Note: Descriptions are shown in the official language in which they were submitted.
1336960
--1--
INHIBITION OF SERINE PROTEASES IN THE
TREATMENT OF DEGENERATIVE DISORDERS
Technical Field
This invention is directed to pharmaceutical
compositions, diagnostic methods and treatment methods
that utilize compounds which function as inhibitors of
serine proteases.
Background
The inhibition of proteolytic enzymes by non-
toxic reagents is useful in the treatment of
degenerative disorders for which proteolysis is a
substantive element.
Protease inhibitors are widely utilized in
biomedical research. Serine proteases are the most
widely distributed class of proteolytic enzymes. Some
serine proteases are characterized as chymotrypsin-like
or elastase-like, based upon their substrate
specificity.
Chymotrypsin and chymotrypsin-like enzymes
normally cleave peptide bonds in proteins at a site at
which the amino acid residue on the carbonyl side is
typically Trp, Tyr, Phe, Met, Leu or another amino acid
residue which contains aromatic or large alkyl side
chains.
Elastase and elastase-like enzymes normally
cleave peptide bonds at a site at which the amino acid
residue on the carbonyl side of the bond is typically
Ala, Val, Ser, Leu or other similar, smaller amino
acids.
Both chymotrypsin-like and elastase-like
enzymes are found in leukocytes, mast cells, and
pancreatic juice in higher organisms, and are secreted
by many types of bacteria, yeast and parasites.
-2- 133 69 60
Several compounds are known which are
inhibitors of serine protease activity. U.S. Patent No.
4,659,855; U.S. Patent No. 4,623,645; U.S. Patent No.
4,547,371; Teshima et al (1982) J. of Biol. Chem.
257:5085-5091; Cha (1975) Biochem. Pharmacol. 24:2177-
2185.
Several substituted saccharin derivatives are
known. Some of these derivatives can be utilized as
enzyme inhibitors and others are useful as photographic
reagents.
Some 2-substituted saccharin derivatives have
been disclosed to function as inhibitors for elastase-
like and chymotrypsin-like enzymes. U.S. Patent No.
4,195,023 discloses 2-N-acyl derivatives of saccharin as
inhibitors of human leukocyte elastase (HLE) activity.
U.S. Patent No. 4,276,298 discloses 2-N-phenyl
substituted saccharin derivatives that can function as
elastase inhibitors. Powers et al. (1985) Biochemistry
24:2048-2058 discloses N-furoylsaccharin and N-(2,4-
dicyanophenyl)saccharin inhibitors for chymotrypsin-like
enzymes.
Svoboda et al. (1986) Collect. Czech. Chem.
Commun. 51:1133-9 discloses a saccharin derivative
containing an acetyl ester substituent in the
2-position. Similar compounds are disclosed as
photographic reagents in U.S. Patent No. 4,350,752, U.S.
Patent No. 4,263,393, U.S. Patent No. 4,410,618, and
U.S. Patent No. 4,363,865. There is no disclosure that
the therein described 2-substituted saccharin compounds
act as enzyme inhibitors.
U.S. Patent No. 3,314,960. discloses a
2-substituted saccharin derivative which may be used as
a sedative. The compound has a 2-glutaric acid group
attached to the nitrogen in the 2-position of saccharin.
1336960
--3--
Inhibitors of HLE and chymotrypsin-like
enzymes are useful for treating degenerative diseases
such as emphysema, rheumatoid arthritis and
- pancreatitis. Clinical symptoms found in these diseases
are believed to be controlled in large part by
uncontrolled elastase in the affected tissues, as well
as by uncontrolled chymotrypsin-like enzymes such as
cathepsin G and mast cell chymase. Thus the production
and utilization of specific inhibitors for these
proteolytic enzymes enables the diagnosis and treatment
of degenerative diseases. These inhibitors may also be
utilized to prevent the degradation of stored peptides
and proteins by specific proteolytic enzymes.
Summary
The present invention is directed to
pharmaceutical compositions, methods of inhibiting
proteolytic enzyme activity, diagnostic methods and a
method for treatment of degenerative disorders.
This invention is directed to pharmaceutical
compositions that contain a 2-substituted saccharin
derivative as an active agent together with a
pharmaceutically acceptable carrier. The 2-substituted
saccharin derivatives present in the pharmaceutical
compositions are represented by the general structural
formula tI):
R3
~1 \I
- S /
O ~ (I)
_4_ 1336960
where R1 is selected from the group consisting
of halogen, ester, aryloxy, alkylthio, arylthio,
sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl
and substituted derivatives thereof;
R2 is hydrogen or an electron-withdrawing
group; and
R3 and R4 are each independently selected from
the group consisting of hydrogen, halogen, cyano, nitro,
amino, amido and a straight, cyclic or branched-chain
carbon-containing group having from 1 to 20 carbon
R2
atoms; wherein the -CH- group is always appended to a
heteroatom on R1.
This invention is also directed to a method of
inhibiting proteolytic enzyme activity in which a liquid
sample containing proteolytic enzyme activity is
contacted with a 2-substituted saccharin derivative
corresponding to Formula I.
This invention is also directed to a
diagnostic method in which a body fluid sample is
contacted with a composition containing a 2-substituted
saccharin derivative corresponding to Formula I for a
time period sufficient to permit enzyme inhibition, and
then measuring the concentrations of inhibited and
uninhibited proteolytic enzyme remaining in the sample.
Preferred proteolytic enzymes monitored are human
leukocyte elastase and chymotrypsin-like enzymes. The
diagnostic method of the present invention enables the
detection of abnormal levels of the inhibited
proteolytic enzyme which is indicative of physiological
disorders.
This invention is further directed to a method
of treatment for degenerative disorders for such
disorders as emphysema, adult respiratory distress
syndrome, pancreatitis, rheumatoid arthritis and other
-
-5- 1336g 60
inflammatory disorders. In this treatment method, a
therapeutically effective amount of a pharmaceutical
composition containing a 2-substituted saccharin
derivative corresponding to Formula I as an active agent
is administered to a mammal with the degenerative
disorder.
Description of Preferred Embodiments
The present invention is directed to
pharmaceutical compositions that contain 2-substituted
saccharin derivatives as active ingredients and
pharmaceutically acceptable carriers, where the
2-substituted saccharin derivative is a compound
represented by the general structural formula (I):
R~
F~4-- 1 \N--CH--R1
~ S
~J~
O O
where R1 is selected from the group consisting
of halogen, ester, aryloxy, alkylthio, arylthio,
sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl
and substituted derivatives thereof;
R2 is hydrogen or an electron-withdrawing
group and
R3 and R4 are each independently selected from
the group consisting of hydrogen, halogen, cyano, nitro,
amino, amido and a straight, cyclic or branched-chain
carbon-containing group having from 1 to 20 carbon
-
-6- 1336960
R2
I
atoms; wherein the -CH- group is always appende~ to a
heteroatom on R1.
S R1 groups are preferably leaving groups having
a pKa of less than about 7 that carry away an electron
pair (March, Advanced Organic Chemistry, 3rd edition,
1985 (Wiley and Sons, N.Y.) page 219).
As used herein "substituted derivatives"
include the attachment to a group of a substituent
selected from alkyl, aryl, cycloalkyl alkylamidoaryl,
heterocyclyl, arylthio, nitro, amino, amido, sulfinyl
and sulfonyl groups.
Exemplary halogens include fluorine, chlorine,
bromine and iodine derivatives and exclude astatine
derivatives that are radioactive.
As used herein, the terms "alkyl" and "lower
alkyl" include C1 to C6 lower aliphatic groups, as for
example, methyl, ethyl, propyl, iso-propyl, n-butyl,
sec-butyl, t-butyl, n-pentyl, 2-methyl-3-butyl,
l-methylbutyl, 2-methylbutyl, neopentyl, n-hexyl,
1-methylpentyl, 3-methylpentyl, l-ethylbutyl,
2-ethylbutyl, 2-hexyl, 3-hexyl, and the like.
As used herein, the term "cycloalkyl" includes
C3 to C6 carbocyclic ring groups, as for example,
cyclopropyl, cyclopentyl, cyclohexyl and the like.
As used herein, the term "aryl" includes
aromatic rings that are fused, unfused or linked and can
include one or more heteroatoms, for example, phenyl,
naphthyl, biphenylyl, anthracenyl, quinolyl,
pyrimidinyl, and the like.
As used herein, the phrase "a straight, cyclic
or branched chain carbon-containing group having 1 to 20
carbon atoms" includes alkyl, cycloalkyl and aryl groups
that can include one or more heteroatoms, as for
1~36960
--7--
example, 3,3-diphenylpropanamido, isopropyl, 1,3,3-
trimethylcyclopentyl, and the like.
As used herein, the term "aryloxy" includes
fused or unfused aromatic rings, that can include one or
more heteroatoms, which form an ether linkage, such as
p-nitrophenoxy, biphenylyloxy, 2-indolyloxy, and the
like.
As used herein, the term "alkylthio" includes
C1 to C6 lower alkylthio groups that form sulfide
ethers, as for example, methylsulfide, propylsulfide,
hexylsulfide, and the like.
As used herein, the term "arylthio" includes
fused or unfused aromatic rings, that can include one or
more heteroatoms, that form a thioether linkage through
a thio group. Exemplary arylthio groups include
tetrazolylthio groups such as:
l-phenyl-lH-tetrazol-5-ylthio; l-methyl-lH-
tetrazol-5-ylthio; 1-(3-acetamidophenyl)-lH-
tetrazol-5-ylthio;
1-cyclohexyl-lH-tetrazol-5-ylthio;
1-(3-heptanamidophenyl)-1-H-tetrazol-
5-ylthio; and phenylthio.
As used herein, the term "ester" includes
acyloxy radicals and thioesters, such as acylthio groups
that are formed between a mercapto group and a lower
alkyl or aryl carboxylic acid. Exemplary acyloxy or
acylthio groups are acyclic, cyclic or aromatic. When
acyclic, the acyl groups preferably have about 1 to
about 20 carbon atoms in the alkyl portion. When
cyclic, the ring contains 3 to 6 carbon atoms. When
aromatic, the aryl groups preferably are substituted or
unsubstituted aryl groups and can be a heterocyclic
aromatic group having about 5 to about 10 nuclear carbon
-
13369fiO
--8--
and hetero atoms. Exemplary acylthio radicals are
acetylthio, propionylthio, hexanoylthio, and the like.
As used herein, the term "thionyl" includes
groups containing the sulfinyl group, as for example,
methylsulfinyl, phenylsulfinyl, tetrazolylsulfinyl, and
the like.
As used herein, the term "heterocyclyl"
includes fused or unfused cyclic organic groups that
contain one or more heteroatoms such as N, S, or 0 in
the ring structure such as phthalimidyl, benzoxazolyl,
benzothiazolyl, and the like.
As used herein, the term "alkylamidoaryl"
includes C1 to C12 lower aliphatic carbonyl groups
coupled in an amide bond with arylamine groups, such as,
for example, heptanamidophenyl, and the like.
As used herein, the term "sulfonyl" includes
alkyl, aryl and heterocyclic groups that contain a
sulfonyl group attached thereto, such as phenylsulfonyl,
2-pyrimidinylsulfonyl, and the like.
The R2 group is preferably hydrogen or an
electron-withdrawing group such as trifluoromethyl.
As used herein an "electron-withdrawing group"
includes a substituent group that will manifest greater
inductive effect to draw electrons to itself than would
a hydrogen atom if it occupied the same position on the
molecule. Exemplary electron-withdrawing groups include
cyano, halo, nitro, carboxy, acyl, arylthio, e.g.,
phenylthio, and the like.
Suitable R3 and R4 groups are preferably
substituents such as hydrogen, halogen, methyl, cyano,
nitro, amino, amido, cyanato, thiocyanato, hydroxy,
alkoxy, straight, cyclic and branched chain carbon-
containing groups having from 1 to 20 carbon atoms and
the like. The R3 and R4 groups of the present invention
can be unsubstituted or can contain a substituent
` -
I336960
selected from the group consisting of alkyl, phenyl and
nitro.
The present invention provides a
- pharmaceutical composition comprising a compound of
formula (I), or a pharmaceutically acceptable salt
thereof, hereinafter referred to as the "active
compound" or "agent", in association with a
pharmaceutically acceptable carrier.
As used herein, the phrase "pharmaceutically 10 acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar
untoward reaction, such as gastric upset, dizziness and
the like, when administered to a mammal. The
physiologically tolerable carrier may take a wide
variety of forms depending upon the preparation desired
for administration and the intended route of
administration. A carrier is a material useful for
administering the active compound and must be
"acceptable" in the sense of being compatible with the
other ingredients of the composition and not deleterious
to the recipient thereof.
The pharmaceutical compositions are prepared
by any of the methods well known in the art of pharmacy
all of which involve bringing into association the
active compound and the carrier therefor.
For therapeutic use, the agent utilized in the
present invention can be administered in the form of
conventional pharmaceutical compositions. Such
compositions can be formulated so as to be suitable for
oral or parenteral administration, or as suppositories.
In these compositions, the agent is typically dissolved
or dispersed in a physiologically tolerable carrier.
As an example, the compounds of the present
invention can be utilized in liquid compositions such as
sterile suspensions or solutions, or as isotonic
- 1336960
--10--
preparations containing suitable preservatives.
Injectable media containing aqueous injectable isotonic
and sterile saline or glucose solutions may be utilized.
Additional liquid forms in which the present compounds
may be incorporated for administration include flavored
emulsions with edible oils such as cottonseed oil,
sesame oil, coconut oil, peanut oil, and the like, as
well as elixirs and similar pharmaceutical vehicles.
The present agents can also be administered in
the form of liposomes. As is known in the art,
liposomes are generally derived from phospholipids or
other lipid substances. Liposomes are formed by mono-
or multi-lamellar hydrated liquid crystals that are
dispersed in an aqueous medium. Any non-toxic,
physiologically acceptable and metabolizable lipid
capable of forming liposomes can be used. The present
compositions in liposome form can contain, in addition
to the agent of the present invention, stabilizers,
preservatives, excipients and the like. The preferred
lipids are the phospholipids and the phosphatidyl
cholines (lecithins), both natural and synthetic.
Methods to form liposomes are known in the
art. See, for example, Prescott, Ed., Methods in Cell
Biology, Volume XIV, Academic Press, New York, N.Y.
(1976), p. 33 et seq.
The present compounds can also be used in
compositions such as tablets or pills, preferably
containing a unit dose of the compound. To this end,
the agent (active ingredient) is mixed with conventional
tableting ingredients such as corn starch, lactose,
sucrose, sorbitol, talc, stearic acid, magnesium
stearate, dicalcium phosphate, gums, or similar
materials as non-toxic, physiologically tolerable
carriers. The tablets or pills of the present
compositions can be laminated or otherwise compounded to
13369~0
--11--
provide unit dosage forms affording prolonged or delayed
action.
It should be understood that in addition to
the aforementioned carrier ingredients the
pharmaceutical formulation described herein can include,
as appropriate, one or more additional carrier
ingredients such as diluents, buffers, flavoring agents,
binders, surface active agents, thickeners, lubricants,
preservatives (including antioxidants), and the like,
and substances included for the purpose of rendering the
formulation isotonic with the blood of the intended
recipient.
The tablets or pills can also be provided with
an enteric layer in the form of an envelope that serves
to resist disintegration in the stomach and permits the
active ingredient to pass intact into the duodenum or to
be delayed in release. A variety of materials can be
used for such enteric layers or coatings, including
polymeric acids or mixtures of such acids with such
materials as shellac, shellac and cetyl alcohol,
cellulose acetate, and the like. A particularly
suitable enteric coating comprises a styrene-maleic acid
copolymer together with known materials that contribute
to the enteric properties of the coating.
The 2-substituted saccharin compounds utilized
in the pharmaceutical compositions of the present
invention inhibit the activity of serine proteases,
specifically human leukocyte elastase and chymotrypsin-
like enzymes.
The present invention is further directed to a
method of inhibiting proteolytic enzymes, such as human
leukocyte elastase and chymotrypsin-like enzymes. A
composition containing an effective amount of a
1336960
-12-
2-substituted saccharin derivative corresponding to
Formula I is contacted with a liquid sample containing
such proteolytic enzyme activity.
- The present invention is also directed to a
diagnostic method which enables the detection of
proteolytic enzymes such as human leukocyte elastase and
chymotrypsin-like enzymes which can indicate the
presence of physiological disorders. In the diagnostic
method, a body fluid sample is contacted with an
effective amount of a composition containing a
2-substituted saccharin derivative corresponding to
Formula I for a time period sufficient to permit
inhibition of appropriate proteolytic activities. The
concentration of inhibited proteolytic enzyme in the
sample is then measured.
The present invention is further directed to a
method of treatment of degenerative disorders produced
by the activity of proteolytic enzymes. Such disorders
include emphysema, adult respiratory distress syndrome,
pancreatitis, rheumatoid arthritis and other
inflammatory disorders. In the treatment method, a
therapeutically effective amount of a 2-substituted
saccharin derivative corresponding to Formula I is
administered, preferably in a pharmaceutical
composition, to a mammal such as a human with such a
degenerative disorder.
Preparation of the compounds of the present
invention is carried out by known methods. 5-nitro
saccharin derivatives are prepared as described in
D'Alelio et al, J. Macromol. Sci-Chem. A3(5):941 (1969)
and in Saari et al., J. Heterocyclic Chem. 23:1253
(1986).
4- or 5- Substituted saccharin derivatives are
prepared from appropriate 6- or 5- substituted
anthranilic acids as described in the above-referenced
1336960
-13-
article by Saari, et al. Basically, a methyl ester is
prepared by conventional means from the substit=uted
anthranilic acid and then diazotized. The diazonium
- salt is then reacted with sulfur dioxide and cupric
chloride to produce a sulfonyl chloride which is then
reacted with concentrated ammonium hydroxide to produce
the substituted saccharin derivative. This reaction is
schematically shown for the production of a
4-substituted saccharin from a 6-substituted anthranilic
acid as follows:
S~EME I
P
~ - ~
.~ ~
IV nl
The present invention is further illustrated
by the following examples which are not intended to
limit the scope of the invention in any way.
EXAMPLE 1: 2-AcetoxYmethyl-4-methyl saccharin
(a) MethYl 6-methylanthranilate
Powdered KOH (7.4 g; 132 mmol; 2 equiv.) was
admixed with dimethyl sulfoxide (DMSO) (100 mL) and the
mixture was stirred for 5 minutes. 6-Methylanthranilic
acid (10.0 g; 66 mmol) was then added to the mixture,
1336960
- -14-
followed dropwise by iodomethane (4.52 mL; 73 mmol; 1.1
equiv.). The reaction mixture was stirred for 30
minutes at room temperature and then diluted with ether
- (250 mL), washed with water (3 x 100 mL), dried (MgSO4)
and concentrated. The crude product was filtered
through a pad of flash grade (32-63) silica gel and
eluted with 1:9 ether:hexanes to afford 4.23 g (3g%) of
the anthranilate ester as an oil. lH nmr (300 MHz,
CDCl3): 7.078 (lH, t, J=7.67 Hz); 6.529 (2H, d, J=7.79
Hz); 5.111 (2H, br s); 3.887 (3H, s); 2.424 (3H, s). IR
(neat film, cm~1): 3480 (m), 3380 (m), 2950 (w), 1690
(s); 1605 (s).
(b) 4-Methylsaccharin
The anthranilate ester, methyl
6-methylanthranilate prepared in (a) (4.23 g; 25.6 mmol)
was dissolved in acetic acid (25 mL) and cooled to 0 C.
Concentrated hydrochloric acid (45mL) was added to
produce a tan slurry. Sodium nitrite (1.89 g; 27.4
mmol; 1.07 equiv.) dissolved in water (8 mL) was added
slowly, dropwise, and the resulting orange solution was
stirred at 0 C for 1 hour. This solution was then added
in 6 portions to a 0 C mixture of cupric chloride
dihydrate (2.18 g; 12.8 mmol; 0.5 equiv.) and sulfur
dioxide (6.3 g; excess) in acetic acid (33 mL) and water
(6 mL). The dark green solution was stirred at room
temperature overnight, poured into ice-water (300 mL),
and the solid was collected and dried by suction to
3~ provide 1.11 g of the sulfonyl chloride. This was
immediately added to ice cold ammonium hydroxide (100
mL) and stirred at room temperature overnight. The
solution was acidified to pH 1 with concentrated HCl and
the resulting precipitate was collected and air-dried to
provide 729 mg (12%) of 4-methylsaccharin, mp 224-226 C.
-
1336960
--15--
1H nmr (300 MHz, CD3CN): 9.5 (lH, br s); 7.782 (2H, d,
J=4.35 Hz); 7.644 (lH, t, J=4.20 Hz); 2.683 (3H, s). IR
(KBr, cm1): 3400 (w); 3100 (s); 3000 (s); 1720 (s);
1580 (m). FDMS: m/e 197 (M ).
s
(c) 2-Hydroxymethyl-4-methylsaccharin
4-Methylsaccharin prepared in (b) (500 mg;
2.54 mmol) was dissolved in 2.53 mL of warm ethanol
(steam bath). Once a homogeneous solution was achieved,
formalin (37% in methanol; 1.76 mL; excess) was added
dropwise. The solution was allowed to cool to room
temperature and then chilled to 0 C for 4 days. The
resulting solid was collected and air-dried to afford
476 mg (82%) of 2-hydroxymethyl-4-methylsaccharin, mp
196-198 C. ~H nmr (300 MHz, CDCl3): 7.767 (lH, t,
J=6.75 Hz); 7.732 (lH, d, J=7.72 Hz); 7.600 (lH, d,
J=6.64 Hz); 5.361 (2H, d, J=8.00 Hz); 3.296 (lH, t,
J=8.16 Hz); 2.793 (3H, s). IR (KBr, cm1): 3505 (s);
3070 (w); 1735 (s); 1580 tm).
(d) 2-Acetoxymethyl-4-methylsaccharin
2-Hydroxymethyl-4-methylsaccharin produced in
(c) (76 mg; 0.33 mL) was admixed into acetic anhydride
(1 mL; excess) and concentrated sulfuric acid (2 drops)
was added. The reaction mixture was stirred for 2 hours
at room temperature, at which time a non-polar spot was
observed by thin-layer chromatography (tlc) analysis.
The reaction mixture was diluted with dichloromethane
(50 mL) and washed with saturated NaHCO3 (2 x 15 mL).
After drying (Na2SO4), the solvent was removed to afford
64 mg (72%) of 2-acetoxy-4-methylsaccharin, mp 198-205 C
(decomp.) 1H nmr (300 MHz, CDCl3): 7.8 (2H, m); 7.64
(lH, d, J=6.18 Hz); 5.84 (2H, s); 2.82 (3H, s); 2.15
-
13369 60
-16-
(3H, s). IR (KBr, cm1): 2920 (w); 1745 (s); 1735 (s);
1630 (w). FDMS: m/e 269 (M).
- EXAMPLE 2: 2-Acetoxymethyl-4-chlorosaccharin
(a) Methyl 6-chloroanthranilate
This compound was prepared by the same method
as used for preparing methyl-6-methylanthranilate in
EXAMPLE 1, (a), using powdered KOH (4.08 g; 72.7 mmol;
2.5 equiv.), 6-chloroanthranilic acid (5.00 g; 29.2
mmol), and iodomethane (2.75 mL; 44 mmol; 1.5 equiv) to
give 4.22 g (78%) of the compound as an oil. 1H nmr
(300 MHz, CDCl3): 7.077 (lH, t, J=8.06 Hz); 6.744 (lH,
d, J=6.7 Hz); 6.575 (lH, d, J=8.25 Hz); 4.871 (lH, br
s); 3.929 (3H, s). IR (neat film, cm1): 3480 (m); 3380
(m); 2950 (w); 1705 (s); 1610 (s).
(b) 4-Chlorosaccharin
4-Chlorosaccharin was prepared by the same
method as used for preparing 4-methylsaccharin using
methyl 6-chloroanthranilate ((a) above) (4.22 g; 22.7
mmol) in AcOH (22 mL) and conc. HCl (40 mL) and sodium
nitrite (1.68 g; 24.3 mmol) in water (7 mL) to prepare
the diazonium salt. This was added to cupric chloride
dihydrate (1.93 g: 11.4 mmol; 0.5 equiv) and sulfur
dioxide (6.5 g; excess) in AcOH (30 mL)/water (5 mL).
The resulting sulfonyl chloride was treated with
ammonium hydroxide (150 mL) as previously described to
afford 3.07 g (62%) of 4-chlorosaccharin as a pale
yellow solid, mp 245-246 C. 1H nmr (300 MHz, CD3CN):
7.918 (lH, dd, J=7.39, 1.91 Hz); 7.865 (lH, t, J=7.52
Hz); 7.829 (lH, br d, J=7.30 Hz). IR (KBr, cm1): 3570
1336960
-17-
(s); 3520 (s); 2950 (s,b); 1735 (s); 1630 (m). FDMS:
m/e 217 (M).
(c) 2-Hydroxymethyl-4-chlorosaccharin
This compound was prepared in the same manner
as 2-hydroxymethyl-4-methylsaccharin, in EXAMPLE 1, (c),
from 4-chlorosaccharin (1.00 g; 4.60 mmol) and formalin
(37%; 3.22 mL; excess). Unfortunately, this product did
not crystallize from solution, but rather formed a
separate layer of viscous oil. This oil was not
characterized, but was used as is, since any attempt to
isolate 2-hydroxymethyl-4-chlorosaccharin resulted in
its reversion to 4-chlorosaccharin.
(d) 2-Acetoxymethyl-4-chlorosaccharin
Acetoxymethyl-4-chlorosaccharin was prepared
in the same manner as used for 2-acetoxymethyl-4-
methylsaccharin, in EXAMPLE 1, (d), from the crude
hydroxymethyl compound of (c) (0.34 g max; 1.4 mmol max)
and acetic anhydride (2.5 mL) with 2 drops of sulfuric
acid. In this case, after isolation, the product was
purified by filtration through a pad of silica gel and
elution with 1:1 ether:hexanes to afford 2-acetoxy-4-
chlorosaccharin (35 mg, 9% yield) as a white solid, mp
138-142 C. 1H nmr (300 MHz, CDCl3): 7.921 (lH dd,
J=6.54, 2.63 Hz); 7.874 (lH, t, J=7.98 Hz); 7.842 (lH,
dd, J-6.70, 2.20 Hz); 5.869 (2H, s); 2.172 (3H, s). IR
(KBr, cm1): 1745 (s); 1735 (m, shoulder); 1575 (w).
Comb. anal.:
Theor C, 41.46; H, 2.78; N, 4.83;
Found C, 41.17; H, 2.81; N, 4.75.
13~6960
-18-
EXAMPLE 3: 2-Chloromethyl-4-chlorosaccharin
Crude 2-hydroxymethyl-4-chlorosaccharin, from
EXAMPLE 2, (c), (609 mg; 2.46 mmol max) was admixed into
diethyl ether (5 mL), and thionyl chloride (3 mL;
excess) was added. The resulting mixture was heated
until homogeneity was achieved and then stirred at room
temperature overnight. It was then diluted with ether
(20 mL) and filtered through a pad of celite topped with
sand and eluted with ether. Removal of the solvent
afforded 430 mg of crude chloromethyl derivative. A
portion (225 mg) was removed for further reactions. The
remainder (205 mg) was chromatographed on flash silica
gel and eluted with 40% ether/pentane to provide 137 mg,
mp 135-136 C. 1H nmr (300 MHz, CDC13): 7.925 (lH, dd,
J=6.62, 2.26 Hz); 7.882 (lH, t, J=8.18 Hz); 7.846 (lH,
dd, J=7.42, 2.36 Hz); 5.561 (2H, s). IR (KBr, cm1):
3090 (w); 3050 (w); 1750 (s); 1575 (m). FDMS: m/e 265
(M).
EXAMPLE 4: 4-Chloro-2-(1-phenyl-lH-
tetrazol-5-ylthiomethyl)saccharin
The chloromethyl derivative prepared in
EXAMPLE 3 (225 mg; 0.85 mmol) and sodium
N-phenylmercaptotetrazole (200 mg; 1.01 mmol; 1.2 equiv)
were dissolved in acetone (5 mL) to give a tan solution.
After about 10 minutes a precipitate was observed, and
after stirring overnight at room temperature no
2-chloromethyl-4-chlorosaccharin was present by tlc
analysis. The reaction mixture was poured into water
and extracted with dichloromethane (3 x 25 mL). The
combined extracts were dried (Na2SO4), concentrated and
the residue was chromatographed on flash silica gel and
eluted with 1:1 ether:hexanes. The major spot was
-
1336960
--19--
collected to afford 122 mg of product as a white solid,
mp 175-177 C. lH nmr (300 MHz, CDCl3): 7.813 (3H, m);
7.515 (5H, s); 5.710 (2H, s). IR (KBr, cm1): 3080 (w);
1740 (s); 1590 (w). FDMS: m/e 407 (M); 230 (M -PMT);
178 (1%, PMT).
EXAMPLE 5: 4-Chloro-2-(4-phenyl-5-
thioxo-2-tetrazolin-1-ylmethyl)saccharin
The chloromethyl derivative prepared as in
EXAMPLE 3, (337 mg crude; maximum 1.27 mmol) was
dissolved (as much as possible) in acetone (10 mL).
Sodium N-phenylmercaptotetrazole (304 mg; 1.52 mmol; 1.2
equiv) was added and the reaction mixture was stirred at
room temperature for 3 days. The mixture was diluted
with dichloromethane (50 mL), washed with water (3 x 25
mL), dried (Na2S04), concentrated and filtered through a
pad of silica gel (1:1 ether:hexanes elution). The
material thus obtained was chromatographed on flash
silica gel and eluted with 1:1 ether:hexanes to afford
44 mg (8.5~) of the nitrogen-linked PMT derivative, mp
158-162 C. lH nmr (300 MHz, CDCl3): 7.981 (lH, d,
J=7.12Hz); 7.95 (2H, m); 7.887 (lH, t, J=6.74 Hz); 7.864
(lH, d, J=7.32Hz); 7.567 (3H, m); 6.392 (2H, s). IR
(KBr, cm1): 1745 (s); 1185 (s). FDMS: m/e 407 (M); 230
(M -PMT).
EXAMPLE 6: 2-[1-(3-Acetamidophenyl)-lH-tetrazol-5-
ylthiomethyl~saccharin
A mixture of 2-(chloromethyl)saccharin (0.98
g, 4.2 mmol), 1-(m-acetamidophenyl)-5-mercaptotetrazole
(1 g, 4.2 mmol) and potassium bicarbonate (0.84 g, 8.4
mmol) in methyl ethyl ketone (50 mL) was heated at 50 C
under N2 overnight. The reaction mixture was cooled and
-
13369 60
-20-
poured into dilute HCl/ice water (300 mL). The water
was decanted from the semi-solid which was solidified by
stirring in hot EtOAc. The resultant white solid
isolated by filtration was treated with NoritR, filtered
and then was recrystallized from acetonitrile (MeCN), to
afford 0.82 g of the desired product as small white
needles, mp 195-196 C decomp. 1H nmr (90 MHz, CDCl3) 2.05
(3H, s); 5.65 (2H, s). FDMS: m/e 430 (M).
Theor C, 47.43; H, 3.28; N, l9.S2;
Found C, 47.02; H, 3.27; N, 19.53.
EXAMPLE 7: 2-[1-(3-Heptanamidophenyl)-lH-tetrazol-
5-ylthiomethyl~saccharin
A mixture of 2-(bromomethyl)saccharin (2.7 g,
9.8 mmol), 1-(m-heptanamidophenyl)-5-mercaptotetrazole
(3 g, 9.8 mmol) and potassium carbonate (3.4 g, 24.5
mmol) was heated at reflux in methyl ethyl ketone (50
mL) under nitrogen for 1 hour. The mixture was cooled
and poured into a NaHCO3/ice solution. The water layer
was decanted from the resultant white semi-solid. The
semi-solid was washed with water then dissolved in hot
MeCN, treated with NoritR and filtered. The filtrate
was freed of solvent under vacuum and the resultant
solid chromatographed (silica gel-95:5 CH2Cl2:acetone)
to give a clear oil. The oil was crystallized from hot
EtOH to afford 1.6 g of the desired product as a white
solid, mp 146-147.5 C. 1H nmr (90 MHz, CDCl3) 5.65 (2H,
s). FDMS: m/e 500 (M).
Theor C, 52.79; H, 4.83; N, 16.79;
Found C, 52.44; H, 4.75; N, 16.64.
1336960
-21-
EXAMPLE 8: 2-(1-Methyl-lH-tetrazol-5-ylthiomethyl)-
saccharin
A mixture of 2-(bromomethyl)saccharin (3 g,
10.8 mmol) and 5-mercapto-1-methyltetrazole, sodium salt
(1.49 g, 10.8 mmol) was heated at reflux in methyl ethyl
ketone (75 mL) for 2 hours. The reaction mixture was
cooled, poured into dilute sodium bicarbonate/ice
solution and extracted with methylene chloride (2x's).
The combined organic extracts were dried (Na2SO4) and
freed of solvent under vacuum. The crude product was
chromatographed (silica gel-95:5 CH2Cl2:ether) and the
resultant oil was crystallized from hot isopropanol to
afford 2.7 g (80%) of the desired product as a white
solid, mp 106-110 C. 1H nmr (90 MHz, CDCl3) 5.55 (2H, s).
FDMS: m/e 311 (M).
Theor C, 38.58; H, 2.91; N, 22.49;
Found C, 38.58; H, 2.79; N, 22.60.
EXAMPLE 9: 2-(1-Cyclohexyl-lH-tetrazol-5-
ylthiomethyl)saccharin
A mixture of 2-(chloromethyl)saccharin (3 g,
12.9 mmol), 1-cyclohexyl-5 mercaptotetrazole (2.37 g,
12.9 mmol) and potassium carbonate (4.45 g, 32.2 mmol)
was heated at reflux in methyl ethyl ketone (50 mL) for
1 hour. The reaction mixture was cooled, poured into
dilute sodium bicarbonate/ice solution and extracted
with EtOAc (2x's). The combined organic layers were
washed with water, dried (Na2SO4) and freed of solvent
under vacuum. Chromatography (silica gel; CH2Cl2)
afforded 2 g of the desired product as a white foam that
could be crystallized from hot cyclohexane, mp 103-
105 C. lH nmr (90 MHz, CDC13) 5.65 (2H, s). FDMS: m/e
379 (M ).
1336960
-22-
Theor C, 47.48; H, 4.52; N, 18.46;
Found C, 47.84; H, 4.61; N, 18.36.
- EXAMPLE 10: 2-(1-Phenyl-lH-tetrazol-5-
ylsulfinylmethyl)saccharin
A mixture of m-chloroperbenzoic acid (0.43 g,
2.67 mmol) and 2-(1-phenyl-5-tetrazolyl-
thiomethyl)saccharin(l g, 2.67 mmol) in methylene
chloride was stirred at room temperature (RT) for 24
hours. TLC (95:5 CH2Cl2:ether) revealed the presence of
starting sulfide. Additional peracid (0.2 g) was added
and the mixture stirred for an additional 2 days. The
reaction mixture was washed with sodium bicarbonate
solution, dried (Na2S04) and freed of solvent under
vacuum. Chromatography (silica gel-95:5 CH2Cl2:ether)
afforded a foam that was crystallized from ether to
afford 0.52 g of the desired product as a white solid,
mp 161-162 C. 1H nmr (90 MHz, CDCl3) 5.5-6.0 (2H, q).
FDMS: m/e 196 (M -PMT), 389 (M).
Theor C, 46.27; H, 2.85; N, 17.98;
Found C, 46.00; H, 2.83; N, 17.76.
EXAMPLE 11: 5-Nitro-2-(1-phenyl-lH-tetrazol-
5-Ylthiomethyl)saccharin
A mixture of 2-bromomethyl-5-nitrosaccharin (2
g,6.2 mmol) and 1-phenyl-5-mercaptotetrazole, sodium
salt in methyl ethyl ketone (40 mL)/DMF (10 mL) was
heated at reflux for 2 hours. The reaction mixture was
cooled and poured into a dilute sodium bicarbonate/ice
solution. The resultant white solid, isolated by
filtration, was washed with water and air dried. The
compound was sonicated with 50:50 CH2Cl2:acetone and
filtered to remove soluble impurities. The remaining
-23- 1336960
solid was recrystallized from 2:1 MeCN:EtOH to afford
1.5 g of the desired product as an off white solid, mp
189-190 C. 1H nmr (90 MHz, DMSO-d6) 5.75 (2H, s). FDMS:
m/e 418 (M ).
Theor C, 43.06; H, 2.41; N, 20.09;
Found C, 42.29; H, 2.43; N, 20.13.
EXAMPLE 12: 2-(Phenylsulfonylmethyl)saccharin
A mixture of m-chloroperbenzoic acid (2.2 g,
12.8 mmol) and 2-(phenylsulfinylmethyl)saccharin (3.75
g, 11.6 mmol) in methylene chloride (50 mL) was stirred
at room temperature for 2 hours. An additional spatula
of peracid was added and stirring was continued for an
additional 1 hour. m-Chlorobenzoic acid was removed by
filtration and the solid was washed with a small amount
of methylene chloride. The filtrate was washed with
sodium bicarbonate solution, dried (Na2SO4) and freed of
solvent under vacuum. The resultant solid was
recrystallized from 50:50 EtOH:MeCN to afford the
desired product as a white solid, mp 169-171 C. 1H nmr
(90 MHz, DMSO-d6, CDCl3) 5.15 (2H, s). FDMS: m/e 196
(M).
Theor C, 49.84; H, 3.29; N, 4.15;
Found C, 49.92; H, 3.24; N, 4.13.
EXAMPLE 13: 2-(2-PYrimidylsulfinylmethyl)saccharin
A mixture of m-chloroperbenzoic acid (0.9 g,
5.37 mmol) and 2-(2-pyrimidylthiomethyl)saccharin
prepared by procedures similar to those of Examples 9
and 11 (1.5 g, 4.8 mmol) in methylene chloride (75 mL)
was stirred overnight at room temperature. The reaction
mixture was washed with sodium bicarbonate solution,
dried (Na2SO~) and freed of solvent under vacuum. Part
1336960
-24-
of this crude product (0.5 g) was saved for direct
conversion to the sulfone; the remaining material was
chromatographed (silica gel-95:5 CH2Cl2:acetone).
Recrystallization (EtOH/MeCN) afforded 0.95 g of white
crystals, mp 197-198 C. decomp. 1H nmr (90 MHz, CDCl3,
DMSO-d6) 5.1-5.5 (2H, q).
Theor C, 44.57; H, 2.81; N, 13.00;
Found C, 44.67; H, 2.84; N, 12.97.
EXAMPLE 14: 2-(2-Pyrimidylsulfonylmethyl)saccharin
A mixture of m-chloroperbenzoic acid (0.4 g,
2.3 mmol) and the sulfoxide prepared in EXAMPLE 13 (0.75
g, 2.3 mmol) in methylene chloride (50 mL) was stirred
at room temperature with TLC (95:5 CH2Cl2:acetone)
monitoring. After 2 hours, some of the starting
sulfoxide still remained; an additional spatula of
peracid was added and the reaction was stirred
overnight. Methylene chloride (100 mL) was added and
the mixture was washed with sodium bicarbonate solution.
The organic layer was dried (Na2SO4) and solvent was
removed under vacuum. Recrystallization (MeCN/EtOH)
afforded 0.95 g of white solid, mp 225-227 C decomp. 1H
nmr (90 MHz, DMSO-d6) 5.78 (2H, s). FDMS: m/e 196
(M -PMT), 339 (M ).
Theor C, 42.47; H, 2.67; N, 12.38;
Found C, 42.20; H, 2.62; N, 12.46.
EXAMPLE 15: 2-(4-Nitrophenoxymethyl)saccharin
A mixture of 2-(chloromethyl)saccharin (3 g,
12.9 mmol) and sodium p-nitrophenoxide (2.55 g, 12.9
mmol) in tetrahydrofuran (THF) (50 mL) was heated
overnight at 50C and then was refluxed for 45 minutes.
The reaction mixture was cooled, poured into a dilute
1336960
sodium bicarbonate/ice solution and extracted with EtOAc
(2x's). The combined organic layers were washed with
sodium bicarbonate solution and water, dried (Na2SO4)
and then solvent was removed under vacuum.
Chromatography (silica gel; CH2Cl2) afforded an oil that
was crystallized from hot cyclohexane/ether. The
resultant solid was recrystallized from EtOH to afford
0.92 g of the desired product as white shiny platlets,
mp 162-164 C. H nmr (90 MHz, CDCl3, DMSO-d6) 5.95 (2H,
s). FDMS: m/e 334 (M).
Theor C, 50.30; H, 3.02; N, 8.38;
Found C, 50.06; H, 2.91; N, 8.28.
EXAMPLE 16: 5-(3,3-Diphenylpropionamido)-2-
(1-phenyl-lH-tetrazol-5-ylthiomethyl)-
saccharin
5-Nitro-2-(1-phenyl-lH-tetrazol-5-
ylthiomethyl)saccharin (4 g, 9.56 mmol) was dissolved in
THF (250 ml) and placed in a Parr shaker bottle. 10~
Pd/C (2 spatulas) was added under N2and the mixture was
shaken under hydrogen (55 psi) for 2.5 days. The
reaction mixture was filtered through Celite
diatomaceous earth. The filtrate was added to water and
extracted with methylene chloride. The organic layer
was dried (Na2SO4) and freed of solvent under vacuum.
The resultant yellow foam was sonicated with warm
ethanol, cooled, and filtered. The desired
5-aminosaccharin derivative, 0.5 g, was isolated as a
cream colored solid. FDMS: m/e 388 (M ).
A mixture of the above 5-aminosaccharin
derivative (0.5 g, 1.29 mmol) and 3,3-diphenylpropanoyl
chloride (0.315 g, 1.29 mmol) in acetonitrile (50 ml)
was heated at reflux for 2.5 hours. TLC (95:5 methylene
chloride:acetone) analysis revealed the presence of some
-
1336960
-26-
starting amine. A small additional amount of acid
chloride was added and reflux was continued for 1.5
hours. The reaction mixture was cooled and poured into
ice water (400 ml). After 30 minutes, the mixture was
filtered and the resultant tan colored solid was washed
with water and air dried. Chromatography on silica gel
(95:5 methylene chloride:ether) produced a foam that was
crystallized from hot ethanol to yield 0.68 g of a white
solid, mp 92-93 C decomp. FDMS: m/e 596(M ). 1H nmr
(90 MHz, CDCl3); 3.25 (lH, d); 4.8 (2H, t); 5.6 (2H, s);
6.9-8.2 (m, Ar). NMR also revealed approximately two
ethanol molecules of crystallization: 1.25 (t); 3.7
(q). Comb. anal.:
Theor for C3oH24N6o4s2+ 2C2HsH: C~
H, 5.27; N, 12.2;
Found: C, 58.09; H, 5.15; N, 12.09.
EXAMPLE 17: Methyl 2-(1-Phenyl-lH-tetrazol-5-
ylthio)-2-(2-saccharinyl)acetate
Methyl l-chloro-l-thiophenylacetate was
prepared as reported in the literature: I. Fleming and
J. Iqbal, Tetra. Lett., 24, 327 (1983); M. Campbell, et
al., Tetra. Lett., 21, 3305 (1980).
Saccharin (10 g, 54.6 mmol) was dissolved in
EtOH (500 ml) with slight warming. Thallous ethoxide
(13.6 g, S4.6 mmol) was added dropwise and the resultant
heterogeneous mixture was stirred at room temperature
for 2 hours. The mixture was cooled and filtered and
the solid was washed with cold ethanol. The grey,
white, crystalline solid was dried under vacuum in a
desiccator to yield 19.4 g (92%) of the thallium salt of
saccharin.
A mixture of the thallium salt of saccharin
(1.78 g, 4.6 mmol) and methyl 2-chloro-2-thiophenyl
13~6960
-27-
acetate (1 g, 4.6 mmol) in DMF (25 ml) was stirred at
60 C for 7 hours. The mixture was cooled and poured
into ice water (400 ml). After 30 minutes, the mixture
was filtered and the solid was washed with water and
air-dried. Chromatography on silica (methylene
chloride) afforded a clear oil that was crystallized
from hot EtOH to yield 0.87 g (51%) of white needles of
methyl 2-phenylthio-2-(2-saccharinyl)acetate, mp 144-
146 C. 1H nmr (90 MHz, CDCl3): 3.8 (3H, s); 5.95 (lH, s);
7.2-8.15 (9H, m). FDMS: m/e 363(M).
A solution of methyl 2-phenylthio-2-(2-
saccharinyl)acetate (2 g, 5.5 mmol) and sulfuryl
chloride (0.74 g, 5.5 mmol) in methylene chloride (50
ml) was stirred at room temperature for 2 hours.
Solvent was removed under vacuum and the yellow oil was
crystallized from warm ethanol to give 0.94 g of
product. NMR revealed greater than 50% starting
material. An additional amount of starting material (1
g, 2.75 mmol) was added to the crude product mixture and
it was redissolved in methylene chloride. Sulfuryl
chloride (0.5 ml) was again added and the mixture was
stirred at room temperature for about 12 hours. Work up
as above afforded 0.66 g of crude chloride that was used
immediately in the next step.
A mixture of this chloride (0.66 g crude
mixture) and l-phenyl-5-mercapto-lH-tetrazole, sodium
salt (0.44 g, 2.2 mmol) was heated at reflux in methyl
ethyl ketone (25 ml) for 4 hours. After stirring at
room temperature for 2 days, the reaction mixture was
poured into ice water. The tan solid isolated by
filtration was washed with water and air-dried.
Chromatography on silica gel (methylene chloride)
yielded an off-white foam that was crystallized from
ethanol to yield 0.36 g of white crystalline solid, mp
-
1336960
-28-
160-162 C. lH nmr (9o MHz, CDCl3); 3.8 (3H, s); 7.05
(lH, s); 7.4-8.1 (9H, m). FDMS: m/e 431 (M ).
Theor for C17H~3N5O5S2: C~ 47-33;
- N, 16.23;
Found: C, 47.15; H, 3.09; N, 16.30.
Other compounds of the present invention are
prepared by syntheses similar to those described above.
Table 1 lists the compounds corresponding to
the above-described examples, together with other
exemplary compounds of the present invention.
Measurement of the inhibition constant, Kj, of
a HLE-inhibitor complex has been described for "truly
reversible inhibition constants" usually concerning
competitive inhibitors. The compounds of the present
invention, however, do not form truly reversible
inhibitor complexes. The compounds of the present
invention are consumed by the enzyme to some extent.
Thus, instead of measuring a Kj, a Kj* is calculated
which is defined as the ratio of the koff/kon, the rate of
reactivation of the enzyme to the rate of inactivation
of the enzyme. The values of koff and kon are measured
and Kj* is then calculated.
The rate of inactivation, kon, of enzymatic
activity was determined for the compounds tested by
measuring the enzyme activity of an aliquot of the
respective enzyme as a function of time after addition
of the test compound. By plotting the log of the enzyme
activity against time, an observed rate of inactivation,
kobs~ is obtained which can be represented as kobS =
ln2/t~/z where t1/Z is the time required for the enzyme
activity to drop by 50%. The rate of inactivation is
then equal to
-
-29- 1336960
kobs
kon
[I]
where [I] is the concentration of the inhibiting
compound.
The reactivation constant, koff, is similarly
determined and the inhibition constant, Kj*, is then
calculated as
Kj* = koff/kon
The values obtained for kon and koff/kon for
specific substituted saccharin derivatives are shown in
TABLE 1.
1336960
_30-
TABLE I
Example Elastase Alpha-ChymotrYpsin
Structure10 3 X kon koff/kon 10 3 X kon koff/kon
(M1 sec1) (nM) (Ml sec~) (nM)
. 1 0.63 102 1.2 917
u O O
~ oJ~
2 4.9 45 2.9 51
3 450 0.5 5.8 26
4 20 12
~b
"=,,
44 6 6.0 25
6 5.5 lS 3.7 100
7 5.2 15
1n
1336960
-31-
TABLE I (Continued~
Example Elastase Alpha-Chymotrypsin
Structure 103 x kon koff/kon 10-3 x k koff/kon
sec1) (nM) (M1 sec1) (nM)
8 1.0 81 2.1 523
o
~_11
o~ o ~ 9 2.5 32
7.0 11 7.0 157
~
-~ 11 4.6
~ r 12 0.97 82 1.1 1000
O~O ~ ~ 13 0-3 285 2.6 423
14 0.6 138 2.8 392
1336960
32
TABLE I (Continued)
Example Elastase Alpha-Chymotrypsin
Structure 10-3 x k kff/k~ 103 x kon koff/kon
(M1 secl) (nM) (M1 secl) (nM)
3.2 69
16 2,600
" _".
17 2.9 270
18 1.0 100 0.21 620
19 50 4.4
'~, f~
~ ~ ~ 10,500
-
1336960
TABLE I (Continued)
ExamPle Elastase Alpha-Chymotrypsin
$tructure lO 3 X kon koff/kon 10 3 X kon kOff/kon
(M1 sec1) (nM) (M1 sec1) (nM)
/~ 8,500
o
22 500
23 Z.5 17
'~ ~ ~ 24 7.4 250
1336960
- -34-
TABLE I (Continued)
Example Elastase AlDha-chymotrypsin
Structure 10-3 x kon koff/kon 103 x k koff/kon
(M1 secl) (nM) (M1 sec1) (nM)
~-o
~ ~ 26 .02 40,000
o
27 44 1.8 6.0 180
28 5.6 lS 4.3 254
-
~35~ 1 33 6g60
The foregoing description and examples are
intended as illustrative and are not to be taken as
limiting. Still other variables within the spirit and
scope of this invention are possible and will readily
present themselves to those skilled in the art.
