.60 ml, 4.30 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 1.32 g, 3.91
mmole) in acetonitrile (30 ml) and water (30 ml) was stirred at
room temperature overnight. TLC analysis of the reaction the next
day indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (0.25 g), triethylamine
(0.6 ml) and acetonitrile (5 ml) was added and the mixture was
stirred at room temperature for another day. The reaction mixture
was concentrated in vacuo to remove most of the acetonitrile,
acidified to pH .about.3 with 10% aqueous citric acid solution, and
the white emulsion extracted twice with methylene chloride. The
combined organic layers were washed with water, brine, dried over
magnesium sulfate. Filtration and concentration gave a crude oil
which was purified by column chromatography (eluted with
2.fwdarw.5.fwdarw.10% methanol/methylene chloride) to give racemic
Fmoc-5-bromo-2 aminotetraline-2-carboxylic acid (1.09 g, 68% yield)
as a white solid. HRMS (FAB): C.sub.26H.sub.22BrNNaO.sub.4 (M+Na)
calc. 514.0630; observed: 514.0643.
EXAMPLE 10
Preparation of Fmoc-(D,L)-5-chloro-2 aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-ClAtc-OH)
[0246] Step 1: 92
[0247] A mixture of 3-(2-chlorophenyl)propanoic acid (5.0 g, 27.1
mmole), thionyl chloride (10.9 ml, 149 mmole) and toluene (75 ml)
was refluxed for two hours. Concentration in vacuo gave
3-(2-chlorophenyl)propanoyl chloride which was taken up in
methylene chloride and used in the next step without further
purification.
[0248] Step 2: 93
[0249] A solution of the above acid chloride (crude, 27.1 mmole) in
methylene chloride was slowly added to a solution of diazomethane
(generated from 17.8 g of 1-methyl-3-nitro-1-nitrosoguanidine) in
ether (120 ml) cooled in an ice bath. The mixture was then warmed
up to room temperature and stirred overnight. The mixture was
concentrated in vacuo to give 1-diazo-4-(2-chlorophenyl)butan-2-one
(5.87 g, >100% over 2 steps) as a bright yellow oil. The
compound was used in the next step without further purification.
.sup.1H NMR (CDCl.sub.3) 6 7.05-7.32 (4H, m, phenyl), 5.13 (1H,
broad s, diazo), 3.00 (2H, t, benzylic), 2.57 (2H, m).
[0250] Step 3 94
[0251] To a mixture of rhodium (II) acetate dimer (60 mg, 0.27
mmole) in methylene chloride (400 ml) under reflux was slowly added
a solution of crude 1-diazo-4-(2-bromophenyl)butan-2-one (5.87 g,
27.1 mmole theoretical) in methylene chloride (50 ml). After the
addition was complete, the mixture was refluxed for an extra twenty
minutes. The mixture was cooled to room temperature,
trifluoroacetic acid (6.0 ml) was added and the mixture was stirred
at room temperature for two hours. The reaction was quenched with
saturated sodium bicarbonate solution. The layers were separated
and the methylene chloride layer was washed once more with
saturated sodium bicarbonate solution. The combined aqueous layers
were back-extracted with methylene chloride. The combined organic
layers were dried over magnesium sulfate, filtered and concentrated
in vacuo to give a brown oil. Purification by column chromatography
(10.fwdarw.15% ethyl acetate/hexanes) gave
5-chloro-.beta.-tetralone (3.32 g, 68% yield for steps a through c)
as a light brown oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.30 (1H, m,
phenyl), 7.15 (1H, t, phenyl), 7.05 (1H, d, phenyl), 3.60 (2H, s,
benzylic), 3.22 (2H, t, benzylic), 2.56 (2H, t).
[0252] Step 4: 95
[0253] A mixture of 5-chloro-.beta.-tetralone (880 mg, 4.87 mmole),
potassium cyanide (500 mg, 7.67 mmole), ammonium carbonate (2.85 g,
29.7 mmole), ethanol (24 ml) and water (6 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 66
hours. After cooling to room temperature, the slurry was poured
into ice-water and stirred at room temperature for a couple of
hours. Filtration followed by air-drying gave hydantoin (0.92 g,
75%) as a light beige solid. .sup.1H NMR (DMSO-d.sub.6) .delta.
10.70 (1H, broad, NH), 8.25 (1H, broad s, NH), 7.0-7.3 (3H, m,
phenyl). LRMS (Electrospray): C.sub.12H.sub.11ClN.sub.2O.- sub.2,
calc. 250; observed: 249 (M-H), 251 (M-H).
[0254] Step 5: 96
[0255] A mixture of hydantoin (880 mg, 3.51 mmole), Ba(OH).sub.2.
H.sub.2O (3.40 g, 18.0 mmole) in water (50 ml, too dilute) in a
sealed, thick walled pressure flask was heated in a 125.degree. C.
oil bath for 2 days. The reaction mixture was cooled to room
temperature, acidified to .about.pH 3 using 4N sulfuric acid while
being stirred vigorously. The suspension was stirred in a boiling
water bath for two hours and cooled to room temperature. The white
suspension was filtered and the precipitates rinsed with water. The
combined filtrate and washings were concentrated in vacuo to
.about.50 ml. Neutralization with concentrated ammonium hydroxide
solution gave white precipitate which were filtered, washed with
water and dried in vacuo overnight to give racemic
5-chloro-2-aminotetraline-2-carboxylic acid (788 mg, 99% yield).
LRMS (Electrospray): C.sub.11H.sub.12ClNO.sub.2, calc. 225;
observed: 226 (M+H), 228 (M+H), 224 (M-H), 226 (M-H).
[0256] Step 6: 97
[0257] A mixture of racemic 5-chloro-2-aminotetraline-2-carboxylic
acid (402 mg, 1.78 mmole), triethylamine (0.38 ml, 2.73 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 904 mg, 2.68
mmole) in acetonitrile (20 ml) and water (20 ml) was stirred at
room temperature for two days. TLC analysis of the reaction after
two days indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (0.12 g) and triethylamine
(0.1 ml) was added and the mixture was stirred at room temperature
for another day. The reaction mixture was concentrated in vacuo to
remove most of the acetonitrile, acidified to pH .about.3 with 10%
aqueous citric acid solution, and the white emulsion extracted
three times with ethyl acetate. The combined organic layers were
washed with water, brine, dried over magnesium sulfate. Filtration
and concentration gave a crude oil which was purified by column
chromatography (eluted with 3.fwdarw.6.fwdarw.8% methanol/methylene
chloride) to give racemic
Fmoc-5-chloro-2-aminotetraline-2-carboxylic acid (540 mg, 68%
yield) as a white solid. HRMS (EI): C.sub.26H.sub.22ClNO.sub.4 (M)
calc. 447.1237; observed: 447.1234.
EXAMPLE 11
Preparation of Fmoc-(D,L)-5-methoxy-2-aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-MeOAtc-OH)
[0258] Step 1: 98
[0259] A mixture of racemic 5-methoxy-2-aminotetraline-2-carboxylic
acid (prepared according to Obrecht, D. et. al. Helv. Chim Acta.
1992, 75, 1666) (802 mg, 3.62 mmole), triethylamine (0.62 ml, 4.45
mmole), 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 1.47 g,
4.36 mmole) in acetonitrile (25 ml) and water (25 ml) was stirred
at room temperature for 30 hours. TLC analysis of the reaction
indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (370 mg) and triethylamine
(0.6 ml) were added and the mixture was stirred at room temperature
for another 24 hours. The reaction mixture was concentrated in
vacuo to remove most of the acetonitrile, acidified to pH .about.3
with 10% aqueous citric acid solution, and the white emulsion was
extracted three times with ethyl acetate. The combined organic
layers were washed with water, brine and dried over magnesium
sulfate. Filtration and concentration gave a crude oil which was
purified by column chromatography (eluted with
1.fwdarw.3.fwdarw.5.fwdarw.10% methanol/methylene chloride) to give
racemic Fmoc-5-methoxy-2-aminotetral- ine-2-carboxylic acid (1.14
g, 71% yield) as an off-white solid. HRMS (FAB):
C.sub.27H.sub.26NO.sub.5 (M+H) calc. 444.1812; observed:
444.1814.
EXAMPLE 12
Preparation of Fmoc-(D,L)-5-ethoxy-2-aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-EtOAtc-OH)
[0260] Step 1: 99
[0261] A mixture of 1,6-dihydroxynaphthalene (5.02 g, 31.3 mmole),
anhydrous potassium carbonate (52.0 g, 376 mmole),
N,N-dimethylformamide (50 ml) and iodoethane (15 ml, 188 mmole) was
stirred in a 35.degree. C. oil bath for 24 hours. The reaction
mixture was filtered and the solid residue was rinsed thoroughly
with ethyl ether. The filtrate and the washings were combined and
concentrated in vacuo to remove most of the solvents. The brown
residue was partitioned between water and ether and the layers were
separated. The ether layer was washed with water. The combined
aqueous layers were back extracted with ether. The ether extracts
were combined, washed with brine and dried over magnesium sulfate.
Filtration and concentration gave a crude brown solid (6.74 g, 99%
yield). Recrystallization of the crude product from hot methanol
gave 1,6-diethoxynaphthalene (4.36 g, 64% yield, first crop) as a
light brown solid. .sup.1H NMR (CDCl.sub.3) .delta. 8.20 (1H, d,
phenyl), 7.06-7.36 (4H, m, phenyl), 6.66 (1H, dd, phenyl),
4.10-4.23 (4H, 2 sets of q, 2 CH.sub.2), 1.45-1.56 (6H, 2 sets of
t, 2 CH.sub.3).
[0262] Step 2: 100
[0263] To a refluxing solution of 1,6-diethoxynaphthalene (4.15 g,
19.2 mmole) in absolute ethanol (100 ml) was carefully added small
pieces of sodium metal (6.8 g, 296 mmole) over 60 minutes. The
mixture was refluxed for another 90 minutes. TLC indicated the
presence of unreacted starting material. Extra sodium metal (1.0 g,
43.5 mmole) was added and the reaction mixture was refluxed for
another 60 minutes. The reaction was cooled to room temperature,
quenched with water and acidified with concentrated hydrochloric
acid. The mixture was concentrated in vacuo to remove most of the
ethanol. The aqueous mixture was extracted three times with ether.
The combined organic layers were washed with water and dried over
sodium sulfate. Filtration and concentration gave a brown solid
which was dissolved in 1:1 ethanol/water (200 ml), then
p-toluenesulfonic acid (400 mg) was added. The mixture was refluxed
for 210 minutes. Extra p-toluenesulfonic acid (100 mg) was added
and the mixture was refluxed for another 60 minutes. After cooling
to room temperature, most of the ethanol was removed under reduced
pressure. The aqueous mixture was extracted three times with ether
and the combined organic layers were washed with water, saturated
sodium chloride solution and dried over sodium sulfate. Filtration
and concentration gave a brwon oil which was purified by column
chromatography (7% ethyl acetate/hexanes) to give
5-ethoxy-.beta.-tetralone (2.43 g, 67% yield) as a light yellow
oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.15 (1H, t, phenyl), 6.76
(1H, d, phenyl), 6.72 (1H, d, phenyl), 4.05 (2H, q, CH.sub.2), 3.56
(2H, s, benzylic), 3.10 (2H, t, benzylic), 2.53 (2H, t), 1.44 (3H,
t, CH.sub.3).
[0264] Step 3: 101
[0265] A mixture of 5-ethoxy-.beta.-tetralone (2.23 g, 11.7 mmole),
potassium cyanide (1.20 g, 18.4 mmole), ammonium carbonate (6.75 g,
70.2 mmole), ethanol (80 ml) and water (20 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 3
days. After cooling to room temperature, the slurry was poured into
ice-water and stirred at room temperature for a couple of hours.
Filtration followed by air-drying gave hydantoin (2.69 g, 88%) as a
beige solid. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.65 (1H, broad s,
NH), 8.22 (1H, broad s, NH), 7.06 (1H, t, phenyl), 6.75 (1H, d,
phenyl), 6.65 (1H, d, phenyl), 3.98 (2H, q, CH.sub.2), 1.32 (3H, t,
CH.sub.3). LRMS (Electrospray): C.sub.14H.sub.16N.sub.2O.sub.3,
calc. 259; observed: 258 (M-H).
[0266] Step 4: 102
[0267] A mixture of hydantoin (2.57 g, 9.87 mmole), Ba(OH).sub.2.
H.sub.2O (9.40 g, 49.6 mmole) in water (200 ml, too dilute) in a
sealed, thick walled pressure flask was heated in a 105.degree. C.
oil bath for 39 hours. Extra Ba(OH).sub.2. H.sub.2O (9.40 g, 49.6
mmole) was added and the mixture was heated in a 125.degree. C. oil
bath for an additional 21 hours. The reaction mixture was cooled to
room temperature, acidified to .about.pH 3 using 4N sulfuric acid
while being stirred vigorously. The suspension was stirred in a
boiling water bath for one hour and cooled to room temperature. The
white suspension was filtered and the precipitates rinsed with
water. The combined filtrate and washings were concentrated in
vacuo to .about.75 ml. Neutralization with concentrated ammonium
hydroxide solution gave white precipitate which were filtered,
washed with water and air-dried to give racemic
5-ethoxy-2-aminotetraline-2-carb- oxylic acid (2.34 g, quantitative
yield) as a light beige solid. LRMS (Electrospray):
C.sub.13H.sub.17NO.sub.3, calc. 235; observed: 236 (M+H), 234
(M-H).
[0268] Step 5: 103
[0269] A mixture of racemic 5-ethoxy-2-aminotetraline-2-carboxylic
acid (2.22 g, 9.44 mmole), triethylamine (2.00 ml, 14.3 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 4.81 g, 14.3
mmole) in acetonitrile (75 ml) and water (75 ml) was stirred at
room temperature for two days. TLC analysis of the reaction
indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (645 mg) and triethylamine
(1.0 ml) was added and the mixture was stirred at room temperature
for another day. The reaction mixture was concentrated in vacuo to
remove most of the acetonitrile, acidified to pH .about.3 with 10%
aqueous citric acid solution, and the white emulsion extracted
three times with ethyl acetate. The combined organic layers were
washed with water, brine, dried over magnesium sulfate. Filtration
and concentration gave a crude oil which was purified by column
chromatography (eluted with 3.fwdarw.5.fwdarw.10%
methanol/methylene chloride) to give racemic
Fmoc-5-ethoxy-2-aminotetraline-2-carboxylic acid (4.66 g,
>quantitative yield) as a white solid. HRMS (FAB):
C.sub.28H.sub.28NO.sub.5 (M+H) calc. 458.1967; observed:
458.1985.
EXAMPLE 13
Preparation of
Fmoc-(D,L)-5-isopropoxy-2-aminotetraline-2-carboxylic acid
(Fmoc-(D,L) 5-iPrOAtc-OH)
[0270] Step 1: 104
[0271] A mixture of 6-methoxy-1-tetralone (5.07 g, 28.8 mmole), 10%
Pd/C (3.53 g, 3.32 mmole) in dry p-cymene (250 ml) was heated to
reflux under argon for 38 hours. The reaction mixture was cooled to
room temperature, filtered over celite and the residue rinsed
thoroughly with p-cymene. The filtrate and the washings were
combined and extracted twice with 1N sodium hydroxide solution
(2.times.70 ml). The combined aqueous extracts were acidified with
6N hydrochloric acid to pH .about.3 and extracted three times with
ether. The combined organic layers were washed with water, dried
over anhydrous sodium sulfate. Filtration and concentration gave
crude 5-hydroxy-6-methoxynaphthalene (2.31 g, 46% yield) as a light
brown solid which was used in the next step without further
purification. LRMS (Electrospray): C.sub.11H.sub.10O.sub.2, calc.
174; observed: 173 (M-H).
[0272] Step 2: 105
[0273] A mixture of 5-hydroxy-6-methoxynaphthalene (2.10 g, 12.1
mmole), cesium carbonate (19.7 g, 60.5 mmole),
N,N-dimethylformamide (12 ml) and 2-bromopropane (3.50 ml, 36.9
mmole) was stirred in a 40.degree. C. oil bath overnight. The
reaction mixture was filtered and the solid residue was rinsed
thoroughly with ethyl ether. The filtrate and the washings were
combined and concentrated in vacuo to remove most of the solvents.
The brown residue was partitioned between water and ether and the
layers were separated. The ether layer was washed with water. The
combined aqueous layers were back extracted with ether. The ether
extracts were combined, washed with brine and dried over sodium
sulfate. Filtration and concentration gave a crude which was
purified by column chromatography (2.5.fwdarw.5% ethyl
acetate/hexanes) to give 1-isopropoxy-6-methoxynapht- halene (2.23
g, 86% yield) as a light brown oil. .sup.1H NMR (CDCl.sub.3)
.delta. 8.17 (1H, d, phenyl), 7.05-7.38 (4H, m, phenyl), 6.72 (1H,
dd, phenyl), 4.73 (1H, m, CH of iPr), 3.92 (3H, s, OCH.sub.3), 1.42
(6H, d, 2 CH.sub.3 of iPr).
[0274] Step 3: 106
[0275] To a refluxing solution of 1-isopropoxy-6-methoxynaphthalene
(2.23 g, 10.3 mmole) in absolute ethanol (50 ml) was carefully
added small pieces of sodium metal (3.6 g, 157 mmole) over 45
minutes. The mixture was refluxed for a further 120 minutes. The
reaction was cooled to room temperature, quenched with water and
acidified with concentrated hydrochloric acid. The mixture was
concentrated in vacuo to remove most of the ethanol. The aqueous
mixture was extracted three times with ether. The combined organic
layers were washed with water and dried over sodium sulfate.
Filtration and concentration gave a reddish oil which was dissolved
in 1:1 ethanol/water (90 ml), then p-toluenesulfonic acid (200 mg)
was added. The mixture was refluxed for 60 minutes. After cooling
to room temperature, most of the ethanol was removed under reduced
pressure. The aqueous mixture was extracted twice with ether and
the combined organic layers were washed with water, saturated
sodium chloride solution and dried over sodium sulfate. Filtration
and concentration gave a reddish oil which was purified by column
chromatography (8.fwdarw.15 % ethyl acetate/hexanes) to give
5-isopropoxy-.beta.-tetralone (1.37 g, 65% yield) as a colorless
oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.16 (1H, t, phenyl), 6.78
(1H, d, phenyl), 6.71 (1H, d, phenyl), 4.53 (1H, m, CH of iPr),
3.56 (2H, s, benzylic), 3.08 (2H, t, benzylic), 2.50 (2H, t), 1.37
(6H, d, 2 CH.sub.3 of iPr).
[0276] Step 4: 107
[0277] A mixture of 5-isopropoxy-.beta.-tetralone (1.37 g, 6.71
mmole), potassium cyanide (660 mg, 10.1 mmole), ammonium carbonate
(3.87 g, 40.3 mmole), ethanol (44 ml) and water (9 ml) in a sealed,
thick walled pressure flask was heated in a 80.degree. C. oil bath
for 42 hours. After cooling to room temperature, the slurry was
poured into ice-water and stirred at room temperature for a couple
of hours. Filtration followed by air-drying gave hydantoin (1.64 g,
89%).
[0278] Step 5: 108
[0279] A mixture of hydantoin (1.64 g, 5.98 mmole),
Ba(OH).sub.2.H.sub.2O (5.66 g, 29.9 mmole) in water (25 ml) in a
sealed, thick walled pressure flask was heated in a 100.degree. C.
oil bath for 70 hours. The reaction mixture was cooled to room
temperature, neutralized to .about.pH 7 using 4N sulfuric acid
while being stirred vigorously. The suspension was stirred in a
boiling water bath for one hour and cooled to room temperature.
Basified with 1N sodium hydroxide solution and the white suspension
was filtered and the precipitates rinsed with water. The combined
filtrate and washings were concentrated in vacuo to .about.75 ml.
Neutralization with concentrated hydrochloric acid solution gave
white precipitate which were filtered, washed with water and
air-dried to give racemic
5-isopropoxy-2-aminotetraline-2-carboxylic acid (3.48 g, wet and
containing inorganic salt, >quantitative yield). LRMS
(Electrospray): C.sub.14H.sub.19NO.sub.3, calc. 249; observed: 248
(M-H).
[0280] Step 6: 109
[0281] A mixture of racemic
5-isopropoxy-2-aminotetraline-2-carboxylic acid (3.48 g, 5.98 mmole
theoretical), triethylamine (1.10 ml, 7.89 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 2.62 g, 7.77
mmole) in acetonitrile (30 ml) and water (30 ml) was stirred at
room temperature for one day. TLC analysis of the reaction
indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (500 mg) was added and the
mixture was stirred at room temperature for another day. The
reaction mixture was concentrated in vacuo to remove most of the
acetonitrile, acidified to pH .about.3 with 10% aqueous citric acid
solution, and the white emulsion extracted three times with
methylene chloride. The combined organic layers were washed with
water, brine, dried over magnesium sulfate. Filtration and
concentration gave a crude oil which was purified by colunm
chromatography (eluted with 1.fwdarw.2.fwdarw.5.fwdarw.8%
methanol/methylene chloride) to give racemic
Fmoc-5-isopropoxy-2-aminotet- raline-2-carboxylic acid (0.50 g, 18%
yield over 2 steps) as a white solid. HRMS (FAB):
C.sub.29H.sub.30NO.sub.5 (M+H) calc. 472.2124; observed:
472.2117.
EXAMPLE 14
Preparation of
Fmoc-(D,L)-5-dimethylamino-2-aminotetraline-2-carboxylic acid
(Fmoc-(D,L) 5-DmaAtc-OH)
[0282] Step 1: 110
[0283] A mixture of 5-amino-2-naphthol (2.97 g, 18.6 mmole),
potassium carbonate (37.0 g, 268 mmole), acetone (100 ml) and
iodomethane (10.0 ml, 161 mmole) was refluxed for 2 days. The
reaction mixture was cooled to room temperature, filtered and the
solid residue was rinsed thoroughly with ethyl ether and acetone.
The filtrate and the washings were combined and concentrated in
vacuo to remove most of the solvents. The brown residue was
partitioned between water and ether and the layers were separated.
The ether layer was washed with water. The combined aqueous layers
were back extracted with ether. The ether extracts were combined,
washed with brine and dried over sodium sulfate. Filtration and
concentration gave crude 1-dimethylamino-6-methoxynaphthalene (3.54
g, 94% yield) as a dark brown oil. .sup.1H NMR (CDCl.sub.3) .delta.
8.16 (1H, t, phenyl), 7.30-7.50 (2H, m, aromatic), 7.10-7.20 (2H,
m, aromatic), 6.96 (1H, d, aromatic), 3.93 (3H, s, OCH.sub.3), 2.89
(6H, S, N(CH.sub.3).sub.2).
[0284] Step 2: 111
[0285] To a refluxing solution of
1-dimethylamino-6-methoxynaphthalene (2.99 g, 14.9 mmole) in
absolute ethanol (100 ml) was carefully added small pieces of
sodium metal (5.76 g, 251 mmole) over 45 minutes. The mixture was
refluxed for another 45 minutes. TLC indicated the presence of
unreacted starting material. Extra sodium metal (7.09 g, 308 mmole)
was added and the reaction mixture was refluxed until TLC indicated
the complete consumption of all the starting material. The reaction
was cooled to room temperature and pH adjusted to .about.9-10 with
concentrated hydrochloric acid. The mixture was concentrated in
vacuo to remove most of the ethanol. The aqueous mixture was
extracted four times with ethyl acetate. The combined organic
layers were washed with saturated sodium bicarbonate and dried over
sodium sulfate. Filtration and concentration gave a dark brown oil
which was dissolved in 1:1 ethanol/water (150 ml), then
p-toluenesulfonic acid (3.05 g) was added to bring the pH to
.about.2-3. The mixture was refluxed for 3 hours. After cooling to
room temperature, most of the ethanol was removed under reduced
pressure. The pH of the mixture was adjusted to .about.9-10 with 2N
sodium hydroxide solution and the aqueous mixture was extracted
four times with ethyl acetate. The combined organic layers were
washed with saturated sodium bicarbonate solution and dried over
sodium sulfate. Filtration and concentration gave a dark brown oil
which was purified by column chromatography (15% ethyl
acetate/hexanes) to give 5-dimethylamino-.beta.-tetralone (834 mg,
30% yield) as a brown oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.18
(1H, t, phenyl), 6.96 (1H, d, phenyl), 6.82 (1H, d, phenyl), 3.57
(2H, s, benzylic), 3.10 (2H, t, benzylic), 2.70 (6H, S,
N(CH.sub.3).sub.2), 2.48 (2H, t). LRMS (Electrospray):
C.sub.12H.sub.15NO, calc. 189; observed: 190 (M+H).
[0286] Step 3: 112
[0287] A mixture of 5-dimethylamino-.beta.-tetralone (0.97 g, 5.13
mmole), potassium cyanide (510 mg, 7.82 mmole), ammonium carbonate
(2.98 g, 31.0 mmole), ethanol (40 ml) and water (10 ml) in a
sealed, thick walled pressure flask was heated in a 80.degree. C.
oil bath for 29 hours. After cooling to room temperature, the dark
brown slurry was poured into ice-water and stirred at room
temperature for a couple of hours. Filtration followed by
air-drying gave hydantoin (885 mg, 67%) as a dark brown solid. LRMS
(Electrospray): C.sub.14H.sub.17N.sub.3O.sub.2, calc. 259;
observed: 260 (M+H), 258 (M-H).
[0288] Step 4: 113
[0289] To a solution of hydantoin (832 mg, 3.21 mmole) in THF (25
ml) was added di-t-butyl dicarbonate (2.51 g, 11.5 mmole),
triethylamine (0.50 ml, 3.59 mmole) and 4-dimethylamninopyridine
(17 mg, 0.14 mmole). The mixture was stirred at room temperature
overnight. The solvents were removed in vacuo and the crude was
purified using column chromatography (15% ethyl acetate/hexanes) to
give bis-Boc hydantoin (1.02 g, 69% yield) as a yellow foam. LRMS
(Electrospray): C.sub.24H.sub.33N.sub.3O.sub.6, calc. 459;
observed: 919 (2M+H).
[0290] Step 5: 114
[0291] To a solution of bis-Boc hydantoin (988 mg, 2.15 mmole) in
dimethoxyethane (15 ml) was added 1N sodium hydroxide solution (20
ml). The mixture was stirred at room temperature overnight. The
reaction mixture was concentrated in vacuo to remove most of the
solvents and water was added to the resulting light brown mixture.
The aqueous mixture was extracted twice with methylene chloride and
twice with ethyl acetate. The aqueous layer was concentrated to
.about.20 ml, neutralized to pH .about.7 with 1N hydrochloric acid
to give a slurry. The slurry was filtered to give racemic
5-dimethylamino-2-aminotetraline-2-carboxylic acid (1.33 g, still
wet, >quantitative yield) as a off-white solid. LRMS
(Electrospray): C.sub.13H.sub.18N.sub.2O.sub.2, calc. 234;
observed: 235 (M+H).
[0292] Step 6: 115
[0293] A mixture of 5-dimethylamino-2-aminotetraline-2-carboxylic
acid (1.33 g, 2.15 mmole theoretical), triethylamine (0.40 ml, 2.87
mmole), 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 0.92 g,
2.73 mmole) in acetonitrile (10 ml) and water (10 ml) was stirred
at room temperature for one day. TLC analysis of the reaction
indicated the presence of starting material amino acid.
9-fluorenylmethyl succinimidyl carbonate (400 mg) and triethylamine
(0.2 ml) were added and the mixture was stirred at room temperature
for another day. The reaction mixture was concentrated in vacuo to
remove most of the acetonitrile and the almost neutral mixture was
extracted three times with ethyl acetate. The combined organic
layers were washed with water, brine, dried over sodium sulfate.
Filtration and concentration gave a crude which was purified by
column chromatography (eluted with
2.5.fwdarw.6.fwdarw.10.fwdarw.15.fwdar- w.20% methanol/methylene
chloride) to give racemic Fmoc-5-dimethylamino-2--
aminotetraline-2-carboxylic acid (602 mg, 61% yield over 2 steps)
as an off-white solid. HRMS (FAB): C.sub.28H.sub.28N.sub.2O.sub.4
(M) calc. 456.2049; observed: 456.2056.
EXAMPLE 15
Preparation of Fmoc-(D,L)-5-methyl-2-aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-MeAtc-OH)
[0294] Step 1: 116
[0295] A mixture of 2-methylhydrocinnamic acid (3.0 g, 18.3 mmole),
oxalyl chloride (3.19 ml, 36.6 mmole) and methylene chloride (30
ml) was cooled in an ice bath and N,N-dimethylformamide (0.14 ml,
1.81 mmole) was added dropwise. The mixture was stirred at room
temperature overnight. Concentration in vacuo gave
3-(2-methylphenyl)propanoyl chloride which was taken up in
methylene chloride and used in the next step as a crude.
[0296] Step 2: 117
[0297] A solution of the above acid chloride (crude, 18.3 mmole) in
methylene chloride was slowly added to a solution of diazomethane
(generated from 11.9 g of 1-methyl-3-nitro-1-nitrosoguanidine) in
ether (80 ml) cooled in an ice bath. The mixture was then warmed up
to room temperature and stirred overnight. The mixture was
concentrated in vacuo and purified by column chromatography
(10.fwdarw.20% ethyl acetate/hexanes) to give
1-diazo-4-(2-methylphenyl)butan-2-one (2.08 g, 60% over 2 steps) as
a bright yellow oil.
[0298] Step 3: 118
[0299] To a mixture of rhodium (II) acetate dimer (24 mg, 0.109
mmole) in methylene chloride (200 ml) under reflux was slowly added
a solution of 1-diazo-4-(2-methylphenyl)butan-2-one (2.08 g, 11.1
mmole) in methylene chloride (50 ml) over 180 minutes. After the
addition was complete, the mixture was refluxed for an extra twenty
minutes. The mixture was cooled to room temperature,
trifluoroacetic acid (2.40 ml) was added and the mixture was
stirred at room temperature for an hour. The reaction was quenched
with saturated sodium bicarbonate solution. The layers were
separated and the methylene chloride layer was washed once more
with saturated sodium bicarbonate solution. The combined aqueous
layers were back-extracted with methylene chloride. The combined
organic layers were dried over magnesium sulfate, filtered and
concentrated in vacuo to give a crude brown oil. Purification by
column chromatography (15% ethyl acetate/hexanes) gave
5-methyl-.beta.-tetralone (1.48 g, 84% yield) as a light brown oil.
.sup.1H NMR (CDCl.sub.3) .delta. 6.90-7.20 (3H, m, phenyl), 3.58
(2H, s, benzylic), 3.03 (2H, t, benzylic), 2.55 (2H, t), 2.34 (3H,
s, CH.sub.3).
[0300] Step 4: 119
[0301] A mixture of 5-methyl-.beta.-tetralone (1.48 g, 9.24 mmole),
potassium cyanide (902 mg, 13.9 mmole), ammonium carbonate (5.33 g,
55.5 mmole), ethanol (45 ml) and water (9 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 3
days. After cooling to room temperature, the slurry was poured into
ice-water and stirred at room temperature for a couple of hours.
Filtration followed by air-drying gave crude hydantoin (1.81 g, 85%
yield) as a beige solid. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.66
(1H, broad s, NH), 8.22 (1H, broad s, NH), 6.85-7.05 (3H, m,
phenyl), 2.17 (3H, s, CH.sub.3).
[0302] Step 5: 120
[0303] A mixture of hydantoin (1.80 g, 7.82 mmole), Ba(OH).sub.2.
H.sub.2O (7.40 g, 39.1 nimole) in water (28 ml) in a sealed, thick
walled pressure flask was heated in a 125.degree. C. oil bath for
88 hours. The reaction mixture was cooled to room temperature,
acidified to .about.pH 3 using 4N sulfuric acid while being stirred
vigorously. The suspension was stirred in a boiling water bath for
an hour and cooled to room temperature. The white suspension was
filtered and the precipitates rinsed with water. The combined
filtrate and washings were concentrated in vacuo to .about.50 ml.
Neutralization with concentrated ammonium hydroxide solution gave
white precipitate which were filtered, washed with water and
air-dried to give racemic 5-methyl-2-aminotetraline-2-carboxylic
acid (1.05 g, 65% yield) as a beige solid. LRMS (Electrospray):
C.sub.12H.sub.15NO.sub.2, calc. 205; observed: 206 (M+H).
[0304] Step 6: 121
[0305] A mixture of racemic 5-methyl-2-aminotetraline-2-carboxylic
acid (1.05 g, 5.12 mmole), triethylamine (0.93 ml, 6.67 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 2.24 g, 6.64
mmole) in acetonitrile (30 ml) and water (30 ml) was stirred at
room temperature for 2 days. TLC analysis of the reaction indicated
the presence of starting material amino acid. 9-fluorenylmethyl
succinimidyl carbonate (520 mg) was added and the mixture was
stirred at room temperature for another 24 hours. The reaction
mixture was concentrated in vacuo to remove most of the
acetonitrile, acidified to pH .about.3 with 10% aqueous citric acid
solution, and the white emulsion was extracted twice with methylene
chloride. The combined organic layers were washed with water, brine
and dried over magnesium sulfate. Filtration and concentration gave
a crude oil which was purified by column chromatography (eluted
with 2.fwdarw.5.fwdarw.8% methanol/methylene chloride) to give
racemic Fmoc-5-methyl-2-aminotetraline-2-carboxylic acid (1.62 g,
74% yield) as an light brown solid. HRMS (FAB):
C.sub.27H.sub.26NO.sub.4 (M+H) calc. 428.1862; observed:
428.1844.
EXAMPLE 16
Preparation of Fmoc-(D,L)-5-ethyl-2 aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-EtAtc-OH)
[0306] Step 1: 122
[0307] A mixture of 3-(2-ethylphenyl)propanoic acid (prepared in 3
steps from 1-ethyl-2-iodobenzene, 4.24 g, 23.8 mmole), thionyl
chloride (9.50 ml, 130 mmole) and toluene (100 ml) was refluxed for
2 hours. Concentration in vacuo gave 3-(2-ethylphenyl)propanoyl
chloride which was taken up in methylene chloride and used in the
next step as a crude.
[0308] Step 2: 123
[0309] A solution of the above acid chloride (crude, 23.8 mmole) in
methylene chloride was slowly added to a solution of diazomethane
(generated from 15.6 g of 1-methyl-3-nitro-1-nitrosoguanidine) in
ether (100 ml) cooled in an ice bath. The mixture was then warmed
up to room temperature and stirred overnight. The mixture was
concentrated in vacuo and purified by column chromatography
(10.fwdarw.20% ethyl acetate/hexanes) to give
1-diazo-4-(2-ethylphenyl)butan-2-one (3.47 g, 72% over 2 steps).
.sup.1H NMR (CDCl.sub.3) .delta. 7.1-7.25 (4H, m, phenyl), 5.21
(1H, broad s, diazo), 2.97 (2H, m, CH.sub.2 of ethyl), 1.20 (3H, t,
CH.sub.3).
[0310] Step 3: 124
[0311] To a mixture of rhodium (II) acetate dimer (38 mg, 0.172
mmole) in methylene chloride (300 ml) under reflux was slowly added
a solution of 1-diazo-4-(2-ethylphenyl)butan-2-one (3.47 g, 17.2
mmole) in methylene chloride (50 ml) over 90 minutes. After the
addition was complete, the mixture was refluxed for an extra twenty
minutes. The mixture was cooled to room temperature,
trifluoroacetic acid (3.75 ml) was added and the mixture was
stirred at room temperature for an hour. The reaction was quenched
with saturated sodium bicarbonate solution. The layers were
separated and the methylene chloride layer was washed once more
with saturated sodium bicarbonate solution. The combined aqueous
layers were back-extracted with methylene chloride. The combined
organic layers were dried over magnesium sulfate, filtered and
concentrated in vacuo to give crude 5-ethyl-.beta.-tetralone (3.09
g, >quantitative yield) as a reddish-brown oil. The compound was
used in the next step without further purification. .sup.1H NMR
(CDCl.sub.3) .delta. 6.9-7.2 (3H, m, phenyl), 3.58 (2H, s,
benzylic), 3.08 (2H, s, benzylic), 2.70 (2H, q, CH.sub.2 of ethyl),
2.52 (2H, t, benzylic), 1.20 (3H, t, CH.sub.3 of ethyl).
[0312] Step 4: 125
[0313] A mixture of 5-ethyl-.beta.-tetralone (3.09 g, 17.7 mmole),
potassium cyanide (1.73 g, 26.6 mmole), ammonium carbonate (10.2 g,
106 mmole), ethanol (80 ml) and water (16 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 48
hours. After cooling to room temperature, the white slurry was
poured into ice-water and stirred at room temperature for a couple
of hours. Filtration followed by air-drying gave hydantoin (3.85 g,
92% yield over 2 steps) as a light beige solid. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.67 (1H, broad s, NH), 8.26 (1H, broad s,
NH), 6.8-7.1 (3H, m, phenyl), 1.13 (3H, t, CH.sub.3). LRMS
(Electrospray): C.sub.14H.sub.16N.sub.2O.sub.2, calc. 244;
observed: 243 (M-H).
[0314] Step 5: 126
[0315] A mixture of hydantoin (1.00 g, 4.09 mmole), Ba(OH).sub.2.
H.sub.2O (4.00 g, 21.1 mmole) in water (20 ml) in a sealed, thick
walled pressure flask was heated in a 125.degree. C. oil bath for
48 hours. The reaction mixture was cooled to room temperature,
acidified to .about.pH 3 using 4N sulfuric acid while being stirred
vigorously. The suspension was stirred in a boiling water bath for
two hours and cooled to room temperature. The white suspension was
filtered and the precipitates rinsed with water. The combined
filtrate and washings were concentrated in vacuo to 50 ml.
Neutralization with concentrated ammonium hydroxide solution gave
white precipitate which were filtered, washed with water and dried
in vacuo overnight to give racemic
5-ethyl-2-aminotetraline-2-carboxylic acid (796 mg, 89% yield).
LRMS (Electrospray): C.sub.13H.sub.17NO.sub.2, calc. 219; observed:
220 (M+H).
[0316] Step 6: 127
[0317] A mixture of racemic 5-ethyl-2-aminotetraline-2-carboxylic
acid (765 mg, 3.49 mmole), triethylamine (1.0 ml, 7.17 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 1.79 g, 5.31
mmole) in acetonitrile (40 ml) and water (40 ml) was stirred at
room temperature for 2 days. The reaction mixture was concentrated
in vacuo to remove most of the acetonitrile, acidified to pH
.about.3 with 10% aqueous citric acid solution, and the white
emulsion extracted twice with methylene chloride, twice with ethyl
acetate. The methylene chloride extracts were washed with water,
brine and dried over magnesium sulfate. The ethyl acetate extracts
were washed with water, brine and dried over magnesium sulfate.
Filtration and concentration gave a crude oil which was purified by
column chromatography (eluted with 2.fwdarw.5.fwdarw.8%
methanol/methylene chloride) to give racemic
Fmoc-5-ethyl-2-aminotetralin- e-2-carboxylic acid (330 mg, 21%
yield) as a white solid. HRMS (FAB): C.sub.28H.sub.28NO.sub.4 (M+H)
calc. 442.2018; observed: 442.2010.
EXAMPLE 17
Preparation of Fmoc-(D,L)-5-isopropyl-2-aminotetraline-2-carboxylic
acid (Fmoc-(D,L) 5-iPrAtc-OH)
[0318] Step 1: 128
[0319] A mixture of 3-(2-isopropylphenyl)propanoic acid (prepared
in 3 steps from 1-isopropyl-2-iodobenzene, 2.01 g, 10.5 mmole),
thionyl chloride (4.30 ml, 59.0 mmole) and toluene (40 ml) was
refluxed for 2 hours. Concentration in vacuo gave
3-(2-isopropylphenyl)propanoyl chloride which was taken up in
methylene chloride and used in the next step as a crude.
[0320] Step 2: 129
[0321] A solution of the above acid chloride (crude, 10.5 mmole) in
methylene chloride was slowly added to a solution of diazomethane
(generated from 6.95 g of 1-methyl-3-nitro-1-nitrosoguanidine) in
ether (50 ml) cooled in an ice bath. The mixture was then warmed up
to room temperature and stirred overnight. The mixture was
concentrated in vacuo and purified by column chromatography (20%
ethyl acetate/hexanes) to give
1-diazo-4-(2-isopropylphenyl)butan-2-one (1.87 g, 82% over 2 steps)
as a bright yellow oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.10-7.30
(4H, m, phenyl), 5.21 (1H, broad s, diazo), 3.15 (1H, m, CH of
iPr), 3.00 (2H, t, benzylic), 2.57 (2H, m), 1.24 (6H, d, 2 CH.sub.3
of iPr).
[0322] Step 3: 130
[0323] To a mixture of rhodium (II) acetate dimer (20 mg, 0.091
mmole) in methylene chloride (160 ml) under reflux was slowly added
a solution of 1-diazo-4-(2-bromophenyl)butan-2-one (1.87 g, 8.65
mmole) in methylene chloride (25 ml) over 60 minutes. After the
addition was complete, the mixture was refluxed for an extra
fifteen minutes. The mixture was cooled to room temperature,
trifluoroacetic acid (1.90 ml) was added and the mixture was
stirred at room temperature for 45 minutes. The reaction was
quenched with saturated sodium bicarbonate solution. The layers
were separated and the methylene chloride layer was washed once
more with saturated sodium bicarbonate solution. The combined
aqueous layers were back-extracted with methylene chloride. The
combined organic layers were dried over magnesium sulfate, filtered
and concentrated in vacuo to give a crude brown oil. Purification
by column chromatography (5% ethyl acetate/hexanes) gave
5-isopropyl-.beta.-tetralone (1.57 g, 96% yield) as a light yellow
oil. .sup.1H NMR (CDCl.sub.3) .delta. 6.93-7.22 (3H, m, phenyl),
3.59 (2H, s, benzylic), 3.24 (1H, m, CH of iPr), 3.12 (2H, t,
benzylic), 2.52 (2H, t), 1.27 (6H, d, 2 CH.sub.3 of iPr).
[0324] Step 4: 131
[0325] A mixture of 5-isopropyl-.beta.-tetralone (1.57 g, 8.34
mmole), potassium cyanide (0.82 g, 12.6 mmole), ammonium carbonate
(4.81 g, 50.1 mmole), ethanol (40 ml) and water (10 ml) in a
sealed, thick walled pressure flask was heated in a 80.degree. C.
oil bath for 48 hours. After cooling to room temperature, the brown
slurry was poured into ice-water and stirred at room temperature
for a couple of hours. Filtration followed by air-drying gave crude
hydantoin as a beige solid which was used in the next step without
further purification. .sup.1H NMR (DMSO-d.sub.6) .delta. 10.69 (1H,
broad s, NH), 8.30 (1H, broad s, NH), 6.85-7.32 (3H, m, phenyl),
1.15 (6H, t, CH.sub.3). LRMS (Electrospray):
C.sub.15H.sub.18N.sub.2O.sub.2, calc. 258; observed: 539
(2M+Na).
[0326] Step 5: 132
[0327] A mixture of hydantoin (crude, 8.34 mmole theoretical),
Ba(OH).sub.2. H.sub.2O (7.90 g, 41.7 mmole) in water (40 ml) in a
sealed, thick walled pressure flask was heated in a 125.degree. C.
oil bath for 38 hours. The reaction mixture was cooled to room
temperature, acidified to .about.pH 3 using 4N sulfuric acid while
being stirred vigorously. The suspension was stirred in a boiling
water bath for two hours and cooled to room temperature. The white
suspension was filtered and the precipitates rinsed with water. The
combined filtrate and washings were concentrated in vacuo to 50 ml.
Neutralization with concentrated ammonium hydroxide solution gave
white precipitate which were filtered, washed with water and dried
in vacuo overnight to give racemic
5-isopropyl-2-aminotetraline-2-carboxylic acid (1.23 g, 63% yield
over 2 steps) as a beige solid. LRMS (Electrospray):
C.sub.14H.sub.19NO.sub.2, calc. 233; observed: 232 (M-H).
[0328] Step 6: 133
[0329] A mixture of racemic
5-isopropyl-2-aminotetraline-2-carboxylic acid (250 mg, 1.07
mmole), triethylamine (1.2 ml, 8.61 mmole), 9-fluorenylmethyl
succinimidyl carbonate (Fmoc-OSu, 2.70 g, 8.00 mmole) in
acetonitrile (30 ml) and water (30 ml) was stirred at room
temperature for 2 days. The reaction mixture was concentrated in
vacuo to remove most of the acetonitrile, acidified to pH .about.3
with 10% aqueous citric acid solution, and the white emulsion was
extracted with ethyl acetate. The organic layer was washed with
water, brine and dried over sodium sulfate. Filtration and
concentration gave a crude oil which was purified by column
chromatography (eluted with 2.fwdarw.5.fwdarw.8% methanol/methylene
chloride) to give racemic Fmoc-5-isopropyl-2-aminotetr-
aline-2-carboxylic acid (208 mg, 43% yield) as an off-white foam.
HRMS (FAB): C.sub.29H.sub.30NO.sub.4 (M+H) calc. 456.2175;
observed: 456.2184.
EXAMPLE 18
Preparation of Fmoc-4-amino-1-phenylpiperidine-4-carboxylic acid
(Fmoc-Appc-OH)
[0330] Step 1: 134
[0331] To a solution of iodobenzene (6.37 g, 3.5 mL, 31.2 mmole),
1,4-dioxa-8-azaspiro [4.5] decane (10.32 g, 9.3 mL, 72.2 mmole, 2.3
equiv) and sodium tert-butoxide (8.0 g, 83.3 mmole, 2.7 equiv) in
dry dioxane (120 mL) were added
tris(dibenzylideneacetone)dipalladium(0) (91 mg, 0.1 mmol) and
tri-o-tolylphosphine (180 mg, 0.591 mmol). The reaction was heated
at 90.degree. C. for 26 hrs. The resulting reaction mixture was
concentrated to remove solvent. The residue was treated with water
and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give a brown oil. This crude product was purified
on flash chromatography (hexane/EtOAc, 95/5 to 75/25) to provide
the pure product as a slightly yellow solid (6.08 g, 89%). .sup.1H
NMR (CDCl.sub.3), 7.25 (ddt, 2H), 6.95 (dd, 2H), 6.84 (t, 1H), 4.00
(s, 4H), 3.32 (t, 4H) and 1.84 (t, 4H); MS (electrospray) m/e 220
(M+H), Calcd for C.sub.13H.sub.17NO.sub.2, 219.
[0332] Step 2: 135
[0333] To a solution of the ketal (3.22 g, 15.16 mmol) in acetone
(100 mL) was added 6N hydrochloric acid (50 mL) and the reaction
was heated at reflux overnight. The resulting reaction mixture was
concentrated to remove solvent. The residue was taken up in EtOAc
and neutralized with aqueous 6N NaOH solution. The layers were
separated and the aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 80/20.fwdarw.60/40) to give
the product as a yellow oil (2.58 g, 97%). MS (electrospray) m/e
176 (M+H), Calcd for C.sub.11H.sub.13NO, 175.
[0334] Step 3: 136
[0335] To a solution of the ketone (2.53 g, 14.46 mmol) in ethanol
(75 mL) and water (25 mL) in a glass pressure bottle, were added
ammonium carbonate (12.9 g, 134.3 mmole, 9 equiv.) and potassium
cyanide (2.11 g, 32.5 mmol, 2 equiv.). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was
concentrated in vacuo and the residue was treated with water,
extracted with EtOAc (4x). The combined organic extracts were
washed with water, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to give the spectroscopically pure hydantoin as a
white solid (3.36 g, 95% yield). MS (electrospray) m/e 246 (M+H),
Calcd for C.sub.13H.sub.15N.sub.3O.sub.2, 245.
[0336] Step 4: 137
[0337] The hydantoin (3.36 g) was suspended in aqueous NaOH (6N,
100 mL) and heated at 130.degree. C. for 2-3 days. Upon completion
(by HPLC) of the hydrolysis, the reaction mixture was neutralized
with conc. HCl to slightly acidic (pH .about.6). The resulting
slurry was filtered, washed with water and dried to give
4-amino-1-phenylpiperidine-4-carboxylic acid (APPC) as a white
solid (5.26 g, >100% yield, wet and contaminated with inorganic
salt), which showed a single peak on HPLC and used directly for the
next step. MS (electrospray) m/e 221 (M+H), Calcd for
C.sub.12H.sub.16N.sub.2O.sub.2, 220.
[0338] Step 5: 138
[0339] The crude amino acid APPC from the last step was suspended
in dioxane (80 mL) and aqueous 10% Na.sub.2CO.sub.3 (40 ml),
treated with Fmoc-Cl (5.3 g, 20.57 mmole, 1.5 equiv) and was
stirred vigorously overnight. The reaction mixture was then
concentrated to remove dioxane, neutralized with 6N HCl to slightly
acidic (pH 6) and extracted with EtOAc. The combined organic
extracts were washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave the crude product which was purified on
flash chromatography (hexane/EtOAc to CH.sub.2Cl.sub.2/MeOH) to
give pure APPC (4.91 g, 81% overall yield for two steps). .sup.1H
NMR(DMSO-d.sub.6), 7.88 (d, 2H), 7.74 (d, 2H), 7.19-7.42 (m, 8H),
4.20-4.31 (m, 3H); HRMS m/z 465.1788, Calcd for
C.sub.27H.sub.26N.sub.2O.sub.4Na, 465.1791
EXAMPLE 19
Preparation of
Fmoc-4-amino-1-(4-methylphenyl)piperidine-4-carboxylic acid
(Fmoc-4-MeAppc-OH)
[0340] Step 1: 139
[0341] To a solution of 4-iodotoluene (2.12 g, 9.7 mmol),
1,4-dioxa-8-azaspiro[4.5]decane (2.8 mL, 3.12 g, 21.82 mmol, 2.2
equiv) and sodium tert-butoxide (2.6 g, 27.08 mmol, 2.8 equiv) in
dry dioxane (40 mL) were added
tris(dibenzylideneacetone)dipalladium (0) (44.4 mg, 0.0485 mmol)
and tri-o-tolylphosphine (59.0 mg, 0.194 mmol). The reaction was
heated at 90.degree. C. for 26 hrs. The resulting reaction mixture
was concentrated to remove solvent. The residue was treated with
water and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give brown oil. This crude product was purified on
flash chromatography (hexane/EtOAc, 95/5 to 75/25) to provide the
pure product as a slightly yellow solid (1.937 g, 85%). .sup.1H NMR
(CDCl.sub.3), 7.06 (d, 2H), 6.87 (d, 2H), 3.99 (s, 4H), 3.26 (t,
4H), 2.26 (s, 3H) and 1.85 (t, 4H).
[0342] Step 2: 140
[0343] To a solution of the ketal (1.58 g, 6.79 mmol) in acetone
(50 mL) was added 6N hydrochloric acid (25 mL) and the reaction was
heated at reflux overnight. The resulting reaction mixture was
concentrated to remove solvent. The residue was taken up in EtOAc
and neutralized with aqueous 6N NaOH solution. The layers were
separated and the aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 90/10.fwdarw.70/30) to give
the product as a yellow oil (1.27 g, 98%). MS (electrospray) m/e
190 (M+H), Calcd for C.sub.12H.sub.15NO, 189.
[0344] Step 3: 141
[0345] To a solution of the ketone (1.17 g, 6.18 mmol) in ethanol
(60 mL) and water (20 mL) in a glass pressure bottle, were added
ammonium carbonate (4.74 g, 49.44 mmole, 8 equiv.) and potassium
cyanide (1.01 g, 15.54 mmol, 2.5 equiv.). The mixture was heated at
90.degree. C. for 22 hrs. The cooled reaction mixture was
concentrated in vacuo and the residue was treated with water,
extracted with EtOAc (4x). The combined organic extracts were
washed with water, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to give the spectroscopically pure hydantoin as a
white solid (1.554 g, 97% yield). MS (electrospray) m/e 260 (M+H),
Calcd for C.sub.14H.sub.17N.sub.3O.sub.2, 259.
[0346] Step 4: 142
[0347] The hydantoin (1.502 g) was suspended in aqueous NaOH (6N,
40 mL) and heated at 130.degree. C. for 4 days. Upon completion (by
HPLC) of the hydrolysis, the reaction mixture was neutralized with
conc. HCl to slightly acidic (pH .about.6). The resulting slurry
was filtered, washed with water and dried to give
4-amino-1-(4-methylphenyl)piperidine-4-carbo- xylic acid (4-MeAPPC)
as a white solid (2.10 g, >100% yield, wet and contaminated with
inorganic salt), which showed a single peak on HPLC and used
directly in the next step. MS (electrospray) m/e 235 (M+H), Calcd
for C.sub.13H.sub.18N.sub.2O.sub.2, 234.
[0348] Step 5: 143
[0349] The crude amino acid 4-MeAPPC from the last step was
suspended in dioxane (80 mL) and aqueous 10% Na.sub.2CO.sub.3 (40
ml), treated with Fmoc-Cl (2.2 g, 8.59 mmole, 1.5 equiv) and was
stirred vigorously overnight. The reaction mixture was then
concentrated to remove dioxane, neutralized with 6N HCl to slightly
acidic (pH 6) and extracted with EtOAc. The combined organic
extracts were washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave the crude product which was purified on
flash chromatography (hexane/EtOAc to CH.sub.2Cl.sub.2/MeOH) to
give pure Fmoc-4-MeAPPC (2.16 g, 82% overall yield for two steps).
.sup.1H NMR (DMSO-d.sub.6): 7.88 (d, 2H), 7.72 (d, 2H), 7.39 (t,
2H), 7.30 (td, 2H), 6.99 (d, 2H), 6.82 (d, 2H), 2.18 (s, 3H); MS
(electrospray) m/e 457 (M+H), Calcd for C.sub.28H.sub.28N.sub.2O.-
sub.4, 456.
EXAMPLE 20
Preparation of
Fmoc-4-amino-1-(4-chlorophenyl)piperidine-4-carboxylic acid
(Fmoc-4-CIAppc-OH)
[0350] Step 1: 144
[0351] To a solution of 1-chloro-4-iodobenzene (2.38 g, 10.0
mmole), 1,4-dioxa-8-azaspiro [4.5] decane (3.1 mL, 3.44 g, 24.0
mmole, 2.4 equiv) and sodium tert-butoxide (2.68 g, 28.0 mmole, 2.8
equiv) in dry dioxane (40 mL) were added
tris(dibenzylideneacetone)dipalladium(0) (45.5 mg, 0.0497 mmol) and
tri-o-tolyl-phosphine (61 mg, 0.20 mmol). The reaction was heated
at 90.degree. C. for 9 hrs. The resulting reaction mixture was
concentrated to remove solvent. The residue was treated with water
and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na2SO4 and concentrated to
give a brown oil. This crude product was purified on flash
chromatography (hexane/EtOAc, 95/5 to 75/25) to provide the pure
product as a slightly yellow solid (2.17 g, 86%). .sup.1H
NMR(CDCl.sub.3), 7.18 (dt, 2H), 6.85 (dt, 2H), 3.98 (s, 4H), 3.28
(t, 4H) and 1.82 (t, 4H).
[0352] Step 2: 145
[0353] To a solution of the ketal (2.123 g, 8.39 mmole) in acetone
(75 mL) was added 6N hydrochloric acid (30 mL) and the reaction was
heated at reflux overnight. The resulting reaction mixture was
concentrated to remove solvent. The residue was taken up in EtOAc
and neutralized with aqueous 6N NaOH solution. The layers were
separated and the aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 95/5.fwdarw.70/30) to give
the product as a yellow solid (1.515 g, 86%). MS (electrospray) m/e
210 (M+H), Calcd for C.sub.11H.sub.12ClNO, 209.
[0354] Step 3: 146
[0355] To a solution of the ketone (1.465 g, 6.986 mmole) in
ethanol (75 mL) and water (25 mL) in a glass pressure bottle, were
added ammonium carbonate (5.36 g, 55.88 mmole, 8 equiv.) and
potassium cyanide (1.135 g, 17.46 mmol, 2.5 equiv.). The mixture
was heated at 80-90.degree. C. for 18 hrs. The cooled reaction
mixture was concentrated in vacuo and the residue was treated with
water, extracted with EtOAc (4x). The combined organic extracts
were washed with water, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to give the spectroscopically pure hydantoin as a
white solid (1.817 g, 93% yield). MS (electrospray) m/e 280 (M+H),
Calcd for C.sub.13H.sub.14ClN.sub.3O.sub.2, 279.
[0356] Step 4: 147
[0357] The hydantoin (1.768 g) was suspended in aqueous NaOH (6N,
50 mL) and heated at 130.degree. C. for 4 days. Upon the completion
(by HPLC) of the hydrolysis, the reaction mixture was neutralized
with conc. HCl to slightly acidic (pH .about.6). The resulting
slurry was filtered, washed with water and dried to give
4-amino-1-(4-chlorophenyl)piperidine-4-carbo- xylic acid (4-ClAPPC)
as a white solid (2.05 g, >100% yield, wet and contaminated with
inorganic salt), which showed a single peak on HPLC and used
directly for the next step. MS (electrospray) m/e 253 (M-H), Calcd
for C.sub.12H.sub.15ClN.sub.2O.sub.2, 254.
[0358] Step 5: 148
[0359] The crude amino acid 4-CIAPPC from the last step was
suspended in dioxane (100 mL) and aqueous 10% Na.sub.2CO.sub.3 (50
ml), treated with Fmoc-Cl (2.0 g, 7.75 mmole, 1.2 equiv) and was
stirred vigorously overnight. The reaction mixture was then
concentrated to remove dioxane, neutralized with 6N HCl to slightly
acidic (pH 6) and extracted with EtOAc. The combined organic
extracts were washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave the crude product which was purified on
flash chromatography (hexane/EtOAc to CH.sub.2Cl.sub.2/MeOH) to
give pure Fmoc-4-ClAPPC (1.18 g, 81% overall yield for two steps).
.sup.1H NMR (DMSO-d.sub.6): 7.87 (d, 2H), 7.71 (d, 2H), 7.39 (td,
2H), 7.30 (td, 2H), 7.20 (d, 2H), 6.92 (d, 2H), 3.44 (d, 2H), 2.93
(t, 2H); MS (electrospray) m/e 477 (M+H), Calcd for
C.sub.27H.sub.25N.sub.2O.sub.4, 476.
EXAMPLE 21
Preparation of
Fmoc-4-amino-1-(4-phenoxyphenyl)piperidine-4-carboxylic acid
(Fmoc-4-PhOAppc-OH)
[0360] Step 1: 149
[0361] To a solution of 1-iodo-4-phenoxybenzene (3.15 g, 10.6
mmol), 1,4-dioxa-8-azaspiro [4.5] decane (3.3 mL, 3.66 g, 25.6
mmole, 2.4 equiv) and sodium tert-butoxide (2.85 g, 29.7 mmol, 2.8
equiv) in dry dioxane (40 mL) were added tris
(dibenzylideneacetone) dipalladium (0) (48.5 mg, 0.053 mmol) and
tri-o-tolyl- phosphine (64 mg, 0.4 mmol). The reaction was heated
at 90.degree. C. for 9 hrs. The resulting reaction mixture was
concentrated to remove solvent. The residue was treated with water
and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give a brown oil. This crude product was purified
on flash chromatography (hexane/EtOAc, 95/5 to 80/20) to provide
the pure product as a slightly yellow solid (2.805, 85%). .sup.1H
NMR (CDCl.sub.3), 7.26-7.32 (m, 2H), 7.03 (t, 1H), 6.92-6.97 (m,
6H), 4.00 (s, 4H), 3.26 (t, 4H), 1.86 (t, 4H).
[0362] Step 2: 150
[0363] To a solution of the ketal (2.755 g, 8.86 mmol) in acetone
(90 mL) was added 6N hydrochloric acid (45 mL) and the reaction was
heated at reflux overnight. The resulting reaction mixture was
concentrated to remove solvent. The residue was diluted with EtOAc
and neutralized with aqueous 6N NaOH. The layers were separated and
the aqueous layer was extracted with EtOAc. The combined organic
extracts were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give the crude product which was purified on flash
chromatography (hexane/EtOAc, 90/10 to 70/30) to give the product
as a yellow oil (2.21 g, 93%). MS (electrospray) m/e 268 (M+H),
Calcd for C.sub.17H.sub.17ClNO.sub.2, 267.
[0364] Step 3: 151
[0365] To a solution of the ketone (2.01 g, 7.52 mmol) in ethanol
(80 mL) and water (25 mL) in a glass pressure bottle, were added
ammonium carbonate (5.78 g, 60.0 mmol, 8 equiv.) and potassium
cyanide (1.22 g, 18.80 mmol, 2.5 equiv.). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was
concentrated in vacuo and the residue was treated with water,
extracted with EtOAc (4x). The combined organic extracts were
washed with water, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to give the spectroscopically pure hydantoin as a
white solid (2.34 g, 95% yield). MS (electrospray) mle 338 (M+H),
Calcd for C.sub.19H.sub.19N.sub.3O.sub.3, 337.
[0366] Step 4: 152
[0367] The hydantoin (2.28 g, 6.76 mmole) was suspended in aqueous
NaOH (6N, 60 mL) and heated at 130.degree. C. for 4 days. Upon
completion (by HPLC) of the hydrolysis, the reaction mixture was
neutralized with conc. HCl to slightly acidic (pH .about.6). The
resulting slurry was filtered, washed with water and dried to give
4-amino-1-(4-phenoxyphenyl)piperidine- -4-carboxylic acid
(4-PhOAPPC) as a white solid (2.53 g, >100% yield, wet and
contaminated with inorganic salt), which showed a single peak on
HPLC and used directly for the next step. MS (electrospray) m/e 313
(M+H), Calcd for C.sub.18H.sub.20N.sub.2O.sub.3, 312.
[0368] Step 5: 153
[0369] The crude 4-PhOAPPC from the last step was treated with
Fmoc-Cl (2.6g, 1.25 equiv) in dioxane (50 L) and aqueous 10%
Na.sub.2CO.sub.3 (50 ml) and stirred vigorously overnight. The
reaction mixture was concentrated to remove dioxane, neutralized
with 6N HCl to slightly acidic (pH 6) and extracted with EtOAc. The
combined organic extracts were washed with brine and dried over
Na.sub.2SO.sub.4. Removal of the solvent gave the crude product
which was purified on flash chromatography (hexane/EtOAc to
CH.sub.2Cl.sub.2/MeOH) to give pure 4-PhOAPPC (2.18 g, 60% overall
yield for two steps). .sup.1H NMR (DMSO-d.sub.6): 7.87 (d, 2H),
7.72 (d, 2H), 7.38 (t, 2H), 7.30 (td, 4H), 7.02 (dt, 1H), 6.86-6.96
(m, 6H), 3.35 (m, 2H), 2.94 (t, 2H); MS (electrospray) m/e 535
(M+H), Calcd for C.sub.33H.sub.30N.sub.2O.sub.5, 534.
EXAMPLE 22
Preparation of
Fmoc-4-amino-1-(2-methylphenyl)piperidine-4-carboxylic
acid(Fmoc-2-MeAppc-OH)
[0370] Step 1: 154
[0371] To a solution of 2-iodotoluene (4.36 g, 2.5 mL, 20.0 mmol),
1,4-dioxa-8-azaspiro[4.5]decane (6.88 g, 6.2 mL, 48.1 mmol, 2.4
equiv) and sodium tert-butoxide (5.3 g, 55.2 mmol, 2.8 equiv) in
dry dioxane (80 mL) were added
tris(dibenzylideneacetone)dipalladium(0) (91 mg, 0.1 mmol) and
tri-o-tolylphosphine (122 mg, 0.4 mmol). The reaction was heated at
90.degree. C. for 26 hrs. The resulting reaction mixture was
concentrated to remove solvent. The residue was treated with water
and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give brown oil. This crude product was purified on
flash chromatography (hexane/EtOAc, 95/5 to 75/25) to provide the
pure product as a slightly yellow solid (2.66 g, 57%). .sup.1H NMR
(CDCl.sub.3), 7.12-7.18 (m, 2H), 6.94-7.06 (m, 2H), 4.01 (s, 4H),
2.98 (t, 4H) and 1.88 (t, 4H).
[0372] Step 2: 155
[0373] To a solution of the ketal (2.66 g, 11.4 mmol) in acetone
(70 mL) was added 6N hydrochloric acid (35 mL) and the reaction was
heated at 85.degree. C. overnight. The resulting reaction was
concentrated to remove solvent. The residue was diluted with EtOAc
and neutralized with aqueous NaOH (6N). The layers were separated
and the aqueous layer was extracted with EtOAc. The combined
organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 90/10 to 70/30) to give the
product as a yellow oil (2.04 g, 95%). MS (electrospray) m/e 190
(M+H), Calcd for C.sub.12H.sub.15NO, 189.
[0374] Step 3: 156
[0375] To a solution of the ketone (1.54 g, 8.15 mmol) in ethanol
(60 mL) and water (20 mL) in a glass pressure bottle, were added
ammonium carbonate (4.69 g, 48.9 mmol, 6 equiv.) and potassium
cyanide (800 g, 12.2 mmol, 1.5 equiv.). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was added
to icy water (300 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (2.01 g,
95% yield). MS (electrospray) m/e 260 (M+H), Calcd for
C.sub.14H.sub.17N.sub.3O.sub.2, 259.
[0376] Step 4: 157
[0377] To a suspension of the hydantoin (1.07 g, 4.13 mmol) in dry
THF (25 mL) were added di-tert-butyl dicarbonate (2.25 g, 10.32
mmol, 2.5 equiv), triethylamine (0.63 mL, 460 mg, 4.54 mmol, 1.1
equiv) and DMAP (36 mg, 0.29 mmol) in succession. About 15 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (300 mL), washed with 1N HCl
(3.times.30 mL), saturated aqueous Na.sub.2CO.sub.3 (2.times.30 mL)
and brine (2.times.30 mL), dried over anhydrous Na.sub.2SO.sub.4
and concentrated under reduced pressure. The crude light yellow
product was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.80/20) to give the pure bis-Boc hydantoin as a white
solid (1.71 g, 90%). MS (electrospray) m/e 460 (M+H), Calcd for
C.sub.24H.sub.33N.sub.3O.sub.6, 459.
[0378] Step 5: 158
[0379] The bis-Boc hydantoin (1.71 g, 3.72 mmol) was dissolved in
DME (23 mL) to give a clear solution. To this solution was added 1N
NaOH (33 mL, 33 mmol) and the reaction was stirred overnight at
room temperature, giving a fairly clear mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
4-amino-1-(2-methylphenyl)piperidine-4-carboxyli- c acid (2-MeAPPC)
was treated with 6N HCl to adjust the pH to 11-12. This solution
(30 mL) was then diluted with 1,4-dioxane (30 mL) and treated with
Fmoc-Cl (1.28 g, 4.96 mmol, 1.3 equiv) and stirred overnight at
room temperature. The reaction mixture was concentrated under
reduced pressure to remove dioxane, neutralized with 1N HCl and
extracted with EtOAc. The combined organic extracts were washed
with brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated.
The crude product was purified through flash chromatography
(hexane/EtOAc.fwdarw.CH.sub.2Cl.sub.2/MeOH) to give the pure
product as a white solid (1.09 g, 64% yield from the bis-Boc
hydantoin). .sup.1H NMR (DMSO-d.sub.6): 7.87 (d, 2H), 7.74 (d, 2H),
7.40 (td, 2H), 7.31 (td, 2H), 7.12 (m, 2H), 6.97 (d, 1H), 6.92 (td,
1H), 2.72-2.88 (m, 4H) and 2.22 (s, 3H); MS (electrospray) m/e 457
(M+H), Calcd for C.sub.28H.sub.28N.sub.2O.sub.4, 456.
EXAMPLE 23
Preparation of
Fmoc-4-amino-1-(2-isopropylphenyl)piperidine-4-carboxylic acid
(Fmoc-2-iPrAppc-OH)
[0380] Step 1: 159
[0381] To a solution of 1-iodo-2-iso-propylbenzene (10.0 g, 40.7
mmol), 1,4-dioxa-8-azaspiro[4.5]decane (12.0 mL, 13.3 g, 93.0 mmol,
2.3 equiv) and sodium tert-butoxide (10.0 g, 104.2 mmol, 2.6 equiv)
in dry dioxane (160 mL) were added
tris(dibenzylideneacetone)dipalladium(0) (180 mg, 0.197 mmol) and
tri-o-tolyl-phosphine (244 mg, 0.80 mmol) and the reaction was
heated at 90.degree. C. for 26 hrs. The resulting reaction mixture
was concentrated to remove solvent, treated with water and
extracted with EtOAc. The combined organic extracts were combined,
washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to
give a brown oil. This crude product was purified on flash
chromatography (hexane/EtOAc, 95/5.fwdarw.75/25) to provide the
pure product as a slightly yellow solid (3.61 g, 35% yield). MS m/z
262 (M+H), Calcd for C.sub.16H.sub.23NO.sub.2, 261.
[0382] Step 2: 160
[0383] To a solution of the ketal (3.24 g, 12.4 mmol) in acetone
(90 mL) was added 6N hydrochloric acid (45 mL) and the reaction was
heated at reflux overnight. The resulting reaction mixture was
concentrated to remove solvent and the residue was diluted with
EtOAc, neutralized with aqueous NaOH (6N). The layers were
separated and the aqueous layer was extracted with EtOAc. The
combined organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 90/10.fwdarw.70/30) to give
the product as a yellow oil (2.42 g, 89%). .sup.1H NMR
(CDCl.sub.3): 7.27 (m, 1H), 7.04-7.19 (m, 3H), 3.58 (m, 1H), 3.20
(t, 4H), 2.60 (t, 4H) and 1.25 (d, 6H); MS m/z 218 (M+H), Calcd for
C.sub.14H.sub.19NO, 217.
[0384] Step 3: 161
[0385] To a solution of the ketone (2.30 g, 10.6 mmol) in ethanol
(90 mL) and water (20 mL) in a glass pressure bottle, were added
ammonium carbonate (8.1 g, 84.3 mmol, 8 equiv) and potassium
cyanide (1.72 g, 26.5 mmol, 2.5 equiv). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was added
to icy water (400 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (2.78 g,
91% yield). MS m/z 288 (M+H), Calcd for
C.sub.16H.sub.21N.sub.3O.sub.2, 287.
[0386] Step 4: 162
[0387] To a suspension of the hydantoin (2.74 g, 9.54 mmol) in dry
THF (100 mL) were added di-tert-butyl dicarbonate (5.2 g, 24.24
mmol, 2.5 equiv), triethylamine (1.5 mL, 1.07 g, 10.5 mmol, 1.1
equiv) and DMAP (46 mg, 0.29 mmol) in succession. About 15 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (300 mL), washed with brine
(3.times.30 mL), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The crude light yellow product
was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.80/20) to give the pure bis-Boc hydantoin as a white
solid (4.39 g, 94% yield). MS m/z 488 (M+H), Calcd for
C.sub.26H.sub.37N.sub.3O- .sub.6, 487.
[0388] Step 5: 163
[0389] The bis-Boc hydantoin (2.34 g, 4.8 mmol) was dissolved in
DME (30 mL) to give a clear solution. To this solution was added 1N
NaOH (45 mL, 45 mmol) and the reaction was stirred overnight at
room temperature, giving a fairly clear mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
4-amino-1-(2-isopropylphenyl)piperidine-4-carbox- ylic acid
(2-iPrAPPC) was treated with 6N HCl to adjust the pH to 11-12. This
solution (.about.45 mL) was then diluted with 1,4-dioxane (45 mL)
and treated with Fmoc-Cl (1.78 g, 6.89 mmol, 1.5 equiv) and stirred
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove dioxane, neutralized
with 1N HCl and extracted with EtOAc. The combined organic extracts
were washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (hexane/EtOAc.fwdarw.CH- .sub.2Cl.sub.2/MeOH) to
give the pure product as a white solid (1.46 g, 63% yield from the
bis-Boc hydantoin). HRMS m/z 507.2263, Calcd for
C.sub.30H.sub.32N.sub.2O.sub.4Na, 507.2260.
EXAMPLE 24
Preparation of
Fmoc-4-amino-1-(3-methylphenyl)piperidine-4-carboxylic acid
(Fmoc-3-MeAppc-OH)
[0390] Step 1: 164
[0391] To a solution of 3-iodotoluene (4.36 g, 2.6 mL, 20.0 mmol),
1,4-dioxa-8-azaspiro [4.5] decane (6.88 g, 6.2 mL, 48.1 mmol, 2.4
equiv) and sodium tert-butoxide (5.3 g, 55.2 mmol, 2.8 equiv) in
dry dioxane (80 mL) were added tris (dibenzylideneacetone)
dipalladium (0) (91 mg, 0.1 mmol) and tri-o-tolylphosphine (122 mg,
0.4 mmol). The reaction was heated at 90.degree. C. for 26 hrs. The
resulting reaction mixture was concentrated to remove solvent. The
residue was treated with water and extracted with EtOAc. The
combined organic extracts were combined, washed with brine, dried
over Na.sub.2SO.sub.4 and concentrated to give a brown oil. This
crude product was purified on flash chromatography (hexane/EtOAc,
95/5 to 75/25) to provide the pure product as a slightly yellow
solid (3.21 g, 69%).
[0392] Step 2: 165
[0393] To a solution of the ketal (1.25 g, 5.36 mmol) in acetone
(20 mL) was added 6N hydrochloric acid (10 mL) and the reaction was
heated at reflux overnight. The. resulting reaction was
concentrated to remove solvent. The residue was diluted with EtOAc
and neutralized with aqueous NaOH (6N). The layers were separated
and the aqueous layer was extracted with EtOAc. The combined
organic extracts were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
on flash chromatography (hexane/EtOAc, 90/10 to 70/30) to give the
product as a yellow oil (843 mg, 83% yield). MS m/z 190 (M+H),
Calcd for C.sub.12H.sub.15NO, 189.
[0394] Step 3: 166
[0395] To a solution of the ketone (763 g, 4.03 mmol) in ethanol
(45 mL) and water (15 mL) in a glass pressure bottle, were added
ammonium carbonate (3.09 g, 32.21 mmol, 8 equiv) and potassium
cyanide (675 mg, 10.38 mmol, 2.5 equiv). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was added
to icy water (200 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (930 mg,
89% yield). MS m/z 260 (M+H), Calcd for
C.sub.14H.sub.17N.sub.3O.sub.2, 259.
[0396] Step 4: 167
[0397] To a suspension of the hydantoin (780 mg, 3.012 mmol) in dry
THF (22 mL) were added di-tert-butyl dicarbonate (1.64 g, 7.52
mmol, 2.5 equiv), triethylamine (0.42 mL, 305 mg, 3.01 mmol, 1.0
equiv) and DMAP (20 mg, 0.164 mmol) in succession. About 5 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (300 mL), washed with brine
(3.times.30 mL), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The crude light yellow product
was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.80/20) to give the pure bis-Boc hydantoin as a white
solid (1.37 g, quantitative). HRMS m/z 482.2261 (M+Na), Calcd. for
C.sub.24H.sub.33N.sub.3O.sub.6Na, 482.2267.
[0398] Step 5: 168
[0399] The bis-Boc hydantoin (1.29 g, 2.818 mmol) was dissolved in
DME (20 mL) to give a clear solution. To this solution was added 1N
NaOH (25 mL, 25 mmol) and the reaction was stirred overnight at
room temperature, giving a fairly clear mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
4-amino-1-(3-methylphenyl)piperidine-4-carboxyli- c acid (3-MeAPPC)
was treated with 6N HCl to adjust the pH to 11-12. This solution
(30 mL) was then diluted with 1,4-dioxane (30 mL) and treated with
Fmoc-Cl (1.46 mg, 5.65 mmol, 2.0 equiv) and stirred overnight at
room temperature. The reaction mixture was concentrated under
reduced pressure to remove dioxane, neutralized with 1N HCl and
extracted with EtOAc. The combined organic extracts were washed
with brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated.
The crude product was purified through flash chromatography
(hexane/EtOAc.fwdarw.CH.sub.2Cl.sub- .2/MeOH) to give the pure
product as a white solid (1.002 g, 78% yield from the bis-Boc
hydantoin). HRMS m/z 479.1940 (M+Na), Calcd. for
C.sub.28H.sub.28N.sub.2O.sub.4Na, 479.1947.
EXAMPLE 25
Preparation of
Fmoc-4-amino-1-(3-methoxyphenyl)piperidine-4-carboxylic acid
(Fmoc-3-MeOAppc-OH)
[0400] Step 1: 169
[0401] To a solution of 3-iodoanisole (4.68 g, 2.4 mL, 20.0 mmol),
1,4-dioxa-8-azaspiro [4.5] decane (6.2 mL, 6.88 g, 48.1 mmol, 2.4
equiv) and sodium tert-butoxide (5.3 g, 55.2 mmol, 2.8 equiv) in
dry dioxane (80 mL) were added
tris(dibenzylideneacetone)dipalladium(0) (91 mg, 0.1 mmol) and
tri-o-tolylphosphine (122 mg, 0.4 mmol) and the reaction was heated
at 90.degree. C. for 26 hrs. The resulting reaction mixture was
concentrated to remove solvent and the residue was treated with
water and extracted with EtOAc. The combined organic extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give brown oil. This crude product was purified on
flash chromatography (hexane/EtOAc, 95/5 to 75/25) to provide the
pure product as a slightly yellow solid (3.10 g, 62% yield). MS m/z
(M+H), 250 (M+H), Calcd for C.sub.14H.sub.19NO.sub.3, 249.
[0402] Step 2: 170
[0403] To a solution of the ketal (3.10 g, 12.45 mmol) in acetone
(90 mL) was added 6N hydrochloric acid (45 mL) and the reaction was
heated at reflux overnight. The resulting reaction was concentrated
to remove solvent. The residue was diluted with EtOAc and
neutralized with aqueous NaOH (6N). The layers were separated and
the aqueous layer was extracted with EtOAc. The combined organic
extracts were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated. The crude product was purified on flash
chromatography (hexane/EtOAc, 90/10 to 70/30) to give the product
as a yellow oil (2.53 g, 99% yield). .sup.1H NMR (CDCl.sub.3): 7.20
(m, 1H), 6.58 (d, 1H), 6.39-6.56 (m, 2H), 3.80 (s, 3H), 3.59 (m,
4H) and 2.58 (m, 4H).
[0404] Step 3: 171
[0405] To a solution of the ketone (1.81 g, 8.82 nmuol) in ethanol
(60 mL) and water (20 mL) in a glass pressure bottle, were added
ammonium carbonate (6.77 g, 70.52 mmol, 8 equiv) and potassium
cyanide (1.14 g, 17.6 mmol, 2.0 equiv). The mixture was heated at
80-90.degree. C. for 18 hrs. The cooled reaction mixture was added
to icy water (200 ml) and stirred vigorously for 30 min. The
resulting precipitate was suction filtered, washed thoroughly with
water and dried to yield the hydantoin as a white solid (2.23 g,
92% yield). MS m/z 276 (M+H), Calcd for
C.sub.14H.sub.17N.sub.3O.sub.3, 275.
[0406] Step 4: 172
[0407] To a suspension of the hydantoin (1.10 g, 4.00 mmol) in dry
THF (50 mL) were added di-tert-butyl dicarbonate (2.18 g, 10.0
mmol, 2.5 equiv), triethylamine (0.62 mL, 445 mg, 4.4 mmol, 1.1
equiv) and DMAP (20 mg, 0.164 mmol) in succession. About 15 minutes
after the addition, the reaction turned into a clear yellow
solution and was stirred overnight at room temperature. The
reaction mixture was concentrated under reduced pressure to yield a
solid that was then taken up in EtOAc (300 mL), washed with brine
(3.times.30 mL), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The crude light yellow product
was purified through flash chromatography (hexane/EtOAc,
90/10.fwdarw.80/20) to give the pure bis-Boc hydantoin as a white
solid (1.90 g, quantitative). .sup.1H NMR (CDCl.sub.3): 7.16 (t,
1H), 6.57 (d, 1H), 6.24 (s, 1H), 6.19 (d, 1H), 3.77 (s, 3H), 1.58
(s, 9H), 1.42 (s, 9H); MS m/z 476 (M+H), Calcd for
C.sub.24H.sub.33N.sub.3O.sub.7, 475.
[0408] Step 5: 173
[0409] The bis-Boc hydantoin (1.06 g, 2.23 mmol) was dissolved in
DME (20 mL) to give a clear solution. To this solution was added 1N
NaOH (20 mL, 20 mmol) and the reaction was stirred overnight at
room temperature, giving a fairly clear mixture. HPLC showed
completion of the reaction. The reaction mixture was concentrated
under reduced pressure to remove DME and extracted with Et.sub.2O.
Without purification, the resulting aqueous layer containing
4-amino-1-(3-methoxyphenyl)piperidine-4-carboxyl- ic acid
(3-MeOAPPC) was treated with 6N HCl to adjust the pH to 11-12. This
solution (35 mL) was then diluted with 1,4-dioxane (35 mL) and
treated with Fmoc-Cl (755 mg, 2.93 mmol, 1.3 equiv) and stirred
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure to remove dioxane, neutralized
with 1N HCl and extracted with EtOAc. The combined organic extracts
were washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The crude product was purified through flash
chromatography (hexane/EtOAcO CH.sub.2Cl.sub.2/MeOH) to give the
pure product as a white solid (668 mg, 63% yield from the bis-Boc
hydantoin). .sup.1H NMR (CDCl.sub.3): 7.83 (d, 2H), 7.72 (d, 2H),
7.41 (td, 2H), 7.34 (dt, 2H), 7.16 (t, 1H), 6.52 (d, 1H), 6.42 (s,
1H), 6.36 (d, 1H), 4.25 (m, 3H), 3.68 (s, 3H), 3.23-3.40 (m, 2H),
2.96 (t, 2H) and 1.86-2.18 (m, 4H). HRMS m/z 495.1901 (M+Na),
Calcd. for C.sub.28H.sub.28N.sub.2O.sub.5Na, 495.1896.
EXAMPLE 26
Preparation of Fmoc-1-amino-4-cyclohexylcyclohexane-1-carboxylic
acid (Fmoc-Achc-OH)
[0410] Step 1: 174
[0411] A mixture of 4-cyclohexylcyclohexanone (3.00 g, 16.6 mmole),
potassium cyanide (1.63 g, 25.0 mmole), ammonium carbonate (9.59 g,
99.8 mmole), ethanol (75 ml) and water (15 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 15
hours. After cooling to room temperature, the white slurry was
poured into ice-water and stirred at room temperature for a couple
of hours. Filtration and air-drying gave hydantoin (6.10 g, still
wet, >100% yield) as a white solid. .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.52 (1H, broad, NH), 8.43 (1H, broad s, NH), 0.80-1.80
(20H, m). LRMS (APCI): C.sub.14H.sub.22N.sub.2O.sub.2, calc. 250;
observed: 249 (M-H), 251 (M+H).
[0412] Step 2: 175
[0413] A mixture of hydantoin (1.39 g, 5.55 mmole) and 6N sodium
hydroxide solution (50 ml) in a sealed, thick walled pressure flask
was heated in a 130.degree. C. oil bath for 2 days. The reaction
mixture was cooled in an ice bath, neutralized to .about.pH 7 using
concentrated hydrochloric acid. The white slurry was filtered and
the precipitates rinsed with water to give crude
1-amino-4-cyclohexylcyclohexane-1-carboxylic acid (48.3 g, wet and
containing inorganic salts, >100% yield). LRMS (Electrospray):
C.sub.13H.sub.23NO.sub.2, calc. 225; observed: 226 (M+H).
[0414] Step 3: 176
[0415] A mixture of crude
1-amino-4-cyclohexylcyclohexane-1-carboxylic acid (48.3 g, 5.55
mmole theoretical), triethylamine (1.0 ml, 7.17 mmole),
9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 2.43 g, 7.20
mmole) in acetonitrile (75 ml) and water (75 ml) was stirred at
room temperature for 24 hours. The reaction mixture was
concentrated in vacuo to remove most of the acetonitrile, acidified
to pH .about.3 with 10% aqueous citric acid solution, and the white
emulsion extracted three times with methylene chloride. The
combined organic layers were washed with water, brine, dried over
magnesium sulfate. Filtration and concentration gave a crude oil
which was purified by column chromatography (eluted with
1.fwdarw.5.fwdarw.8% methanol/methylene chloride) to give
Fmoc-1-amino-4-trans-cyclohexylcyclohexane-1-carboxylic acid (250
mg, 10% yield for two steps). HRMS (FAB): C.sub.28H.sub.34NO.sub.4
(M+H) calc. 448.2488; observed: 448.2497.
EXAMPLE 27
Preparation of Fmoc-1-amino-4,4-diphenylcyclohexane-1-carboxylic
acid (Fmoc-Adpc-OH)
[0416] Step 1: 177
[0417] A mixture of 4,4-diphenylcyclohexanone (prepared by
hydrogenation of 4,4-diphenylcyclohexenone according to the
procedures of Freeman, P. K. et. al. J. Org. Chem. 1989, 54,
782-789) (1.55 g, 6.19 mmole), potassium cyanide (0.65 g, 9.97
mmole), ammonium carbonate (3.60 g, 37.5 mmole), ethanol (48 ml)
and water (12 ml) in a sealed, thick walled pressure flask was
heated in a 80.degree. C. oil bath for 24 hours. After cooling to
room temperature, the white slurry was poured into ice-water and
stirred at room temperature for a couple of hours. Filtration and
air-drying gave hydantoin (1.89 g, 95% yield) as a white solid.
.sup.1H NMR (DMSO-d.sub.6) .delta. 10.57 (1H, broad, NH), 8.59 (1H,
broad s, NH), 7.00-7.50 (1OH, m, phenyl). LRMS (Electrospray):
C.sub.20H.sub.20N.sub.2O- .sub.2, calc. 320; observed: 319
(M-H).
[0418] Step 2: 178
[0419] A mixture of hydantoin (1.88 g, 5.87 mmole), barium
hydroxide monohydrate (5.60 g, 29.6 mmole) and water (100 ml, too
dilute!) in a sealed, thick walled pressure flask was heated in a
105.degree. C. oil bath for 2 days. More barium hydroxide
monohydrate (5.60 g, 29.6 mmole) was added and the mixture was
heated in a 105.degree. C. oil bath for another 24 hours. The
reaction mixture was cooled to room temperature, acidified to
.about.pH 3 using 4N sulfuric acid while being stirred vigorously.
The suspension was stirred in a boiling water bath for two hours
and cooled to room temperature. The white suspension was filtered
and the precipitates rinsed with water. The combined filtrate and
washings were concentrated in vacuo to .about.30 ml. Neutralization
with concentrated ammonium hydroxide solution gave white
precipitates which were filtered, washed with water and dried in
vacuo overnight to give crude
1-amino-4,4-diphenylcyclohexane-1-carboxylic acid (0.52 g, 30%
yield) as a white solid. LRMS (Electrospray):
C.sub.19H.sub.21NO.sub.2, calc. 295; observed: 294 (M-H), 296
(M+H).
[0420] Step 3: 179
[0421] A mixture of crude
1-amino-4,4-diphenylcyclohexane-1-carboxylic acid (510 mg, 1.73
mmole), triethylamine (0.37 ml, 2.65 mmole), 9-fluorenylmethyl
succinimidyl carbonate (Fmoc-OSu, 880 mg, 2.61 mmole) in
acetonitrile (25 ml) and water (25 ml) was stirred at room
temperature overnight. TLC analysis of the reaction indicated the
presence of starting material amino acid. 9-fluorenylmethyl
succinimidyl carbonate (200 mg) and acetonitrile (5 ml) were added
and the mixture was stirred at room temperature for another 24
hours. The reaction mixture was concentrated in vacuo to remove
most of the acetonitrile, acidified to pH .about.3 with 10% aqueous
citric acid solution, and the white emulsion extracted three times
with ethyl acetate. The combined organic layers were washed with
water, brine, dried over sodium sulfate. Filtration and
concentration gave a crude oil which was purified by column
chromatography (eluted with 1.fwdarw.4.fwdarw.8% methanol/methylene
chloride) to give Fmoc-1-amino-4,4-diphenylcyclohexane-1-carboxylic
acid (350 mg, 39% yield) as a white solid. HRMS (FAB):
C.sub.34H.sub.32NO.sub.- 4 (M+H) calc. 518.2331; observed:
518.231
EXAMPLE 28
Preparation of Fmoc-1-amino-4-trans-t-butylcyclohexane-1-carboxylic
acid (Fmoc-Abc-OH)
[0422] Step 1: 180
[0423] A mixture of 4-t-butylcyclohexanone (2.00 g, 13.0 mmole),
potassium cyanide (1.27 g, 19.5 mmole), ammonium carbonate (7.48 g,
77.8 mmole), ethanol (60 ml) and water (12 ml) in a sealed, thick
walled pressure flask was heated in a 80.degree. C. oil bath for 15
hours. After cooling to room temperature, the white slurry was
poured into ice-water and stirred at room temperature for a couple
of hours. Filtration gave hydantoin (2.78 g, 96% yield) as a white
solid which was used in the next step as a crude. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.52 (1H, broad, NH), 8.50 (1H, broad s,
NH), 0.81 (9H, s, t-Bu).
[0424] Step 2: 181
[0425] A mixture of hydantoin (2.78 g, 12.4 mmole), barium
hydroxide monohydrate (11.74 g, 62.0 mmole) and water (50 ml) in a
sealed, thick walled pressure flask was heated in a 120.degree. C.
oil bath for 2 days. The reaction mixture was cooled to room
temperature, acidified to .about.pH 3 using 4N sulfuric acid while
being stirred vigorously. The suspension was stirred in a boiling
water bath for one hour and cooled to room temperature. The white
suspension was filtered and the precipitates rinsed with water. The
combined filtrate and washings were concentrated in vacuo to
.about.30 ml. Neutralization with concentrated ammonium hydroxide
solution gave white precipitates which were filtered, washed with
water and dried in vacuo overnight to give
1-amino-4-trans-t-butylcy- clohexane-1-carboxylic acid (2.10 g, 85%
yield) as a white solid.
[0426] Step 3: 182
[0427] A mixture of crude
1-amino-4-trans-t-butylcyclohexyl-1-carboxylic acid (2.10 g, 10.54
mmole), 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 6.33 g,
7.20 mmole) in dioxane (150 ml) and 10% sodium carbonate solution
(120 ml) was stirred at room temperature for 24 hours. The reaction
mixture was concentrated in vacuo to remove most of the dioxane,
acidified to pH .about.3 with 3N HCl, and the white emulsion
extracted twice with methylene chloride. The combined organic
layers were washed with water, brine, dried over magnesium sulfate.
Filtration and concentration gave a crude which was purified by
column chromatography (eluted with 1.fwdarw.4.fwdarw.5%
methanol/methylene chloride) to give
Fmoc-1-amino-4-trans-t-butylcyclohexane-1-carboxylic acid (1.42 g,
32% yield). HRMS (FAB): C.sub.26H.sub.32NO.sub.4 (M+H) calc.
422.2331; observed: 422.23
EXAMPLE 29
Preparation of Fmoc-Linker-BHA Resin
[0428] Benzhydrylamine copolystyrene-1% divinylbenzene cross-linked
resin (10.0 g, 9.3 mequiv, 100-200 ASTM mesh, Advanced ChemTech)
was swelled in 100 mL CH.sub.2Cl.sub.2, filtered and washed
successively with 100 ml each of CH.sub.2Cl.sub.2, 6%
DIPEA/CH.sub.2Cl.sub.2 (two times), CH.sub.2Cl.sub.2 (two times).
The resin was treated with p-[(R,
S)-.alpha.-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]-ph-
enoxyacetic acid (Fmoc-Linker) (7.01 g, 13.0 mmole),
N-hydroxybenzotriazole (2.16 g, 16.0 mmole), and
diisopropylcarbodiimide (2.04 ml, 13.0 mmol) in 100 mL 25%
DMF/CH.sub.2Cl.sub.2 for 24 hours at room temperature. The resin
was filtered and washed successively with 100 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol (two times), DMF, and
CH.sub.2Cl.sub.2 (three times). A Kaiser ninhydrin analysis was
negative. The resin was dried under vacuum to yield 16.12 g of
Fmoc-Linker-BHA resin. A portion of this resin (3.5 mg) was
subjected to Fmoc deprotection and quantitative UV analysis
indicated a loading of 0.56 mmol/g.
EXAMPLE 30
Preparation of Bu-His-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0429] 183
[0430] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-His (Trt) (300 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Bu-Pentapeptide resin.
[0431] The Bu-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 130 mg of an
off-white solid.
[0432] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 52 mg (34%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray C.sub.38H.sub.50N.sub.12O.sub.6 cal: 770 observed:
m/z (771 M+H).
EXAMPLE 31
Penta-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0433] 184
[0434] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin was
filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0435] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0436] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 61 mg (36%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.6, cal: 849
observed: m/z (850 M+H).
EXAMPLE 32
Phenylacetyl-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0437] 185
[0438] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with phenylacetic
acid (82 mg, 0.6 mmole) and HBTU (226 mg, 0.6 mmol) in DMF. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Phenylacetyl-Pentapeptide resin.
[0439] The Phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 155 mg of an
off-white solid.
[0440] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.49H.sub.58N.sub.10O.sub.6, cal: 883
observed: m/z (884 M+H).
EXAMPLE 33
Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0441] 186
[0442] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mal)
and HBTU (226 mg, 0.6 mal), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated n-butyl isocyante
(5 eq) in 6% DIPEA/DMF for 12 hours. The resin was filtered and
washed successively with 20 ml each of CH.sub.2Cl.sub.2 (two
times), isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin
was dried under vacuum to yield 550 mg of Butyl urea-Pentapeptide
resin.
[0443] The Butyl urea-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0444] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal: 864
observed: m/z (865 M+H).
EXAMPLE 34
Preparation of Penta-Apc-(D)Phe-Arg-Trp-NH.sub.2
[0445] 187
[0446] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using Protocol 1 above. All
couplings were performed using HBTU in DMF as the coupling agent
and DIPEA (3 equiv.) as base. Four coupling cycles were performed
of one cycle each with), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226 mg,
0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin was
filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
pentyl-tetrapeptide resin.
[0447] The Pentyl-tetra peptide resin was treated with, 40 .mu.L
dimethylsulfide, 120 .mu.L anisole, and 4 mL trifluoroacetic acid
at room temperature for 180 min. The resin was filtered off, washed
with .about.2 ml TFA and the filtrates precipitated in chilled
ethyl ether. The precipitates were centrifuged and the ether layer
decanted. The residue was washed with two or three volumes of
Et.sub.2O and recentrifuged and the crude product was dried under
vacuum to yield 110 mg of an off-white solid.
[0448] This material was purified by preparative HPLC on a Vydac
C18-column (2.5.times.20 cm) and eluted with a linear gradient of
10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 40 mg ( 25%) of a white
powder. This compound was homogeneous by HPLC. LR-Electrospray
C.sub.44H.sub.57N.sub.9O.sub.5cal: 792 observed: m/z (793 M+H)
EXAMPLE 35
Penta-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0449] 188
[0450] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-2-Nal (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin was
filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0451] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuiged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0452] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 61 mg (36%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.61N.sub.9O.sub.6, cal: 860 observed:
m/z (861 M+H).
EXAMPLE 36
Bu-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0453] 189
[0454] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
acetic anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Butyl Pentapeptide resin
[0455] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 144 mg of an
off-white solid.
[0456] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (32%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.59N.sub.9O.sub.6, cal 846 observed:
m/z (847 M+H).
EXAMPLE 37
Ac-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0457] 190
[0458] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
acetic anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Ac-Pentapeptide resin.
[0459] The Ac-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 150 mg of an
off-white solid.
[0460] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 62 mg (38%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.55N.sub.9O.sub.6, cal: 818 observed:
m/z (819 M+H).
EXAMPLE 38
Bu-Carbamoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0461] 191
[0462] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmo-Gly (180 mg, 0.6 mal) and
HBTU (226 mg, 0.6 mal), Fmoc-(2)Nal (265 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated n-butyl isocyante
(5 eq) in 6% DIPEA/DMF for 12 hours. The resin was filtered and
washed successively with 20 ml each of CH.sub.2Cl.sub.2 (two
times), isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin
was dried under vacuum to yield 550 mg of Butyl
carbamoyl-Pentapeptide resin.
[0463] The Butyl carbamoyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0464] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.62N.sub.10O.sub.6, cal: 875
observed: m/z (876 M+H).
EXAMPLE 39
Benzoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0465] 192
[0466] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmo-Gly (180 mg, 0.6 mal) and
HBTU (226 mg, 0.6 mal), Fmoc-(2)Nal (265 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated benzoic anhydride
in 6% DIPEA/DMF for 12 hours. The resin was filtered and washed
successively with 20 ml each of CH.sub.2Cl.sub.2 (two times),
isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin was
dried under vacuum to yield 570 mg of benzoyl-Pentapeptide
resin.
[0467] The benzoyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 130 mg of an
off-white solid.
[0468] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 50 mg (28%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.50H.sub.57N.sub.9O.sub.6, cal: 880 observed:
m/z (881 M+H).
EXAMPLE 40
3-carboxylpropanoyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0469] 193
[0470] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
succinic acid (71 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in DMF.
The resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 550 mg of
3-carboxypropanoyl-Pentapeptide resin.
[0471] The 3-carboxypropanoyl-Pentapeptide resin was treated with
40 .mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L
anisole, and 4 mL trifluoroacetic acid at room temperature for 180
min. The resin was filtered off, washed with 2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 136 mg of an
off-white solid.
[0472] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 52 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.57N.sub.9O.sub.8, cal: 876 observed:
m/z (877 M+H).
EXAMPLE 41
Phenylacetyl-Apc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0473] 194
[0474] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
inmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
phenylacetic acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in
DMF. The resin was filtered and washed successively with 20 ml each
of CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Phenylacetyl-Pentapeptide resin.
[0475] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuiged and the
crude product was dried under vacuum to yield 132 mg of an
off-white solid.
[0476] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 49 mg (29%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.51H.sub.59N.sub.9O.sub.6, cal: 894 observed:
m/z (895 M+H).
EXAMPLE 42
Penta-4-ClApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0477] 195
[0478] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-ClApc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin.
[0479] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 141 mg of an
off-white solid.
[0480] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 45 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.59N.sub.10O.sub.6Cl, cal: 883
observed: m/z (884 M+H).
EXAMPLE 43
Penta-4-HOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0481] 196
[0482] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-HOApc (280 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin.
[0483] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 150 mg of an
off-white solid.
[0484] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.7, cal: 865
observed: m/z (866 M+H).
EXAMPLE 44
Penta-4-MeOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0485] 197
[0486] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Oly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and LIBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-MeOApc (300 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0487] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L ani sole, and 4
mL trifluoro acetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Bt.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 152 mg of an
off-white solid.
[0488] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 59 mg (33 %) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.7, cal: 879
observed: 880 m/z (M+H).
EXAMPLE 45
Penta-3-MeOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0489] 198
[0490] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-3-MeOApc (300 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol),. The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0491] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 152 mg of an
off-white solid.
[0492] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 59 mg (33 %) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.7, cal: 879
observed: 880 m/z (M+H).
EXAMPLE 46
Penta-4-EtOApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0493] 199
[0494] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-EtOApc (320 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 615 mg of
Pentyl-Pentapeptide resin.
[0495] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 160 mg of an
off-white solid.
[0496] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 63 mg (35 %) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.64N.sub.10O.sub.7, cal: 893
observed: 894 m/z (M+H).
EXAMPLE 47
Penta-4-iPrOApc-(D)Phe-Arg-Trp-Gly
[0497] 200
[0498] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-iPrOApc (285 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0499] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0500] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 45 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.49H.sub.66N.sub.10O.sub.7, cal: 907
observed: m/z (908 M+H).
EXAMPLE 48
Penta-4-MeApc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0501] 201
[0502] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-MeApc (280 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 590 mg of
Pentyl-Pentapeptide resin.
[0503] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with.about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 139 mg of an
off-white solid.
[0504] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 51 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.6, cal: 863
observed: m/z (864 M+H).
EXAMPLE 49
Penta-Apc-(D)Phe-Arg-Trp-Sar-NH.sub.2
[0505] 202
[0506] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Sar (187 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin.
[0507] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 175 mg of an
off-white solid.
[0508] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 69 mg (40%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.6, cal: 863
observed: 864 m/z (M+H).
EXAMPLE 50
Penta-Apc-(D)Phe-Arg-N-methyl (2)Nal-Gly-NH.sub.2
[0509] 203
[0510] Fmoc-Linker-BHA resin (700 mg, 0.385 mmol) synthesized using
the procedure in Example 29 was subjected to solid phase synthesis
using DIC/HOBT coupling conditions and washings were performed as
shown in protocol 1. All amino acid couplings were performed using
DIC (5 eq.), HOBT (2.5 eq.) as the coupling reagents and the
Fmoc-amino acid (2.5 eq.). The resin was subjected to washing steps
1-6 as shown in protocol 1, after each peptide coupling. Two
coupling cycles were performed, one each with Fmoc-Gly (286 mg,
0.96 mmol) followed by Fmoc-(2)Nal (421 mg, 0.96 mmol). After Fmoc
removal from 2-Nal residue, the resulting amine was converted to
it's 2-nitrobenzene sulfonyl derivative using
2-nitrobenzenesulfonyl chloride (5 eq., 426 mg, 1.93 mmol) and
DIPEA (5 eq.) as the base in DMF. Washings were performed using DMF
(6.times.30 ml) followed by CH.sub.2Cl.sub.2 (3.times.30 ml) and
the resin was dried under vacuum. The sulfonamide obtained was
subjected to methylation using triphenylphosphine (5 eq., 505 mg,
1.93 mmol), N,N-diethylazodicarboxylat- e (5 eq., 303 .mu.l, 1.93
mmol) and methanol (10 eq. 156 .mu.l, 3.85 mmol) in THF. Washings
were performed using THF (6.times.30 ml) followed by
CH.sub.2Cl.sub.2 (5.times.30 ml) and the resin was dried under
vacuum. The 2-nitrobenzene sulfonyl group was then removed using
1,8-Diazabicyclo [5.4.0] undec-7-ene (3 eq., 173 .mu.l, 1.16 mmol),
2-mercaptoethanol (5eq. 135 .mu.l, 1.93 mmol) in DMF. Washings were
performed using DMF (3.times.30 ml), isopropanol (3-.times.30 ml)
followed by ethyl ether (3-.times.30 ml) and the resin was dried
under vacuum. The resulting N-Me-(2)Nal residue was subjected to
three coupling cycles, one cycle each with Fmoc-Arg (Pmc) (638 mg,
0.96 mmol), Fmoc-(D)Phe (373 mg, 0.96 mmol) and Fmoc-Apc (170 mg,
0.96 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 300 .mu.l valeric anhydride, 245 .mu.l pyridine in 15 ml DMF
for 5 h. The resin was filtered and washed successively with 30 ml
each of DMF (three times), isopropanol, CH.sub.2Cl.sub.2 (three
times) and ethyl ether (3 times). The resulting pentyl-peptide
resin was dried under vacuum and treated with 7 ml of 60%
trifluoroacetic acid in CH.sub.2Cl.sub.2, 1% water and 615 ml
triethylsilane (10 eq., 3.85 mmol) for 160 minutes. The resin was
filtered off, washed with 5-7 ml CH.sub.2Cl.sub.2, and the
filtrates were concentrated on a Savant speed vacuum pump to yield
the crude product.
[0511] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 30 mg (.about.10%) of a
white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.49H.sub.63N.sub.9O.sub.6, cal: 873 observed:
m/z (874 M+H).
EXAMPLE 51
Penta-Apc-(D)Phe-Arg-N-methyl (2) Nal-NH.sub.2
[0512] 204
[0513] Fmoc-Linker-BHA resin (700 mg, 0.385 mmol) synthesized using
the procedure in Example 29 was subjected to solid phase synthesis
using DIC/HOBT coupling conditions and washings were performed as
shown in protocol 1. All amino acid couplings were performed using
DIC (5 eq.), HOBT (2.5 eq.) as the coupling reagents and the
Fmoc-amino acid (2.5 eq.) The resin was subjected to washing steps
1-6 as shown in protocol 1, after each peptide coupling. One
coupling cycle was performed with Fmoc-(2)Nal (421 mg, 0.96 mmol).
After Fmoc removal from (2)Nal residue, the resulting amine was
converted to it's 2-nitrobenzene sulfonyl derivative using
2-nitrobenzenesulfonyl chloride (5 eq., 426 mg, 1.93 mmol) and
DIPEA (5 eq.) as the base in DMF. Washings were performed using DMF
(6.times.30 ml) followed by CH.sub.2Cl.sub.2 (3.times.30 ml) and
the resin was dried under vacuum. The sulfonamide obtained was
subjected to methylation using triphenylphosphine (5 eq., 505 mg,
1.93 mmol), N,N-diethylazodicarboxylate (5 eq., 303 .mu.l, 1.93
mmol) and methanol (10 eq. 156 .mu.l, 3.85 mmol) in THF. Washings
were performed using THF (6.times.30 ml) followed by
CH.sub.2Cl.sub.2 (5.times.30 ml) and the resin was dried under
vacuum. The 2-nitrobenzene sulfonyl group was then removed using
1,8-diazabicyclo [5.4.0] undec-7-ene (3 eq., 173 .mu.l, 1.16 mmol),
2-mercaptoethanol (5eq. 135 .mu.l, 1.93 mmol) in DMF. Washings were
performed using DMF (3.times.30 ml), isopropanol (3.times.30 ml)
followed by ethyl ether (3.times.30 ml) and the resin was dried
under vacuum. The resulting N-Me-(2)Nal residue was subjected to
three coupling cycles, one cycle each with Fmoc-Arg (Pmc) (638 mg,
0.96 mmol), Fmoc-(D)Phe (373 mg, 0.96 mmol) and Fmoc-Apc (170 mg
0.96 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 300 .mu.l valeric anhydride, 245 .mu.l pyridine in 15 ml DMF
for 5 h. The resin was filtered and washed successively with 30 ml
each of DMF (three times), isopropanol, CH.sub.2Cl.sub.2 (three
times) and ethyl ether (3 times). The resulting pentyl-peptide
resin was dried under vacuum and treated with 7 ml of 60%
trifluoroacetic acid in CH.sub.2Cl.sub.2, 1% water and 615 ml
triethylsilane (10 eq., 3.85 mmol) for 160 minutes. The resin was
filtered off, washed with .about.5-7 ml CH.sub.2Cl.sub.2, and the
filtrates were concentrated on a Savant speed vacuum pump to yield
the crude product.
[0514] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 43 mg (14%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.60N.sub.8O.sub.5, cal: 817 observed:
m/z (818 M+H).
EXAMPLE 52
Penta-Apc-(D)Phe-Arg-N-methylTrp-Gly-NH.sub.2
[0515] 205
[0516] Fmoc-Linker-BHA resin (700 mg, 0.385 mmol) synthesized using
the procedure in Example 29 was subjected to solid phase synthesis
using DIC/HOBT coupling conditions and washings were performed as
shown in the protocol 1. All amino acid couplings were performed
using DIC (5 eq.), HOBT (2.5 eq.) as the coupling reagents and the
Fmoc-amino acid (2.5 eq.) The resin was subjected to washing steps
1-6 as shown in protocol 1, after each peptide coupling. Two
coupling cycles were performed, one each with Fmoc-Gly (286 mg,
0.96 mmol) followed by Fmoc-Trp (461 mg, 0.96 mmol). After Fmoc
removal from Trp residue, the resulting amine was converted to its
2-nitrobenzene sulfonyl derivative using 2-nitrobenzenesulfonyl
chloride (5 eq., 426 mg, 1.93 mmol) and DIPEA (5 eq.) as the base
in DMF. Washings were performed using DMF (6.times.30 ml) followed
by CH.sub.2Cl.sub.2 (3.times.30 ml) and the resin was dried under
vacuum. The sulfonamide obtained was subjected to methylation using
triphenylphosphine (5 eq., 505 mg, 1.93 mmol),
N,N-diethylazodicarboxylat- e (5 eq., 303 .mu.l, 1.93 mmol) and
methanol (10 eq. 156 .mu.l, 3.85 mmol) in THF. Washings were
performed using THF (6.times.30 ml) followed by CH.sub.2Cl.sub.2
(5.times.30 ml) and the resin was dried under vacuum. The
2-nitrobenzene sulfonyl group was then removed using
1,8-diazabicyclo [5.4.0] undec-7-ene (3 eq., 173 .mu.l, 1.16 mmol),
2-mercaptoethanol (5eq. 135 .mu.l, 1.93 mmol) in DMF. Washings were
performed using DMF (3.times.30 ml), isopropanol (3-.times.30 ml)
followed by ethyl ether (3-.times.30 ml) and the resin was dried
under vacuum. The resulting N-MeTrp residue was subjected to three
coupling cycles, one cycle each with Fmoc-Arg (Pmc) (638 mg, 0.96
mmol), Fmoc-(D)Phe (373 mg, 0.96 mmol) and Fmoc-Apc (170 mg 0.96
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 300
.mu.l valeric anhydride, 245 .mu.l pyridine in 15 ml DMF for 5 h.
The resin was filtered and washed successively with 30 ml each of
DMF (three times), isopropanol, CH.sub.2Cl.sub.2 (three times) and
ethyl ether (3 times). The resulting pentyl-peptide resin was dried
under vacuum and treated with 7 ml of 60% trifluoroacetic acid in
CH.sub.2Cl.sub.2, 1% water and 615 ml triethylsilane (10 eq., 3.85
mmol) for 160 minutes. The resin was filtered off, washed with
.about.5-7 ml CH.sub.2Cl.sub.2, and the filtrates were concentrated
on a Savant speed vacuum pump to yield the crude product.
[0517] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 30 mg (.about.10%) of a
white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.6- , cal: 863
observed: m/z (864 M+H).
EXAMPLE 53
Penta-Apc-(D)Phe-Arg-N-methylTrp-NH.sub.2
[0518] 206
[0519] Fmoc-Linker-BHA resin (700 mg, 0.385 mmol) synthesized using
the procedure in Example 29 was subjected to solid phase synthesis
using DIC/HOBT coupling conditions and washings were performed as
shown in the protocol 1. All amino acid couplings were performed
using DIC (5 eq.), HOBT (2.5 eq.) as the coupling reagents and the
Fmoc-amino acid (2.5 eq.) The resin was subjected to washing steps
1-6 as shown in protocol 1, after each peptide coupling. One
coupling cycle was performed with Fmoc-Trp (461 mg, 0.96 mmol).
After Fmoc removal from Trp residue, the resulting amine was
converted to it's 2-nitrobenzene sulfonyl derivative using
2-nitrobenzenesulfonyl chloride (5 eq., 426 mg, 1.93 mmol) and
DIPEA (5 eq.) as the base in DMF. Washings were performed using DMF
(6.times.30 ml) followed by CH.sub.2Cl.sub.2 (3.times.30 ml) and
the resin was dried under vacuum. The sulfonamide obtained was
subjected to methylation using triphenylphosphine (5 eq., 505 mg,
1.93 mmol), N,N-diethylazodicarboxylate (5 eq., 303 .mu.l, 1.93
mmol) and methanol (10 eq. 156 .mu.l, 3.85 mmol) in THF. Washings
were performed using THF (6.times.30 ml) followed by
CH.sub.2Cl.sub.2 (5.times.30 ml) and the resin was dried under
vacuum. The 2-nitrobenzene sulfonyl group was then removed using
1,8-diazabicyclo [5.4.0] undec-7-ene (3 eq., 173 .mu.l, 1.16 mmol),
2-mercaptoethanol (5eq. 135 .mu.l, 1.93 mmol) in DMF. Washings were
performed using DMF (3.times.30 ml), isopropanol (3.times.30 ml)
followed by ethyl ether (3.times.30 ml) and the resin was dried
under vacuum. The resulting N-MeTrp residue was subjected to three
coupling cycles, one cycle each with Fmoc-Arg (Pmc) (638 mg, 0.96
mmol), Fmoc-(D)Phe (373 mg, 0.96 mmol) and Fmoc-Apc (170 mg 0.96
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 300
.mu.l valeric anhydride, 245 .mu.l pyridine in 15 ml DMF for 5 h.
The resin was filtered and washed successively with 30 ml each of
DMF (three times), isopropanol, CH.sub.2Cl.sub.2 (three times) and
ethyl ether (3 times). The resulting pentyl-peptide resin was dried
under vacuum and treated with 7 ml of 60% trifluoroacetic acid in
CH.sub.2Cl.sub.2, 1% water and 615 ml triethylsilane (10 eq., 3.85
mmol) for 160 minutes. The resin was filtered off, washed with 5-7
ml CH.sub.2Cl.sub.2, and the filtrates were concentrated on a
Savant speed vacuum pump to yield the crude product.
[0520] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 43 mg (14%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.59N.sub.9O.sub.5, cal: 806 observed:
m/z (807 M+H).
EXAMPLE 54
Bu-Apc-(D)Phe-Arg-Trp-Ala-NH.sub.2
[0521] 207
[0522] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Ala (187 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL butyric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin was
filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
butyl-Pentapeptide resin.
[0523] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0524] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 61 mg (36%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.6, cal: 849
observed: m/z (850 M+H).
EXAMPLE 55
Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-Ala-NH.sub.2
[0525] 208
[0526] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Ala (187 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with n-butyl
isocyanate (5 equ.) in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes.
The resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Bu-carbamoyl Pentapeptide resin.
[0527] The Bu-carbamoyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 143 mg of an
off-white solid.
[0528] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 65 mg (37%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.63N.sub.11O.sub.6, cal: 878
observed: m/z (879 M+H).
EXAMPLE 56
Phenylacetyl-Apc-(D)Phe-Arg-Trp-Ala-NH.sub.2
[0529] 209
[0530] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Ala (187 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with phenylacetic
acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in DMF. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
phenylacetyl-Pentapeptide resin.
[0531] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 138 mg of an
off-white solid.
[0532] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 53 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.50H.sub.60N.sub.10O.sub.6, cal: 897
observed: m/z (898 M+H).
EXAMPLE 57
Bu-Apc-(D)Phe-Arg-Trp-.beta.-Ala-NH.sub.2
[0533] 210
[0534] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-.beta.-Ala (186 mg, 0.6
mal) and HBTU (226 mg, 0.6 mal), Fmoc-Trp (260 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
butyric anhydride in 6% DIPEA/DMF for 12 hours. The resin was
filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 550 mg of
Butyl-Pentapeptide resin.
[0535] The Butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0536] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (32%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.6, cal: 848
observed: m/z (850 M+H).
EXAMPLE 58
Bu-Carbamoyl-Apc-(D)Phe-Arg-Trp-.beta.-Ala-NH.sub.2
[0537] 211
[0538] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-p-Ala (186 mg, 0.6 mal)
and HBTU (226 mg, 0.6 mal), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated n-butyl isocyanate
(5eq) in 6% DIPEA/DMF for 12 hours. The resin was filtered and
washed successively with 20 ml each of CH.sub.2Cl.sub.2 (two
times), isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin
was dried under vacuum to yield 550 mg of Butyl
carbamoyl-Pentapeptide resin.
[0539] The Butyl carbamoyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0540] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.63N.sub.11O.sub.6, cal: 878
observed: m/z (879 M+H).
EXAMPLE 59
Phenylacetyl-Apc-(D)Phe-Arg-Trp-.beta.-Ala-NH.sub.2
[0541] 212
[0542] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-.beta.-Ala (186 mg, 0.6
mal) and HBTU (226 mg, 0.6 mal), Fmoc-Trp (260 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
phenylacetic acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in
DMF. The resin was filtered and washed successively with 20 ml each
of CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 550 mg of
phenylacetyl-Pentapeptide resin.
[0543] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 129 mg of an
off-white solid.
[0544] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 49 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.50H.sub.60N.sub.10O.sub.6, cal: 897
observed: m/z (898 M+H).
EXAMPLE 60
Bu-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0545] 213
[0546] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL butyric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
butyl-Pentapeptide resin.
[0547] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0548] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 47 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.50H.sub.60N.sub.10O.sub.6, cal: 897
observed: m/z (898 M+H).
EXAMPLE 61
Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0549] 214
[0550] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with n-butyl
isocyanate (5eq) in 6% DIPEA/CH.sub.2Cl.sub.2 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
butyl-carbamoyl Pentapeptide resin.
[0551] The butyl-carbamoyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 152 mg of an
off-white solid.
[0552] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.51H.sub.63N.sub.10O.sub.6, cal: 926
observed: m/z (927 M+H).
EXAMPLE 62
Phenylacetyl-Apc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0553] 215
[0554] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with phenylacetic
acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in DMF. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 615 mg of
phenylacetyl-Pentapeptide resin.
[0555] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 142 mg of an
off-white solid.
[0556] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 53 mg (29%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.54H.sub.60N.sub.10O.sub.6, cal: 945
observed: m/z (955 M+H).
EXAMPLE 63
Bu-Apc-(D)Phe-Arg-Trp-3-Amb-NH.sub.2
[0557] 216
[0558] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-3-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL of
butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 590 mg of
butyl-Pentapeptide resin.
[0559] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0560] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 50 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.51H.sub.62N.sub.10O.sub.6, cal: 911
observed: m/z (912 M+H).
EXAMPLE 64
Bu-carbamoyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH.sub.2
[0561] 217
[0562] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-3-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with n-butyl
isocyanate (5eq) in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
butyl-carbamoyl Pentapeptide resin.
[0563] The butyl-carbamoyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 143 mg of an
off-white solid.
[0564] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 53 mg (28%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.52H.sub.65N.sub.10O.sub.6, cal: 940
observed: m/z (941 M+H).
EXAMPLE 65
Phenylacetyl-Apc-(D)Phe-Arg-Trp-3-Amb-NH.sub.2
[0565] 218
[0566] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-3-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with phenylacetic
acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in DMF. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
phenylacetyl-Pentapeptide resin.
[0567] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0568] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 49 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.55H.sub.62N.sub.10O.sub.6, cal: 959
observed: m/z (960 M+H).
EXAMPLE 66
Bu-Apc-(D)Phe-Arg-Trp-4-Amb-NH.sub.2
[0569] 219
[0570] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-4-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL butyric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 615 mg of
butyl-Pentapeptide resin.
[0571] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 153 mg of an
off-white solid.
[0572] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.51H.sub.62N.sub.10O.sub.6, cal: 911
observed: m/z (912 M+H).
EXAMPLE 67
Phenylacetyl-Apc-(D)Phe-Arg-Trp-4-Amb-NH.sub.2
[0573] 220
[0574] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-4-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with phenylacetic
acid (82 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol) in DMF. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 585 mg of
phenylacetyl-Pentapeptide resin.
[0575] The phenylacetyl-Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 142 mg of an
off-white solid.
[0576] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 47 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.55H.sub.62N.sub.10O.sub.6, cal: 959
observed: m/z (960 M+H).
EXAMPLE 68
Bu-Apc-(D)Phe-Arg-(2)Nal-Ala-NH.sub.2
[0577] 221
[0578] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Ala (187 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
of butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes.
The resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Butyl Pentapeptide resin.
[0579] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 149 mg of an
off-white solid.
[0580] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 57 mg (33%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.61N.sub.9O.sub.6, cal 860 observed:
m/z (861 M+H).
EXAMPLE 69
Bu-Apc-(D)Phe-Arg-(2)Nal-beta-Ala-NH.sub.2
[0581] 222
[0582] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-beta-Ala (187 mg, 0.6
mmol) and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg,
0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 1 mL Butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30
minutes. The resin was filtered and washed successively with 20 ml
each of CH.sub.2Cl.sub.2 (two times), isopropanol, and
CH.sub.2Cl.sub.2 (three times). The resin was dried under vacuum to
yield 605 mg of Butyl Pentapeptide resin.
[0583] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 142 mg of an
off-white solid.
[0584] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 54 mg (32%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.61N.sub.9O6, cal 860 observed: m/z
(861 M+H).
EXAMPLE 70
Bu-Apc-(D)Phe-Arg-(2)Nal-3-Amb-NH.sub.2
[0585] 223
[0586] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-3-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Ape (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
Butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 550 mg of
Butyl Pentapeptide resin.
[0587] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 125 mg of an
off-white solid.
[0588] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 44 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.53H.sub.63N.sub.9O.sub.6, cal 922 observed:
m/z (923 M+H).
EXAMPLE 71
Bu-Apc-(D)Phe-Arg-(2)Nal-2-Aba-NH.sub.2
[0589] 224
[0590] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the0
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 510 mg of
Butyl Pentapeptide resin.
[0591] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 114 mg of an
off-white solid.
[0592] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 36 mg (20%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.52H.sub.61N.sub.9O.sub.6, cal 908 observed:
m/z (909 M+H).
EXAMPLE 72
Bu-Apc-(D)Phe-Arg-(2)Nal-4-Amb-NH.sub.2
[0593] 225
[0594] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-4-Amb (230 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Butyl Pentapeptide resin.
[0595] The butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 139 mg of an
off-white solid.
[0596] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 56 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.53H.sub.63N.sub.9O.sub.6, cal 922 observed:
m/z (923 M+H).
EXAMPLE 73
Penta-Apc-(D)Phe-acylguanidine-Trp-Gly-NH.sub.2
[0597] 226
[0598] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Glu(allyl) (250 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield
Pentyl-Pentapeptide resin.
[0599] The allyl protecting group was removed using
PdCl.sub.2/Triphenyl-phosphine/tributyltin hydride under Argon in
DMF. The guanidinylation was achieved using Boc-Guanidine. HCl (100
mg, 0.6 mmol ) and HBTU (226 mg, 0.6 mmol).
[0600] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0601] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 30 mg (15%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.58N.sub.10O.sub.7, cal 977 observed:
m/z (978 M+H).
EXAMPLE 74
Bu-Apc-(D)Phe-PhenylhomoArg-Trp-Gly-NH2
[0602] 227
[0603] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Phenyl homo Arg (295 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg,
0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 1 mL butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30
minutes. The resin was filtered and washed successively with 20 ml
each of CH.sub.2Cl.sub.2 (two times), isopropanol, and
CH.sub.2Cl.sub.2 (three times). The resin was dried under vacuum to
yield 570 mg of Butyl-Pentapeptide resin.
[0604] The Butyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0605] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 54 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.52H.sub.64N.sub.10O.sub.6, cal 925 observed:
m/z (926 M+H).
EXAMPLE 75
Penta-Apc-(D)Phe-Cit-Trp-Gly-NH.sub.2
[0606] 228
[0607] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Cit (240 mg, 0.6 mmol) and HBTU (226 mg,
0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Apc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 590 mg of
Pentyl-Pentapeptide resin.
[0608] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 152 mg of an
off-white solid.
[0609] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 65 mg (38%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.56N.sub.9O.sub.7, cal: 850 observed:
m/z (851 M+H).
EXAMPLE 76
Penta-Adpc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0610] 229
[0611] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Adpc (320 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0612] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 142 mg of an
off-white solid.
[0613] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 47 mg (26%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.52H.sub.64N.sub.10O.sub.6, cal: 925
observed: m/z (926 M+H).
EXAMPLE 77
Penta-Ape-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 1)
[0614] 230
[0615] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Ape (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0616] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0617] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The first main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 25 mg (15%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.58N.sub.10O.sub.6, cal: 847
observed: m/z (948 M+H).
EXAMPLE 78
Penta-Ape-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 2)
[0618] 231
[0619] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Ape (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0620] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0621] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The second main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 22 mg (14%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.58N.sub.10O.sub.6, cal: 847
observed: m/z (948 M+H).
EXAMPLE 79
Penta-Abc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0622] 232
[0623] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Abc (270 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0624] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA, and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 155 mg of an
off-white solid.
[0625] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 61 mg (36%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.44H.sub.64N.sub.10O.sub.06, cal: 829
observed: m/z (830 M+H).
EXAMPLE 80
Penta-Achc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0626] 233
[0627] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Achc (278 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0628] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0629] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 65 mg (38%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.66N.sub.10O.sub.6, cal: 855
observed: m/z (856 M+H).
EXAMPLE 81
Preparation of Bu-Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0630] 234
[0631] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using Protocol 1 above. All
couplings were performed using HBTU in DMF as the coupling agent
and DIPEA (3 equiv.) as base. Five coupling cycles were performed
of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6 mmol),
Fmoc-(D,L)Atc (252 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL butyric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 550 mg of
Bu-Pentapeptide resin.
[0632] The Bu-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 110 mg of an
off-white solid.
[0633] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280
.mu.m. The second main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 42 mg (26%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.43H.sub.54N.sub.10O.sub.6, cal 807 observed:
m/z (808 M+H).
EXAMPLE 82
Penta-5-Br-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0634] 235
[0635] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-Br-(D,L)Atc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0636] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 135 mg of an
off-white solid.
[0637] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 45 mg (25%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.44H.sub.55N.sub.10O.sub.6Br, cal 900
observed: m/z (901 M+H).
EXAMPLE 83
Penta-5-Br-Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 1)
[0638] 236
[0639] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D,L)5-BrAtc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 590 mg of
Pentyl-Pentapeptide resin.
[0640] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 130 mg of an
off-white solid.
[0641] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The first main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 40 mg (22%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.44H.sub.55N.sub.10O.sub.6Br, cal 900
observed: m/z (901 M+H).
EXAMPLE 84
Penta-5-BrAtc-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 2)
[0642] 237
[0643] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0644] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0645] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The second main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 55 mg (30%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.44H.sub.55N.sub.10O.sub.6Br, cal 900
observed: m/z (901 M+H).
EXAMPLE 85
Penta-5-Cl-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0646] 238
[0647] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-ClAtc (290 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin.
[0648] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 150 mg of an
off-white solid.
[0649] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 48 mg (28%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.44H.sub.55N.sub.10O.sub.6Cl, cal 855
observed: m/z (856 M+H).
EXAMPLE 86
Penta-5-MeO-(D,L)Atc-(D)Phe-Arg-Trp-Gly -NH.sub.2
[0650] 239
[0651] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-MeO(D,L)Atc (300 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0652] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 155 mg of an
off-white solid.
[0653] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 46 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.58N.sub.10O.sub.7, cal 851 observed:
m/z (852 M+H).
EXAMPLE 87
Penta-5-EtO-(D,L)Atc-(D)Phe-Arg-Trp-Gly -NH.sub.2
[0654] 240
[0655] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-EtO(D,L)Atc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 594 mg of
Pentyl-Pentapeptide resin.
[0656] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0657] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 41 mg (24%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.7, cal 865 observed:
m/z (866 M+H).
EXAMPLE 88
Penta-5-iPrO-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0658] 241
[0659] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling * cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-iPrO(D,L)Atc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0660] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 142 mg of an
off-white solid.
[0661] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 43 mg (25%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.7, cal 879 observed:
m/z (880 M+H).
EXAMPLE 89
Penta-5-Me-(D,L)Atc-(D)Phe-Arg-Trp-Gly -NH.sub.2
[0662] 242
[0663] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-Me(D,L)Atc (290 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol).The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0664] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 143 of an off-white
solid.
[0665] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 40 mg (24%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.58N.sub.10O.sub.6, cal 835 observed:
m/z (836 M+H).
EXAMPLE 90
Penta-5-Et-(D,L)Atc-(D)Phe-Arg-Trp-Gly -NH.sub.2
[0666] 243
[0667] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-Et(D,L)Atc (285 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin
[0668] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 154 mg of an
off-white solid.
[0669] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 53 mg (31%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.60N.sub.10O.sub.6, cal 849 observed:
m/z (850 M+H).
EXAMPLE 91
Penta-5-iPr-(D,L)Atc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0670] 244
[0671] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-iPr(D,L)Atc (300 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0672] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 149 mg of an
off-white solid.
[0673] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 47 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.62N.sub.10O.sub.6, cal 863 observed:
m/z (864 M+H).
EXAMPLE 92
Penta-5-DmaAtc-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 1)
[0674] 245
[0675] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-Dma(D,L)Atc (300 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0676] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 149 of an off-white
solid.
[0677] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The first main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 22 mg (13%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal 864 observed:
m/z (865 M+H).
EXAMPLE 93
Penta-5-DmaAtc-(D)Phe-Arg-Trp-Gly-NH.sub.2 (peak 2)
[0678] 246
[0679] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-5-Dma(D,L)Atc (300 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0680] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 149 of an off-white
solid.
[0681] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The second main peak was cut by analytical HPLC analysis of
collected fractions, pooled and lyophilized to yield 27 mg (16%) of
a white, amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal 864 observed:
m/z (865 M+H).
EXAMPLE 94
Bu-(D,L)5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0682] 247
[0683] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D,L) 5-BrAtc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 ml
of butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes.
The resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
butyl Pentapeptide resin.
[0684] The butyl Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 141 mg of an
off-white solid.
[0685] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 35 mg (19%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.55N.sub.10O.sub.6Br, cal: 948
observed: m/z (949 M+H).
EXAMPLE 95
Bu-carbamoyl-(D,L)-5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0686] 248
[0687] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
n-butyl isocyanate (5 eq) in 6% DIPEA/CH.sub.2Cl.sub.2 for 12 hours
. The resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
butyl carbamoyl-Pentapeptide resin.
[0688] The butyl-carbamoyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 153 mg of an
off-white solid.
[0689] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 41 mg (21%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.49H.sub.58N.sub.11O.sub.6Br, cal: 977
observed: m/z (978 M+H).
EXAMPLE 96
Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-Trp-2-Aba-NH.sub.2
[0690] 249
[0691] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with
phenylacetic acid, HBTU in DMF. The resin was filtered and washed
successively with 20 ml each of CH.sub.2Cl.sub.2 (two times),
isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin was
dried under vacuum to yield 610 mg of Phenylacetyl Pentapeptide
resin.
[0692] The phenylacetyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 148 mg of an
off-white solid.
[0693] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 38 mg (19%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.52H.sub.55N.sub.10O.sub.6Br, cal: 996
observed: m/z (997 M+H).
EXAMPLE 97
Penta-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0694] 250
[0695] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226
mg, 0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 1 mL valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30
minutes. The resin was filtered and washed successively with 20 ml
each of CH.sub.2Cl.sub.2 (two times), isopropanol, and
CH.sub.2Cl.sub.2 (three times). The resin was dried under vacuum to
yield 620 mg of Pentyl-Pentapeptide resin.
[0696] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 162 mg of an
off-white solid.
[0697] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 60 mg (33%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.56N.sub.9O.sub.6Br , cal: 911
observed: m/z (912 M+H).
EXAMPLE 98
3-carboxylpropanoyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH2
[0698] 251
[0699] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226
mg, 0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with succinic acid, HBTU in DMF. The resin was filtered and washed
successively with 20 ml each of CH.sub.2Cl.sub.2 (two times),
isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin was
dried under vacuum to yield 610 mg of
3-carboxylpropanoyl-Pentapeptide resin.
[0700] The 3-carboxylpropanoyl Pentapeptide resin was treated with
40 .mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L
anisole, and 4 mL trifluoroacetic acid at room temperature for 180
min. The resin was filtered off, washed with 2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 158 mg of an
off-white solid.
[0701] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.52N.sub.9O.sub.8Br, cal: 927
observed: m/z (928 M+H).
EXAMPLE 99
Phenylacetyl-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0702] 252
[0703] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226
mg, 0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with phenyl acetic acid, HBTU in DMF. The resin was filtered and
washed successively with 20 ml each of CH.sub.2Cl.sub.2 (two
times), isopropanol, and CH.sub.2Cl.sub.2 (three times). The resin
was dried under vacuum to yield 600 mg of phenylacetyl Pentapeptide
resin.
[0704] The phenylacetyl Pentapeptide resin was treated with 40
.mu.L ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole,
and 4 mL trifluoroacetic acid at room temperature for 180 min. The
resin was filtered off, washed with .about.2 ml TFA and the
filtrates precipitated in chilled ethyl ether. The precipitates
were centrifuged and the ether layer decanted. The residue was
washed with two or three volumes of Et.sub.2O and recentrifuged and
the crude product was dried under vacuum to yield 161 mg of an
off-white solid.
[0705] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 58 mg (30%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.49H.sub.54N.sub.9O.sub.6Br, cal: 945
observed: m/z (946 M+H).
EXAMPLE 100
Bu-(D,L)-5-BrAtc-(D)Phe-Arg-(2)Nal-2-Aba-NH.sub.2
[0706] 253
[0707] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-2-Aba (215 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D,L)-5-BrAtc (310 mg 0.6 mmol) and HBTU (226
mg, 0.6 mmol). The peptide resin was carried through steps 1-5 of
protocol 1, washed with CH.sub.2Cl.sub.2 (three times) and treated
with 1 ml of butyric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30
minutes. The resin was filtered and washed successively with 20 ml
each of CH.sub.2Cl.sub.2 (two times), isopropanol, and
CH.sub.2Cl.sub.2 (three times). The resin was dried under vacuum to
yield 590 mg of butyl Pentapeptide resin.
[0708] The butyl Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0709] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 30 mg (16%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.50H.sub.56N.sub.9O.sub.6Br, cal: 959
observed: m/z (960 M+H).
EXAMPLE 101
Penta-Appc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0710] 254
[0711] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-Appc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol). The
peptide resin was carried through steps 1-5 of protocol 1, washed
with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL valeric
anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The resin
was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 620 mg of
Pentyl-Pentapeptide resin.
[0712] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 153 mg of an
off-white solid.
[0713] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 65 mg (38%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.59N.sub.11O.sub.6, cal: 850
observed: m/z (851 M+H).
EXAMPLE 102
Penta-Appc-(D)Phe-Arg-(2)Nal-Gly-NH.sub.2
[0714] 255
[0715] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-(2)Nal (265 mg, 0.6 mmol) and
HBTU (226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-Appc (275 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0716] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0717] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (32%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.47H.sub.60N.sub.10O.sub.6, cal: 861
observed: m/z (862 M+H).
EXAMPLE 103
Penta-2-MeAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0718] 256
[0719] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-2-MeAppc (285 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0720] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 145 mg of an
off-white solid.
[0721] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 59 mg (35%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal: 864
observed: m/z (865 M+H).
EXAMPLE 104
Penta-2-iPrAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0722] 257
[0723] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-2-iPrAppc (295 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0724] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 147 mg of an
off-white solid.
[0725] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 49 mg (27%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.48H.sub.65N.sub.11O.sub.6, cal: 892
observed: m/z (893 M+H).
EXAMPLE 105
Penta-3-MeAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0726] 258
[0727] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-3-MeAppc (285 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 595 mg of
Pentyl-Pentapeptide resin.
[0728] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0729] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 55 mg (32%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal: 864
observed: m/z (865 M+H).
EXAMPLE 106
Penta-3-MeOAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0730] 259
[0731] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-3-MeOAppc (290 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0732] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 154 mg of an
off-white solid.
[0733] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 50 mg (29%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.7, cal: 880
observed: m/z (881 M+H).
EXAMPLE 107
Penta-4-MeAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0734] 260
[0735] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-MeAppc (285 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 600 mg of
Pentyl-Pentapeptide resin.
[0736] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 150 mg of an
off-white solid.
[0737] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 57 mg (33%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.46H.sub.61N.sub.11O.sub.6, cal: 864
observed: m/z (865 M+H).
EXAMPLE 108
Penta-4-ClAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0738] 261
[0739] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-ClAppc (290 mg 0.6 mmol) and HBTU (226 mg, 0.6 mmol).
The peptide resin was carried through steps 1-5 of protocol 1,
washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 580 mg of
Pentyl-Pentapeptide resin.
[0740] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with .about.2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 140 mg of an
off-white solid.
[0741] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 49 mg (28%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.45H.sub.58N.sub.11O.sub.6Cl, cal: 884
observed: m/z (885 M+H).
EXAMPLE 109
Penta-4-PhOAppc-(D)Phe-Arg-Trp-Gly-NH.sub.2
[0742] 262
[0743] Fmoc-Linker-BHA resin (360 mg, 0.2 mmol) from Example 29
were subjected to solid phase synthesis using protocol 1 described
above. All couplings were performed using HBTU in DMF as the
coupling agent and DIPEA (3 equiv.) as base. Five coupling cycles
were performed of one cycle each with Fmoc-Gly (180 mg, 0.6 mmol)
and HBTU (226 mg, 0.6 mmol), Fmoc-Trp (260 mg, 0.6 mmol) and HBTU
(226 mg, 0.6 mmol), Fmoc-Arg (Pmc) (400 mg, 0.6 mmol) and HBTU (226
mg, 0.6 mmol), Fmoc-(D)Phe (240 mg, 0.6 mmol) and HBTU (226 mg, 0.6
mmol), Fmoc-4-PhOAppc (325 mg 0.6 mmol) and HBTU (226 mg, 0.6
mmol). The peptide resin was carried through steps 1-5 of protocol
1, washed with CH.sub.2Cl.sub.2 (three times) and treated with 1 mL
valeric anhydride in 6% DIPEA/CH.sub.2Cl.sub.2 for 30 minutes. The
resin was filtered and washed successively with 20 ml each of
CH.sub.2Cl.sub.2 (two times), isopropanol, and CH.sub.2Cl.sub.2
(three times). The resin was dried under vacuum to yield 610 mg of
Pentyl-Pentapeptide resin.
[0744] The Pentyl-Pentapeptide resin was treated with 40 .mu.L
ethanedithiol, 40 .mu.L dimethylsulfide, 120 .mu.L anisole, and 4
mL trifluoroacetic acid at room temperature for 180 min. The resin
was filtered off, washed with 2 ml TFA and the filtrates
precipitated in chilled ethyl ether. The precipitates were
centrifuged and the ether layer decanted. The residue was washed
with two or three volumes of Et.sub.2O and recentrifuged and the
crude product was dried under vacuum to yield 143 mg of an
off-white solid.
[0745] This crude material was purified by preparative HPLC on a
Vydac C18-column (2.5.times.20 cm) and eluted with a linear
gradient of 10-60% B (buffer A: 0.1% TFA/H.sub.2O, buffer B: 0.1%
TFA/CH.sub.3CN) in 60 min., flow rate 8 ml/min, detection 280 nm.
The main peak was cut by analytical HPLC analysis of collected
fractions, pooled and lyophilized to yield 41 mg (22%) of a white,
amorphous powder. This compound was homogeneous by HPLC.
LR-Electrospray: C.sub.15H.sub.63N.sub.11O.sub.7, cal: 942
observed: m/z (943 M+H).
BIOLOGICAL ACTIVITY EXAMPLE
EXAMPLE A
Agonist Assay
[0746] Method
[0747] Description: 293 cells transfected with either the MC-4
receptor or MC-1 receptor were grown in 96 well plates. The cells
were stimulated with either 100 nM NDP-.alpha.MSH or screening
compounds. Cyclic AMP was extracted from the cells and
concentrations were determined using a Biotrak-cAMP SPA assay.
Agonists were identified as those compounds causing an increase in
cAMP.
[0748] Cell Culture: 293MC4 cells (obtained from Dr. Wei Gu,
Millenium) were cultured in 75 cm.sup.2flasks in D-MEM supplemented
with 10% FCS and 500 .mu.g/ml G418. Cells were trypsinized and
split 1:3 into 96 well flat-bottom tissue culture treated plates.
Cells were stimulated at confluence (day 2-4).
[0749] cAMP Response: Compounds serially diluted in 100% DMSO were
further diluted 1:200 (2.5.mu.l compound dilution +500 .mu.l media)
in D-MEM containing 10% FBS and 0.1 mM IBMX. For unstimulated
cells, 2.5 .mu.l of DMSO was added to 500 .mu.l of media. For
NDP-.alpha.MSH stimulated cells, 2.5 .mu.l of 20 .rho.M
NDP-.alpha.MSH in 100% DMSO was added to 500 .mu.l of media (final
conc. 100 nM).
[0750] Final concentration of DMSO in all wells was 0.5%.
[0751] Note: Each sample was run in duplicate on separate
plates
[0752] Culture medium was removed from confluent 96 well culture
plates and replaced with 200 .mu.l of above dilutions into the
appropriate wells . The plates were incubated for 1 hr at RT. The
media was removed, and the plates were washed 1.times. with 200
.sup..mu.l well of PBS. CAMP was extracted by the addition of 60
.mu.l 70% ethanol (stored in the refrigerator). After a 30 min
extraction period, 10 .mu.l ethanol extract was transferred to the
cAMP assay plate or samples were stored at -20.degree. C. until the
cAMP assay was performed.
[0753] cAMP Assay: The extracted samples and all reagents included
in the kit were brought to room temperature. To a 96 well
OptiPlate, 10 .mu.l ethanol extract, 40 .mu.l assay buffer, 50 ul
[1251]cAMP, 50 .mu.l antiserum and 50 .mu.l SPA beads were added.
The total well volume after addition was 200 .mu.l. The plates were
sealed and incubated for 15-20 hr at room temperature. [125I]cAMP
binding to the SPA beads was determined by counting each plate for
2 minutes on a Packard TopCount.TM..
[0754] Note: Each plate contained samples of controls for
unstimulated cells and NDP-.alpha.MSH for stimulated cells.
* * * * *