U.S. patent application number 10/137119 was filed with the patent office on 2003-03-20 for guanidinylation reagents.
This patent application is currently assigned to The Regents of The University of California. Invention is credited to Feichtinger, Konrad, Goodman, Murray, Romoff, Todd T..
Application Number | 20030055214 10/137119 |
Document ID | / |
Family ID | 25337171 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030055214 |
Kind Code |
A1 |
Goodman, Murray ; et
al. |
March 20, 2003 |
Guanidinylation reagents
Abstract
Trisubstituted N-protected guanidines and methods for use as
guanidinylating reagents to yield N-protected guanidine
derivatives.
Inventors: |
Goodman, Murray; (La Jolla,
CA) ; Feichtinger, Konrad; (San Diego, CA) ;
Romoff, Todd T.; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
The Regents of The University of
California
|
Family ID: |
25337171 |
Appl. No.: |
10/137119 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10137119 |
Apr 30, 2002 |
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09452895 |
Dec 1, 1999 |
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6380358 |
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09452895 |
Dec 1, 1999 |
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08861942 |
May 22, 1997 |
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6072075 |
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Current U.S.
Class: |
530/332 ;
560/158 |
Current CPC
Class: |
C07K 1/006 20130101;
Y02P 20/55 20151101; C07K 1/064 20130101; C07C 279/24 20130101;
C07C 279/14 20130101; C07C 311/64 20130101 |
Class at
Publication: |
530/332 ;
560/158 |
International
Class: |
C07K 005/00; C07C
279/14 |
Claims
What is claimed is:
1. A protected guanidine of the structure 18wherein P.sub.1,
P.sub.2, and P.sub.3 are the same or different urethane protecting
groups, and salts and solvates thereof.
2. A protected arginine homologue of the structure 19wherein Q,
P.sub.1, P.sub.2, and P.sub.3 are the same or different urethane
protecting groups, and n is an integer of 0-3, and salts and
solvates thereof.
3. The protected molecules of claims 1-2 wherein the urethane
protecting groups are selected from the group consisting of (a)
tert-butyloxycarbonyl (Boc) (b) benzyloxycarbonyl (Cbz), (c)
allyloxycarbonyl (Alloc) (d) 2,2,2-trichloroethyloxycarbonyl
(Troc), (e) 2-chlorobenzyloxycarbonyl; and (f)
4-methoxy-benzyloxycarbonyl (Moz).
4. N-,N'-,N"-Tri-tert-butyloxycarbonyl-guanidine
5. N-,N'-,N"-Tri-benzyloxycarbonyl-guanidine
6. N-Methyl-N-,N'-,N"-tri-tert-butyloxycarbonyl-guanidine
7. N-Methyl-N-,N'-,N"-tri-tert-benzyloxycarbonyl-guanidine
8. A protected guanidine of the structure 20wherein P.sub.1 and
P.sub.2 are the same or different urethane protecting groups, and R
is a substituted or unsubstituted alkyl or aryl group or a
heterocyclic group, and salts and solvates thereof.
9. A protected guanidine of the structure 21wherein P.sub.1 and
P.sub.2 are the same or different urethane protecting groups, and
salts and solvates thereof.
10. N-N'-Di-Boc-N"-trifluoromethanesulfonyl-guanidine
11. N-N'-Di-Cbz-N"-Trifluoromethanesulfonyl-guanidine
12. N-Boc-N'-Cbz-N"-Trifluoromethanesulfonyl-guanidine
13. A method for guanidinylation comprising reacting a protected
guanidine of the structure 22wherein P.sub.1, P.sub.2 and P.sub.3
are the same or different urethane groups, with an alcohol of the
structure 23wherein R.sub.1 and R.sub.2 are selected from the group
consisting of H, substituted or unsubstituted alkyl, alkenyl, aryl,
arylalkyl or R.sub.1, and R.sub.2 are members of a 3-6 member
cycloalkyl structure, to yield a product of the structure 24
14. A method according to claim 13, further comprising the step of
reacting a primary or secondary alcohol having the structure
25wherein R.sub.3 and R.sub.4 are independently the same as or
different from R.sub.1 or R.sub.2 with the product of the method of
claim 13 to yield a compound having the structure 26
15. A method for preparing arginine homologs, comprising reacting a
protected guanidine of the structure 27wherein P.sub.1, P.sub.2 and
P.sub.3 are the same or different urethane groups with a protected
amino acid of the structure 28wherein R.sub.1 is a substituted or
unsubstituted alkyl or aryl group, Q is a urethane protecting group
and n is 0-3, to yield a product of the structure 29
16. A method for guanidinylating amines, comprising reacting a
protected guanidine of the structure 30wherein P.sub.1, P.sub.2 and
P.sub.3 are the same or different urethane groups, and R.sub.1 is a
substituted or unsubstituted alkyl group, and a primary or
secondary amine of the structure 31wherein R.sub.2 and R.sub.3 are
independently H, or a substituted or unsubstitued alkyl group, or
an amino acid or a polypeptide, or R.sub.2 and R.sub.3 are part of
a cyclic structure as in piperidine, pyrrolidine, or morpholine, to
yield a product of the structure 32
17. The method of claim 16 wherein the sulfonyl guanidinylating
group has the structure 33
18. Guanidinylated compounds prepared by the method of any one of
claims 15-17.
19. Guanidinylated compounds prepared by reacting a guanidinylating
reagent selected from the group consisting of
N-N'-di-Boc-N"-trifluoromet- hanesulfonyl-guanidine,
N-N'-di-Cbz-N"-trifluoromethanesulfonyl-guanidine, and
N-Boc-N'-Cbz-N"-trifluoromethanesulfonyl-guanidine with a primary
amine.
20. Guanidinylated compounds prepared by reacting a guanidinylating
reagent selected from the group consisting of
N-N'-di-Boc-N"-trifluoromet- hanesulfonyl-guanidine,
N-N'-di-Cbz-N"-trifluoromethanesulfonyl-guanidine, and
N-Boc-N'-Cbz-N"-trifluoromethanesulfonyl-guanidine with a secondary
amine.
21. A method for the solid phase synthesis of a peptide comprising
one or more arginine homologs, comprising the steps of: (a)
assembling a sequence (Xaa).sub.n of amino acids on a solid phase
peptide synthesis resin, where Xaa denotes any appropriately
protected amino acid. (b) attaching an amino acid having the
structure 34wherein Q is Boc or Fmoc and P is any protecting group
that can be selectively removed, and n=0-3, to the amino terminus
of the product of step (a); (c) coupling any sequence of suitably
protected amino acids to the amino terminus of the product of step
(b); (d) selectively removing all the protecting groups P as
defined in (b) from the product of step (c) (e) reacting a
protected guanidine having the structure 35with the product of step
(d) to guanidinylate all unprotected amino functions present; and
(f) removing all protecting groups and cleaving the peptide from
the resin to yield the product 36
Description
FIELD OF THE INVENTION
[0001] The invention relates to reagents and methods for the
synthesis of organic molecules containing a guanidine group. It
relates particularly to reagents useful for introducing a protected
guanidine group into a molecule.
BACKGROUND OF THE INVENTION
[0002] Many natural compounds that bear a guanidine function have
biological activity that make them useful as pharmaceuticals. Among
these compounds are antimicrobials, antifungals, antivirals,
neurotoxins, hormones, and agents that act as agonists or
antagonists to biological signals. A review of these natural
products is presented in Progress in the Chemistry of Organic
Natural Products (1995) 66:119 and Berlinck, R. G. S. (1996) Nat.
Prod. Reports 13(5):377409. Much effort has been directed to
developing routes for preparing these compounds or their analogues
synthetically.
[0003] Guanidine-containing bioactive molecules, particularly the
analogs or derivatives of the natural products, are now significant
targets for drug design and discovery. The guanidine moiety in the
bioactive compound frequently occurs in arginine-containing
polypeptide chains which may comprise the entire biomolecule or
exist as an incorporated moiety. Arginine, together with lysine,
another amino acid with a positively charged side chain, plays an
important role in biologically active proteins and peptides.
Various arginine analogues and derivatives have been synthesized
and incorporated into peptides and peptidomimetics to study the
structure-activity relationships of arginine-containing molecules.
These residues are frequently the critical amino acid residues in
peptidomimetics.
[0004] A completely satisfactory guanidinylating reagent has not
yet been achieved. More effective guanidinylation reagents are
useful in improving the synthesis of arginine analogues and other
guanidine-containing molecules.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIG. 1: X-ray structure of di-Boc-trifl-guanidine
[0006] FIG. 2: Comparison of N-,N'-di-Boc-N"-triflyl-guanidine with
two commercially available guanidinylation reagents. All three
reactions were carried out in an NMR-instrument and the formation
of product was followed by integration of the signals of the
benzylic CH.sub.2-groups. In all reactions the concentration of the
guanidinylating agent was 100 mM and the concentration of
benzylamine was 90 mM. Benzene-d6 was used as a solvent. Similar
results were obtained in deuterated chloroform and in deuterated
acetonitrile.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0007] Definitions
[0008] The term "alkyl" used herein refers to a monovalent straight
or branched chain radical of from one to ten carbon atoms,
including, but not limited to methyl, ethyl, n propyl, isopropyl,
n-butyl, isobutyl, tert-butyl, n-hexyl, and the like. Alkyl also
represents cyclic radicals, including, but not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
[0009] The term "perfluoroalkyl" as used herein refers to a
monovalent straight chain radical of from one to four carbon atoms,
in which all hydrogen atoms are substituted by fluorine. A typical
perfluorinated alkyl group is the trifluoromethyl group.
[0010] The term "aryl" when used alone refers to an aromatic
radical whether or not fused. Preferred aryl groups include phenyl,
naphthyl, biphenyl and the like. Aryl also refers to heteroaromatic
groups including, but not limited to, furanyl, pyrrolyl, thienyl,
pyrazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, indolyl, and
the like.
[0011] The term "substituted aryl" denotes an aryl group
substituted with one, two or three substituents chosen from
halogen, cyano, nitro, C1-C10 alkyl, C1-C10-alkyloxy,
trifluoromethyl, alkyloxycarbonyl, and the like. Examples of such
groups are 4 chlorophenyl, 2-methylphenyl, and 3-ethoxyphenyl.
[0012] The term "arylalkyl" means one, two or three aryl groups
having the designated number of carbons, appended to an alkyl chain
having the number of carbons designated. A typical arylalkyl group
is the benzyl group.
[0013] The term "alkenyl" refers to a straight or branched chain
group of from two to ten carbon atoms containing a carbon-carbon
double bond, including, but not lirnited to allyl, vinyl, and the
like.
[0014] Guanidinylation Reagents
[0015] We have discovered two types of guanidinylation reagents
that allow the synthesis of protected guanidines. Compounds of type
I comprise guanidines with three symmetrically arranged
electron-withdrawing protecting groups (P) and have the structure
1
[0016] wherein P.sub.1, P.sub.2 and P.sub.3 are the same or
different urethane protecting groups, each having the general
structure 2
[0017] wherein R is a substituted or unsubstituted alkyl or aryl
group or heterocyclic group.
[0018] P is chosen from urethane protecting groups which are
conveniently removable. These groups are available in an almost
limitless number. Reviews of urethane groups and their use in
peptide synthesis are provided by Geiger, R. and Konig, W. in "The
Peptides" (Gross, E. Meienhofer, I., eds) Vol.3, p3. New York, N.Y.
1981 and in Winsch, E. Methoden der Org. Chem. (Houben-Weyl) Vol.
15/1 (Wunsch, E.,ed.), p.46, Stuttgart: Thieme 1974. Particularly
preferred are the urethane groups containing a substituted or
unsubstituted benzylic carbon atom. Urethane-type protecting groups
having a benzylic carbon atom are described by Bodanszky, M. (1984)
Principles of Peptide Synthesis Chap. III Sec. C, Springer-Verlag,
New York 1984. Such groups are removable by hydrogenolysis and by
acidolysis, as well as by base-induced .beta.-elimination.
Preferably, the protecting group P is an alkyloxylcarbonyl group
such as Boc (P=tert-butyloxycarbonyl), Cbz (P=benzyloxycarbonyl),
Alloc (P=allyloxycarbonyl), Troc
(P=2,2,2-trichloroethyloxycarbonyl), or Moz
(P=4-methoxybenzyloxycarbonyl- ). The protecting groups Boc and Cbz
are particularly preferred.
[0019] The protected type I guanidines of the invention are weak
acids and can be used to guanidinylate primary or secondary
alcohols in a Mitsunobu-reaction to produce triprotected alkyl
guanidines (scheme 1). The product of such a reaction still
possesses one acidic hydrogen atom which can be exploited in a
second Mitsunobu-reaction to produce protected dialkylated
guanidines. 3
[0020] R.sub.1, R.sub.2, R.sub.3, and R.sub.4 can be hydrogen or
any substituted or unsubstituted alkyl, alkenyl, aryl, or arylalkyl
group as described earlier. R.sub.1 and R.sub.2 (and/or R.sub.3 and
R.sub.4) may be part of a ring structure as in cyclopropanol,
cyclobutanol, cyclopentanol, cyclohexanol and the like.
[0021] Compounds of type II comprise guanidines with a sulfonyl
group in addition to two urethane protecting groups, having the
structure 4
[0022] wherein P.sub.1 and P.sub.2 are as defined above and R.sub.1
is a substituted or unsubstituted alkyl or aryl group.
Perfluoroalkyl groups are preferred. Type II protected guanidines
react with primary or secondary amines to produce diprotected alkyl
guanidines (scheme 2). 5
[0023] R.sub.2 and R.sub.3 can be hydrogen or any substituted or
unsubstituted alkyl, alkenyl, aryl, or arylalkyl group as described
earlier. R.sub.2 and R.sub.3 may be part of a ring structure as in
aziridine, azetidine, pyrrolidine, piperidine, morpholine, and the
like.
[0024] Preferably, P is Boc or Cbz and R.sub.1 is phenyl,
4-methyiphenyl, methyl, or trifluoromethyl. Analogs with other
protecting groups such as Troc, Alloc or Moz at the P-position are
expected to show the same kind of reaction. Because of the
exceptionally strong electron-withdrawing character of the triflyl
group, the triflyl-guanidines (R.sub.1=trifluoromethyl) are the
most reactive among the compounds synthesized so far and these are
therefore preferred. They have been shown to be superior to
previously described guanidinylating reagents.
Synthesis Procedures
[0025] Synthesis of Guanidinylation Agents of Type I
[0026] A general route towards symmetrical trisubstituted
guanidines is shown in scheme 3. The introduction of the first two
protecting groups into guanidine hydrochloride is accomplished in
one step. Yields between 50 and 80% are usually obtained. The
diprotected guanidine is then treated with two equivalents of
sodium hydride under anhydrous conditions. Acylation of the
resulting anion then completes the synthesis. Preferably, R is
benzyl, 2-chlorobenzyl, 4-methoxylbenzyl, 2,2,2-trichloroethyl,
allyl, or tert-butyl and X is chloro, azido, succinimidyloxy, or
alkoxycarbonyloxy. 6
[0027] Alternatively, symmetrical triprotected guanidines can be
synthesized in one step from guanidine hydrochloride by phase
transfer catalysis (scheme 4). Acylating reagents wherein R=benzyl,
R=allyl, and R=2,2,2-trichloroethyl are preferred. 7
[0028] Synthesis of Guanidinylation Agents of Type II
[0029] Guanidinylation agents of type II can be synthesized by
deprotonation of diprotected guanidines with sodium hydride in an
inert solvent and reaction of the resulting anion with a sulfonyl
chloride (scheme 5). This method was successfully applied in the
synthesis of N-,N'-di-Cbz-N"-methylsulfonyl-guanidine
(R.sub.1=benzyl, R.sub.2=Methyl),
N-,N'-di-Cbz-N"-phenylsulfonyl-guanidine (R.sub.1=benzyl,
R.sub.2=phenyl), and N-,N'-di-Cbz-N"-tosyl-guanidine
(R.sub.1=benzyl, R.sub.2=tosyl). 8
[0030] Instead of sulfonyl chlorides, sulfonyl anhydrides can be
used as shown in the synthesis of N-,N'-di-Cbz-N-triflyl-guanidine
(scheme 6). 9
[0031] In some cases triethylamine can be used as a base instead of
sodium hydride. An example is given in scheme 7 with the synthesis
of N-,N'-di-Boc-N"-triflyl-guanidine. 10
[0032] Reactions of Guanidinylation Agents of Type I
[0033] Guanidinylation reagents of type I react with primary and
secondary alcohols in a Mitsunobu-reaction to produce protected
alkylated guanidines. This is exemplified in the synthesis of
several orthogonally protected arginine analogs (scheme 8) from
suitable precursor molecules. The reactions with
N-,N'-,N"-tri-Boc-guanidine are preferably carried out in refluxing
THF and yields of up to 70% can be obtained. If
N-,N'-,N"-tri-Cbz-guanidine is used as the guanidinylating species,
the reaction can be carried out at room temperature. In addition,
the yields are usually somewhat higher (up to 86%) than in
comparable reactions with N-,N'-,N"-tri-Boc-guanidine. 11
[0034] Many biologically interesting guanidines contain two
different alkyl substituents connected to two different N-atoms of
the guanidine nucleus. Compounds of this type are accessible from
triprotected guanidines by two consecutive Mitsunobu-reactions. An
example is given in scheme 9 with the synthesis of protected
derivative of .omega.-methyl-arginine, an important inhibitor of
nitric oxide synthethase. 12
[0035] Reactions of Guanidinylation Agents of Type II
[0036] N-,N'-Di-Boc-N"-triflyl-guanidine reacts rapidly and under
mild conditions with primary (scheme 10) and secondary amines
(scheme 11). The reactions are carried out at room temperature and
are usually complete within 1 h. Succesful guanidinylation
reactions have been performed in a wide range of solvents such as
benzene, chloroform, or dichloromethane, acetonitrile or DMSO.
Unpolar solvents such as benzene, chloroform, or dichloromethane
are preferred.[U6] Compounds that are insoluble in one of the
preferred solvents can in many cases be converted into a more
soluble derivative which can then be succesfully guanidinylated.
This is demonstrated in scheme 10 with the synthesis of a
homoarginine derivative from N-.alpha.-Fmoc-lysine. In this
procedure N-.alpha.-Fmoc-lysine is first silylated with MSTFA
(N-methyl-N-trimethylsilyl-trifluoroacetarnide- ) to generate a
derivative that is soluble in dichloromethane. This derivative is
then guanidinylated in the same pot with
N-,N'-di-Boc-N"-triflyl-guanidine. The silyl-groups used to
solubilize the starting material are removed again during the
workup procedure. Other protected diamino acids such as
N-.alpha.-Fmoc-ornithine, N-.alpha.-Fmoc-2,4-diamino-butyric acid
or N-.alpha.-Fmoc-2,3-diamino-pro- pionic acid are expected to show
the same kind of reaction The arginine analogues produced by this
methodology are orthogonally protected and can be used for peptide
coupling reactions without further modifications. 13
[0037] Exceptionally good yields of protected guanidines are
obtained by guanidinylation of secondary amines (scheme 11). Even
with divalent amines such as piperazine the reaction is extremely
facile. 14
[0038] N-,N'-Di-Cbz-N"-triflyl-guanidine is an excellent reagent
for the guanidinylation of unreactive aromatic amines. The reaction
with aniline is complete after 1 h at room temperature (scheme 12).
15
[0039] Guanidinylations with N-,N'-Di-Boc-N"-Triflyl-Guanidine on
Solid Phase
[0040] Reactions on solid phase are usually slower than comparable
reactions in solution. Much effort is currently directed to adapt
useful chemical reactions to the unique conditions of solid phase
synthesis. Such optimized reactions are especially important for
the construction of chemical libraries by parallel and
combinatorial methods.
[0041] The high reactivity of N-,N'-di-Boc-N"-triflyl-guanidine
allows guanidinylations on solid phase to be performed
successfully. This is demonstrated by the conversion of an
ornithine residue in a peptide sequence to arginine (scheme 13).
The peptide was assembled on a PAM-resin (PAM:
phenylacetamidomethyl) by standard methods. Omithine, the
.epsilon.-amino group protected by Fmoc, was incorporated in place
of arginine. After complete assembly of the sequence the Fmoc-group
on the ornithine side chain was removed selectively and the free
amino group was guanidinylated with
N-,N'-di-Boc-N"-triflyl-guanidine. The unprotected
arginine-containing peptide was then obtained after removal of the
Boc-groups and cleavage of the peptide from the resin with HF.
Analysis of the crude peptide by FAB-MS indicated a homogeneous
product. No peaks suggesting incomplete guanidinylation could be
detected.
[0042] The strategy as outlined in scheme 13 could prove to be very
valuable for synthesis of peptides containing multiple arginine
residues. Such peptides are often difficult to synthesize by
conventional methods. 16
[0043] Comparison of N-,N'-Di-Boc-N'-triflyl-Guanidine With Other
Guanidinylating Reagents
[0044] The guanidinylation of benzylarnine in benzene was chosen as
a model reaction to compare two commercially available
guanidinylating agents with N-,N'-di-Boc-N"-triflyl-guanidine
(scheme 14). All three reactions were carried out in an
NMR-instrument and the formation of product was followed by
integration of the signals of the benzylic CH.sub.2-groups. Under
the conditions chosen, N-,N'-di-Boc-N"-triflyl-gua- nidine proved
superior to the other reagents. Similar results were obtained in
deuterated chloroform and ind deuterated acetonitrile. 17
Experimental Procedures
EXAMPLE 1
N-,N'-,N"-Tri-Boc-Guanidine
[0045] Potassium hydroxide pellets (2.81 g, 50 mmol) and sodium
carbonate (5.30 g, 50 mmol) are finely ground in a mortar and
transferred into a 250 ml round bottomed flask equipped with a
magnetic stirrer and a reflux condenser. DMSO (50 ml) is added and
the resulting suspension is stirred for 5 min at room temperature.
Guanidine hydrochloride (4.78 g, 50 mmol) is added and the mixture
is again stirred for 5 min After the addition of
di-tert-butyl-dicarbonate (51.7 ml, 225 mmol) the mixture is
stirred for 60 h at 40.degree. C. The colorless precipitate
obtained by pouring the cold reaction mixture into 11 water is
collected by filtration on Buechner funnel, washed with water and
dried overnight in vacuo. Recrystallization from acetonitrile
yields colorless needles (14.9 g, 83%): mp 147-150.degree. C.
(dec); .sup.1H NMR (360 MHz, CDCl.sub.3) .delta. 1.48 (s, 27H);
FAB-MS m/e (relative intensity) 360 (100, M+H.sup.+), 304 (34), 260
(10), 248 (74); Anal. Calc. for: C, 53.47%; H, 8.13%;.N, 11.69%;
Found: C, 53.48%; H, 8.34%;.N, 11.86%.
EXAMPLE 2
N-,N'-,N"-Tri-Cbz-Guanidine
[0046] Sodium hydride (400 mg, 60% dispersion in mineral oil) is
added in small portions to a suspension of N-,N'-di-Cbz-guanidine
(1.65 g, 5.0 mmol) in anhydrous THF (20 ml) at -45.degree. C. under
an atmosphere of argon. After the addition is completed, the
mixture is stirred for 1 h at -45.degree. C. Benzyl chloroformate
(0.82 ml, 5 mmol) is added, the mixture is allowed to warm up to
room temperature and stirred overnight. The solvent is removed
under reduced pressure and the residue is dissolved in a mixture of
dichloromethane (50 ml) and water (25 ml). The phases are separated
and the aqueous layer is extracted twice with dichloro methane (50
ml each). The extracts are combined, washed with 1N hydrochloric
acid and water and dried with magnesium sulfate. After filtering
and removal of the solvent under reduced pressure the crude product
is purified by flash chromatography on silica gel (eluent:
dichloromethane-ethyl ether 98:2). N-,N'-N"-tri-Cbz-guanidine (2.07
g, 90%) is obtained as a white powder: mp: 111-112.degree. C.;
.sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta. 10.55, (s, 2H), 7.36
(s, 10H), 5.22 (br s, 6H); FAB-MS m/e (relative intensity) 506 (5,
M-H.sup.++2Na.sup.+), 484 (100, M+Na.sup.+), 462 (24, M+H.sup.+);
Anal. Calc. for C, 65.07%; H, 5.02%;.N, 9.11%; Found: C, 64.89%; H,
4.74%;.N, 8.82%.
EXAMPLE 3
N-Methyl-N-,N'-,N"-Tri-Boc-Guanidine
[0047] A solution of anhydrous methanol (0.04 ml, 1.0 mmol),
N-,N'-,N"-tri-Boc-guanidine (1.80 g, 5.0 mmol), and
triphenylphoshine (393 mg, 1.5 mmol) in anhydrous THF (50 ml) is
cooled to -5.degree. C. under an atmosphere of argon.
Diethylazodicarboxylate (DEAD, 0.22 ml, 1.5 mmol) is added dropwise
at a rate such that the reaction mixture is completely colorless
before addition of the next drop. After the addition is completed,
the reaction mixture is refluxed for 15h. The solution is then
cooled to room temperature, and hexanes (50 ml) is added. A
precipitate of excess N-,N'-,N"-tri-Boc-guanidine forms which is
collected by filtration on a Buechner-funnel and washed with a
mixture of THF/hexanes 1:1. The filtrate is concentrated under
reduced pressure and the product (colorless oil, 182 mg, 49%)
isolated by flash chromatography on silica gel (eluent:
dichloromethane-ethyl ether 98:2): .sup.1H NMR (360 MHz,
DMSO-d.sub.6) .delta. 10.17 (s, 1H), 2.94 (s, 3H), 1.43-1.36 (27H);
FAB-MS m/e (relative intensity) 396 (100, M+Na.sup.+).374 (91,
M+H.sup.+).
EXAMPLE 4
L-N-Cbz-.delta.,.omega.,.omega.'-Tri-Boc-Arginine Methyl Ester
[0048] A solution of S--N-Cbz-2-amino-5-hydroxy-valeric acid methyl
ester (0.56 g, 2.0 mmol), N-,N'-,N"-tri-Boc-guanidine (3.59, 10.0
mmol), and triphenylphoshine (0.79 g, 3.0 mmol) in anhydrous THF
(100 ml) is cooled to -5.degree. C. under an atmosphere of argon.
Diethylazodicarboxylate (DEAD, 0.45 ml, 3.0 mmol) is added dropwise
at a rate such that the reaction mixture is completely colorless
before addition of the next drop. After the addition is completed,
the reaction mixture is stirred for 18 h at 45.degree. C. The
solution is then cooled to room temperature, and hexanes (100 ml)
is added. A precipitate of excess N-,N'-N"-tri-Boc-guanidine forms
which is collected by filtration on a Buechner-funnel and washed
with a mixture of THF/hexanes 1:1. The filtrate is concentrated
under reduced pressure and the product (colorless oil, 0.87 g, 70%)
isolated by flash chromatography on silica gel (eluent:
dichloromethane-ethyl ether 9:1): 1H NMR (360 MHz, DMSO-d.sub.6)
.delta. 10.18 (s, 1H), 7.72 (d, 1H, J=7.9 Hz), 7.40-7.26 (m, 5H),
5.01 (s, 2H), 4.03-3.94 (m, 1H), 3.60 (s, 3H), 3.45 (t, 2H, J=5.8
Hz), 1.73-1.45 (m, 4H), 1.39 (s, 18H), 1.37 (s, 9H); FAB-MS m/e 623
(M+H.sup.+).
EXAMPLE 5
L-N-Cbz-.omega.-Methyl-.delta.,.omega.,.omega.'-Tri-Boc-Arginine
Benzyl Ester
[0049] A solution of S--N-Cbz-2-amino-5-hydroxy-valeric acid methyl
ester (143 mg, 0.4 mmol), N-methyl-N-,N'-,N"-tri-Boc-guanidine (150
mg, 0.4 mmol), and triphenylphoshine (105 mg, 0.4 mmol) in
anhydrous THF (2 ml) is cooled to -5.degree. C.
Diethylazodicarboxylate (DEAD, 0.06 ml, 0.38 mmol) is added
dropwise at a rate such that the reaction mixture is completely
colorless before addition of the next drop. After the addition is
completed, the reaction mixture is refluxed for 3 h. The solvent is
removed under reduced pressure and the product (colorless oil, 181
mg, 63%) is isolated by flash chromatography on silica gel (eluent:
ethyl acetate-hexanes 1:3): .sup.1H NMR (360 MHz, DMSO-d.sub.6)
.delta. 7.80 (d, 2H, J=7.9 Hz), 7.39-7.28 (m, 10H), 5.10 (s, 2H),
5.06-4.94 (m, 2H), 4.11-4.00 (m, 1H), 3.53-3.44 (m, 2H), 2.89 (s,
3H), 1.75-1.50 (m, 4H), (1.40-1.34 (27H); FAB-MS z/e 845
(M+Cs.sup.+).
EXAMPLE 6
N-,N'-Di-Boc-Guanidine
[0050] 1,4-Dioxane (50 ml) is added to a solution of guanidine
hydrochloride (2.39 g, 25 mmol) and sodium hydroxide (4.0 g, 0.1
mol) in water (25 ml) and the resulting mixture is cooled to
0.degree. C. Di-tert-butyl-pyrocarbonate (12.0 g, 55 mmol) is added
in one portion while stirring. The reaction mixture is allowed to
warm to room temperature within 2 h. After stirring for 20 h the
mixture is concentrated in vacuo to one third of its original
volume. The resulting suspension is diluted with water (50 ml) and
extracted three times with ethyl acetate (5 ml each). The combined
extracts are washed with 10% citric acid, water and brine and dried
with magnesium sulfate. After filtering and removal of the solvent
under reduced pressure the crude product is purified by flash
chromatography on silica gel (eluent: dichloromethane-methanol
97:3). N-,N'-di-Boc-guanidine (3.84 g, 59%) is obtained as a
colorless powder: mp: 144.degree. C.; .sup.1H NMR (360 MHz,
DMSO-d.sub.6) .delta. 10.42 (br s, 1H), 8.47 (br s, 2H), 1.39 (s,
18H); FAB-MS m/e (relative intensity) 260 (50, M+H.sup.+), 204
(48), 148 (100); Anal. Calc. for: C, 50.95%; H, 8.16%;.N, 16.21%;
Found: C, 50.83%; H, 8.04%;.N, 16.26%.
EXAMPLE 7
N'-Di-Boc-N"-Trifluorometbanesulfonyl-Guanidine
[0051] A solution of N-,N'-di-Boc-guanidine (0.52 g, 2.0 mmol) and
triethyl amine (0.29 ml) in anhydrous dichloromethane (10 ml) is
cooled to -78.degree. C. under an atmosphere of argon. Triflic
anhydride (0.35 ml, 2.1 mmol) is added dropwise at a rate such that
reaction temperature does not exceed -65.degree. C. After the
addition is completed, the mixture is allowed to warm to room
temperature within 4 h. The solution is transferred to a separation
funnel, washed with 2M sodium bisulfate and water and dried with
anhydrous sodium sulfate. After filtering and removal of the
solvent under reduced pressure the crude product is purified by
flash chromatography on silica gel (eluent: dichloromethane).
N-N'-Di-Boc-N'"-trifluoromethanesulfonyl-guanidine (686 mg, 88%) is
obtained as pale yellow crystals. The product can be further
purified by recrystallization from hexanes: mp: 115.degree. C.;
.sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta. 11.45 (br s, 2H), 1.45
(s, 18H). FAB-MS m/e (relative intensity) 414 (16, M+Na.sup.+), 392
(13, M+H.sup.+), 336 (43), 280 (100), 236 (9); Anal. Calc. for C,
36.83%; H, 5.15%;.N, 10.74%; F, 14.56%; S, 8.19%; Found: C, 36.93%;
H, 5.21%;.N, 10.66%; F, 14.80%; S, 8.33%.
EXAMPLE 8
N-,N'- Di-Cbz-Guanidine
[0052] Dichloromethane (80 ml) is added to a solution of guanidine
hydrochloride (3.82 g, 40 mmol) and sodium hydroxide (8 g, 0.2 mol)
in water (40 ml) and the resulting mixture is cooled to 0.degree.
C. Benzyloxycarbonyl chloride (17.1 ml, 120 mmol) is added dropwise
with vigorous stirring over a period of 45 min. After the addition
is completed, stirring is continued for 20 h at 0.degree. C. The
mixture is diluted with dichloromethane (100 ml), the layers are
separated and the aqueous layer is extracted with dichloromethane
(100 ml). The extracts are combined, washed with water and dried
with magnesium sulfate. After filtering and removal of the solvent
under reduced pressure the crude product is recrystallized from
methanol. N-,N'-Di-Cbz-guanidine (9.85 g, 75%) is obtained as
colorless crystals: mp: 149-150.degree. C.; H NMR (360 MHz,
DMSO-d.sub.6) .delta. 10.88 (br s, 1H), 8.67 (br s, 2H), 7.40-7.25
(m, 10H), 5.10 (s, 4H); Anal. Calc. for C, 62.38%; H, 5.23%;.N,
12.84%. Found: C, 62.26%; H, 5.01%;.N, 12.79%.
EXAMPLE 9
N-N'-Di-Cbz-N"-Trifluoromethanesulfonyl-Guanidine
[0053] Sodium hydride (400 mg, 60 dispersion in mineral oil) is
added to a solution of N-,N'-di-Cbz-guanidine (1.65 g, 5.0 mmol) in
anhydrous chlorobenzene (50 ml) at 0.degree. C. under an atmosphere
of argon. After stirring for 1 h at 0.degree. C., the mixture is
cooled to -45.degree. C. Triflic anhydride (0.82 ml, 5 mmol) is
added, the mixture is allowed to warm up to room temperature and
stirred overnight. The solvent is removed under reduced pressure
and the residue is dissolved in a mixture of ethyl acetate (100 ml)
and 2M sodium bisulfate (25 ml). The phases are separated and the
organic layer is washed with water and brine and dried with
magnesium sulfate. After filtering and removal of the solvent under
reduced pressure the crude product is purified by flash
chromatography on silica gel (eluent: dichloromethane-ethyl ether
95:5). N-N'-Di-Cbz-N'"-trifluoromethanesulfonyl guanidine (1.58 g,
69%) is obtained as a pale oil that crystallizes in vacuo: mp:
74-75.degree. C.; .sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta. 11.55
(br s, 2H), 7.45-7.28 (m, 10H), 5.20 (s, 4H); Electrospray-MS m/le
(relative intensity) 498 (30, M+K.sup.+), 482 (100, M+Na.sup.+),
460 (2, M+H.sup.+); Anal. Calc. for C, 47.06%; H, 3.51%;.N, 9.15%;
F, 12.41%; S, 6.98%; Found: C, 47.37%; H, 3.35%;.N, 8.67%; F,
12.79%; S, 6.92%.
EXAMPLE 10
N-Cbz-Guanidine
[0054] 1,4-Dioxane (20 ml) is added to a solution of guanidine
hydrochloride (0.96 g, 10 mmol) and sodium hydroxide (0.8 g, 20
mmol) in water (10 ml) and the resulting mixture is cooled to
0.degree. C. Benzyloxycarbonyl chloride (1.1 ml, 7.7 mmol) is added
dropwise with vigorous stirring over a period of 10 min. After the
addition is completed, the ice-bath is removed and stirring is
continued for 1 h at room temperature. The mixture is concentrated
in vacuo to one third of its original volume and extracted three
times with ethyl acetate (20 ml each). The combined extracts are
washed with brine (20 ml) and dried with anhydrous sodium sulfate.
After filtering and removal of the solvent under reduced pressure
N-Cbz-guanidine (1.31 g, 88%) is obtained as a white powder: mp:
120-122.degree. C.; .sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta.
7.35-7.25 (m, 5H), 6.88 (br s, 4H), 4.95 (s, 2H); Electrospray-MS
m/e 194 (M+H.sup.+).
EXAMPLE 11
N-Boc-N'-Cbz-Guanidine
[0055] A solution of di-tertbutyl-pyrocarbonate (1.32 g, 6.05 mmol)
in acetone (5 ml) is added in one portion to a stirred solution of
N-Boc-N'-Cbz-guanidine (1.30 g, 6.73 mmol) and triethyl amine (0.94
ml) in acetone (15 ml). After stirring for 48 h at room temperature
the solvent is removed under reduced pressure and the resulting
residue is dissolved in a mixture of ethyl acetate (100 ml) and
water (50 ml). The phases are separated and the organic layer is
washed with 2M sodium bisulfate, water and brine and dried with
anhydrous sodium sulfate. After filtering and removal of the
solvent under reduced pressure the crude product is purified by
flash chromatography on silica gel (eluent: dichloromethane-ethyl
ether 9:1). N-Boc-N'-Cbz-guanidine (1.44 g, 82%) is obtained as a
white powder: mp: 125-126.degree. C.; .sup.1H NMR (360 MHz,
DMSO-d.sub.6) .delta. 10.59 (br s, 1H), 8.69 (br s, 1H), 8.50 (br
s, 1H), 7.40-7.25 (m, 5H), 5.04 (s, 2H), 1.42 (s, 9H).
EXAMPLE 12
N-tert-Butoxycarbonyl-N'-Cbz-N"-Trifluoromethanesulfonyl-Guanidine
[0056] A solution of N-Boc-N'-Cbz-guanidine (586 mg, 2.0 mmol) and
triethyl amine (0.42 ml) in anhydrous dichloromethane (20 ml) is
cooled to -78.degree. C. under an atmosphere of argon. Triflic
anhydride (0.42 ml, 2.5 mmol) is added dropwise at rate such that
reaction temperature does not exceed -65.degree. C. After the
addition is completed, the mixture is allowed to warm to room
temperature within 4 h. The solution is transferred to a separation
funnel, washed with 2M sodium bisulfate and water and dried with
anhydrous sodium sulfate. After filtering and removal of the
solvent under reduced pressure the crude product is purified by
flash chromatography on silica gel (eluent: dichloromethane).
N-Boc-N'-Cbz-N"-trifluoromethanesulfonyl-guanidine (699 mg, 82%) is
obtained as a pale oil that crystallizes upon drying in vacuo: mp:
95-97.degree. C.; .sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta. 11.49
(br s, 1H), 11.17 (br s, 1H), 7.40 (m, 5H), 5.21 (s, 1H), 1.43 (s,
9H); FAB-MS m/e (relative intensity) 448 (23, M+Na.sup.+), 426 (44,
M+H.sup.+), 329 (5), 370 (100), 348 (15), 326 (15).
EXAMPLE 13
N,N'-Bis(tert-Butyloxycarbonyl)-Pyrrolidine-1-Carboxamidine
[0057] N-N'-Di-Boc-N"-trifluoromethanesulfonyl-guanidine (235 mg,
0.6 mmol) is added to a solution of pyrrolidine (0.042 ml, 0.5
mmol) and triethyl amine (0.083 ml) in chloroform (1 ml). After
stirring for 4 h at room temperature, the product is isolated by
flash chromatography on silica gel (eluent: ethyl acetate-hexane
2:3). The product (146 mg, 93%) is obtained as a colorless oil that
crystallizes in vacuo: mp: 88-91.degree. C.; .sup.1H NMR (360 M
CDCl.sub.3) .delta. 3.58-3.53 (m, 4H), 1.90-1.83 (m, 4H), 1.46 (s,
18H); FAB-MS m/e (relative intensity) 649 (13, 2M+Na.sup.+), 627
(5, 2M+H+), 336 (29, M+Na.sup.+), 314 (100, M+H.sup.+), 258 (28),
202 (94).
EXAMPLE 14
N-N'-Di-Boc-N"-Phenyl-Guanidine
[0058] Aniline (0.055 ml, 0.6 mmol) is added to a solution of
N-N'-di-Cbz-N"-trifluoromethanesulfonyl-guanidine in chloroform and
the mixture is stirred for 1 h at room temperature. The solvent is
removed under reduced pressure the residue is dissolved in ethyl
ether (10 ml). The solution is washed with 10% citric acid, water
and brine, and dried with magnesium sulfate. After filtration and
removal of the solvent under reduced pressure
N-N'-di-Boc-N"-phenyl-guanidine (198 mg, 98%) is obtained as a
colorless oil that crystallizes upon drying in vacuo: mp:
105-108.degree. C.; .sup.1H NMR (360 MHz, DMSO-d.sub.6) .delta.
11.34 (br s, 1H), 9.99 (s, 1H), 7.56-7.11 (m, 15H), 5.23 (s, 2H),
5.02(s, 1H). FAB-MS m/e (relative intensity) 426 (M+Na.sup.+), 404
(M+H.sup.+).
[0059] The procedures of the invention as described above are to be
understood as exemplary and do not indicate limitations of the
invention, which is to be understood as limited only by the scope
of the following claims.
* * * * *