close

Вход

Забыли?

вход по аккаунту

?

Non-thiol Farnesyltransferase InhibitorsUtilization of the Far Aryl Binding Site by 5-Cinnamoylaminobenzophenones.

код для вставкиСкачать
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Andreas Mitschb,
Pia Wißnerb,
Markus Böhmb,*
Katrin Silberb,
Gerhard Klebeb,
Isabel Sattlerc,
Martin Schlitzera
Department für Pharmazie ⫺
Zentrum für
Pharmaforschung,
Ludwig-MaximiliansUniversität München,
D-81377 München, Germany
b
Institut für Pharmazeutische
Chemie, Philipps-Universität
Marburg,
D-35032 Marburg, Germany
c
Hans-Knöll-Institut für
Naturstoff-Forschung e.V.,
D-07745, Jena, Germany
a
Non-thiol Farnesyltransferase Inhibitors 493
Non-thiol Farnesyltransferase Inhibitors:
Utilization of the Far Aryl Binding Site by
5-Cinnamoylaminobenzophenones
We recently described two novel aryl binding sites of farnesyltransferase. In this
study, the cinnamoyl residue was designed as an appropriate substituent for our
benzophenone-based AAX-peptidomimetic compound capable of occupying the
far aryl binding site.
Keywords: Non-thiol farnesyltransferase inhibitors; Structure-activity relationships; Aryl binding site
Received: January 1, 2004; Accepted: July 12, 2004 [FP871]
DOI 10.1002/ardp.200400871
Introduction
Farnesyltransferase has been one of the prime targets
in the development of novel anticancer drugs.
Farnesyltransferase catalyzes the covalent modification of proteins carrying the so-called CAAXsequence at their C terminus by transfer of a farnesyl
residue from farnesylpyrophosphate to the thiol of a
cysteine side chain. In the CAAX sequence, C represents a cysteine, the side chain of which is farnesylated; AA are amino acids which normally, but not
necessarily, carry aliphatic side chains; and X, in most
cases, stands for methionine or serine [1, 2]. Several
farnesyltransferase inhibitors are in advanced stages
of clinical trials for the therapy of different types of cancer [3⫺12].
Later generations of farnesyltransferase inhibitors are
different from the early inhibitors in that they lack a
free thiol, a moiety associated with several adverse
drug effects [13]. Most of these so-called non-thiol farnesyltransferase inhibitors have nitrogen-containing
heterocycles, the ring nitrogen of which coordinates
the enzyme-bound zinc, similarly to the cysteine thiol
group [14]. However, it has been shown that nitrogen
heterocycles can be replaced by aryl residues, which
lack the ability to coordinate metal atoms, without
Correspondence: Martin Schlitzer, Department für Pharmazie ⫺ Zentrum für Pharmaforschung, Ludwig-MaximiliansUniversität München, Butenandtstraße 5⫺13, D-81377 München, Germany. Phone: +49 89 2180-77804; Fax +49 89
2180-79992, e-mail: martin.schlitzer@cup.uni-muenchen.de
losing too much of the farnesyltransferase inhibitory
activity [15, 16]. Based on these observations, the
existence of at least one aryl binding region in the active site of farnesyltransferase has been postulated
[17, 18].
Using docking studies of model compounds of nonthiol farnesyltransferase inhibitors as well as GRID
analyses of the binding region of farnesyltransferase,
we have located two different aryl binding clefts in the
active site of farnesyltransferase, which we suggest to
be the postulated aryl binding regions (Figure 1) [19].
One aryl binding region (“near aryl binding site”) is located in close proximity to the zinc ion. Recently, we
have described some arylacetyl-substituted benzophenones targeting this binding site [20].
The second region (“far aryl binding site”) is defined
by the side chains of Tyr 300β, Leu 295β, Lys 294β,
Lys 353β, and Lys 356β. In the present study, we intended to fit an appropriate substituent to our AAXpeptidomimetic benzophenone scaffold which is able
to place an aromatic moiety into the far aryl binding
site.
Chemistry
Synthesis of most of the target compounds 2 was
accomplished by acylation of 2-tolylacetylamino-5* Present address: Pfizer Global Research and Development,
Groton, CT 06340, USA.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
494 Schlitzer et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Molecular modeling
Figure 1. Postulated aryl binding sites of farnesyltransferase: near aryl binding site (small box) and far
aryl binding site (big box).
aminobenzophenone 1 [21], using appropriate cinnamic acid chlorides. Commercially unavailable cinnamic acids were prepared via Knoevenagel condensation from the corresponding benzaldehydes. The
cinnamic acids were activated as acid chlorides using
thionyl chloride (Scheme 1). Pyridylacrylic acid derivatives 3 were prepared in the same way, starting from
the appropriate formylpyridines.
Farnesyltransferase inhibition assay
The inhibitory activity was determined using the fluorescence enhancement assay as described by
Pompliano [22]. The assay employs yeast farnesyltransferase fused to Glutathione S-transferase at the
N terminus of the β subunit [23]. Farnesylpyrophosphate and the dansylated pentapeptide Ds-GlyCysValLeuSer were used as substrates. Upon farnesylation
of the cysteine thiol the dansyl residue is placed in a
lipophilic environment. This results in an enhancement
of fluorescence at 505 nm, which is used to monitor
the enzymatic reaction.
Scheme 1. (I) Ar-HC=CH-COCl, toluene/dioxane,
reflux, 2 h.
Flexible docking was performed using the program
FlexX [24]. Based on the coordinates of the published
crystal structure [25] of a ternary complex of farnesyltransferase, a farnesylpyrophosphate analog, and Nacetyl-Cys-Val-Ile-selenoMetOH (PDB-code 1QBQ),
we have calculated the solvent-accessible surface of
the farnesyltransferase active site using the program
MOLCAD as implemented in the molecular modeling
software package SYBYL [26]. For initial evaluation of
the cinnamoyl-substituted lead compound 2a, the position of the benzophenone peptidomimetic substructure calculated in a previous study was used as a
starting fragment for the docking of this inhibitor. Subsequently, the remaining fragments of the inhibitors
were placed in the active site using the incremental
construction algorithm of FlexX. Subsequent docking
of inhibitors 2p and 2ab was performed with AutoDock
3.0 [27, 28]. Docking solutions from 50 individual runs
were clustered using a mutual rmsd <1 Å as criterion.The docking runs provided sets of solutions that
were inspected according to their calculated energy
score. The resulting geometries were evaluated using
the knowledge-based scoring function DrugScore [29].
Results and discussion
As stated above, we were looking for a substituent for
our benzophenone-based AAX-peptidomimetic substructure to place an aromatic moiety into parts of the
far aryl binding site. From modeling studies, the cinnamoyl moiety seemed appropriate, especially because
the trans-configurated double bond would direct the
phenyl residue into the desired direction (Figure 2). Indeed, the lead structure 2a (IC50 = 2.4 µM) proved to
be about threefold more active than the corresponding
3-phenylpropionyl derivative [19] lacking these conformational restraints. In the following, extensive substitution was carried out on the phenyl residue of the cinnamoyl moiety (Table 1). While a methyl substituent
(2c) did not significantly alter the activity, larger alkyl
residues led to compounds with enhanced activity
(2d⫺f) (IC50: 140⫺235 nM). The same holds true for
the methylthio residue (2g) (IC50 = 113 nM). Replacement of the innermost methylene or sulfur by an oxygen (2h) resulted in a markedly decreased activity
(IC50 = 6.5 µM). However, larger alkyl residues proved
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Non-thiol Farnesyltransferase Inhibitors 495
Figure 2. Representative docking solutions for inhibitor 2a showing the terminal phenyl residue in the leftmost part of the far aryl binding site.
to be able to successively overcome this effect (2iⴚk)
(IC50: 122⫺600 nM). An explanation for the enhanced
activity of these alkyl and ether derivatives may be that
these residues occupy a larger portion of the aryl binding site. However, this effect is limited since the considerably larger compounds 2l⫺p showed a markedly
decreased activity (IC50: 4.6⫺10.5 µM). These compounds are obviously too large to fit into the binding
site. This is exemplified with the styrylcinnamoyl-substituted inhibitor 2p. Docking shows that the substituted cinnamoyl moiety is indeed too large to fit into
the far aryl binding site (Figure 3). Furthermore, the
large styrylcinnamoyl substituent forces the remaining
benzophenone core into a conformation that leaves
large portions of the active site unoccupied. In the
series of halogen-substituted inhibitors 2q⫺s, activity
decreased with increasing size of the halogen (IC50:
284⫺7000 nM). Activity is also low for the trifluoromethyl derivative 2t (IC50: 4.2 µM). Not surprisingly for a
pseudo-halogen, the cyano substituent led to an inhibitor (2u) equipotent to the chloro derivative 2r. The
2,2-dicyanovinyl moiety has been suggested as a non-
Table 1. Structure and farnesyltransferase inhibitory activity of cinnamoyl-substituted benzophenones 2a⫺ac and
pyridyl derivatives 3a⫺c.
Compd.
R
IC50 (nM)
Compd.
R
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
4-H
4-NH2
4-CH3
4-CH2-CH3
4-CH(CH3)2
4-C(CH3)3
4-S-CH3
4-O-CH3
4-O-CH2-CH3
4-O-(CH2)2-CH3
4-O-(CH2)3-CH3
4-O-CH2-C6H5
3,4-bis(O-CH2-C6H5)
3-O-C6H5
4-C6H5
4-HC=CH-C6H5
2400 ± 900
680 ± 60
4100 ± 400
230 ± 50
140 ± 12
235 ± 41
113 ± 15
6500 ± 400
600 ± 50
154 ± 4
122 ± 17
4700 ± 500
7600 ± 200
10,500 ± 300
4600 ± 400
9300 ± 300
2q
2r
2s
2t
2u
2v
2w
2x
2y
2z
2aa
2ab
2ac
3a
3b
3c
4-F
4-Cl
4-Br
4-CF3
4-CN
4-HC=C(CN)2
4-CHO
4-COOCH3
4-NO2
3-NO2
2-NO2
4-SO2-CH3
4-SO2-CF3
2-pyridyl
3-pyridyl
4-pyridyl
IC50 (nM)
284
630
7000
4200
910
3900
2900
1450
235
700
565
12
540
1040
3560
4400
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
24
50
200
600
55
80
300
450
20
25
35
2
128
130
1590
200
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
496 Schlitzer et al.
Figure 3. Docking solution of inhibitor 2p (see text).
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Figure 4. Docking solution of inhibitor 2ab (see text).
aromatic replacement for a phenyl residue [30]. Indeed, compound 2v shows nearly the same activity as
the biphenyl derivative 2o.
Introduction of formyl (2w) and methyloxycarbonyl (2x)
into the para position of the terminal phenyl residue
led to inhibitors without improved potency compared
to the unsubstituted lead compound 2a. In contrast, a
nitro group in this position resulted in a tenfold enhancement in activity (2y, IC50 = 235 nM). This effect
proved to be sensitive to the position of the nitro group
on the phenyl residue, since the meta and ortho derivatives (2z, 2aa) are considerably less active. The
highest activity in this series of compounds was obtained with the methylsulfonyl derivative 2ab displaying an IC50 value of 12 nM. Replacement of the
methyl residue by a trifluoromethyl group considerably
reduced the inhibitory activity (2ac, IC50 = 540 nM). A
common feature of the inhibitors 2w⫺ac is the electron-withdrawing property of their substituents, forming
a comparatively electron-poor aromatic residue. Flexible docking shows this residue in the vicinity of the
electron-rich aromatic structure of Tyr 300β to enable
a potential charge transfer interaction between this
residue and the terminal phenyl of the cinnamic acid
substructure. However, the low activity of the formyl
and methoxycarbonyl derivatives and the sensitivity of
the activity to the position of the nitro group argue
against this possibility. A further argument against a
charge transfer interaction is provided by the three pyridyl derivatives 3a⫺c that are more or less equipotent
to the unsubstituted cinnamoyl derivative. Since the
electronic effect of a nitrogen atom inside an aromatic
ring system is equivalent to that of a nitro group attached to the ring, the electronic properties of the nitro
derivatives 2y⫺aa and the pyridyl derivatives 3 should
be similar; however, their activity is markedly different.
Figure 5. Amino acid side chains able to form hydrogen bonds with the sulfonyl moiety of inhibitor 2ab.
Hence, not only interactions of the phenyl residue with
the side chain of Tyr 300β, but also interactions of the
substituents with additional parts of the binding site
seem to be important. Another common feature for the
substituents of inhibitors 2w⫺ac is their hydrogen
bond acceptor property. Lysine side chains with their
hydrogen bond donor properties constitute a considerable part of the far aryl binding site; therefore, hydrogen bonds may be formed between the lone pairs of
the oxygen atoms of the substituents and the terminal
amino groups of the lysine residues. This explanation
would account for the sensitivity of activity to the nitro
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Non-thiol Farnesyltransferase Inhibitors 497
position and the decreased potency of the pyridyl derivatives. Docking of the methylsulfonyl-substituted inhibitor 2ab supports this explanation. Figure 4 shows
this inhibitor occupying the far aryl binding site and, in
contrast to inhibitor 2p (Figure 3), also large portions
of the active site. Three amino acid side chains with
hydrogen bond donor groups (Lys 164α, Arg 291β and
Lys 294β) are in bonding distance to oxygen atoms of
the sulfonyl moiety (Figure 5). The low activity of the
trifluoromethylsulfonyl derivative 2ac may also be explained on this basis, since the electron-withdrawing
effect of the trifluoromethyl group should considerably
reduce the hydrogen bond acceptor properties of the
sulfonyl group. The relatively low activities of the formyl and methoxycarbonyl derivatives remain to be explained. However, the substituents of these two inhibitors are different from those of the others in that they
have only one oxygen capable of acting as a hydrogen
bond acceptor, in contrast to the two of the nitro or
sulfonyl group, and therefore a lower potential to interact with the lysine side chains of the aryl binding site.
In summary, appropriately substituted cinnamoyl residues proved to be promising moieties for occupying
the far aryl binding site, leading to active non-thiol
benzophenone-based farnesyltransferase inhibitors.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]cinnamic acid amide (2a)
From cinnamic acid chloride (270 mg, 1 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 214 mg (45 %). Mp 87 °C. IR (KBr): ν = 3440, 3260,
3085, 1665, 1635, 1505 cm⫺1. 1H NMR (CDCl3): δ 2.31 (s,
3H), 3.67 (s, 2H), 6.45 (d, J = 16 Hz, 1H), 7.15 (m, 3H), 7.22
(m, 3H), 7.34 (m, 3H), 7.46 (m, 4H), 7.56 (m, 2H), 7.68 (m,
2H), 8.01 (m, 1H), 8.51 (m, 1H), 10.51 (s, 1H). MS (EI):
m/z = 342 (39), 474 (100) M+. Anal. calcd. for C31H26N2O3:
C, 78.46; H, 5.52; N, 5.90; found: C, 78.71; H, 5.52; N, 5.38.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4aminocinnamic acid amide (2b)
Compound 2y (480 mg, 0.93 mmol) was dissolved in EtOAc,
and 1.05 g SnCl2 ⫻ 2H2O were added. The mixture was
heated under reflux for 2 h. Then, NaHCO3 solution was added until pH 7⫺8 was reached, and the organic layer was
separated. The aqueous layer was extracted two times with
EtOAc. The combined organic layers were washed with brine
and dried over MgSO4. The solvent was removed in vacuo,
and the residue was purified by flash-chromatography
(EtOAc: n-hexane 3:2). Yield: 420 mg (93 %). Mp 220 °C. IR
(KBr): ν = 3302, 3070, 2925, 2856, 1687, 1630, 1597, 1553,
1511 cm⫺1. 1H NMR (DMSO-d6): δ 2.24 (s, 3H), 3.36 (s, 2H),
6.92 (d, J = 16 Hz, 1H), 6.99 (m, 2H), 7.04 (m, 2H), 7.48 (m,
2H), 7.60⫺7.64 (m, 2H), 7.67 (m, 2H), 7.76 (m, 1H),
7.83⫺7.89 (m, 4H), 8.25 (m, 2H), 10.04 (s, 1H), 10.40 (s,
1H). MS (EI): m/z = 41 (51), 105 (100), 294 (80), 489 (1) M+.
Anal. calcd. for C31H27N3O3: C, 76.05; H, 5.56; N, 8.58;
found: C, 75.79; H, 5.64; N, 8.33.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4methylcinnamic acid amide (2c)
Experimental
1
13
H- and C-NMR spectra were recorded on a Jeol JMN-GX400 and a Jeol JMN-LA-500 spectrometer (Jeol USA Inc.,
Peabody, Massachusetts, USA). Mass spectra were obtained
with a Vacuum Generator VG 7070 H (Vacuum Generators,
Manchester, UK) using a Vector 1 data acquisition system
from Teknivent (Teknivent Corp., Maryland Heights, Missouri,
USA) or a AutoSpec mass spectrometer from Micromass
(Micromass, Manchester, UK). IR spectra were recorded on
a Nicolet 510P FT-IR spectrometer (Thermo Nicolet Corporation, Madison, Wisconsin, USA). Microanalyses were obtained from a CH analyzer according to Dr. Salzer from Labormatic and from a Hewlett Packard CHN analyzer type 185
(Hewlett Packard, Palo Alto, California, USA). Melting points
were obtained with a Leitz microscope (Leitz, Wetzlar, Germany) and are uncorrected. Column chromatography was
carried out using silica gel 60 (0.062⫺0.200 mm) from Merck
(Merck AG, Darmstadt, Germany). The preparation of compounds 2l⫺2p has been described elsewhere [31].
General procedure for the preparation of the target compounds 2 and 3
The various carboxylic acids were dissolved in toluene, and
0.1 mL SOCl2 per mmol acid was added. The mixture was
heated under reflux for 2 h, and the volatiles were evaporated
in vacuo. The resulting acyl chlorides were dissolved in toluene or dioxane (approx. 10 mL) and added to a solution of
the appropriate aromatic amine in hot toluene (approx. 50
mL). The mixtures were heated under reflux for 2 h. Then the
solvent was removed in vacuo to give the crude products.
From 4-methylcinnamic acid (194 mg, 1.2 mmol), according
to general procedure. Purification: recrystallization from toluene. Yield: 170 mg (29 %). Mp 172 °C. IR (KBr): ν = 3358,
2056, 2917, 1655, 1509 cm⫺1. 1H NMR (CDCl3): δ 2.25 (s,
3H), 2.28 (s, 3H), 3.61 (s, 2H), 6.35 (d, J = 16 Hz, 1H),
7.07⫺7.09 (m, 4H), 7.17 (m, 2H), 7.28 (m, 2H), 7.36⫺7.41
(m, 2H), 7.50 (m, 3H), 7.58 (m, 1H), 7.63 (m, 2H), 7.95 (s,
1H), 8.45 (m, 1H), 10.45 (s, 1H). MS (EI): m/z = 105 (44), 145
(100), 212 (55), 488 (40) M+. Anal. calcd. for C32H28N2O3: C,
78.67; H, 5.78; N, 5.73; found: C, 78.35; H, 5.74; N, 5.48.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4ethylcinnamic acid amide (2d)
From 4-ethylcinnamic acid chloride (234 mg, 1.2 mmol), according to general procedure. Purification: MPLC EtOAc : nhexane 1:1. Yield: 362 mg (72 %). Mp 135 °C. IR (KBr): ν =
3230, 1665, 1655, 1620, 1605, 1595, 1550 cm⫺1. 1H NMR
(CDCl3): δ 1.21 (t, J = 7 Hz, 3H), 2.31 (s, 3H), 2.62 (q, J = 7
Hz, 2H), 3.67 (s, 2H), 6.42 (d, J = 16 Hz, 1H), 7.13⫺7.15 (m,
4H), 7.21⫺7.23 (m, 2H), 7.32⫺7.34 (m, 2H), 7.40⫺7.43 (m,
2H), 7.51⫺7.54 (m, 2H), 7.62 (d, J = 16 Hz, 1H), 7.66⫺7.68
(m, 2H), 8.00 (s, 1H), 8.06 (s, 1H), 8.46 (d, J = 9 Hz, 1H),
10.54 (s, 1H). MS (EI): m/z = 159 (74), 212 (32), 344 (43),
502 (100) M+, 503 (37) M++1. Anal. calcd. for C33H30N2O3:
C, 78.85; H, 6.02; N, 5.57; found: C, 78.59; H, 5.81; N, 5.28.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-isopropylcinnamic acid amide (2e)
From 4-isopropylcinnamic acid chloride (250 mg, 1.2 mmol),
according to general procedure. Purification: MPLC EtOAc:
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
498 Schlitzer et al.
n-hexane 1:1. Yield: 434 mg (70 %). Mp 125 °C. IR (KBr): ν =
3260, 2960, 1670, 1640, 1630, 1595, 1550, 1500 cm⫺1. 1H
NMR (CDCl3): δ 1.21 (d, J = 7 Hz, 6H), 2.30 (s, 3H), 2.87 (q,
J = 7 Hz, 1H), 3.67 (s, 2H), 6.43 (d, J = 16 Hz, 1H), 7.12⫺7.14
(m, 2H), 7.15⫺7.17 (m, 2H), 7.21⫺7.23 (m, 2H), 7.31⫺7.33
(m, 2H), 7.38⫺7.41 (m, 2H), 7.49⫺7.52 (m, 2H), 7.62 (d, J =
16 Hz, 1H), 7.65⫺7.67 (m, 2H), 8.06 (s, 1H), 8.18 (s, 1H),
8.44 (d, J = 9 Hz, 1H), 10.54 (s, 1H). MS (EI): m/z = 131 (43),
173 (47), 212 (32), 344 (48), 516 (100) M+, 517 (37) M++1.
Anal. calcd. for C34H32N2O3: C, 79.04; H, 6.24; N, 5.42;
found: C, 78.99; H, 6.18; N, 5.68.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-tertbutylcinnamic acid amide (2f)
From 4-tert-butylcinnamic acid chloride (267 mg, 1.2 mmol),
according to general procedure. Purification: MPLC EtOAc:
n-hexane 1:1. Yield: 427 mg (67 %). Mp 175 °C. IR (KBr): ν =
3230, 2965, 1665, 1640, 1620, 1595, 1550, 1515 cm⫺1. 1H
NMR (CDCl3): δ 1.29 (s, 9H), 2.31 (s, 3H), 3.67 (s, 2H), 6.44
(d, J = 16 Hz, 1H), 7.13⫺7.15 (m, 2H), 7.21⫺7.23 (m, 2H),
7.32⫺7.34 (m, 2H), 7.35⫺7.37 (m, 2H), 7.40⫺7.43 (m, 2H),
7.51⫺7.54 (m, 2H), 7.63 (d, J = 16 Hz, 1H), 7.66⫺7.68 (m,
2H), 8.02 (s, 1H), 8.06 (s, 1H), 8.47 (d, J = 9 Hz, 1H), 10.54
(s, 1H). MS (EI): m/z = 187 (36), 344 (47), 530 (100) M+, 531
(39) M++1. Anal. calcd. for C35H34N2O3: C, 79.22; H, 6.46; N,
5.28; found: C, 79.33; H, 6.40; N, 5.48.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4methylthiocinnamic acid amide (2g)
From 4-methylsulfonylcinnamic acid (192 mg, 1 mmol), according to general procedure. Purification: recrystallization
from toluene. Yield: 315 mg (61 %). Mp 210 °C. IR (KBr): ν =
3229, 3057, 2922, 1681, 1627, 1615, 1554, 1506, 1289, 1171
cm⫺1. 1H NMR (DMSO-d6): δ 2.26 (s, 3H), 2.49 (s, 3H), 3.37
(s, 2H), 6.71 (d, J = 16 Hz, 1H), 7.00⫺7.06 (m, 4H), 7.30 (m,
2H), 7.48⫺7.53 (m, 5H), 7.63 (m, 2H), 7.68 (m, 2H), 7.77 (m,
1H), 7.87 (m, 1H), 10.01 (s, 1H), 10.20 (s, 1H). MS (EI): m/
z = 105 (51), 177 (100), 212 (32), 344 (25), 520 (36) M+. Anal.
calcd. for C32H28N2O3S: C, 73.82; H, 5.42; N, 5.38; found: C,
73.84; H, 5.46; N, 5.27.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4methoxycinnamic acid amide (2h)
From 4-methoxycinnamic acid (214 mg, 1.2 mmol), according
to general procedure. Purification: recrystallization from toluene. Yield: 60 mg (10 %). Mp 89 °C. IR (KBr): ν = 3274, 1664,
1603, 1549, 1510 cm⫺1. 1H NMR (DMSO-d6): δ 2.25 (s, 3H),
3.36 (s, 2H), 3.79 (s, 3H), 6.60 (d, J = 16 Hz, 1H), 6.99 (m,
4H), 7.04 (m, 2H), 7.47⫺7.54 (m, 5H), 7.57⫺7.64 (m, 2H),
7.68 (m, 2H), 7.76 (m, 1H), 7.85⫺7.88 (m, 1H), 10.02 (s, 1H),
10.18 (s, 1H). MS (EI): m/z = 105 (30), 161 (100), 212 (29),
504 (21) M+. Anal. calcd. for C32H28N2O4: C, 76.17; H, 5.59;
N, 5.55; found: C, 75.85; H, 5.55; N, 5.52.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4ethoxycinnamic acid amide (2i)
From 4-ethoxycinnamic acid chloride (253 mg, 1.2 mmol),
according to general procedure. Purification: MPLC EtOAc:
n-hexane 3:2. Yield: 392 mg (63 %). Mp 175 °C. IR (KBr): ν =
3310, 1680, 1670, 1645, 1620, 1605, 1515 cm⫺1. 1H NMR
(DMSO-d6): δ 1.33 (t, J = 7 Hz, 3H), 2.25 (s, 3H), 3.38 (s, 2H),
4.07 (q, J = 7 Hz, 2H), 6.60 (d, J = 16 Hz, 1H), 6.95⫺6.97 (m,
2H), 7.00⫺7.02 (m, 2H), 7.04⫺7.06 (m, 2H), 7.48⫺7.53 (m,
5H), 7.61⫺7.64 (m, 2H), 7.68⫺7.70 (m, 2H), 7.76⫺7.78 (m,
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
1H), 7.87⫺7.89 (m, 1H), 10.02 (s, 1H), 10.16 (s, 1H). MS
(EI): m/z = 175 (100), 344 (40), 518 (90) M+, 519 (34) M++1.
Anal. calcd. for C33H30N2O4 : C, 76.42; H, 5.84; N, 5.40;
found: C, 76.40; H, 5.69; N, 5.41.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4propoxycinnamic acid amide (2j)
From 4-propoxycinnamic acid chloride (270 mg, 1.2 mmol),
according to general procedure. Purification: MPLC EtOAc:
n-hexane 1:1. Yield: 383 mg (60 %). Mp 175 °C. IR (KBr): ν =
3230, 1665, 1605, 1550, 1510 cm⫺1. 1H NMR (DMSO-d6): δ
0.93 (t, J = 7 Hz, 3H), 1.67⫺1.77 (m, 2H), 2.26 (s, 3H), 3.37
(s, 2H), 3.97 (t, J = 6 Hz, 2H), 6.60 (d, J = 16 Hz, 1H),
6.96⫺6.98 (m, 2H), 7.00⫺7.02 (m, 2H), 7.04⫺7.06 (m, 2H),
7.50⫺7.53 (m, 5H), 7.60⫺7.70 (m, 4H), 7.77 (s, 1H), 7.88 (d,
J=9 Hz, 1H), 10.02 (s, 1H), 10.16 (s, 1H). MS (EI): m/z = 43
(100), 60 (97), 73 (93), 83 (38), 189 (39), 532 (10) M+, 533
(4) M++1. Anal. calcd. for C34H32N2O4: C, 76.66; H, 6.07; N,
5.26; found: C, 76.26; H, 5.88; N, 5.56.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4butoxycinnamic acid amide (2k)
From 4-butoxycinnamic acid chloride (286 mg, 1.2 mmol),
according to general procedure. Purification: MPLC EtOAc:
n-hexane 1:1. Yield: 466 mg (71 %). Mp 152 °C. IR (KBr): ν =
3230, 1665, 1650, 1620, 1605, 1550, 1510 cm⫺1. 1H NMR
(CDCl3): δ 0.96 (t, J = 7 Hz, 3H), 1.42⫺1.52 (m, 2H),
1.71⫺1.80 (m, 2H), 2.30 (s, 3H), 3.67 (s, 2H), 3.93 (t, J = 6
Hz, 2H), 6.32 (d, J = 16 Hz, 1H), 6.79⫺6.81 (m, 2H),
7.12⫺7.14 (m, 2H), 7.21⫺7.23 (m, 2H), 7.31⫺7.33 (m, 2H),
7.40⫺7.42 (m, 3H), 7.50⫺7.52 (m, 2H), 7.58 (d, J = 16 Hz,
1H), 7.65⫺7.67 (m, 2H), 8.05 (s, 1H), 8.47 (d, J = 9 Hz, 1H),
10.53 (s, 1H). MS (EI): m/z = 147 (40), 203 (100), 344 (36),
546 (62) M+. Anal. calcd. for C35H34N2O4: C, 76.89; H, 6.27;
N, 5.13; found: C, 76.50; H, 6.12; N, 5.33.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4fluorocinnamic acid amide (2q)
From 4-fluorocinnamic acid (166 mg, 1 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 441 mg (90 %). Mp 193 °C. IR (KBr): ν = 3358, 2961,
1675, 1629, 1597, 1559, 1511 cm⫺1. 1H NMR (CDCl3): δ 2.25
(s, 3H), 3.61 (s, 2H), 6.31 (d, J = 16 Hz, 1H), 6.96 (m, 2H),
7.09 (m, 2H), 7.17 (m, 2H), 7.34⫺7.47 (m, 4H), 7.49 (m, 2H),
7.55 (d, J = 16 Hz, 1H), 7.62 (m, 3H), 7.95 (s, 1H), 8.43 (m,
1H), 10.45 (s, 1H). MS (EI): m/z = 149 (48), 212 (53), 360
(47), 492 (100) M+. Anal. calcd. for C31H25FN2O3: C, 75.59;
H, 5.12; N, 5.69; found: C, 75.34; H, 5.15; N, 5.76.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4chlorocinnamic acid amide (2r)
From 4-chlorocinnamic acid chloride (223 mg, 1.2 mmol), according to general procedure. Purification: recrystallization
from toluene. Yield: 70 mg (11 %). Mp 85 °C. IR (KBr): ν =
3358, 1673, 1632, 1563, 1510 cm⫺1. 1H NMR (CDCl3): δ 2.25
(s, 3H), 3.63 (s, 2H), 6.36 (d, J = 16 Hz, 1H), 7.08 (m, 2H),
7.16 (m, 2H), 7.24 (m, 2H), 7.29 (m, 2H), 7.37⫺7.41 (m, 2H),
7.46⫺7.52 (m, 2H), 7.56 (m, 1H), 7.63 (m, 3H), 7.95 (s, 1H),
8.43 (m, 1H), 10.45 (s, 1H). MS (EI): m/z = 105 (89), 165
(64), 212 (100), 508 (43) M+, 510 (18) M+. Anal. calcd. for
C31H25N2O3Cl: C, 73.15; H, 4.95; N, 5.50; found: C, 72.89;
H, 4.92; N, 5.22.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Non-thiol Farnesyltransferase Inhibitors 499
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4bromocinnamic acid amide (2s)
From 4-bromocinnamic acid (227 mg, 1 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 452 mg (82 %). Mp 190 °C. IR (KBr): ν = 3358, 3050,
2918, 1676, 1647, 1692, 1595, 1561, 1511 cm⫺1. 1H NMR
(CDCl3): δ 2.32 (s, 3H), 3.68 (s, 2H), 6.46 (d, J = 16 Hz,
1H), 7.15 (m, 2H), 7.22⫺7.30 (m, 4H), 7.45⫺7.47 (m, 4H),
7.53⫺7.60 (m, 3H), 7.70 (m, 2H), 7.77 (m, 1H), 8.02 (s, 1H),
8.49 (m, 1H), 10.51 (s, 1H). MS (EI): m/z = 105 (72), 212
(100), 552 (23) M+, 554 (25) M+. Anal. calcd. for
C31H25BrN2O3: C, 67.28; H, 4.55; N, 5.06; found: C, 66.90;
H, 4.53; N, 5.23.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4trifluoromethylcinnamic acid amide (2t)
From 4-trifluoromethylcinnamic acid (259 mg, 1.2 mmol), according to general procedure. Purification: recrystallization
from toluene. Yield: 200 mg (31 %). Mp 126 °C. IR (KBr): ν =
3342, 3058, 1675, 1511, 1324 cm⫺1. 1H NMR (DMSO-d6): δ
2.24 (s, 3H), 3.36 (s, 2H), 6.87 (d, J = 16 Hz, 1H), 7.02 (m,
4H), 7.48 (m, 2H), 7.59⫺7.64 (m, 3H), 7.67 (m, 2H),
7.75⫺7.81 (m, 5H), 7.87 (m, 1H), 10.02 (s, 1H), 10.34 (s,
1H). MS (EI): m/z = 60 (68), 73 (76), 98 (33), 129 (33), 542
(4) M+. Anal. calcd. for C32H25N2O3F3: C, 70.84; H, 4.64; N,
5.16; found: C, 70.79; H, 4.44; N, 5.12.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4cyanocinnamic acid amide (2u)
From 4-cyanocinnamoyl chloride (230 mg, 1.2 mmol), according to general procedure. Purification: recrystallization from
n-hexane/acetone. Yield: 402 mg (67 %). Mp 241 °C. IR (KBr):
ν = 3375, 2225, 1670, 1630, 1520 cm⫺1. 1H NMR (DMSOd6): δ 2.26 (s, 3H), 3.38 (s, 2H), 6.89 (d, J = 16 Hz, 1H),
7.00⫺7.05 (m, 4H), 7.48⫺7.52 (m, 2H), 7.60⫺7.64 (m, 3H),
7.67⫺7.70 (m, 2H), 7.73⫺7.77 (m, 3H), 7.87⫺7.90 (m, 3H),
10.05 (s, 1H), 10.37 (s, 1H). MS (EI): m/z = 367 (96), 394
(51), 499 (100) M+. Anal. calcd. for C32H25N3O3: C, 76.93; H,
5.05; N, 8.41; found: C, 76.43; H, 5.02; N, 8.22.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-(2,2dicyanoethenyl)cinnamic acid amide (2v)
4-Formylcinnamic acid (270 mg, 1.5 mmol) and malonodinitrile (109 mg, 1.5 mmol) were dissolved in a mixture of
acetic acid and dioxane (1:1). Sodium acetate (500 mg) was
added, and the mixture was heated under reflux for 1 h and
stirred at room temperature overnight. The gelatinous mixture
was gently heated to obtain a clear solution. The solvent was
distilled in vacuo until the product began to precipitate. Of this
product, 134 mg (0.6 mmol) was used according to general
procedure. Purification: recrystallization from toluene/THF.
Yield: 89 mg (27 %). Mp 250 °C. IR (KBr): ν = 3307, 3050,
2933, 2229, 1679, 1586, 1551, 1508 cm⫺1. 1H NMR (DMSOd6): δ 2.25 (s, 3H), 3.36 (s, 2H), 6.91 (d, J = 16 Hz, 1H), 7.00
(m, 2H), 7.04 (m, 2H), 7.49 (m, 2H), 7.62 (m, 3H), 7.68 (m,
2H), 7.77 (m, 1H), 7.81 (m, 2H), 7.88 (m, 1H), 7.98 (m, 2H),
8.50 (s, 1H), 10.03 (s, 1H), 10.41 (s, 1H). MS (EI): m/z = 44
(100), 54 (57), 73 (60), 105 (61), 212 (48), 550 (10) M+. Anal.
calcd. for C35H26N4O3: C, 76.35; H, 4.76; N, 10.18; found: C,
75.98; H, 4.84; N, 9.82.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4formylcinnamic acid amide (2w)
From 4-formylcinnamic acid (170 mg, 1 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 143 mg (28 %). Mp: 189 °C. IR (KBr): ν = 3366, 3287,
3023, 2836, 1694, 1632, 1559, 1510 cm⫺1. 1H NMR (DMSOd6): δ 2.25 (s, 3H), 3.37 (s, 2H), 6.94 (d, J = 16 Hz, 1H), 7.00
(m, 2H), 7.04 (m, 2H), 7.49 (m, 2H), 7.63 (m, 4H), 7.70 (m,
2H), 7.80 (m, 2H), 7.89 (m, 1H), 7.96 (m, 2H), 10.02 (s, 1H),
10.05 (s, 1H), 10.44 (s, 1H). MS (EI): m/z = 44 (93), 105 (96),
212 (100), 370 (76), 502 (56) M+. Anal. calcd. for
C32H26N2O4: C, 76.48; H, 5.21; N, 5.57; found: C, 76.20; H,
5.15; N, 5.66.
4-{2-[3-Benzoyl-4-(4-tolylacetylamino)phenylaminocarbonyl]vinyl}benzoic acid methyl ester (2x)
From 4-methylcarboxycinnamic acid (264 mg, 1.2 mmol) according to general procedure. Purification: flash-chromatography EtOAC: n-hexane 2:3 and recrystallization from toluene. Yield: 410 mg (63 %). Mp 205 °C. IR (KBr): ν = 3345,
1666, 1634, 1557, 1511 cm⫺1. 1H NMR (DMSO-d6): δ 2.26
(s, 3H), 3.38 (s, 2H), 3.87 (s, 3H), 6.89 (d, J = 16 Hz, 1H),
7.01 (m, 2H), 7.05 (m, 2H), 7.48⫺7.52 (m, 2H), 7.59⫺7.65
(m, 3H), 7.68⫺7.74 (m, 4H), 7.78 (m, 1H), 7.88 (m, 1H), 8.00
(m, 2H), 10.04 (s, 1H), 10.35 (s, 1H). MS (EI): m/z = 44 (100),
60 (72), 73 (76), 532 (12) M+. Anal. calcd. for C33H28N2O5:
C, 74.42; H, 5.30; N, 5.26; found: C, 74.14; H, 5.35; N, 5.06.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4nitrocinnamic acid amide (2y)
From 4-nitrocinnamic acid chloride (382 mg, 1.5 mmol), according to general procedure. Purification: recrystallization
from toluene. Yield: 560 mg (72 %). Mp 224 °C. ⫺ IR (KBr):
ν = 3430, 3070, 1685, 1665, 1505 cm⫺1. 1H NMR (DMSOd6): δ 2.22 (s, 3H), 3.35 (s, 2H), 6.90 (d, J = 16 Hz, 1H), 7.00
(m, 4H), 7.48 (m, 3H), 7.61 (m, 3H), 7.66 (m, 2H), 7.75 (m,
1H), 7.82 (m, 2H), 8.22 (m, 2H), 10.00 (s, 1H), 10.37 (s, 1H).
MS (EI): m/z = 212 (75), 387 (100), 519 (68) M+. Anal. calcd.
for C31H25N3O5: C, 71.67; H, 4.85; N, 8.09; found: C, 71.58;
H, 4.75; N, 8.20.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3nitrocinnamic acid amide (2z)
From 3-nitrocinnamic acid (193 mg, 1 mmol), according to
general procedure. Purification: flash-chromatography
EtOAC: n-hexane 2:3. Yield: 255 mg (55 %). Mp 202 °C. IR
(KBr): ν = 3381, 2924, 1688, 1635, 1509, 1351 cm⫺1. 1H
NMR (DMSO-d6): δ 2.23 (s, 3H), 3.35 (s, 2H), 6.92 (d, J = 16
Hz, 1H), 6.98 (m, 2H), 7.02 (m, 2H), 7.48 (m, 2H), 7.59⫺7.71
(m, 6H), 7.75 (m, 1H), 7.86 (m, 1H), 8.00 (m, 1H), 8.19 (m,
1H), 8.40 (m, 1H), 10.01 (s, 1H), 10.32 (s, 1H). MS (EI):
m/z = 105 (47), 212 (39), 387 (100), 414 (58), 519 (91) M+.
Anal. calcd. for C31H25N3O5: C, 71.76; H, 4.85; N, 8.09;
found: C, 71.44; H, 4.58; N, 8.10.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-2nitrocinnamic acid amide (2aa)
From 2-nitrocinnamic acid (290 mg, 1.5 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 183 mg (29 %). Mp 95 °C. IR (KBr): ν = 3250, 2915,
1667, 1523, 1344 cm⫺1. 1H NMR (DMSO-d6): δ 2.26 (s, 3H),
3.38 (s, 2H), 6.77 (d, J = 16 Hz, 1H), 6.99 (m, 2H), 7.03 (m,
2H), 7.50 (m, 2H), 7.62⫺7.70 (m, 5H), 7.79 (m, 3H), 7.84 (m,
1H), 7.88 (m, 1H), 8.05 (m, 1H), 10.05 (s, 1H), 10.41 (s, 1H).
MS (EI): m/z = 105 (98), 212 (70), 399 (100), 519 (1) M+.
Anal. calcd. for C31H25N3O5: C, 71.76; H, 4.85; N, 8.09;
found: C, 71.63; H, 4.95; N, 7.82.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
500 Schlitzer et al.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4methylsulfonylcinnamic acid amide (2ab)
From 4-metyhlsulfonylcinnamic acid (81 mg, 0.36 mmol), according to general procedure. Purification: recrystallization
from toluene/ethanol. Yield: 32 mg (16 %). Mp 185 °C. IR
(KBr): ν = 3435, 3020, 2923, 1687, 1639, 1430, 1311, 1150
cm⫺1. 1H NMR (DMSO-d6): δ 2.21 (s, 3H), 3.17 (s, 3H), 3.32
(s, 2H), 6.87 (d, J = 16 Hz, 1H), 6.97 (m, 4H), 7.45 (m, 2H),
7.59 (m, 3H), 7.64 (m, 2H), 7.73 (m, 1H), 7.79 (m, 2H), 7.84
(m, 1H), 7.92 (m, 2H), 9.99 (s, 1H), 10.32 (s, 1H). MS (EI):
m/z = 105 (47), 211 (55), 344 (45), 420 (45), 552 (26) M+.
Anal. calcd. for C32H28N2O5S: C, 69.95; H, 5.11; N, 5.07;
found: C, 69.69; H, 4.98; N, 5.19.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
474 (100), 501 (42), 580 (42), 606 (68) M+. Anal. calcd. for
C32H25F3N2O5S: C, 63.36; H, 4.15; N, 4.62; found: C, 63.13;
H, 4.19; N, 4.66.
Enzyme preparation
Yeast farnesyltransferase was used as a fusion protein to
glutathione S-transferase at the N terminus of the b subunit.
Farnesyltransferase was expressed in Escherichia coli DH5a
grown in LB media containing ampicillin and chloramphenicol
for co-expression of pGEX-DPR1 and pBC-RAM2 [23]. The
enzyme was purified by standard procedures with glutathione-agarose beads for selective binding of the target protein.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-(2pyridyl)acrylic acid amide (3a)
From 3-(2-pyridyl)acrylic acid (149 mg, 1 mmol), according to
general procedure. Purification: recrystallization from toluene.
Yield: 99 mg (20 %). Mp 197 °C. IR (KBr): ν = 3256, 3023,
1679, 1576, 1509, 1408, 1259 cm⫺1. 1H NMR (DMSO-d6): δ
2.20 (s, 3H), 3.31 (s, 2H), 6.93⫺7.01 (m, 4H), 7.24 (d, J = 16
Hz, 1H), 7.41 (m, 3H), 7.46 (m, 1H), 7.52⫺7.65 (m, 5H), 7.77
(m, 1H), 7.83⫺7.91 (m, 2H), 8.62 (m, 1H), 10.07 (s, 1H),
10.54 (s, 1H). MS (EI): m/z = 105 (58), 132 (100), 475 (37)
M+. Anal. calcd. for C30H25N3O3: C, 75.77; H, 5.30; N, 8.84;
found: C, 75.58; H, 5.29; N, 8.62.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-(3pyridyl)acrylic acid amide (3b)
From 3-(3-pyridyl)acrylic acid (179 mg, 1.2 mmol), according
to general procedure. Purification: recrystallization from toluene. Yield: 338 mg (71 %). Mp 202 °C. IR (KBr): ν = 3432,
3284, 3055, 1700, 1684, 1633, 1563, 1507 cm⫺1. 1H NMR
(DMSO-d6): δ 2.24 (s, 3H), 3.36 (s, 2H), 6.85 (d, J = 16 Hz,
1H), 6.99⫺7.05 (m, 4H), 7.43⫺7.50 (m, 3H), 7.61 (m, 3H),
7.67 (m, 2H), 7.76 (m, 1H), 7.88 (m, 1H), 7.98 (m, 1H), 8.56
(m, 1H), 8.77 (m, 1H), 10.01 (s, 1H), 10.31 (s, 1H). MS (EI):
m/z = 105 (35), 132 (35), 212 (48), 343 (75), 370 (69), 475
(100) M+. Anal. calcd. for C30H25N3O3: C, 75.77; H, 5.30; N,
8.84; found: C, 75.48; H, 5.37; N, 8.48.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-(4pyridyl)acrylic acid amide (3c)
From 3-(4-pyridyl)acrylic acid (149 mg, 1 mmol), according to
general procedure. Purification: recrystallization from ethanol.
Yield: 116 mg (24 %). Mp 221 °C. IR (KBr): ν = 3410, 3281,
3026, 1684, 1670, 1638, 1596, 1560, 1502, 1403 cm⫺1. 1H
NMR (DMSO-d6): δ 2.25 (s, 3H), 3.36 (s, 2H), 6.98⫺7.05 (m,
4H), 7.15 (m, 1H), 7.49 (m, 3H), 7.59⫺7.63 (m, 2H), 7.68 (m,
2H), 7.81 (m, 3H), 7.90 (m, 1H), 8.75 (m, 2H), 10.09 (s, 1H),
10.66 (s, 1H). MS (EI): m/z = 77 (43), 105 (100), 132 (46),
343 (55), 475 (36) M+. Anal. calcd. for C30H25N3O3: C, 75.77;
H, 5.30; N, 8.84; found: C, 75.29; H, 5.11; N, 8.54.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4trifluoromethylsulfonyl-cinnamic acid amide (2ac)
From 4-trifluorometyhlsulfonylcinnamic acid (280 mg, 1 mmol),
according to general procedure. Purification: recrystallization
from toluene. Yield: 188 mg (31 %). Mp 122 °C. IR (KBr): ν =
3280, 3087, 1674, 1656, 1551, 1507, 1219 cm⫺1. 1H NMR
(DMSO-d6): δ 2.24 (s, 3H), 3.35 (s, 2H), 6.97 (m, 2H), 7.02
(m, 2H), 7.48 (m, 2H), 7.58 (m, 2H), 7.62⫺7.70 (m, 3H), 7.79
(m, 1H), 7.88 (m, 1H), 8.00 (m, 2H), 8.16 (m, 2H), 8.28 (m,
1H), 10.12 (s, 1H), 10.54 (s, 1H). MS (EI): m/z = 448 (89),
Farnesyltransferase assay
The assay was conducted as described [22]. Farnesylpyrophosphate (FPP) was obtained as a solution of the ammonium salt in methanol : 10 mM aqueous NH4Cl (7 : 3) from
Sigma-Aldrich. Dansyl-GlyCysValLeuSer (Ds-GCVLS) was
custom-synthesized by ZMBH, Heidelberg, Germany. The assay mixture (100 µL) contained 50 mM Tris/HCl pH 7.4, 5 mM
MgCl2, 10 µM ZnCl2, 5 mM dithiothreitol (DTT), 7 µM DsGCVLS, 20 µM FPP and 5 nmol (approx.) yeast GST-farnesyltransferase and 1 % of various concentrations of the test
compounds dissolved in dimethylsulfoxide (DMSO). The progress of the enzyme reaction was followed by monitoring the
enhancement of the fluorescence emission at 505 nm (excitation 340 nm). The reaction was started by addition of the
enzyme and run in a quartz cuvette thermostatted at 30 °C.
Fluorescence emission was recorded with a Perkin Elmer
LS50B spectrometer (Perkin Elmer, Shelton, Connecticut,
USA). IC50 values (concentrations resulting in 50 % inhibition)
were calculated from the initial velocity of three independent
measurements at four to five different concentrations of the
respective inhibitor.
Molecular modeling
Molecular modeling of inhibitor 2a was carried out using
SYBYL [26] version 6.8/6.9 running on a Silicon Graphics O2
(R10000). Flexible docking was performed using FlexX [24]
version 1.11. During ligand construction, the FlexX command
MAPREF and the perturbate algorithm was used for the
placement of the base fragment. Default parameters were
employed, except for the MAXENERGY value which was set
to 10 kJ mol⫺1.
For the use within AutoDock 3.0 [27, 28], polar hydrogens
were added with the PROTONATE utility from AMBER [32].
AMBER-united atom force field charges were assigned [33],
and solvation parameters were added using the ADDSOL
utility from AutoDock 3.0. Ligand structures were built in mol2
format, Gasteiger partial atomic charges were assigned [34],
and all bonds except for amides were kept rotatable. Docking
runs were performed with the Lamarckian genetic algorithm
included in AutoDock 3.0 [35], performing 50 independent
runs per ligand, using an initial population of 50 randomly
placed individuals, a maximum number of 1.5 ± 106 energy
evaluations, a mutation rate of 0.02, a crossover rate of 0.80,
and an elitism value of 1. Resulting ligand conformations that
differ by less than 1 Å rmsd from each other were clustered
together and were represented by the solution with the best
docking energy.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 493−501
Non-thiol Farnesyltransferase Inhibitors 501
Acknowledgments
The pGEX-DPR1 and pBC-RAM2 plasmids were
kindly provided by Prof. F. Tamanoi (UCLA). I. S.
wishes to thank Prof. Dr. S. Grabley for generous
support and Ms. S. Egner for technical assistance.
References
[17] M. J. Breslin, J. deSolms, E. A. Giuliani, G. E. Stokker,
S. L. Graham, D. L. Pompliano, S. D. Mosser, K. A.
Hamilton, J. H. Hutchinson, Bioorg. Med. Chem. Lett.
1998, 8, 3311⫺3316.
[18] T. M. Ciccarone, S. C. MacTough, T. M. Williams, C. J.
Dinsmore, T. J. O’Neill, D. Shah, J. C. Culberson, K. S.
Koblan, N. E. Kohl, J. B. Gibbs, A. I. Oliff, S. L. Graham,
G. D. Hartman, Bioorg. Med. Chem. Lett. 1999, 9,
1991⫺1996.
[1] F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996,
65, 241⫺269.
[19] M. Böhm, A. Mitsch, P. Wißner, I. Sattler, M. Schlitzer, J.
Med. Chem. 2001, 44, 3117⫺3124.
[2] H.-W. Fu, P. J. Casey, Rec. Prog. Hormon Res. 1999,
54, 315⫺343.
[20] A. Mitsch, M. Böhm, I. Sattler, M. Schlitzer, Arch. Pharm.
Pharm. Med. Chem. 2004, 337, 213⫺218.
[3] F. Tamanoi, C.-L. Gau, C. Jiang, H. Edamatsu, J. KatoStankiewicz, Cell Mol. Life Sci. 2001, 58, 1636⫺1649.
[21] J. Sakowski, M. Böhm, I. Sattler, H.-M. Dahse, M.
Schlitzer, J. Med. Chem. 2001, 44, 2886⫺2899.
[4] G. C. Prendergast, N. Rane, Expert Opin. Investig.
Drugs 2001, 10, 2105⫺2116.
[22] D. L. Pompliano, R. P. Gomez, N. J. Anthony, J. Am.
Chem. Soc. 1992, 114, 7945⫺7946.
[5] M. Crul, G. J. de Klerk, J. H. Beijnen, J. H. M. Schellens,
Anti-Cancer Drugs 2001, 12, 163⫺184.
[23] K. Del Villar, H. Mitsuzawa, W. Yang, I. Sattler, F. Tamanoi, J. Biol. Chem. 1997, 272, 680⫺687.
[6] W. T. Purcell, R. C. Donehower, Curr. Oncol. Rep. 2002,
4, 29⫺36.
[24] G. Klebe, T. Mietzner, F. Weber, J. Comput.-Aided Mol.
Des. 1994, 8, 751⫺787.
[7] A. D. Cox, C. J. Der, Curr. Opin. Pharmacol. 2002, 2,
388⫺393.
[25] C. L. Strickland, W. T. Windsor, R. Syto, L. Wang, R.
Bond, R. Wu, J. Schwartz, H. V. Le, L. S. Beese, P. C.
Weber, Biochemistry 1998, 37, 16601⫺16611.
[8] A. Wittinghofer, H. Waldmann, Angew. Chem. 2000, 112,
4360⫺4383; Angew. Chem. Int. Ed. 2000, 39,
4192⫺4214.
[26] SYBYL molecular modeling software; Tripos Inc., 1699
South Hanley Rd, Suite 303, St. Louis, MO 63144.
[9] E. K. Rowinsky, A. Patnaik, Emerging Drugs 2000, 5,
161⫺199.
[10] S. M. Sebti, A. D. Hamilton, Oncogene 2000, 19,
6584⫺6593.
[27] D. S. Goodsell, A. J. Olson, Proteins 1990, 8, 195⫺202.
[28] G. M. Morris, D. S. Goodsell, R. Huey, A. J. Olson, J.
Comput.-Aided Mol. Des. 1996, 10, 293⫺304.
[11] A. D. Cox, Drugs 2001, 61, 723⫺732.
[29] H. Gohlke, M. Hendlich, G. Klebe, J. Mol. Biol. 2000,
295, 337⫺356.
[12] I. M. Bell, Exp. Opin. Therp. Patents 2000, 10,
1813⫺1831.
[30] C. Haubmann, H. Hübner, P. Gmeiner, Bioorg. Med.
Chem. Lett. 1999, 9, 1969⫺1972.
[13] Martindale The Extra Pharmacopeia, 31st ed., J. E.
F.Reynolds (Ed.); Royal Pharmaceutical Society of
Great Britain: London, 1996; p. 821.
[31] J. Wiesner, P. Wißner, H.-M. Dahse, H. Jomaa, M.
Schlitzer, Bioorg. Med. Chem. 2001, 9, 785⫺792.
[14] J. T. Hunt, V. G. Lee, K. Leftheris, B. Seizinger, J. Carboni, J. Mabus, C. Ricca, N. Yan, V. Manne, J. Med.
Chem. 1996, 39, 353⫺358.
[15] S. J. O’Connor, K. J. Barr, L. Wang, B. K. Sorensen, A.
S. Tasker, H. Sham, A.-C. Ng, J. Cohen, E. Devine, S.
Cherian, B. Saeed, H. Zhang, J. Y. Lee, R. Warner, S.
Tahir, P. Kovar, P. Ewing, J. Alder, M. Mitten, J. Leal,
K. Marsh, J. Bauch, D. J. Hoffman, S. M. Sebti, S. H.
Rosenberg, J. Med. Chem. 1999, 42, 3701⫺3710.
[16] D. J. Augeri, D. Janowick, D. Kalvin, G. Sullivan, J.
Larsen, D. Dickman, H. Ding, J. Cohen, J. Lee, R.
Warner, P. Kovar, S. Cherian, B. Saeed, H. Zhang, S.
Tahir, S.-C. Ng, H. Sham, S. H. Rosenberg, Bioorg. Med.
Chem. Lett. 1999, 9, 1069⫺1074.
[32] D. A. Case, D. A. Pearlman, J. W. Caldwell, T. E. Cheatham III, J. Wang, W. S. Ross, C. L. Simmerling, T. A.
Darden, K. M. Merz, R. V. Stanton, A. L. Cheng, J. J.
Vincent, M. Crowley, V. Tsui, H. Gohlke, R. J. Radmer,
Y. Duan, J. Pitera, I. Massova, G. L. Seibel, U. C. Singh,
P. K. Weiner, P. A. Kollman, (2002), AMBER 7; University of California, San Francisco.
[33] S. J. Weiner, P. A. Kollman, D. A. Case, U. C. Singh, C.
Ghio, G. Alagona, S. Profeta, P. Weiner, J. Am. Chem.
Soc. 1984, 106, 765⫺784.
[34] J. Gasteiger, M. Marsili, Tetrahedron 1980, 36,
3219⫺3228.
[35] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W.
E. Hart, R. K. Belew, A. J. Olson, J. Comput. Chem.
1998, 19, 1639⫺1662.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Документ
Категория
Без категории
Просмотров
3
Размер файла
182 Кб
Теги
inhibitorsutilization, site, cinnamoylaminobenzophenones, thiol, farnesyltransferase, non, far, binding, aryl
1/--страниц
Пожаловаться на содержимое документа