Methanesulfonamido-cyclohexylamine derivatives of 2,4-diaminopyrimidine as potent ALK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.040

The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group into a methanesulfonamide. Tumor growth inhibition wa

Full text of DOI:10.1016/j.bmcl.2011.05.040

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3877–3880
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Bioorganic & Medicinal Chemistry Letters
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Methanesulfonamido-cyclohexylamine derivatives of 2,4-diaminopyrimidine
as potent ALK inhibitors
⇑
Craig A. Zificsak , Jay P. Theroff, Lisa D. Aimone, Thelma S. Angeles, Mark S. Albom, Mangeng Cheng,
Eugen F. Mesaros, Gregory R. Ott, Matthew R. Quail, Ted L. Underiner, Weihua Wan, Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a
number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group
into a methanesulfonamide. Tumor growth inhibition was observed when an orally bioavailable analog
was evaluated in a Karpas-299 tumor xenograft mouse model.
Received 25 April 2011
Revised 9 May 2011
Accepted 10 May 2011
Available online 18 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
ALK
Diaminopyrimidine
Inhibitors
Anaplastic Lymphoma Kinase (ALK) is an insulin receptor tyro-
sine kinase family member which has been identified as a compo-
nent of several fusion proteins with relevance to cancer,
particularly with nucleophosmin (NPM), to form the chimeric fu-
sion protein NPM–ALK.1 This fusion kinase is constitutively active
and plays an oncogenic role in the majority of anaplastic large cell
lymphomas; as such it has been the target of several medicinal
chemistry programs.2 Notably, Pfizer has reached phase III clinical
trials with a dual ALK–cMet inhibitor, crizotinib (PF-02341066), in
nonsmall cell lung cancer.3
2,4-Diaminopyrimidines have been incorporated as kinase
hinge-binding motifs in a number of discovery programs, leading
to ALK preclinical candidates such as NVP-TAE684 (1) from Novar-
tis4 and 2 from Cephalon (Fig. 1).5 A common structural feature in a
number of potent ALK inhibitors is a C4 aniline bearing an ortho-
sulfonyl group. A new class of inhibitors was envisioned which
would maintain a sulfonyl group vicinal to the C4-nitrogen, but
would break away from the common anilines and be replaced by
a sulfonylated 1,2-diaminocyclohexane.
Initial screening results for 6a–c are shown in Table 1; racemic
diamine 6a showed sub-micromolar inhibition of ALK whereas the
R,R-enantiomer 6b showed more potent inhibitory activity in the
enzyme assay (IC50 = 10 nM) and in the cellular assay (IC50
=
65 nM).8 Counterscreening of 6b against the insulin receptor (IR)
showed good selectivity.9 On the other hand, the S,S-enantiomer
6c displayed no significant activity in the ALK enzyme assay (IC50
N
N
N
Cl
N
N
N
N
H
H
O
O S
O
1, NVP-TAE684
Intermediates 5a–c were prepared in a two-step, one-pot oper-
ation from the three stereoisomers of 1,2-diaminocyclohexane (4a
meso cis, 4b R,R-trans or 4c S,S-trans) and 2,4,5-trichloropyrimidine
(Scheme 1).6 Treatment of 5a–c with an appropriately substituted
aniline (similar to the 2-methoxyaniline in 1 and in NVP-TAE2267)
furnished diaminopyrimidine analogs 6a–c.
O
N
Cl
N
N
N
N
H
H
O
O
S
O
N
2
⇑
Corresponding author. Tel.: +1 610 738 6745; fax: +1 610 738 6643.
E-mail address: czificsa@cephalon.com (C.A. Zificsak).
Figure 1. Novartis (1) and Cephalon (2) ALK inhibitors.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.040

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C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3877–3880
Cl
MeO
NH2
Cl
Cl
N
N
N
a, b
N
+
NH2
H2N
Cl
N
N
H
Cl
N
NH2
HN
7a
MeO
7b
SO2Me
3
4a, meso cis
5a-c
4b, R,R-trans
4c, S,S-trans
OMe
NH2
c
N
O
N
7c
Cl
N
Figure 2. Benzazepine anilines.
N
N
N
H
H
OMe
HN
SO2Me
over IR (11 vs 10). Further exploration of the substituent effects
of the cyclohexane ring were explored by utilizing a trifluoroacetyl
protecting group after addition of the diamine to 2,4,5-trichloro-
pyrimidine. After addition of 7a or 7b to the C2 position of the
pyrimidine, the trifluoroacetyl group could be readily removed
and reacted with other sulfonyl chlorides or dimethylcarbamoyl
chloride. The incorporation of larger sulfonyl groups (15–18) re-
sulted in marginal reduction of activity against ALK (IC50 = 31–
130 nM) whereas the trifluoroacetyl group resulted in a two-fold
loss in ALK potency (12 and 13). The primary amine 14
(IC50 = 680 nM) and dimethylurea analog 19 (IC50 = 370 nM) were
significantly less active, whereas the acetyl derivative 20 main-
tained ALK potency but was less selective against IR.
6a-c
Scheme 1. (a) THF, 1.1 equiv diamine; (b) MsCl, Et3N; (c) 2-methoxy-4-morphol-
inoaniline, i-PrOH, 120 °C, microwave.
Table 1
Diaminocyclohexane methanesulfonamide SAR
Compd.
Diamine
IC50a, nM
ALK cell
ALK enzyme
330
IR enzyme
180
H2N
( )-6a
NT
65
NT
In addition to 1,2-diaminocyclohexane, other cyclohexyl groups
were prepared and screened (Table 3). Use of trans-2-hydrox-
ycyclohexylamine furnished 21 which was inactive against ALK.
Cyclohexylamine analog 22 (IC50 = 78 nM) was 10-fold less active
than 11 which highlights the impact of the methanesulfonamide
fragment on potency. N-Alkylation12 of the methanesulfonamide
with methyl (23) or ethyl (24) gave potent analogs, but selectivity
against IR dropped. In addition, broader kinase selectivity also fell
with incorporation of an N-methyl group. As measured in the Am-
bit Bioscience KINOMEscan™ assay,13 11 showed >90% inhibition
HN
SO2Me
SO2Me
SO2Me
H2N
H2N
6b
6c
10
1700
6300
HN
HN
>10 lM
at 1 l
M against 5% of 63 kinases,14 whereas 23 inhibited 27%.
Lastly, a substitution pattern scan of the methanesulfonamide
group on the cyclohexane ring was undertaken. Incorporation of
either the cis or the trans isomer of 1,4-diaminocyclohexane or
1,3-diaminocyclohexane furnished chloropyrimidine fragments
that were coupled with 7b to afford 25–28. The 1,4-diamines
proved weakly active (IC50 = 100–200 nM), and the 1,3-diamine
NT = not tested.
a
IC50 values are reported as the average of at least two separate determinations.
>10 lM). Based upon these results, we proceeded to further inves-
products were inactive in the enzyme assay (IC50 >3 lM).
tigate the R,R-enantiomer series.
Moving forward with the most potent and selective analog, the
PK properties of 11 were evaluated (Table 4). Whereas low intrinsic
and oral exposure were observed in rat, sufficient exposures in
mouse were observed to warrant further evaluation in murine
xenograft models. Sulfonamide 11 was evaluated in a single-dose
PK/PD experiment (30 mg/kg, po) in mice bearing Karpas-299 tu-
Earlier results from our labs had shown the utility of benzaze-
pine anilines 7a–c (Fig. 2) as replacements of 4-amino-substituted
anilines in the diaminopyrimidine scaffold.10,11 Reaction of these
preferred amines with R,R-chloropyrimidine 5b (Scheme 2) affor-
ded analogs equal in potency to 6b (Table 2, 9 and 10, Table 3,
11), with the o-methoxy aniline conferring improved selectivity
Cl
R2
Cl
R1
Cl
N
a, b
N
N
c
+
N
N
9-11, R = SO2Me
12, 13, R = COCF3
Cl
N
N
H
H2N
4b
Cl
N
Cl
N
H
N
N
N
H
HN
NH2
HN
R
3
20, R = Ac
R
5b, R = SO2Me
8a, R = COCF3
8b, R = Ac
R1 = H or OMe
R2 = H or OMe
d, e
R2
R1
Cl
N
14, R = H;
15-19, R = SO2Et, SO2i-Pr,
N
H
N
H
SO2CH2CF3, SO2c-Pr, CONMe2
HN
R1 = H or OMe
R2 = H or OMe
R
Scheme 2. (a) THF, 1.1 equiv 4b; (b) MsCl, TFAA, or Ac2O, Et3N; (c) aniline 7a–c, 2-methoxyethanol, HCl, 120 °C; (d) 13, KOH, MeOH; (e) R-Cl, Et3N, CH2Cl2.

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3877–3880
3879
Table 2
Table 3
Biological data for compounds 9, 10, 12–20
Biological data for compounds 11, 21–28
R2
R1
Cl
Cl
O
N
N
N
R3
N
R3
N
H
N
N
H
N
H
N
N
H
Compd. R1
H2NR3
IC50a, nM
Compd. R2
H2NR3
IC50a, nM
ALK
enzyme
ALK
cell
IR
enzyme
ALK
enzyme
ALK
cell
IR
enzyme
H2N
H2N
H2N
H2N
9
10
12
H
9
60
920
540
740
H2N
H2N
11
MeO
6
65
1400
HN
HN
HN
HN
HN
SO2Me
SO2Me
COCF3
COCF3
SO2Me
( )-21
H
H
>3000
78
NT
NT
NT
77
MeO
9
73
OH
22
H2N
H2N
H
26
200
23
24
MeO
MeO
3
15
460
430
N
N
SO2Me
SO2Me
13
14
15
MeO
MeO
MeO
22
680
50
100
NT
710
NT
H2N
H2N
20
150
H2N
H2N
NHSO2Me
NH2
25
26
H
H
100
220
NT
NT
NT
NT
NHSO2Me
NT
930
HN
HN
HN
HN
HN
HN
SO2Et
SO2
H2N
H2N
H2N
( )-27
( )-28
H
H
>3000
>3000
NT
NT
NT
NT
H2N
H2N
H2N
H2N
H2N
16
17
18
19
20
MeO
MeO
MeO
H
130
31
NT
300
NT
NT
65
NT
NHSO2Me
NHSO2Me
CF3
SO2
840
NT
NT = not tested.
a
IC50 values are reported as the average of at least two separate determinations.
110
370
9
Table 4
Pharmacokinetic parameters of 11 in Sprague–Dawley rats and in CD-1 micea
SO2
PK prameters
S-D rat
CD-1 mouse
i.v.
Dose (mg/kg)
t1/2 (h)
AUC0–t (ng h/mL)
Vd (L/kg)
1
1
0.6 0.2
72 21
10.1 2.6
217 86
0.9
2931
0.4
5.6
150
150
CONMe2
CL (mL/min/kg)
p.o.
Dose (mg/kg)
Cmax (ng/mL)
tmax (h)
t1/2 (h)
AUC0–t (ng h/mL)
F%
5
23
10
1
2877
1
1.0
8200
28
H
1.8 1.1
n.d.
Ac
110
24
7
2
NT = not tested.
a
IC50 values are reported as the average of at least two separate determinations.
a
Values are means of data from three animals. In mouse, three animals were
tested per time point.
mor xenografts. After 2 h, a significant (75%) decrease in NPM-ALK
autophosphorylation was observed, but this inhibition was not
maintained beyond 6 h (Fig. 3).
Analysis of plasma and tumor samples showed levels of 11 in
plasma far exceeding the ALK cellular IC50, but levels in tumor were

3880
C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3877–3880
tolerated and no overt toxicity or body weight loss was observed.
The high plasma exposures observed in mouse raised expectations
for a sustained PD response leading to strong TGI. However, the ob-
served attenuated and fleeting PD response and modest TGI ap-
peared more consistent with tumor PK rather than plasma PK.
The low tumor concentrations of 11 (relative to its cellular ALK
IC50) resulted in only transient inhibition of ALK-phosphorylation
that proved insufficient to generate a more robust TGI response.
In conclusion, the incorporation of R,R-1,2-diaminocyclohexane
at C4 of the 2,4-diaminopyrimidine scaffold provided potent inhib-
itors of ALK in both enzyme and cellular assays. Capping of the
2-amino group with a methanesulfonamide provided the most ac-
tive analogs while maintaining selectivity over IR. Analog 11
showed bioavailability in mouse and demonstrated in vivo tumor
growth inhibition in a Karpas-299 tumor xenograft mouse model.
Figure 3. Inhibition of NPM–ALK phosphorylation in Karpas-299 tumor xenografts
in mice by 11 with a single oral dose.
Supplementary data
Plasma (ng/mL)
Tumor (ng/g)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.040.
10000
3685
2650
2740
References and notes
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Karpas-299 Tumor Xenografts in Mice
8
Vehicle, PO, bid
30 mg/kg, PO, bid
6
55 mg/kg, PO, bid
#
8. For in vitro and in vivo assay conditions, see Ref. 5.
4
9. As a key regulator of glucose metabolism, selectivity against IR was desired. For
an account of a small molecule kinase inhibitor’s effect on glucose homeostasis,
see: Sabbatini, P.; Korenchuk, S.; Rowand, J. L.; Groy, A.; Liu, Q.; Leperi, D.;
Atkins, C.; Dumble, M.; Yang, J.; Anderson, K.; Kruger, R. G.; Gontarek, R. R.;
Maksimchuk, K. R.; Suravajjala, S.; Lapierre, R. R.; Shotwell, J. B.; Wilson, J. W.;
Chamberlain, S. D.; Rabindran, S. K.; Kumar, R. Mol. Cancer Ther. 2009, 8, 2811.
10. Mesaros, E. F.; Burke, J. P.; Parrish, J. D.; Dugan, B. J.; Anzalone, A. V.; Angeles, T.
S.; Albom, M. S.; Aimone, L. D.; Quail, M. R.; Wan, W.; Lu, L.; Huang, Z.; Ator, M.
A.; Ruggeri, B. A.; Cheng, M.; Ott, G. R.; Dorsey, B. D. Bioorg. Med. Chem. Lett.
2011, 21, 463.
11. Zificsak, C. A.; Theroff, J. P.; Aimone, L. D.; Albom, M. S.; Angeles, T. S.; Brown,
R.; Galinis, D.; Grobelny, J. V.; Herbertz, T.; Husten, J.; Kocsis, L. S.; LoSardo, C.;
Miknyoczki, S. J.; Murthy, S.; Rolon-Steele, D.; Underiner, T. L.; Wells-Knecht, K.
J.; Worrell, C. S.; Zeigler, K.; Dorsey, B. D. Bioorg. Med. Chem. Lett. 2011, 21, 660.
12. Compound 5b was treated with methyl or ethyl iodide and the product was
reacted with 7c to furnish 23 or 24.
#
#
2
0
#
#
*
#
0
3
6
9
12
Days in treatment
Initiation of cmpd-11 treatment
# P < 0.05
*
Figure 5. Effect of 11 on Karpas-299 xenograft tumors.
13. Fabian, M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. R.; Azimioara, M. D.;
Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.;
Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.;
Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L.
M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
14. A table of kinase targets and inhibition data is in the Supplementary data.
Analog 11 inhibited ALK, KIT(D816V) and PLK1; 23 inhibited ALK, CSF1R,
ERBB4, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, JNK1, JNK3, KIT, KIT(D816V),
KIT(V559D, T670I), LKB1, PDFGRB, PIM1 and PLK1.
significantly lower than the plasma levels at all three time points
(2, 6 and 12 h) (Fig. 4). Compound 11 was also evaluated for tumor
growth inhibition (TGI) in a 10 day Karpas-299 tumor xenograft
model at two doses (30 and 55 mg/kg, p.o., bid, Fig. 5). A shallow
dose response between the 30 and 55 mg/kg groups was observed
leading to 45% and 54% TGI, respectively. The compound was well