Non-thiol farnesyltransferase inhibitors: N-(4-Acylamino-3-benzoylphenyl)-4-nitrocinnamic acid amides

Article abstract of DOI:10.1016/S0968-0896(01)00274-7

We have developed a 4-nitrocinnamoyl substituted benzophenone as a novel non-thiol farnesyltransferase inhibitor. Replacement of the p-tolyl moiety of our initial lead structure by different para and ortho substituted phenyl residues as well as by 1-naphthyl resulted in derivatives with considerably enhanced activity displaying IC₅₀ values between 42 and 52 nM. These compounds represent novel, readily accessible non-thiol farnesyltransferase inhibitors being more active than the corresponding thiol-containing analogues. Copyright

Full text of DOI:10.1016/S0968-0896(01)00274-7

Bioorganic & Medicinal Chemistry 10 (2002) 233–239
Non-thiol Farnesyltransferase Inhibitors:
N-(4-Acylamino-3-benzoylphenyl)-4-nitrocinnamic Acid Amides
Jacek Sakowski,a Isabel Sattlerb and Martin Schlitzera,*
aInstitut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
¨
¨
bHans-Knoll-Institut fur Naturstoff-Forschung e.V., Beutenbergstraꢀe 11, D-07745, Jena, Germany
¨
¨
Received 4 April 2001; revised 13July 2001; accepted 13July 2001
Abstract—We have developed the 4-nitrocinnamoyl substituted benzophenone 4a as a novel non-thiol farnesyltransferase inhibitor.
Replacement of the p-tolyl moiety of our initial lead structure 4a by different para and ortho substituted phenyl residues as well as
by 1-naphthyl resulted in derivatives with considerably enhanced activity displaying IC50 values between 42 and 52 nM. These
compounds represent novel, readily accessible non-thiol farnesyltransferase inhibitors being more active than the corresponding
thiol-containing analogues.
Introduction
Most inhibitors described in literature are peptidomi-
Inhibition of farnesyltransferase has received consider-
able interest in recent years as a strategy for the devel-
opment of novel potential anti-cancer drugs.1À6
Farnesyltransferase catalyzes the transfer of a farnesyl
residue from farnesylpyrophosphate to the thiol of a
cysteine side chain of proteins which bear the CAAX-
tetrapeptide sequence (C: cysteine, A: aliphatic amino
acid, X: serine or methionine) at their C-terminus.7,8
The rationale for using inhibitors of farnesyltransferase
as anti-cancer agents stems from the observation that
farnesylation is a pre-requisite for the transforming
activity of oncogenic Ras which is found in approxi-
mately 30% of all cancers in humans. However, there is
accumulating evidence that prevention of Ras farnesy-
lation may not be the crucial cellular event responsible
for the antiproliferative effect of farnesyltransferase
inhibitors.9 Focus has shifted to the prenylation of
RhoB, another member of the class of small GTPases
which is involved in receptor trafficking.10,11 Disregard-
ing of the unresolved mechanism by which farnesyl-
transferase inhibitors exert their antiproliferative effects,
the efficacy of these compounds and their low toxicity
has been demonstrated12 and, therefore, administration
of such compounds is regarded as a major strategy
emerging in cancer therapy.
metics resembling the CAAX-tetrapeptide recognition
sequence of farnesylated proteins. The majority of these
CAAX-peptidomimetics exhibit a free thiol group1À6
which is believed to coordinate the enzyme-bound zinc
ion as it has been shown for the native peptide sub-
strate.13 However, free thiols are associated with several
adverse drug effects14 and, therefore, the development
of farnesyltransferase inhibitors is clearly directed
towards the so-called non-thiol farnesyltransferase inhi-
bitors. The most frequently used replacement for
cysteine are nitrogen-containing heterocycles. The ring
nitrogen is supposed to coordinate to the enzyme-bound
zinc similar to the cysteine thiol group.15 However, it
has been shown that nitrogen heterocycles can be
replaced by aryl residues lacking the ability to coordi-
nate metal atoms without loosing too much of their
farnesyltransferase inhibitory activity.16,17 Therefore,
the existence of at least one hitherto unknown aryl
binding region in the farnesyltransferase’s active site has
been postulated.18,19
Using our benzophenone-based AAX-peptidomimetic
scaffold,20 we have introduced para-substituted cinna-
moyl moieties as cysteine replacements designed to uti-
lize one of these aryl binding sites. Early investigations
on the structure–activity relationships of this novel class
of non-thiol farnesyltransferase inhibitors revealed the
nitrocinnamoyl-substituted benzophenone 4a as a par-
ticular potent congener.21 In this study, we addressed
*Corresponding author. Tel.: +49-6421-282-5825; fax: +49-6421-
282-7052; e-mail: schlitze@mailer.uni-marburg.de
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00274-7

234
J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
the question on how the replacement of the p-tolylacetyl
residue at the 2-amino group of the AAX-peptidomi-
metic benzophenone core structure of 4a would
influence farnesyltrasferase inhibitory activity.
compounds 4g and 4j (Scheme 2). First, the 2-amino
group of 1 was protected as trifluoroacetamide (5).
After reduction of the 5-nitro group, the resulting amine
6 was acylated by 4-nitrocinnamic acid chloride. After
removal of the protective group from 7, the resulting
intermediate 8 could by acylated by 2- and 4-tri-
fluoromethylphenylacetic acid chloride, respectively,
yielding compounds 4g and 4j.
Chemistry
Synthesis of most of the target compounds 4 was
accomplished by acylation of the appropriate 2-acyla-
mino-5-aminobenzophenones 3 using 4-nitrocinnamic
acid chloride. Intermediates 3 were prepared from 2-
amino-5-nitrobenzophenone 1 as described previously22
(Scheme 1). However, since acylation of 1 by 2- or 4-tri-
fluoromethylphenylacetic acid chloride failed, an alter-
native route had to be developed for the preparation of
Farnesyltransferase Inhibition Assay
The inhibitory activity of the inhibitors was determined
using the fluorescence enhancement assay as described
by Pompliano.23 The assay employed yeast farnesyl-
transferase (FTase) fused to glutathione S-transferase at
Scheme 1. (i) R–CO–Cl, toluene/dioxane, reflux, 2 h; (ii) SnCl2Â2H2O, EtOAc, reflux 2 h; (iii) 4-nitrocinnamic acid chloride, toluene/dioxane,
reflux, 2 h.
Scheme 2. (i) TFAA, DCM/pyridine, 0 ꢀC, 2 h; (ii) SnCl2Â2H2O, EtOAc, reflux 2 h; (iii) 4-nitrocinnamic acid chloride, toluene/dioxane, reflux, 2 h;
(iv) K2CO3, dioxane/H2O, reflux, 3h; (v) R–C 6H4–CH2–COCl, toluene/dioxane, reflux, 2 h.

J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
235
the N-terminus of the b-subunit.24 Farnesylpyrophos-
phate and the dansylated pentapeptide Ds-Gly-
CysValLeuSer were used as substrates. Upon
farnesylation of the cysteine thiol, the dansyl residue is
placed in a lipophilic environment. The resulting
enhancement of fluorescence at 505 nm is used to
monitor the enzyme reaction.
derivatives with different substituents in the para posi-
tion, the methoxy (4d: IC50=925 nM) and the chloro
substitutions (4e: IC50=875 nM) yielded compounds
with a considerable drop in activity. In contrast, the
para bromo (4f: IC50=46 nM) and trifluoromethyl (4g:
IC50=50 nM) substituted derivatives displayed farne-
syltransferase inhibitory activity with IC50 values equal
to or less than 50 nM. The same was true for the bulky
phenyl residue (4h: IC50=44 nM). In case of the methyl
residue, shifting this substituent from para into the meta
or ortho position resulted in an increased activity.
However, this effect could not seen with the bromo or
the trifluoromethyl residue. While the ortho bromo
derivative (4i: IC50=65 nM) was less active than its para
analogue 4f, the trifluoromethyl group in the ortho
position produced a slightly more active inhibitor (4j:
IC50=42 nM) than the para substituted derivative 4g.
With compounds 4k and 4l, the phenyl residue was
replaced by a naphthyl moiety. Here, the position by
which the naphthyl is attached turned out to be crucial
for farnesyltransferase inhibitory activity. While the
compound with attachment at C-1 of the naphthyl
group (4k: IC50=68 nM) displayed considerable activ-
ity, the attachment at C-2 (4l: IC50=950 nM) led to a
significant drop in activity. The same was found for the
analogues compounds 4m (IC50=52 nM) and 4n
(IC50=210 nM) in which the methylene spacer between
the aryl moiety and the amide group had been omitted.
Results and Discussion
We have developed a novel class of non-thiol farnesyl-
transferase inhibitors in which a cinnamoyl moiety has
been introduced as cysteine replacement designed to fit
into a recently discovered aryl binding site next to the
farnesyltransferase’s active site. From early investiga-
tions on the structure activity relationships of these
inhibitors, the p-nitrocinnamoyl derivative 4a turned
out to be the most active example.21 In this study we
addressed the question of how variations of the acyl
moiety at the 2-amino group of the AAX-peptidomi-
metic benzophenone core structure would influence far-
nesyltransferase inhibitory activity of these compounds
(Table 1). Thus, we were aiming at developing more
potent inhibitors. Shifting the methyl group of the lead
structure 4a from the para into the meta (4b: IC50=88
nM) or ortho position (4c: IC50=66 nM) resulted in an
enhancement of inhibitory potency. From several
In summary, replacement of the p-tolyl moiety of our
initial lead structure by different para and ortho sub-
stituted phenyl residues as well as by 1-naphthyl resul-
ted in derivatives with considerably enhanced activity
displaying IC50 values between 42 and 52 nM. These
compounds represent novel, readily accessible non-thiol
farnesyltransferase inhibitors being more active than the
corresponding thiol-containing analogues.
Table 1. Farnesyltransferase inhibitory activity of compounds 4a–n
Compd
R
IC50 (nM) Compd
R
IC50 (nM)
Experimental
4a
1H and 13C NMR spectra were recorded on a Jeol
JMN-GX-400 and a Jeol JMN-LA-500 spectrometer.
Mass spectra were obtained with a Vacuum Generators
VG 7070 H using a Vector 1 data acquisition system
from Teknivent or a AutoSpec mass spectrometer from
Micromass. IR spectra were recorded on a Nicolet 510P
FT-IR-spectrometer. Microanalyses were obtained from
a CH analyzer according to Dr. Salzer from Labormatic
and from a Hewlett Packard CHN-analyzer type 185.
Melting points were obtained with a Leitz-microscope
and are uncorrected. Column chromatagraphy was car-
ried out using silica gel 60 (0.062–0.200 mm) from
Merck. The preparation of the following compounds has
been described: 4a,25 2b-f, 2h, 2k-m, 3b-f, 3h, 3k-m.22
235Æ20
88Æ18
66Æ4
4h
4i
44Æ5
4b
4c
4d
4e
4f
65Æ4
42Æ2
4j
975Æ65
825Æ5
46Æ4
4k
4l
68Æ4
950Æ50
52Æ5
4m
4n
General procedure 1: acylation of aromatic amines by
acyl chlorides
Appropriate 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.
4g
50Æ3
210Æ30

236
J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
Then, the solvent was removed in vacuo to give the
crude products.
mmol) according to general procedure 1. Yield 437 mg
(81%); mp: 224 ꢀC. IR (KBr): n=3322, 3070, 2925,
1687, 1633, 1597, 1552, 1512, 1403, 1341, 1291 cmÀ1. 1H
NMR (DMSO-d6): d 3.41 (s, 2H), 6.93 (d, J=16 Hz,
1H), 7.11 (m, 2H), 7.27 (m, 2H), 7.47 (m, 2H), 7.59 (m,
2H), 7.63(m, 1H), 7.66 (m, 2H), 7.77 (m, 1H), 7.84 (m,
2H), 7.89 (m, 1H), 8.25 (m, 2H), 10.06 (s, 1H), 10.39 (s,
1H). MS (EI): m/z 539 (21), 146 (100), 212 (86), 121
(58), 77 (57), 105 (57), 387 (56), 91 (49), 414 (30). Anal.
calcd for C30H22ClN3O5: C, 66.73; H, 4.11; N, 7.78;
found: C, 66.43; H, 4.03; N, 7.78.
Acyl chlorides not commercially available were pre-
pared form the corresponding carboxylic acids. These
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
residue obtained was used for the acylation step as
described above.
N-[3-Benzoyl-4-[(3-methylphenyl)acetylamino]phenyl]-4-
nitrocinnamic acid amide (4b). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-(3-methylphenyl)acetamide (344 mg, 1.0
mmol) according to general procedure 1. Yield 478 mg
(92%); mp 219 ꢀC. IR (KBr): n=3310, 3065, 1685, 1667,
1638, 1595, 1553, 1513, 1403, 1341 1290 cmÀ1. 1H NMR
(DMSO-d6): d 2.22 (s, 3H), 3.36 (s, 2H), 6.91 (m, 2H),
6.99 (m, 1H), 7.10 (m, 1H), 7.47 (m, 2H), 7.60 (m, 3H),
7.66 (m, 3H), 7.76 (m, 1H), 7.83 (m, 2H), 7.87 (m, 1H),
8.24 (m, 2H), 10.03(s, 1H), 10.40 (s, 1H). MS (EI): m/z
519 (29) [M+], 44 (100), 73(86), 105 (67), 212 (65), 387
(55), 129 (49), 414 (29). Anal. calcd for C31H25N3O5: C,
71.67; H, 4.85; N, 8.09; found: C, 71.43; H, 4.86; N,
7.99.
N-[3-Benzoyl-4-[(4-bromophenyl)acetylamino]phenyl]-4-
nitrocinnamic acid amide (4f). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-(4-bromophenyl)acetamide (409 mg, 1.0
mmol) according to general procedure 1. Yield 0.427 g
(73%); mp 231 ꢀC. IR (KBr): n=3398, 1668, 1635, 1596,
1
1553, 1513, 1403, 1341, 1290 cmÀ1. H NMR (DMSO-
d6): d 3.40 (s, 2H), 6.94 (d, J=16 Hz, 1H), 7.05 (m, 2H),
7.42 (m, 2H), 7.48 (m, 2H), 7.57 (m, 1H), 7.64 (m, 2H),
7.67 (m, 2H), 7.78 (m, 1H), 7.85 (m, 2H), 7.90 (m, 1H),
8.26 (m, 2H), 10.10 (s, 1H), 10.43(s, 1H). MS (EI): m/z
583(51) [M +], 585 (15, M++2), 146 (100), 212 (72),
387 (43), 105 (33), 175 (31), 414 (21). Anal. calcd for
C30H22BrN3O5: C, 61.66; H, 3.79; N, 7.19; found: C,
61.58; H, 3.87; N, 7.18.
N-[3-Benzoyl-4-[(2-methylphenyl)acetylamino]phenyl]-4-
nitrocinnamic acid amide (4c). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-(2-methylphenyl)acetamide (344 mg, 1.0
mmol) according to general procedure 1. Yield 500 mg
(96%); mp 260 ꢀC. IR (KBr): n=3434, 3071, 1685, 1666,
N-(2-Benzoyl-4-nitrophenyl)trifluoroacetamide (5). 2-
Amino-5-nitrobenzophenone (1.2 g, 5 mmol) was dis-
solved in a mixture of dry dichloromethane (50 mL) and
dry pyridine (4.5 mL). The solution was cooled to 0 ꢀC
and trifluoroacetic anhydride (0.75 mL) was added. The
mixture was left to warm to room temperature for 2 h.
Then, the solution was diluted with dichloromethane
and washed with water, brine, satd NaHCO3 solution
and dried over MgSO4. The residue obtained after
removal of the solvent was recrystallized from EtOH.
Yield 1.420 g (83%); mp 135 ꢀC. IR (KBr): n=3432,
1735, 1643, 1619, 1587, 1558, 1522, 1448, 1417, 1353,
1634, 1596, 1544, 1505, 1401, 1339, 1290 cmÀ1 1H
.
NMR (DMSO-d6): d 2.13 (s, 3H), 3.44 (s, 2H), 6.91 (d,
J=16 Hz, 1H), 7.03(m, 2H), 7.09 (m, 2H), 7.49 (m,
2H), 7.62 (m, 2H), 7.66 (m, 3H), 7.76 (m, 1H), 7.84 (m,
2H), 7.88 (m, 1H), 8.25 (m, 2H), 10.05 (s, 1H), 10.41 (s,
1H). MS (EI): m/z 519 (53) [M+], 105 (100), 146 (75),
212 (64), 387 (63), 211 (57). Anal. calcd for
C31H25N3O5: C, 71.67; H, 4.85; N, 8.09; found: C,
71.49; H, 4.89; N, 8.02.
1
1324 cmÀ1. H NMR (CDCl3): d 7.56 (m, 2H), 7.71 (m,
3H), 8.50 (m, 1H), 8.57 (m, 1H), 8.87 (m, 1H), 12.27 (s,
1H). MS (EI): m/z 105 (100), 77 (69), 338 (55) [M+], 269
(34), 191 (32), 145 (16), 339 (10), 139 (8), 106 (8), 241
(7). Anal. calcd for C15H9F3N2O4: C, 53.27; H, 2.68; N,
8.28; found: C, 53.03; H, 2.91; N, 8.28.
N-[3-Benzoyl-4-[(4-methoxyphenyl)acetylamino]phenyl]-
4-nitrocinnamic acid amide (4d). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-(4-methoxyphenyl)acetamide (360 mg, 1.0
mmol) according to general procedure 1. Yield: 416 mg
(78%); mp 214 ꢀC. IR (KBr): n=3421, 3070, 2840, 1665,
N-(4-Amino-2-benzoylphenyl)trifluoroacetamide (6). To
a
solution of the N-(2-benzoyl-4-nitrophenyl)tri-
1633, 1597, 1551, 1511, 1402, 1340, 1291 cmÀ1
.
1H
fluoroacetamide (5) (1.400 g, 4.1 mmol) in EtOAc (25
mL) SnCl2Â2H2O (4.62 g) was added. Then, the solu-
tion was refluxed for 2 h. The cooled solution was dilu-
ted with water and the pH was adjusted to 7–8 by
addition of sat. NaHCO3 solution. The aqueous phase
was extracted with EtOAc (3 Â 100–200 mL) and the
combined organic extracts were thoroughly washed with
brine and dried over MgSO4. The product obtained
after the removal of the solvent was used without fur-
ther purification. Yield 1.205 g (95%); mp 108 ꢀC. IR
(KBr): n=3445, 3389, 3058, 2975, 2874, 1714, 1653,
NMR (DMSO-d6): d 3.32 (s, 2H), 3.69 (s, 3H), 6.77 (m,
2H), 6.91 (d, J=16 Hz, 1H), 7.01 (m, 2H), 7.47 (m, 2H),
7.60 (m, 2H), 7.66 (m, 2H), 7.75 (m, 1H), 7.82 (m, 3H),
7.86 (m, 1H), 8.23(m, 2H), 10.00 (s, 1H), 10.73(s, 1H).
MS (EI): m/z 535 (7) [M+], 121 (100), 387 (96), 212 (58),
211 (44), 77 (30), 105 (33), 414 (17). Anal. calcd for
C31H25N3O5: C, 69.52; H, 4.71; N, 7.85; found: C,
69.39; H, 4.83; N, 7.51.
N-[3-Benzoyl-4-[(4-chlorophenyl)acetylamino]phenyl]-4-
nitrocinnamic acid amide (4e). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-2-(4-chlorophenyl)acetamide (364 mg, 1.0
1
1595, 1539, 1438, 1329 cmÀ1. H NMR (CDCl3): d 6.83
(m, 1H), 6.87 (m, 1H), 7.43(m, 2H), 7.55 (m, 1H), 7.65
(m, 2H), 8.32 (m, 1H), 11.47 (s, 1H). MS (EI): m/z 308

J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
237
(100) [M+], 105 (91), 77 (54), 211 (29), 161 (21), 239
(18), 309 (17), 106 (12), 210 (10), 78 (10). C15H11F3N2O2
2H), 10.22 (s, 1H), 10.37 (s, 1H). MS (EI): m/z 581 (29)
[M+], 414 (100), 182 (32), 105 (29), 415 (24), 265 (22).
Anal. calcd for C36H27N3O5: C, 74.34; H, 4.68; N, 7.22;
found: C, 74.15; H, 4.81; N, 7.28.
N-[3-Benzoyl-4-(trifluoroacetylamino)phenyl]-4-nitrocin-
namic acid amide (7). From 4-nitrocinnamoyl chloride
(633 mg, 3.0 mmol) and N-(4-amino-2-benzoylphenyl)-
trifluoroacetamide (6) (925 mg, 3.0 mmol) according to
general procedure 1. Yield 1.228 g (82%); mp 210 ꢀC.
IR (KBr): n=3396, 1725, 1685, 1635, 1598, 1558, 1522,
1448, 1408, 1342 cmÀ1. 1H NMR (DMSO-d6): d 6.94 (d,
J=16 Hz, 1H), 7.52 (m, 3H), 7.65 (m, 1H), 7.69 (m,
3H), 7.83 (m, 1H), 7.87 (m, 2H), 7.99 (m, 1H), 8.25 (m,
2H), 10.59 (s, 1H), 11.31 (s, 1H). MS (EI): m/z 59 (100),
72 (46), 69 (39), 73 (35), 83 (27), 105 (24), 483 (10) [M+].
Anal. calcd for C24H16F3N3O5: C, 59.63; H, 3.34; N,
8.69; found: C, 59.83; H, 3.59; N, 8.95.
N-(2-Benzoyl-4-nitrophenyl)-2-(2-bromophenyl)acetamide
(2i). From 2-amino-5-nitrobenzophenone (0.969 g, 4.0
mmol) and 2-(2-bromophenyl)acetyl chloride (0.935 g,
4.0 mmol) according to general procedure 1. The pro-
duct crystallized upon cooling and was recrystallized
from EtOH. Yield 1.023g (58%); mp 96 ꢀC. IR (KBr):
n=3203, 3108, 1701, 1642, 1615, 1597, 1579, 1539, 1506,
1
1442, 1411, 1348 cmÀ1. H NMR (CDCl3): d 3.96 (s,
2H), 7.23(m, 1H), 7.37 (m, 1H), 7.41 (m, 1H), 7.52 (m,
2H), 7.60 (m, 1H), 7.66 (m, 3H), 8.38 (m, 1H), 8.42 (m,
1H), 8.89 (m, 1H), 11.01 (s, 1H). MS (EI): m/z 359
(100), 269 (43), 169 (32), 171 (32), 105 (29), 191 (26), 77
(22), 438 (0.5) [M+], 440 (0.5, M++2). Anal. calcd for
C21H15BrN2O4: C, 57.42; H, 3.44; N, 6.38; found: C,
57.54; H, 3.56; N, 6.58.
N-(4-Amino-3-benzoylphenyl)-4-nitrocinnamic acid amide
(8).
N-[3-Benzoyl-4-(trifluoroacetylamino)phenyl]-4-
nitrocinnamic acid amide (7) (1.162 g, 2.4 mmol) was
dissolved in a 1:1 mixture of dioxane/satd K2CO2 solu-
tion (30 mL) and refluxed for 3 h. Then, the solution
was diluted with water and extracted with EtOAc (3 Â
50 mL). The combined organic extracts were thoroughly
washed with water, brine and dried over MgSO4. The
residue obtained after removal of the solvent was
recrystallized from toluene. Yield 0.790 g (85%); mp
242 ꢀC. IR (KBr): n=3397, 1725, 1685, 1635, 1598,
N-(4-Amino-2-benzoylphenyl)-2-(2-bromophenyl)aceta-
mide (3i). As described for compound 6 from N-(2-ben-
zoyl-4-nitrophenyl)-2-(2-bromophenyl)acetamide (2m)
(1.008 g, 2.3mmol) and SnCl Â2H2O (2.58 g). Yield
2
0.847 g (87%); Oil. IR (KBr): n=3438, 3357, 3282,
1658, 1594, 1559, 1518, 1471, 1446, 1434, 1325 cmÀ1. 1H
NMR (CDCl3): d 3.83 (s, 4H), 6.73(d, J=3Hz, 1H),
6.85 (dd, J=3, 7 Hz, 1H), 7.15 (m, 1H), 7.31 (m, 1H),
7.36 (m, 1H), 7.44 (m, 2H), 7.56 (m, 2H), 7.65 (m, 2H),
8.26 (d, J=9 Hz, 1H), 10.04 (s, 1H). MS (EI): m/z 212
(100), 408 (70), 410 (70), 211 (50), 409 (17) [M+], 41
(16), 239 (16), 213 (15), 105 (14), 169 (9), 171 (8), 210
(8).
1
1556, 1519, 1448, 1411, 1342 cmÀ1. H NMR (DMSO-
d6): d 6.93(d, J=16 Hz, 1H), 7.56 (m, 2H), 7.63(m,
3H), 7.71 (m, 1H), 7.79 (m, 1H), 7.86 (m, 2H), 8.28 (m,
2H), 10.03(s, 1H). MS (EI): m/z 387 (100) [M+], 211
(50), 212 (46), 388 (25), 176 (11), 193 (10), 105 (6), 213
(6), 102 (6), 77 (5), 166 (5), 130 (4).
N-[3-Benzoyl-4-[(4-trifluormethylphenyl)acetylamino]phe-
nyl]-4-nitrocinnamic acid amide (4g). From 2-(4-tri-
fluoromethylphenyl)acetyl chloride (0.156 g, 0.7 mmol)
and N-(4-amino-3-benzoylphenyl)-4-nitrocinnamic acid
amide (8) (271 mg, 0.7 mmol) according to general pro-
cedure 1. Yield 0.293 g (73%); mp 231 ꢀC. IR (KBr):
n=3439, 3072, 2924, 1685, 1668, 1633, 1597, 1550, 1514,
N-[3-Benzoyl-4-[(2-bromophenyl)acetylamino]phenyl]-4-
nitrocinnamic acid amide (4i). From 4-nitrocinnamoyl
chloride (211 mg, 1.0 mmol) and N-(4-amino-2-ben-
zoylphenyl)-2-(2-bromophenyl)acetamide (3m) (409 mg,
1.0 mmol) according to general procedure 1. Yield 403
mg (69%); mp 248 ꢀC. IR (KBr): n=3432, 1686, 1631,
1596, 1541, 1507, 1443, 1403, 1340 cmÀ1 1H NMR
.
1
1448, 1403, 1341, 1291 cmÀ1. H NMR (DMSO-d6): d
(DMSO-d6): d 3.57 (s, 2H), 6.91 (d, J=16 Hz, 1H), 7.16
(m, 2H), 7.26 (m, 2H), 7.51 (m, 3H), 7.62 (m, 1H), 7.66
(m, 4H), 7.77 (m, 1H), 7.82 (m, 2H), 7.88 (m, 1H), 8.23
(m, 2H), 10.08 (s, 1H), 10.39 (s, 1H). MS (EI): m/z 44
(100), 73 (76), 69 (56), 71 (43), 129 (38), 585 (8, M++2),
583(8) [M +]. Anal. calcd for C30H22BrN3O5: C: 61.66;
H: 3.79; N: 7.19; found: C: 62.06; H: 3.94; N: 7.21.
3.50 (s, 2H), 6.93 (d, J=16 Hz, 1H), 7.29 (m, 2H), 7.43
(m, 2H), 7.54 (m, 4H), 7.64 (m, 3H), 7.74 (m, 1H), 7.83
(m, 2H), 7.88 (m, 1H), 8.23(m, 2H), 10.11 (s, 1H), 10.42
(s, 1H). MS (EI): m/z 146 (100), 573(51) [M +], 212 (42),
211 (41), 176 (40), 387 (28), 159 (28), 118 (23), 398 (19),
129 (19), 105 (18), 574 (18). Anal. calcd for
C31H22F3N3O5: C: 64.92; H: 3.82; N: 7.33; found: C:
64.79; H: 3.98; N: 7.38.
N-[3-Benzoyl-4-[(2-trifluormethylphenyl)acetylamino]phe-
nyl]-4-nitrocinnamic acid amide (4j). From (2-tri-
fluoromethylphenyl)acetyl chloride (0.156 g, 0.7 mmol)
and N-(4-amino-3-benzoylphenyl)-4-nitrocinnamic acid
amide (8) (271 mg, 0.7 mmol) according to general pro-
cedure 1. Yield 0.271 g (72%); mp 224 ꢀC. IR (KBr):
n=3431, 1686, 1665, 1637, 1597, 1559, 1512, 1403, 1342,
N-[3-Benzoyl-4-(4-biphenylylacetylamino)phenyl]-4-nitro-
cinnamic acid amide (4h). From 4-nitrocinnamoyl chlo-
ride (211 mg, 1.0 mmol) and N-(4-amino-2-
benzoylphenyl)-(4-biphenylyl)acetamide (406 mg, 1.0
mmol) according to general procedure 1. Yield 420 mg
(72%); mp 239 ꢀC. IR (KBr): n=3427, 3066, 1686, 1658,
1
1317, 1292 cmÀ1. H NMR (DMSO-d6): d 3.64 (s, 2H),
1636, 1599, 1554, 1516, 1401, 1339, 1287 cmÀ1
.
1H
6.94 (d, J=16 Hz, 1H), 7.28 (m, 1H), 7.45 (m, 1H), 7.52
(m, 2H), 7.57 (m, 1H), 7.65 (m, 3H), 7.69 (m, 2H), 7.80
(m, 1H), 7.86 (m, 2H), 7.91 (m, 1H), 8.27 (m, 2H), 10.10
(s, 1H), 10.42 (s, 1H). MS (EI): m/z 146 (100), 212 (60),
73 (60), 84 (53), 211 (51), 159 (41), 105 (41), 91 (38), 129
NMR (DMSO-d6): d 4.94 (s, 1H), 6.87 (d, J=16 Hz,
1H), 7.07 (m, 4H), 7.13(m, 2H), 7.19 (m, 4H), 7.38 (m,
2H), 7.53(m, 1H), 7.57 (m, 1H), 7.61 (m, 2H), 7.71 (m,
1H), 7.77 (m, 1H), 7.79 (m, 1H), 7.82 (m, 1H), 8.19 (m,

238
J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
(35), 573 (27) [M+]. Anal. calcd for C31H22F3N3O5: C:
64.92; H: 3.82; N: 7.33; found: C: 64.79; H: 4.19; N:
7.26.
C24H16N2O4: C: 72.72; H: 4.07; N: 7.07; found: C:
72.55; H: 4.22; N: 6.99.
N-[(4-Amino-2-benzoyl)phenyl]-2-naphthoyl amide (3n).
As described for compound 6 from N-(2-benzoyl-4-
nitrophenyl)-2-naphthoyl amide (2b) (0.450 g, 1.1
mmol) and SnCl2Â2H2O (1.25 g). Yield 0.385 g (95%);
mp 81 ꢀC. IR (KBr): n=3433, 3233, 1661, 1620, 1595,
1529, 1506, 1433, 1321, 1305 cmÀ1. 1H NMR (CDCl3): d
3.63(s, 2H), 6.84 (m, 1H), 6.94 (m, 1H), 7.43(m, 2H),
7.50 (m, 3H), 7.68 (m, 2H), 7.81 (m, 1H), 7.87 (m, 1H),
7.94 (m, 1H), 8.00 (m, 1H), 8.47 (m, 1H), 8.617 (m, 1H),
11.53(s, 1H). MS (EI): m/z 155 (100), 127 (71), 366 (43)
[M+], 59 (42), 41 (25), 43 (23), 57 (23), 44 (22), 55 (21),
771 (19), 105 (18), 211 (15). C24H18N2O2
N-[3-Benzoyl-4-(1-naphthylacetylamino)phenyl]-4-nitro-
cinnamic acid amide (4k). From 4-nitrocinnamoyl chlo-
ride (211 mg, 1.0 mmol) and N-(4-amino-2-
benzoylphenyl)-1-naphthylacetamide (380 mg, 1.0
mmol) according to general procedure 1. Yield 494 mg
(89%); mp 229 ꢀC. IR (KBr): n=3420, 3062, 1663, 1597,
1
1557, 1512, 1402, 1341, 1237 cmÀ1. H NMR (DMSO-
d6): d 3.92 (s, 2H), 6.92 (d, J=16 Hz, 1H), 7.31 (m, 1H),
7.40 (m, 1H), 7.48 (m, 4H), 7.61 (m, 2H), 7.69 (m, 3H),
7.81 (m, 2H), 7.84 (m, 2H), 7.88 (m, 2H), 7.93(m, 1H),
8.25 (m, 2H), 10.19 (s, 1H), 10.40 (s, 1H). MS (EI): m/z
555 (11) [M+], 44 (100), 387 (86), 73 (81), 141 (70), 129
(54), 256 (51), 212 (43), 414 (36), 105 (29). Anal. calcd
for C34H25N3O5: C, 73.50; H, 4.54; N, 7.56; found: C,
73.29; H, 4.60; N, 7.44.
N-[3-Benzoyl-4-(2-naphthoylamino)phenyl]-4-nitrocinnamic
acid amide (4n). From 4-nitrocinnamoyl chloride (211
mg, 1.0 mmol) and N-(4-amino-2-benzoylphenyl)-2-
naphthoyl amide (366 mg, 1.0 mmol) according to gen-
eral procedure 1. Yield: 108 mg (20%); mp 262 ꢀC. IR
(KBr): n=3339, 3059, 1665, 1631, 1596, 1550, 1517,
1403, 1341, 1295 cmÀ1. 1H NMR (DMSO-d6): d 6.98 (d,
J=16 Hz, 1H), 7.49 (m, 2H), 7.59 (m, 3H), 7.68 (d,
J=16 Hz, 1H), 7.76 (m, 3H), 7.86 (m, 3H), 7.91 (m,
1H), 7.97 (m, 5H), 8.22 (m, 1H), 8.27 (m, 2H), 10.51 (s,
1H), 10.72 (s, 1H). MS (EI): m/z 541 (22) [M+], 155
(100), 127 (46), 73 (32), 366 (10). Anal. calcd for
C33H23N3O5: C, 73.19; H, 4.28; N, 7.76; found: C,
73.19; H, 4.27; N, 7.66.
N-[3-Benzoyl-4-(2-naphthylacetylamino)phenyl]-4-nitro-
cinnamic acid amide (4l). From 4-nitrocinnamoyl chlo-
ride (211 mg, 1.0 mmol) and N-(4-amino-2-
benzoylphenyl)-2-naphthylacetamide (380 mg, 1.0
mmol). Yield 272 mg (49%); mp 225 ꢀC. IR (KBr):
n=3297, 3063, 1686, 1663, 1595, 1550, 1507, 1402, 1341,
1
1290 cmÀ1. H NMR (DMSO-d6): d 3.57 (s, 2H), 6.91
(d, J=16 Hz, 1H), 7.24 (m, 1H), 7.44 (m, 4H), 7.55 (m,
1H), 7.60 (m, 3H), 7.65 (m, 2H), 7.76 (m, 3H), 7.82 (m,
3H), 7.87 (m, 1H), 8.23 (m, 2H), 10.12 (s, 1H), 10.39 (s,
1H). MS (EI): m/z 555 (9) [M+], 387 (100), 212 (98), 211
(71), 141 (51), 105 (33), 238 (27), 414 (21). Anal. calcd
for C34H25N3O5: C, 73.50; H, 4.54; N, 7.56; found: C,
73.20; H, 4.69; N, 7.55.
Enzyme preparation
Yeast farnesyltransferase was used as a fusionprotein to
glutathione S-transferase at the N-terminus of the b-sub-
unit. Farnesyltransferase was expressed in Escherichia
coli DH5a grown in LB media containing ampicillin
and chloramphenicol for co-expression of pGEX-DPR1
and pBC-RAM2 for farnesyltransferase production.24
The enzyme was purified by standard procedures with
glutathione–agarose beads for selective binding of the
target protein.
N-[3-Benzoyl -4-(1-naphthoylamino)phenyl]-4-nitrocin-
namic acid amide (4m). From 4-nitrocinnamoyl chloride
(0.211 g, 1.0 mmol) and N-(4-amino-2-benzoylphenyl)-
1-naphthoyl amide (0.366 g, 1.0 mmol) according to
general procedure 1. Yield 0.380 g (70%); mp 230 ꢀC.
IR (KBr): n=3427, 1680, 1633, 1596, 1514, 1402, 1341,
1
1292 cmÀ1. H NMR (DMSO-d6): d 6.96 (d, J=16 Hz,
1H), 7.28 (m, 1H), 7.48 (m, 2H), 7.51 (m, 3H), 7.62 (m,
1H), 7.67 (m, 2H), 7.77 (m, 2H), 7.86 (m, 3H), 7.92 (m,
1H), 7.98 (m, 2H), 7.99 (m, 1H), 8.25 (m, 2H), 10.47 (s,
1H), 10.57 (s, 1H). MS (EI): m/z 541 (16) [M+], 155
(100), 127 (56), 63 (33), 366 (10). Anal. calcd for
C33H23N3O5: C, 73.19; H, 4.28; N, 7.76; found: C,
72.92; H, 4.46; N, 7.51.
Farnesyltransferase assay
The assay was conducted as described.23 Farnesylpyro-
phosphate (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, Hei-
delberg, Germany. The assay mixture (100 mL volume)
contained 50 mM Tris–HCl pH 7.4, 5 mM MgCl2, 10
mM ZnCl2, 5 mM dithiothreitol (DTT), 7 mM Ds-
GCVLS, 20 mM 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 fol-
lowed by monitoring the enhancement of the fluor-
escence 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. Fluor-
escence emission was recorded with a Perkin-Elmer
LS50B spectrometer. IC50 values (concentrations
N-(2-Benzoyl-4-nitrophenyl)-2-naphthoyl amide (2n).
From 2-amino-5-nitrobenzophenone (0.726 g, 3.0
mmol) and 2-naphthoyl chloride (0.570 g, 3.0 mmol)
according to general procedure 1. The products crystal-
lized upon cooling and were recrystallized from EtOH.
Yield 0.475 g (40%); mp 211 ꢀC. IR (KBr): n=3447,
3219, 1695, 1630, 1579, 1559, 1546, 1509, 1470, 1426,
1
1340, 1285 cmÀ1. H NMR (CDCl3): d 7.51 (m, 4H),
7.61 (m, 1H), 7.68 (m, 2H), 7.79 (m, 1H), 7.91 (m, 1H),
7.97 (m, 1H), 8.03(m, 1H), 8.42 (m, 1H), 8.50 (m, 1H),
8.53(m, 1H), 9.11 (m, 1H), 12.34 (s, 1H). MS (EI): m/z
155 (100), 396 (41) [M+], 156 (13), 397 (11), 366 (4), 291
(3), 127 (2), 398 (1), 367 (1), 157 (1). Anal. calcd for

J. Sakowski et al. / Bioorg. Med. Chem. 10 (2002) 233–239
239
resulting in 50% inhibition) were calculated from initial
velocity of three independent measurements of four to
five different concentrations of the respective inhibitor.
Bond, R.; Wu, R.; Schwartz, J.; Le, H. V.; Beese, L. S.; Weber,
P. C. Biochemistry 1998, 37, 16601.
14. Reynolds, J. E. F., Ed. Martindale The Extra Pharmaco-
peia. 31st ed.; Royal Pharmaceutical Society of Great Britain:
London, 1996, p. 821.
15. Hunt, J. T.; Lee, V. G.; Leftheris, K.; Seizinger, B.; Car-
boni, J.; Mabus, J.; Ricca, C.; Yan, N.; Manne, V. J. Med.
Chem. 1996, 39, 353.
16. O’Connor, S. J.; Barr, K. J.; Wang, L.; Sorensen, B. K.;
Tasker, A. S.; Sham, H.; Ng, A.-C.; Cohen, J.; Devine, E.;
Cherian, S.; Saeed, B.; Zhang, H.; Lee, J. Y.; Warner, R.;
Tahir, S.; Kovar, P.; Ewing, P.; Alder, J.; Mitten, M.; Leal, J.;
Marsh, K.; Bauch, J.; Hoffman, D. J.; Sebti, S. M.; Rosen-
berg, S. H. J. Med. Chem. 1999, 42, 3701.
Acknowledgements
The pGEX-DPR1 and pBC-RAM2 plasmids were
kindly provided by Prof. F. Tamanoi (UCLA). Finan-
cial support by the Deutsche Pharmazeutische Gesell-
schaft is gratefully acknowledged. I. S. wishes to thank
Prof. Dr. S. Grabley for generous support and Ms. S.
Egner for technical assistance.
17. Augeri, D. J.; Janowick, D.; Kalvin, D.; Sullivan, G.;
Larsen, J.; Dickman, D.; Ding, H.; Cohen, J.; Lee, J.; Warner,
R.; Kovar, P.; Cherian, S.; Saeed, B.; Zhang, H.; Tahir, S.;
Ng, S.-C.; Sham, H.; Rosenberg, S. H. Bioorg. Med. Chem.
Lett. 1999, 9, 1069.
18. Breslin, M. J.; deSolms, J.; Giuliani, E. A.; Stokker, G. E.;
Graham, S. L.; Pompliano, D. L.; Mosser, S. D.; Hamilton,
K. A.; Hutchinson, J. H. Bioorg. Med. Chem. Lett. 1998, 8,
3311.
19. Ciccarone, T. M.; MacTough, S. C.; Williams, T. M.;
Dinsmore, C. J.; O’Neill, T. J.; Shah, D.; Culberson, J. C.;
Koblan, K. S.; Kohl, N. E.; Gibbs, J. B.; Oliff, A. I.; Graham,
S. L.; Hartman, G. D. Bioorg. Med. Chem. Lett. 1999, 9, 1991.
20. Schlitzer, M.; Sattler, I.; Dahse, H.-M. Arch. Pharm.
Pharm. Med. Chem. 1999, 332, 124.
References and Notes
1. Leonard, D. M. J. Med. Chem. 1997, 40, 2971.
2. Qian, Y. S.; Sebti, M.; Hamilton, A. D. Biopolymers 1997,
43, 25.
3. Williams, T. M. Exp. Opin. Therp. Pat. 1998, 8, 553.
4. Williams, T. M. Exp. Opin. Therp. Pat. 1999, 9, 1263.
5. Williams, T. M.; Dinsmore, C. J. Adv. Med. Chem. 1999, 4,
273.
6. Wittinghofer, A.; Waldmann, H. Angew. Chem. 2000, 112,
4360. Austin, S Angew. Chem., Int. Ed. 2000, 39, 4192.
7. Zhang, F. L.; Casey, P. J Annu. Rev. Biochem. 1996, 65,
241.
21. Schlitzer, M.; Bohm, M.; Mitsch, A.; Sakowski, J.; Wib
¨
ner, P.; Sattler, I. Short communication presented at the
annual meeting of the German Pharmaceutical Society,
8. Fu, H.-W.; Casey, P. J. Rec. Prog. Hormon Res. 1999, 54,
315.
Munster, Oct 5–7, 2000; Abstr.: Arch. Pharm. Pharm. Med.
¨
Chem. 2000, 333 (Suppl. 2), 12; D 1.62.
9. Cox, A. D.; Der, C. J. Biochim. Biophys. Acta 1997, 1333,
F51.
22. Sakowski, J.; Bohm, M.; Sattler, I.; Dahse, H.-M.; Schlitzer,
¨
M. J. Med. Chem. 2001, 44, 2886.
10. Du, W.; Lebowitz, P. F.; Prendergast, G. C. Mol. Cell.
Biol. 1999, 19, 1831.
23. Pompliano, D. L.; Gomez, R. P.; Anthony, N. J. J. Am.
Chem. Soc. 1992, 114, 7945.
11. Prendergast, G. C. Curr. Opin. Cell Biol. 2000, 12, 166.
12. Oliff, A. Biochim. Biophys. Acta 1999, 1423, C19.
13. Strickland, C. L.; Windsor, W. T.; Syto, R.; Wang, L.;
24. Del Villar, K.; Mitsuzawa, H.; Yang, W.; Sattler, I.;
Tamanoi, F. J. Biol. Chem. 1997, 272, 680.
25. Manuscript in preparation.