Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro- N 2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)- N 4-(2-(isopropylsulfonyl) phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials

Article abstract of DOI:10.1021/jm400402q

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

Full text of DOI:10.1021/jm400402q

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Article
Synthesis, Structure-Activity Relationships and In Vivo Efficacy
of the Novel Potent and Selective Anaplastic Lymphoma Kinase
(ALK) Inhibitor LDK378 Currently In Phase 1 and 2 Clinical Trials
Tom H. Marsilje, Wei Pei, Bei Chen, Wenshuo Lu, Tetsuo Uno, Yunho Jin, Tao Jiang, Sungjoon Kim,
Nanxin Li, Markus Warmuth, Yelena Sarkisova, Fangxian Sun, Auzon Steffy, AnneMarie C. Pferdekamper,
Sean B Joseph, Young Kim, Tove Tuntland, Xiaoming Cui, Nathanael S Gray, Ruo Steensma, Yongqin
Wan, Jiqing Jiang, Jie Li, Greg Chopiuck, W. Perry Gordon, Allen G Li, Wendy Richmond, Johathan
Chang, Todd Groessl, You-Qun He, Bo Liu, Andrew Phimister, Alex Aycinena, Badry Bursulaya,
Christian Lee, Donald S Karanewsky, H Martin Seidel, Jennifer L Harris, and Pierre-Yves Michellys
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400402q • Publication Date (Web): 06 Jun 2013
Downloaded from http://pubs.acs.org on June 13, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
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Journal of Medicinal Chemistry
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Jiang, Jiqing; Genomics Institute of the Novartis Research Foundation,
Li, Jie; Genomics Institute of the Novartis Research Foundation, Medicinal
Chemistry
Chopiuck, Greg; Investors Group,
Gordon, W.; Genomics Institute of the Novartis Research Foundation,
Li, Allen; PhaseRx Inc.,
Richmond, Wendy; Genomics Institute of the Novartis Research
Foundation,
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Chang, Johathan; Genomics Institute of the Novartis Research Foundation,
Groessl, Todd; Genomics Institute of the Novartis Research Foundation,
He, You-Qun; Genomics Institute of the Novartis Research Foundation,
Liu, Bo; Genomics Institute of the Novartis Research Foundation,
Phimister, Andrew; Novartis Vaccines and Diagnostics,,
Aycinena, Alex; Novartis Vaccines and Diagnostics,,
Bursulaya, Badry; Genomics Institute of the Novartis Research Foundation,
Lee, Christian; Genomics Institute of the Novartis Research Foundation,
Protein Science
Karanewsky, Donald; Senomyx Inc.,
Seidel, H; Genomics Institute of the Novartis Research Foundation,
Harris, Jennifer; Genomics Institute of the Novartis Research Foundation,
Michellys, Pierre-Yves; Genomics Institute of the Novartis Research
Foundation, Medicinal chemistry
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Synthesis, Structure-Activity Relationships and In
Vivo Efficacy of the Novel Potent and Selective
Anaplastic Lymphoma Kinase (ALK) Inhibitor 5-
chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-
yl)phenyl)-N4-(2-
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(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine
(LDK378) Currently In Phase 1 and 2 Clinical Trials
Thomas H. Marsilje, Wei Pei, Bei Chen, Wenshuo Lu, Tetsuo Uno, Yunho Jin, Tao Jiang, Sungjoon
Kim, Nanxin Li, Markus Warmuth, Yelena Sarkisova, Frank Sun, Auzon Steffy, AnneMarie C.
Pferdekamper, Allen G. Li, Sean B. Joseph, Young Kim, Bo Liu, Tove Tuntland, Xiaoming Cui,
Nathanael S Gray, Ruo Steensma, Yongqin Wan, Jiqing Jiang, Greg Chopiuck, Jie Li, W. Perry Gordon,
Wendy Richmond, Kevin Johnson, Jonathan Chang, Todd Groessl, You-Qun He, Andrew Phimister,
Alex Aycinena, Christian C. Lee, Badry Bursulaya, Donald S. Karanewsky, H. Martin Seidel, Jennifer
L. Harris, Pierre-Yves Michellys.*
KEYWORDS. ALK, ALKi, SAR, ALCL, NSCLC, Karpas299, NCIꢀH2228, kinase.
ABSTRACT. The synthesis, preꢀclinical profile and in vivo efficacy in rat xenograft models of the novel
and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) is described. In this initial report,
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preliminary structureꢀactivity relationships (SAR) are described as well as the rational design strategy
employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684).
Compound 15b is currently in Phase 1 and 2 clinical trials with substantial antitumor activity being
observed in ALKꢀpositive cancer patients.
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Introduction
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase of the insulin receptor superfamily.
1
Expression of ALK in normal human tissues is only found in a subset of neural cells. However, altered
expression and hyperactivation of ALK as a consequence of translocations or point mutations has been
2ꢀ4
demonstrated to be an essential oncogenic lesion in a number of cancers. To date, no essential role has
been found for ALK in mammals. Mice deficient in ALK have normal development and display an
antiꢀdepressive phenotype with enhanced performance in hippocampusꢀdependent tasks, potentially due
5
to increased hippocampal progenitor cells.
Deregulation of ALK was first identified in anaplastic large cell lymphoma (ALCL), where the
tyrosine kinase domain is fused to nucleophosmin (NPM), a product of recurrent t(2;5)(p23;q35)
6
chromosomal translocation. Subsequently, chromosomal rearrangements resulting in ALK fused to
various partner genes have been found in nearly 70% of ALCL, 40ꢀ60% of inflammatory
7
myofibroblastic tumors (IMT), a few dozen cases of diffuse large Bꢀcell lymphoma (DLBCL), and,
8
ꢀ10
most recently, in 2ꢀ7% of nonꢀsmall cell lung cancer (NSCLC).
Among fusion partner genes
identified to date, NPM is the most common partner in ALCL and echinoderm microtubuleꢀassociated
proteinꢀlikeꢀ4 (EML4) is the main partner in NSCLC. In addition to the chromosomal rearrangements
that result in ALK fusion genes, amplification of ALK gene and activating point mutations in the full
1
1ꢀ13
14
length ALK gene have recently been reported in neuroblastoma,
inflammatory breast cancer, and
15
ovarian cancer.
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Multiple pharmaceutical companies have described ALK inhibitors (ALKi) at various stages of
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development.
Structures of ALKi in clinical development are depicted in Figure 1. Compound 1
2
5ꢀ28
(PFꢀ02441066, crizotinib)
was approved in 2011 by the FDA to treat ALKꢀpositive NSCLC
29ꢀ30
31
patients. Other ALK inhibitors, including 3 (CH5424802, Roche/Chugai),
5 (ASP3026, Astellas),
3
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2
(Xꢀ396, XꢀCovery), AP26113 (Ariad Pharmaceuticals. Structure undisclosed; series exemplified as
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3ꢀ34
35a
compound 6),
Tesaro (structure undisclosed) and 15b (LDK378, Novartis), are currently in Phase
1
/2 clinical trials.
Figure 1. Structures of selected ALK inhibitors.
In this report, we describe the work we performed around the previously disclosed ALKi 4
3
5b
(
TAE684) . This research allowed us to find novel and selective ALK inhibitors with potent in vivo
efficacy in ALCL and NSCLC rat xenograft models as well as improved development characteristics,
leading to the discovery of compound 15b.
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Compound 4 is a potent ALK inhibitor; however, it was found to form an extensive number of
1
2
reactive adducts upon metabolic oxidation, which creates the potential for significant toxicological
liabilities. Semiꢀquantitative LCꢀMS analysis indicated that approximately 20% of 4 is converted into
reactive species when incubated in liver microsomes (Table 2), and these reactive species can be trapped
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using glutathione (GSH). Although no correlation was found between hepatotoxic drugs and the
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formation of GSH adducts, it has been postulated that reactive metabolites may have a role in
38
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idiosyncratic and/or other toxicities.
A systematic evaluation of various compounds from the historical SAR dataset revealed that the
reactive metabolite formation was primarily correlated with the presence of a solubilizing group
connected by a nitrogen atom onto the central aniline moiety (Figure 2). It is hypothesized that
metabolic oxidation of the electronꢀrich aromatic ring undergoes the formation of a 1,4ꢀdiiminoquinone
moiety (A), which is highly reactive and forms adducts in the presence GSH (B).
Figure 2. Hypothesized mechanism of reactive adduct formation from 4.
Results and Discussion
3
5b
Analogue Design and Chemistry. Based on modeling information,
previous work in our
laboratories have focused on increasing the kinase selectivity of 4 by modifying the methoxy moiety.
This work resulted in finding that other alkoxy groups, such as an isoꢀpropoxy moiety (7), was also well
tolerated for ALK (Table 1) while having a positive effect on improved overall kinase selectivity
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(
supporting information, Figure S9) and reducing the potential metabolic liability associated with
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methoxy moieties. However, it was also found the formation of reactive adducts observed with 7 and
closely related analogues (unpublished results) was independent of the nature of alkoxy moiety present
on the molecules (Table 2) confirming our hypothesis stated in Figure 2. With this information in hand,
we took the decision to reꢀevaluate the SAR around this scaffold and focus our efforts towards the
eradicating the reactive adducts that prevented 4 and some close analogues like 7 to enter into
development. In order to reach this goal, we chose to retain the iso-propylsulfoneaniline as well as the
isoꢀpropoxy moieties, since these were shown to be important for potent and selective ALK inhibition.
Variations on this theme were synthesized and profiled (e.g., cyclobutylsulfoneaniline, N,Nꢀ
dimethylsulfonamide) to determine the moiety with the best overall development profile. We also
replaced the iso-propoxy with a cyclobutyloxy moiety, which is wellꢀtolerated by ALK. For the
compounds described in this report, we also kept the pyrimidine hingeꢀbinding core constant, changing
only the 5ꢀchloro substituent to a 5ꢀmethyl in the case of compounds 18a-d. As an additional point of
comparison, 23, which replaces the pyrimidine core with a pyridine, was synthesized. The focus of this
report is on the SAR of the 4ꢀposition aniline ‘tail’ containing the solubilizing moiety. The SAR
changes are highlighted in Figure 3. Additional, broader SAR explorations will be discussed in future
publications.
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Figure 3. Structureꢀactivity relationship overview.
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With the aim of designing novel derivatives that would not form reactive adducts, we made the
1
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rational design decision to reverse the piperidine at the para position of the aniline moiety present in the
series of compounds represented by 4 in order to remove the possibility of a 1,4ꢀdiiminoquinone
formation. We also designed the inclusion of a methyl group para to the isoꢀpropoxy moiety in order to
further reduce metabolism on the interior phenyl ring, thereby further reducing the possibility of
forming reactive metabolites.
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The syntheses of the compounds 15a-e, 16a,b, 17a-i, 18a-d¸19, 20 and 23 are shown in Schemes 1
and 2. The aniline moieties 8a-b were synthesized in 2 steps from 2ꢀfluoronitrobenzene according to a
4
0
known procedure.
8c was synthesized in
a
very straightforward way from 2ꢀ
4
1
nitrobenzenesulfonylchloride. 8a-c were then coupled to 2,3,5ꢀtrichloropyrimidine or 2,4ꢀdichloroꢀ5ꢀ
methylpyrimidine to afford compounds 9a-d in acceptable overall yield (40ꢀ61%). Anilines 13a-e were
synthesized in 4ꢀ8 synthetic steps from 2ꢀchloroꢀ4ꢀfluorotoluene or 3ꢀchlorofluorobenzene, while 13f
3
9
was synthesized from 2,4ꢀdichloroꢀ5ꢀnitropyrimidine using an identical synthetic route. Nitration of 2ꢀ
chloroꢀ4ꢀfluorotoluene or 3ꢀchlorofluorobenzene using KNO in H SO yielded the corresponding nitro
3
2
4
derivatives 10a,b. Condensation of 10a,b with iso-propanol or cyclobutanol (60°C in the presence of
K CO in DMF/DMSO) afforded 10a-c in excellent yield (>80%). Suzuki coupling of 11a-c with 4ꢀ
2
3
pyridineboronic acid afforded 12a-c in >70% yield. Nꢀmethylation of 12b followed by global reduction
(NaBH followed by PtO /H ) generated 13d. Alternatively, concomitant reduction of the pyridine and
4
2
2
the nitro group present in 12a-c using PtO /H afforded the piperidineꢀaniline intermediates, which were
2
2
subsequently protected, generating the Boc derivatives 13a-c in good yields (>60%). Compound 13e
was synthesized by condensation of 2ꢀmethoxyꢀ4ꢀpyridylboronic acid onto 11b, followed by
demethylation of the methoxy group (refluxing conc. HCl in dioxane) and reduction of the pyridine ring
(PtO /H ). For the syntheses of 15a-e, 16a,b, 17a-i, 18a-d and 19-20, 9aꢀd were typically coupled to the
2
2
aniline 13a-f using a modified Buchwald coupling (Xantphos (10%), Pd(OAc) (5%), Cs CO (3 eq.),
2
2
3
THF, MW, 150°C). Aside from 16a,b and 19, this was followed by Boc deprotection (TFA, CH Cl ).
2
2
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Finally, subsequent piperidine alkylation or acylation afforded 17a-i and 18a-d in good overall yields.
Compounds 19 and 20 followed a similar synthetic route, except 9a was coupled to 2ꢀiso-propoxyꢀ5ꢀ
methylꢀ4ꢀ(2ꢀoxopiperidinꢀ4ꢀyl)benzenaminium 2,2,2ꢀtrifluoroacetate for 19 and 13f in the case of 20. In
order to synthesize compound 23, we had to reverse the synthetic sequence. Amination of 4ꢀbromoꢀ2,5ꢀ
dichloropyridine with 13a (Xantphos, Pd(OAc) , Cs CO , THF, MW, 150°C) afforded 22 in 41% yield.
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2 was subsequently aminated with 8a (Pd (dba) , dicyclohexyl(2,4,6ꢀ triisoꢀpropylphenyl)phosphine,
2 3
NaOtBu THF, MW, 150°C ) to afford 23 in 11% yield.
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R2
N
1
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NH2
O
S
O
a
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R1
HN
O
S
N
Cl
N
O
R1
+
Cl
N
Cl
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8a, R = iso-propyl
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a, R = iso-propyl; R = Cl
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b, R = cyclobutyl
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9b, R = iso-propyl; R = CH
3
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c, R = N(Me)2
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9c, R = cyclobutyl; R = Cl,
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9d, R = N(Me) ; R = Cl
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R3
O
R3
R3
Boc
R3
R3
N
N
Cl
e, f
Cl
Cl
d
b
c
O N
O N
2
2
O N
H N
2
2
F
F
R4
O
O
R4
R4
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3
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R = H or CH3
10a, R = H
11a, R = H; R = iso-propyl
12a, R = H; R = iso-propyl
13a, R = H; R = iso-propyl
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0b, R = CH3
11b, R = CH3; R = iso-propyl 12b, R = CH3; R = iso-propyl
13b, R = CH ; R = iso-propyl
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4
3
4
3
4
11c, R = CH ; R = cyclobutyl 12c, R = CH ; R = cyclobutyl
13c, R = CH ; R = cyclobutyl
3
3
3
1
2b, g, h, e
N
H N
2
1
3d
O
O
1
1b, i, j, e
NH
3e
H N
2
1
O
Boc
N
Cl
N
Cl
N
c
d, e, f
N
O
N
N
O N
O N
2
2
N
Cl
O
H N
2
1
3f
14
Scheme 1. Reagents and conditions: a. NaH, DMF/DMSO , 0°C to 20°C ; b. KNO , H SO , 0°C to
3
2
4
20°C c. Alcohol, Cs CO , 60°C. d. 4ꢀPyridineboronic acid, dioxane/H O or 1ꢀBuOH (Pd (dba) , 2ꢀ
2
3
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2
3
dicyclohexylphosphineꢀ2’ꢀ6’ꢀdimethoxy biphenyl, MW, 150°C. e. AcOH/TFA; PtO , H . f. Anhydrous
2
2
o
o
CH Cl , TEA, Boc O, 20°C. g. CH I, THF, 40°C, sealed tube. h. NaBH , CH OH, 0 C to 20 C. i. (2ꢀ
2
2
2
3
4
3
methoxypyridinꢀ4ꢀyl)boronic acid, Pd dba , 2ꢀdicyclohexylphosphineꢀ2’ꢀ6’ꢀdimethoxy biphenyl,
2
3
o
K PO , 1ꢀBuOH, 150 C. j. Concentrated HCl, dioxane, reflux.
3
4
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Reagents and conditions: a. Xantphos, Pd(OAc) , Cs CO , THF, MW, 150°C. b. TFA,
2
2
3
·
CH Cl , 20°C. c. RꢀBr (or RꢀI), Et N, DMF, 20°C. d. ClC(O)CH N(Me) HCl, pyridine, 20°C. e.
2
2
3
2
2
Pd (dba) , dicyclohexyl(2,4,6ꢀtriisopropylphenyl)phosphine, NaOtBu THF, MW, 150°C.
2
3
In Vitro SAR.
Table 1 shows the in vitro cellular activity of compounds 15a-e, 16a,b, 17a-i, 18a-d,19-20 and 23
against Ba/F3ꢀNPMꢀALK, Ba/F3ꢀTelꢀInsR and in Karpas299 cells. Since we had very potent starting
points (compounds 4 and 7) for this secondꢀgeneration ALK inhibitor project, we decided to evaluate
ALK inhibition directly in a cellular context by measuring the proliferation of Ba/F3 cells expressing
the NPMꢀALK or TelꢀInsR fusion protein. Wildꢀtype (WT) Ba/F3 cells were used as counterscreen in
order to check for nonspecific cytotoxicity or interference with coꢀexpressed luciferase which was used
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to
monitor
cell
proliferation.
1
2
Antiꢀproliferative activity was monitored using the Karpas299 cell line which is a wellꢀestablished line
isolated from ALKꢀpositive ALCL tumors possessing the NPMꢀALK fusion. The data for the
compounds described in this report is summarized in Table 1 below.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ba/F3ꢀNPMꢀALK
150.8 ± 17.8
3.7 ± 0.5
Ba/F3ꢀTelꢀInsR
1643 ±272.0
43.7 ± 7.3
Ba/F3ꢀWT
3479±180.0
1336±279.1
3395±600
Karpas299
64.2±6.0
2.4±0.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
7
1
1
24.8±7.9
414.0±81.0
281.8±28.2
319.5 ±23.5
541.5±54.0
197.2±26.2
510.0±50.0
464 ± 42.0
339.0±33.0
344.5±40.0
240.1±53.6
326.5±58.5
349.0±38.5
1186±92.5
4874±290.0
891.5±106.0
361.1±42.3
416.0±49.0
375.3±24.6
401.0±32.0
25.5±7.6
7.8±2.8
5a
5b
10.6±1.7
3013±154.0
2477±448.0
2884±194.0
2460±260.0
2517±406.0
3997±24.6
1190±110.0
4120±475.0
3855±215.0
4336±285.0
4973±310.0
1420±107.5
>10000
26.0±0.3
22.8±0.3
13.1±1.3
13.5±2.4
13.1±9.0
11.2 ± 0.1
14.3±0.7
11.1±0.9
14.5±1.1
8.1±0.8
15c
40.6 ± 5.5
38.1±2.2
1
1
1
1
5d
5e
6a
6b
17.3±5.9
11.5 ± 0.2
25.0±5.3
17a
32.1±3.8
1
1
7b
7c
29.9±4.4
8.1±2.3
17d
43.5±6.0
16.1±1.6
100.3±19.3
293.5±81.0
15.3±0.6
7.9±0.3
1
1
1
1
7e
7f
258.4±49.7
305.7±105.2
88.9±20.9
16.5±3.2
7g
7h
>10000
7543±1050
2737±113.0
4077±173.3
4005±480.0
17i
25.8±3.4
21.7±5.9
15.6±1.8
10.5±0.6
1
1
8a
8b
14.6±2.6
29.4±3.6
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1
1
8c
63.6±6.9
44.0±5.0
48.3 ±7.8
36.4±4.6
4240±1360
1643±126.0
418.0±31.0
784.0±112.0
564.0±74.0
1920±890.0
>10000
23.8±2.0
22.4±1.6
38.4±4.0
33.3±4.3
5510±2190
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8d
4407±290.0
> 10000
19
2
2
0
3
3800±200.0
5412±330.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Activity profile of compounds 1, 4, 7, 15a-e, 16a,b, 17a-i, 18a-d, 19-20 and 23. All data
given in nM and are an average of at least duplicate measurements.
Compound
GSHꢀtrapping result
4
7
~20%
~19%
<1%
<1%
<1%
<1%
<1%
1
1
1
1
5b
5e
7a
7e
18a
Table 2. GSHꢀtrapping results of derivatives 4, 7, 15b, 15e, 17a, 17e and 18a.
Overall, most substitutions on the nitrogen of the piperidine were fairly wellꢀtolerated (most cellular
IC ’s against ALK ranged from 10 to 50 nM). Among the compounds tested, derivatives such as 15a,
50
16a and 17c approached the potency of compound 4 and were more potent than 7. They were only
approximately 2ꢀ3ꢀfold less potent in the Ba/F3ꢀNPMꢀALK assay, and they possessed a better
selectivity window versus the insulin receptor compared to 4 (InsR, 25ꢀ to 40ꢀfold versus 11 and 15ꢀfold
for 4 and 7 respectively). Most compounds tested had an IC for NPMꢀALK below 50 nM. However, it
5
0
is interesting to note that addition of pKaꢀmodulating moieties [such as a 2,2ꢀtrifluoroethyl group (17f)
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Journal of Medicinal Chemistry
or an acetic acid (17g)] decreased ALK inhibition activity. In contrast, addition of an acetamide (17h), a
N,Nꢀdimethylaminoacetate (17i), or conversion to a cyclic lactam (19) were wellꢀtolerated for ALK
inhibition and retained selectivity versus InsR. A similar trend was observed in the Karpas299 cell line.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3, in which we replaced the pyrimidine core with a pyridine, resulted in a large loss of potency. This
3
observation is consistent with the binding mode of these molecules in the ATPꢀbinding site. The N of
the pyrimidine make a key contact at the hinge (Figure 4) and therefore, removing this key interaction
considerably lowers affinity of compound 23. We synthesized and profiled a number of minor variations
of 15b to identify the best development candidate. Substitutions in 15d, 15e, 16b, and 20 were tolerated
but did not show any significant development advantages over 15b. A selected set of the compounds
from Table 1 was tested in the GSHꢀtrapping assay to evaluate their potential to form reactive adducts.
The data are shown in Table 2. All the derivatives tested were found to have close to undetectable levels
of GSH adducts (<1%) when submitted to the same assay conditions as 4 and 7 (supporting information,
Figures S1ꢀS4), experimentally confirming the validity of our rational designs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Modeling and docking
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Docking of 15b in the ALK kinase domain.
We performed molecular modeling of 15b based upon the reported crystal structure of TAE684 with
42
the kinase domain of ALK. As expected, the ligand makes hydrogen bonds at the hinge area via the
pyrimidine and amino nitrogen atoms onto the backbone nitrogen and oxygen of Met1199 respectively.
Our model, shown in Figure 4, does not show the hydrogen bond between the carbonyl oxygen of
Met1199 and the amino nitrogen of the inhibitor, even though they are within the range of a typical
hydrogen bond, both distanceꢀ and angleꢀwise. The reason is that these two atoms occupy different
planes, probably due to constraints of the crystal structure. We expect that they form a hydrogen bond in
the context of a conformationally flexible protein in solution. The central pyrimidine ring of the
inhibitor is sandwiched between Ala1148 and Leu1256 and its chlorine substituent is directed towards the
back of the pocket making hydrophobic contact with gatekeeper Leu1196. The piperidine ring extends to
the solvent and is engaged in a salt bridge with Glu . The sulfonyl makes an intramolecular Hꢀbond
1
210
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with the amine and is directed at Lys1150. The isopropyl substituent bends down into the cavity formed
by amino acids Arg1253, Asn1254, Cys1255, Leu1256, Gly1260, and Asp1270. The iso-propoxy group fits into a
pocket formed by side chains of Arg1120 and Glu1132 and the Leu1198ꢀAla1200ꢀ Gly1201ꢀGly1202 hinge
segment.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
While this model was not primarily used for compound optimization, it does correlate well with some
of the SAR observations. For example, the lower potency of 17e compared to 15b may be explained by
a greater number of rotatable bonds (entropic effect) present in 17e as well as its greater mobility in the
bound state. The latter follows from a difference in docking poses. In 15b, the terminal piperidine fits
closely to the protein surface and makes a salt bridge with E1210. In 17e, by contrast, the corresponding
central piperidine ring is pushed up into the solvent. This allows the formation of a salt bridge between
its terminal piperidine and E1210 of the enzyme. The fact that 17e has reduced surface contact with
protein (see Figure S7 in supplementary information material) leads to its greater mobility. This can be
demonstrated by conducting mixed Monte Carlo Multiple Minimum (MCMM)/LLMOD (Large Scale
Low Mode) conformational search available in MacroModel 9.9 (Schrodinger, Inc, Portland, OR, 2012).
The MCMM/LLMOD simulations revealed much greater mobility in the 17e complex, especially in its
terminal piperidine and the side chain of E1210 (see Figure S8 in supplementary information material).
We hypothesize that this greater mobility of 17e translates into its weaker binding potency.
ADME SAR. A selected set of these derivatives were evaluated for their ADME properties. Table 3
shows a summary of liver microsome intrinsic clearance, CYP3A4 inhibition, high throughput (HT)
solubility as well as hERG inhibition (dofetilide binding assay) for 15a,b, 15e, 17a, 16c, 16i, 17a and
43
1
9. The selected compounds have relatively good metabolic stability when tested in liver microsomes.
Moderate CYP3A4 (Midazolam substrate) inhibition was observed for these derivatives without a clear
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SAR trend. A wide range of solubility values was observed, which was largely dependent on the pKa of
the amine appended to the molecule. As expected, some hERG inhibition was also seen (again
correlating well with the pKa of the amine moiety). hERG patch clamp experiments showed an IC of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
50
4
6 and 20 µM respectively for 15b and 18a. Compound 15b was further evaluated in vivo in both dog
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and monkey telemetry studies, and no evidence of QTc prolongation was observed (data not shown).
2
3
4
compound
CLint
CYP3A4
HTsol.
hERG
5.3
1
m, r, h
1
5a
15, 15, 15
15, 10, 19
15, 15, 15
15, `4, 17
15, 14, 15
45, 21, 46
15, 14, 8
1.4 ± 0.2
1.5 ± 0.3
7.3± 0.8
2.1± 0.4
2.5± 0.2
0.6± 0.1
9.8 ± 1.2
9.3± 0.8
4
5
15b
24
<2
64
36
4
1.25
1
5e
6a
3.02
3.35
2.64
24.5
1
17c
1
1
7i
5
8a
144
138
1.1
20
21, 18, 16
0.4
1
Table 3. ADME profile of selected derivatives. Clearance (CL ) is in µL/min/mg in liver microsomes.
int
2
3
Data in µM. Inhibition measured using midazolam as substrate. Data in µM measured from DMSO
4
5
solution in buffer adjusted to pH 6.8. Data in µM in hERG dofetilide binding assay. Followꢀup hERG
patch clamp data is shown in the body of paper.
Full In Vitro and In Vivo Profiling of 15b Additional profiling of compound 15b for its kinase
selectivity and pharmacokinetic profile was performed. Tables 4 and 5 display a selected enzymatic and
cellular kinase profile of 15b and Table 6 displays its PK profile in mouse, rat, dog and monkey.
Compound 15b displays a remarkable selectivity profile when tested against an enzyme panel of 46
kinases, showing biochemical inhibition below 100 nM for only three kinases: IGFꢀ1R, InsR and
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STK22D with IC ’s of 8, 7 and 23 nM respectively. Compound 15b is very potent against ALK at the
5
0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
enzymatic level (IC of 200 pM) thus its selectivity against these offꢀtargets is 80ꢀ, 70ꢀ and 230ꢀfold,
5
0
respectively. When tested in a cellular proliferation panel of 39 kinases, 15b proved to be very selective.
Aside from ALK, no inhibition below 100 nM was observed for all kinases tested and the potent
inhibitions observed for IGFꢀ1R and FLT3 at the enzymatic level proved to be nonꢀreproducible in a
cellular context (IC of 410 and 3143 nM respectively). Likewise, cellular inhibition of FGFR2 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
FGFR4 was found to be > 2 ꢀM. We could not establish a cellular assay for STK22D but its inhibition
was deemed to not be prohibitive for clinical development.
kinase
IC50
(ꢀM)
Kinase
IC50 (ꢀM)
kinase
IC50 (ꢀM)
ABL
AKT
ALK
1.25
FGFR4
FLT3
0.95
0.06
>10
LYN
0.84
3.17
2.24
>10
cMET
MKNK2
0.0002
GSK3β
AURORA
BTK
0.66
3.36
GSK3β
>10
PAK2
>10
IGFꢀ1R
0.008
PDGFRα
RET
1.14
CDK2
CDK4
EGFR
FGFR2
FGFR3
3.97
4.72
0.9
InsR
JAK1
JAK2
cKit
0.007
3.73
0.61
1.28
0.56
0.4
ROCK2
SYK
1.27
3.05
0.023
9.7
0.26
0.43
STK22D
ZAP70
LCK
Table 4. Enzymatic kinase profile of 15b.
kinase
IC50 (nM)
40.7±5.3
Kinase
TelꢀJAK2
IC50 (nM)
2160±320
kinase
IC50 (nM)
2304±459
TelꢀALK
TelꢀRET
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EML4ꢀALK
TelꢀFGFR3
TelꢀFGR
2.2±0.5
TelꢀKDR
Telꢀckit
TelꢀLck
TelꢀLYN
TelꢀMet
4210±170
TelꢀRos
141.8±22.7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
>10000
2855±215
672±264
2306±552
1339±74
TelꢀSrc
1694±276
2727±222
1829±80
1947±433
3143±933
410±10
TelꢀTRKA
TelꢀTRKB
TelꢀWT
TelꢀFLT3
TelꢀIGFꢀ1R
3250±710
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Cellular kinase profile of 15b in Ba/F3 cells.
Compound 15b was tested in mouse, rat, dog and monkey for its PK profile (Table 6). The compound
exhibited low plasma clearance in animals (mouse, rat, dog and monkey) compared to liver blood flow.
The volume of distribution at steady state (V ) was high and approximately 10ꢀfold greater than total
ss
body water. Halfꢀlife (T ) ranged from moderate to long (6.2 to 26 hrs). Following a single oral
1
/2
administration of 15b as a solution or suspension, the oral bioavailability was good (≥55%) in mouse,
rat, dog and monkey. Time of maximal concentration (Tmax) occurred consistently late in animal species
tested, indicating slow oral absorption. Overall, 15b displays a consistent PK profile across all species
tested. Although compound 15a, which deletes the methyl para to the alkoxy group in 15b, was fully
tolerated by ALK, 15a displayed significantly worse oral bioavailability in rat compared to 15b (12%F
vs. 66%F).
Species
Mouse
Rat
dog
Monkey
1
1
1
1
2
3
2
4
Parameter
IV
PO
20
IV
PO
10
IV
PO
20
IV
PO
60
Dose (mg/kg)
AUC (hrs*nM)
5
3
5
5
5634
12296
2779
6092
6790
11305
763
25
18096
4
CL
26.6
ꢀ
36.8
ꢀ
9.2
ꢀ
12.8
ꢀ
(
mL/min/kg)
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Vss (L/kg)
9.7
ꢀ
21.2
770
ꢀ
13.5
ꢀ
15
ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cmax (nM)
Tmax (h)
T1/2 (h)
F(%)
1756
696
259
2329
1899
2526
169
7
0.03
6.2
ꢀ
7.0
ꢀ
0.03
9.1
ꢀ
7.0
ꢀ
0.03
21
8.0
13
56
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
ꢀ
55
66
ꢀ
119
1
Table 6. Mouse, rat, dog and monkey PK parameters of 15b. Formulated in a solution of 75%
2
polyethylene300 and 25% dextrose (5%) in water. Formulated in a solution of 30% propylene glycol
3
and 5% Solutol® in phosphateꢀbuffered saline Formulated in a suspension of 0.5% (w/v) aqueous
4
methylcellulose and 0.5% Tween 80. Formulated in a suspension of 0.5% (w/v) aqueous
methylcellulose.
Compound 15b was tested for in vivo efficacy in 2ꢀweek Karpas299 (sc injection of Karpas299
cells possessing the NPMꢀALK fusion) and H2228 (sc injection of H2228 cells possessing the
EML4ꢀALK fusion) rat xenograft models. Doses were chosen to be 6.25, 12.5, 25 and 50 mg/kg
daily dosing for the Karpas299 study and 5, 10, and 25 mg/kg daily dosing for the H2228 study.
The efficacy of 15b in these two models is summarized in Figures 6a and 6b. In the Karpas299
study, 15b induced a doseꢀdependent growth inhibition (Tumor growth inhibition factor [T/C] =
74% at 6.25 mg/kg; T/C = 30% at 12.5 mg/kg) and tumor regression (T/C = ꢀ33% at 25 mg/kg
and T/C = ꢀ66% at 50 mg/kg). In the H2228 study, 15b induced a doseꢀdependent growth
inhibition (T/C = 67% at 5 mg/kg; T/C = 2% at 10 mg/kg) and complete tumor regression at
25mg/kg (T/C = ꢀ100% at 25 mg/kg). In both models, 15b was well tolerated and no bodyꢀ
weight loss was observed at all doses tested (Figures S5 and S6, supporting information). These
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results are consistent with our previous observations (Karpas299 is a much more aggressive in
vivo model than H2228) as well with the relative potency of 15b against the NPMꢀALK and
EML4ꢀALK fusions (26.0 and 2.2 nM respectively)
0
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0
A singleꢀdose PK/PD experiment was performed in the rat Karpas299 model at 50 mg/kg, and
the biomarker pSTAT3 response correlated well with the tumor regression observed in the
xenograft study. Figure 7 shows the plot of plasma and tumor drug concentrations (leftꢀhand side
of the graph) versus pSTAT3 inhibition (rightꢀhand side of the graph). Upon singleꢀdose oral
administration at 50 mg/kg, sustained pSTAT3 inhibition (about 80%) was observed through 72
hours, which paralleled the sustained tumor concentrations of 15b.
In contrast to the firstꢀgeneration ALK inhibitor 4, compound 15b showed no impact on
insulin levels or plasma glucose utilization (tested via an oral glucose tolerance test) in the
mouse upon chronic dosing up to 100 mg/kg (data not shown). This lack of in vivo effect on
insulin receptor was confirmed using a homeostasis model assessment of insulin resistance
(HOMAꢀIR; OGTT/ITT ratio) in the same animals (Figure 8). This in vivo selectivity is not fully
understood at this time (the in vitro selectivity versus the InsR is comparable between 4 and 15b)
but indicates an additional advantage of 15b over 4.
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Figure 6a
Figure 6b
Figure 6. a. Efficacy of 15b in a 14ꢀday rat xenograft Karpas299 model. b. Efficacy of 15b in
a 14ꢀday rat xenograft H2228 model.
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Figure 7. Singleꢀdose PK/PD relationship of 15b (50 mg/kg) in the rat Karpas299 model.
Figure 8. HOMAꢀIR result of 15b and 4 after 7 days of dosing in mice.
Conclusions
In conclusion, we have described the synthesis of novel, selective and potent ALK inhibitors.
Medicinal chemistry intuition and rational design were used to specifically and successfully
address the extensive reactive adduct formation liability present in the firstꢀgeneration clinical
candidate 4. Compound 15b proved to have an acceptable overall profile (selectivity, ADME), a
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consistent PK profile across species (mouse, rat, dog and monkey) and displayed strong in vivo
efficacy in ALCL and NSCLC cancer xenograft models at wellꢀtolerated doses. Compound 15b
also showed an improved profile in terms of in vivo glucose homeostasis over 4 when examined
in mouse OGTT experiments. For these reasons, compound 15b was chosen to move into
10
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4
4
development and is currently being evaluated in ALKꢀpositive patients where it has displayed
4
5
substantial preliminary antitumor clinical activity.
Experimental section
Unless otherwise noted, materials were obtained from commercial suppliers and were used
without further purification. Removal of solvent under reduced pressure or concentration refers
to distillation using Bu� chi rotary evaporator attached to a vacuum pump (3 mmHg). Products
obtained as solids or high boiling oils were dried under vacuum (1 mmHg).
Purification of compounds by preparative RPꢀHPLC was achieved using a Waters
autopurification system consisting of a 2767 autosampler/fraction collector, a 2525 binary
gradient module, a 2487 UV detector, and a ZQ mass spectrometer. Compounds were purified
using flow rate of 30 mL/min with a 50 mm × 20 mm i.d. Ultra 120 5 ꢁm C18Q column (Peeke
Scientific, Novato, CA). A 7.5 min linear gradient from 10% solvent A (acetonitrile with 0.035%
trifluoroacetic acid) in solvent B (water with 0.05% trifluoroacetic acid) to 30ꢀ90% A was used,
followed by a 2.5 min hold at 90% A. Silica gel chromatography was performed either by
CombiFlash (separation system Sg. 100c, ISCO) or using silica gel (Merck Kieselgel 60, 230–
4
00 mesh). Elemental analyses were carried out by Midwest Microlabs LLC, Indianapolis, IN.
1
H NMR spectra were recorded on Bruker XWINꢀNMR (400 MHz) spectrometer. Proton
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2
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2
2
2
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5
5
6
1
resonances are reported in parts per million (ppm) downfield from tetramethylsilane (TMS). H
NMR data are reported as multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint,
quintuplet; sept septuplet; dd, doublet of doublets; dt, doublet of triplets; bs, broad singlet). For
spectra obtained in CDCl3, DMSOꢀd6, CD3OD, the residual protons (7.27, 2.50, and 3.31 ppm,
respectively) were used as the internal reference.
0
1
2
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0
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0
1
2
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9
0
Purity of Compounds
All compounds were analyzed with a Waters ZQ 2000 LC/MS system, which used an Agilent
binary pump and photodiode array detector (PDA) along with a Sedere 75 evaporative light
scattering detector (ELSD). Samples were injected from 96ꢀwell or 384ꢀwell plates with a Leap
Technologies HTS Pal autosampler. The mobile phases used were (A) H2O + 0.05% TFA and
(B) acetonitrile + 0.035% TFA. A gradient HPLC method with flow of 1.0 mL/min started at 5%
B, with a hold of 0.1 min before a linear increase to 95% B at 2.70 min. At 2.71 min the B
mobile phase was increased to 100% and held until 2.98 min. Total run time was 3.0 min. The
column used was a Waters Atlantis dC18 2.1 mm × 30 mm, 3 ꢁm. The mass spectrometer was
operated in positive mode, with a spray voltage of 3.2 kV and cone voltage of 30 V. The source
and desolvation temperatures were 130 and 400 °C, respectively, with 600 L/h of nitrogen
desolvation flow. The progress of reactions and the purity of products were measured using 254
and 220 nm wavelengths and electrospray ionization (ESI) positive mode. Mass spectra were
obtained in ESI positive mode. All the reported compounds were found to be >95% when
analyzed at the 254 nm wavelength.
Synthesis of 2ꢀchloroꢀNꢀ(2ꢀ(isoꢀpropylsulfonyl)phenyl)ꢀ5ꢀmethylpyrimidinꢀ4ꢀamine 9b
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To a suspension of 730 mg of NaH in a mixture of DMF/DMSO (25/2.5 ml) was added dropꢀ
o
wise at 0 C 2.53 g (12.69 mmol) of 2ꢀ(iso-propylsulfonyl)benzenamine 8a in DMF/DMSO (10
o
ml, ratio 9/1). The solution was stirred for 30 minutes at 0 C and then 4.11 g (25.3 mmol, 2 eq.)
10
11
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of 2,4ꢀdichloroꢀ5ꢀmethylpyrimidine diluted in 10 ml of DMF/DMSO (ratio: 9/1) was added
slowly. The solution was warmed to room temperature and stirred overnight. After aqueous
workꢀup, the crude product was directly crystallized from cold CH CN in several batches to
3
afford 2.53
g
(7.75 mmol, 61%) of 2ꢀchloroꢀNꢀ(2ꢀ(isoꢀpropylsulfonyl)phenyl)ꢀ5ꢀ
+
1
methylpyrimidinꢀ4ꢀamine 9b as pale creamy colored crystals: ESMS m/z 326.1 (M + H ). H
NMR (400 MHz, DMSOꢀd6): δ 9.29 (s, 1H), 8.41 (dd, J = 0.9, 8.3 Hz, 1H), 8.23 (d, J = 0.8 Hz,
1H), 7.93 – 7.78 (m, 2H), 7.48 – 7.37 (m, 1H), 3.58 – 3.43 (m, 1H), 2.18 (s, 3H), 1.16 (d, J = 6.8
Hz, 6H).
Synthesis of 2,5ꢀdichloroꢀNꢀ(2ꢀ(isoꢀpropylsulfonyl)phenyl)pyrimidinꢀ4ꢀamine 9a
Using the same procedure described for the synthesis of 2ꢀchloroꢀNꢀ(2ꢀ(iso-
propylsulfonyl)phenyl)ꢀ5ꢀmethylpyrimidinꢀ4ꢀamine
9b,
2,5ꢀdichloroꢀNꢀ(2ꢀ(isoꢀ
propylsulfonyl)phenyl)pyrimidinꢀ4ꢀamine 9a was isolated in 60% yield as a creamy colored
+
1
solid from 8a and 2,4,5ꢀtrichloropyrimidine: ESMS m/z 346.0 (M + H ). H NMR (400 MHz,
DMSOꢀd6): δ 9.81 (s, 1H), 8.57 (s, 1H), 8.32 (d, J = 8.3 Hz, 1H), 7.96 – 7.82 (m, 2H), 7.56 –
7.42 (m, 1H), 3.61 – 3.46 (m, 1H), 1.16 (d, J = 6.8 Hz, 6H).
Synthesis of 2ꢀchloroꢀ4ꢀfluoroꢀ5ꢀnitrotoluene 10b.
To a solution of 100 g (0.7 mol) of 2ꢀchloroꢀ4ꢀfluorotoluene in 250 ml of concentrated H SO
2
4
3
o
was added portionꢀwise 85 g (0.875 mol) of KNO at 0 C (addition of the total amount of KNO
3
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was complete in about 1 hour). The reddish mixture was slowly warmed to room temperature,
stirred overnight, then poured over crushed ice and extracted with EtOAc. The organic layers
were combined, dried over MgSO and concentrated. The crude oil was purified over a large
4
0
1
2
3
4
5
6
7
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9
0
1
2
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0
silica plug (eluent: 97/3 hexanes/EtOAc) to afford 94.7 g (0.5 mol, yield: 71%) of 2ꢀchloroꢀ4ꢀ
1
fluoroꢀ5ꢀnitrotoluene 10b as a pale yellow oil that solidifies upon standing. H NMR (CDCl ,
3
400 Mz): δ 7.97 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 10.4 Hz, 1H), 2.43 (s, 3H).
Synthesis of 2ꢀchloroꢀ4ꢀiso-propoxyꢀ5ꢀnitrotoluene 11b
To a solution of 25 g (0.131 mol) of 2ꢀchloroꢀ4ꢀfluoroꢀ5ꢀnitrotoluene 10b in 250 ml of 2ꢀ
o
propanol was added 208 g (0.659 mol, 5 eq.) of Cs CO . The mixture was stirred at 60 C
2
3
overnight and after cooling to room temperature, most of the 2ꢀpropanol was evaporated under
reduced pressure. Water was added and the solution was extracted with EtOAc. The organic
layers were combined, dried over MgSO , concentrated and the crude product filtered through a
4
silica plug (eluent: 95/5 hexanes/EtOAc) to afford 28.7 g (0.125 mol, 95%) of 2ꢀchloroꢀ4ꢀisoꢀ
1
propoxyꢀ5ꢀnitrotoluene 11b as a pale yellow fluffy solid. H NMR (400 MHz, DMSOꢀd6): δ
7.90 (d, J = 0.6 Hz, 1H), 7.52 (s, 1H), 4.92 – 4.76 (m, 1H), 2.30 (s, 3H), 1.27 (d, J = 6.0 Hz, 6H).
Synthesis of 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀphenyl)ꢀpyridine 12b
4ꢀPyridineboronic acid (147 mg, 1.20 mmol, 1.1 eq.) was dissolved in a 2:1 v/v mixture of
dioxane and H O (15 mL) and N was bubbled through for 5 minutes. Pd dba (100 mg, 0.109
2
2
2
3
mmol, 0.1 eq.), 2ꢀdicyclohexylphosphineꢀ2’ꢀ6’ꢀdimethoxy biphenyl (112 mg, 0.272 mmol, 0.25
eq.), 1ꢀchloroꢀ5ꢀisoꢀpropoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀbenzene (11b, 250 mg, 1.09 mmol, 1.0 eq.) and
K PO (462 mg, 2.18 mmol, 2.0 eq.) were added under a N blanket. The reaction vessel was
3
4
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9
o
sealed and heated with microwave irradiation to 150 C for 20 min. After cooling to room
temperature, the reaction was diluted with ethyl acetate and washed twice with 1 N aqueous
NaOH. The organic layer was then dried over Na SO and filtered. After concentration, the
2
4
10
11
12
13
14
15
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crude product was purified by silica gel chromatography (gradient from hexanes to 30% ethyl
acetate in hexanes) to give 217 mg (0.798 mmol, 73%) of 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀ
+
phenyl)ꢀpyridine (12b) as a brown solid. ESMS m/z 273.1 (M + H ).
Synthesis of 2ꢀiso-propoxyꢀ5ꢀmethylꢀ4ꢀ(1ꢀmethylꢀpiperidinꢀ4ꢀyl)ꢀphenylamine (13d)
4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀphenyl)ꢀpyridine (12b, 217 mg, 0.798 mmol) was dissolved
in anhydrous THF (9 mL). Iodomethane (0.10 mL, 1.61 mmol, 2 eq.) was added and the
o
reaction was stirred at 40 C in a sealed tube for 2 days. The volatiles were removed under
vacuum generating 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀphenyl)ꢀ1ꢀmethylꢀpyridinium iodide as a
brown solid directly used in the next step: ESMS m/z 287.1 (M+).
4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitroꢀphenyl)ꢀ1ꢀmethylꢀpyridinium iodide (0.80 mmol) was
o
dissolved in CH OH (20 mL) and cooled to 0 C. NaBH (302 mg, 8.0 mmol, 10 eq.) was slowly
3
4
added. After this addition was complete, the cooling bath was removed and the reaction was
stirred at room temperature for 2.5 h. The reaction was quenched by the slow addition of 1N
aqueous HCl (14 mL). The CH OH was partially removed by vacuum and the resulting residue
3
was partitioned between EtOAc and 1 N aqueous NaOH. Additional 50% aqueous NaOH was
added until the aqueous layer pH>12. The EtOAc layer was washed twice with 1 N aqueous
NaOH, the organic layer was then dried over Na SO , filtered, and concentrated under vacuum.
2
4
After concentration, the crude product (175 mg) was dissolved in acetic acid (10 mL). TFA
(
0.15 mL, 3 eq.) and PtO (53 mg, 30% w/w) were added and the reaction was placed under 50
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psi H gas in a Parr Shaker for 14 h. The reaction mixture was filtered and the filtrate
2
concentrated under vacuum. The resulting residue was partitioned between EtOAc and 1 N
aqueous NaOH. Additional 50% aqueous NaOH was added until the aqueous layer pH>12. The
EtOAc layer was washed with 1 N aqueous NaOH (2x), and then the organic layer was dried
over Na SO , filtered, and concentrated under vacuum to give 2ꢀiso-propoxyꢀ5ꢀmethylꢀ4ꢀ(1ꢀ
0
1
2
3
4
5
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8
9
0
1
2
3
4
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3
4
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7
8
9
0
2
4
+
methylꢀpiperidinꢀ4ꢀyl)ꢀphenylamine 13b (161 mg, 77%): ESMS m/z 263.2 (M + H ).
Synthesis of 4ꢀ(4ꢀaminoꢀ5ꢀiso-propoxyꢀ2ꢀmethylphenyl)piperidinꢀ2ꢀone 13e
1ꢀchloroꢀ5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitrobenzene 11b (275 mg, 1.20 mmol, 1.0 eq.) was
dissolved in 1ꢀbutanol (6 mL) and N is bubbled through for 5 minutes. Pd dba (55 mg, 0.06
2
2
3
mmol, 0.05 eq.), 2ꢀdicyclohexylphosphineꢀ2’ꢀ6’ꢀdimethoxy biphenyl (50 mg, 0.12 mmol, 0.1
eq.), (2ꢀmethoxypyridinꢀ4ꢀyl)boronic acid (239 mg, 1.56 mmol, 1.3 eq.) and K PO (497 mg, 2.4
3
4
mmol, 2.0 eq.) were added under a N blanket. The reaction vessel was sealed and heated at
2
o
1
50 C overnight. After cooling to room temperature, the solvent was removed under reduced
pressure and the residue was purified by silica gel chromatography (gradient from 0% to 20%
ethyl acetate in hexanes) to give 130 mg of 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitrophenyl)ꢀ2ꢀ
+
1
methoxypyridine as yellow oil. ESMS m/z 303.1 (M + H ). H NMR (CD OD, 400 Mz): δ 8.21
3
(
d, J = 5.6 Hz, 1H), 7.70 (s, 1H), 7.08 (s, 1H), 6.96 (dd, J = 5.6, 1.6 Hz, 1H), 6.79 (s, 1H), 4.75
m, 1H), 3.97 (s, 3H), 2.22 (s, 3H), 1.34 (d, J = 6.0 Hz, 6H).
(
Synthesis of 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitrophenyl)ꢀ2ꢀmethoxypyridine (130 mg, 0.43
mmol) was dissolved in 1,4ꢀdioxane (8.0 mL) and concentrated HCl (1.0 mL) was added. The
resulting mixture was refluxed overnight. After cooling to room temperature, the solvent was
removed under reduced pressure and the crude product, 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀ
nitrophenyl)pyridinꢀ2ꢀol, was used directly for the next step without further purification. ESMS
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Journal of Medicinal Chemistry
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+
1
m/z 289.1 (M + H ). H NMR (CD OD, 400 Mz): δ 8.00 (d, J = 6.4 Hz, 1H), 7.65 (s, 1H), 7.12
3
(s, 1H), 7.05 (dd, J = 6.4, 1.6 Hz, 1H), 6.92 (d, J = 1.6 Hz, 1H), 4.70 (m, 1H), 2.19 (s, 3H), 1.26
(
d, J = 6.0 Hz, 6H).
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To the above product, 4ꢀ(5ꢀiso-propoxyꢀ2ꢀmethylꢀ4ꢀnitrophenyl)pyridinꢀ2ꢀol, in TFA (8.0 mL)
was added PtO (9.8 mg). The mixture was purged with H and stirred at room temperature
2
2
overnight under 1 atm. H (g). After filtration through celite, the filtrate was concentrated to
2
yield the crude product, 4ꢀ(4ꢀaminoꢀ5ꢀiso-propoxyꢀ2ꢀmethylphenyl)piperidineꢀ2ꢀone 13e, which
+
was used directly for the next step without further purification. ESMS m/z 263.1 (M + H ).
Synthesis of tertꢀbutyl 4ꢀ(5ꢀaminoꢀ4ꢀiso-propoxypyrimidinꢀ2ꢀyl)piperidineꢀ1ꢀcarboxylate 13f
A degazed mixture of 109 mg (0.5 mmol) of 2ꢀchloroꢀ4ꢀisoꢀpropoxypyrimidinꢀ5ꢀamine, 230
mg (0.75 mmol) of tertꢀbutyl 4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ5,6ꢀ
dihydropyridineꢀ1(2H)ꢀcarboxylate, Pd(OAc) (10 mg, 0.044 mmol), Xantphos (20 mg, 0.034
2
o
mmol), K PO (350 mg, 1.5 mmol) in 1.5 mL of THF was heated to 85 C for 24 hrs. After
3
4
cooling at RT, the mixture was filtered, concentrated, dissolved in 5 mL of dry MeOH and 5 mg
of Pd/C was added. The resulting mixture was flushed with N and placed under H atmosphere
2
2
(
balloon) ON. After filtration and concentration, the residue was purified over SiO column to
2
afford 20 mg (0.059 mmol, yield: 12%) of tertꢀbutyl 4ꢀ(5ꢀaminoꢀ4ꢀiso-propoxypyrimidinꢀ2ꢀ
+
yl)piperidineꢀ1ꢀcarboxylate 13f as an oil. ESMS m/z 337.2 (M + H ).
Synthesis of 4ꢀ(4ꢀAminoꢀ5ꢀiso-propoxyꢀ2ꢀmethylꢀphenyl)ꢀpiperidineꢀ1ꢀcarboxylic acid tertꢀ
butyl ester 13b
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