3-Acyl-2,6-diaminopyridines as cyclin-dependent kinase inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2005.03.024

A novel series of 2,6-diamino-3-acylpyridines were designed and synthesized as cyclin-dependent kinase (CDK) inhibitors. The representative compounds 2r and 11 showed potent CDK1 and CDK2 inhibitory activities and inhibited cellular proliferation in HeLa,

Full text of DOI:10.1016/j.bmcl.2005.03.024

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2221–2224
3-Acyl-2,6-diaminopyridines as cyclin-dependent kinase
inhibitors: synthesis and biological evaluation
Ronghui Lin,* Yanhua Lu, Steven K. Wetter, Peter J. Connolly, Ignatius J. Turchi,
William V. Murray, Stuart L. Emanuel, Robert H. Gruninger, Angel R. Fuentes-Pesquera,
Mary Adams, Niranjan Pandey, Sandra Moreno-Mazza,
Steven A. Middleton and Linda K. Jolliffe
Johnson & Johnson Pharmaceutical Research & Development L.L.C., Drug Discovery, 1000 Route 202, Raritan,
New Jersey 08869, USA
Received 15 February 2005; revised 4 March 2005; accepted 7 March 2005
Abstract—A novel series of 2,6-diamino-3-acylpyridines were designed and synthesized as cyclin-dependent kinase (CDK) inhibi-
tors. The representative compounds 2r and 11 showed potent CDK1 and CDK2 inhibitory activities and inhibited cellular prolif-
eration in HeLa, HCT116, and A375 tumor cells.
Ó 2005 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs), such as CDK1,
CDK2, and CDK4, constitute a class of serine–threo-
nine protein kinases that plays an important role in regu-
lation of the cell cycle.1 Abnormal CDK control of the
cell cycle has been strongly linked to the molecular
pathology of cancer. CDKs have thus become attractive
therapeutic targets for cancer therapy.2 The CDKs regu-
late cell cycle progression through complexes with their
corresponding cyclin partners such as cyclin A, B, D,
and E. For example, CDK1 associated with cyclin B regu-
lates the cell cycle at the G2 and mitosis (cell division)
phases. CDK1 inhibitors could block mitosis entry
and arrest cell growth, and therefore may be useful ther-
apeutic agents with potentially fewer side effects than
conventional cytotoxic drugs targeting DNA synthesis.
387252 (1) are potent and selective CDK inhibitors
and anti-proliferative agents.5 To investigate the role
of the central heterocyclic core in the biological activity
of the acyl-diaminotriazoles, we designed a new series of
3-acyl-2,6-diaminopyridines (2) as potential CDK inhibi-
tors. Herein we report the synthesis of these novel com-
pounds and their biological evaluation as inhibitors of
CDK activity and tumor cell proliferation.
Synthesis of 3-acyl-N6-aryl-2,6-diaminopyridines (2)
unsubstituted at the 4-position is outlined in Scheme 1.
The key steps involved ortho-metalation–acylation of
an N,N-diprotected 2,6-diaminopyridine, followed by
nucleophilic substitution on activated aryl or heteroaryl
halides with the less sterically-hindered 6-amino group
of the deprotected 3-acyl-2,6-diaminopyridine products
(3). Specifically, ortho-metalation–acylation of 2,6-bis-
(pivaloylamino)pyridine (4)6 was achieved by first treat-
ing with n-butyl lithium at ꢀ78 °C in tert-butyl methyl
ether (MTBE) containing (N,N,N0,N0-tetramethyl)ethyl-
enediamine (TMEDA), followed by coupling with an
acyl chloride. Subsequent hydrolysis of the pivaloyl pro-
tecting groups with aqueous KOH afforded the 3-acyl-
2,6-diaminopyridines (3). Selective nucleophilic substitu-
tion of aryl iodides or bromides with the 6-amino group
of 3 was generally performed under palladium-catalyzed
conditions to give the desired compounds (2a–b, 2e, 2j–l,
and 2y).
Several CDK inhibitors have entered clinical evaluation
for the treatment of cancer.3 These include flavopiridol,
7-hydroxystaurosporine
(UCN-01),
roscovitine
(CYC202), and an aminothiazole compound (BMS-
387032).4 In our program to develop CDK inhibitors
as anti-cancer agents, we recently discovered that acyl-
substituted diaminotriazole derivatives such as RWJ-
Keywords: Synthesis; Cyclin-dependent kinase inhibitor; CDK inhibi-
tor; Cell proliferation inhibitor.
*
Corresponding author. Tel.: +1 908 704 5268; fax: +1 908 526
6465; e-mail: rlin@prdus.jnj.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.024

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R. Lin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2221–2224
Ar
Ar
O O
S
R
Ar
R′2N
O
O
F
F
a
O
O
O
S
O
H2N
N
NH2
N
H
N
NH2
H2N
N N
(21-80%)
Ar′
3a-e
N
H
N
NH2
2p-q, 2s-x
NH2
N
H
N
2
1
R′ = Me, Et, or Bn
F
F
O O
S
Ar
H2N
O
b, c
2q
O
a, b
Piv
Piv
N
H
N
NH2
Piv
Piv
N
N
N
(21%)
N
H
N
N
H
R′ = Bn
Ar = 2,6-F2-C6H3
H
H
(40-60%)
4
5
2r
Piv = pivaloyl
c
(60-80%)
Scheme 2. General synthetic route toward 2,6-diamino-3-acylpyridine
with benzenesulfonamide group. Reagents and conditions: (a) 4-I-
C6H4SO2NR02,9 Pd2(dba)3, BINAP, Cs2CO3, dioxane, 95 °C; (b) BBr3,
CH2Cl2, reflux, 6 h; (c) aqueous HI, HOAc, reflux, 4 h.
Ar
Ar
d
O
O
Ar′
(26-90%)
N
H
N
NH2
NH2
H2N
N
3a-e
2a-b, 2e, 2j-l, 2y
n-BuO
OH
Scheme 1. General synthetic route toward 2,6-diamino-3-acylpyri-
dines. Reagents and conditions: (a) n-BuLi, MTBE/TMEDA, ꢀ15 to
0 °C, 16 h; (b) ArC(O)Cl, 0 °C, 3 h; (c) 1:1 2 MKOH/dioxane, reflux,
6 h; (d) Ar0-I or Ar0-Br, Pd2(dba)3, BINAP, Cs2CO3, dioxane, 95 °C.
a
n-BuO2C
N
CO2-n-Bu
HO2C
N
CO2H
(14%)
7
6
b, c, d
(15%)
Cyano and nitro groups on 2b, 2e, and 2l were further
derivatized. For example, hydrolysis of nitrile 2b with
hydrochloric acid afforded amide 2c (23%) and acid 2d
(27%). Catalytic hydrogenation of nitro compounds 2e
and 2l gave the corresponding amine compounds 2f
(90%) and 2m (46%). The amino group of 2f was selec-
tively derivatized by acetyl chloride, benzoyl chloride,
and benzenesulfonyl chloride to give 2g (82%), 2h
(99%), and 2i (40%), respectively. Similarly, reductive
amination of 2m with ammonium formate and reaction
with benzenesulfonyl chloride afforded compounds 2n
(14%) and 2o (73%), respectively.
n-BuO
n-BuO
Ar
O
e, f
(37%)
BocHN
N
NHBoc
BocHN
N
NHBoc
8
9
g
(86%)
n-BuO
Ar
O O
S
R′2
N
O
N
H
N
NH2
n-BuO
Ar
h (30%)
i, j (23%)
10, R
′
= Me, Ar = 2,6-F2-C6H3
O
Because many potent CDK inhibitors contain a benz-
enesulfonamide group,5a,7,8 this moiety was incorpo-
rated to the 3-acyl-2,6-diaminopyridine scaffold.
Aromatic nucleophilic substitution of para-iodo- or
para-bromobenzenesulfonamide with (2,6-diamino-pyri-
din-3-yl)-(2,6-difluoro-phenyl)-methanone (3a) using the
palladium-catalyzed conditions described above failed
to give the amination product. Nevertheless, N,N-dial-
kyl para-iodobenzenesulfonamides, including N,N-di-
methyl, N,N-diethyl, and N,N-dibenzyl derivatives,9
were successfully coupled with 3-acyl-2,6-diaminopyri-
dines (3a–e) via the palladium-catalyzed amination pro-
tocol (Scheme 2). Stepwise deprotection of the benzyl
groups of 2q to give free sulfonamide 2r was achieved
in 21% combined yield by treatment with BBr3 in meth-
ylene chloride, followed by aqueous HI in acetic acid.
H2N
N
NH2
11, R
′
= H, Ar = 2,6-F2-C6H3
3f, Ar = 2,6-F2-C6H3
Scheme 3. General synthetic route toward 4-substituted 2,6-diamino-
3-acyl-pyridine analogs. Reagents and conditions: (a) excess n-BuI,
K2CO3, DMF; (b) N2H4, EtOH; (c) HCl, NaNO2, 0 °C, 1 h; (d) t-
BuOH, reflux, 5 h; (e) n-BuLi, MTBE/TMEDA, ꢀ15 to 0 °C, 16 h; (f)
ArCOCl, 0 °C, 3 h; (g) 1:1 DCM/TFA, rt, 5 h; (h) 4-I-C6H4SO2NMe2,9
Pd2(dba)3, BINAP, Cs2CO3, dioxane, 95 °C; (i) 4-I-C6H4SO2NHBoc,12
Pd2(dba)3, BINAP, Cs2CO3, dioxane, 95 °C; (j) 1:1 DCM/TFA, rt, 3 h.
converted to the corresponding di-n-butyl ester n-butyl
ether (7) in the presence of excess iodobutane (14%). Di-
phenyl phosphorylazide (DPPA)-mediated double Cur-
tius rearrangement10 using the diacid hydrolysis
product of intermediate 7 proved to be problematic.
An alternative stepwise procedure was then employed.11
Toward that end, the diester ether 7 was first converted
to the corresponding dihydrazide using hydrazine. The
dihydrazide was then carefully converted to diazide at
low temperature using nitrous acid generated in situ.
The resulting diazide was then transformed to the
To evaluate the effect of introducing an additional func-
tional group on the scaffold, substitution at the 4-posi-
tion of the core pyridine ring was investigated. The
synthesis of 4-substituted diamino pyridine analogues
is outlined in Scheme 3. Chelidamic acid (6) was first

R. Lin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2221–2224
2223
BOC-protected diaminopyridine 8 by heating at reflux
in anhydrous tert-butanol (15%). ortho-Metalation–
acylation at the 3-position of bis-Boc-protected 2,6-di-
aminopyridine 8 followed by deprotection afforded 3,4-
disubstituted diaminopyridine 3f (32% for two steps).
Aromatic nucleophilic substitution of N,N-dimethyl-
para-iodobenzenesulfonamide with compound 3f gave
compound 10 (30%). Similarly, palladium-catalyzed
amination of N-Boc-para-iodobenzenesulfonamide12
with compound 3f followed by facile deprotection of
the Boc group provided 11 (23%).
groups like N,N-dibenzylaminosulfonyl (2q) and N,N-
diethylaminosulfonyl (2u and 2w). With 2,6-difuoro-
benzoyl at 3-position of the core pyridine ring, amino-
sulfonyl (2r) and N,N-dimethylaminosulfonyl groups
(2p) in the para-position of the N6-phenyl ring produce
the most potent CDK1 inhibitors. Also, replacement
of the unsubstituted phenyl ring of 2a with a 2-pyridinyl
group (2y) improves CDK1 potency two-fold. On the
other hand, replacement of the 2,6-difluorobenzoyl
substituent of compound 2p with 2-furoyl or 2-thienoyl
groups (i.e., 2v or 2x) or removal of one or both
fluorines (i.e., 2s or 2t) is detrimental to CDK1 potency.
Table 1 shows the structures and CDK1 inhibitory
activities for the 3-acyl-2,6-diaminopyridine analogues.
Examination of substitution on the 6-anilino group re-
veals that the ortho-position (compounds 2e–i) is the
most sensitive site for maintaining CDK1 potency com-
pared to the meta and para positions. The para-position
is generally tolerant of the size and electronic character-
istics of the substituent, except in the case of very large
As shown by the CDK1 data for compounds 3f, 10, and
11, the 4-position at the pyridine core tolerates a lipo-
philic n-butoxy substituent, indicating that this could
be another location to introduce additional functional
groups to improve kinase inhibitory potency or ADME
properties. Interestingly, the intermediate N6-unsub-
stituted 3-benzoyl-2,6-diaminopyridines (3a–c, 3f)
Table 1. Structures and CDK1 inhibitory activity for the 2,6-diamino-3-acyl-pyridine analogs
R
Ar
O
Ar
′
N
H
N
NH2
2a-y, 3a-f, 10,11
Compd #
Ar
Ar0
Ph
R
CDK1 IC50, lMa
2a
2b
2c
2d
2e
2f
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
2-F–C6H4
Ph
H
21.7
1.37
0.95
ꢁ10
>10
11.6
>100
>100
>100
2.67
6.16
0.96
0.75
1.14
0.74
0.59
>10
0.36
1.96
2.19
>100
10.6
>100
12.7
12.3
2.6
4-NC–C6H4
H
4-H2NCO–C6H4
4-HO2C–C6H4
2-O2N–C6H4
2-H2N–C6H4
2-AcHN–C6H4
2-PhC(O)HN–C6H4
2-PhS(O2)HN–C6H4
3-O2N–C6H4
3-Cl–C6H4
H
H
H
H
2g
2h
2i
H
H
H
2j
H
2k
2l
H
4-O2N–C6H4
4-H2N–C6H4
4-MeHN–C6H4
4-PhS(O2)HN–C6H4
4-Me2NS(O2)–C6H4
4-Bn2NS(O2)–C6H4
4-H2NS(O2)–C6H4
4-Me2NS(O2)–C6H4
4-Me2NS(O2)–C6H4
4-Et2NS(O2)–C6H4
4-Me2NS(O2)–C6H4
4-Et2NS(O2)–C6H4
4-Me2NS(O2)–C6H4
2-Pyridinyl
H
2m
2n
2o
2p
2q
2r
2s
2t
H
H
H
H
H
H
H
H
2u
2v
2w
2x
2y
3a
3b
3c
3d
3e
3f
Ph
2-Furyl
H
H
2-Furyl
2-Thienyl
H
H
2,6-F2–C6H3
2,6-F2–C6H3
2-F–C6H4
Ph
H
H
H
H
H
8.2
1.1
H
H
2-Furyl
2-Thienyl
H
H
>100
>100
1.2
H
H
2,6-F2–C6H3
2,6-F2–C6H3
2,6-F2–C6H3
H
n-BuO
n-BuO
n-BuO
10
11
4-Me2NS(O2)–C6H4
4-H2NS(O2)–C6H4
1.0
0.26
a See Ref. 5a for a description of the CDK1 assay. IC50 data are the average of at least two separate experiments. IC50 values listed as >10 or >100
indicate no observed 50% inhibition at the highest dose tested, nor was an inhibition maximum observed.

2224
R. Lin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2221–2224
Table 2. Inhibitory activity on other kinases and anti-proliferative
activity on various tumor cells for representative compounds 2p, 2r,
and 11
3. (a) Fischer, P. M.; Gianella-Borradori, A. Expert Opin.
Investig. Drugs 2003, 12, 955; (b) Sausville, E. A. Curr.
Med. Chem.—Anti-Cancer Agents 2003, 3, 47.
a
4. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.;
Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.;
Ahmed, S. J.; Qian, L.; Chen, B.; Zhao, R.; Bednarz, M.
S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.;
Roongta, U.; Kamath, A.; Marathe, P.; Ranadive, S. A.;
Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.;
Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47,
1719.
5. (a) Lin, R.; Connolly, P. J.; Wetter, S.; Huang, S.;
Emanuel, S.; Gruninger, R.; Middleton, S. PCT Int.
Appl. 2002, WO 2002057240; Chem. Abstr. 2002, 137,
125160; (b) Emanuel, S. L.; Rugg, C.; Lin, R.; Connolly,
P. J.; Napier, C.; Hollister, B.; Hall, C.; Middleton, S.
Proc. Am. Assoc. Can. Res. 2004, 45, 191, 95th Annual
Meeting of the American Association for Cancer
Research, Orlando, Florida, March 27–31, 2004; Abstract
#833; (c) Emanuel, S.; Gruninger, R.; Lin, R.; Rugg, C.;
Fuentes-Pesquera, A. R.; Connolly, P.; Wetter, S.; Holl-
ister, B.; Kruger, W. W.; Napier, C.; Johnson, D.; Jolliffe,
L.; Middleton, S. Proc. Am. Assoc. Can. Res. 2003, 44,
162, 94th Annual Meeting of the American Association
for Cancer Research, Washington DC, July 11–14, 2003;
Abstract #707.
Kinase or tumor cell
Inhibition IC50 (lM)
2p
2r
11
CDK1
CDK2
VEGF
HER2
EGFR
HeLa
0.59
0.50
0.36
0.18
0.26
0.084
>100
>100
>100
>10
>100
>100
>100
>100
>100
>100
17.2
10.7
HCT116
A375
>10
>10
16.5
6.5
6.7
2.9
a See Ref. 5a for descriptions of kinase and cellular anti-proliferation
assays. IC50 data are the average of at least two separate experiments.
IC50 values listed as >10 or >100 indicate no observed 50% inhibition
at the highest dose tested, nor was an inhibition maximum observed.
maintain modest CDK1 potency whereas CDK1 IC50
values for similar 2-furoyl (3d) and 2-thienoyl (3e) ana-
logues could not be determined at the highest dose
tested. In addition, the enhancement of CDK1 potency
by 2,6-difluoro substituents was observed for the subset
of compounds 2 having the aryl group at N-6; a similar
trend was not seen in the subset of N6-unsubstituted
intermediate compounds 3.
6. 2,6-Bis-(pivaloylamino)pyridine (4) was prepared accord-
ing the literature procedure: Fenlon, E. E.; Murray, T. J.;
Baloga, M. H.; Zimmerman, S. C. J. Org. Chem. 1993, 58,
6625.
7. (a) Chong, W. K. M.; Shao, S.; Duvadie, R. K.; Li, L.;
Xiao, W.; Yang, Y. PCT Int. Appl. 1999, WO 99/21845;
(b) Chong, W. K. M.; Chu, S.; Duvadie, R. K.; Li, L.; Na,
J.; Schaffer, L.; Yang, Y. PCT Int. Appl. 2004, WO
2004072070.
Table 2 shows a comparison of CDK1 with CDK2 and
other kinase inhibitory activities as well as in vitro anti-
proliferative activities in human tumor cells for three
representative compounds (2p, 2r, and 11). These com-
pounds are more potent against CDK2 than CDK1
and are inactive against VEGF-R2, HER2, and EGFR
kinases at the highest concentration tested. Compounds
2r and 11 also proved to be active in vitro as anti-prolif-
eratives in various human tumor cell lines, such as HeLa
(cervical carcinoma), HCT116 (colon carcinoma), and
A375 (melanoma).
8. (a) Anderson, M.; Beattie, J. F.; Breault, G. A.; Breed, J.;
Byth, K. F.; Culshaw, J. D.; Ellston, R. P. A.; Green, S.;
Minshull, C. A.; Norman, R. A.; Pauptit, R. A.; Stanway,
J.; Thomas, A. P.; Jewsbury, P. J. Bioorg. Med. Chem.
Lett. 2003, 13, 3021; (b) Byth, K. F.; Culshaw, J. D.;
Green, S.; Oakes, S. E.; Thomas, A. P. Bioorg. Med.
Chem. Lett. 2004, 14, 2245; (c) Byth, K. F.; Cooper, N.;
Culshaw, J. D.; Heaton, D. W.; Oakes, S. E.; Minshull, C.;
Norman, R. A.; Pauptit, R. A.; Tucker, J. A.; Breed,
J.; Pannifer, A.; Rowsell, S.; Stanway, J.; Valentine, A. L.;
Thomas, A. P. Bioorg. Med. Chem. Lett. 2004, 14, 2249;
(d) Griffin, R. J.; Calvert, A. H.; Curtin, N. J.; Golding,
B. T.; Hardcastle, I. R.; Newell, D. R.; Jewsbury, P. J.
PCT Int. Appl. 2002, WO 2002059125.
9. N,N-Dimethyl, N,N-diethyl, and N,N-dibenzyl para-iodo-
benzenesulfonamide were conveniently prepared from
para-iodobenzenesulfonyl chloride and corresponding di-
alkyl amine.
10. For an example of one-pot DPPA-mediated Curtius
rearrangement see: Eaton, P. E.; Shankar, B. K. R.; Price,
G. D.; Pluth, P. J.; Gilbert, E. E.; Alster, J.; Sandus, O. J.
Org. Chem. 1984, 49, 185.
In summary, we have discovered a novel series of 3-
acyl-2,6-diaminopyridine derivatives that are effective
cyclin-dependent kinase inhibitors. The key steps for
the synthesis employed an ortho-metalation–acylation
of diprotected 2,6-diaminopyridines and a selective
palladium-catalyzed amination on activated aryl or
heteroaryl halides using the intermediate 3-acyl-2,6-di-
aminopyridines. Representative compounds 2r and 11
showed potent CDK1 and CDK2 inhibitory activities
and inhibited in vitro cellular proliferation in HeLa,
HCT116, and A375 human tumor cell lines. Future
progress on related series will be reported in due course.
11. For an example of stepwise Curtius rearrangement see:
Feibush, B.; Figueroa, A.; Charles, R.; Onan, K. D.;
Feibush, P.; Karger, B. L. J. Am. Chem. Soc. 1986, 108,
3310.
References and notes
1. Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101,
2511.
2. (a) Sielecki, T. M.; Boylan, J. F.; Benfiled, P. A.; Trainor,
G. T. J. Med. Chem. 2000, 43, 1; (b) Toogood, P. I. Med.
Res. Rev. 2001, 21, 487.
12. N-(tert-Butyloxycarbonyl)-4-iodobenzenesulfonamide was
`
prepared as described in: Cerezo, S.; Cortes, J.; Galvan,
D.; Lago, E.; Marchi, C.; Molins, E.; Moreno-Man˜as, M.;
´
Pleixats, R.; Torrejon, J.; Vallribera, A. Eur. J. Org.
Chem. 2001, 329.