Note: Descriptions are shown in the official language in which they were submitted.
~094/25049 2161216 PCT~S94/04058
T;tle
Amidino and Guanidino Substituted Boronic Acid
Inhibitors of Trypsin-~ike Enzymes
~ Cross Reference to Rel~te~ A~pllc~t;ons
This application is a continuation-in-part of
Application Serial Number 08/052,835, filed April 27,
1993.
Fiel~ of the Invention
The present invention relates generally to a-
aminoboronic acids and corresponding peptide analogs in
which the alpha substituent is either an aromatic
guanidino, isothiouronium, amidino group, halogen, cyano
1~ group or an aliphatic amidino, isothiouronium, or
formamidino group.
R~ck~rol~n~ of the Invention
Simple boronic acids are inhibitors of serine
proteases. For example, Koehler et al. Biochemistry 10:
2477 (1971) reports that 2-phenylethane boronic acid
inhibits chymotrypsin at millimolar levels. The
synthesis of boronic acid analogs of N-acyl--amino
acids has yielded more effective inhibitors. Ac-
2~ boroPhe-OH, ~-1-acetamido-2-phenylethane boronic acid,
inhibits chymotrypsin with a Ki of 4 ~M Matteson et al.
J. Am. C~em. Soc. 103: 5241 (1981). More recently,
Shenvi, US 4,537,773 (1985) disclosed that boronic acid
analogs of a - amino acids, containing a free amino group,
were effective inhibitors of aminopeptidases. Shenvi,
US 4,499,082 (1985) discloses that peptides containing
an a - aminoboronlc acid with a neutral side chain were
more effective inhibitors of serine proteases exceeding
inhibitors disclosed earlier by as much as 3 orders of
3~ magnitude in potency. The chemistry of a - aminoboronic
acids was furthe- expanded to the synthesis of peptide
wog4/25049~6 ~2 ~6 PCT~S94/0~58
analogs containing boronic acid with positive charged
sidechains, boro~ysine, boroArginine, boroOrnithine, and
isothiouronium analogs (EPA 0 293 881, 12/7/88). This
series of compounds have provided highly effective
inhibitors of thrombin and other trypsin-like enzymes.
The boroArginine analogs specifically designed as
thrombin inhibitors are highly effective in the
inhibition of blood coagulation both in vitro and in
vivo. In the present invention, this group of compounds
is extended to aliphatic amidino and formamidino, to
aromatic amidino and guanidino, and to cyano and halogen
substituted aromatic boronic acid analogs.
It should be noted that additional boronic acids
have been disclosed. Metternich (EP 0471651) have
described peptides containing boroArginine and
boro~ysine which contain at least one unnatural amino
acid residue. Elgendy et al. Tetrahedron Lett., 33,
4209-4212 ~1992) have described peptides containing a -
aminoboronic acids with aliphatic neutral sidechains
which are thrombin inhibitors. Kakkar in (WO 92/07869)
has claimed peptide thrombin inhibitors of the general
structure, X-Aa1-Aa2-NH-CH(Y)-Z where Aa1 and Aa2 are
unnatural amino acid residues. Z is -CN, -COR,
-B(R2)(R3), -P(O)(R)(R), and Y is -[CH2]n-Q or -CH2-Ar-Q
where Q = H, amino, amidino, imidazole, guanidino or
isothioureido and n=1-5 and where R2 and R3 are the same
or different and are selected from the group consisting
of OH, oR6, and NR6R7, or R2 and R3 taken together
represent the residue of a diol. This specialized group
of compounds where Z is -B(R2)(R3) fall within the scope
of our present application. It should be noted that
this is a narrow subset of Kakkar et al. However,
rather specialized chemical transformations are required
to prepare these compounds and Kakkar et al. does not
make an enabling disclosure.
W094/25049 ~ 2 1 6 1 2 1~ ` P,~ ~ 94/04058
Sl~mm~ry of the Invention
A compound of formula (I)
R2 y1
R ~ ~ N ~ ~ B ~ 2
R1
wherein
Rl is
a) C1-C12-alkyl substituted with -CN, -C(NH)NHR6,
-NHC(NH)H, -NHC(NH)NHR6, -SC(NH)NHR6, -NHC(NH)NHOH,
-NHC(NH)NHCN, -NHC(NH)NHCOR6, or
b)
~ (CH2)q ~
-- (CH2)pX
~ ;
X is
a) halogen (F, Cl, Br, I)
b) -CN,
c) -NO2,
d) -CF3,
e) -NH2
f) -NHC(NH)H,
g) -NHC(NH)NHOH,
h) -NHC(NH)NHCN,
i) -NHC(NH)NHR6,
j) -NHC(NH)NHCOR6,
k) -C(NH)NHR
1) -C(NH)NHCOR
m) -C(O)NHR2,
n) -CO2R2,
o) -oR2, or
p) -OCF3
W094/25049 ~ ~G ~ 2 ~6 ~ PCT~S94104058
q) -SC(NH)NHR6;
R2 is
a) H,
b) Cl-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents
selected from the group consisting of halo (F, Cl,
Br, I), Cl-C4-alkyl, Cl-C4-alkoxy, -NO2, -CF3,
-S(O)r-Cl-C4-alkyl, -OH, -NH2, -NH(Cl-C4-alkyl),
-N(Cl-C4-alkyl)2, -C02R4, or
d) -Cl-C4-alkylaryl, where aryl is defined above;
R3 is H, alkyl, aryl, alkylaryl, or an NH2-blocking
group comprised of 1-20 carbon atoms;
R4 and R5 are independently
a) H,
b) Cl-C4-alkyl, or
c) -CH2-aryl, where aryl is defined above;
R6 is
a) H,
b) Cl-C4-alkyl,
c) aryl, wherein aryl is phenyl or napthyl
optionally substituted with one or two substituents
selected from the group consisting of halo (F, Cl,
Br, I), Cl-C4-alkyl, Cl-C7-alkoxy, -NO2, -CF3,
-S(O)r-Cl-C4-alkyl, -OH, -NH2, -NH(Cl-C4-alkyl),
-N(Cl-C4-alkyl)2, -Co2R4l or
d) -Cl-C4-alkylaryl, where aryl is defined above;
A is an amino acid residue or a peptide comprised of 2-
20 amino acid residues;
yl and y2 are
a) -OH,
b) -F,
c) Cl-C8-alkoxy, or
when taken together yl and y2 form a
W094/25049 2 1 5 12 16 ~ ~ ~ PCT~S94/04058
d) cyclic boron ester where said chain or ring
contains from 2 to 20 carbon atoms and, optionally,
1-3 heteroatoms which can be N, S, or O,
n is 0 or 1;
p is 0 to 3;
q is 0 to 4;
r is 0 to 2i
and pharmaceutically acceptable salts thereof, with the
proviso that when R1 is aliphatic, an R6 substituent on
-NHC(NH)NHR6 cannot be H.
Preferred are those compounds of formula(I) where
yl and y2 are
a) -OH,
when taken together yl and y2 form a
b) cyclic boron pinacol ester, or
c) cyclic boron pinanediol ester;
Rl is
a) -(CH2)3NHC(NH)H,
b) -(CH2)4C(NH)NH2
c)
-~- CH2 ~
CN
,~
d)
-~- CH2 ~
C(NH)NH2
,~
, or
e)
CH2 ~
--NHC(NH)NH2
,~
W094/25049 21612 1 B PCT~S94/0~58
R2 is H;
A is Pro or (D)Phe-Pro;
R3 is
a) H,
b) Boc,
c) Z, or
d) Ac, or
e) hydrocinnamoyl
f) Cl-Cl0 alkyl sulfonyl
g) Cl-Cl5 alkylaryl sulfonyl
Illustrative of the preferred compounds of this
invention are the following: ~
Ac-(D)Phe-Pro-NH-CH[(CH2)4CN]BO2-cloHl6
Ac-(D)phe-pro-NHcH[(cH2)4c(NH)NH2]Bo2-cloHl6
Ac-(D)phe-pro-NHcH[(cH2)3-NHc(NH)H]B(oH)2
Boc-(D)Phe-Pro-NHCH[(CH2)3-NHc(NH)H]B(OH)2
Ac-(D)Phe-Pro-boroPhe[m-C(NH)NH2]-Cl0Hl6
Ac-(D)Phe-Pro-boroPhe(m-CH2NH2)-cl0Hl6
Ac-(D)Phe-Pro-boroPhe(m-Br)-Cl0Hl6
Ac-(D)phe-pro-boroArg(cN)-cloHl6
Ac-(D)Phe-Pro-boroPhe(p-CN)-ClOHl6
Bcc-(D)Phe-Pro-boroPhe-(m-cN)-cloHl6
N,N-(CH3)2-(D)Phe-Pro-~oroPhe-(m-CN)-OH-HCl (ISOMER
I)
Ac-(D)Phe-Pro-boroPhe-(m-CN)-OH-HCl
Ms-(D)Phe-Pro-boroPhe-(m-CN)-OH-HCl
Boc-(D)Thiazolylalanine-Pro-boroPhe-(m-CN)-CloHl6
Boc-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-CloHl6
Ms-(D)3-Pyridylalanine-Pro-boroPhe-(m-CN)-CloHl6
Boc-(D)2-Pyridylalanine-PrO-borophe-(m-cN)-cloHl6
Boc-(D)2-Thienylalanine-pro-borophe-(m-cN)-cloHl6
Ms-(D)2-Thienylalanine-Pro-boroPhe-(m-CN)-Cl0Hl6
Boc-(D)Phe-Aze-boroPhe-(m-CN)-Cl0Hl6
Hydrocinnamoyl-Pro-boroIrg(CH3)-OH-HBr
--6--
W094/25049 21612 i ~ PCT~S94/0~58
Ac-(D)Phe-Pro-boroArg(CH3)-OH-HCl
PhCH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH-HCl
CH3CH2CH2SO2-(D)Phe-Pro-boroOrn(CH=NH)-OH-HCl
CH3CH2CH2SO2-(D)Phe-Pro-boroArg(CH3)-OH-HCl
Ac-(D)Phe-Sar-boroOrn(CH=NH)-OH-HCl
Boc-(D)Phe-Sar-borOphe(mcN)-cloHl6
Boc-(D)Phe-Aze-boroOrn(CH=NH)-OH-HCl
4-(Phenyl)benzoyl-boroOrn(CH=NH)-CloHl6-HCl
This invention also provides compositions
comprising one or more of the foregoing compounds and
methods of using such compositions in the treatment of
aberrant proteolysis such as thrombosis in mammals or as
reagents used as anticoagulants in the processing of
blood to plasma for diagnostic and other commercial
purposes.
Det~il DescriDt;on of the Invention
As used throughout the specifications, the
following abbreviations for amino acid residues or amino
acids apply:
Ala = L-alanine
Arg = L-arginine
Asn = L-asparagine
25 Asp = L-aspartic acid
Aze = azedine-2-carboxlic acid
Cys = L-cysteine
Gln = L-glutamine
Glu = L-glutamic acid
30 Gly = glycine
His = L-histidine
HomoLys = L-homolysine
Ile = L-isoleucine
Irg = isothiouronium analog of L-Arg
3~ Leu = L-leucine
Lys = L-lysine
W094l25049 ~ PCT~S94/04058
"; ~ . .. .
~161216
Met = L-methionine
Orn = L-ornithine
Phe = L-phenylalanine
Pro = L-proline
5 Ser = L-serine
Thr = L-threonine
Trp = L-tryptophan
Tyr = L-tyrosine
Val = L-valine
10 Sar = L-sarcosine
Phe(4-fluoro)= para-fluorophenylalanine
The "D" prefix for the foregoing abbreviations
indicates the amino acid is in the D-configuration.
"D,L" indicates the amino is present in mixture of the
D- and the L-configuration. The prefix "boro" indicates
amino acid residues where the carboxyl is replaced by a
boronic acid or a boronic acid ester. For example, if
R1 is isopropyl and yl and y2 are OH, the C-terminal
residue is abbreviated "boroVal-OH" where "-OH"
indicates the boronic acid is in the form of the free
acid. The pinanediol boronic acid ester and the pinacol
boronic acid ester are abbreviated "-C1oH16" and
"-C6H12", respectively. Examples of other useful diols
for esterification with the boronic acids are
1,2-ethanediol, 1,3-propanediol, 1,2-propanediol,
2,3-butanediol, 1,2-diisopropylethanediol,
5,6-decanediol, and 1,2-dicyclohexylethanediol. The
formamidino modified amino group is abbreviated (CH=NH).
For example, the formamidino analog of -boroOrn-OH {-NH-
CH[(CH2)3-NH-CH(NH)H]B(OH)2 }is -boroOrn(CH=NH)-OH.
Analogs containing sidechain substituents are described
by indicating the substituent in parenthesis following
the name of the parent residue. For example the analog
of boroPhenylalanine containing a meta cyano group is
-boroPhe(mCN)-. N-alkyl substituents on the guanidino
W094/25049 2161216 PCT~S94/0~58
~ , .
group of boroArg- or on the isothiouronium analogs
(boroIrg) are also put in parenthesis in a similar
manner. Other abbreviations are: Z, benzyloxycarbonyl;
BSA, benzene sulfonic acid; THF, tetrahydrofuran; Boc-,
t-butoxycarbonyl-; Ac-, acetyl; pNA, p-nitro-aniline;
DMAP, 4-N,N-dimethylaminopyridine; Tris,
Tris(hydroxymethyl)aminomethane; MS, mass spectrometry;
FAB/MS, fast atom bombardment mass spectrometry.
LRMS(NH3-CI) and HRMS(NH3-CI) are low and high
resolution mass spectrometry, respectively, using NH3 as
an ion source.
It is understood that many of the compounds of the
present invention contain one or more chiral centers and
that these stereoisomers may possess distinct physical
and biological properties. The present invention
comprises all of the stereoisomers or mixtures thereof.
If the pure enantiomers or diasteromers are desired,
they may be prepared using starting materials with the
appropriate stereochemistry, or may be separated from
mixtures of undesired stereoisomers by standard
techniques, including chiral chromatography and
recrystalization of diastereomeric salts.
"NH2-blocking group" as used herein, refers to
various acyl, thioacyl, alkyl, sulfonyl, phosphoryl, and
phosphinyl groups comprised of 1 to 20 carbon atoms.
Substitutes on these groups maybe either alkyl, aryl,
alkylaryl which may contain the heteroatoms, O, S, and N
as a substituent or as inchain component. A number of
NH2-blocking groups are recognized by those skilled in
the art of organic synthesis. By definition, an NH2-
blocking group may be removable or may remain
permanently bound to the NH2. Examples of suitable
groups include formyl, acetyl, benzoyl, trifluoroacetyl,
and methoxysuccinyl; alkyl and alkylaryl sulfonyl
groups, such as n-propylsulfonyl, phenylmethyl and
benzylsulfonyl; aromatic urethane protecting groups,
W094/25049 2 ~ 6 1216 , PCT~S94/04058
such as, benzyloxycarbonyl; and aliphatic urethane
protecting groups, such as t-butoxycarbonyl or
adamantyloxycarbonyl. Gross and Meinhoffer, eds., The
Peptides, Vol 3; 3-88 (1981), Academic Press, New York,
and Greene and Wuts Protective Groups in Organic
Synthesis, 315-405 (1991), J. Wiley and Sons, Inc., New
York disclose numerous suitable amine protecting groups
and they are incorporated herein by reference for that
purpose.
"Amino acid residues" as used herein, refers to
natural or unnatural amino acids of either D- or L-
configuration. Natural amino acids residues are Ala,
Arg, Asn, Asp, Aze,Cys, Gln, Glu, Gly, His, Ile, Irg
~eu, Lys, Met, Orn, Phe, Phe(4-fluoro), Pro, Sar, Ser,
1~ Thr, Trp, Tyr, and Val. Roberts and Vellaccio, The
Peptides, Vol 5; 341-449 (1983), Academic Press, New
York, discloses numerous suitable unnatural amino acids
and is incorporated herein by reference for that
purpose. Additionally, said reference describes, but
does not extensively list, acylic N-alkyl and acyclic
a,~-disubstituted amino acids. Included in the scope of
the present invention are N-alkyl, aryl, and alkylaryl
analogs of both in chain and N-terminal amino acid
residues. Similarly, alkyl, aryl, and alkylaryl maybe
substituted for the alpha hydrogen. Illustrated below
are examples of N-alkyl and alpha alkyl amino acid
residues, respectively.
CH3 CH3
s~5S~N~ ~, SSSS~N~
Me
--10--
~ W094l2~049 2 ~ 61216 ~ PCT~S94/0~58
,
~ Amino acids residues" also refers to various amino
acids where sidechain functional groups are coupled with
appropriate protecting groups known to those skilled in
the art. "The Peptides", Vol 3, 3-88 (1981) discloses
numerous suitable protecting groups and is incorporated
- herein by reference for that purpose.
Synthesis
Novel peptide boronic acids containing aliphatic
sidechains were prepared by the series of reactions
outlined in Scheme I. First, the precursor, NH2-
CH[(CH2)nBr]BO2-CloHl6~ n = 3 or 4, was prepared and
coupled with an N-terminal protecting group or with an
N-terminal and sidechain protected peptide by the
procedure we have described previously [Kettner et al.
J. Biol . Chem. 265 18289-18297 (1990)]. An example of
this product is 1 where the above intermediate is
coupled to Ac-(D)Phe-Pro-OH. 1 was converted to the
corresponding alkyl cyanide ~ by treatment with
tetrabutyl ammonium cyanide in THF at 55 C for 2 hours.
This appears to be a general method for introducing the
cyano group. In contrast, other common methods of
introducing this group can be applied only with limited
success. For example, the reaction of Ac-(D)Phe-Pro-NH-
2~ CH[(CH2)4-Br]BO2-CloHl6 with KCN in N,N-
dimethylformamide failed to yield a detectable product.
Our data are consistent with the formation of a cyclic
product arising from the nucleophilic displacement of
the sidechain bromide by the adjacent amide NH.
Treatment of Z-NH-CH[(CH2)4-Br]BO2-C1oH16 with NaCN in
N,N-dimèthylformamide gave the cyano compound, but only
in low yield, indicating that cyclization does not occur
quite so readily when the urethane protecting group (Z)
is present. Typically, 2 was purified by standard
techniques such as silica gel chromatography. The
corresponding amidine, 3, was prepared by treating the
W094/25049 2 1 6 12 16 PCT~S94/04058 ~
nitrile with a saturated solution of a mineral acid such
as HCl in methanol. Excess solvent and acid were
removed by evaporation and the residue was allowed to
react with anhydrous ammonia to yield the desired
product.
Scheme 1
Ac-(D)Phe-Pro-OH + H2N-CH--B'
or N-Terminal Protecting I ~~V
~Group J (CHI2)3
Br -Cl0~6
R3-[A~-oH
R3-[A]n--NH-CH BO2-C10H16 R3-[A~ NH-CH--BO2-C10H~6
(CH2)3Br (Bu)4N ' CN (CH2)3cN
1 MeOH, HCI R3-~A},--NH-CIH--BO2-C10H16
(CH2)3C(NH)NH2
The formamidino substituted boronic acid, 5, was
prepared by the synthesis of the corresponding alkyl
amine such as Ac-(D)Phe-Pro-boroOrn-C1oH16 9, Scheme 2.
This in turn was prepared by treating 1 with sodium
azide followed by hydrogenation (Kettner et al., 1990).
The amine, 4, was treated with ethyl formimidate to
yield the formamidino compound, 5.
-12-
~ W094l25049 216121~ PCT~S94/0~58
Scheme 2
2 Ha P3d/C R3-[A~n--NH-CH--BO2-CloHl6
(CH2)3NH2
R3-[A]n--NH--CI H--BO2-cloH16
(CH2)3NHC(NH)H
N-substituted isothiouronium derivatives and N-
substituted guanidines are readily prepared as shown in
Scheme 2a. Treatment of bromide 1 with a thiourea
produces directly the isothiouronium 21. Alternatively 1
can be converted to the amine 4 as shown in Scheme 2.
Employing a method originally described by Kim et al.,
Tetrahedron Lett. 29, 3183 (1988), the amine 4 then is
heated with a formamidinesulfonic acid in the presence
of 4-DMAP to afford the guanidine ~. The required
formamidinesulfonic acids can be prepared by oxidation
of the corresponding thioureas, see: Walter and Randau,
Liebigs Ann. Chem. 722, 98 (1969).
WO9~/25049 Z 16 12 I G PCT~S94/040~8
Scheme 2a
R3-[A~,- N~ CH--BO2 -Cl oHl 6 S R3-[A~,- NH - CH--BO2 -C,oH16
H2N C-NHR
(CH2)3Br (CH2)3SC(NH)NHR6
1 21
I
R3-[A]n- NH- CH--Bo2-c10H16 SO3H ~3-[A},- NH- CH--BO2-CloH16
HN -C -NHR6
(CH2)3NH2 (CH2)3NHC(NH)NHR6
4 22
The substituted boronic acid, l, is prepared by
treating 4 with dimethyl cyanodithioiminocarbonate or
diphenyl cyanodicarbonimiate to yield the S-methyl
isourea (~) or O-phenyl isourea, respectively, using a
procedure similar to that reported by Barpill et al. J.
Hereocyclic Chem. 25, 1698 (1988), Scheme 3. This
intermediate is treated with ammonia in either THF or
alcohol to yield the desired product.
Scheme 3
MeSC(NCN)SMe R3-[A]n--NH-CH--BO2-C~OH16 3
(CH2)3NHC(NCN)SMe
R3-lA]n--NH ~ I H--Bo2-c1 oH 16
(cH2)3NHc(NcN)NH2
-14-
216121~
W094/2~049 . PCT~S94/0~58
r
Hydroxyguanidino inhibitors are prepared by
treating 9 with cyanogen bromide or cyanogen chloride
followed by hydroxylamine to yield 8, Scheme 4. These
are known chemical transformations, Nakahara et. al.
Tetrahedron, 33, 1591 (1977) and Belzecki et al. J.
Chem. Soc. Chem. Commun., 806 (1970).
Scheme 4.
1. BrCN
2. NH2OH R3-[A]n--NH--I H--BO2-C~0H~6
(C~)3NHC(NOH)N~
The preparation of new aromatic boronic acids are
shown in Scheme 5. Functionalized benzylic anions
containing either a halogen or cyano substituent (the
cyano group is shown for illustration) are obtained by
treatment with activated Zn metal in THF or other inert
solvent and then with CuCN-2LiCl [Berk et al.
Organometallics 9, 3053-3064 (1990)]. Dichloromethyl
boronic acid pinanediol was prepared by the method
described by Tsai et al. Organometallics 2, 1543-1545
(1983). It was allowed to react with the transmetalated
anion to yield ~. This was the only acceptable method
of preparing these functionalized benzylic anions. For
example, treatment of p-nitobenzyl chloride with lithium
metal using the method of Michel et al. J.
Organometallic Chem. 204, 1-12 (1981) failed to yield an
identifiable product. Similarly, treatment of p-
cyanobenzyl chloride with lithium naphthalenide in the
presence of ZnCl2 using the conditions of Zhu et al. J.
Org. Chem. 56, 1445-1453 (1991) did not give the desired
product.
-15-
W094/25049 2 ~6 ~2 ~6 ` PCT~S94/04058
The a-aminoboronic acid, 10, was obtained by
treating ~ with the lithium salt of hexamethyldisilazane
and removing the trimethylsilanyl groups by treatment
with anhydrous HCl. lQ was coupled to either an N-
terminal protecting group or to a peptide using known
techniques.
The aromatic substituted cyanides, 11, wereconverted to the corresponding amidino compound, 1~,
using the same sequence of reactions described for
preparation of the aliphatic amidino compound, ~.
Scheme 5
CH2Br Cl- Cl H--BO2-C10H 16
1. Zn CH 1. (Me3Si)2N Li
2. CuCN-2LiCI ~ 1 2 2. HCI
CN Cl2CH- B o~ ¢~CN
H2N CIH--BO2-C10H~6 R3-[A]n-NH CIH--BO2-C10H16
CH2 Ac-(D)Phe-Pro-OH CH2
or N-Terminal Plol~;til~g
+ ~Group J ~
CN R3-lAh-oH CN
11
R3-[A]n-NH Cl H--BO2-c10H16
1. MeOH, HCI CH2
2. NH3
C(NH)NH2
11 can be hydrogenated to yield the corresponding
aminomethyl group as an aromatic substituent 1~, Scheme
6. The corresponding formamidino, cyanoguanidino,
hydroxyguanidino and guanidino compounds, 1
-16-
~ W094/25049 2161216 - PCT~S94/04058
and 17, respectively, are prepared by the procedures
described for the aliphatic series, Scheme l.
Sch~me6
R3-[Al"-NH-CH-BQ2-C,oHl6 R3-[Ah-NH-CIH-Bo2-c1oH16
11 H2, 10~ Pd/C CH2 ' CH2
NH2C(NH)SO~H,
13 CH2NH! CH2NHC(NH)NH2
EtOC(NH)H / \ 17
1. BrCN \
2. NH20H \
/ 1. M~SC(NCN)SMe
~/ 2. NH3 \~
R3-[Al,,-NH-ClH-BO2-c1OHl6 '' R3-[Alr,-NH-CHBO2-ClOH16
CH2 R3-[Al~-NH- ~CHBO 2-C1oH16 CH2
14 CH2NHc(NH)H ~ J 16 CH2NHC(NOH)NH2
CH2NHC(NCN)NH2
Aromatic guanidino inhibitors, 20, were prepared
from precursor R-boroPhe-C1oH16, Scheme 7. The aromatic
ring was nitrated by reaction with NO+BF4- to yield 1
which was reduced to the corresponding amine, 1~. The
amine is converted to the guanidine by reaction with
aminoiminomethane sulfonic acid [Mosher et al.
Tetrahedral Lett. 29 3183 (1988)] or cyanamide (Kettner
et al. 1990).
W094/25049 PCT~S94/04058 ~
2 1 ~
Scheme 7
R3-[A]n-NH ~CI H-Bo2-c1oH16 R3-[A]n-NH ~ I H-Bo2-c1oH16
l H2 No2+BFi IH2
~ ~ .
\~ \~ NO2
18
R3-[A]n-NH-CI H-Bo2-c1oH16
H2, Pd/C CH2 NH2C(NH)SO3H
~ DMAP
NH2
19
R3-[A]n-NH-CI H-B2-C10H16
CH2
\ ~ NHC(NH)NH2
NMR, proton nuclear magnetic resonance, chemical
shifts are reported in ~ units, parts per million
downfield from the internal tetramethylsilane standard.
Elemental analyses were conducted by Galbraith
Laboratories Inc., Knoxville, TN and Microanalysis Inc.,
Wilmington, DE. FAB/MS samples of free boronic acids
did not give consistent results making it difficult to
-18-
W094/25049 2161216 PCT~S94/04058
monitor the removal of ester protecting groups by this
means. However, the presence of the pinanediol and the
pinacol groups are readily observed in NMR spectra. For
the pinanediol ester, a methyl group is observed at
0.9 and the methyl groups of the pinacol groups are
observed as singlet at ~ l.l. Following the removal of
pinanediol protecting group, MS were run by treating the
sample with -2 equivalents of pinacol in methanol for 5
minutes and evaporating the solvent. Similarly, MS
samples of free boronic acid, obtained by removal of the
pinacol, were prepared by treating with pinanediol. In
some cases, ethylene glycol was used as a matrix for
mass spectroscopy to yield the boronic acid-
ethyleneglycol ester (designated EG ester). For the
subsequent Example see Table l for analytical data.
Example l
Synthesis of Ac-(D)Phe-Pro-NH-CH~(CH2)4CN1B02-C~
The intermediate, Ac-(D)Phe-Pro-NH-CH[(CH2)4Br]BO2-
CloHl6, was prepared using the mixed anhydride
procedure. Ac-(D)Phe-Pro-OH (3.04 g, l0 mmol) was
dissolved in 50 mL of THF and N-methylmorpholine (l.l
mL, l0 mmol) was added. The solution was cooled to
-20C using a CCl4 dry ice bath and isobutyl
chloroformate (l.30 mL, l0 mmol) was added. After 5 min
at -20C, the mixture was added to NH2-CH[(CH2)4Br]BO2-
CloHl6-HCl (3.81 g, l0 mmol) which was dissolved in 20 mL
of THF and precooled to -20C. Triethylamine ( 1.39 mL,
l0 mmol) was added and the mixture was allowed to stir
for l h at -20C and 2 h at room temperature. Insoluble
material was removed by filtration and the filtrate was
evaporated under a reduced pressure. The residue was
dissolved in 50 mL of ethyl acetate and washed
subsequently with 75 mL of 0.2 N HCl, 5% NaHCO3, and
saturated aqueous sodium chloride. The organic phase
was dried over Na2SO4 and concentrated in vacuo to give
--1 9-
WO 94/25049 PCT/US94/04058
216 121G
Ac-(D)Phe-Pro-NHCH[(CH2)4Br]BO2_CloHl6 (6.01 g, 95%
yield).
The bromide (1.89 g, 3.0 mmol) and tetra--n-butyl
ammonium cyanide (3.2 g, 11.8 mmol, 4 eq) were dissolved t
in 50 mL of acetonitrile. This solution was heated at
90C for 3 h and solvent was removed under reduced
pressure. The residue was dissolved in 50 mL of ethyl
acetate and was washed with three 50 mL portions of
saturated aqueous NaCl. The ethyl acetate solution was
dried over anhydrous Na2SO4 and evaporated to give 2.5 g
of crude product. It was purified by silica gel
chromatography using 5% MeOH in CHCl3 as an eluent to
yield the desired product (0.50 g, 29% yield).
LRMS (NH3-CI) m/e calcd. for M (C32H4sN4OsB) + NH4+:
594.4. Found: 594. HRMS(NH3-CI) m/e calcd. for M
(C32H4sN4OsB) + H+: 577.3561. Found: 577.3555.
Example 2
Synthesis of Ac-(D)Phe-Pro-NE~CH r (cH2)4c(NH)NH2l-Bo2
20 C~ 6-henzene sulfonic ~ci~
The nitrile, (Example 1, 0.40 g, 0.70 mmol), was
dissolved in 50 mL of a cold solution of saturated HCl
in methanol and the solution was stirred overnight at
4C. The solution was then concentrated under reduced
25 pressure. The residue was dissolved in anhydrous
methanol (50 mL), gaseous NH3 was bubbled through the
solution for 1 h, and the solution was heated at 50 C
for 3 h. Solvent was evaporated, the residue was
suspended in minlmum volume of methanol, and 0.11 g of
30 benzenesulfonic acid (1 eq) was added. Methanol was
evaporated and the residue was triturated with hexane to
yield the desired product as a pale yellow powder (0.52
g, 99 % yield).
FABMS: m/e calculated for M (C32H4gNsOsB) + H+:
594.38. Found: 594.14. HRMS(NH3-CI) m/e calcd for M
(C32H48NsOsB) + H+: 594.3827. Found: 594.3824.
--20--
WO94/250JY 21612 1 6 rCT~594/~4058
Example 3
Synthesis of Ac-(D)Phe-Pro-NHCH~(CH2)~NHC(NH)HlBO2-Cl~H
or Ac-(D)Phe-Pro-horoOrn(CH=NH)-C~
Ethyl formimidate-HCl was prepared by the procedure
- of Ohme and Schmitz Angew. C~em. Internat. Edit. 6 566
(1967) and Ac-(D)Phe-Pro-boroOrn-CloH16 was prepared by
the procedure of Kettner et al. (1990). The formimidate
(1.29 g, 11.7 mmol) and 4-N,N-dimethylaminopyridine
(1.44 g) were added to a solution of Ac-(D)Phe-Pro-
boroOrn-C1oH16-BSA (2.78 g, 3.92 mmol) dissolved in g0 mL
of ethanol. The resulting solution was refluxed for 8
h. After removal of solvent, the residue was purified
by chromatography using a column of Sephedex~LH 20 and
methanol as a solvent to give pure product (1.28 g, 56 %
yield).
HRMS(NH3-CI) m/e calcd. for M (C3lH46BN5O5) + H :
580.3670. Found: 580.3679.
Example 4
Synthesis of Ac-(D)Phe-Pro-NHCH~(CH~)3-NHC(NH)HlB(OH)~
The pinanediol protecting group on the boronic acid
portion of Ac-(D)Phe-Pro-NHCH[(CH2)3-NHC(NH)H]-BO2-
C1oH16-HCl (Example 3) was removed by
transesterification using the procedure we have
described previously in U.S.Application 08/010731. The
pinanediol ester (0.30 g, 0.51 mmol) and phenyl boronic
acid (0.31 g, 2.6 mmol) were suspended in 10 mL of a 1:
1 mixture of ether and water and was allowed to stir for
2.5 h at room temperature. The phases were separated
and the aqueous phase was extensively washed with ether.
The aqueous phase was evaporated to yield a solid. This
material was triturated with ether to give the desired
product as an amorphous white solid, 0.20 g (83 %
yield). LRMS (NH3-CI) m/e calcd. for the pinacol ester
M (C27H42N5O5B) + H+: 528.3. Found: 528. HRMS (NH3-
-21-
~'094/25049 ~ ` PCT~S94/04058
~6i2i6
CI) m/e calcd. for the pinacol ester M (C27H42NsOsB)
H+: 528.3357. Found: 528.3347.
Example 5
Synthes;s of Roc-Pro-N~C~ r (CH2)~NHC(NH)HlRO2-ClQ~1~
Boc-Pro-boroOrn-C1oH16-BSA was also prepared by the
procedure described previously (Kettner et al. 1990).
This peptide (3.0 g, 6.5 mmol) was dissolved in 25 mL of
absolute ethanol, 4-N,N-dimethylaminopyridine (1.6 g,
12.9 mmol) and ethyl formimidate-HCl (1.4 g, 12.9 mmol)
were added. The solution was heated on a 85 C oil bath
for 1 h. Solvent was evaporated and the residue was
dissolved in methanol and was chromatogramed on a 2.5 X
100 cm column of LH20 in methanol to yield 1.3 g of the
desired product.
LRMS (NH3-CI) m/e calcd. for M (C2sH43N4OsB) + H~:
491.5. Found: 491.
Example 6
Synthesis of Roc- (D)Phe-Pro-NHCH r (CH~L~-NHC(NH)HlRO2-
~10~16
The reaction was run using the procedure described
for Example 3. Boc-(D)Phe-Pro-boroOrn-C1oH16-BSA (3.7
g, 4.78 mmol), 4-N,N-dimethylaminopyridine (1.71 g, 13.8
mmol), and ethyl formimidate-HCl (1.54 g, 13.8 mmol)
were dissolved in 50 mL of absolute ethanol and was
heated at 85 C for 7 h. The desired product was
obtained by chromatography on a column of LH 20 in a
yield of 1.56 g.
HRMS (NH3-CI) m/e calcd for M (C34Hs2NsO6B) + H+:
638.4089. Found: 638.4082.
Example 7
Synthesis of Roc-(D)Phe-Pro-NHCH r (CH~L3_
3~ NHC(NH)HlR(OH)2 Boc-(D)Phe-Pro-NHcH[(cH2)3-
NHC(NH)H]BO2-C1oH16- 0.40 BSA, 0.60 HC1 (Example 6, 0.16
-
W094/25049 ~161216 PCT~S94/0~58
g, 0.22 mmol) and phenyl boronic acld tO.13g, 1.1 mmol)
were placed in mixture of 5 mL of ether and 5 mL of
water and was allowed to stir for 4 h at room
temperature. The phases were separated and the organic
phase was washed with 5 mL of water. The combined
aqueous phases were extensively washed with ether. The
aqueous phase was evaporated and the residue triturated
with ether to yield the desired product as a white
solid, 0.10 g. LRMS (NH3-CI) m/e calcd. for the pinacol
ester M (C30H4gNsO6B) + H+: 586.4. Found: 586. HRMS
(NH3-CI) m/e calcd. for the pinacol ester M
(C30H48Nso6B) + H+: 586.3776. Found: 586.3772.
Example 8
Synthesis of H-(D)Phe-Pro-NHCH~ICH~)~-NHC(NH)HlB02-
QHl~-2Hcl
Boc-(D)phe-pro-NHcH[(cH2)3-NHc(NH)H]Bo2-cloHl6-o-4o
BSA, 0.60 HCl (Example 6, 0.20 g, 0.25 mmol) was
dissolved in 2 mL of 4 N HCl: dioxane and was allowed to
stir for 1 h at room temperature. Solvent was
evaporated and the residue was triturated with ether to
yield 0.18 g of the desired product.
HRMS (NH3-CI) m/e calcd for M (C2gH44NsO4B) ~ H~:
538.3565. Found: 538.3569.
Example 9
Synthesis of H-tD)Phe-Pro-NHCH~(CH~)~-NHC(NH)HlB(OH)2
H-(D)phe-pro-NH-cH[(cH2)3-NH-c(NH)H]Bo2-cloHl6-o-35
BSA, 0.65 HCl (Example 8, 0.10 g, 0.16 mmol) was allowed
to react with phenyl boronic acid according to the
procedure in Example 4 to yield the desired product,
O.053 g. LRMS (NH3-CI) m/e calcd. for the pinacol ester
M (C2sH40NsO4B) + H+: 486.3. Found: 486. HRMS (NH3-
CI) m/e calcd for pinacol ester M (C2sH40NsO4B) + H~:
486.3251. Found: 486.3255.
-23-
WO 94/25049 PCT/US94/04058
',~ '' ' ' '~ '
21fil216 Example lo
Synthesis of H2NCHrCH2C~ m-CNlB02C~Hl6 HC1 or
H-horoPhe(m-CN)-C;LQ~16 HCl
The first intermediate, Cl-CH[CH2-(m-
~; cyanophenyl)]BO2-C1OH16, was prepared from m-cyanobenzyl
bromide and dichloromethyl boronate pinanediol. Zinc
dust (1.0 g) in 1 mL of THF was cooled to 0-5C and a
solution of m-cyanobenzyl bromide (1.37 g, 7.0 mmol) in
7 mL of THF was added dropwise (5 sec/drop). The
reaction mixture was allowed to stir at 5C for 2 h. A
mixture consisting of LiBr (1.22 g, 14 mmol), CuCN (0.63
g, 7.0 mmol), and 6 mL of THF was placed in a 50 ml
flask and cooled to -40C; then the benzylic organozinc
reagent was added by cannulation. The mixture was
allowed to warm to -20C and stir for 5 min. It was
cooled to -78C and neat dichloromethyl boronic acid
pinanediol (1.47 g, 5.6 mmol) was added dropwise. The
resulting mixture was stirred at -78C for 2 h and at
room temperature for 2 days. Saturated aqueous NH4Cl
(20 mL) was added to the mixture and the aqueous
solution was extracted with three 20 ml portions of
ether. The combined organic layers was dried over
anhydrous MgSO4 and evaporated ln vacuo to give crude
compound (1.8 g) . It was purified by silica gel
chromatography where the column was stepwise eluted with
hexane (100 mL) and then 15% ether in hexane (200 mL) to
give the desired product 0.53 g (27% yield). LRMS(NH3-
CI) m/e calcd. for M (C1gH23NO2BCl)+NH4+: 361.2. Found:
361.1.
To a solution of hexamethyldisilazane (0.21 mL,
O.98 mmol) in 2 mL of THF at -78C was added n-
butyllithium (1.45 M, 0.67 mL, 0.98 mmol). The solution
was allowed to slowly warm to room temperature to ensure
the anion generation was complete. The resulting
solution was then cooled to -78C and Cl-CH[CH2- ~m-
cyanophenyl)]BO2-CloHl6 (0.33 g, 0.98 mmol) in 2 mL of
-24-
~ WO 94/250~9 2161216 PCT/US94/04058
THF was added. The mixture was allowed to warm to room
temperature and to stir overnight. Solvent was
evaporated and 8 mL of hexane was added to give a
suspension. HCl in dioxane (4.1 N, 1.5 mL, 6.0 mmol)
was added at -78C. The mixture was slowly warmed to
room temperature and stirred for 2 h. Additional hexane
(6 mL) was added and crude product was isolated as a
precipitate. This product was dissolved in chloroform
and insoluble material was removed by filtration. The
filtrate was evaporated at a reduced pressure to give an
oil (~0.2 g). Final purification was achieved by
chromatography on a column of SephedexlM LH 20 column
using methanol as a solvent. H-boroPhe(m-CN)-C1oH16-HCl
was obtained as an oil (0.12 g, 34% yield) . HRMS(NH3-
CI) m/e calcd. for M (C1gH26BN2O2) + H~: 325.2087.
Found: 325.2094.
Example 11
Synthesis of Ac-(D)Phe-Pro-horoPhe(m-CN)-C~H~
Ac-(D)Phe-Pro-OH (0.10 g, 0.33 mmol) and N-
methylmorpholine (0.037 mL, 0.33 mmol) were allowed to
react with isobutyl chloroformate (0.043 mL, 0.33 mmol)
in 5 mL of THF at -20C. After 5 min, H-boroPhe(m-CN)-
C1QH16-HCl, (Example 10, 0.12 g, 0.33 mmol) dissolved in
3 mL of cold THF and triethylamine (0.046 mL, 0.33 mmol)
were added. The mixture was allowed to stir at -20C
for 1 h and to stir at room temperature for an
additional hour. Insoluble material was removed by
filtration and solvent was evaporated. The residue was
dissolved in ethyl acetate and was washed with 0.20 N
HCl, 5 % NaHCO3, and saturated aqueous NaCl. The
organic layer was dried over anhydrous Na2SOg and was
evaporated in vacuo to give 0.2 g of an oil. It was
purified by chromatography on a column of SephedexTM LH
20 yielding 0.13 g of desired product (65% yield).
--25--
W094/25049 PCT~S94/0~58
HRMS(NH3-CI ~ ~e calcd. for M (C3sH43BN40s) + H+:
611.3405. Found: 611.3416.
Example 12
Synthesis of Ac-(D)Phe-Pro-horoPherm-C(NH)NH~l-C~
Ac-(D)Phe-Pro-boroPhe(m-CN)-CloHl6, Example ll, (50
mg) was dissolved in 5 mL of saturated solution of HCl
in methanol. The solution was allowed to stir overnight
at 4 C. After removal of solvent, the residue was
resuspended in 5 mL of anhydrous methanol, cooled to
O~C, and anhydrous NH3 was bubbled through the solution
for 0.5 h. It was heated at 60C for 6.2 h. Solvent
was evaporated and one equivalent of benzene sulfonic
acid (13 mg) and 1 mL of methanol were added. Solvent
was evaporated under N2 and the product was triturated
with ether to give the desired product as a pale brown
powder (65 mg, 100% yield). HRMS(NH3-CI) m/e calcd. for
M (C3sH47BNsOs) + H+: 628.3670. Found: 628.3688.
Example 13
Synthesis of Ac-(D)Phe-Pro-horoPhe(m-CH2NH2)-C1o~l~
Ac-(D)Phe-Pro-boroPhe(m-CN)-CloH16 was placed in 5
mL of methanol, 10% Pd/C (25 mg) and O.lN HCl (0.41 mL)
were added, and the mixture was stir under H2 at room
temperature for 2.5 h. The solution was filtered
through Celite and washed with 20 mL of methanol. The
filtrate was concentrated under a reduced pressure and
the residue was triturated with ether to give pure
product as white powder (15.6 mg, 59% yield). HRMS(NH3-
CI) m/e calcd. for M (C35H47N40sB) + H+: 615.3718.
Found: 615.3700.
Example 14
Syn~hesis of Ac-(D)Phe-Pro-boroPhe(m-Rr)-C10~16
Cl-CH[CH2-~m-bromo-phenyl)]B02-CloHl6 was prepared
making the anion of m-~romobenzyl bromide and coupling
-26-
~ WO 94/25049 2161216 PCT/US94/04058
it to dichloromethyl boronic acid pinanediol. This
intermediate and the corresponding amine were prepared
using the procedure described for Example 10. The amine
was coupled to Ac- (D)Phe-Pro-OH using the method
described in Example 11.
LRMS (NH3-CI) m/e calcd. for M (C34H43N3OsBrB) + H+:
666.3. Found: 666.2.
Example 15
Synthesis of Ac- ~D)Phe-Pro-horoArg (CN)-Cm~l~
Ac- (D)Phe-Pro-boroOrn-CloH16-HCl ~0.15 g, 0.25
mmol), triethylamine (0.035 mL, 0.25 mmol), and diphenyl
cyanocarbonimidate (Aldrich, 0.060 g, 0.25 mmol) were
heated at a gentle reflux for 5 h in THF and then
stirred overnight at room temperature. The sample was
diluted with chloroform and washed with water and
saturated aqueous NaCl. It was dried over K2CO3 and
purified by silica gel chromatgraphy using methanol:
chloroform (1:9) as a solvent to yield 80 mg of Ac-
(D)Phe-Pro-NH-CH [(cH2) 3-NH-C (N-CN) O-ph]Bo2-cloHl6.
LRMS (NH3-CI) m/e calcd. for M (C38H49N6O6B) + H :
697.7. Found: 697.
The above product (0.060 g, 0.080 mmol) was
dissolved in 0.5 mL of THF and was allowed to react with
1 equivalent of 30% aqueous ammonia for 30 min at room
temperature. Four additional equivalent of ammonia were
added and the solution was allowed to stir overnight at
room temperature. A large excess of ammonia was added
and the reaction mixture was allowed to stir 2 days at
room temperature. The reaction mixture was diluted with
methylene chloride and was washed with water and
saturated aqueous NaCl. It was dried over K2CO3 and
purified by chromatography on a silica gel column using
methanol and chloroform (1:9) as a solvent to yield 15
3~ mg of the desired product. I.RMS(NH3-CI) m/e calcd. for
M (C32H46N7O53) + H~: 619.5. Found: 620.
W094/2i049 PCT~S94/04058
216~21~ -
Example 16
Synthesis of Ac-(D)Phe-Pho-boroPhe(p-CN)-ClQH16
ClCH[CH2C6H4-p-CN]BO2C1oH16 was prepared by making
the anion of p-cyanobenzyl bromide and coupling it to
dichloromethyl boronate pinanediol. This intermediate
and the corresponding amine were prepared using the
procedure described for Example 10. NH2CH[CH2C6H4-p-
CN]BO2C1oH16 (Example 78) was coupled to Ac-(D)Phe-Pro-
OH using the method described in Example 11.
HRMS (NH3-Cl)m/e calcd. for M (C3sH43N4OsB) + H~:
611.3405. Found: 611.3408.
Example 17
Synthesis of Boc-(D)Phe-Pro-horoPhe(mC~)-C1nH16
Boc-(D)Phe-Pro-boroPhe(mCN)-C1oH16 was prepared by
reacting Boc-(D)Phe-Pro-OH (0.43 g, 1.2 mmol), H-
boroPhe(mCN)-C1oH16-HCl (0.42 g, 1.2 mmol), N-
methylmorpholine (0.26 mL, 2.4 mmol),
hydroxybenzotriazole-H2O (0.36 g, 2.4 mmol), and
dicyclohexylcarbodiimide (0.25 g, 1.2 mmol) in 20 mL of
dichloromethane overnight at room temperature. The
reaction mixture was filtered and the filtrate was
chromatogramed on a 2.5 X 100 cm column of Sephedex LH-
20 in methanol to yield 0.36 g of the desired product.
Example 18
Synthesis of H-(D)Phe-Pro-horoPhe(mCN)-C1QH1~ HCl
Boc-(D)Phe-Pro-boroPhe(mCN)-C1oH16 (0.21 g) was
allowed to react with 2 mL of 4 N HCl dioxane for 2 h at
room temperature. Solvent was removed by evaporation
and the residue was triturated with ether to yield 0.11
g of the desired product as a white solid.
Example 19
Synthesis of H-(D)Phe-Pro-boroPhe(mCN)-OH-HCl
-28-
W094/2i049 PCT~S94/040~8
2161216
H-(D)Phe-Pro-boroPhe(mCN)-C1oH16-HCl (0.63 g, 1.0
mmol) was allowed to react with 5 equivalents of
phenylboronic acid using the procedure described for
Example 7 to yield 0.46 g of product.
- Example 20
Synthesis of N.N Dimethyl-(D)Phe-Pro-horoPhe(mCN)-OH-HCl
H-(D)Phe-Pro-boroPhe(mCN)-OH-HCl (0.20 g, 0.42
mmol), 37% aqueous formaldehyde (0.34 mL, 4.2 mmol) were
dissolved in 2 mL of acetonitrile. Sodium
cyanoborohydride (0.080 g, 1.3 mmol) was added and after
5 min glacial acetic acid (20~L) were added. The
reaction pH was ~7. After S h, additional acetic acid
(20 ~L) were added and the mixture was stirred for 1 h.
The reaction mixture was poured into 20 mL of ethyl
acetate and the organic phase was washed with 10 mL of
saturated aqueous sodium chloride and dried over
anhydrous sodium sulfate. Evaporation of solvent
yielded 0.16 g of an oil which was triturated with ether
to give a white solid.
Example 52
Synthesis of Ac-(D)Phe-Pro-NH-H r ~CH~ c (NH)NHCH3l R (OH)2
The intermediate, Ac-(D)Phe-Pro-NH-
CH[(CH2)3Br]BO2C1oH16, was prepared using the mixed
anhydride procedure of example 1. A solution of this
bromide (0.35 g, 0.57 mmol) and 1-methyl-2-thiourea
(0.077 g, 0.85 mmol) in 10 mL of absolute ethanol was
refluxed for 18 hours. After cooling the solvent was
removed under vacuum, and the product was separated from
excess thiourea employing chromatography (elution:
methanol) on Sephadex~ LH-20 gel to provide 0.31 g (77~)
of the isothiouronium product. This boronic acid ester
(0.28 g) was then deprotected as described in example 4
to afford 0.13 g (57%) of the desired product. L~S
(ESI) m/e calcd. for M (C22H34BNsOsS) + H+: 492. Found:
-29-
W094/25049 2161216 PCT~S94/04058
492. HRMS (NH3-CI) m/e calcd. for ethylene glycol ester
M (C24H36BNsOsS) + H+: 518.260847. Found: 518.261656.
Example 54
Synthesis of Ac- (D ) Phe-Pro-NH-C~ r (CH2)3NHC(NH)NHCH3l-
R ( OH)~
A solution of Ac-(D)Phe-Pro-boroOrn-BO2C10H16 HCl
[0.50 g, 0.85 mmol, prepared by the procedure of Kettner
et al.(1990)], 4-methylaminopyridine (0.21 g, 1.7 mmol),
N-methylamino-iminomethanesulfonic acid (0.24 g, 1.7
mmol), and 10 mL of absolute ethanol was refluxed for 18
hours. After cooling the mixture was filtered and the
precipitate was washed with chloroform. The combined
filtrates were concentrated under vacuum, and the
residue was dissolved in 10 mL of chloroform. The
chloroform solution was washed with ice-cold 0.1 N
hydrochloric acid (2 X 3 mL), ice-cold water (2 X 3 mL),
and brine. The resulting organic solution was then dried
over anhydrous magnesium sulfate, filtered, and
concentrated. The product was purified employing
chromatography (elution: methanol) on Sephadex~ LH-20
gel to provide 0.30 g (55%) of the guanidine. This
boronic acid ester was then deprotected as described in
example 4 to afford 0.14 g (59%) of the desired product.
LRMS (NH3-CI) m/e calcd. for ethylene glycol ester M
(C24H37BN6Os) + H+: 501. Found: 501. HRMS (NH3-CI) m/e
calcd. for ethylene glycol ester M (C24H37BN6O5) + H+:
501.299674. Found: 501.300760.
The examples of Table 1 can be prepared by the
schemes and procedures described above using the
appropriate starting materials.
-30-
~ wo 94/2~049 2 ~ ~12316~ ~ PCT/US94/04058
Table 1.
EX MS Method LRMS LRMS
# Compound CALC'D FOUND
NH3/CI 594.4 594
Ac-(D)Phe-Pro-NH- (M+NH4)
CH[(CH2)4CN]BO2C1 oH 16
2 NH3/CI 594.4 594
Ac-(D)phe-pro-NH-cH[(cH2)4- (M+H)
C(NH)NH2]B02C1 oH1 6-BSA
3 NH3/CI 580.4 580
Ac-(D)Phe-Pro-boroOrn(CH=NH)]- (M+H)
C1 oH 16-HCI
4 NH3/CI 528.3 528
Ac-(D)Phe-Pro-boroOrn(CH=NH)]-OH HCI pinacol
ester+H
NH3/CI 491.5 491
Boc-Pro-boroOrn(CH=NH)-C10H16 HCI (M+H)
6 NH3/CI 638.4 638
Boc-(D)Phe-Pro-boroOrn(CH=NH)]- (M+H)
C10H16-o.5 HCI 0.5 BSA
7 NH3/CI 586.4 586
Boc-(D)Phe-Pro-boroOrn(CH=NH)]-OH-0.6 pinacol
HCI 0.4 BSA ester+H
8 NH3/CI 538.4 538
H-(D)Phe-Pro-boroOrn(CH=NH)]- (M+H)
C10H16-o 5 HCI 0.5 BSA
9 NH3/CI 486.3 486
H-(D)Phe-Pro-boroOrn(CH=NH)]-OH-0.65 pinacol
HCI-0.35 BSA ester+H
1 0
H-boroPhe(mCN)-C1 oH1 6-HCI
1 1 NH3/CI 611.3 611
Ac-(D)Phe-Pro-boroPhe-(m-CN)-c10H16 (M+H)
1 2 NH3/CI 628.4 628
Ac-(D)Phe-Pro-boroPhe-(m-C(NH)NH2)- (M+H)
C1 oH1 6-BSA
1 3 NH3/CI 615.4 615
Ac-(D)Phe-Pro-boroPhe-(m-CH2NH2)- (M+H)
C1 oH1 6-Hcl
1 4 NH3/CI 683.4 683
Ac-(D)Phe-Pro-boroPhe-(m-Br)-C10H16 (M+NH4)
1 5 NH3/CI 619.5 620
Ac-(D)Phe-Pro-boroArg(CN)-C10H16-HCI (M+H)
1 6 NH3/CI 628.4 628
Ac-(D)Phe-Pro-boroPhe-(P-CN)-c10H16 (M+NH4)
1 7 NH3/CI 686.4 686
Boc-(D)Phe-Pro-boroPhe-(m-CN)-C10H16 (M+NH4)
--31--
WO 94/25049 PCT/US94/04058
2~
1 8 NH3/CI 569.3 569
H-(D)Phe-Pro-boroPhe-(m-CN)- (M+H)
C10H 16-HCI
1 9 NH3/CI 461.2 461
H-(D)Phe-Pro-boroPhe-(m-CN)-OH HCI EG ester+H
2 0 NH3/CI 489.3 489
N,N-(CH3)2-(D)Phe-Pro-boroPhe-(m-CN)- EG ester+H
OH-HCI (ISOMER 1)
2 1 NH3/CI 615.4 615
Ac-(D)Phe-Pro-boroPhe-(p-CH2NH2)- (M+H)
C1oH16- BSA
2 2 FAB 628.37 628.44
Ac-(D)Phe-Pro-boroPhe-(p-C(NH)NH2)- (M+H)
C10H16- BSA
2 3 NH3/CI 520.3 520
Ac-(D)Phe-Pro-boroPhe-(m-CN)-OH HCI EG
ester+NH4
2 4 NH3/CI 556.2 556
Ms-(D)Phe-Pro-boroPhe-(m-CN)-OH-HCI EG
ester+NH4
2 5 NH3/CI 583.4 583.3
N-CH3-(D)Phe-Pro-boroPhe-(m-CN)- (M+H)
c- oH 1 6-HCI
2 6 NH3/CI 422.3 422
H-Pro-boroPhe-(m-CN)-C10H16-HCI (M+H)
2 7 NH3/CI 676.4 676.4
Boc-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H 16
2 8 NH3/CI 670.4 670.4
Boc-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16
2 9 NH3/CI 576.3 576
H-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 oH1 6-HCI
3 0 NH3/CI 570.3 570
H-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 oH16 HCI
3 1 NH3/CI 654.3 654
Ms-(D)Thiazolylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16
3 2 NH3/CI 648.3 648
Ms-(D)3-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H 16
3 3 NH3/CI 700.4 700
N-Boc-N-CH3-(D)Phe-Pro-boroPhe-(m- (M+NH4)
CN)-C10H16
--32--
WO 94/25049 2161216 7 ' 'i'' PCT/US94/04058
3 4 NH3/CI 670.4 670
Boc-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16
3 5 NH3/CI 481.3 481
Ac-pro-borophe-(m-cN)-c1 oH 16 (M+NH4)
3 6 NH3/CI 692.4 692
Boc-(D)2-Thienylalanine-Pro-boroPhe-(m- (M+NH4)
CN)-C10H 16
3 7 NH3/CI 570.3 570
H-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H16 HCI
3 8 NH3/CI 575.3 575
H-(D)2-Thienylalanine-Pro-boroPhe-(m- (M+H)
CN)-C1 oH1 6-HCI
3 9 NH3/CI 648.3 648
Ms-(D)2-Pyridylalanine-Pro-boroPhe-(m- (M+H)
CN)-C10H 16
4 0 NH3/CI 670.3 670
Ms-(D)2-Thienylalanine-Pro-borOPhe-(m- (M+NH4)
CN)-C10H16
4 1 NH3/CI 574.3 574
(2-Pyrimidylthio)acetyl-Pro-boroPhe-(m- (M+H)
CN)-C10H16
4 2 NH3/CI 553.3 553
trans-3-(3-pyridyl)acryl-Pro-boroPhe-(m- (M+H)
CN)-C10H 16
4 3 NH3/CI 573.3 573
(4-Pyridylthio)acetyl-Pro-boroPhe-(m-CN)- (M+H)
C10H16
4 4 NH3/CI 578.3 578
Succinyl-(D)Phe-Pro-boroPhe-(m-CN)-OH EG
ester+NH4
4 5 NH3/CI 553.3 555
3-Pyridylpropionyl-Pro-boroPhe-(m-CN)- (M+H)
C10H16
4 6 NH3/CI 672.4 672
Boc-(D)Phe-Aze-boroPhe-(m-CN)-C10H16 (M+NH4)
4 7 NH3/CI 555.3 555
H-(D)Phe-Aze-boroPhe-(m-CN)- (M+H)
C10H 16-HCI
4 8 FAB 445.5 445
Hydrocinnamoyl-Pro- EG ester+H
boroOrn(CH=NH)]OH-BSA
4 ~ ESI 461 461
Hydrocinnamoyl-Pro- (M+H)
borolrg(CH2CH=CH2)-OH HBr
5 0 ESI 435 435
Hydrocinnamoyl-Pro-borolrg(CH3)-OH- (M+H)
HBr
WO 94/25049 PCT/US94/04058
~l6~2~
5 1 NH3/CI 718 718
Cbz-(D)Phe-Pro-borolrg(CH3)-C10H16 (M+H)
HBr
5 2 ESI 492 492
Ac-(D)Phe-Pro-borolrg(CH3)-OH HBr (M+H)
5 3 ESI 449 449
Hydrocinnamoyl-Pro-borolrg(CH2CH3)-OH (M+H)
HBr
5 4 NH3/CI 501 501
Ac-(D)Phe-Pro-boroArg(CH3)-OH HCI EG es~er+H
ESI 418 418
Hydrocinnamoyl-Pro-boroArg(CH3)-OH- (M+H)
HCI
5 6 ESI 511 511
Ms-(D)Phe-Pro-boroArg(CH3)-OH- HCI (M+H)
5 7 ESI 482 482
Ms-(D)Phe-Pro-boroOrn(CH=NH)-OH HCI (M+H)
5 8 ESI 573 573
PhSO2-(D)Phe-Pro-boroArg(CH3)-OH (M+H)
HCI
5 9 ESI 544 544
PhSO2-(D)Phe-Pro-boroOrn(CH=NH)-OH (M+H)
HCI
6 0 ESI 500 500
Ms-(D)Phe(4-fluoro)-Pro- (M+H)
boroOrn(CH=NH)-OH HCI
6 1 ESI 587 587
PhCH2SO2-(D)Phe-Pro-boroArg(CH3)-OH (M+H)
HCI
6 2 ESI 558 558
PhCH2SO2-(D)Phe-Pro-boroOrn(CH=NH)- (M+H)
OH HCI
63 ESI 510 510
CH3CH2CH2SO2-(D)Phe-Pro- (M+H)
boroOrn(CH=NH)-OH HCI
6 4 ESI 539 539
CH3CH2CH2SO2-(D)Phe-Pro- (M+H)
boroArg(CH3)-OH HCI
6 5 ESI 553 553
CH3(CH2)3SO2-(D)Phe-Pro- (M+H)
boroArg(CH3)-OH HCI
6 6 ESI 524 524
CH3(CH2)3SO2-(D)Phe-Pro- (M+H)
boroOrn(CH=NH)-OH HCI
67
Ac-(D)Phe-Sar-boroOrn(CH=NH)-OH HCI
68
Ms-(D)Phe-Sar-boroOrn(CH=NH)-OH-H
-34-
WO 94/~5049~, PCT 1594/1)4058
6 9
Phenethyl-S02-(D)Phe-Sar-
boroOrn(CH=NH)-OH HCI
Boc-(D)Phe-Sar-boroOrn(CH=NH)-OH HCI
71
N-alpha-[boroOrn(CH=NH)-OH]-(2-trans
benzylcarboxamido)-cyclopentane-1 -
carboxamide HCI
72
H-(D)Phe-Sar-boroOrn(CH=NH)-
C1 oH 1 6-2HCI
73
Boc-(D)Phe-Sar-boroPhe(m-CN)-C1 oH1 6
74
Boc-(D)Phe-Aze-boroOrn(CH=NH)-
OH HCI
H-(D)Phe-Sar-boroPhe(m-CN)-
C 1 oH 1 6-2HCI
76
4-(Phenyl)benzoyl-boroOrn(CH=NH)-
C1 oH 1 6-HCI
77 NH3/CI 620 58 620 36
Z-(D)Phe-Pro-boroOrn(CH=NH)-OH HCI pinacol
ester+H
78
H-boroPhe-(p-CN)-C1 oH 1 6-HCI
7 9Boc-(D)Phe-Pro-
N(CH3)CH[(CH2)3NHC(NH)H]-B(OH)2
8 0Boc-(D)Phe-Pro-
N(Phenyl)CH[(CH2)3NHC(NH)H]-B(OH)2
8 1Boc-(D)Phe-Pro-
N(benzyl)CH[(CH2)3NHC(NH)H]-B(OH)2
8 2Boc-(D)Phe-Pro-
N(CH3)CH[(CH2)3NHC(NH)H]-B(OMe)2
8 3Boc-(D)Phe-Pro-
N(CH3)CH[(CH2)3NHC(NH)H]-B[N(Me)]2
8 4Boc-(D)Phe-Pro-
N(CH3)CH[(CH2)3NHC(NH)H]-B(F)2
8 5FMoc-(D)Phe-Pro-
NHCH[(CH2)3NHC(NH)H]-B(OC1 oH1 6)2
--35--
WO 94/25049 i PCT/US94/04058
2~6 ~2~6
8 6 Ac-(D)cyclohexylalanyl-Pro-
NHCH[(CH2)3NHC(NH)H]-B(Oc1 oH1 6)2
8 7 Ac-(D)Phe-Gly-NHCH[(CH2)3NHC(NH)H]-
B(OC1 oH1 6)2
8 8Ac-(D)Phe-Pro-
NHcH[(cH2)3NHc(NoH)NH2]
B(OC1 oH1 6)2
9 1 Ac- (D)phe-pro-borophe-(p-Br)-Gl oH 16
92 Ac- (D)Phe-Pro-boroPhe-(p-NH2)-C1oH16
93Ac- (D)Phe-Pro-boroPhe-(p-
NHC(NH)NH2)-C1 oH1 6
9 5Ac- (D)Phe-Pro-boroPhe-(p-
CH2NHC(NH)NH2)-C1 oH 16
9 6Ac- (D)Phe-Pro-boroPhe-(m-
CH2N HC(N H)N H2)-c1 oH 16
9 7Ac- (D)Phe-Pro-boroPhe-(m-
CH2NHC(NH)NHCN)-C1 oH 1 6
9 8Z-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)-
Asn-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)-
Asn-NHCH[(CH2)3NHC(NH)H]-
B(OC1 oH1 6)2
9 9H-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)-
Asn-Leu-Ser(Ot-Bu)-Asn-Leu-Ser(Ot-Bu)-
Asn-NHCH[(CH2)3NHC(NH)H]-
B(OC1 oH1 6)2
100 Z-Leu-Ser-Asn-Leu-Ser-Asn-Leu-Ser-Asn-
Leu-Ser-Asn-NHCH[(CH2)3NHC(NH)H]-
B(OC1 oH16)2
1 01 H-Leu-Ser-Asn-Leu-Ser-Asn-Leu-Ser-Asn-
Leu-Ser-Asn-NHCH[(CH2)3NHC(NH)H]-
B(OC1 oH1 6)2
--36--
W094/25049 2161216 . PCT~S94/04058
Utility
N-Acyl and N-peptide boronic acids which are
described in the present invention represent a novel
class of potent, reversible inhibitors of trypsin-like
enzymes. Trypsin-like enzymes are a group of proteases
which hydrolyzed peptide bonds at basic residues
liberating either a C-terminal arginyl or lysyl residue.
Among these are enzymes of the blood coagulation and
fibrinolytic system required for hemostasis. They are
Factors II, X, VII, IX, XII, kallikrein, tissue
plasminogen activators, urokinase-like plasminogen
activator, and plasmin. Enzymes of the complement
system, acrosin (required for fertilization), pancreatic
trypsin are also in this group. Elevated levels of
proteolysis by these proteases can result in disease
states. For example, consumptive coagulopathy, a
condition marked by a decrease in the blood levels of
enzymes of both the coagulation system, the fibrinolytic
system and accompanying protease inhibitors is often
fatal. Intervention by a synthetic inhibitor would
clearly be valuable. More specifically, proteolysis by
thrombin is required for blood clotting. Inhibition of
thrombin results in an effective inhibitor of blood
clotting. The importance of an effective inhibitor of
thrombin is underscored by the observation that
conventional anticoagulants such as heparin (and its
complex with the protein inhibitor, antithrombin III)
are ineffective in blocking arterial thrombosis
associated with myocardial infractions and other
clotting disorders. However, a low molecular weight
thrombin inhibitor, containing a different
functionality, was effective in blocking arterial
thrombosis [Hanson and Harker (1988) Proc. Natl. Acad.
-37-
W094l25049 ~ 2 ~ 6 PCT~S94/04058
Sci. U.S.A. 85, 3184-3188]. Therefore, we have chosen
to demonstrate utility of compounds in the inhibition of
thrombin, both as in buffered solutions and in plasma.
Specifically, the compounds have utility as drugs for
the treatment of diseases arising from elevated thrombin
activity such as myocardial infarction, and as reagents
used as anticoagulants in the processing of blood to
plasma for diagnostic and other commercial purposes.
When used in the processing of blood products, the
compounds of this invention may be mixed with whole
blood without the need for any additional
anticoagulants. The compounds of this invention serve
to inhibit blood coagulation thereby facilitating the
processing of whole blood into desired cellular
components or plasma proteins. Once the processing is
complete, the compounds may be removed, if so desired,
by membrane ultrafiltration, dialysis, or diafiltration
to afford the desired product. The low molecular weight
of these compounds relative to conventional
anticoagulants like heparin allow them to be separated
from desired products more easily.
Compounds of the present invention are expected to
be effective in the control of aberrant proteolysis and
a number of accompanying disease states such as
inflammation, pancretitis, and heritary angioedema.
The in vitro effectiveness of compounds of the
present invention as inhibitors of the blood coagulation
protease thrombin was determined under two different
conditions: (1) measurements in buffered solutions
using a synthetic substrate; ~2) measurement in plasma
where the rate of blood clotting is determined. For the
former, the chromogenic substrate S2238 (H-(D)Phe-Pip-
Arg-PNA, where PIP refers to pipecolic acid; Helena
Laboratories, Beaumonl, TX) was used following
procedures similar to those described in Kettner et al.
J. ~iol. Chem. 265 18289-18297 (1990). Here hydrolysis
-38-
W094/25049 ~ 61216 PCT~S94/04058
resulted in the release of pNA which was monitored
spectrophotometricaly by measuring the increase in
absorbance at 405 nm. The Michaelis constant, Km~ for
substrate hydrolysis was determined at 25 C in O.lO M
sodium phosphate buffer, pH 7.5, containing 0.20 M NaCl,
and 0.5 % PEG 8000 using the method of Lineweaver and
Burk.
Values of Ki were determined by allowing thrombin
(O.l9 nM) to react with substrate (0.20 mM) in the
presence of inhibitor. Reactions were allowed to go for
30 minutes and the velocities (rate of absorbance change
vs time) were measured in the time frame of 25-30
minutes. The following relationship was used to
calculate Ki values.
yQ-v~_ = I
v5 Ki(l + S/Km)
where
vO is the velocity of the control in the absence of
inhibitor;
VS is the velocity in the presence of inhibitor;
I is the concentration of inhibitor;
Ki is the dissociation constant of enzyme: inhibitor
complex;
S is the concentration of substrate;
Km is the Michaelis constant.
Inhibition of blood clotting activity of thrombin
in plasma was determined in rabbit plasma. Plasma was
prepared by diluting blood l:lO with 3.2% aqueous citric
acid and centrifuging. Buffer consisted of O.lO M Tris,
pH 7.4, containing 0.9% sodium chloride, and 2.5 mg/mL
bovine serum albumin. Bovine thrombin was obtained from
Sigma and was diluted to 24 NIH units/mL. Plasma (200
~L) and buffer (50 ~L) containing inhibitor were
incubated 3 min at 37 C in a fibrameter. Reactions
_3 G_
W094/25049 ~ 2 ~ ~ PCT~S94/0~58
were initiated by adding thrombin (50~L) and clotting
times measured. Controls were run under identical
conditions except in the absence of inhibitor. The
final concentration of thrombin was 4 NIH units/mL.
Table 2 - Inhibition of Thrombin.
Ex Kj a
# (nM)
750
2 0.26
3 0.38
4 0.28
6 0.085
7 0.040
8 0.18
9 .05
11 3.2
12 2.8
13 4.83
14 10
1~ 40
16 134
17 0.27
0.14
23 0.55
24 0.059
27 0.17
28 0.37
32 0.48
34 0.33
36 0.381
0.19
46 0.55
~859
54
-40-
W094/25049 PCT~S94/0~58
2161216 '~
62 0.03
63 0.5
64 0.~
67 8.2
73 81
74 <0.5
76 110
a Ki values were measure at 25 C at pH 7.5.
Another measure of compound effectiveness toward
prolonging clotting times can be reported as IC50 (level
of inhibitor required to prolong clotting to the time
observed for 2 NIH units/mL thrombin in the absence of
inhibitor). Representative of data for compounds of the
present invention, Examples 3, 7, 9, 11, and 12
increased thrombin clotting times 2-fold at 0.25,
<0.075, 0.10, 0.60, and 0.85 ~M, respectively.
The effectiveness of compounds of the present
invention as anticoagulants in vivo was demonstrated by
the prolongation of the activated partial thromboplastin
time of samples of blood taken from conscious dogs or
anesthetized rats after either oral or intravenous
administration at doses of the compounds from 0.5 to 10
mg/kg. Arterial or venous blood was withdrawn by
syringe and mixed with l/10 volume 3.2% sodium citrate.
Plasma was obtained after centrifugation and a standard
clinical activated partial thromboplastin time (APTT
reagent, Sigma Chemical Co., St. Louis, Mo.) determined
at 37C in a fibrometer. Results from blood samples
obtained at various times after dosing showed an
effective anticoagulant response which was at least
equivalent to doubling of activated partial
thromboplastin time as compared to the value obtained
prior to dosing. In this model, Examples 4, 57, and 77
-41-
W094/25049 PCT~S94/04058
2161216
were shown to be effective following i.v. dosing andExamples 4, 56, 57, 60, and 66 effective following oral
dosing. Similarly, oral administration of Examples 31
and 54 resulted in at least a 2-fold elevation in
anticoagulant activity in an identical model except
activity was measured by increases in thrombin clotting
times.
WO 94/25049 PCT/US94/04058
2 1 ~ 1 2 1 ~
~FoUFNCF I ISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Sheng-Lian O. Lee
John Matthew Fevig
Charles Adrian Kettner
David L. Carini
(ii) TITLE OF INVENTION: Amidino and Guanidino
Substituted Boronic Acid Inhibitors of Trypsin-Like Enzymes
( i i i ) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCEADDRESS:
(A) ADDRESSEE: The Du Pont Merck Pharmaceutical
Company
(B) STREET: 1007 Market Street, Legal Department
(C) CITY: Wilmington
(D) STATE: DE
(E) COUNTRY: U.S.
(F) ZIP: 19898
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.50 inch disk
(B) COMPUTER: Apple Macintosh
(C) OPERATINGSYSTEM: Apple Macintosh
(D) SOFTWARE: Microsoft Word
( v i ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/052,835
(B) FILING DATE:
(C) CLASSIFICATION: unknown
(v i i ) PRIOR APPLICATION DATA: None
W094125049 PCT~S94/04058
21612~6
( v i i i ) ATTORNEY/AGENT INFORMATION:
(A) NAME: Reinert, Norbert, F.
(B) REGISTRATION NUMBER: 18,926
(C) REFERENCE/DOCKETNUMBER: DM-6567-A
( i x ) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 302-892-8867
(B) TELEFAX: 302-892-8536
10 (2) INFORMATION FOR SEQ ID NO:1:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acids
(C) TOPOLCGY: linear
( i i ) MOLECULAR TYPE: peptide
(vi) ORIGINALSOURCE: synthetic
( i x ) FEATURE:
(D) OTHER INFORMATION: Example Number 98
at page 36 and within Table 1
( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Xaa Xaa A~n Leu Xaa A~n ~eu Xaa A~n Leu Xaa Asn
(2) INFORMATION FOR SEQ ID NO:2:
( i ) SEQUENCE CHARACTERISTICS:
( A ) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: linear
-44-
WO 94/25049 PCT/US94/04058
216121~
( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Leu Xaa Asn LQU Xaa A n Leu Xaa A~n Leu Xaa Asn
(3) INFORMATION FOR SEQ ID NO:3:
, ( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOLOGY: I i near
( i i ) MOLECULAR TYPE: peptide
(vi) ORIGINALSOURCE: synthetic
( i x ) FEATURE:
(D) OTHERINFORMATION: Example Number 100
at page 36 and within Table 1
( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Xaa Ser Asn Leu Ser A~n Leu Ser Asn Leu Ser Asn
1 5 10
(3) INFORMATION FOR SEQ ID NO:4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 2
(B) TYPE: amino acids
(C) TOPOL~GY: I i near
( i i ) MOLECULAR TYPE: peptide
(vi) ORIGINALSOURCE: synthetic
( i x ) FEATURE:
(D) OTHERINFORMATION: Example Number 101
at page 36 and within Table 1
( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Leu Ser Asn Leu Ser Asn Leu Ser Asn Leu Ser Asn
1 5 10
--45--
