close

Вход

Забыли?

вход по аккаунту

?

Syntheses and Selective Inhibitory Activities of Terphenyl-Bisamidines for Serine Proteases.

код для вставкиСкачать
73
Terphenyl-Bisamidines
Syntheses and Selective Inhibitory Activities of Terphenyl-Bisamidines
for Serine Proteases*[l]
Wolfgang von der Saal*a),Richard A. Enghb),Andreas Eichingerb),Bernhard Gabriela'c),Ralf Kucznierza), and Jiirgen Sauerc)
a)
Chemical Research Department, Boehringer Mannheim GmbH, D-68298 Mannheim, Germany
b,
Max-Planck-Institut fiir Biochemie, D-82152 Martinsried, Germany
Institut fur Organische Chemie der UniversiCat, D-93040 Regensburg, Germany
Key Words: blood-coagulation inhibitors; trypsin inhibitors; Suzuki coupling; Stille coupling
Summary
Biphenyl nitriles 5a-c. terphenyl dinitriles lla-d. and naphthalene-bis(benzonitri1e) l l e were prepared by palladium-catalyzed
cross coupling reactions and subsequently converted to biphenyl
amidines 8a-c and bis(benzamidines) 4a-e. Among the biphenyl
amidines 8 only the mefa-derivative 8b inhibits factor Xa and
tqpsin (Ki= 10@
TheIterphenyl
).
bisamidine 4c does not inhibit
factor Xa,trypsin, thrombin, and plasmin. while 4a and 4d are
almost equipotent inhibitors of theses enzymes (Ki 1-6 pM), and
4b and 4e are selective for trypsin (K,= 0.2 and 0.3 pM; but K,>
1 pM for factor Xa, thrombin, and plasmin). X-ray analysis of
crystals of 4b complexed with bovine trypsin revealed a unique
binding mode: one benzamidino group binds in the S 1 site to the
side chain carboxylate of Arg189. The central phenyl group is
twisted away from the SUS3 sites and the second amidino group
contacts the Asn143 side chain.
1
2
3
Introduction
The pancreatic enzyme trypsin, enzymes of the coagulation
cascade (such as thrombin and factor Xa), and the clot lysing
enzyme plasmin function similarly. They hydrolyze their
respective substrates at a precisely defined bond at the carboxy side of an arginine residue. The side chain of this arginine
extends into a narrow pocket of the enzyme (S 1 site) and its
guanidino group binds ionically to the carboxylate group of
Asp189 [21. The discovery that the benzamidinium ion (1,
Scheme 1) is an unselective inhibitor of these enzymesL3]and
occupies in t sin the same site as the arginine side chain of
the substrater$ lead to the development of potent inhibitors.
The S1 site is very much conserved in these enzymes, and
selectivity is brought about by interactions with further sites
(S2, S3) which differ among the enzymes.
The two branches of the coagulation cascade converge on
factor Xa, which therefore became a major target for the
design of selective inhibitors for the development of anticoagulant drugs [51. The observation that a second benzamidine
moiety in an inhibitor enhances potency for several serine
proteases but not for trypsint6] lead to the development of
bisamidines, e.g. zt7] and 3@],as factor-Xa inhibitors. The
structure of factor Xa has been solved,[91but structures of
inhibitodfactor Xa complexes remain unknown. One basic
group, e.g. a benzamidino group of 2 or the naphthamidine
moiety of 3, seems to bind in the S 1 site, but the binding site
Arch. Pharm. Pharm. Med. Chem.
4
Scheme 1
of the second basic group has been controversial in several
molecular modelling studies 16, lo]. Further progress in the
understanding of the structural basis for factor Xa inhibition
is hampered mainly by two facts: (1) the conformational
flexibility of the currently available dibasic inhibitors impedes the molecular modelling studies, and ( 2 ) a systematic
variation of compounds like 2 and 3 is still lacking. The
objective of our work was to develop a short synthetic route
to such compounds, i.e. the bis(benzamidines) 4 with various
spacer groups. Although compounds like 2 fall into this
category and 3 is also related to it, their use is limited by the
fact that only symmetrical congeners of 2 could be prepared
and there was no control over the configuration of the double
bond, whereas 3 has to be prepared in more than 20 steps. We
focused our attention on palladium catalyzed cross coupling
reactions because these are applicable to carbon-carbon bond
formation with aryl-, heteroaryl-, allyl-, alkenyl-, alkinyl-,
vinyl-, and benzylgroups, and are prone to stereoselective
syntheses by the use of optically active ligands on the palladium t1 'I. We present here our first results for compounds of
0VCH Verlagsgesellschaft mbH, D-6945 1 Weinheim, 1996
0365-6233/96/0202-0073$5.00 + ,2570
14
von der Saal and co-workers
type 4 with phenylene- and a naphthalene group as spacers.
Inhibition studies revealed an unexpected selectivity shift
towards trypsin, and we present an X-ray structure of an
inhibitor-trypsin complex which revealed the reason for that
selectivity.
Biphenylamidines are a partial structure of the proposed
terphenyl-bisamidines. Their binding to serine proteases is a
prerequisite for the binding of the proposed compounds of
type 4. Molecular modelling studies of the binding of the
biphenylamidines 8 in thrombin suggested[12] that only the
meta compound 8b.H' would fit into the S 1 site and achieve
an ionic contact with Aspl89. Inhibition constants for thrombin, trypsin, and plasmin were available for 8b only['31, while
8c had not even been prepared previously.
Consistent with modelling studies only the metu-compound
8b inhibited the enzymes (see later). Therefore we focused
on the preparation of therphenyls 4a-d which bear at least
one rnetu-biphenylamidine moiety. The symmetrical dicyanoterphenyls l l a - c were prepared by double Suzuki coup l i n g s of metu-cyanophenyl boronic acid 9[15] and
dihalobenzenes 10. The reaction of para-dibromobenzene
with 9 lead to intractable mixtures of oligo-phenylene nitriles['6] as jugded by mass spectra, but the more reactive
diiodobenzene gave l l a in 90% yield (Scheme 3).
..
CN
Results and Discussion
Chemistry.We obtained the precursors, biphenyl-carbonitriles 5, in almost quantitative yield by Suzuki coupling of
phenylboronic acid with bromobenzonitriles. The nitriles
were converted into 8a (43% overall yield based onparu-bromobenzonitrile) and 8b (55% yield) via thioamides 6a,b, and
S-methyl-thioimidates 7a,b. (Scheme 2). The last step in this
reaction sequence failed for the ortho-compound: Reaction
of 7c with ammonium acetate afforded almost quantitatively
the starting material (nitrile 5c).We speculated that the elimination of methanethiol was much faster than the substitution
by ammonia because in the transition state leading to 8c the
hybridization of the attacked carbon atom changes from sp2
to sp3. The sterically demanding transition state would then
interfere with the ortho-phenyl group. Thus, the addition of
ammonia would be slowed down, whereas the elimination of
methanethiol would not. The direct amination of the nitrile
5c with methylchloroaluminum amide[14] which avoids this
transition state gave 8c in 57% yield (55% based on orthobromobenzonitrile).
R
5a-c
-
sc
R
6a-c
Ja
t
R = -C(SMe)=NH2+ I-
-
R = -C(=NH)NH2:
d
5c
8a, b
8c
a) H S ; b) Mel; c) NH4+0Ac-; d) MeCIAINH2;
Scheme 2
fi
\
\
R
R
a: para
b: mefa
c: ortho
R = -CN:
11a-c
R = -C(=S)NH2:
12a-c
R = -C(SMe)=NH2+ I- :
13a-c
R = -C(=NH)NH2:
-
Me/
JNH4+0Ac-
4a-c
Scheme 3
a: para
b: meta
c: ortho
R = -CN:
= -C(=S)NH2:
X = l : 10a
X = Br: lob, c
9
For the preparation of the unsymmetrical dicyano-terphenyl
l l d we exploited the difference in the reaction rates of Stille
vs Suzuki couplings. In the para-borostannyl benzene 14 ["I,
the stannyl moiety reacted faster (Stille coupling) than the
boryl moiety with para-bromobenzonitrile to give 15 in 48%
yield. This compound was then converted to l l d by Suzuki
coupling with metu-bromobenzonitrile (Scheme 4).
The all-rnetu-bisamidine 4b turned out to be the strongest
inhibitor (see later). As a congener of this compound with a
naphthalene spacer we prepared derivative 4e. We wanted to
retain the overall geometry of the terphen 1 4 b , and so the
2,7-dihydroxynaphthalene derivative 10d 81 was used as
starting material (2,7-dihalo-naphthalenes are not commercially available). Suzuki coupling with 9 gave the dinitrile l l e
in 80% yield (Scheme 5).
The bisamidines 4a-e were prepared from the requisite
dinitriles 1la-e in 4660% overall yield via bis-thioamides
(12a-e) and S-methylated thioimidates (13a-e). Here again,
in the final step, elimination of methanethiol competed with
substitution by ammonia: The resulting crude bisamidines
4a-e contained trace amounts of mono-amidino-nitriles,
which were detected by a weak C=N signal at 2225 cm-' in
6
Arch. Pharm. P h a m Med. Chern. 329,7342 (1996)
75
Terphenyl-Bisamidines
-
Pd(PPhd4
4-bromobenzonitrile
Me
Me
Table 1. Structures of the bis(benzamidines) 4 and inhibition constants at
25H. 1 “Cof 4, benzamidine (l),and the biphenylamidines 8 for some serine
proteases.
3-bromobenzonitrile
OmB.0
14
15
Me Me
6
4b
R
($,
R=-CN:
lld
R = -C(=S)NH2:
12d
R = -C(SMe)=NH2+ I- :
13d
R = -C(=NH)NH2:
4d
2
Me1
A
NH4+OAc-
NH
4d
4c
R
Scheme 4
H2N$
9, Pd(PPhd4
F,CO,SO
OSO,CF,
1Od
Ki [PMI
actor Xa
k
R = -CN:
R
,[I 91
lle
12e
-
J
13e
35
thrombin
220
plasmin
350
4a
3.8
1.3
4.8
2.1
4b
2.8
0.2
7.5
8.3
H2s
7
R = -C(SMe)=NH2+ I- :
410
trypsin
J
Me’
4c
>I 00
>I 00
>50
>I 00
4d
4.8
2.9
5.2
6.2
4e
1.2
0.3
4.1
2.4
NH4+OAcR = -C(=NH)NH2:
4e
Scheme 5
the IR spectra. This signal was very strong in the dinitriles
l l a - e but was absent in the intermediates 12a-e and 13a-e.
The signal disappeared only on repeated recrystallizations of
the amidines from ethyl acetate.
Enzyme Inhibition. Among the proteases tested, benzamidine (1) is selective for trypsin[”] (Table 1). Adding one
phenyl group in the metu position (8b) increases the potency
for trypsin by a factor of 3.5~.[’~]
Surprisingly, factor Xa is
Arch. P h a n Pharm.Med. Chem. 329,7342 (1996)
8a
2500
>I 00
>50
>I 00
8b
10
10
>50
>I 00
8c
>500
>I 00
>50
>I00
now inhibited 40 times better, and hence the structure of 8b
was retained as partial structure in all other compounds. The
ortho- and para-biphenyl amidines @a,c) do not inhibit the
76
von der Saal and co-workers
Figure 1. Stereo drawing of the three-dimensional structure of the terphenyl-bisamidine 4b
in trypsin. Dashed lines, distances (A) between amidino nitrogen atoms of 4b (bottom) and
side chain oxygen atoms of Am143 and Asp189 oftrypsin (top).
enzymes, as expected. In the terphenyl series (4a-d), the
potency and selectivity strongly depends on the position of
the second phenyl group. 4a inhibits all enzymes with approximately equal potency. Potency is increased for all enzymes, but much more for thrombin and plasmin (factor
10-50) than for trypsin, as expected from recent studies with
other mono- to tetra-amidines.[61 Inhibition of factor Xa is
also slightly improved (factor 7). In the all-meta-terphenyl
4b, the potency for trypsin again increased by a factor of 6
compared to 4a, but did not change for factor Xa, whereas
potency decreased slightly for thrombin and plasmin. This
shift towards trypsin selectivity was unexpected and contrary
to previous observations with other bisamidines
Actually, when comparing benzamidine to the terphenyl 4b we
gained nothing in selectivity which remains in favor of trypsin
by more than a factor of 10. The bisamidine 4c is completely
devoid of any activity. This is most probably due to steric
hindrance as suggested from molecular modelling of the
complexes with trypsin, factor Xa, and thrombin: it is impossible for an amidino group of 4c to make an ionic contact with
Asp189 in the S1 site, which is the primary force governing
the binding of benzamidine to these enzymes. The relative
potencies of the bisamidine 4d are quite comparable with
those of 4a, but the absolute inhibition constants are higher
by factors of 1.5 to 3. This difference between 4a and 4d may
be caused solely by a statistical factor: 4a can bind in two
identical modes to the enzymes because its benzamidino
moieties are identical. Not so 4d: Only the meta-benzamidine
moiety can bind to the S1 site, but not the para-benzamidine.
This follows immediately from the fact that the parubiphenylamidine 8a does not inhibit the enzymes. The same
trypsin selectivity as the terphenyl 4b is also caused by the
naphthalene derivative 4e. The overall geometry of these two
compounds is quite similar. The amidino groups are a little
farther apart due to the larger spacer group.
In order to delineate the chemical basis of this peculiar
increase in selectivity for trypsin over factor Xa, we prepared
crystals of the 4b/trypsin complex and determined the structure by x-ray crystallography (Fig 1).
The x-ray analysis of the 4b/trypsin complex showed that
4b binds in an unusual way: One amidino group provides the
expected contacts to Asp189 in the S1 site, but the other part
of the molecule is twisted in such a way that it directs the
[6371.
second amidino group away from the S2IS3 sites. This amidino group contacts the amide group of the Asn143 side chain.
This is the reason for the trypsin selectivity: in factor Xa, the
amino acid 143 is an arginine. The positive charge and longer
side chain prevents the amidino group from binding in that
area.
Conclusion
The recent discovery of a one-stey y p a r a t i o n of 9 at
-100 "C from metu-bromobenzonitrile
makes it a convenient starting material for the synthesis of his-benzamidines 4
via palladium-catalyzed cross-coupling reactions. We have
prepared 4a-e as first examples of a new class of serine
proteinase inhibitors, which differ from previous bisbenzamidines in that they are conformationally more restricted
and are thus better managable in molecular modelling studies.
Such studies are of current interest in the interpretation of the
binding mode of selective factor Xa inhibitors, e.g. 3, and may
help in the development of more potent inhibitors for the use
as antithrombotic drugs. Usually, his-benzamidines are
known to be less potent trypsin than factor Xa inhibitors
but 4b and e were more potent for trypsin than for factor Xa,
thrombin, and plasmin. This is due to a unique binding mode
of 4b (and probably of 4e as well) in trypsin. We do not expect
that compounds of type 4 with spacers different from those
reported here will bind to trypsin in this way. Thus, such
compounds remain valuable target structures for selective
factor Xa inhibitors, and we intend to fully exploit the synthetic potential of the palladium catalyzed cross coupling
reactions for the preparation of such compounds.
[6371,
Experimental
' H NMR: [D6]DMSO, Bruker AC 250 spectrometer, TMS as internal
standard, chemical shifts in F ppm.- "C NMR: [DhlDMSO, Bruker AC 250
spectrometer at 63 MHz, TMS as internal standard, chemical shifts in F ppm.
J-modulated spin-echo spectra were recorded to differentiate between quaternary/secondary C atoms (positive signals) and primary/tertiary C atoms
(negative signals).- "B NMR spectra: [D6]DMSO, Bruker AMX 500 spectrometer at 161 MHz, BF3 etherate as external standard, chemical shifts in 6
ppm.- Mass spectra: Finnigan MAT 3 12, Micro PDP- 11 computer, Finnigan
program SS300.- IR spectra: KBr pellets, Bruker IFS 48, programm Opus.Melting points: Biichi model 530 apparatus, uncorrected.- TLC: TLC plates
Merck Darmstadt; boronic acid derivatives were
0.25 mm silica gel 60 Fz~A,,
Arch. Pharm. Pharm.Med. Chem 329, 73-82 (1996)
77
Terphenyl-Bisamidines
detected by spraying with diphenylcarbazone in methanol (0.2% wh),stannyl compounds by spraying with phosphomolybdate (1% in 95% ethan01)‘*”~.-Dry solvents: Acetone allowed to stand over P205 for at least one
hour, then distilled over a mirrored Vigreux column and kept over molecular
sieve (0.4 nm). Diethyl ether, dichloromethane, tetrahydrofuran, and toluene
kept over A1203 (alumina N, activity grade I, ICN Biomedicals). Methanol
distilled from sodium methanolate (1 0 g sodium per L methanol).- Reagents:
Trimethyl borate (Merck) distilled prior to each experiment using a 20 cm
mirrored Vigreux column ””. Typically, starting with 81.3 g (87.4 mL) of
this trimethyl borate, 12.1 g boiled between 5 9 4 6 “C (azeotrope of methanolhrimethyl borate) and were discarded; 62.3 g trimethylhorate boiled at
67-69 “C and were used in the reaction, while 10.3 g of yellow turbid residue
werediscarded.-n-Butyllithiumin hexane was titratedusing [ 1,1’]-hi henyl4-methanol [221.0.67 M methylchloroaluminum amide in toluene ”’]: At
0-5 “C and under nitrogen atmosphere, a suspension of 270 mg (5.05 mmol)
ammonium chloride in 5.0 mL dry toluene was added to 2.50 mL of 2 M
trimethyl aluminum in toluene. The solution was strirred at room temperature
until methane evolution ceased.
3-Cyano-phenylboronic
acid (9)was prepared from reaction of 3-bromobenzonitrile with n-butyllithium in n-hexane at -100 “C, followed by
trimethyl borate in toluene and acid hydrolysis
Extraction of the crude
product in a Soxleth apparatus gave a somewhat purer product asjudged from
the higher melting point as that re orted in the literature: Colorless needles;
yield 68%; mp 341-344 “C (ref.“”: 320-322 “C); “B NMR: 28.3 ppm.
4‘-(5,5-Dimethyl[l,3,2/dioxahorinan-2-yl)biphenyl-4-carbon~trile
(15)
[ I , I‘]-Biphenyl-2-carbonitrile(5c)
Method A. 2.73 g (15.0 mmol) 3-bromobenzonitrile in 50 mL toluene,
2.20 g (18.0 mmol) phenylboronic acid in 50 mL ethanol, 30 mL 2 M sodium
carbonate, 16 h reflux; cc (4 x 35 cm, silicagel, i-hexaneldiethy1ether 1:l);
yield 2.57 g (96%) oil; (ref.[261mp 35-37°C).
[ I , 1‘;4‘,I”]-Terphenyl-3,3“-dicarbonitrile (lla)
Method B. 2.47 g (7.50 mmol) 10a in 70 mL toluene; 2.65 g (18.0 mmol)
9 in 50 mL ethanol; 30 mL 2 M aqueous sodium carbonate, 15 reflux; yield:
1.89 g (90%) colorless crystals; mp 241-242 “C; IR: v = 3097 (w), 3069 (w).
3040 (w, =C-H); 2231 (s, C=N); 1597 (w), 1578 (w), 1477 (m, v C=C).- ‘H
NMR: 6 = 7.69 (t, 2H, 5-H, 5”-H, 3J = 7.9 Hz, 5J = 0.6 Hz), 7.82 (dt, 2H,
4-H, 4”-H, 3J = 7.9 Hz, 4J = 1.4 Hz), 7.87 (s, 4H, 2’-H, 3’-H, 5’-H, 6’-H,
AA’A”A”’),8.07(ddd,2H,6-H,6”-H,3J=7.9Hz,4J=
1.2Hz),8.18(t,2H,
2-H, 2”-H, 4J = 1.5 Hz).- I3C-NMR: 6 = 112.43 (pos, C-3, C-3”), 118.60
(pos, GIN), 127.60 (neg, C-Y,C-3’,C-5’,C-6‘), 130.14 (neg, C-2,C-2”,C-5,CS”), 131.17 (neg, C-4, C-4”), 131.37 (neg, C-6, C-6”), 138.04 (pos, C-l’,
C-4’), 140.71 (pos, C-1, C-I”).- EI-MS: m/z (%) = 280 (100) [M+].- Anal.
(CzoH12N2).
[ I , I‘;3’,l“]-Terphenyl-3,3“-dicarbonitrile(llb)
Method B. 1.20 mL (2.34 g, 9.92 mmol) 10b in 120 mL toluene; 3.58 g
(24.4 mmol) 9 in 40 mL ethanol; 40 mL 2 M aqueous sodium carbonate; 4 d
reflux; yield: 2.37 g (85%) colorless crystals; mp 164-165 “C; IR: v = 3083
(w), 3061 (m), 3031 (w, =C-H), 2232 (m, v CzN), 1601 (w). 1578 (w), 1490
(w), 1479 (w, v C=C).- ’H NMR: 6 = 7.58-7.66 (m, lH, 5’-H), 7.69 (2H,
5-H, 5”-H), 7.78-7.83 (m, 2H, 4’-H,6’-H), 7.85 (2H, 4-H, 4”-H), 8.11 (1H,
2’-H), 8.13-8.19 (m, 2H, 6-H, 6”-H), 8.35 (2H, 2-H, 2”-H).- I3C NMR: 6 =
112.04 (pos, C-3,C-3”). 118.71(pos, C=N), 125.56 (neg, C-2’), 126.74 (neg,
C-4’, C-6’), 129.79 (neg, C-5’), 129.96 (neg, C-5, C-5”), 130.53 (neg, C-2,
C-2”), 131.14 (neg, C-4, C-4”), 131.66 (neg, C-6, C-6”), 138.76 (pos,
C-l’,C-3’), 140.80(pos, C-1, C-1”).-ELMS: m/z (%) = 280 (100) [M’], 251
(7) [M’-HCN1.- Anal. (C2oHizN2).
Under nitrogen, 1.82 g (10.0 mmol) 4-bromohenzonitrile, 3.20 g (11.0
mmol 5,5 dimethyl-2-(4’-(tributylstannanyl)-phenyl-[1,3,2]-dioxaborinane
(14)“ and Pd(PPhs)4 (15 mg) in 80 mL toluene were heated to reflux until
14 had disappeared (50 h; TLC: toluene/methanol = 100 1). The solvent was
removed in vacuo and the residue was purified (cc, 3 x 35 cm, silica gel,
toluene/methanol 100:1). Yield: 0.94 g (48%). mp 170-72 T-IR: v = 3047
(w, =C-H), 2959 (m), 2225 (m, v C=N), 1590 (m), 1604 (m),1521 (w). 1476
(m. C=C).- ‘H NMR: 6 = 0.98 (s, 6H, =C(-CH3)2), 3.78 (s, 4H, -O-CH2-),
7.71 (d, 2H, H 3‘/5‘, BB’), 7.82 (d, 2H, H 2‘/6‘, AA’), 7.84-7.96 (m, 4H, H
2/3/5/6, AA’BB’).- 13C NMR: 6 = 21.24 (neg, =C(-CHs)z), 31.40 (pos,
=C(-CH3)2), 71.38 (POS,O-CH2), 110.08 (POS,4’-C), 118.69 (POS,G N ) ,
126.14 (neg, C-2, C-6), 127.15 (neg, C-2‘,C-6‘), 133.73 (neg, C-3’, C-5’),
[ I , 1’;2’,I“]-Terphenyl-3,3”-dicarbonitrile
(llc)
134.25 (neg, C-3,C-5), 140.20 (pos, C-I), 144.38 (pos, C-l’).- “B NMR: 6
= 26.7.-EI-MS: m/z (%) = 291 (100) [M’], 205 (52) [NC-(C~H~)~-BEO+].- MethodB.0.91 mL(1.77g,7.5Ommol)lOcin25mLtoluene;2.65g(18.0
mmol) 9 in 30 mL ethanol; 20 mL 2 M aqueous sodium carbonate; 26 h reflux;
Anal. (CixHixBN02).
yield: 1.79 g (85%) colorless crystals; mp 1 1 6 118 “C.- IR: v = 3065 (w),
3031 (w, =C-H), 2230 (s, C=N), 1602 (w), 1579 (w), 1468 (m, C=C).- ‘H
Suzuki Couplings
NMR: 6 = 7.37 (dt, 2H, 4-H, 4”-H, ’J = 7.6 Hz, 4J = 1.5 Hz), 7.45 (t, 2H,
5-H, 5”-H, 3 J = 7.6 Hz), 7.47-7.59 (m, 4H, 3’-H, 4’-H, 5’-H, 6’-H, AA’BB’),
Method A: Under nitrogen, a heterogeneous mixture of 1 equiv haloben1.5
=
Hz), 7.72 (dt, 2H, 6-H, 6‘-H, 3 J = 7.6 Hz, ‘J
7.63 (t, 2H, 2-W2“-H, ‘.I
zene in toluene, 15 mg palladium tetrakis(triphenylphosphine), 1.2 equiv
= 1.5 Hz).- I3C NMR: 6 = 111.38 (pos, C-3, C-3”), 118.61 (pos, CN), 128.74
phenylboronic acid in ethanol, and 2 M aqueous sodium carbonate, was
(neg, C-3’, C-6’), 129.31 (neg, C-4’, C-S’), 130.56 (neg, C-5, C-S”), 130.76
refluxed for several hours until educts had disappeared (TLC). The organic
layer was seperated, the aqueous layer was extracted three times with dietheyl
(neg, C-2, C-2”), 133.12 (neg, C-4, C-4”), 134.67 (neg, C-6, C-6”), 138.11
ether, the combined organic phases were dried over sodium sulfate, filtered,
(pos, C-l’, C-2’), 141.58 (pos, C-I, C-I”).-EI-MS: m/z(%) =280 (100) [M’],
and the solvent removed in vacuo.The residue was purified chromatographi253 (26) [M+-HCN], 226 (7) [M+-2HCN].- Anal. (CmHi2N2).
cally (silica gel, i-hexaneldichloromethane 1:4).- Method B: 2.4 equiv
phenylboronic acid, all other reagents and methods the same as method A.
[ I , l’:4’, I “]-Terphenyl-4,3”-dicarbonitrile
(lld)
4
-
’
[ I , l’]-Biphenyl-4-carbonitrile
(5a)
Method A. 1.82 g (10.0 mmol) 3-bromobenzonitrile in SO mL toluene,
1.46 g (12.0 mmol) phenylboronic acid in 20 mL ethanol, 20 mL 2 M sodium
carbonate, 6 h reflux; cc (2 x 20 cm, silica gel, i-hexaneldiethyl ether 1 : I ) ;
yield 1.74 g (97%) colorless crystals; mp 87-88 “C (ref.‘24185-86 “C).
[ I , I’]-Biphenyl-3-carbonitrile(5b)
Method A. I .82 g (10.0 mmol) 3-bromohenzonitrile in 50 mL toluene,
1.46g (12.0 mmol) phenylboronic acid in 20 mL ethanol, 20 mL 2 M aqueous
sodium carbonate, 6 h reflux; cc (2 x 20 cm, silicagel, i-hexane/diethyl ether
2:3); yield 1.78 g (99%) colorless crystals; mp 46-48 “C (ref.[25149 “C).
Arch. Phaim. Phaim. Med Chem.329,7342(19%)
Method A. 550 mg (3.02 mmol) 3-bromobenzonitrile in 50 mL toluene;
440 mg (3.00 mmol) 15 in 30 mL ethanol; 20 mL 2 M aqueous sodium
carbonate; 26 h reflux; yield: 638 mg (80%) colorless crystals; mp 201202 “C.- IR: N = 3078 (w), 3038 (w, v =C-H), 2228 (s, v C=N), 1604 (m),
1582 (w), 1500 (w), 1477 (m, v C=C).- ‘H NMR: 6 = 7.69 (t. lH, S”-H),
7.85 (dt, 1H, 4”-H, 3J = 7.9 Hz, 4J = 1.4 Hz), 7.87-7.97 (m, 8H, 2-H, 3-H,
5-H, 6-H, 2’-H, 3’-H, 5’-H, 6’-H, 2 AA’BB’), 8.068.12 (m, lH, 6”-H), 8.23
(t, lH, 2”-H, 4J = 1.5 Hz).- I3C NMR: 6 = 110.20 (pos, C-4), 112.16 (pos,
C-3”), 118.63 (pos, CIN), 118.70 (pos, C=N), 127.34 (neg, C-3’, C-5’),
127.55 (neg, C-2’, C-6’), 127.65 (neg, C-2, C-6), 130.10 (neg, C-5”), 130.15
(neg, C-2”), 131.22 (neg, C-4”), 131.33 (neg, C-6”), 132.77 (neg, C-3, C-5),
137.99 (pos. C-l’), 138.10 (pos, C-4’), 140.30 (pos, C-I”), 143.70 (pos,
C-I).- EI-MS: m/z (%) = 280 (100) [M’].- Anal. (C2oHnNz).
78
von der Saal and co-workers
3‘,3“-(Nuphthulene-2,7-diyl)-bisbenzonitrile
(He)
7.86 (s, 4H, 2‘-H, 3’-H, 5’-H, 6’-H, AA’A’””’), 7.94 (dt, 2H, 6-H, 6”-H, 3J
MethodB.2.63g,6.20mmol) 10d1171in5SmLtoluene;2.18g(14.8mmol)= 7.9 Hz, 4J = 1.4 Hz), 8.19 (t. 2H, 2-H, 2”-H, 4J = 1.5 Hz), 9.65 (s, 2H,
-C=S(-NHz)), 9.96 (s, 2H, -C=S(-NHz)).- I3C NMR: 6 = 125.04, 127.15
9 in 40 mL ethanol; 25 mL 2 M aqueous sodium carbonate; 16 hreflux; yield:
(neg, C-2, C-2”, C-4,C-4”), 127.47 (neg, C-2’,C-3’,C-5’,C-6‘), 128.69 (neg,
1.29 g (63%) colorless crystals; mp 198-199 “C.- IR: v = 3070 (w). 3057
C-5, C-S”), 129.26 (neg, C-6, C-6”), 138.91 (pos, C-3, C-3”), 139.19 (pos,
(w), 3023 (w, =C-H), 2230 (s, CEN), 1600 (w), 1579 (w), 1483 (w, C=C).C-l’, C-4’), 140.15 (POS,C-I, C-I”), 199.98 (POS,C=S).- (+)-FAB-MS,
‘H NMR: 6 = 7.73 (t, 2H, 5”H, 5”-H, 3J = 7.8 Hz), 7.86 (dt, 2H, 4’-H, 4”-H,
glycerol/3-nitrobenzylalcohol: m/z (%) = 349 (100) [M+H]+, 332 (13)
3 J = 7.8 Hz), 7.94 (dd, 2H, 3-H, 6-H, ’ J = 8.5 Hz, ‘.I=
1.5 Hz), 8.08 (d, 2H,
[M+H+-NH3], 315 (21) [M+H+-H2S], 298 (13) [M+H+-NH3-HzS].-Anal.
4-H, 5-H, 3J = 8.5 Hz), 8.16 (dt, 2H, 6’-H, 6”-H, ’J = 7.8 Hz), 8.29 (t, 2H,
(CzoH I 6NS).
2’-H, 2”-H), 8.39 (d, 2H, 1-H, 8-H).- 13CNMR: 6 = 112.20(pos, C-3’, C-3”),
118.66 (pos, C=N), 125.36 (neg, C-3, C-6), 126.41 (neg, C-1, C-8), 128.40
( I , 1’;3’, 1”]-Terphenyl-3,3”-dicarbothioamide
(12b)
(neg, C-4, C-5), 130.16 (neg, C-5’, C-5”), 130.39 (neg, C-Y, C-Y), 131.08
(neg, C-4‘, C-4”), 131.53 (neg, C-6‘, C-6”), 132.07 (pos, C-8a), 133.31 (pos,
1.06 g (3.78 mmol) llb in 30 mL pyridine, 3.00 mL (3.10 g, 21.6 mmol)
TEA; 8 d standing at room temperature; recrystallized from diethyl ether.
C-4a), 135.82 (pos, C-2, C-7), 140.88 (pos, C-l’, C-l”).- EI-MS: m/z (%) =
330 (100) [M+].- Anal. (Cz4Hi4Nz).
Yield: 0.85 g (65 %) mp 209-210 “C.- IR: v = 3300 (s), 3287 (s), 3144 (s,
NHz), 1638 (s), 1614 (s, NHz); 1569 (sh), 1456 (w, C=C).- ‘H NMR: 6 =
7.54 (m. 2H, 5-H, 5”-H, 3J = 7.8 Hz), 7.58-7.65 (m, 1H, S’-H), 7.71-7.77
Thioamidesfrom Nitriles
(m, 2H, 4’-H, 6’-H), 7.90 (m, 2H, 4-H, 4”-H. 3J = 7.9 Hz, 4J = 1.4 Hz), 7.97
For 15-30 min, hydrogen sulfide was bubbled through a solution of
(m,2H,6-H,6”-H,3J=7.9Hz,4J=1.2Hz),8.04(t,1H,2’-H,4~=1.4H
carbonitrile and 3 equiv for each cyano group of triethylamine (TEA) in dry
8.18 (t, 2H, 2-H, 2”-H, 4J= 1.7 Hz), 9.65 (s, 2H, -C=S(-NHz)), 9.95 (s, 2H,
pyridine. The solution immediately turned green. It was allowed to stand at
-C=S(-NHz)).- 13C NMR: 6 = 124.93 (neg), 125.42 (neg), 126.24 (neg),
room temperature until no more educt was detected (TLC, toluene/methanol
127.32 (neg), 128.49 (neg), 129.49 (neg), 139.59 (pos), 139.94 (pos), 140.32
5: I), then nitrogen was bubbled through the solution to remove hydrogen
(pos), 199.78 (-C=S).- (+)FAB-MS, dithiothreitol: m/z (%) = 349 (70)
sulfide, the solution was evaporated in vucuo, the residue was dissolved in
[M+H]+, 332 (31) [M+H+-NH3], 315 (14) [M+H+-H2S], 298 (19) [M+H+ethyl acetate (75 mL), extracted three times with 50 mL each of I N HCI and
NH3-HzSI.- Anal. (CzoHi6NS).
three times with 50 mL each of water. The organic layer was dried over
sodium sulfate, filtered, and the solvent was removed in vucuo. The light
yellow powder was recrystallized and dried in vucuo at SO “C.
[ I , 1’;2’, I”]-Terphenyl-3,3”-dicarbothioamide
(12c)
[ I , l’]-Biphenyl-4-carbothioumide(6a)
0.80 g (4.46 mmol) Sa, 20 mL pyridine, 2.00 mL (1.46 g, 14.4 mmol) TEA,
8 d standing at room temperature: recrystallized from acetone. Yield 0.66 g
(69%) mp 224-225 T - ‘H NMR 6 = 7.367.53 (m, 3H, 3’-H, 4’-H),
7.68-7.76 (m, 4H, 2-H, 3-H), 7.98-8.05 (m, 2H, 2’-H), 9.54, 9.88 (2s, 2H,
C(=S)-NHz).- I3C NMR: 6 = 125.95, 126.70,127.93, 128.90 (neg, C-2, C-3,
C-2’, C-3’), 137.95 (POS,C-4), 138.98 (POS,C-l’), 142.59 (C-I), 199.26(POS,
C=S).- EI-MS: m/z (%) = 213 (100) [M’], 197 (21) [M+-NHz], 180 (72)
[M+-SH].- Anal. ( C I ~ HINS).
I
[ I , I’]-Biphenyl-3-carbothioumide
(6b)
0.90 g (3.21 mmol) l l c in 30 mL pyridine, 3.00 mL (2.19 g, 21.6 mmol)
TEA; 3 d standing at room temperature: recrystallized from diethyl ether.
Yield: 0.92 g (82 %) mp 217-218 “C (dec).- IR: v = 3420,3272,3174 (m,
NHz), 1611 (s, NHz); 1479 (w, C=C).- ‘HNMR: 6 = 7.02-7.08 (m, 2H, 4-H,
4”-H), 7.19 (t. 2H, 5-H, 5”-H, ’J = 7.8 Hz), 7.51 (s, 4H, 3’-H, 4‘-H, 5’-H,
6’-H, AA’BB’), 7.74-7.79 (m, 2H, 6-H, 6”-H), 7.87 (t, 2H, 2-H/2’-H, ‘J =
~ . ~ H ~ ) , ~ . ~ ~ ( ~ , ~ H , N H Z ) , ~ . ~ ~ ( S ,125.88,
~ H , 127.20
NH~).-’~CN
(neg, ‘2-2, C-2”, C-4, C-4”), 127.92 (neg, C-3’, C-6’), 128.09 (neg, C-4’,
C-S’), 130.52 (neg, C-5, C-S”), 132.31 (neg, C-6, C-6”), 139.14 (pos, C-3,
C-3”), 139.16, 140.21 (pos, C-l’, C-2’, C-1, C-1”), 199.47 (pos, C=S).(+)FAB-MS, glycerolkhioglycerol: d z (%) = 349 (100) [M+H]+, 332 (37)
[M+H+-NH3], 317 (22) [M+Hf-S], 315 (11) [M+H+-HzS], 298 (47)
[M+H+-NH3-H2S].- Anal. (CzoHi6NS).
1.05g(5.86mmoi)Sb,25mLpyridine,2.50mL(l.83g,
18.0mmol)TEA,
3 d standing at room temperature; recrystallized from acetone. Yield 1.25 g
( I , 1’;4’,
I”]-Terphenyl-4,3“-dicarbothioamide
(12d)
(99%) mp 104-106 “C. ‘H NMR 6 = 7.35-7.43 (m, IH, 4-H), 7.46-7.54 (m,
0.56 g (2.00 mmol) l l d in 30 mL pyridine, 3.00 mL (3.10 g, 21.6 mmol)
IB,6-H),7.89-7.95
3H, 3’-H,4‘-H),7.69-7.74(m,2H,2’-H),7.76-7.84(m,
TEA; 4 d standing at room temperature; recrystallized from diethyl ether.
(m, IH,5-H),8.12(t, lH,2-H,‘J= 1.8Hz),9.17,9.93(2~,2H,C(=S)-NHz).Yield: 610 mg (88 %) mp 258-260 “C (dec).- IR: v = 3159 (s, NHz); 1619
I3C NMR: 6 = 125.09, 126.75, 127.62, 128.45, 128.85, 129.18 (neg, C-2,
(s), 1603 (s, NHz), 1479 (w. C=C).- ‘HNMR: 6 = 7.53 (t. lH, 5”-H, 3J= 7.8
C-4, ‘2-5, C-6, C-2’, C-3’ C-4’), 139.48 (POS,C-3), 139.70 (POS,C-l), 139.93
Hz), 7.80 (d, 2H, 2-H, 6-H, BB’), 7.83-7.90 (m, 5H, 2’-H, 3’-H, S-H, 6’-H,
(C-l’), 199.85 (pos, C=S).- +FAB-MS: m/z (%) = 213 (100) [M+H+], 197
AA’BB’, and 6”-H), 7.95 (d, lH, 4”-H, ’5 = 7.8 Hz, 4J = 1.4 Hz), 8.03 (d,
(81) [M+H+-NH’], 180 (76) [M+H+-HzS1.- Anal. (CI~HIINS).
2H, 3-H, 5-H, AA’), 8.19 (t, IH, 2”-H, 4 J = 1.7 HL), 9.56 (s, 1H, NHz), 9.66
(s, 1H, NHz), 9.89 (s, lH, NHz), 9.96 (s, 1H, NHz).- 13C NMR: 6 = 124.91
( I , l‘]-Biphenyl-2-carbothioamide (6c)
(neg), 125.86 (neg), 127.09 (neg), 127.27 (neg), 127.34 (neg), 127.99 (neg),
1.05 g (5.86 mmol) Sc, 25 mL pyridine, 2.50 mL (1.83 g, 18.0 mmol) TEA,
128.55 (neg), 129.11 (neg), 138.03 (pos), 138.21 (pos), 138.98 (pos), 139.03
(pos), 139.98 (pos), 141.88 (pos), 199.19 (pos, C=S), 199.79 (pos, C=S).8 d standing at room temperature: cc (2.5 x40cm, silica gel, toluene/methanol
EI-MS: m/z (%) = 348 (68) [M+], 314 (45) [M+-HzS], 297 (16) [M+-NH35:l). Yield 1.15 g (92%) mp 162-163 “C. ‘H NMR 6 = 7.26-7.46 (m, 7H),
HzS], 280 (100) [M+-2H2S].- Anal. (CzoHi6NS).
7.47-7.53 (m, 2H), 9.25,9.75 (2s, 2H, C(=S)-NHz).- I3C NMR: 6 = 126.82,
127.14, 127.63, 128.02, 128.49, 128.71, 129.92 (neg, C-3, C-4, C-5, C-6,
C-Y, C-Y, C-4’), 136.88 (POS,C-2), 140.17 (POS,C-I), 142.85 (C-l’), 203.26
3,3’-(Nuphthalene-2,7-diyl)-bisbenzthioamide
(12e)
(pos, C=S).- EI-MS: m/z (96)= 213 (100) [M’], 180 (48) [M+-SH].- Anal.
1.02
g
(3.09
mmol)
lle
in
30
mL
pyridine,
3.50
mL (2.56 g, 25.3 mmol)
(C iiHi INS).
TEA: 3 d standing at room temperature: recrystallized from ethyl acetate.
Yield: 1.17 g (95 %) mp 236-237 “C (dec).- IR: v = 3 140 (s, NH2), 1639 (s),
[1,1’;4’, I”]-Terphenyl-3,3”-dicarbothioamide
(12a)
1624 (sh), 1605 (m, NHz), 1475 (w, C=C).- ‘H NMR: 6 = 7.58 (t, 2H, 5’-H,
5”-H, 3J = 7.8 Hz), 7.91-8.01 (m, 6H, 4’-H, 4”-H, 6’-H, 6”-H, 3-H, 6-H),
1.11 g (3.96 mmol) l l a in 25 mL pyridine, 5.00 mL (3.65 g, 36.0 mmol)
8.08 (d, 2H, 4-H, 5-H, 3J = 8.5 Hz), 8.34 (t, 2H, 2’-H, 2”-H, 4 J = 1.7 Hz),
TEA: 5 d standing at room temperature: recrystallized from diethyl ether.
Yield: 1.05 g (76 %) mp 230 “C (dec).- IR: v = 3162 (s, NHz); 3055. 3016
8.39 (bs, 2H, 1-H, 8-H), 9.69 (s, 2H, NHz), 9.99 (s, 2H, NHz).- 13CNMR: 6
(sh, =C-H), 1632 (s, NHz); 1477 (w, C=C).- ‘H NMR: 6 = 7.53 (t, 2H, 5-H,
= 124.05 (neg, C-2’, C-2”), 125.53 (neg, C-3, C-6), 126.02 (neg, C-1, C-8),
127.08 (neg, C-4’, C-4”). 128.41 (neg, C-4, C-5), 128.76 (neg, C-5’, C-5”),
S”-H,’J=7.8H~).7.84-7.89(dt,2H,4-H,4”-H,~J=7.9Hz,~J=
1.4Hz),
Arch. Pharm. P h a m Med Chem 329,7342 (19%)
Terphenyl-Bisamidines
129.59(neg,C-6‘, C-6”), 131.78 (pos, C-8a). 133.64(pos, C-4a). 137.47(pos,
C-2, C-7), 139.58 (POS, C-3’, C-3”), 140.25 (POS,C-1’, C-1”), 200.02 (POS,
C=S), (+)FAB-MS, glycerolkhioglycerol: d z (%) = 399 (32) [M+H]+.Anal. (Cz4HisNzSz).
Methyl Curboximidothionutesfrom Curbothioamides
Six equiv. iodomethane and 1 equiv. dicarhothioamide in acetone were
refluxed for several hours. When the educt had disappeared (TLC, toluene/methanol 5:1), the solvent was removed in vucuo and the residue was
dissolved in hot acetone, filtered hot, and left standing to crystallize as yellow
powders.
Methyl [I,I’]-Biphenyl-4-curboximidothionute
Hydroiodide (7a)
0.50 g (2.34 mmol) 6a, 0.73 mL (1.66 g, 11.7 mmol) iodomethane, 20 mL
acetone; 5 d room temperature. Yield 0.67 g (81%); mp 213-215 “C. IR. v
= 3153 (m, NHz); 1672 (m, NHz); 1603 (s), 1562 (m), 1485 (m, C=C).- ‘H
NMR: 6 = 2.88 (s, 3H, S-CH3), 7.44-7.59 (m, 3H, 3’-H, 4’-H, 5’-H),
7.77-7.83 (m, 2H, 2’-H, 6’-H), 7.97-8.00 (s, 4H, 2-H, 3-H, 5-H, 6-H,
AA’BB’).- I3C-NMR: 6 = 15.58 (neg. SCH3), 127.01 (neg, C-2’, C-6‘),
127.44 (neg, C-2, C-6), 128.70 (neg, C-3, C-5), 128.87 (neg, C-4’), 129.09
(neg, C-3’. C-5’), 129.68 (pos, C-4), 137.91 (pos, C-1), 146.47 (pos, C-1’),
188.02 (pos, C=NH).- (+)FAB-MS, 3-nitrobenzylalcohol: m/z (%) = 228
(100) [M+H]+, 181 (20) [M+H+-SCH3].- Anal. (C14HidNS).
Methyl [ I , I‘]-Biphenyl-3-curboximidothionateHydroiodide (7b)
1.00 g (4.69 mmol) 6b, 1.33 mL (3.03 g, 21.3 mmol) iodomethane, 25 mL
acetone; 5 d room temperature. Yield 1.15 g (69%); mp 176-177 “C.- IR: v
= 3138 (sh, NHz), 291 1 (s, CH3); 1675 (sh, NHz); 1595 (m), 1585 (m), 1477
(m, C=C); 1449 (w, CH3).- ‘H NMR: 6 = 2.89 (s, 3H, SCH3), 7.44-7.59 (m,
3H), 7.77-7.83 (m, 4H), 8.08-8.17 (m, 2H).- 13C NMR: 6 = 15.90 (neg,
SCH3), 126.13, 127.03, 127.17 (neg, (2-2, C-4, C-2’, C-6’), 128.49, (neg,
C-4’). 129.21 (neg, C-3’, C-5’), 130.32 (neg, C-5), 131.76 (pos, C-3), 133.28
(neg, C-6), 138.34 (pos, C-l‘), 141.29 (pos, C-1), 189.21 (pos, C=NHz’).EI-MS: d z (%) = 227 (10) [M+], 180 (100) [M+-SCH3].- Anal.
(Ci4HidNS).
Methyl [ I , I’]-Biphenyl-2-curboximidothionuteHydroiodide (7c)
0.94 g (4.41 mmol) 612, 1.33 mL (3.03 g, 21.3 mmol) iodomethane, 25 mL
acetone; 5 d room temperature. Yield 0.92 g (58%); mp 202-204 “C.- IR: v
= 3445 (m), 3125 (sh, NHz), 2965 (s, CH3), 1646 (w, NHz), 1594 (w),1472
(m, C=C); 1448 (w, CH3).- ‘H NMR: 6 = 2.59 (s, 3H, SCH3), 7.37-7.54 (m,
5H), 7.567.67 (m. 3H), 7.74-7.82 (m. lH).- 13C NMR: 6 = 16.54 (neg,
SCH?), 128.31 (neg), 128.66 (neg), 128.94 (neg, C-2’, C-6‘), 129.11 (neg,
C-3’, C-5’), 129.29 (neg), 131.23 (pos, C-2), 131.36(neg), 133.27 (neg, C-5),
138.38 (pos, C-l’), 140.25 (pos, C-I), 191.37 (pos, C=NHz’).-EI-MS: d z
(%) = 227 (15) [M’], 180 (100) M’-SCH3].-Anal.
(Ci4HidNS).
79
Methyl [I,1‘;3’,I”]-Terphenyl-3,3”-di~urboximidothionute
Dihydroiodide
(13b)
0.60 g (1.72 mmol) 12b, 0.64 mL (1.47 g, 10.3 mmol) iodomethane, 25
mL acetone; 3 h reflux. Yield 1.01 g (93%); mp 222-223 “C (dec).- IR: v
= 3427 (m, NHz), 3055 (sh, =C-H), 2981 (s, CH3), 1652 (m. NHz); 1593 (w),
1573 (m), 1514 (w, C=C).- ‘H NMR 6 = 2.90 (s, 6H, CH3), 7.71 (t, lH,
5’-H, 3J = 7.8 Hz), 7.80 (t, 2H, 5-H, 5”-H, 3J= 7.6 Hz), 7.84-7.94 (m, 4H,
4’-H, 6’-H, 6-H, 6”-H), 8.15 (t. IH, 2’-H, 4J = 1.5 Hz), 8.21-8.29 (m, 4H,
2-H, 2”-H, 4-H, 4”-H).- 13C NMR: 6 = 15.94 (neg, SCH3). 125.84 (neg,
C-2’), 126.43 (C-4’, C-6’), 127.12 (neg, C-2, C-2”), 127.45 (neg, C-4, C-4”),
130.09 (neg, C-5’), 130.31 (neg, C-5, C-5”), 131.95 (pos, C-3, C-3”), 133.53
(neg, C-6, C-6”), 139.38 (pos, C-l’, C-3’), 140.94 (pos, C-I, C-1”), 188.89
(pos, C=NHz’).- (+)FAB-MS, DMS0/3-nitrobenzylalcohol: d z (%) = 377
(71) [M+H+], 329 (15) [M+H+-SCH3], 281 (11) [M+H+-2SCH3].- Anal.
(CzzHz2IzNzSz).
Methyl [I,I’;2~,I”]-Terphenyl-3,3”-dicurboximidothionute
Dihydroiodide
(13c)
1.00 g (2.87 mmol) 12c, 1.07 mL (2.44 g, 17.2mmol) iodomethane, 30 mL
acetone; 4 h reflux. Yield 1.67 g (92%); mp 214-216 “C (dec).- IR: v = 3426
(w, NHz), 3047 (sh, =C-H), 2973 (s, CH3), 1648 (m, NHz), 1593 (sh), 1570
(m), 1511 (sh), 1469 (w, C=C).- ’H NMR: 6 = 2.81 (s, 6H, SCH3), 7.45 (dt,
2H, 4-H, 4“-H, 3J = 7.6 Hz, 4J = 1.5 Hz), 7.52 (t, 2H, 5-H, 5“-H, 3J = 7.6
Hz), 7.59-7.61 (m, 4H, 3’-H, 4’-H, 5’-H, 6’-H, AA’BB’), 7.68 (t, 2H, 2-H,
2”-H, 4J = 1.5 Hz), 7.75 (dt, IH, 6-H, 6”-H, 3 J = 7.6 Hz, 4J= 1.5 Hz), 13C
NMR: 6 = 15.70 (neg, SCH3), 126.52 (neg, CH), 128.74 (neg, CH), 128.98
(neg, CH), 129.29 (neg, CH), 130.48 (neg, C-5, C-5”), 131.12 (pos, C-3,
C-3”), 135.97 (neg, C-6, C-6”), 138.05 (pos. C-l’, C-21, 141.26 (pos, C-1,
C-I”), 188.28 (pos, C=NHz+).- (+)FAB-MS, 3-nitrobenzylalcohol:m/z (%)
= 377 (82) [M+H+], 329 (17) [M+H+-SCH31, 281 (21) [M+H+-2SCH3].Anal. (C22HzzIzNzSz).
Methyl [I,1’;4~,I”]-Terphenyl-4,3“-dicurboximidothionate
Dihydroiodide
(13d)
554 mg (1.59 mmol) 12d, 0.60 mL (1.35 g, 9.45 mmol) iodomethane,
40 mL acetone; 3 h reflux. Yield 741 mg (74%); mp 210-213 “C (dec).- IR:
v = 3419 (w, NH2), 3027 (s, =C-H), 2984 (sh, CH3), 1652 (m, NHz), 1601
(s), 1472 (w, C=C).- ‘H NMR: 6 = 2.89 (s, 6H, SCH3), 7.76 (t. lH, 5”-H, 3J
= 7.9 Hz), 7.85-8.1 1 (m, 10H, 4”-H, 6”-H, 2-H, 6-H, and 2‘-H, 3’-H, 5’-H,
6’-H, AA’BB’), 8.19 (m, lH, 2’-H).- I3C NMR: 6 = 15.71 (neg, SCH3), 15.81
(neg, SCH3). 126.08 (neg, C-2”), 127.48 (neg, C-2, C-6), 127.56 (neg, C-4”),
127.81 (neg, C-2’, C-6‘), 127.94 (neg, C-3’, C-5’), 128.89 (neg, C-3 C-5),
130.37 (neg, C-5”), 132.08, 133.11, 133.62 (neg, C-3”, C-6”, C-4), 137.91,
138.80 (POS,C-4’, C-l’), 140.33 (POS,C-1”), 145.65 (POS,C-1). 187.91 (POS,
C=NHz+),188.61 (pos, C=NHz+).- (+)FAB-MS glycerol/3-nitrobenzylalcohol: m/z (%) = 377 (100) [M+H’], 329 (21) [M+H+-SCH3].- Anal.
(C2zHzzIzNzSz).
Dimethyl 3,3“-(nuphthulene-2,7-diyl)-benzcurboximidothionuteDihydroiodide (13e)
Methyl [1,1’;4f,I”]-Terphenyl-3,3“-dicurboximidothionute
Dihydroiodide
(134
0.98 g (2.81 mmol) 12a, 1.05 mL (2.40 g, 16.9mmol) iodomethane, 75 mL
acetone; 3 h reflux. Yield 1.57 g (88%); mp 219-220 “C (dec).- IR: v = 3428
(w, NHz), 3023 (sh, C-H), 2989 (rn, CH3), 1652 (m, NHz), 1585 (m), 1580
(m), 1507 (w), 1477 (w, v C=C).- ‘H NMR: 6 = 2.92 (s, 6H, SCH3), 7.79 (t,
2H, 5-H, 5”-H, 3J= 7.7 Hz), 7.86-8.00 (m, 6H, 2’-H, 3’-H, 5’-H, 6’-H,
AA’BB’ and 4-H, 4”-H), 8.17-8.26 (m, 4H, 2-H, 2”-H, 6-H, 6”-H).- 13CNMR: 6 = 15.83 (neg, SCH3), 125.95 (neg, C-2, C-2”), 127.42 (neg, C-4,
C-4”), 127.62 (neg, C-2’, C-3’, C-5’, C-6‘), 130.24 (neg, C-5, C-5”), 131.69
(pos, C-3, C-3”), 133.06 (neg, C-6, C-6”), 138.08 (pos, C-l’, C-4’), 140.25
(pos, C-I, C-1”), 189.05 (pos, C=NHz+).- (+)FAB-MS, glycerol/3-nitrobenzyl alcohol: m/z (%) = 377 (100) [M+H+],363 (21) [M+H+-CH2], 329 (13)
[M+H+-SCH3].- Anal. (CZ~HZ~IZNZSZ).
Arch. Phan Phan Med. Chem 329,73-82 (19%)
750 mg (1.88 mmol) 12e, 0.94 mL (2.14 g, 15.1 mmol) iodomethane,
30 mL acetone; 2.5 h reflux. Yield 1.19 g (92%); mp 224 “C (dec).- IR: v =
3426 (w. NH2), 3047 (sh, =C-H), 2973 (s, CH3), 1648 (m, NHz), 1593 (sh),
1570 (m), 1511 (sh), 1469 (w, C=C).- ‘HNMR: 6 = 2.93 (s, 6H, SCH3). 7.84
(t, 2H, 5’-H, 5”-H, 3J = 7.9 Hz), 7.93 (dt, 2H, 4’-H, 4”-H, 3J = 7.9 Hz, 4J =
1.4 Hz), 8.02 (dd, 2H, 3-H, 6-H, 3J=8.5 Hz, 4J= 1.5 Hz), 8.17 (d, 2H, 4-H,
5-H, 3J= 8.5 Hz), 8.28-8.32 (m, 4H, 2’-H, 2”-H, 6’-H, 6”-H), 8.47 (d, 2H,
I-H, 8-H,4J= 1.2Hz).-I3CNMR: 6 = 15.82(neg,SCH3), 125.52(neg,(C-3,
C-6), 126.25 (neg, C-1, C-8), 126.35, 127 24 (neg, C-2’, C-2”, C-4’, C-4”),
128.58 (neg, C-4, C-5), 130.32 (pos, C-3, C-3”). 131.87 (pos, C-ga), 132.12
(neg. C-5’, C-5”), 133.29 (neg, C-6‘, C-6”), 133.33(pos, C-4a), 136.22 (pos,
C-2, C-7), 140.77 (POS,C-1’, C-1”), 188.87 (pos, C=NHz+).- (+)FAB-MS,
glycerol: d z (%) = 427 (18) [M+H+], 379 (4) [M+H+-SCH3].- Anal.
(Cz6Hz4IzNzSz).
80
Ainidines from Methyl Curhoximidothionutes
One equiv. methyl carhoximidothionate hydroiodide and 3 equiv. ammonium acetate in dry methanol were refluxed for several hours until educt had
disappeared (TLC, 2-propanol/25% aqueous ammonidwater 6:3: 1), the solvent was removed and the residue recrystallized from acetone to give
colorless powders.
[ I , S;4’, I”]-Terphenyl-.?,3”-dicarbamidiniumDiacetute (4a2HOAc)
0.50 g (0.73 mmol) 13a, 0.45 g (5.86 mmol) ammonium acetate, 40 mL
methanol, 3 h reflux. Yield 155 mg (49%); mp 258-260 “C.- IR: v = 3391
(w, NH2), 3063 (w, =C-H), 2984 (sh, CH3), 1678 (m, C=O), 1571 (s), 1526
(m), 1473 (m, C=C).- ‘H NMR, CF3COOD: 6 = 1.93 (s, 6H, CH3), 7.74 (t,
2H, 5-H, 5”-H, ’J = 7.8 Hz), 7.88 (dt, 2H, 4-H, 4”-H, 3J = 7.8 Hz, 4J = 1.5
Hz), 7.99 (s,4H, 2’-H, 3’-H, 5’-H, G-H, AA’A”A”’), 8.12 (dt, 2H, 6-H, 6”-H,
’J = 7.8 Hz, 4J = 1.5 Hz), 8.22 (bs, 2H, 2-H, 2”-H).- ”C NMR: 6 = 20.46
(neg, CH?), 126.50, 127.08 (neg, C-2, C-2”, (2-4, C-4”), 127.53, (neg, C-2’,
C-3’. C-5’, C-6’). 128.61 (pos, C-3, C-3”), 129.47 (neg. C-5, C-S’j, 131.66
(neg, C-6. C-6“), 138.50, 140.15 (pos, C-l’, C-4’, C-1, C-I”), 165.33 (pos,
C=NH2+),171.82 (pos, COO).- (+)FAB-MS, glycerol/thioglycerol: d z (%)
= 315 (26) [M+H+],298 (76) [M+H+-NH31.- Anal. (C24HztiN404).
[I,lr;3‘,I”]-Terphenyl-3,3”-dicarbumidinium
Acetute Iodide
(4b. HOAC HI)
von der Saal and co-workers
/1,1’;4’, l“l-Terphenyl-4,3”-dicarbamidiniumDiacetate (4d.2HOAc)
614 mg (0.97 mmol) 13d, 0.60 g (7.77 mmol) ammonium acetate, 45 mL
methanol, 6 h reflux. Yield 340 mg (81%); mp 219-221 “C.- IR: v = 3266
(w, NH2); 3055 (w, =C-H): 2930 (sh, CH3),1672 (s, C=O); 1610 (m, NH2),
1565 (w), 1546 (m), 1474 (s, C=C).- ‘H NMR, +CF3COOD: 6 = 1.92 (s, 6H,
CH3),7.75(t, 1H,5”-H,3J=7.9H~),7.86(dt,~ H , ~ ” - H , ’ J = ~ . ~ H Z , ~ J =
1.5 Ha), 7.92-8.07 (m, 8H, 2-H, 3-H, 5-H, 6-H, 2’-H, 3’-H, S-H, 6’-H,
AAfBB’),8.12(dt, ~ H , ~ ” - H , ’ J = ~ . ~ H 1.5Hz),8.20(t,
z,~J=
1H,2”-H,4J
= 1.5 Hz), 9.12 (s, 1H, C=NH2+), 9.19 (s, IH, C=NHz+), 9.41 (s, lH,
C=NH2+),9.47 (s, 1H, C=NH2+).- I3C NMR: 6 = 20.98 (neg, CH31, 126.54
(neg), 126.79 (posj, 126.96 (neg), 127.24 (negj,127.67 (neg), 128.14 (neg),
128.66 (pos), 128.85 (neg), 129.74 (neg), 131.78 (neg), 138.08 (pos), 138.91
(pos), 140.04 (pos), 144.48 (pos),165.14 (pos, C=NH2+), 165.55 (pos,
C=NH2+)), 171.92 (pos, COO).- (+)FAB-MS, glycerol-3-nitrohenzylalcohol: m/z (%) = 315 (44) [M+H+], 298 (22) [M+H+-NH’].- Anal.
(C24H26N404).
3’,3”-(Nuphthulene-2,7-diyl)-bisbenzcarbamidinium
Diacetute (4e)
0.60 g (0.88 mmol) 13e, 0.54 g (7.03 mmol) ammonium acetate, 30 mL
methanol, 5 h reflux. Yield 308 mg (72%): mp 277-278 T - IR: v = 3200
(sh, NH2): 3063 (sh, =C-H); 2993 (w, CH’j, 1681 (m, C=O); 1624 (sh, NHz),
1568 (s), 1522 (s), 1480 (m, C=C).- ‘H NMR, +CF3COOD: 6 = 1.92 (s, 6H,
CH3), 7.79 (t, 2H, 5’-H, 5”-H, 3J = 7.8 Hz), 7.90 (dt, 2H, 4’-H, 4”-H, 3J=
7.9 Hz). 8.04 (dd, 2H, 3-H, 6-H, ’ J = 8.4 Hz, 4J= 1.4 Hz), 8.14 (d, 2H, 4-H,
5-H, ’J = 8.5 Hz), 8.22 (dt, 2H, 6’-H, 6”-H, 3J = 7.9 Hz), 8.32 (t, 2H, 2’-H,
2”-H, 4 J = 1.4 Hz), 8.45 (bs, 2H, 1-H, 8-H), 9.22 (s, IH, C=NH2+). 9.51 (s,
lH, C=NH2+).- 13C NMR: 6 = 20.51 (neg,
- CH3), 125.53 (neg, C-3, C-6),
126.23 (neg, C-I, C-8). 126.82, 127.11 (neg, C-2’, C-2”, C-4’, C-4”), 128.45
(neg, C-4, C-S), 128.79 (C-3’, C-3”), 129.59 (neg, C-S’, C-S”), 131.97 (neg,
c-6‘,c-6”), 132.10 (POS, C&), 133.49 (POS, C-4a), 136.77 (PO?, C-2, c-7),
140.60 (C-1’, C-l”), 165.37 (pos, C=NH2+), 171.82 (pos, COO).- (+)FABMS, glyceroVthioglycerol: d z (%) = 365 (48) [M+H+], 348 (29) [M+H+NH31.- Anal. (C28H28N404).
0.70 g (1.1 I mmol) 13b, 0.68 g (8.86 mmol) ammonium acetate, 50 mL
methanol, 8.S h reflux. Yield 0.33 g (68%); mp 247-249.- Anal calcd for
C22H23INi02: C 52.60 H 4.61 N 1 I. 15 125.26 found C 50.99 H 4.46 N 10.55
1 22.30.- ~1,1’;3’,I”j-Terphenyl-3,3”-dicarbamidine
(4bj precipitated immediately when 3 mL conc sodium hydoxide was added to a solution of 66.0
mg (0.21 mmol) 4b.kIOAcHI. The precipitate was washed with methanol
and dried in vacuo at 50°C. Yield 66 mg (84%): mp 133-134 “C.- IR: v =
[ I , lf]-Biphen~l-4-caibamidinium
acetate (8a2HOAc)
3413 (m, NH2), 3055 (w, =C-H), 1635 (m, NH2); 1568 (m), 1453 (m, C=C).‘H NMR: 6 = 6.77 (bs, 2H, -C=NH(-NH2)),7.53 (t. 2H, 5-H, 5”-H, 3J = 7.8
0.50 g (1.41 mmol) 7a, 0.44 g (5.63 mmol) ammonium acetate, 20 mL
Hz),7.60(m, lH,S’-H),7.71-7.87(m,6H,4-H,4”-H,6-H,6”-H,4’-H,6’-H),
methanol, 5.5 h reflux. Yield 290 mg (80%), mp 241-242 “C ( ref.‘271
8.04 (t, IH, 2’-H, ‘J = 1.5 Hz), 8.10 (t, 2H, 2-H, 2”-H, 4J= 1.5 Hz).-- ”C
250-251 “C). +FAB-MS, 3-nitrohenzylalcohol: d z ( W ) = 197 (100)
NMR: 6 = 125.05 (neg, C-6, C-6”), 125.35 (neg, C-2’), 125.80 (neg, CH),
[M+H+], 180 (6), [M+H+-NH’].
126.09 (neg, CH), 128.39 (neg, CH), 128.63 (neg, CH), 129.43 (neg, C-S’,),
136.35 (POS, C-3, C-3”), 139.85 (POS, C-l’, C-3’). 140.54 (POS,C-I, C-1”).
[ I , I‘]-Biphenyl-3-curbamidiniumacetate (8b.2HOAc)
162.57 (pos, C=NH).- (+)FAB-MS, glycerol/3-nitrobenzylalcohol:
d z (%)
0.80 g (2.25 mmol) 7b, 0.70 g (9.08 mmol) ammonium acetate, 25 mL
= 315 (100) [M+H+], 298 (48) [M+H+-NH’], 281 (14) [M+H+-2NH’].methanol, 8.5 h reflux. Yield 460 mg (80%). mp 240-241 “C.- IR: v = 3210
Anal. (CZOHIXNJ).
(m, NH2), 3063 (w). 3031 (w, =C-H); 2968 (m, CH3), 1679 (sh, C=O); 1608
(m, NH2), 1569 (s), 1519 (s), 1470 (m, C=C): 1447 (w, CH3).- ‘H NMR 6
= 1.72 (s, 3H, CH3), 7.42 (m, IH, 4‘-Hj, 7.48-7.56 (m, 2H, 3’-H, 5’-H, 3J =
[ I , 1‘;2’, l”~-Ter~~henyl-3,3”-dicurbamidinium
Diacetate (4c2HOAc)
7.6 Hz), 7.66 (t, IH, 5-H, ’/= 7.6 Hz), 7.75-7.81 (m, 3H, 2‘-H, 6‘-H, 6-H),
7.96(dt, 1H,4-H,3J=7.6Hz,4J=1.4H~j,8.07(t,1H,2-H,4J=1.7Hz).
1.00 g ( I .58 mmol) 13c, 0.98 g ( 12.7 mmol) ammonium acetate, 40 mL
I3C NMR: 6 = 24.68 (neg, CH’), 125.83, 126.28 (neg, 2-H, 4-H), 126.87
methanol, 8 h reflux. Yield 0.57 g (83%); mp 251-252 “C.- Anal calcd for
(neg, 2’-H, 6‘-H), 127.98 (neg, 4’-H), 128.93 (neg, 3’-H, S’-H), 129.38 (neg,
C24H26N404: C 66.34 H 6.03 N 12.89 10.00 found C 63.77 H 5.81 N 12.14
5-H), 130.37 ( ~ o s3-H),
,
130.90 (neg, 6-H), 138.85, 140.62 (pos, l’-H, I-H),
I 3.16.- 1,1‘:2‘, I”]-7erplienyl-3,3”-dicurbumidine
(4c) precipitated imme165.82 (pos, C=NHZ+j, 176.52 (pos, COO).- (+)FAB-MS, glycerollthiodiately when 3 mLconc sodium hydroxide was added to a solution of 208 mg
glycerol: d z (9%) = 197 (100) [M+H+].- Anal. ( C I ~ H I ~ N ~ O Z ) .
(0.48 mmol) 4e2HOAc in 8 mL water methanol 1 :1. The precipitate was
washed with methanol and dried in vacuo at 50 “C. Yield 136mg (90%); mp
138-139 “C.- IR: v = 3466 (w), 3334 (m, NHz), 3062 (w, =C-H), 1632 (s,
[ I , I’]-biphenyl-2-carbumidine
(8,)
NMR: 6 = 124.78 (neg, C-6, C-6”), 127.39 (neg, C-5, C-S”), 127.54 (neg,
C-2, C-2”), 127.71, 130.43 (neg, C-4’, C-5’, C-3’, C-6‘), 130.93 (neg, C-4,
c - q ) , 136.09 (POS,c-3, c-3”), 139.56 (pas, c-l’, c-2’), 140.55 (pas, C-1,
C-I”), 162.37 (pos, C=NHj.- (+)FAB-MS, glyceroVthioglycerol: 315 (100)
[M+H+], 298 (59) [M+H+-NH’], 281 (10) [M+H+-2NH3].- Anal.
“GoH I 8N4).
10 g silica gel in 50 mL dichloromethane, filtered, washed with 50 mL
methanol, the solvent was removed in vacuo, the residue was suspended in
50 mL water, 15 mL 2 N HCI was added and extracted with ethyl acetate (2
X 40 mL). 30 mL 2 N NaOH was added to the aqueous suspension and
extracted with dichloromethane (2 x SO mL). The organic layer was separated, dried (NazS04), filtered and the solvent removed in varuo Yield:
Arch. P h a m P h u m Med. Chem 329, 73-82 (1996)
81
Terphenyl-Bisamidines
0.75 g (57%) colorless needles.- mp 149-151 "C.- IR: v = 3410 (m), 3324
(w. N-H), 3102 (w), 3060 (m. =C-H), 1675 (m), 1640 (m, N-H), 1601 (m),
1588 (m), 1577 (w), 1564 (w), 1481 (m. C=C).- 'HNMR: 6 = 5.60-6.40 (bs,
3H, C=NH(-NH2), 7.28-7.36 (m. 3H), 7.37-7.44 (m, 4H) 7.44-7.51 (m.
2H).- 13C NMR: 6 = 126.88 (neg), 126.92 (neg) 127.67 (neg), 127.91 (neg,
C-2', C-6'), 128.31 (neg, C-3', C-5'), 128.46 (neg), 129.84 (neg), 138.33
(pos). 138.50 (pos), 140.38 (pos), 165.28 (pos. C=NH).- (+)FAB-MS, 3 4 trobenzylalcohol: d z (%) =197 (100) [M+H+], 180 (11) [M+Hf-NH3].Anal. (Ci3HizNz).
References
Dedicated to Prof. Dr. Richard Neidlein, Heidelberg, on the occasion
of his 65th birthday.
The results of the synthetic and enzymatic part are from the Diploma
thesis of B. Gabriel, University of Regensburg, 1995.
M. T. Stubbs, W. Bode, Current Opinion Struct. Biol. 1994 4,823-832;
M. T. Stubbs, W. Bode, Trends in Cardiovascular Med. 1995 5,
157-166.
M. Mares-Guia, E. Shaw, J. Biol. Chem. 1965 240, 1579-1585.
Enzyme Kinetics
Enzyme kinetics were carried out at 25 f 0.1 "C in 0.1 M phosphate buffer
saline, pH=7.5,0.2MNaCI: 8.90gNazHP04.2H20,5.84gNaCl,
and2.50g
polyethyleneglycol 8000 were dissolved in 500 mL water (solution I). 1.36 g
KHzP04, 1.17 g NaCl and 0.50 g PEG were dissolved in 100 mL water
(solution 11). Solution I1 (approx. 85 mL) was added to solution I until pH =
7.5 was reached.- Enzyme stock solutions: 10 mg bovine trypsin (Sigma)
were dissolved in 100mL 1 mM hydrochloric acid and stored at 2-8 "C. Prior
to use, 990
1 mM HCI and 10 pL of that solution were mixed and stored
on ice.- Human thrombin (100 NIH units; Sigma; specific activity
2000 U/mg) was dissolved in 1mL water and stored in 20 pL aliquots at
-18 "C. Prior to use, 1.48 mL phosphate buffer saline was added to an
aliquote and stored on ice.- Human plasmin (Sigma, 10 U) was dissolved in
water and stored in 20 pL aliquots at -1 8 "C. Prior to use, 1.98 mL phosphate
buffer saline was added to one aliquote and stored on ice.- 990 pL phosphate
buffer saline was added to 10 pL human factor Xa (Boehringer Mannheim,
10 U, suspension) and stored on ice.- Substrates: For trypsin and thrombin,
25.0 mg (40.0 mmol) H-(D)-Phe-Pip-Arg-pNA.2HCl
(S-2238, Chromogenix AB) was dissolved in 4.0 mL water. KM 40 pM (trypsin), 4 pM
(thrombin).- For plasmin, 25.0 mg (45.3 mmol) H-(D)-Val-Leu-LyspNA.2HC1 (S-2251, Chromogenix AB) was dissolved in 11.3 mL water. KM
250 pM.- For factor Xa, 23.9 mg N-methoxycarbonyl-D-norleucyl-glycylL-arginine-4-nitroaniline(Chromozym X, Boehringer Mannheim) was dissolved in 4.1 mL water. KM400 pM.- Inhibitor stock solutions were 10 mM
in dimethyl sulfoxide.
Kinetic measurements were performed in semi micro polystyrene cuvettes
in a total volume of 1 mL, composed of 850 pL phosphate buffer saline,
1OOpL substrate, 25 pL inhibitor solution or 25 pL dimethyl sulfoxide
(blank). The solutions were thermostatted to 2.5 "C in the photometer, and
reactions were started by the addition of 25 pL enzyme solution. For each
Ki, 16 measurements (4 different substrate concentrations and 4 different
inhibitor concentrations each) were performed twice.The reaction was monitored at 405 nm, where the product (para-nitroaniline) has an E of 8800
[W' cm-'1 [281. Reactions were followed for 10 min. Data (absorbance vs
time) were stored in a personal computer. Inhibition constants (Ki)were
calculated from nonlinear fits'291of the data to the Michaelis-Menten equation Vo = Vm,-[sl/(KM.(l+[rl/Ki)+[sl],
where V, is the observed rate, Vmax
is the rate at [S] +-, [S] is the substrate concentration, which was assumed
to remain constant throughout the reaction (data used for the calculation were
from the start of the reaction until 4 0 % of substrate was converted to
product), [I] is the inhibitor concentration, KM is the Michaelis constant. The
Ki of the two determinations were within 10%. Results see Table 1.
Crystallization
Bovine P-trypsin (20 mg/mL in0.8 M ammonium sulfate, pH = 6.0/50 mM
imidazoIe/HzSO4)was crystallized by hanging drop vapor diffusion against
a reservoir of 1.6M ammonium sulfate at pH 6.0 (100 mM imidazole/HzSO4)
and with microseeds from trypsin crystals of space group P212121 with cell
axes 63.6, 63.4, 69.1. These crystals were soaked overnight in a 2mM
inhibitor (4b) solution in 2.5 M ammonium sulfate buffered with 200 mM
imidazole/HzSOr at pH = 8.0. The inhibitor structure was built and refined
with the program MAIN (details will be published elsewhere).
Arch. Pharm. Pharm. Med Chem. 329,7332 (19%)
F. C. Bemstein, T. F. Koetzle, G. J. B. Williams, E. F. Meyer Jr., M.
D. Brice, J. R. Rodgers, 0. Kennard, T. Shimanouchi, M. Tasumi, J.
Mol. Biol. 1977 112,535
B. Kaiser, J. Hauptmann, Cardiovasc. Drug Rev. 1994 12, 225-236;
S . - S . Mao, Perspectives in Drug Discovery andDesign, 1994,423430;
N. A. Prager, D. R. Abendschein, C. R. McKenzie, P. R. Eisenberg,
Circulation 1995 92, 962-967.
P. Ascenzi, R. Fruttero, G. Amiconi, L. Pugliese, M. Bolognesi, M.
Coletta, S. Onesti, M. Guameri, E. Menegatti, J. Enzyme Inhibition
1992 6, 131-139; E. Casale, C. Collyer, P. Ascenzi, G. Balliano, P.
Milla, F. Viola, M. Fasano, E. Menegatti, M. Bolognesi, Biophys.
Chem. 1995 54,75-8 1.
J. Sturzebecher, U. Sturzebecher, H. Vieweg, G. Wagner, J.
Hauptmann, F. Markwardt, Thromb. Res. 198954,245-252.
S-I. Katakura, T. Nagahara, T. Hara, M. Iwamoto, Biochem. Biophys.
Res. Commun. 1993,197,965-972; T. Hara, A. Yokoyama, H. Ishihara,
Y. Yokoyama, T. Nagahara, M. Iwamoto, Thromb. Haemostasis 1994
71,314319; T. Yokoyama, A. B. Kelly, U. M. Marzec, S . R. Hanson,
S. Kunitada, L. A. Harker, Circulation 1995 92,485491; T. Hara, A.
Yokoyama, K. Tanabe, H. Ishihara, M. Iwamoto, Thromb. Haemostasis
1995 74,635-639.
K. Padmanabhan, K.P. Padmanabhan, A. Tulinsky, C.H. Park, W.
Bode, R. Huber, D. T. Blankenship, A. D. Cardin, W. Kiesiel, J. Mol.
Biol. 1993 232, 947-966.
[lo] T. Nagahara, Y. Yokoyama, K. Inamura, S . Katakura, M. Iwamoto, J.
Med. Chem. 1994 37, 1200-1207; S. Katakura, T. Nagahara, T. Hara,
S . Kunitada, M. Iwamoto, Eur. J. Med. Chem. 1995 30,387-394; Z. L.
Lin, M. E. Johnson, FEBS Letters 1995 370, 1-5.
For reviews see: K. Ritter, Synthesis 1993 8,735-762; A. de Meijere,
F. E. Meyer, Angew. Chem. 1994 106, 2437-2506; A. R. Martin, Y.
Yang,Actu Chem. Scand. 199347,221-230; J. K. Stille, Angew. Chem.
1986 98,504-519.
For the modelling studies we used the x-ray structure of trypsin complexed with a benzamidine derivative: W. Bode, D. Turk, J. Stiirzebecher, Eur. J. Biochem. 1990 193, 175-182.
J. M. Andrews, D. P. Roman Jr, D. H. Bing, M. Cory, J. Med. Chem.
1978 21, 1202-1207.
R. S . Garigipati, Tetrahedron Lett. 1990 31, 1969-1972.
D. P. Kelly, S . A. Bateman, R. F. Martin, M. E. Reum, M. Rose, R. D.
Whittacker, Aust. J. Chem. 1994 47,247-262.
Side reactions of Suzuki couplings: Z . 2. Song, H. N. C. Wong, J. Org.
Chem. 199459,3341;T. I. Wallow, B. M. Novak, J. Org. Chem. 1994
59,50345037; G. B. Smith, G. C. Dezeny, D. L. Hughes, A. 0. King,
T. R. Verhoeven, J. Org. Chem. 1994 59,8151-8156.
Y. Yamamoto, T. Seko, H. Nemoto, J. Org. Chem. 1989 54,4734-4736.
M. Takeuchi, T. Tuihiji, J. Nishimura, J. Org. Chem. 1993 37, 73887392.
F. Markwardt, H. Landmann, P. Walsmann, Eur. J. Biochem. 1968 6,
502.
B.M. Kinsey, A.I. Kassis, Nucl. Med. Biol. 1993 20, 13-22.
82
1211 Trimethyl borate hydrolyzes upon standing: R. M. Washburn, E.
Levena, C . F. Albright, F. A. B h g , Org Synth. Coll. Vol IV, 1963,
68-72.
1221 E. Juaristi, A. Martinez-Richa, A. Garcia-Rivera, J. S. Cruz-SancheL,
J. Org. Chem. 1983 48,2603-2606.
[23] J.I. Levin, E. Tyros, S.M. Weinreb, Synth. Commun. 1982 12,989-993.
[24] L. Bauer, J. Cymerman, J. Chem. SOC. 1950, 2078-2080.
[25] M. Gomberg, W.E. Bachmann, J. Am. Chem. SOC.192446,2339-2343.
von der Saal and co-workers
[26] M. Pestemer, E. Mayer-Pritsch, Monutsh. Chem. 1937 70, 104-112.
[27] J. Krechl, S. SmrskovB, F. PavlkovB, J. Kuthan, Coll. Czech. Chem.
Commun. 1989 54.2415-2422.
[28] B. E. Erlanger, N. Kokowsky, W. Cohn, Arch. Biochem. Biophys. 1961
95, 271-278.
[29] The program used was Sigmaplot from Jandel Scientific, 40699 Erkrath, Germany.
Received: September 29, 1995 [FP062]
Arch. Phurm.Phurni. Med. Chem. 329, 73-82 (1996)
Документ
Категория
Без категории
Просмотров
1
Размер файла
1 021 Кб
Теги
serine, bisamidine, synthese, inhibitors, selective, protease, terphenyl, activities
1/--страниц
Пожаловаться на содержимое документа