Macrocyclic piperazinones as potent dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I

Article abstract of DOI:10.1016/j.bmcl.2003.11.051

A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.

Full text of DOI:10.1016/j.bmcl.2003.11.051

Bioorganic & Medicinal Chemistry Letters 14 (2004) 639–643
Macrocyclic piperazinones as potent dual inhibitors of
farnesyltransferase and geranylgeranyltransferase-I
Christopher J. Dinsmore,a,* C. Blair Zartman,a Jeffrey M. Bergman,a Marc T. Abrams,b
Carolyn A. Buser,b J. Christopher Culberson,c Joseph P. Davide,b
Michelle Ellis-Hutchings,b Christine Fernandes,b Samuel L. Graham,a
George D. Hartman,a Hans E. Huber,b Robert B. Lobell,b Scott D. Mosser,b
b
a
RonaldG. Robinson andTheresa M. Williams
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
Received29 September 2003; accepted18 November 2003
Abstract—A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory
activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for
the binding mode of compound 3o in FPTase is proposed.
# 2003 Elsevier Ltd. All rights reserved.
There has been considerable interest in protein pre-
nyltransferase inhibitors over recent years, due to the
importance of Ras proteins in cell growth andoncogen-
esis,1 coupledwith the observation that Ki-Ras requires
prenylation for biological function.2 Selective farnesyl-
transferase (FPTase) inhibitors show promising activity
in preclinical andclinical studies, 3 but they are incapable
of blocking the function of Ki-Ras, since they fail to
inhibit its alternative geranylgeranylation by geranylger-
anyl-transferase-I (GGPTase-I). While non-Ras pro-
teins may therefore be important for the biological
effects of FPTase inhibitors,4 blocking Ki-Ras function
remains an important strategy in cancer research. Dual
FPTase-GGPTase-I inhibitors (FTI-GGTIs) have been
shown to inhibit the prenylation of Ki-Ras in cell cul-
ture andin animals, albeit with a narrow in vivo ther-
apeutic index.5
macrocyclic constraint (Fig. 1).6,7 Macrocycle
emergedas a highly potent FTI with only moderate
2
activity versus GGPTase-I. Unexpectedly, whereas 1
inhibits GGPTase-I in a GGPP-competitive manner,8
constrained 2 was foundto be protein substrate-com-
petitive, suggesting different GGPTase-I binding modes
for 1 and 2.6 By replacing the naphthyl ring in 2 with a
substitutedbenzyl group, we have discovereda strategy
for modulating the relative prenyltransferase inhibitory
activities in this series. Here, we describe the synthesis
andbiological activities of macrocyclic compounds ( 3)
with dramatically increased GGPTase-I inhibition,
leading to highly potent macrocyclic dual FTI-GGTIs.9
We recently discovered that N-arylpiperazinone FTI-
GGTIs such as compound 1 (L-778,123) bindto
FPTase in a folded conformation, and that potency and
selectivity enhancements are achievedby enforcing a
Figure 1. Piperazinone prenyltransferase inhibitors.
Compounds 3a–s (Table 1) in this study were prepared
6,10
Keywords: Inhibitor; Farnesyltransferase; Geranylgernyltransferase.
* Corresponding author. Tel.: +215-652-8257; fax: +215-652-7310;
e-mail: christopher_dinsmore@merck.com
in analogy to recently reportedsyntheses.
nol 4 was convertedin four steps to piperazinone
The phe-
6
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.051

640
C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 14 (2004) 639–643
Table 1. FPTase and GGPTase-I inhibition data and cell culture activity for compounds 1, 2, and 3a–s
Compds
R (in 3)
FPTase
in vitro
IC50 (nM)a
GGPTase-I
in vitro
IC50 (nM)b
GGTase-I
Slope log(IC50
FPTase binding
in cell culture
IC50 (nM)e
Rap1a processing
in cell culture
MIC (nM)f
)
vs log[GGPP]d
1
—
—
H
F
Cl
Br
2
0.1
2.2
<1
<1
0.45
0.26
0.66
0.1
98
301
3780
2750
450
100
502
124
107
97
1.42
ꢀ0.44
0.08
3.6
0.29
3.3
nd
nd
10,000
nd
1.2
1000
0.44
1000
10,000
30,000
(+)-2
3a
3b
3c
3d
3e
3f
3g
3h
nd1
nd0.30
nd1.1
nd0.33
ꢀ0.16
nd0.59
ꢀ0.12
Methyl
Ethyl
Allyl
n-Propyl
1000
1000
0.31
3i
0.5
38
212
55 (135)c
2.4 (26)c
534
nd1.4
3000
3j
0.32
10
1000
300
3k
364
645
0.66
19
0.68
170
3l
ndnd nd
nd2.2
0.48
3m
3n
238
10,000
28
2.1 (18)c
1.7
100
30
3o
1.6
6.8
0.5
24
ꢀ0.25
nd40
0.25
5.7
(ꢁ)-3p
(ꢁ)-3q
3r
1050
nd
46 (36)c
420
0.86
3000
30
<300
nd20
0.28
3s
20
43
100
a Concentration requiredto reduce the human FPTase-catalyzedincorporation of [ 3H]FPP into recombinant Ras-CVIM by 50%.11a
b Concentration requiredto reduce the human GGPTase-I-catalyzedincorporation of [ 3H]GGPP into biotinylated peptide corresponding to the C-
8a
terminus of human Ki-Ras by 50%. Assay run with 30 min preincubation of enzyme andinhibitor in the presence of 5 mM ATP.
c In parentheses: same as footnote b without prior incubation of enzyme andinhibitor.
d Qualitative measure of GGPP competition, with slopes of 0 and1 indicating noncompetitive andcompetitive binding, respectively. IC
were determined at various GGPP concentrations in the presence of 5 mM ATP.8a
values
50
e Concentration required to displace 50% of a radiolabeled FTI from FPTase in cultured Ha-ras transformedRAT1 cells. 11b
8a
f Minimum concentration of compoundrequiredto inhibit Rap1a processing in PSN-1 cells.
(Scheme 1), which was reductively alkylated with alde-
hyde 7 to give the cyclization precursor 8. Intramole-
cular SNAr reaction provided 3d. Compounds 3a–c were
preparedin an analogous fashion. The bromide 3d was
used in palladium-mediated cross-coupling reactions to
afford, with additional straightforward transformations,
3e–s.
Assessment of the in vitro inhibitory activities for com-
pounds (Table 1) revealeda clear trendrelating
increasedsubstituent size (R in 3) to enhancedpotency
versus GGPTase-I, without significant effects on
FPTase inhibition. This was true of both halogen sub-
stituents (3a, 3b–d) andsimple alkyl groups ( 3a, 3e–h).
The addition of bulky aliphatic substituents to a series

C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 14 (2004) 639–643
641
of alkynes (3i–3k) enhancedGGPTase-I inhibition and
decreased potency versus FPTase, providing selective
GGTI 3k (IC50 2.4 nM), which displayed slow tight
binding to GGPTase-I. These results suggest that the
GGPTase-I active site is better able to accommodate
bulky hydrophobic groups in this region of the inhibitor
than is FPTase. The phenylacetylide 3l was poorly tol-
eratedby both enzymes. Potent dual FPTase-GGPTase-
I inhibition couldbe attainedby removing the acetylene
conformational constraint (3m–o), with the 2-(cyclo-
hexyl)ethyl FTI-GGTI 3o being the most potent overall
(IC50 FPTase 1.6 nM, GGPTase-I 1.7 nM). The incor-
poration of polar functionality causededcreases in
inhibitory activity for both FPTase andGGPTase-I ( 3p,
3r), but this effect was attenuated by the addition of
aliphatic functionality (3q versus 3p, 3s versus 3r).
A preliminary kinetic analysis of GGPTase-I inhibition
by compounds was undertaken. A plot of log (IC50)
versus log [GGPP] provided a qualitative assessment of
inhibitory mechanism8a (Table 1), andindicatedthat
compounds of structure 3 spannedthe range from not
competitive with respect to GGPP (presumably compe-
titive with respect to protein substrate) to mixedtype.
Interestingly, while the selective GGTI 3k displayed
mixedtype inhibition (slope 0.68), the most potent FTI-
GGTI 3o was not competitive with GGPP
Scheme 1. Reagents andconditions: (a) MsCl, Et 3N, DCM, 0 ꢂC; (b)
NBS, AIBN, CCl4, reflux, 61% for 2 steps; (c) N-Boc-piperazinone,
NaH, DMF, 0 ꢂC, 92%; (d) HCl, EtOAc, 0 ꢂC, 98%; (e) 7, Na(A-
cO)3BH, 4 A sieves, DCE, 69% (f) Cs2CO3, 0.05 M DMSO, 80 ꢂC,
74%; (g) Me4Sn, PdBn(PPh3)2Cl, HMPA, 70 ꢂC, 72%; (h) R0CꢃCH,
Pd(PPh3)2Cl2, CuI, Et3N, DMF, 90 ꢂC; (i) H2, Pd/C, EtOH; (j) allyl-
SnBu3, Pd(PPh3)4, DMF, 110 ꢂC, 99%; (k) CH2N2, Pd(OAc)2,
.
CHCl3, 9%; (l) K2OsO4 2H2O, K3Fe(CN)6, K2CO3, quinuclidine, 1:1
t-BuOH:H2O, 0 ꢂC, 68%; (m) Acetone, CSA, reflux, 100%; (n) CO,
Pd(OAc)2, dppp, Et3N, DMSO, 110 ꢂC; (Me3Si)CHN2, 35%; (o) 2-
.
adamantyl-NH2, EDC HCl, HOBt, DMF, 81%.
Table 2. Inhibition of protein prenylation in PSN-1 cells by 1,2, and 3o,q
Entry
Compds
Protein processing EC50 (nM)a
Rap1a
EC50 ratio
Ki-Ras/HDJ2
HDJ2
Ki-Ras
1
2
3
4
5
1
(+)-2
(+)-2+GGTI
92
2
2b
12
10
6760
>10,000
nd90
6300
>10,000
68
>5000
47b
b
3o
3q
140
690
1065
1515
89
150
5
a Dose of compoundthat causedhalf-maximal inhibition of prenylation in PSN-1 cells, determinedby scanning immunoblots of whole cell extracts.
b Inhibition by (+)-2, adding 1 mM of a selective GGTI (EC50 HDJ2=23 mM; Rap1a=2 nM, Ki-Ras >100 mM).5 The resulting Ki-Ras/HDJ2 ratio
reflects the optimal balance of activities for inhibition of Ki-Ras.
12
.
Figure 2. ProposedFPTase 3o FPP ternary complex structure in cross-eyedstereoview, basedon molecular modeling.
.
Inhibitor 3o is colored
green, the farnesyl group of FPP is magenta, the Zn ion is purple, Zn-ligating residues are cyan, and several key FTase residues are colored gray.

642
C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 14 (2004) 639–643
(slope=ꢀ0.25), despite close structural similarity
Ramjit, C. W. Ross III, B.-L. Wan, andM. M. Zrada
for analytical support.
between the two compounds.
The activities of compounds in cell culture were con-
sistent with their in vitro potencies (Table 1). IC50
values determined in an FPTase in-cell enzyme
occupancy assay were generally within 2–4-foldof the
corresponding in vitro inhibition IC50s, indicating high
cell permeability. The minimal concentrations required
to detect the unprocessed form of the specifically ger-
anylgeranylatedprotein Rap1a roughly correlatedwith
the in vitro GGPTase-I IC50s. In a more detailed study,
the relative FPTase andGGPTase-I inhibition activities
of FTI-GGTIs 3o and 3q were assessedin a uniform cell
background, and compared to the activities of 1 and 2
(Table 2). As detailed elsewhere,5 1 inhibitedthe pro-
cessing of farnesylatedandgeranylgeranylatedproteins
(HDJ2 andRap1a, respectively), andwas therefore able
to suppress the prenylation of Ki-Ras (entry 1). The
more potent FTI 2 was unable to block Ki-Ras pre-
nylation due to insufficient GGTase-I inhibition (entry
2). However, the addition of high concentration of a
potent GGTI provided the necessary inhibition to
observe unprenylatedKi-Ras (entry 3). This experiment
indicated the maximum achievable level of Ki-Ras inhi-
bition by a dual FTI-GGTI with a given level of FPTase
potency, andthus the optimal balance of these enzyme
activities for Ki-Ras inhibition (Ki-Ras/HDJ2 EC50
ratio ꢄ47).5 Compound 3o (entry 4) was shown to
be more potent at inhibiting the prenylation of all
three proteins relative to 1, andis a closely balanced
FTI-GGTI (ratio ꢄ89). Compound 3q is also more
potent than 1, but is slightly excessive in its FTI activity
(entry 5).
References and notes
1. Gibbs, J. B.; Oliff, A.; Kohl, N. E. Cell 1994, 77, 175.
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5. Lobell, R. B.; Omer, C. A.; Abrams, M. T.; Bhim-
nithwala, H. G.; Brucker, M. J.; Buser, C. A.; Davide,
J. P.; deSolms, S. J.; Dinsmore, C. J.; Ellis-Hutchings,
M. S.; Kral, A. M.; Liu, D.; Lumma, W. C.; Machotka,
S. V.; Rands, E.; Williams, T. M.; Graham, S. L.; Hart-
man, G. D.; Oliff, A. I.; Heimbrook, D. C.; Kohl, N. E.
Cancer Res. 2001, 61, 8758.
6. Dinsmore, C. J.; Bogusky, M. J.; Culberson, J. C.; Berg-
man, J. M.; Homnick, C. F.; Zartman, C. B.; Mosser,
S. D.; Schaber, M. D.; Robinson, R. G.; Koblan, K. S.;
Huber, H. E.; Graham, S. L.; Hartman, G. D.; Huff, J. R.;
Williams, T. M. J. Am. Chem. Soc. 2001, 123, 2107.
7. Dinsmore, C. J.; Bell, I. M. Curr. Topics Med. Chem.
2003, 3, 1075.
8. (a) Huber, H. E.; Robinson, R. G.; Watkins, A.; Nahas,
D. D.; Abrams, M. T.; Buser, C. A.; Lobell, R. B.;
Patrick, D.; Anthony, N. J.; Dinsmore, C. J.; Graham,
S. L.; Hartman, G. D.; Lumma, W. C.; Williams, T. M.;
Heimbrook, D. C. J. Biol. Chem. 2001, 276, 24457. (b)
Buser, C. A.; Dinsmore, C. J.; Fernandes, C.; Greenberg,
I.; Hamilton, K.; Mosser, S. D.; Walsh, E. S.; Williams,
T. M.; Koblan, K. S. Anal. Biochem. 2001, 290, 126.
9. (a) Recent reports of dual FTI-GGTIs: Bergman, J. M.;
Abrams, M. T.; Davide, J. P.; Greenberg, I. B.; Robinson,
R. G.; Buser, C. A.; Huber, H. E.; Koblan, K. S.; Kohl,
N. E.; Lobell, R. B.; Graham, S. L.; Hartman, G. D.;
Williams, T. M.; Dinsmore, C. J. Bioorg. Med. Chem.
Lett. 2001, 11, 1411. (b) Nguyen, D. N.; Stump, C. A.;
Walsh, E. S.; Fernandes, C.; Davide, J. P.; Ellis-Hutch-
ings, M.; Robinson, R. G.; Williams, T. M.; Lobell, R. B.;
Huber, H. E.; Buser, C. A. Bioorg. Med. Chem. Lett.
2002, 12, 1269. (c) Tucker, T. J.; Abrams, M. T.; Buser,
C. A.; Davide, J. P.; Ellis-Hutchings, M.; Fernandes, C.;
Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Huber,
H. E.; Liu, D.; Lobell, R. B.; Lumma, W. C.; Robinson,
R. G.; Sisko, J. T.; Smith, A. M. Bioorg. Med. Chem.
Lett. 2002, 12, 2027. (d) deSolms, S. J.; Ciccarone, T. M.;
MacTough, S. C.; Shaw, A. W.; Buser, C. A.; Ellis-
Hutchings, M.; Fernandes, C.; Hamilton, K. A.; Huber,
H. E.; Kohl, N. E.; Lobell, R. B.; Robinson, R. G.; Tsou,
N. N.; Walsh, E. S.; Graham, S. L.; Beese, L. S.; Taylor,
J. S. J. Med. Chem. 2003, 46, 2973.
A hypothesis for the binding mode of the macrocyclic
dual FTI-GGTI 3o in FPTase (Fig. 2) was derived from
molecular modeling studies based on published X-ray
crystal structures of relatedmacrocyclic inhibi-
12
.
.
tor FPTase FPP ternary complexes. As in related
complexes,12b the imidazole moiety is ligated to zinc, the
cyanophenyl group is stackedagainst the isoprenoid
chain of FPP, andthe benzylpiperazinone portion is in
contact with key hydrophobic residues. Notably, the
cyclohexylethyl group is angledtowarda large cavity in
the active site. Given that the mechanism of GGPTase-I
inhibition by 3o is not competitive with respect to
GGPP, it is possible that the inhibitory binding mode of
3o in GGPTase-I is qualitatively similar to the FPTa-
.
.
se 3o FPP complex.
In summary, several of the macrocyclic pre-
nyltransferase inhibitors prepared in this study demon-
strate dual FPTase and GGPTase-I inhibition.
Compound 3o potently inhibits both enzymes in cell
culture with relative activities that are near-optimally
balancedfor the inhibition of Ki-Ras prenylation.
10. Dinsmore, C. J.; Bergman, J. M.; Bogusky, M. J.; Cul-
berson, J. C.; Hamilton, K. A.; Graham, S. L. Org. Lett.
2001, 3, 865.
11. (a) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands,
E.; O’Hara, M. B.; Garsky, V. M.; Marshall, M. S.;
Pompliano, D. L.; Gibbs, J. B. J. Biol. Chem. 1991, 266,
14603. (b) Lobell, R.; Davide, J. P.; Kohl, N.; Burns, D.;
Eng, W.-S.; Gibson, R. J. Biomol. Screening 2003, 8, 430.
Acknowledgements
The authors wish to thank K. D. Anderson, P. A.
Ciecko-Steck, A. B. Coddington, G. M. Smith, H. G.

C. J. Dinsmore et al. / Bioorg. Med. Chem. Lett. 14 (2004) 639–643
643
12. (a) A model of the FPTase active site containing FPP was
derived from the 1LD8 pdb entry (ref 12b). Random
conformations of 3o were generatedusing the distance
geometry methodJG (S. Kearsley, Merck & Co., Inc.,
unpublished). These conformations were energy mini-
mizedin the static active site using Batchmin andthe
MMFFs force field, while restraining the Zn-ligating
nitrogen to its crystallographic position. The lowest
energy structure is shown. (b) Bell, I. M.; Gallicchio, S. N.;
Abrams, M.; Beese, L. S.; Beshore, D. C.; Bhimnathwala,
H.; Bogusky, M. J.; Buser, C. A.; Culberson, J. C.;
Davide, J.; Ellis-Hutchings, M.; Fernandes, C.; Gibbs,
J. B.; Graham, S. L.; Hamilton, K. A.; Hartman, G. D.;
Heimbrook, D. C.; Homnick, C. F.; Huber, H. E.; Huff,
J. R.; Kassahun, K.; Koblan, K. S.; Kohl, N. E.; Lobell,
R. B.; Lynch, J. J., Jr.; Robinson, R.; Rodrigues, A. D.;
Taylor, J. S.; Walsh, E. S.; Williams, T. M.; Zartman,
C. B. J. Med. Chem. 2002, 45, 2388.