Structure-activity relationship of 3-substituted N-(pyridinylacetyl)-4- (8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)- piperidine inhibitors of farnesyl-protein transferase: Design and synthesis of in vivo active antitumor compounds

Article abstract of DOI:10.1021/jm970464g

Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and lode analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amine 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vive antitumor activity, the 3-bromo-substituted pyridyl N- oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.

Full text of DOI:10.1021/jm970464g

4290
J . Med. Chem. 1997, 40, 4290-4301
Str u ctu r e-Activity Rela tion sh ip of 3-Su bstitu ted N-(P yr id in yla cetyl)-4-
(8-ch lor o-5,6-d ih yd r o-11H-ben zo[5,6]cycloh ep ta [1,2-b]p yr id in -11-ylid en e)-
p ip er id in e In h ibitor s of F a r n esyl-P r otein Tr a n sfer a se: Design a n d Syn th esis
of in Vivo Active An titu m or Com p ou n d s
F. George Njoroge,* Bancha Vibulbhan, Dinananth F. Rane, W. Robert Bishop, J oanne Petrin, Robert Patton,
Mathew S. Bryant, K.-J . Chen, Amin A. Nomeir, C.-C. Lin, Ming Liu, Ivan King, J ianping Chen, Suining Lee,
Bohdan Yaremko, J anet Dell, Philip Lipari, Michael Malkowski, Zujun Li, J oseph Catino, Ronald J . Doll,
V. Girijavallabhan, and Ashit K. Ganguly
Departments of Chemistry and Tumor Biology, Schering-Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth, New J ersey 07033
Received J uly 16, 1997X
Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive
structure-activity relationship (SAR) study of compounds arising from substitution at the
3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the
chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the
fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent
such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of
bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted
in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and
hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo
antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent
FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and
52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They
selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also
inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed
cells.
Ras proteins are known to play a major role in
controlling cell growth and differentiation.1 Mutated
Ras genes are found in 50% of lung and colorectal
carcinomas and in up to 95% of pancreatic carcinomas.
This observation has prompted considerable efforts at
elucidating the pathways of Ras transformation and
developing therapeutic agents which might interfere
with this pathway.2 To perform both its normal as well
as its oncogenic functions, the Ras protein must be
bound to the cell membrane. This occurs through a
series of posttranslational modifications which include
the farnesylation of the Ras protein using a farnesyl
pyrophosphate donor and catalyzed by the enzyme
farnesyl-protein transferase (FPT).3 Inhibitors of FPT
would therefore have potential as anticancer agents for
tumors in which the Ras gene is mutated. A number
of potent FPT inhibitors have been reported in the
literature.4-6 Some of these inhibitors have been shown
to inhibit in vitro tumor cell growth7 and have also
shown activity in reducing tumor growth in animal
models.8 However, most of the reported FPT inhibitors
are peptidic in nature or contain a sulfhydryl group.
This laboratory recently reported a number of tricyclic
compounds as novel nonsulfhydryl, nonpeptidic FPT
inhibitors that also showed cellular activity.9-11 One of
these compounds, SCH 56580, a 3-methyl substituted
analogue showed greatly enhanced activity in compari-
son to its unsubstituted analogue SCH 44342. It is for
this reason that we decided to carry out an extensive
study to investigate the effect of introducing other
functionalities at the 3-position of the tricyclic pyridine
ring system.
Ch em istr y
Compounds prepared for this study are shown in
Tables 1-5 and their synthetic routes are outlined in
Schemes 1-7. Most of these compounds were prepared
via carbodiimide-mediated coupling from appropriate
tricyclic piperidine with either 3- or 4-pyridylacetic
acid.9 Chemistry for preparation of the 3-substituted
analogues was greatly facilitated by our previous dis-
covery that nitration of Loratadine12 using tetrabutyl-
ammonium nitrate-trifluoroacetic anhydride nitrating
system exclusively gave the 3-nitrosubstituted carbam-
ate 2 (unpublished results). Hydrolysis of 2 in refluxing
concentrated HCl gave amine 3 which was subsequently
coupled with pyridineacetic acid to give target compound
4. Reduction of 4 with iron powder in refluxing aqueous
ethanol provided the 3-amino pyridylacetamide 5
(Scheme 1).
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0022-2623(97)00464-0 CCC: $14.00 © 1997 American Chemical Society

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4291
Sch em e 1a
Sch em e 2
ethanol as previously described (Scheme 3). The 3-flu-
oro and 3-chloro carbamates 15 and 16, respectively,
were obtained from treatment of the amine 14 with
nitrosonium tetrafluoroborate in dichloromethane/o-
dichlorobenzene solvent system and heating the reaction
mixture to 150 °C. Alternatively, the 3-chloro carbam-
ate 16 could be prepared by treatment of the amino
carbamate 14 with isoamyl nitrite in methylene chloride
medium at refluxing temperatures. Carbamate 15 was
hydrolyzed in refluxing concentrated HCl and subse-
quently coupled either to 4-pyridineacetic acid to give
compound 17 or to 3-pyridineacetic acid to give com-
pound 18. In a similar manner the hydrolysate product
of 3-chloro carbamate 16 was coupled with either 4- or
3-pyridylacetic acid to provide compound 19 or 20,
respectively (Scheme 4).
a
(a) Bu4NNO3, TFAA; (b) concd HCl, reflux; (c) 4-pyridineacetic
acid, DEC, HOBT, NMM; (d) iron fillings, CaCl2.
Condensation of amine 5 with acetaldehyde followed
by reduction with NaBH3CN at pH ∼3 afforded the
mono- and dialkylated amino carbamates 6 and 7.
While treatment of the amine 5 with acetyl chloride in
the presence of triethylamine gave the 3-aminoacetyl
compound 8, reaction with methanesulfonyl chloride in
the presence of potassium carbonate afforded the 3-ami-
nosulfonyl derivative 9 (Scheme 2).
Preparation of the 3-bromo carbamate 21 was achieved
by reacting the diazonium salt from amine 14 with Br2
according to the method of Craig.15 Again standard acid
hydrolysis in refluxing HCl followed by standard DEC-
HOBT coupling with either 4- or 3-pyridineacetic acid
gave the desired 3-bromo target compound 22 or 23,
respectively. Reaction of 3-bromo carbamate 21 with
sodium methylthiolate in the presence of strong UV
light afforded the 3-thiomethyl carbamate 24. The
latter was acid-hydrolyzed and then coupled with 4-py-
ridineacetic acid to give the desired amide 25 (Scheme
4).
Diazotization of amine 5 using NaNO2-HCl (HONO)
followed by treatment with CuCN13 gave the 3-cyano
derivative 10. On the other hand, treatment of the
diazonium salt derived from amine 5 with CuSCN and
KSCN according to the procedure of Burawoy et al.14
gave the thiocyano compound 11. In a similar manner
diazotization of 5 followed by treatment with boiling
CuSO4 afforded the 3-hydroxyl compound 12. Reaction
of 12 with diazomethane gave the 3-methoxy analogue
13 (Scheme 3).
Methods of preparation of compounds 14-29 are as
outlined in Scheme 4; thus, halogenations at the 3-posi-
tion of the tricyclic ring system were conveniently
effected through diazotization of amino carbamate 14
followed by treatment with the appropriate halogenat-
ing reagent. The latter is obtained from the reduction
of the 3-nitro carbamate 2 using iron filings in refluxing
Finally, the 3-iodo and 3-phenyl substitutions were
obtained by treatment of amine 14, dissolved in ben-
zene, with isoamyl nitrite in the presence of iodine. In
this reaction both the 3-iodo and the 3-phenyl carbam-
ates 26 and 27 were formed in a 2.6:1.0 ratio, respec-

4292 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Njoroge et al.
Sch em e 3
Resu lts a n d Discu ssion
Compounds prepared in this study were tested for
their ability to inhibit the transfer of [3H]farnesyl from
farnesyl pyrophosphate to H-Ras-CVLS, a process that
is mediated by FPT using conditions previously de-
scribed.21 Biological and pharmacokinetic data for these
compounds are summarized in Tables 1-5.
In a previous report, we established the importance
of having a pyridinylacetyl functionality, off the amino
end of the piperidinyl moiety of the tricyclic system, in
enhancement of FPT activity.9 We also reported that
introduction of a methyl group at the 3-position of the
tricyclic pyridine gave SCH 56580, a compound with
greatly enhanced FPT activity. We have now under-
taken a study geared toward understanding the struc-
ture-activity relationship (SAR) at the 3-position of the
tricyclic ring system. We also show with a few examples
that the 4-pyridylacetyl group provides more potent
compounds than the 3-pyridylacetyl derivatives in FPT
inhibition.
Introduction of a bulky alkyl substituent such as the
tert-butyl group at the 3-position of the tricyclic ring
system was disadvantageous to FPT activity as dem-
onstrated by compound 33 which did not inhibit this
enzyme even at 4.0 µM. Similarly the 3-phenyl-
substituted analogue 29 was also found to be inactive.
These results suggested that the enzyme pocket at the
3-position of the tricyclic pyridine could only accom-
modate small groups such as methyl but not the bulky
tert-butyl or phenyl groups. Interestingly, the trifluo-
romethyl-substituted analogue, compound 31 (IC50
)
0.43 µM), was found to be 1 order of magnitude less
active than the corresponding methyl analogue SCH
56580; since the size of the fluorine is considered to be
similar in size to hydrogen, it is conceivable that some
electronic factors might be responsible for this unex-
pected result.
tively. It is possible that this reaction went through a
free radical type of chemistry as previously described
by Gokel et al.16 The 3-iodo carbamate 26 and the
3-phenyl carbamate 27 were hydrolyzed and coupled to
4-pyridineacetic acid to give the target compounds 28
and 29, respectively (Scheme 4).
Introduction of the trifluoromethyl moiety at the
3-position was accomplished by treatment of 3-iodo
carbamate 26 with methyl fluorosulfonyldifluoroacetate
in the presence of CuI according to the procedure
described by Chen et al.17 Since the trifluoromethyl
group was labile toward acid hydrolysis,18 transforma-
tion of the 3-trifluoromethyl carbamate 30 to the desired
amine was carried out in aqueous potassium hydroxide.
This reaction was rather sluggish; nevertheless, we
obtained enough of the desired amine that was coupled
with pyridineacetic acid to give the target 3-trifluoro-
methyl compound 31 (Scheme 5).
The 3-tert-butyl-substituted amide 33 was prepared
from the coupling of the previously reported amine 3219
with 4-pyridineacetic acid through similar chemistry as
described above. The 3-methyl-substituted 4-pyridyl-
acetamide 35 was prepared in a similar manner from
the previously reported amine 3420 (Scheme 6).
Preparation of 3-bromopyridine N-oxide acetamide 38
starts with the 3-bromo carbamate 21 which was
hydrolyzed in refluxing concentrated HCl to give amine
36. This amine was then coupled with 4-pyridineacetic
acid N-oxide (37) (prepared by oxidation of ethyl 4-py-
ridylacetate with m-CPBA followed by base hydrolysis)
to give the amide 38 (Scheme 7). Similarly coupling of
amine 39 with 4-pyridineacetic acid N-oxide (37)
provided target 40 (Scheme 7).
We then turned our attention to incorporating halo-
gens at the 3-position. Whereas chloro, bromo, and iodo
derivatives 19, 22, and 28, respectively, were equipotent
(IC50 ∼ 0.07 µM), the fluoro compound 17 was 1 order
of magnitude less potent (IC50 ) 0.65 µM). This further
indicated that both electronic as well as hydrophobic
effects might be at play in the SAR of the 3-substituted
tricyclic analogues.
We next investigated the influence of electron-
withdrawing groups on FPT inhibitory activity. While
the nitro derivative 4 (IC50 ) 0.57 µM) was found to be
2-fold less active than the lead compound SCH 44342,
the cyano compound 10 (IC50 ) 1.4 µM) was found to
be 1 order of magnitude less active than SCH 44342.
Mixed results were obtained when polar electron-
donating groups were introduced. The amino deriva-
tive, compound 5 (IC50 ) 1 µM), was substantially less
active than the lead compound SCH 44342. Monoalky-
lation of the amine 5 to give compound 6 (IC50 ) 1.3
µM) did not affect the FPT activity of the parent amino
compound. However, when the amino 5 was dialkylated
to afford compound 7, FPT activity was greatly reduced;
thus, compound 7 inhibited FPT activity by only 22%
at 4 µM.
Reducing the basicity of the 3-amino group by forming
acetamide 8 resulted in greatly reduced activity, i.e., at
12 µM compound 8 inhibited FPT by only 15%. The

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4293
Sch em e 4a
a
(a) Iron fillings, CaCl2; (b) NOBF4, CH2Cl2, o-dichlorobenzene; (c) NaNO2, Br2-HBr; (d) isoamyl nitrite, I2, C6H6; (e) NaSCH3, hν; (f)
concd HCl, reflux; (g) 3- or 4-pyridineacetic acid, DEC, HOBT, NMM.
sulfonamide 9 (IC50 ) 4.6 µM) was more potent than
the acetamido compound 8 but less potent than the free
amine 5.
The hydroxyl analogue, compound 12 (IC50 ) 0.64
µM), was less active than the lead compound SCH
of 3-pyridylaceticpyridylacetic acid. For example, the
3-methyl-substituted compound 35 was 1 order of
magnitude less potent than SCH 56580 (IC50 ) 0.55
µM), its 4-pyridylacetyl analogue. The fluoro analogue
18 (IC50 ) 3.80 µM) was also substantially less active
than its 4-pyridylacetyl counterpart 17.
44342. Methylation of 12 gave compound 13 (IC50
)
0.49 µM) that was also less active than the lead
compound SCH 44342. The thiomethyl ether analogue
25 exhibited an IC50 of 0.42 µMsone-half as active as
SCH 44342.
In h ibition of F P T ver su s GGP T-1. In accordance
with previous reports from this laboratory,9-11 the
tricyclic benzocycloheptapyridine compounds prepared
in this series have good selectivity on inhibition of FPT
versus the closely related enzyme GGPT-1 (geranylger-
anyl-protein transferase-1). Thus compounds 19, 22, 23,
28, and 35 were found to be inactive in inhibition of
GGPT-1 even at such high concentrations as 40 µM
(Table 3).
In a previous publication,9 we demonstrated that
tricyclic compounds bearing a 4-pyridylacetyl moiety
attached to the piperidine ring were more potent FPT
inhibitors than their 3-pyridylacetamide counterparts.
As shown in Table 2, a similar relationship was ob-
served in the present study; thus analogues of 4-py-
ridylacetic acid were found to be more potent than those
COS Cell In h ibition . Since Ras farnesylation is an
intracellular event and the potential utility of Ras FPT

4294 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Njoroge et al.
Sch em e 5a
Sch em e 7a
a
(a) FO2SCF2CO2Me, CuI, DMF; (b) 1 N KOH; (c) 4-pyridine-
acetic acid, DEC, HOBT, NMM.
Sch em e 6
a
(a) Concd HCl reflux; (b) pyridineacetic acid N-oxide (37), DEC,
HOBT, NMM.
a half-life of 76 min with an AUC of 5.0 µg‚h/mL and a
Cmax of 8 µg/mL (Table 4). Examination of compound
38, the N-oxide analogue of 22, showed that it also had
a relatively good half-life of 48 min, an AUC of 12.9 µg‚h/
mL, and a Cmax of 8.3 µg/mL (Table 4). Our next
objective was to evaluate compounds that showed good
pharmacokinetic profiles in in vivo antitumor models.
In Vivo Activity. To determine the effect of the
tricyclic inhibitors in an animal tumor model, acetamide
38 was evaluated for its ability to block tumor growth
in nude mice. The in vivo efficacy and specificity of 38
were evaluated using a panel of tumor models grown
in nude mice (Table 5). The tumor models included PT-
24 (BALB c/3T3 cells transfected with oncogenic H-ras),
CVLS (NIH3T3 cells transfected with oncogenic H-ras
with its native CVLS C-terminal sequence), CVLL
(NIH3T3 cells transfected with oncogenic H-ras contain-
ing a CVLL C-terminal geranylgeranylation sequence),
MSV-3T3 (NIH3T3 cells transfected with oncogenic
mos), and human colon adenocarcinoma DLD-1 cells
which expressed activated K-ras. As is seen in Table 5
and Figure 1, tricyclic pyridinylacetamide 38, when
inhibitors in cancer therapy depends on their ability to
penetrate the cell membrane and inhibit the posttrans-
lational processing of Ras in vivo, it was necessary that
compounds with good FPT activity be evaluated for their
ability to inhibit the processing of Ras in intact cells.
Compounds 19, 20, 22, 23, 28, and 35 tested in monkey
COS cells were shown to be active in the 1-4 µM range
(Table 3).
P h a r m a cok in etics. We previously disclosed our
findings that SCH 44342 was pharmacokinetically
unstable in the mouse as evidenced by that fact that it
had a half-life of less than 10 min and an AUC of 0.37
µg‚h/mL.11 Close examination of the pharmacokinetic
profile of SCH 44342 indicated that the major metabo-
lite of this compound was the pyridine N-oxide 40.
Compound 40 (IC50 ) 0.53 µM) was subsequently
prepared and found to be slightly less active than SCH
44342, but the two were equipotent in the COS cell
assay with an IC50 of 1 µM. Whereas SCH 44342 had
a short half-life of less than 10 min, compound 40 had

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4295
Ta ble 4. Pharmacokinetic Profile for FPT Compoundsa
Ta ble 1. SAR of 3-Substituted 4-Pyridinylacetyl Tricyclic FPT
Inhibitors
AUC (po)
(µg‚h/mL)
Cmax (po)
(µg/mL)
AUC (iv)
(µg‚h/mL)
t1/2 (iv)
(min)
compd
SCH 44342
38
40
0.37b
12.9c
5.0d
1.02
8.30
8.00
1.75
17.3
11.9
<10
48
76
a
Compounds dosed at 25 mg/kg in mice as solutions of HCl
salts. AUC (0-1 h). c AUC (0-7 h). AUC (0-24 h). Abbrevia-
tions: po, oral; iv, intravenous; AUC, area under the concentra-
tion-time curve.
b
d
entry
X
FPT IC50 (µM)
Ta ble 5. Average Inhibition of Tumor Growth on Various
Tumor Models by Compound 38
SCH 44342
SCH 56580
H
0.25
0.04
0.57
1.0
1.3
22% @4
15% @12
4.6
1.4
1.0
0.64
0.49
0.65
0.072
0.06
CH3
NO2
inhibition (%) at:
4
5
6
7
8
9
10
11
12
13
17
19
22
25
28
29
31
33
5 mpk
10 mpk
20 mpk
50 mpk
NH2
NHEt
N(Et)2
NCOCH3
NSO2CH3
CN
SCN
OH
OCH3
F
CVLL
CVLS
PT-24
DLD-1
MSV-3T3
CVIM
18c
52a
45a
28c
46b
81a
81a
40b
36c
53a
0%
20c
0%
33b
a
b
p , 0.0005. p < 0.005. c 0.1 < p < 0.25.
Cl
Br
SCH3
I
0.42
0.068
0% @12
0.43
Ph
CF3
t-Bu
>4.0
Ta ble 2. SAR of 3-Substituted 3-Pyridinylacetyl Tricyclic FPT
Inhibitors
entry
X
FPT IC50 (µM)
SCH 44324
H
F
Cl
Br
CH3
0.47
3.80
0.58
0.16
0.55
18
20
23
35
F igu r e 1. In vivo antitumor efficacy of compound 38 in CVLS
model. Percent tumor growth inhibition is the average of
percent change in tumor volume between the treated groups
and the vehicle control group measured throughout the experi-
ment period. Compound 38 has an average inhibition of 40%
at 10 mg/kg per dosing versus 80% average inhibition at 50
mg/kg per dosing. Symbols: (0) no-treatment control, (])
vehicle (20% HPâCD) control, (O) 38 at 10 mpk q.i.d., (4) 38
at 50 mpk q.i.d.
Ta ble 3. GGPTase and COS Cell Activity Results
IC50 (µM)
entry
GGPT
COS cell
SCH 44342
SCH 56580
SCH 44324
5
>114
>40
>46
ND
1.0
1.0
3.7
>10
19
20
22
23
28
35
>42
1.0
1.0
3.1
3.5
0.8
3.4
only slightly by compound 38. Compound 38 also
significantly and dose-dependently inhibited the growth
of human colon cancer DLD-1 xenografts. These results
indicate that tricyclic acetamide 38 is an effective Ras
farnesylation inhibitor and a potential anticancer agent
when given orally.
ND
>40
>40
>36
>43
given orally at 10 and 50 mg/kg (four times a day, 7
days a week), significantly inhibited PT-24 and CVLS
tumor growth in a dose-dependent manner; the inhibi-
tion was more than 80% at 50 mg/kg and more than
40% at 10 mg/kg. The rate of tumor growth was
monitored and compared with vehicle control. The
growth of CVLL and MSV-3T3, however, was inhibited
Con clu sion
We have investigated the effect of introducing a
variety of substituents at the 3-position of the benzo-
cycloheptapyridine tricyclic ring system. The need for
a small aliphatic group such as a methyl is evident.
Larger halogens such as chlorine, bromine, or iodine are

4296 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Njoroge et al.
with concentrated NH4OH. The aqueous phase was extracted
with EtOAc. Concentration of the organic phase afforded 8.29
g (99.7% yield) of amine 3. The latter was used for subsequent
reaction without further purification: 1H NMR (200 MHz,
CDCl3) δ 2.17-2.53 (m, 4H), 2.64-3.16 (m, 6H), 3.36-3.59 (m,
2H), 7.03-7.26 (m, 3H), 8.26 (d, J ) 2.5 Hz, 1H), 9.23 (d, J )
2.5 Hz, 1H); MS m/z (rel intensity) 356 (100, MH+).
To a solution of amine 3 (4.72 g, 13.26 mmol) in 40 mL of
CH2Cl2 were added 4-pyridylacetic acid (2.73 g, 19.90 mmol),
HOBT (3.58 g, 26.53 mmol), DEC (5.08, 26.53 mmol), and
N-methylmorpholine (13.42 g, 14.6 mL, 132.65 mmol), and the
mixture was stirred at room temperature for 16 h. The organic
phase was washed with saturated NaHCO3 and brine and then
dried over Na2SO4. It was then concentrated and purified on
silica gel eluting with 3% MeOH (saturated with ammonia)-
CH2Cl2 to afford the amide 4 in 75% yield as a white solid:1H
NMR (200 MHz, CDCl3) δ 2.08-2.64 (m, 4H), 2.76-3.15 (m,
2H), 3.20-3.54 (m, 4H), 3.58-3.72 (m, 1H), 3.75 (s, 2H), 3.88-
4.15 (m, 1H), 7.00-7.33 (m, 5H), 8.24 (br s, 1H), 8.49-8.68
(m, 2H), 9.22 (dd, J ) 5.0, 2.5 Hz, 1H); IR (film) υmax 572, 810,
995, 1340, 1439, 1510, 1641, 2861, 2919, 3067 cm-1; MS m/z
(rel intensity) 475.2 (100, MH+). Anal. (C26H23N4O3Cl‚0.5H2O)
C, H, N.
4-(3-Am in o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (5). 3-Nitro amide 4 (4.70 g, 9.90 mmol) was
dissolved in 200 mL of 85% EtOH-H2O. To this solution were
added iron filings (4.96 g, 88.78 mmol) and CaCl2 (0.49 g, 4.45
mmol), and the reaction mixture was refluxed for 16 h. The
reaction mixture was filtered through Celite and extensively
washed with hot EtOH. The organic solvents were removed,
and the resulting semisolid material was purified on silica gel
eluting with 3% MeOH (saturated with ammonia)-CH2Cl2 to
afford the 3-amino compound 5 in 75% yield as a white solid:
1H NMR (200 MHz, DMSO) δ 2.01-2.36 (m, 4H), 2.58-2.83
(m, 2H), 3.04-3.46 (m, 4H), 3.38 (s, 2H), 3.62-3.89 (m, 2H),
5.28 (br s, 2H), 6.62-6.72 (m, 1H), 6.98-7.08 (m, 1H), 7.15-
7.37 (m, 4H), 7.72 (br s, 1H), 8.42-8.54 (m, 2H); IR (film) υmax
995, 1457, 1631, 2916, 3214, 3438 cm-1; MS m/z (rel intensity)
445.3 (100, MH+). Anal. (C26H25N4OCl 0.65‚H2O‚0.35CH2Cl2)
C, H, N.
4-[8-Ch lor o-3-(eth ylam in o)-5,6-dih ydr o-11H-ben zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e]-1-(4-pyr idin ylacetyl)-
p ip er id in e (6) a n d 4-[8-Ch lor o-3-(d iet h yla m in o)-5,6-
d ih yd r o-11H -b e n zo[5,6]cycloh e p t a [1,2-b]p yr id in -11-
ylid en e]-1-(4-p yr id in yla cet yl)p ip er id in e (7). Tricyclic
amine 5 (1 g, 2.25 mmol) was dissolved in methanol (20 mL)
and cooled in an ice-water bath. The pH of the solution was
adjusted to 3 with the addition of 1 N HCl. Acetaldehyde (1.25
mL) was added followed by sodium cyanoborohydride (1.41 g,
22.4 mmol), and the solution was stirred for 1 h. Solvents were
evaporated off under vacuum, and the residue was extracted
with dichloromethane (100 mL). The organic extract was
washed with 10% sodium bicarbonate (100 mL) and water (100
mL). The extract was dried over magnesium sulfate, and the
solvent was evaporated under vacuum to give an oil. The oil
was purified by silica gel column chromatography using a
solution of 1.5% 10% ammonium hydroxide (in methanol) in
dichoromethane to afford the monoethylamino compound 6 as
the more polar compound (0.158 g, 15% yield): 1H NMR (200
MHz, CDCl3) δ 1.04-1.33 (m, 3H), 2.03-2.51 (m, 4H), 2.59-
2.85 (m, 2H), 2.95-3.40 (m, 6H), 3.50-3.65 (m, 1H), 3.71 (s,
2H), 3.95-4.21 (m, 1H), 6.57 (br s, 1H), 6.90-7.22 (m, 5H),
7.70-7.86 (m, 1H), 8.40-8.62 (m, 2H); MS m/z (rel intensity)
473 (MH+). Anal. (C28H29N4OCl‚0.7H2O) C, H, N, Cl. The
diethylamino compound 7 is the less polar compound (0.198
g, 18% yield): 1H NMR (200 MHz, CDCl3) δ 0.94-1.28 (m, 6H),
2.10-2.54 (m, 6H), 2.62-2.88 (m, 2H), 3.00-3.41 (m, 6H),
3.50-3.67 (m, 1H), 3.71 (s, 2H), 3.94-4.21 (m, 1H), 6.53-6.65
(m, 1H), 6.95-7.22 (m, 5H), 7.78-7.92 (m, 1H), 8.38-8.60 (m,
2H); MS m/z (rel intensity) 501 (MH+). Anal. (C30H33N4OCl)
C, H, N, Cl.
well-tolerated. However, introduction of fluorine seemed
to hurt FPT activity. While very good in vitro potency
was attained in this study, a number of compounds
evaluated were found to be heavily metabolized as
evidenced by their short half-lifes and low AUCs. We,
however, found out that by having a pyridine N-oxide
acetamide attached to the nitrogen of the piperidine,
the pharmacokinetic properties of these compounds
were greatly improved. Information obtained from our
SAR studies that a bromine substitution at the 3-posi-
tion elicited superior FPT potency, coupled to the fact
that a pyridyl N-oxide group gives more pharmacoki-
netically stable compounds, led us to design 38, a
compound that incorporated both of these attributes.
Compound 38 was a potent FPT inhibitor and showed
in vivo efficacy. A key feature of the tricyclic compounds
as FPT inhibitors is that they are nonpeptidic, do not
have a sulfhydryl group, and are orally absorbed. On
the basis of the in vivo data presented in this paper,
these tricyclic inhibitors represent a promising class of
potential cancer chemotherapeutics.
Exp er im en ta l Section
Melting points were determined with an Electrothermal
digital melting point apparatus and are uncorrected. Elemen-
tal analyses were performed by the Physical-Analytical Chem-
istry Department, Schering-Plough Research Institute, on
either a Leeman CE 440 or a FISONS EA 1108 elemental
analyzer. FT-IR spectra were recorded using a BOMEN
Michelson 120 spectrometer. Mass spectra were recorded
using either EXTREL 401 (CI), J EOL, or MAT-90 (FAB), VG
ZAB-SE (SIMS), or Finnigan MAT-CH-5 (EI), spectrometer.
In general, structures of the compounds were determined by
coupling constants, coupling information from the COSY
spectra, and 1D NOE experiments. The 1H and 13C NMR
spectra were obtained on either a Varian VXR-200 (200 MHz,
1H), Varian Gemini-300 (300 MHz, 1H; 75.5 MHz, 13C), or XL-
400 (400 MHz, 1H; 100 MHz, 13C) spectrometer and are
reported as ppm downfield from Me4Si with number of protons,
multiplicities, and coupling constants in hertz indicated
parenthetically. For 13C NMR, a Nalorac Quad nuclei probe
was used. Preparation of SCH 44342, SCH 44324, and SCH
56580 has previously been reported.9 Unless otherwise speci-
fied, all solvents and reagents were obtained from commercial
suppliers and used without further purification.
4 -(8 -C h l o r o -3 -n i t r o -5 ,6 -d i h y d r o -1H -b e n z o [5 ,6 ]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (4). Tricyclic carbamate 1 (5.69 g, 14.9 mmol) was
dissolved in 35 mL of CH2Cl2 under N2 atmosphere and stirred
at ∼0 °C. To this solution was added a mixture of Bu4NNO3
(4.98 g, 16.3 mmol) and trifluoroacetic anhydride (3.12 g, 2.1
mL, 14.9 mmol) dissolved in 20 mL of CH2Cl2 and cooled to 0
°C.22 The reaction was stirred at 0 °C for 2 h and then allowed
to come to room temperature overnight. It was then basified
with saturated NaHCO3 and extracted with CH2Cl2. Com-
bined organic phase was dried over MgSO4 and concentrated.
Purification by flash chromatography eluting first with 10-
20% EtOAc-hexanes afforded the nitro carbamate 2 in 44%
yield: 1H NMR (200 MHz, CDCl3) δ 1.28 (t, J ) 7.5 Hz, 3H),
2.18-2.59 (m, 4H), 2.79-3.10 (m, 2H), 3.15-3.32 (m, 2H),
3.36-3.56 (m, 2H), 3.70-3.89 (m, 2H), 4.16 (q, J ) 7.5 Hz,
2H), 7.06-7.26 (m, 3H), 8.26 (d, J ) 2.5 Hz, 1H), 9.22 (d, J )
2.5 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 14.57, 30.59, 30.72,
30.78, 30.935, 31.57, 44.62, 61.37, 126.59, 129.00, 130.63,
132.33, 132.69, 133.61, 134.48, 136.29, 138.84, 140.19, 141.86,
142.78, 155.31, 162.63; IR (film) υmax 565, 813, 997, 1114, 1230,
1347, 1436, 1516, 1696, 2911, 2980, 3461 cm-1; MS m/z (rel
intensity) 382.3 (10.90), 427.0 (14.55), 428.0 (100, MH+), 429.0
(34.36), 430.0 (36.95). Anal. (C22H22N3O4Cl‚0.2H2O) C, H, N.
To 250 mL of concentrated HCl was added 3-nitro carbam-
ate 2 (10 g, 23.4 mmol). The reaction mixture was refluxed
for 16 h. It was then cooled, poured into ice, and neutralized
N-[8-Ch lor o-6,11-d ih yd r o-11-[1-[1-oxo-2-(4-p yr id in yl)-
eth yl]-4-p ip er id in ylid en e]-5H-ben zo[5,6]cycloh ep ta [1,2-
b]p yr id in -3-yl]a ceta m id e (8). Tricyclic amine 5 (0.3 g, 0.67
mmol) was dissolved in 5 mL of pyridine and acetic anhydride

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4297
(0.102 g, 95 µL, 1.01 mmol). The reaction mixture was stirred
at 60 °C for 16 h. It was then basified with 1 N aqueous NaOH
to pH 11 and extracted with CH2Cl2. The CH2Cl2 fraction was
dried over MgSO4 and concentrated. Purification on normal
phase HPLC eluting with 8% MeOH (saturated with am-
monia)-CH2Cl2 gave 0.22 g (0.45 mmol, 67% yield) of ami-
noacetyl compound 8: 1H NMR (200 MHz, CDCl3) δ 2.17 (s,
3H), 2.21-2.51 (m, 4H), 2.72-2.93 (m, 2H), 3.10-3.42 (m, 4H),
3.57-3.70 (m, 1H), 3.75 (s, 2H), 4.01-4.20 (m, 1H), 6.99-7.27
(m, 5H), 7.94 (d, J ) 2.5 Hz, 1H), 8.07 (s, 1H), 8.27 (d, J ) 7.5
Hz, 1H), 8.54 (d, J ) 2.5 Hz, 2H); IR (film) υmax 995, 1272,
1395, 1455, 1527, 1598, 1631, 2915, 3028, 3079, 3169, 3249,
3295, 3431 cm-1; MS m/z (rel intensity) 487.3 (100, MH+);
HRMS (FAB) cald for C28H27N4O2Cl (MH+) 487.1901, found
487.1903.
3.53-3.68 (m, 1H), 3.75 (s, 2H), 3.92-4.18 (m, 1H), 6.99-7.26
(m, 5H), 7.68 (br s, 1H), 8.44-8.62 (m, 3H); IR (film) υmax 994,
1440, 1478, 1599, 1641, 2157, 2862, 2912, 3439 cm-1; MS m/z
(rel intensity) 487.1 (100, MH+). Anal. (C27H23N4OClS‚1.3H2O)
C, H, N.
4-(8-Ch lor o-3-h yd r oxy-5,6-d ih yd r o-11H -b e n zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (12). Tricyclic amine 5 (0.5 g, 1.12 mmol) was
dissolved in 11 mL of dilute H2SO4 (10%, v/v) at room
temperature. The reaction mixture was then cooled to ∼0-5
°C for 15 min. To this reaction mixture were added NaNO2
(0.083 g, 1.20 mmol) dissolved in 10 mL of H2O and then a
boiling solution of CuSO4‚5H2O (1.13 g, 4.54 mmol) dissolved
in 10 mL of H2O. The reaction mixture was further boiled for
15 min and cooled to room temperature, and pH was adjusted
4-(8-Ch lor o-5,6-d ih yd r o-3-m et h a n esu lfon a m id o-11H -
ben zo[5,6]cycloh ep t a [1,2-b]p yr id in -11-ylid en e)-1-(4-p y-
r id in yla cetyl)p ip er id in e (9). Tricyclic amine 5 (0.3 g, 0.67
mmol) was dissolved in 5 mL of pyridine, and methanesulfonyl
chloride (0.92 g, 62 µL, 0.8 mmol) was added. The reaction
mixture was stirred at 60 °C for 20 h. It was then basified
with 1 N aqueous NaOH to pH 10 and extracted with ethyl
acetate-THF (50/50, v/v) mixture. The organic fraction was
dried over MgSO4 and concentrated. Purification on normal
phase HPLC eluting with 8% MeOH (saturated with am-
monia)-CH2Cl2 gave 0.32 g (0.61 mmol, 92% yield) of amino-
sulfonyl compound 9: 1H NMR (200 MHz, CDCl3) δ 2.16-2.45
(m, 4H), 2.72-2.94 (m, 2H), 3.03 (s, 3H), 3.15-3.44 (m, 4H),
3.55-3.70 (m, 1H), 3.77 (s, 2H), 3.98-4.18 (m, 1H), 7.00-7.27
(m, 5H), 7.42-7.55 (m, 1H), 8.23 (br s, 1H), 8.58 (br s, 2H); IR
(film) υmax 532, 970, 995, 1154, 1324, 1459, 1632, 2861, 2919,
3021, 3088, 3427 cm-1; MS m/z (rel intensity) 523.2 (100,
MH+); Anal. (C27H27N4O3ClS‚1.33H2O) C, H, N, S.
to ∼11 using NaOH. It was extracted twice with CH2Cl2 (2 ×
50 mL), and the pH of the aqueous phase was then adjusted
to ∼7 using 1 N HCl. The aqueous phase was extracted with
CH2Cl2. The CH2Cl2 fraction was dried over MgSO4 and then
concentrated. Purification by flash chromatography eluting
with 5% MeOH (saturated with ammonia)-CH2Cl2 afforded
0.16 g of the hydroxy compound 12 as a light yellow solid (32%
1
yield): mp 157-158 °C; H NMR (300 MHz, CDCl3) δ 2.11-
2.55 (m, 4H), 2.69-2.86 (m, 2H), 3.18-3.39 (m, 4H), 3.55-
3.79 (m, 3H), 3.90-4.12 (m, 1H), 6.94-7.29 (m, 8H), 7.95-
8.05 (m, 1H), 8.52 (br s, 1H);
13C NMR (75.5 MHz, CDCl3) δ
30.62, 31.89, 40.65, 40.79, 43.36, 47.09, 47.22, 126.12, 126.63,
129.37, 130.42, 133.42, 133.79, 134.28, 135.45, 138.17, 139.93,
144.93, 145.40, 14750, 149.73, 150.04, 154.11, 168.05; IR (film)
υ
max 995, 1209, 1296, 1443, 1560, 1601, 1640, 2914, 3028, 3429
cm-1; MS m/z (rel intensity) 327 (63), 446 (100, MH+). Anal.
H24N3O2Cl‚0.2H2O‚0.2CH2Cl2) C, H, N.
(C26
4-(8-Ch lor o-3-m e t h oxy-5,6-d ih yd r o-11H -b e n zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (13). Tricyclic alcohol 12 (0.05 g, 0.11 mmol) was
dissolved in 1 mL of CH2Cl2, and 4 mL of diazomethane
solution in ether was added. After stirring for 72 h the
reaction mixture was concentrated and purified on a prepara-
tive plate eluting with 10% MeOH (saturated with ammonia)-
CH2Cl2 to provide 0.011 g of the methoxy compound 13 as a
light yellow solid (22% yield): mp 93-94 °C; 1H NMR (200
MHz, CDCl3) δ 2.14-2.54 (m, 4H), 2.68-2.95 (m, 2H), 3.09-
3.44 (m, 4H), 3.53-3.79 (m, 3H), 3.83 (s, 3H), 3.97-4.21 (m,
1H), 6.88-7.30 (m, 6H), 8.11 (br s, 1H), 8.55 (br s, 2H); 13C
NMR (75.5 MHz, CDCl3) δ 30.89, 31.43, 40.389, 40.46, 43.05,
43.09, 47.01, 55.62, 122.15, 124.06, 126.33, 128.76, 128.81,
130.13, 132.99, 133.87, 134.08, 134.53, 136.03, 139.58, 139.71,
144.20, 150.12, 154.67, 167.79 cm-1; MS m/z (rel intensity)
341.0 (43), 460.0 (100), 462.0 (37); HRMS (FAB) calcd for
C27H26N3O2Cl (MH+) 460.1787, found 460.1792.
4-(3-C y a n o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (10). Tricyclic amine 5 (0.5 g, 1.12 mmol) was
dissolved in acetone (10 mL), and to this solution was added
230 µL of concentrated HCl. The reaction mixture was cooled
to ∼-10 °C, and then a solution of NaNO2 (0.085 g, 1.23 mmol
dissolved in 4 mL of H2O) was added. To this reaction mixture
was added a solution of CuCN (freshly prepared from dissolv-
ing CuSO4 (0.336 g, 1.34 mmol) in 2 mL of H2O, then cooling
the solution to ∼4 °C with ice, then adding KCN (0.365 g, 5.60
mmol) dissolved in 2 mL of H2O, and heating to ∼60-70 °C)
at temperatures between 60 and 70 °C for a period of 20 min.
The reaction temperature was then raised to ∼70-80 °C, and
heating was continued for 10 min to evaporate most of the
acetone. The reaction mixture was cooled and further diluted
with H2O. It was exhaustively extracted with CH2Cl2 (4 ×
100 mL). The organic phase was dried over Na2SO4 and
concentrated. Purification on normal phase HPLC, eluting
with 3% MeOH (saturated with ammonia)-CH2Cl2 afforded
4-(3-Am in o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh ep t a [1,2-b]p yr id in -11-ylid en e)-1-p ip er id in eca r -
boxylic Acid Eth yl Ester (14). Nitro carbamate 2 (5.99 g,
1
0.25 g of the cyano compound 10 (50% yield): H NMR (200
MHz, CDCl3) δ 2.08-2.60 (m, 4H), 2.72-3.04 (m, 2H), 3.16-
3.50 (m, 4H), 3.54-3.73 (m, 1H), 3.78 (s, 2H), 3.89-4.16 (m,
1H), 6.98-7.33 (m, 5H), 7.74 (s, 1H), 8.48-8.59 (m, 2H), 8.62-
8.72 (m, 1H); IR (film) υmax 995, 1444, 1595, 1642, 2230, 2869,
2912, 3046, 3437 cm-1; MS m/z (rel intensity) 455.2 (100,
MH+). Anal. (C27H23N4OCl‚0.7H2O) C, H, N.
O. To
0.014 mmol) was dissolved in 300 mL of 85% EtOH-H2
this solution were added iron filings (7.01 g, 0.125 mmol) and
CaCl2 (0.5 g, 0.006 mmol), and the reaction mixture was
refluxed for 4 h. The reaction mixture was filtered through
Celite and extensively washed with hot EtOH. It was then
treated with decolorized charcoal and filtered, and the organic
solvents were removed to give amine 14 in 100% yield: 1H
NMR (200 MHz, DMSO) δ 1.16 (t, J ) 7.5 Hz, 3H), 2.04-2.36
(m, 4H), 2.56-2.82 (m, 2H), 3.02-3.21 (m, 4H), 3.53-3.70 (m,
2H), 4.04 (q, J ) 7.5 Hz, 2H), 6.69 (d, J ) 2.5 Hz, 1H), 7.04 (d,
J ) 7.5 Hz, 1H), 7.21 (dd, J ) 7.5 Hz, 2.5 Hz, 1H), 7.31 (d, J
) 2.5 Hz, 1H), 7.73 (d, J ) 2.5 Hz, 1H); IR (film) υmax 768,
998, 1117, 1232, 1440, 1479, 1681, 2919, 2979, 3222, 3350,
3434 cm-1; MS m/z (rel intensity) 362 (17.65), 397 (21.45), 398
(100, MH+), 399 (30.4), 400 (32.59).
4-[8-Ch lor o-3-(th iocya n o)-5,6-d ih yd r o-11H-ben zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e]-1-(4-pyr idin ylacetyl)-
p ip er id in e (11). Tricyclic amine 5 (0.55 g, 1.25 mmol) was
dissolved in 50 mL of dilute H2SO4 (10%, v/v). The reaction
mixture was then cooled to ∼0-5 °C for 15 min. To this
reaction mixture were added NaNO2 (0.092 g, 1.33 mmol
dissolved in 10 mL of H2O), KCN (0.46 g, 4.74 mmol), and
CuSCN (0.3 g, 2.49 mmol) (both dissolved in 15 mL of H2O).
The reaction mixture was stirred for 0.5 h, heated to boiling
for 15-30 min, and then cooled to room temperature. The pH
of the reaction mixture was adjusted to 7, and then the mixture
was extracted with CH2Cl2. The organic phase was dried over
MgSO4 and concentrated. Purification by flash chromatogra-
phy eluting with 5% MeOH (saturated with ammonia)-CH2-
Cl2 afforded 0.19 g of the thiocyano compound 11 as a white
solid (32% yield): mp 97-98 °C; 1H NMR (200 MHz, CDCl3) δ
2.09-2.60 (m, 4H), 2.72-3.02 (m, 2H), 3.15-3.48 (m, 4H),
4-(8-C h lo r o -3-flu o r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh ep t a [1,2-b]p yr id in -11-ylid en e)-1-p ip er id in eca r -
boxylic Acid Eth yl Ester (15) a n d 4-(3,8-Dich lor o-5,6-
d ih yd r o-11H -b e n zo[5,6]cycloh e p t a [1,2-b]p yr id in -11-
ylid en e)-1-p ip er id in eca r boxylic Acid Eth yl Ester (16).
Amino carbamate 14 (16.2 g, 40.83 mmol) was introduced with
stirring to a slurry of nitrosonium tetrafluoroborate (7.14 g,
61.11 mmol) in CH2Cl2 (100 mL), and the reaction mixture

4298 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Njoroge et al.
stirred for 3 h. o-Dichlorobenzene (100 mL) was added to the
reaction mixture, and the solution was heated for 5 h. CH2-
Cl2 was distilled off from the reaction mixture, and the solvents
were then removed under reduced pressure to give rise to a
dark brown oil. The crude product was dissolved in CH2Cl2
(200 mL) and washed with H2O (200 mL). The organic extract
was dried over MgSO4, and the organic solvents were removed
to give a dark brown oil. Purification by flash chromatography
eluting with 20% EtOAc-hexane afforded 4.01 g (25% yield)
of 3-fluoro carbamate 15 as the more polar product: 1H NMR
(400 MHz, CDCl3) δ 1.27 (t, J ) 7.5 Hz, 3H), 2.23-2.39 (m,
3H), 2.42-2.52 (m, 1H), 2.76-2.92 (m, 2H), 3.14-3.23 (m, 2H),
3.31-3.44 (m, 2H), 3.72-3.85 (m, 2H), 4.15 (q, J ) 7.5 Hz,
2H), 7.08-7.21 (m, 4H), 8.27 (d, J ) 2.5 Hz, 1H); MS m/z (rel
intensity) 401 (100, MH+). Also 4.1 g (24% yield) of the less
polar 3-chloro carbamate 16 eluted as a white solid: 1H NMR
(200 MHz, CDCl3) δ 1.23 (t, J ) 7.5 Hz, 3H), 2.17-2.54 (m,
4H), 2.69-2.93 (m, 2H), 3.03-3.21 (m, 2H), 3.22-3.50 (m, 2H),
3.65-3.89 (m, 2H), 4.12 (q, J ) 7.5 Hz, 2H), 7.00-7.20 (m,
3H), 7.44 (d, J ) 2.5 Hz, 1H), 8.34 (d, J ) 2.5 Hz, 1H); MS m/z
(rel intensity) 418 (100, MH+).
4-(8-C h lo r o -3-flu o r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (17). To 100 mL of concentrated HCl was added
3-fluoro carbamate 15 (3.40 g, 0.84 mmol). The reaction
mixture was refluxed for 16 h. It was then cooled, poured into
ice, and neutralized with 50% aqueous NaOH. The aqueous
phase was extracted with CH2Cl2 (2 × 200 mL). Concentration
of the organic phase afforded the hydrolyzed amine that was
used for subsequent reaction without further purification: MS
m/z (rel intensity) 329 (100, MH+).
To a solution of amine obtained above (0.5 g, 1.25 mmol) in
15 mL of DMF were added 4-pyridylacetic acid (0.17 g, 1.2
mmol), HOBT (0.17 g, 1.87 mmol), DEC (0.36 g, 1.9 mmol),
and N-methylmorpholine (0.63 g, 6.2 mmol), and the mixture
was stirred at room temperature for 16 h. The organic phase
was washed with saturated NaHCO3 and brine and then dried
over Na2SO4. It was then concentrated and purified by flash
chromatography (silica gel) eluting with 1.5% MeOH (with
ammonia)-CH2Cl2 to afford 3,10-dichloro pyridylacetamide 17
in 82% yield (0.456 g): 1H NMR (300 MHz, CDCl3) δ 2.05-
2.36 (m, 4H), 2.76-2.92 (m, 2H), 3.12-3.38 (m, 4H), 3.62-
3.90 (m, 2H), 3.28 (s, 2H), 7.09 (dd, J ) 7.5, 5.0 Hz, 1H), 7.20-
7.27 (m, 3H), 7.34 (br s, 1H), 7.58 (dd, J ) 7.5, 5.0 Hz, 1H),
8.32-8.37 (m, 1H), 8.44-8.50 (m, 2H); MS m/z (rel intensity)
MH+ (448, 100). Anal. (C26H23N3OClF‚0.5H2O) C, H, N, Cl,
F.
4-(8-C h lo r o -3-flu o r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(3-pyr idin ylacetyl)-
p ip er id in e (18). Reaction was carried out essentially in the
same way as described for the preparation of compound 17
above replacing 4-pyridylacetic acid with 3-pyridylacetic acid
to obtain the desired amide 18 in 94% yield: 1H NMR (300
MHz, CDCl3) δ 2.08-2.38 (m, 4H), 2.77-2.94 (m, 2H), 3.12-
3.42 (m, 4H), 3.68-3.88 (m, 2H), 3.78 (s, 2H), 7.10 (d, J ) 7.5
Hz, 1H), 7.20-7.28 (m, 1H), 7.30-7.37 (m, 2H), 7.53-7.67 (m,
2H), 8.32-8.36 (m, 1H), 8.40-8.45 (m, 2H); IR (film) υmax 713,
893, 996, 1206, 1272, 1445, 1592, 1641, 2862, 2909, 3020, 3046,
3438 cm-1; MS m/z (rel intensity) 448.1 (100, MH+). Anal.
(C26H23N3OFCl‚0.5H2O) C, H, N.
replacing 4-pyridylacetic acid with 3-pyridylacetic acid to
obtain the desired amide 20 in >95% yield: 1H NMR (400 MHz,
CDCl3) δ 2.19-2.54 (m, 4H), 2.74-2.91 (m, 2H), 3.18-3.42 (m,
4H), 3.62-3.77 (m, 1H), 3.74 (s, 2H), 3.96-4.13 (m, 1H), 7.06
(dd, J ) 10.0, 7.5 Hz, 1H), 7.12-7.21 (m, 2H), 7.23-7.30 (m,
1H), 7.45 (d, J ) 2.5 Hz, 1H), 7.73 (d, J ) 7.5 Hz, 1H), 8.36
(dd, J ) 7.5, 2.5 Hz, 1H), 8.44-8.56 (m, 2H); IR (film) υmax
718, 897, 994, 1135, 1206, 1436, 1479, 1641, 2860, 2908, 2995,
3036, 3432 cm-1; MS m/z (rel intensity) 464 (100, MH+), 465
(42), 466 (69). Anal. (C26H23N3OCl2‚0.3H2O) C, H, N.
4-(3-B r o m o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh ep t a [1,2-b]p yr id in -11-ylid en e)-1-p ip er id in eca r -
boxylic Acid Eth yl Ester (21). Amino carbamate 14 (0.1 g,
0.25 mmol) was dissolved in 2 mL of 48% HBr. The reaction
mixture was then cooled to -5 °C, and molecular bromine (0.05
g, 0.72 mmol) was then added. The reaction mixture was
stirred at that temperature for 15 min after which NaNO2
(0.05 g, 0.72 mmol) dissolved in 5 mL of H2O was slowly added.
The reaction mixture was stirred for 45 min and then neutral-
ized with 40% NaOH. The aqueous phase was extracted with
EtOAc (3 × 60 mL). Combined EtOAc fractions were dried
over Na2SO4 and concentrated to give the 3-bromo carbamate
21 in 88% overall yield: 1H NMR (300 MHz, CDCl3) δ 1.24 (t,
J ) 7.5 Hz, 3H), 2.22-2.52 (m, 4H), 2.71-2.89 (m, 2H), 3.06-
3.20 (m, 2H), 3.25-3.43 (m, 2H), 3.68-3.88 (m, 2H), 4.14 (q,
J ) 7.5 Hz, 2H), 7.04-7.20 (m, 3H), 7.58 (d, J ) 2.5 Hz, 1H),
8.44 (d, J ) 2.5 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) δ 15.02,
30.88, 31.12, 31.60, 31.78, 32.05, 45.06, 61.69, 126.69, 129.25,
130.79, 133.43, 135.55, 137.68, 138.71, 139.57, 140.25, 147.86,
155.66, 155.75; IR (film) υmax 997, 1114, 1229, 1435, 1473, 1698,
2865, 2905, 2979, 3448 cm-1; MS m/z (rel intensity) 463 (100,
MH+).
4-(3-B r o m o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (22). To 75 mL of concentrated HCl was added
3-bromo carbamate 21 (2.70 g, 5.83 mmol). The reaction
mixture was refluxed for 16 h. It was then cooled, poured into
ice, and neutralized with concentrated NH4OH. The aqueous
phase was extracted with EtOAc. Concentration of the organic
phase afforded the hydrolyzed amine that was used for
subsequent reaction without further purification: 1H NMR (200
MHz, CDCl3) δ 2.14-2.56 (m, 4H), 2.65-2.93 (m, 4H), 3.02-
3.18 (m, 2H), 3.26-3.51 (m, 2H), 7.05-7.23 (m, 5H), 7.61 (d,
J ) 2.5 Hz, 1H), 8.47 (d, J ) 2.5 Hz, 1H), 8.55 (d, J ) 5.0 Hz,
2H); MS m/z (rel intensity) 391.1 (100, MH+).
To a solution of amine obtained above (0.5 g, 1.3 mmol) in
15 mL of DMF were added 4-pyridylacetic acid (0.178 g, 1.3
mmol), HOBT (0.17 g, 1.3 mmol), DEC (0.37 g, 1.9 mmol), and
N-methylmorpholine (0.66 g, 0.71 mL, 6.5 mmol), and the
mixture was stirred at room temperature for 16 h. The organic
phase was washed with saturated NaHCO3 and brine and then
dried over Na2SO4. It was then concentrated and purified on
normal phase HPLC (silica gel) eluting with 5% MeOH
(saturated with ammonia)-CH2Cl2 gradient to afford tricyclic
pyridylacetamide 22 in 93% yield as a white solid: 1H NMR
(200 MHz, CDCl3) δ 2.14-2.60 (m, 4H), 2.69-2.96 (m, 2H),
3.15-3.46 (m, 4H), 3.55-3.73 (m, 1H), 3.81 (s, 1H), 3.94-4.19
(m, 1H), 6.98-7.26 (m, 5H), 7.62 (br s, 1H), 8.41-8.67 (m, 3H);
13C NMR (75.5 MHz, CDCl3) δ 30.83, 31.28, 31.59, 31.91,
40.787, 43.39, 47.23, 119.43, 124.60, 126.92, 129.32, 130.21,
133.79, 134.29, 135.68, 137.53, 139.60, 140.52, 145.02, 147.92,
148.05, 150.05, 155.35, 168.05; IR (film) υmax 574, 830, 897,
994, 1205, 1271, 1437, 1639, 2906, 3028 cm-1; MS m/z (rel
intensity) 510.1 (100, MH+). Anal. (C26H23N3OBrCl‚0.5H2O)
C, H, N.
4-(3-B r o m o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(3-pyr idin ylacetyl)-
p ip er id in e (23). Reaction was carried out essentially in the
same way as described for preparation of compound 22 above
replacing 4-pyridylacetic acid with 3-pyridylacetic acid to
obtain the desired amide 23 in 90% yield: 1H NMR (200 MHz,
CDCl3) δ 2.14-2.51 (m, 4H), 2.69-2.94 (m, 2H), 3.12-3.44 (m,
4H), 3.57-3.70 (m, 1H), 3.75 (s, 2H), 3.92-4.18 (m, 1H), 6.99-
7.27 (m, 4H), 7.53-7.70 (m, 2H), 8.37-8.60 (m, 3H); MS m/z
(rel intensity) 510.1 (100, MH+). Anal. (C26H23N3OBrCl‚0.3H2O)
C, H, N.
4-(3,8-Dich lor o-5,6-d ih yd r o-11H-ben zo[5,6]cycloh ep ta -
[1,2-b]p yr id in -11-ylid en e)-1-(4-p yr id in yla cet yl)p ip er i-
d in e (19). Reaction was carried out essentially in the same
way as described for the preparation of compound 17 above to
obtain the desired amide 19 in 33% yield after purification on
normal phase HPLC (silica gel) eluting with 5% MeOH
(saturated with ammonia)-CH2Cl2: mp 113-114 °C; 1H NMR
(400 MHz, CDCl3) δ 2.10-2.50 (m, 4H), 2.70-2.95 (m, 2H),
3.15-3.40 (m, 4H), 3.50-3.70 (m, 1H), 3.80 (s, 2H), 3.95-4.15
(m, 1H), 7.00-7.30 (m, 5H), 745 (br s, 1H), 8.35 (m, 1H), 8.60
(m, 2H); MS m/z (rel intensity) 464 (60, MH+), 466 (45). Anal.
(C26H23N3OCl2‚0.6H2O) C, H, N.
4-(3,8-Dich lor o-5,6-d ih yd r o-11H-ben zo[5,6]cycloh ep ta -
[1,2-b]p yr id in -11-ylid en e)-1-(3-p yr id in yla cet yl)p ip er i-
d in e (20). Reaction was carried out essentially in the same
way as described for the preparation of compound 17 above

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4299
4-[8-Ch lor o-5,6-d ih yd r o-3-(m eth ylth io)-11H-ben zo[5,6]-
cycloh ep t a [1,2-b]p yr id in -11-ylid en e]-1-p ip er id in eca r -
boxylic Acid Eth yl Ester (24). 3-Bromo carbamate 21 was
mixed with CH3SNa (5.0 g, 10.84 mmol), and then 50 mL of
DMF was added. The reaction mixture was illuminated with
a 200-W lamp for 72 h with stirring. The volatiles were then
removed, and the resulting solid was partitioned between 1 N
NaOH and CH2Cl2. The aqueous phase was extracted twice
with CH2Cl2 (2 × 150 mL). Combined CH2Cl2 was dried over
MgSO4 and concentrated. Purification by flash chromatogra-
phy on silica gel first eluting with 20-30% EtOAc-hexane
yielded the 3-methylthio carbamate 24 in 52% yield: 1H NMR
(200 MHz, CDCl3) δ 1.28 (t, J ) 7.5 Hz, 3H), 2.22-2.60 (m,
4H), 2.51 (s, 3H), 2.71-2.94 (m, 2H), 3.04-3.22 (m, 2H), 3.26-
3.49 (m, 2H), 3.67-3.93 (m, 2H), 4.16 (q, J ) 7.5 Hz, 2H), 7.04-
7.22 (m, 3H), 7.34 (d, J ) 2.5 Hz, 1H), 8.31 (d, J ) 2.5 Hz,
1H); IR (film) υmax 998, 1115, 1233, 1435, 1463, 1479, 1696,
2925, 2975, 2987, 3444 cm-1; MS m/z (rel intensity) 429.1 (100,
MH+). Anal. (C23H25N2O2ClS) C, H, N, S.
4-[8-Ch lor o-5,6-d ih yd r o-3-(m eth ylth io)-11H-ben zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e]-1-(4-pyr idin ylacetyl)-
p ip er id in e (25). Reaction was carried out essentially in the
same way as described for the preparation of compound 22
above but replacing 3-bromo carbamate 21 with the 3-meth-
ylthio carbamate 24. Purification on normal phase HPLC
(silica gel) eluting with 3% MeOH (saturated with ammonia)-
CH2Cl2 afforded 3-iodo tricyclic pyridylacetamide 25 in 41%
yield:1H NMR (300 MHz, CDCl3) δ 2.19-2.45 (m, 1H), 2.62-
2.91 (m, 2H), 3.15-3.41 (m, 6H), 3.57-3.78 (m, 4H), 3.96-
4.18 (m, 1H), 7.02-7.36 (m, 8H), 8.27 (dd, J ) 7.5 Hz, 3.75
Hz, 1H); 13C NMR (300 MHz, CDCl3) δ 15.78, 30.43, 30.91,
31.35, 40.35, 42.96, 43.00, 46.86, 126.34, 126.43, 128.88,
130.24, 133.34, 134.02, 136.17, 136.77, 137.49, 139.41, 144.25,
144.38, 144.84, 149.36, 152.93, 167.57; IR (film) υmax 995, 1439,
1478, 1599, 1642, 2860, 2919, 3437 cm-1; MS m/z (rel intensity)
357 (90), 476 (100, MH+). Anal. (C27H26N3OClS‚0.3H2O) C,
H, N.
4-(8-C h lo r o -5,6-d i h y d r o -3-i o d o -11H -b e n z o [5,6]-
cycloh ep t a [1,2-b]p yr id in -11-ylid en e)-1-p ip er id in eca r -
boxylic Acid Eth yl Ester (26) an d 4-(8-Ch lor o-5,6-dih ydr o-
3-p h e n yl-11H -b e n zo[5,6]cycloh e p t a [1,2-b]p yr id in -11-
ylid en e)-1-p ip er id in eca r boxylic Acid Eth yl Ester (27).
Tricyclic amino carbamate 14 (6.0 g, 15.11 mmol) was sus-
pended in 100 mL of benzene, and iodine (2.3 g) followed by
isoamyl nitrite (2.7 g, 3.1 mL, 22.67 mmol) was added. The
reaction mixture was refluxed for 3 h and then diluted with
CH2Cl2 (100 mL). It was then washed with NaHSO3 (100 mL)
and 1 M NaOH. The organic phase was dried over MgSO4
and concentrated. Purification by flash chromatography on
silica gel first eluting with 20-40% EtOAc-hexanes yielded
the 3-iodo carbamate 26 in 42% yield (3.2 g): 1H NMR (200
MHz, CDCl3) δ 1.26 (t, J ) 7.5 Hz, 3H), 2.21-2.57 (m, 4H),
2.71-2.91 (m, 2H), 3.07-3.24 (m, 2H), 3.26-3.48 (m, 2H),
3.71-3.91 (m, 2H), 4.15 (q, J ) 7.5 Hz, 2H), 7.05-7.22 (m,
3H), 7.78 (d, J ) 2.5 Hz, 1H), 8.61 (d, J ) 2.5 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 15.06, 30.93, 31.16, 31.64, 31.74,
45.10, 61.74, 91.74, 126.75, 129.30, 130.84, 133.54, 136.00,
137.69, 138.72, 139.62, 145.91, 152.85, 155.81, 156.06; IR (film)
4.19 (m, 1H), 6.97-7.26 (m, 5H), 7.78 (br s, 1H), 8.42-8.69
(m, 3H); MS m/z (rel intensity) 556.1 (100, MH+). Anal.
(C26H23N3OICl) C, H, N.
4-(8-C h lo r o -3-p h e n y l-5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (29). Reaction was carried out essentially in the
same way as described for the preparation of compound 22
above but replacing 3-bromo carbamate 21 with the 3-phenyl
carbamate 27. Purification on normal phase HPLC (silica gel)
eluting with 5% MeOH (saturated with ammonia)-CH2Cl2
afforded 3-iodo tricyclic pyridylacetamide 29 in 46% yield: 1H
NMR (200 MHz, CDCl3) δ 2.20-2.61 (m, 4H), 2.74-3.06 (m,
2H), 3.12-3.52 (m, 4H), 3.58-3.72 (m, 1H), 3.79 (s, 1H), 4.01-
4.28 (m, 1H), 7.05-7.26 (m, 5H), 7.35-7.61 (m, 5H), 7.64 (br
s, 1H), 8.50-8.69 (m, 3H); MS m/z (rel intensity) 506.2 (100,
MH+); HRMS (FAB) calcd for C32H28N3OCl (MH+) 506.1999,
found 506.2004.
4-[8-Ch lor o-5,6-dih ydr o-3-(tr iflu or om eth yl)-11H-ben zo-
[5,6]cycloh ep t a [1,2-b]p yr id in -11-ylid en e]-1-p ip er id in e-
ca r boxylic Acid Eth yl Ester (30). 3-Iodo carbamate 26
(1.01 g, 1.99 mmol) was dissolved in 30 mL of dry DMF at
room temperature. To this solution were added methyl 2,2-
difluoro-2-(fluorosulfonyl) acetate (1.33 g, 0.88 mL, 6.96 mmol),
and CuI (0.76 g, 3.97 mmol), and the reaction mixture was
heated to between 60 and 80 °C for 8 h. The reaction mixture
was cooled to room temperature, and DMF was removed by
rotary evaporation. It was then partitioned between CH2Cl2
and water. The aqueous phase was further extracted with
CH2Cl2. The CH2Cl2 fraction was dried over MgSO4 and
concentrated. Purification by flash chromatography on silica
gel first eluting with 30% EtOAc-hexanes and then with 10%
MeOH (saturated with ammonia)-CH2Cl2 yielded 0.15 g of
the 3-trifluoromethyl carbamate 30 (17% yield): 1H NMR (200
MHz, CDCl3) δ 1.30 (t, J ) 7.5 Hz, 3H), 2.20-2.60 (m, 4H),
2.70-3.01 (m, 2H), 3.05-3.30 (m, 2H), 3.35-3.55 (m, 2H),
3.70-3.90 (m, 2H), 4.20 (q, J ) 7.5 Hz, 2H), 7.05-7.25 (m,
3H), 7.70 (d, J ) 2.5 Hz, 1H), 8.70 (d, J ) 2.5 Hz, 1H); MS m/z
(rel intensity) 451.1 (100, MH+); HRMS (FAB) calcd for
C23H23N2F3OCl (MH+) 451.1400, found 451.1389.
4-[8-Ch lor o-5,6-dih ydr o-3-(tr iflu or om eth yl)-11H-ben zo-
[5,6]cycloh ep ta [1,2-b]p yr id in -11-ylid en e]-1-(4-p yr id in y-
la cetyl)p ip er id in e (31). 3-Trifluoromethyl carbamate 30
(0.14 g, 0.3 mmol) was dissolved in EtOH (20 mL). To this
reaction mixture was added KOH (0.16 g, 2.7 mmol) dissolved
in 10 mL of H2O. The reaction mixture was heated to reflux
for 16 h. It was then concentrated and partitioned between
CH2
Cl2 and water. The aqueous phase was further extracted
with CH2Cl2. Combined CH2Cl2 was dried over MgSO4 and
concentrated. The resulting amine was used in the next
reaction without further purification.
To the amine derived from hydrolysis of carbamate 30 above
(0.07 g, 0.18 mmol) dissolved in 5 mL of DMF were added
4-pyridylacetic acid (0.05 g, 0.28 mmol), HOBT (0.05 g, 0.37
mmol), DEC (0.071 g, 0.37 mmol), and N-methyl morpholine
(0.093 g, 0.1 mL, 6.5 mmol), and the mixture was stirred at
room temperature for 16 h. The organic phase was washed
with saturated NaHCO3 and brine and then dried over Na2-
SO4. It was then concentrated and purified on normal phase
flash chromatography eluting with 3% MeOH (saturated with
ammonia)-CH2Cl2 gradient to afford 0.029 g of tricyclic
υ
max 996, 1114, 1228, 1434, 1478, 1696, 2860, 2908, 2978, 3447
cm-1; MS m/z (rel intensity) 509.0 (100, MH+). Further elution
with ethyl acetate only gave the 3-phenyl carbamate 27 in 16%
yield (1.1 g): 1H NMR (200 MHz, CDCl3) δ 1.25 (t, J ) 7.5 Hz,
3H), 2.25-2.60 (m, 4H), 2.70-2.95 (m, 2H), 3.05-3.25 (m, 2H),
3.30-3.50 (m, 2H), 3.70-3.95 (m, 2H), 4.15 (q, J ) 7.5 Hz,
2H), 7.00-7.70 (m, 9H), 8.60 (d, J ) 2.5 Hz, 1H); MS m/z (rel
intensity) 459.3 (100, MH+).
1
pyridylacetamide 31 (32% yield): mp 93.6-94.1 °C; H NMR
(200 MHz, CDCl3) δ 2.13-2.67 (m, 4H), 2.76-2.87 (m, 1H),
2.89-2.99 (m, 1H), 3.20-3.45 (m, 4H), 3.57-3.71 (m, 1H), 3.75
(s, 2H), 3.98-4.14 (m, 1H), 7.03-7.24 (m, 5H), 7.68 (s, 1H),
8.55 (d, J ) 5.0 Hz, 2H), 8.67 (d, J ) 10.0 Hz, 1H); MS m/z
(rel intensity) 498.16 (100, MH+); HRMS (FAB) calcd for
C29H23N3F3OCl (MH+) 498.1560, found 498.1551.
4-(8-C h lo r o -3-i o d o -5,6-d i h y d r o -11H -b e n z o [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (28). Reaction was carried out essentially in the
same way as described for the preparation of compound 22
above but replacing 3-bromo carbamate 21 with the 3-iodo
carbamate 26. Purification on normal phase HPLC (silica gel)
eluting with 8% MeOH (saturated with ammonia)-CH2Cl2
afforded 3-iodo tricyclic pyridylacetamide 28 in 66% yield: 1H
NMR (200 MHz, CDCl3) δ 2.12-2.52 (m, 4H), 2.69-2.88 (m,
2H), 3.12-3.40 (m, 4H), 3.53-3.70 (m, 1H), 3.74 (s, 2H), 3.93-
4-(8-Ch lor o-3-ter t-b u t yl-5,6-d ih yd r o-11H -b en zo[5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e (33). Coupling of amine 3219 with 4-pyridylacetic
acid was accomplished in essentially the same manner as
described in the preparation of compound 22 above. Purifica-
tion by flash chromatography on silica gel first eluting with
3% MeOH (saturated with ammonia)-CH2Cl2 yielded the
3-tert-butyl tricyclic pyridylacetamide 33 in 52% yield: 1H
NMR (400 MHz, CDCl3) δ 1.3 (s, 9H), 2.20-2.70 (m, 4H), 2.80-

4300 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Njoroge et al.
2.95 (m, 2H), 3.20-3.45 (m, 4H), 3.60-3.75 (m, 1H), 3.80 (s,
2H), 4.05-4.25 (m, 1H), 7.10-7.30 (m, 5H), 7.45 (br s, 1H),
8.45-8.60 (m, 3H); MS m/z (rel intensity) 486.1 (100, MH+);
HRMS (EI) calcd for C30H32N3OCl (M+) 485.2234, found
485.2229.
by measuring transfer of [3H]farnesyl from [3H]farnesyl pyro-
phosphate to trichloroacetic acid-precipitable Ha-Ras-CVLS as
previously described.21 GGPT-1 activity was similarly deter-
mined using [3H]geranylgeranyl diphosphate and Ha-Ras-
CVLL as substrates.21
4-(8-C h lo r o -3-m e t h y l-5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(3-pyr idin ylacetyl)-
p ip er id in e (35). Coupling of amine 3420 with 3-pyridylacetic
acid was accomplished in essentially the same manner as
described in the preparation of compound 22 above. Purifica-
tion by flash chromatography on silica gel first eluting with
4% MeOH (saturated with ammonia)-CH2Cl2 yielded the
3-methyl tricyclic pyridylacetamide 35 in 99% yield: 1H NMR
(200 MHz, CDCl3) δ 2.20-2.52 (m, 4H), 2.28 (s, 3H), 2.69-
2.90 (m, 2H), 3.07-3.45 (m, 4H), 3.62-3.79 (m, 1H), 3.72 (s,
2H), 3.99-4.20 (m, 1H), 7.02-7.32 (m, 5H), 7.58-7.67 (m, 1H),
8.23 (br s, 1H), 8.44-8.54 (m, 2H); IR (film) υmax 713, 996, 1208,
1272, 1384, 1445, 1479, 1641, 2862, 2918, 3432 cm-1; MS m/z
(rel intensity) 443.25 (18), 444.20 (100, MH+), 445.20 (33),
446.20 (36). Anal. (C27H26N3OCl‚1.2H2O) C, H, N.
4-P yr id yla cetic Acid N-Oxid e (37). Ethyl 4-pyridylac-
etate (6.23 g, 38 mmol) was dissolved in 50 mL of CH2Cl2 under
N2 atmosphere, and the reaction mixture was cooled to - 20
°C with stirring. m-Chloroperbenzoic acid (19.52 g, 110 mmol)
was added to the reaction mixture over a period of 0.5 h. The
reaction mixture was stirred for another 1.0 h and then at 25
°C for 16 h. It was further diluted with CH2Cl2 and washed
with sodium bisulfite and 1 N NaOH. The organic phase was
dried over MgSO4 and concentrated. The resulting semisolid
was purified on silica gel flash chromatography eluting with
10% EtOH-CH2Cl2 to afford 1.05 g of ethyl 4-pyridylacetate
N-oxide (15% yield): 1H NMR (200 MHz, CDCl3) δ 1.30 (t, J
) 7.5 Hz, 3H), 3.60 (s, 2H), 4.20 (q, J ) 7.5 Hz, 2H), 7.25 (d,
J ) 6.0 Hz, 2H), 8.55 (d, J ) 6.0 Hz, 2H); MS m/z (rel intensity)
182 (100, MH+).
Cellu la r Assa ys for In h ibition of Ha -Ra s P r ocessin g
a n d Tr a n sfor m in g F u n ction . Inhibition of intracellular
processing of H-Ras by inhibitors was measured in transfected
COS cells as described previously.21
Cell Lin es for in Vivo Stu d ies. The PT-24 cell line is
derived from BALB c/3T3 cells transfected with activated Ha-
Ras-CVLS. NIH3T3 cells transfected with activated Ha-Ras
containing its native C-terminal sequence (CVLS) or an altered
C-terminal sequence (CVLL) were constructed by removing
H-ras (the coding sequences) from the pSV Sport expression
vectors by restriction digestion.21 These fragments were
incubated with the Klenow fragment of DNA polymerase I and
subcloned into pOPRSVI (Stratagene) by standard methods.23
The resulting pOPRSVI-H-ras-CVLS and pOPRSVI-H-ras-
CVLL plasmids were transfected into NIH3T3 cells using
Lipofectamine (GIBCO-BRL) under the conditions suggested
by the manufacturer. G-418-resistant clones were isolated and
screened for morphological transformation and their ability
to grow in soft agar. NIH3T3 cells transfected with activated
Ki-Ras containing its native C-terminal sequence of CVIM
were similarly constructed. MSV-3T3 cells are NIH3T3 cells
transfected with the mos oncogene. The human colon carci-
noma DLD-1 cell line was obtained from American Type
Culture Collection (Rockville, MD).
In Vivo Effica cy Stu d ies. All animal studies were carried
out in the animal facility of Schering-Plough Research Insti-
tute in accordance with institutional guidelines. All animals
were maintained in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals.
Experimental protocols were reviewed by and the experimental
progress was supervised by the Schering Plough Animal Care
and Use Committee. After 1 week of acclimation, 5-7-week-
old female nude mice (Crl:Nu/Nu-nu Br; Charles River Labo-
ratories, Wilmington, MA) were subcutaneously inoculated
with various cell lines on day 0. The number of cells inoculated
was 3.0 × 105 for NIH3T3 Ki-Ras-CVIM, 2 × 106 for NIH3T3
Ha-Ras-CVLL and CVLS, 3 × 106 for MSV-3T3, and 5 × 106
for DLD-1 and PT-24. Animals were randomly assigned to
control and treatment groups (10 animals/group) before the
first treatment. Drug treatment at either 10 or 50 mg/kg was
initiated on day 1. Compound 38 was dissolved in 20% (w/v)
hydroxypropyl-â-cyclodextrin (HPâCD). Vehicle controls re-
ceived 20% HPâCD. Vehicle or drug solution (0.1 mL) was
administered by oral gavage every 6 h (q.i.d.) for 14-21 days.
A no-treatment control was always included along with the
vehicle control to evaluate the influence of vehicle and of the
q.i.d. gavage treatment. Once palpable, tumor volume was
measured in three dimensions twice weekly and calculated
with the formula of V ) 1/6πLWT, where L, W, and T represent
length, width, and thickness, respectively.21 T/C value in
percent was calculated for each measurement, where T and C
are the median tumor volume of the treated and control
groups, respectively. Average growth inhibition was used to
compare efficacy of various treatments and was derived by
subtracting the average T/C values of each treatment from 100.
Single-tailed Student’s t-test was used for statistical analysis.
Ethyl 4-pyridylacetate N-oxide (0.54 g, 3 mmol) was dis-
solved in 10 mL of EtOH under N2 atmosphere. Lithium
hydroxide (0.5 g, 12 mmol, dissolved in 12 mL of H2O) was
added, and the reaction mixture stirred at room temperature
for 16 h. The organic solvents were then removed, and the
resulting semisolid was neutralized with 20 mL of 1 N HCl.
Volatiles were then removed in vacuo, and the resulting solid
was washed with EtOH. Combined EtOH fractions were then
concentrated to give 0.54 g of compound 37: 1H NMR (200
MHz, DMSO) δ 3.50 (s, 2H), 7.35 (d, J ) 6.5 Hz, 2H), 8.20 (d,
J ) 6.5 Hz, 2H).
4-(3-B r o m o -8-c h lo r o -5,6-d ih y d r o -11H -b e n zo [5,6]-
cycloh epta[1,2-b]pyr idin -11-yliden e)-1-(4-pyr idin ylacetyl)-
p ip er id in e N1-Oxid e (38). Reaction was carried out essen-
tially in the same way as described for the preparation of
compound 22 above but replacing pyridineacetic acid with
pyridineacetic acid N-oxide (37). Purification on normal phase
HPLC (silica gel) eluting with 3% MeOH (saturated with
ammonia)-CH2Cl2 afforded 3-bromo tricyclic pyridylacetamide
1
N-oxide 38 in 63% yield: H NMR (200 MHz, CDCl3) δ 2.21-
2.43 (m, 3H), 2.46-2.63 (m, 1H), 2.72-2.97 (m, 2H), 3.17-
3.46 (m, 4H), 3.57-3.81 (m, 3H), 3.89-4.14 (m, 1H), 7.03-
7.23 (m, 5H), 7.61 (d, J ) 2.5 Hz, 1H), 8.18 (d, J ) 7.5 Hz,
2H), 8.40-8.50 (m, 1H); IR (film) υmax 807, 994, 1176, 1284,
1439, 1478, 1637 cm-1; MS m/z (rel intensity) 526.6 (100,
MH+). Anal. (C26H23N3O2BrCl‚0.6CH2Cl2‚2H2O) C, H, N.
4-(8-Ch lor o-5,6-d ih yd r o-11H-ben zo[5,6]cycloh ep ta [1,2-
b]p yr id in -11-ylid en e)-1-(4-p yr id in yla cetyl)p ip er id in e N1-
Oxid e (40). Reaction was carried out essentially in the same
way as described for the preparation of compound 22 above
by coupling amine 3920 with pyridineacetic acid N-oxide (37).
Purification on normal phase (silica gel) eluting with 3-5%
MeOH (saturated with ammonia)-CH2Cl2 afforded the aceta-
mide pyridyl N-oxide 40 in 17% yield: mp 129-130 °C; 1H
NMR (200 MHz, CDCl3) δ 2.22-2.45 (m, 4H), 2.46-2.64 (m,
1H), 2.72-2.98 (m, 2H), 3.16-3.47 (m, 4H), 3.57-3.78 (m, 2H),
3.92-4.16 (m, 1H), 7.06-7.24 (m, 6H), 7.41-7.52 (m, 1H), 8.15
(d, J ) 5.0 Hz, 2H), 8.36-8.48 (m, 1H); IR (film) υmax 805, 994,
1172, 1249, 1437, 1640, 3438; MS m/z (rel intensity) 446.2 (100,
MH+). Anal. (C26H24N3O2Cl‚0.5H2O‚0.3CH2Cl2) C, H, N.
In Vitr o En zym e Assa ys. FPT activity was determined
P h a r m a cok in etic Stu d ies. Nude mice were also used to
study the pharmacokinetic properties of the tricyclic inhibitors.
Blood samples were collected at nine time points (2 min, 5 min,
15 min, 30 min, 1 h, 2 h, 4 h, 7 h, and 24 h) after a single oral
or intravenous (tail vein) dose of 25 mg/kg inhibitor in 20%
HPâCD. Two mice were used for each time point, and samples
were collected by cardiac puncture after euthanasia with
carbon dioxide. After clotting on ice, serum was isolated by
centrifugation. Quantitation of inhibitor serum levels was
achieved using acetonitrile precipitation followed by high-
performance liquid chromatography-atmospheric pressure
chemical ionization (APCI) tandem mass spectrometry.
A
detailed description of the analytical methodology has been
described for an earlier analogue in this series.24

SAR of FPT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4301
J ames, L.; King, I.; Li, Z.; Lin, C.-C.; Petrin, J .; Remiszewski,
S. R.; Taveras, A. G.; Wang, S.; Wong, J . K.; Catino, J .;
Girijavallabhan, V.; Ganguly, A. K. Antitumor 8-chlorobenzo-
Refer en ces
(1) (a) Barbacid, M. Ras genes. Annu. Rev. Biochem. 1987, 56, 779.
(b) Bourne, H. R.; Saunders, D. A.; McCormick, F. The GTPase
superfamily:
Nature 1990, 348, 125.
cycloheptapyridines:
a new class of selective, nonpeptidic,
a conserved scwith for diverse cell functions.
nonsulfhydryl inhibitors of Ras farnesylation. Bioorg. Med.
Chem. 1997, 5, 93.
(2) (a) Leftheris, K.; Kline, T.; Vite, G. D.; Cho, Y. H.; Bhide, R. S.;
Patel, D. V.; Patel, M. M.; Schmidt, R. J .; Weller, H. N.;
Andahazy, M. L.; Carboni, J . M.; Gullo-Brown, J . L.; Lee, F. Y.
F.; Ricca, C.; Rose, W. C.; Yan, N.; Barbacid, M.; Hunt, J . T.;
Meyers, C. A.; Seizinger, B. R.; Zahler, R.; Manne, V. Develop-
ment of highly potent inhibitors of Ras farnesyltransferase
possessing cellular and in vivo activity. J . Med. Chem. 1996,
39, 224. (b) Gibbs, J . B.; Oliff, A.; Kohl, N. E. Farnesyltransferase
inhibitors: Ras research yields a potential cancer therapeutic.
Cell 1994, 77, 175 (c) Gibbs, J . B Ras C-terminal processing
enzymesnew drug targets? Cell 1991, 65, 1.
(3) Casey, P. J .; Solski, P. A.; Der, C. I.; Buss, J . E. p21 Ras is
modified by a farnesyl isoprenoid. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 8223.
(4) Graham, S. L.; deSolms, S. J .; Giuliani, E. A.; Kohl, N. E.;
Mosser, S. D.; Oliff, A. I.; Pompliano, D. L.; Rands, E.; Breslin,
M. J .; Deana, A. A.; Garsky, V. M.; Scholz, T. H.; Gibbs, J . B.;
Smith, R. L. Pseudopeptide inhibitors of Ras farnesyl-protein
transferase. J . Med. Chem. 1994, 37, 725.
(5) deSolms, S. J .; Deana, A. A.; Giuliani, E. A.; Graham, S. L.; Kohl,
N. E.; Molsser, S. D.; Oliff, A. I.; Pompliano, D. L.; Rands, E.;
Scholz, T. H.; Wiggins, J . M.; Gibbs, J . B.; Smith, R. L.
Pseudodipeptide inhibitor of protein farnesyltransferase. J . Med.
Chem. 1995, 38, 3967.
(6) Singh, S. B.; Lingham, R. B. Farnesyl-protein transferase
inhibitors in early development. Exp. Opin. Invest. Drugs 1996,
5, 1589.
(7) J ames, G. L.; Goldstein, J . L.; Brown, M. S.; Rawson, T. E.;
Somers, T. C.; McDowell, R. S.; Crowley, C.; Lucas, B.; Levinson,
A.; Marsters, J . C. Benzodiazepine peptomimetics: potent
inhibitors of Ras farnesylation in animal cells. Science 1993, 260,
1937.
(8) (a) Nagasu, T.; Yoshimatsu, K.; Rowell, C.; Lewis, M. D.; Garcia,
A. M. Inhibition of human tumor xenograft growth by treatment
with the farnesyl transferase inhibitor B956. Cancer Res. 1995,
55, 5310. (b) Kohl, N. E.; Wilson, F. R.; Mosser, S. D.; Giuliani,
E.; DeSolms, S. J .; Conner, M. W.; Anthony, N. J .; Holtz, W. J .;
Gomez, R. P.; Lee, T.-J .; Smith, R. L.; Graham, S. L.; Hartman,
G. D.; Gibbs, J . B.; Oliff, A. Protein farnesyltransferase inhibitors
block the growth of ras-dependent tumors in nude mice. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 9141.
(12) Schumacher, D. P.; Murphy, B. L.; Clark, J . E.; Tahbaz, P.;
Mann, T. A. Superacid cyclodehydration of ketones in the
production of tricyclic antihistamines. J . Org. Chem. 1989, 54,
2242.
(13) Ishikawa, F.; Saegusa, J .; Inamura, K.; Sakuma, K.; Ashida, S.-
I. Cyclic guanidines: 17. Novel (N-substituted amino)imidazole-
[2,1-b]quinazolin-2-ones: water-soluble platelet aggregation
inhibitors. J . Med. Chem. 1985, 28, 1387.
(14) Burawoy, A.; Turner, C. o-Mercapto-azo-compounds. Part III.
action of thiocyanic acid on diazotized o-nitroarylamines. J .
Chem. Soc. 1953, 959.
(15) Craig, C. C. A study of the preparation of alpha-pyridyl halides
from alpha-aminopyridine by the diazo reaction. J . Am. Chem.
Soc. 1934, 56, 231.
(16) Korzeniowski, S. T.; Gokel, G. W. Crown-cation complex effects.
IX. A phase transfer catalytic synthesis of bromo- and iodoare-
nes. Tetrahedron Lett. 1977, 40, 3519.
(17) Chen, Q.-Y.; Wu, S.-W. Methyl fluorosulphonyldifluoroacetate;
a new trifluoromethylating agent. J . Chem. Soc., Chem. Com-
mun. 1989, 705.
(18) McClinton, M. A.; McClinton, D. A. Trifluoromethylation and
related reactions in organic chemistry. Tetrahedron 1992, 48,
6555.
(19) Piwinski, J . J .; Green, M. J .; Wong, J . K. Bis-benzo or benzopy-
ridocycloheptapiperidene, piperidylidene and piperazine com-
pounds, composition and methods of use. U.S. Patent 5,422,351,
1995.
(20) Wong, J .; Piwinski, J . J .; Green, M. J .; Ganguly, A. K.; Anthes,
J . C.; Billah, M. M. Dual antagonists of platelet activating factor
and histamine. 2. Pyridine ring substitution of N-acetyl-4-(8-
chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-
ylidene)piperidine. Bioorg. Med. Chem. Lett. 1993, 3, 1073.
(21) Bishop, W. R.; Bond, R.; Petrin, J .; Wang, L.; Patton, R.; Doll,
R.; Njoroge, G.; Catino, J .; Shwartz, J .; Carr, D.; J ames, L.;
Kirschmeier, P. Novel tricyclic inhibitors of farnesyl transferase.
J . Biol. Chem. 1995, 270, 30611.
(9) Njoroge, F. G.; Doll, R. J .; Vibulbhan, B.; Alvarez, C. S.; Bishop,
W. R.; Petrin, J .; Kirschmeier, P.; Carruthers, N. I.; Wong, J .
K.; Albanese, M. A.; Piwinski, J . J .; Catino, J .; Girijavallabhan,
V.; Ganguly, A. K. Discovery of novel nonpeptide tricyclic
inhibitors of Ras farnesyl protein transferase. Bioorg. Med.
Chem. 1997, 5, 101.
(10) Njoroge, F. G.; Vibulbhan, B.; Alvarez, C. S.; Bishop, W. R.;
Petrin, J .; Doll, R. J .; Girijavallabhan, V.; Ganguly, A. K. Nove
tricyclic aminoacetyl and sulfonamide inhibitors of Ras farnesyl
protein transferase. Bioorg. Med. Chem. Lett. 1996, 6, 2977.
(11) Mallams, A. K.; Njoroge, F. G.; Doll, R. J .; Snow, M. E.;
Kaminiski, J . J .; Rossman, R. R.; Vibulbhan, B.; Bishop, W. R.;
Kirschmeier, P.; Liu, M.; Bryant, M. S.; Alvarez, C. S.; Carr, D.;
(22) Masci, B. Effect of crown ethers on the selectivity of electrophilic
aromatic nitrations. J . Org. Chem. 1985, 50, 4081.
(23) Sambrook, J .; Fritsch, E. F.; Maniatis, T. Molecular cloning: A
Laboratory Manual, 2nd ed.; Cold Spring Harbor Press: Cold
Spring Harbor, NY, 1989; p 11724.
(24) Bryant, M. S.; Korfmacher, W. A.; Wang, S.; Nardo, C.; Nomeir,
A. A.; Lin, C.-C. Pharmacokinetic screening for selection of new
drug discovery candidates is greatly enhanced through the use
of liquid chromatography-atmospheric pressure ionization tan-
dem mass spectrometry. J . Chromatogr. 1997, 777, 61.
J M970464G