Discovery of the macrocycle 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7, 26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27) ,16,21,23-decaene (SB1518), a potent Janus Kinase 2/Fms-like tyrosine kinase-3 (JAK2/FLT3) inhibitor for the treatment of myelofibrosis and lymphoma

ARTICLE

pubs.acs.org/jmc

Discovery of the Macrocycle 11-(2-Pyrrolidin-1-yl-ethoxy)-14,

19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-

1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a Potent

Janus Kinase 2/Fms-Like Tyrosine Kinase-3 (JAK2/FLT3) Inhibitor

for the Treatment of Myelofibrosis and Lymphoma

Anthony D. William,* Angeline C.-H. Lee, Stꢀephanie Blanchard, Anders Poulsen, Ee Ling Teo,

Harish Nagaraj, Evelyn Tan, Dizhong Chen, Meredith Williams, Eric T. Sun, Kee Chuan Goh,

Wai Chung Ong, Siok Kun Goh, Stefan Hart, Ramesh Jayaraman, Mohammed Khalid Pasha,

Kantharaj Ethirajulu, Jeanette M. Wood, and Brian W. Dymock

S*BIO Pte. Ltd., 1 Science Park Road, #05-09, The Capricorn, Singapore Science Park II, Singapore 117528

S Supporting Information

b

ABSTRACT: Discovery of the activating mutation V617F in Janus Kinase 2

(JAK2^V617F), a tyrosine kinase critically involved in receptor signaling,

recently ignited interest in JAK2 inhibitor therapy as a treatment for

myeloﬁbrosis (MF). Herein, we describe the design and synthesis of a series

of small molecule 4-aryl-2-aminopyrimidine macrocycles and their biological

evaluation against the JAK family of kinase enzymes and FLT3. The most

promising leads were assessed for their in vitro ADME properties culminat-

ing in the discovery of 21c, a potent JAK2 (IC₅₀= 23 and 19 nM for JAK2^WT

and JAK2^V617F, respectively) and FLT3 (IC₅₀= 22 nM) inhibitor with

selectivity against JAK1 and JAK3 (IC₅₀= 1280 and 520 nM, respectively).

Further proﬁling of 21c in preclinical species and mouse xenograft and allograft models is described. Compound 21c (SB1518) was

selected as a development candidate and progressed into clinical trials where it is currently in phase 2 for MF and lymphoma.

’ INTRODUCTION

reduce splenomegaly and improve symptoms in phase 1 and 2

clinical trials in MF patients.^13ꢀ15

The Janus family of kinases (JAK1, JAK2, JAK3, and TYK2)

are intracellular nonreceptor tyrosine kinases that transduce

cytokine-mediated signals via the JAK-STAT (signal transducer

and activator of transcription) pathway and hence play an

important role in the control of cell proliferation, cell diﬀerentia-

tion, and survival.^1,2In 2005, the discovery of the V617F

mutation in JAK2 (JAK2^V617F) catalyzed signiﬁcant eﬀorts in

our laboratories to bring a new small molecule JAK2 inhibitor

therapy to the clinic for the treatment of a wide spectrum of

hematological malignancies, particularly the myeloproliferative

neoplasms (MPNs) and lymphoma.^3ꢀ7The MPNs are a spec-

trum of poorly treated diseases including polycythemia vera

(PV), essential thrombocythemia (ET), and primary, post-PV,

and post-ET myeloﬁbrosis (MF). MF is the most serious disease

with the most severe patients having a life expectancy of between

1.5 to 5 years. The MF disease course is characterized by

symptomatic splenomegaly, progressive anemia leading to fati-

gue, and severe constitutional symptoms including night sweats,

fever, bleeding, bone pain, and frequent infections.⁸Inhibition of

the JAK-STAT signaling pathways⁹in MF patients has given

encouraging initial responses in recent clinical trials,^10ꢀ12and

21c (SB1518) described herein, has demonstrated an ability to

Rationale for use of JAK2 inhibitors in lymphoma is supported

by a number of studies: activation of wild-type JAK2 in lympho-

ma cells by a mutation in the SOCS-1 gene (suppressor of

cytokine signaling); discovery that a high level of a micro-RNA

species targeting JAK2 (miR-135a) correlates with a favorable

prognosis in classical Hodgkin lymphoma patients; and increased

JAK2 gene expression in a number of lymphoma subtypes.^16ꢀ19

Furthermore, JAK2 has been implicated in inﬂammatory disease

such as rheumatoid arthritis and psoriasis with encouraging initial

clinical data.^20,21

Selectivity for JAK2 over JAK3 was strongly desirable due to

the reported immunosuppressive eﬀects of inhibiting JAK3.

Deﬁcient JAK3 signaling in humans and mice causes severe

combined immunodeﬁciency (SCID).²²The eﬀects of adding

JAK1 and/or TYK2 activity to a JAK2 inhibitor are still not well

understood.

Overexpression and activating mutations of receptor tyrosine

kinases (RTKs) are known to be involved in the pathophysiology

Received: March 21, 2011

Published: May 23, 2011

r

2011 American Chemical Society

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|

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ARTICLE

of diverse human cancers. Further to JAK-STAT driven disease,

we sought to broaden the potential application of a JAK2

inhibitor by reﬁning additional RTK activity discovered in our

leads. FLT3 (fms-like tyrosine kinase-3), a class III RTK, is the

most frequently mutated gene in acute myeloid leukemia (AML)

and plays an important role in the maintenance, growth, and

development of hematopoietic and nonhematopoietic cells.

Mutations of the FLT3 receptor can lead to the development

of AML.^23ꢀ27Hence, we were attracted to the potential of a

JAK2/FLT3 combined inhibitor proﬁle. As MF, lymphoma, and

AML continue to present as unmet medical needs in hematology,

JAK2/FLT3 inhibitor therapy could oﬀer new hope for the

development of a safe and eﬀective therapy.

Figure 1. Inception of macrocyclization from an in-house library

screening hit.

therefore, linkers with some hydrophilic character (use of oxygen

atoms) and not containing any groups known to be rapidly

metabolized were prioritized.

Atthe timeof theﬁrst reports ofthe JAK2^V617Fmutation,^4ꢀ6we

wereengagedin a search for novelkinaseinhibitormotifs. Wewere

evaluating a series of compounds which exhibited a unique kinase

inhibitory spectrum with JAK2 and FLT3 inhibition being promi-

nent. Rationale for JAK2 inhibitor therapy in the MPNs was

strengthening, and encouraged by the additional desirable FLT3

activity we were seeing in our leads, we focused our attention on

the rapid identiﬁcation of a clinical candidate which would meet

the desired requirements for MF and lymphoma therapy with

potential for activity in other JAK2 and FLT3-driven diseases:

potent and selective JAK2/FLT3 inhibition with an excellent

safety proﬁle and oral daily dosing. Herein, we describe the

discovery and structureꢀactivity relationship (SAR) of a series

of small molecule macrocycles culminating in the identiﬁcation of

21c, the compound that we selected for preclinical development

and then progressed into clinical trials. Compound 21c is showing

clinical beneﬁt in patients with MF and lymphoma with many

patients remaining on therapy in ongoing phase 1 and 2 trials in

these indications.²⁸

Synthesis of the macrocycles is made possible by taking

advantage of the RCM reaction pioneered by Grubbs. Synthesis

of the precursors for RCM was achieved by the coupling of the

two halves of the molecules via acidic displacement between the

chloropyrimidines and anilines.³⁶In general, RCM of the dienes

was achieved by employing either Grubbs second generation

catalyst ((C₄₆H₆₅Cl₂N₂PRu): [1,3-bis(2,4,6-trimethylphenyl)-

2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexyl-

phosphine)ruthenium) or Zhan-1B catalyst ((RuCl₂[C₂₁

H₂₆N₂][C₁₂H₁₇NO₃S]): 1,3-Bis(2,4,6-trimethylphenyl)-4,5-

dihydroimidazol-2-ylidene[2-(i-propoxy)-5-(N,N-dimethylamino-

sulfonyl)phenyl]methyleneruthenium(II) dichloride).^37,38All

products were isolated as inseparable mixtures of approximately

85:15 trans:cis double bond geometry. Scheme 1 illustrates the

synthetic routes for compounds 16aꢀf.

Left-hand fragments were constructed by Suzuki coupling of

commercially available 2,4-dichloropyrimidine (3a) and boronic

acids 4aꢀb aﬀording the corresponding biaryl alcohols 5aꢀb

(Scheme 1).^39,40Alkylation of the alcohols was achieved either

under phase transfer conditions with allyl bromide, giving

allylethers 6aꢀb, or with cesium carbonate assisted displacement

with 4-bromo-but-1-ene furnishing homoallyl ether, 7.^41,42

Because of their diversity, right-hand fragments were prepared

using a wide variety of methods, as illustrated in Schemes 1 and 2.

Commercially available alcohols 10aꢀb (Scheme 1) were alky-

lated as described above by either allyl bromide or 4-bromo-but-

1-ene to produce the corresponding 11aꢀb and 13, respectively.

Iron-assisted reduction of the nitro group gave key anilines

12aꢀb and 14, respectively, in good to excellent yields.⁴³

Precursors of the RCM reaction, dienes 15aꢀe, were prepared

in moderate yield from coupling of Suzuki products 6aꢀb and 7

and anilines 12aꢀb and 14 in the presence of HCl under reﬂux

conditions. Macrocyclization of 15aꢀe proceeded eﬃciently in

the presence of Grubbs second generation catalyst with hydro-

chloric acid as an additive to aﬀord products 16aꢀf in moderate

yields.

Sequences (1ꢀ8) in Scheme 2 show the synthetic routes to

obtain 12cꢀi. The displacement reaction of 8b with corre-

sponding secondary amines aﬀorded 9aꢀd in quantitative

yields. Alkylation of 8cꢀd proceeded smoothly with 1,2-

dichloroethane to aﬀord 9eꢀf.⁴⁴Benzaldehydes 9aꢀf were

eﬃciently reduced to the corresponding benzylic alcohols

10cꢀh using sodium borohydride. By employing phase trans-

fer conditions with allyl bromide, the alkylated intermediates

were then reduced via the aforementioned iron conditions to

aﬀord anilines 12cꢀh in good to excellent yields. Aniline 12i

was prepared by alkylation of the benzylic alcohol 18 using

’ CHEMISTRY

In-house library screening revealed compound 1 which showed

broad kinase inhibition with reasonable activity in cell lines tested;

however, these rather ubiquitous pyrimidine-based motifs are

heavily patented with very narrow possibilities for developing

proprietary compounds.²⁹We envisioned that by connecting the

open ends of 1, to form macrocycles 2, the binding mode to the

kinase hinge region is not compromised (Figure 1). Thus, we

embarked on the synthesis of small molecule macrocycles by

exploiting the powerful ring-closing metathesis (RCM) reaction

as a synthetic tool.^30ꢀ32We were inspired by macrocyclic natural

products which, although usually complex and diﬃcult to synthe-

size, have evolved to speciﬁcally interact with biological systems in

ways not easily achievable with the much simpler small molecule

compounds found in screening libraries.^33ꢀ35It seemed attractive to

us to explore the potential for diverse but speciﬁc functionalization

possible within the ring structure of 2, conformationally deﬁned by

the semirigid arrangement of the A, B, and C rings. Whereas open

chain compounds will adopt energetically favorable conformations

that are not necessarily complementary to the target binding site, the

ability to position a chosen group at a desired point in space with

limited conformational freedom using a constrained macrocyclic

structure could have beneﬁts leading to greater speciﬁcity between

kinases. The choice of initial linkers, Z, was driven by synthetic

concerns and our desire to avoid very lipophilic and ﬂexible linkers

of straight carbon chains which would dramatically increase log P

and likely be vulnerable to phase 1 metabolism. Solubility and

metabolic stability were primary concerns from the early stages;

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Scheme 1^a

^aReactions and conditions: (a) Pd(PPh₃)₄, Na₂CO₃, 1,2-dimethoxyethane, water, 80 °C, 52ꢀ64%. (b) Allyl bromide, KOH, TBAHSO₄, CH₂Cl₂, rt,

38ꢀ68%. (c) 4-Bromobut-1-ene, CsCO₃, DMF, 40 °C, 52ꢀ64%. (d) Fe powder, NH₄Cl, EtOH, water, 80 °C, 71ꢀ96%. (e) 4 M HCl, n-butanol,

80ꢀ100 °C, 21ꢀ56%. (f) Grubbs second generation catalyst, HCl, CH₂Cl₂, 40ꢀ45 °C, 43ꢀ51%. (g) NaSEt, DMF, 120 °C, 30%.

phase transfer conditions. Reduction of both nitro groups gave

bis-aniline 20, which was monoprotected using Fmoc-Cl

aﬀording a quantitative yield of 12i. Phenol 16f was obtained

though demethylation of 16e using NaSEt.⁴⁵

Compounds 21kꢀl were prepared ﬁrst by basic removal of the

Fmoc group from 21j, prepared as described for 21g, followed by

amidation with 3-diethylamino-propionic acid (sequence 8 in

Scheme 2).

Biaryl 6b underwent chloride displacement with anilines

12cꢀf to furnish the respective dienes that were macrocyclized

with either Grubbs second generation or Zhan-1B catalyst to

aﬀord macrocyles 17a,b,d, and h in moderate yield. Compound

17c was obtained via the removal of the Boc group from 17b. The

free amine on the piperazine ring was further derivatized by

alkylation, acetylation, and sulfonylation to aﬀord 17eꢀg, re-

spectively, with moderate yields. The intermediate 21a, formed

from coupling of 6b and 12g under reﬂux conditions in the

presence of n-butanol, was further derivatized by displacement

reactions with various secondary amines employing microwave

conditions in DMA solvent, aﬀording the more soluble products

21bꢀf. Similarly, 21 hꢀi were obtained from 21g using the same

displacement method. The intermediate 21g was made in

moderate yield by the coupling of meta-aniline 12h and 6b.

Scheme 3 illustrates the synthetic route toward the prepara-

tion of diversely substituted compounds 22aꢀg. Commercially

available 8c and 8e were subjected to the aforementioned

alkylation conditions with 1,2-dichloroethane, followed by

NaBH₄reduction and phase transfer allylation with allyl bromide

to give 11g and 11i, respectively. Pyrrolidine displacement gave

11jꢀk, which were then reduced to the anilines 12jꢀk in

excellent yields.⁴⁶The right-hand fragments were obtained from

diﬀerent commercially available pyrimidines 3aꢀc and boronic

acids 4bꢀf. Following standard Suzuki couping, aldehydes 5d

and 5h and ester 5f were readily reduced to the corresponding

benzyl alcohols, which were alkylated using phase transfer

conditions with allyl bromide to give 6bꢀh in moderate yields.

Compounds 22aꢀg were then prepared using the above-de-

scribed displacement and RCM.

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Scheme 2^a

^aReactions and conditions: (a) K₂CO₃, DMF, 90 °C, quantitative. (b) NaBH₄, THF, water, 0 °C, quantitative. (c) Allyl bromide, KOH, TBAHSO₄,

CH₂Cl₂, rt, 38ꢀ68%. (d) Fe powder, NH₄Cl, EtOH, water, 80 °C, 71ꢀ96%. (e) 1,2-Dichloroethane, K₂CO₃, DMF, 100 °C, 54ꢀ72%. (f) Fmoc-Cl,

DIEA, 1,4-dioxane, rt, 95%. (g) 4 M HCl, n-butanol, 80ꢀ100 °C, 21ꢀ92%. (h) Either Grubbs second generation catalyst or Zhan catalyst-1B, HCl,

CH₂Cl₂, 40ꢀ45 °C, 43ꢀ96%. (i) TFA, CH₂Cl₂, 40 °C, 73%. (j) 2-Bromoethanol, Et₃N, CH₂Cl₂, rt, 34%. (k) Acetyl chloride, Et₃N, CH₂Cl₂, rt, 63%. (l)

Ethanesulfonyl chloride, K₂CO₃, THF, 70 °C, 41%. (m) DMA, microwave 80 °C, 72ꢀ90%. (n) 20% v/v piperidine in CH₂Cl₂, rt, 82%. (o)

3-(Diethylamino)propionic acid hydrochloride, HOBt, EDCI, CH₂Cl₂, 55%.

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Scheme 3^a

^aReactions and conditions: (a) 1,2-dichloroethane, K₂CO₃, DMF, 100 °C, 54ꢀ72%. (b) NaBH₄, THF, water, 0 °C, quantitative. (c) Allyl bromide,

KOH, TBAHSO₄, CH₂Cl₂, rt, 38ꢀ68%. (d) Pyrrolidine, DMA, 90 °C, 66ꢀ76%. (e) Fe powder, NH₄Cl, EtOH, water, 80 °C, 74ꢀ90%. (f) Pd(PPh₃)₄,

Na₂CO₃, 1,2-dimethoxyethane, water, 80 °C, 40ꢀ66%. (g) DIBAL, CH₂Cl₂, 0 °C, 48%. (h) 4 M HCl, n-butanol, 80ꢀ100 °C, 21ꢀ91%. (i) Either

Grubbs second generation catalyst or Zhan catalyst-1B, HCl, CH₂Cl₂, 40ꢀ45 °C, 32ꢀ79%.

’ RESULTS AND DISCUSSION

quite potent against JAK2 and retained good selectivity over

JAK1 and 3 as well as CDK2. For example, 16e has a JAK2 IC₅₀of

70 nM and is 27-, 17-, and 12-fold selective against JAK1, JAK3,

and CDK2, respectively. TYK2 SAR tended to broadly track with

JAK2. We decided to focus on progression of the most potent

dibenzylic compounds 16e and 16f. At this early stage, we were

already concerned about very low solubility in these high cLogP

compounds (16b and 16d were <10 μg/mL in PBS buffered

at pH 7.0); hence, the search for locations to install a solubilizing

group was paramount.

Development of More Soluble Compounds: Exploration

of Substituents on the C-Ring Oriented toward Solvent.

Docking of 16e into the JAK2 ATP binding site supported the

hypothesis that the expected hinge binding mode is preferred with

hydrogen bonding between the amino-pyrimidine and the back-

bone Leu932 residue (Figure 2A). An additional hydrogen bond

between the benzylic ether oxygen and Ser936 could explain the

higher potency of 16cꢀf compared to that of phenolic 16aꢀb.

Clearly, the macrocycle structure fills out the available space in the

Selection of the Preferred Linker. Macrocycles as protein

kinase inhibitors have been explored by few other groups with no

advanced candidates being reported to our knowledge. We aimed

to identify a distinct core structure with clear novelty and

structural rigidity which would in turn reduce conformational

freedom, potentially increasing the likelihood of finding com-

pounds with specific selectivity profiles. Hence, we decided to

focus on linkers retaining the double bond conveniently installed

directly from the RCM reaction. A study of various linkers with

and without R1 groups demonstrated that these templates

inhibited JAK2/FLT3 with selectivity against JAK1/3 and

CDK2 (Table 1). In general, the most potent JAK2 linkers were

8 atoms in length with at least one benzylic ether, as in

compounds 16c, e, and f. Very good selectivity over JAK1,

JAK3, and CDK2 was already apparent especially with bis-

phenolic ether 16a and phenolic-benzylic ether 16c. Symmetrical

dibenzylic linkers, as employed in compounds 16e and 16f, were

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Table 1. SAR Lead Identiﬁcation: Search for a Suitable Linker for Selectivity toward JAK2

^aAll compounds were isolated as inseparable c.85:15 trans/cis mixtures. ^bAll IC₅₀values are the mean ( SD (of at least two independent experiments).

The assays were run at S*BIO using TG101348 as a reference: JAK1 IC₅₀= 120 nM, JAK2 IC₅₀= 7 nM, JAK3 IC₅₀= 2650 nM (published values:⁹JAK1

IC₅₀= 105 nM, JAK2 IC₅₀= 3 nM, JAK3 IC₅₀= 996 nM). ^cNot determined.

Figure 2. Compound 16e docked into the ATP-binding site of JAK2. Compound 16e is shown as a thick tube with green carbon. Hydrogen bonds

between 16e and JAK2 are shown as purple/black dashed lines. (A) JAK2 is shown in thin tube with gray carbon. The backbone of Leu932 forms

two hydrogen bonds with 16e and the side chain of Ser936 hydrogen bonds with an ether-oxygen in the macrocyclic linker. Leu855, Val863, Ala880,

and Leu983 (in the bottom of the binding site) have hydrophobic contacts with the aromatic rings of 16e. (B) JAK2 is shown as a surface. The

methoxy group of 16e is pointing toward a groove on the JAK2 surface that may accommodate a solubility-tag enabling ﬁne-tuning of molecular

properties.

binding site quite well but with opportunities for building

additional interactions with the protein from various sites such

as the pyrimidine, which we hypothesized could potentially

accommodate small lipophilic groups to interact with the

gatekeeper methionine residue Met929. Another possibility

for additional interactions and to attach a solubilizing group

is from the R1 position. Figure 2B shows that the R1 substituent

points directly toward a channel out to the solvent which may

offer potential for additional interactions as well as increase in

solubility by installation of a basic center.

Exploration of this solvent channel from both the R1 and R2

positions proved to be quite fruitful (Table 2). A range of N-

and O-substituents were very well tolerated with, on the whole,

sub-100 nM IC₅₀values against the primary targets JAK2 and

FLT3. Sterically large groups such as morpholine 17a and

piperazines 17cꢀd were potent and selective, although spar-

ingly soluble in aqueous media. Improved solubility was

achieved with the polar hydroxyethyl piperazine 17e; however,

improvement in JAK2 activity seemed elusive with nitrogen-

linked compounds, even with the less sterically encumbered

open-chain analogues 17h and amide 21l. Compound 17h is

noteworthy due to its surprising CDK2 potency, likely due to a

salt bridge interaction with Asp86 made possible by the methyl

substitution on the nitrogen of the side-chain, which allows the

side-chain to adopt an energetically favorable conformation for

interaction between Asp86 and the charged dimethylamine (see

Supporting Information, Figure S1). However, improved po-

tency was achieved with oxygen-linked substituents: compounds

21bꢀf, containing an aminoethylether side-chain, were uni-

formly potent against JAK2 and selective against JAK1/3 and

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Table 2. SAR Optimization: Study of Suitable Solubilizing Groups

c

^aAll IC₅₀values are the mean ( SD (of at least two independent experiments). ^bHigh throughput solubility in PBS buﬀered at pH 7.0. Not

determined.

CDK2 whether or not they contained a basic center. Nonbasic

21e suﬀered a reduction in FLT3 as well as TYK2 activity and was

likely to be less soluble than analogues with charged side chains at

physiological pH; hence, the preferred compounds from this

series were 21b,c,d and f. The direct R2 analogue of 21c, 21h, was

also potent against JAK2 but with submicromolar CDK2 activity.

Morpholine analogue 21i, however, was less CDK2 potent but

suﬀered a 5-fold reduction in JAK2 potency over the most potent

R₁compounds. Nitrogen-linked anline 21k and amide 21l were

also potent JAK2/FLT3 inhibitors but had reduced selectivity

against CDK2. TYK2 SAR was in general quite ﬂat for these

between the amino-pyrimidine and the backbone Leu932

residue as well as the hydrogen bond to Ser936 from one of

the oxygens in the macrocyclic linker. An additional notable

interaction between the basic nitrogen of the pyrrolidine,

forming a salt bridge to Asp939, could explain the improved

potency against JAK2 (Figure 3A). The macrocyclic structure

of 21c with the pendant side-chain fills out the available space

in the binding site with opportunities to fine-tune hydropho-

bic interactions with the protein from various sites of the

molecule, such as the A-ring (biaryl phenyl) and B-ring

(pyrimidine). From the docking analysis, we hypothesized

that these two rings could potentially accommodate small

lipophilic groups that could complement the hydrophobic

area bound by the gatekeeper Met929, Glu930, Ala880, and

Val863 (Figure 3B).

compounds with the notable exception of 21l (TYK2 IC₅₀

=

18 nM). Hence, only a limited study of R₂substituents was carried

out. Aminoethylethers 21bꢀd containing side-chains were prior-

itized for further evaluation in DMPK assays.

Development of More Potent Compounds: Exploration of

A-Ring and B-Ring SARs. Docking of 21c into the JAK2 ATP

binding site confirmed the expected hinge binding mode

Indeed 5⁰-methyl substituted pyrimidine 22b exhibited 4-fold

higher potency for JAK2 (IC₅₀6 nM) as compared to 21c

(Table 3 and Figure 4). The methyl group in the 5-position of the

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Figure 3. Compound 21c docked into the ATP-binding site of JAK2. Compound 21c forms the same hydrogen bonds with JAK2 as 16e. In addition,

the solubilizing pyrrolidine side-chain forms a salt bridge with Asp939. There are opportunities for hydrophobic interactions with (A) Met929 through

derivatization of the 5⁰-pyrimidine position and (B) with Gly993/Asp994 and Lys857 accessible from the A-ring.

Table 3. SAR Exploration of Aromatic Ring Substitutions with Small Groups

JAK2 IC₅₀

(μM)^a

JAK1 IC₅₀

(μM)^a

JAK3 IC₅₀

(μM)^a

TYK2 IC₅₀

(μM)^a

FLT3 IC₅₀

(μM)^a

CDK2 IC₅₀

(μM)^a

solubility

cLogP (μg/mL)^b

cpd

R₂

R₃

R₄

R₅

R₆

22a OCH₃

H

F

H

0.036 ( 0.003 1.7 ( 0.28

0.007 ( 0.000 1.0 ( 0.0

ND^c

0.089 ( 0.004 0.057 ( 0.004 0.019 ( 0.001 >10

0.17 ( 0.021 0.006 ( 0.001 3.4 ( 1.1

4.5

4.6

4.7

4.6

5.2

4.5

4.7

154

22b

22c

22d

22e

22f

H

CH₃

F

178

0.017 ( 0.000 0.83 ( 0.085 1.0 ( 0.00

0.33 ( 0.00 4.9 ( 0.85 7.2 ( 0.21

0.024 ( 0.002 0.38 ( 0.014 ND

0.14 ( 0.028 0.015 ( 0.003 1.55 ( 0.071

0.62 ( 0.064 0.012 ( 0.001 >10

ND

2.61

60.83

H

OCH₃

H

0.036 ( 0.003 0.008 ( 0.004 2.0 ( 0.42

H

OCH₃0.019 ( 0.002 >10

0.025 ( 0.001 4.6 ( 0.35

0.89 ( 0.042 0.18 ( 0.00

0.72 ( 0.11 ND

0.092 ( 0.011 >10

22g

H

F

0.040 ( 0.015 3.25 ( 0.071

ND

^aAll IC₅₀values are the mean ( SD (of at least two independent experiments). ^bHigh throughput solubility in PBS buﬀered at pH 7.0. Not

determined.

c

pyrimidine ﬁts snugly into the hydrophobic area bound by the

gatekeeper Met929, Glu930, Ala880, and Val863. Although

having good solubility, this compound was compromised some-

what by its selectivity against JAK3 which had dropped to about

15-fold. This may be due to the increase in torsion angle between

the two aromatic rings leading to a binding conformation

favorable for JAK3. Furthermore, this conformation docks less

well into the CDK2 binding site with the compound ﬁtting less

well into the hydrophobic area.

hydrophobic groups are ultimately preferred, but selectivity to-

ward JAK1 and JAK3 was retained (Table 3). This compound was

also more potent against CDK2 but retained selectivity of 91-fold.

Substitution on the A ring was studied with a small group of

synthetically accessible compounds. Substitution with methoxy at

R₄(22d) is unfavorable for JAK family activity but suggests that

this area could be an avenue for the preparation of selective FLT3

inhibitors (22d FLT3 IC₅₀12 nM). In contrast, substitution with

electron-withdrawing ﬂuoro at R₅(22e) is well tolerated but

reduces selectivity over JAK1 as well as being of very low solubility.

Small lipophilic groups at the R₆position, such as methoxy 22f,

Replacing the 5⁰-methyl group with electron-withdrawing

ﬂuoro (22c) reduced JAK2 activity by 3-fold, conﬁrming that

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Figure 4. Compound 22b docked into the ATP-binding site of JAK2 (A) and with the surface shown (B). The solvent-accessible surface of JAK2

surrounding the methyl-pyrimidine substituent is shown in peach. The methyl is an excellent ﬁt to this part of the binding site.

Figure 5. Compound 22f docked into JAK2 (A) and CDK2 (B). The 4-methoxy substituent of the A-ring points up into the hydrophobic area in JAK2

while it is in the plane of the A-ring in the CDK2 pose making unfavorable contact with the charged side chains of Lys33 and Asp145.

appear to bewell tolerated on this ringexhibitingexcellentpotency

and selectivity for JAK2 over JAK1. Although the eﬀects of adding

JAK1 activity to a JAK2 inhibitor are poorly understood, we

prioritized more selective compounds in the expectation that they

would have reduced oﬀ-target toxicity. In general, solubility seems

to be lower with substitutions on this ring, possibly as a result of

high log P. The favorable potency and excellent selectivity of 22f

were suggested from Figure 3B and explained in Figure 5 where

the extra methoxy group makes favorable hydrophobic interac-

tions with JAK2 but has unfavorable clashes with Lys33 and

Asp145 of CDK2.

Adding an additional substituent to the right-hand side aniline

ring of 21c, to give the electron rich 22a did not compromise JAK2

activity or selectivity and showed improved FLT3 activity as

compared with 21c and, curiously, solubility. It is not apparent

why there are such solubility diﬀerences within the series. Generally

all compounds in this series were satisfactorily selective against

CDK2 with TYK2 activity being up to 10-fold less active than JAK2.

Further in Vitro Profiling of Preferred Compounds. Com-

pounds with JAK2 and FLT3, IC₅₀<25 nM, SI >25 for JAK1/3

and SI >100 for CDK2, were shortlisted for further celluar and in

vitro ADME profiling (Table 4). Inhibition of the major meta-

bolizing cytochrome P450 isozymes 3A4 and 2D6 were deter-

mined to identify any potential drugꢀdrug interaction liabilities.

Human and mouse liver microsomal stabilities give an indication

of significant phase 1 metabolism events in target species.

Physicochemical measurements assessing solubility and perme-

ability were included as important indicators of oral absorption as

well as cell penetration. Pharmacological activity in cell lines

reflective of the target enzymes focused initially on cell prolifera-

tion using a Ba/F3 cell line harboring the JAK2 V617F mutation

and MV4ꢀ11 cells harboring the drug resistant FLT3 D835Y

mutation. Good potency in these cell lines was observed for all

compounds with IC₅₀values in the range of 100ꢀ180 nM in Ba/

F3 cells and 37ꢀ85 nM in MV4ꢀ11 cells. Furthermore, 21c

inhibited the JAK2-mediated production of p-STAT5 and

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Table 4. In Vitro Proﬁling Leading to Selection of in Vivo Candidate 21c

IC₅₀Selectivity Index

Ba/F3-JAK2

JAK2 IC₅₀JAK1/ JAK3/ TYK2/ CDK2/

cpd (μM)^aJAK2 JAK2 JAK2 JAK2 IC₅₀(μM)^a(μg/mL)^bIC₅₀(μM)^a,cIC₅₀(μM)^a,c(min)

FLT3

solubility CYP3A4

CYP2D6 HLM t_1/2MLM t_1/2V617F GI₅₀MV4ꢀ11 PAMPA P_app

(min)

(μM)

GI₅₀(μM) (x 10^ꢀ6cm/s)

21b 0.024

21c 0.023

21d 0.015

22b 0.007

146

56

26

23

50

13

3.3 196

2.2 170

5.6 180

8.1 >1000

9.5 >500

0.029

0.022

0.026

0.019

0.15

147

2.8

>10

>5^d

>10

44

16

22

11

40

29

0.15

0.16

0.18

0.10

0.037

0.047

ND^e

15.8

9.3

>150

ND^e

>150

60.83

>5^d

0.37

2.5

>60

63

3.93

16.8

10.1

143

0.051

22f

0.019

>500 47

>10

0.085

^aAll IC₅₀values are the means (of at least two independent experiments). ^bHigh throughput solubility in PBS buﬀered at pH 7.0. CYP activity

c

determined in the human liver microsome system, see experimental section for details. ^d20% inhibition at 5 μM. ^eNot determined.

Table 5. Physico-Chemical Properties and in Vitro ADME of

21c

Table 6. Oral Pharmacokinetic Parameters of 21c in Mice,

Rats, and Dogs^a

property

value

mouse^b

parameter

(n = 3/time point)

rat^c(n = 3)

dog^c(n = 4)

mol. wt.

472.58

1

no. of HBD

no. of HBA

cLogP

dose (mg/kg)

C_max(ng/mL)

t_max(h)

30

10

3

6

292

1.0

114 ( 25

4 ( 0

11.5 ( 11.8

2.0^d

4.1

PSA (Å²)

69

t_1/2(h)

AUC_0-t(ng h/mL)^e

^aNote: In preliminary PK screening studies, 21c showed oral bioavail-

ability of 39%, 24%, and 10% in mice, dogs, and rats, respectively.

^bMean. ^cMean ( SD. ^dMedian. ^et = 8 h.

0.84

5.7 ( 1.3

599 ( 111

3.4 ( 0.8

53 ( 53

HLM (t_1/2, min)

>60

41

399

3

DLM (t_1/2, min)

RLM (t_1/2, min)

18

MLM (t_1/2, min)

22

human CYP inhibition IC₅₀(μM)^a

permeability (P_{app, AfB}, ꢁ10^ꢀ6cm/s)^c

plasma protein binding (%)^din human

plasma protein binding (%) in dog

plasma protein binding (%) in mouse

>5^b

22b and 21c are differentiated by a single methyl group which

generates subtle, but important, differences in the compound

profiles. Compound 21c exhibits an overall balanced profile

meeting all target criteria. It was also shown to be active against

the V617F mutant of JAK2 with IC₅₀= 19 nM and the D835Y

mutantof FLT3withIC₅₀= 6 nM, and its proteinkinaseselectivity

was confirmed by testing against more than 50 other protein

kinases covering all major families of the human protein kinome.¹⁹

Thus, 21c was selected for further proﬁling (Table 5). Pre-

ferred properties for an orally administered drug are all exhibited

by 21c: molecular weight <500, number of hydrogen bond

donors <5 and acceptors <10, and cLogP <5 and polar surface

area <120 Å². None of the ﬁve major drug metabolizing human

CYP450s (1A, 3A4, 2D6, 2C9, and 2C19) is inhibited by 21c

implying low drugꢀdrug interaction potential. A Caco-2 bidir-

ectional permeability assay conﬁrmed that 21c has high perme-

ability and was not a substrate for P-glycoprotein (eﬄux ratio of

0.8), hence predicting high intestinal absorption. In vitro plasma

protein binding to human plasma was high (99.9%), but taken

together, the in vitro ADME proﬁle of 21c supports daily oral

dosing.

16

99.88

99.63

99.41

^aHuman CYP3A4, 1A2, 2D6, 2C9, 2C19. ^bPrecipitation observed at

highest concentration of 25 μM. No signiﬁcant inhibition observed at

c

5 μM. Caco-2 bidirectional permeability assay. ^dEquilibrium dialysis

assay in human plasma at 1000 ng/mL.

p-STAT3 dose dependently in Ba/F3 cells, and the production of

p-FLT3 and p-STAT5 in MV4ꢀ11 cells (see Supporting Informa-

tion, Figures S2 ansd S3). This evidence of intracellular target

inhibition was further supported by high permeability and solubility.

Differences in metabolic parameters were more apparent between

compounds: CYP 3A4 was a particular area of differentiation where

22b, although exhibiting desirable single digit nanomolar potency

toward JAK2, had an IC₅₀of 2.5 μM for CYP3A4. Although stable in

liver mircosomes, 22b had lower selectivity for JAK3. CYP3A4

inhibition was clearly an undesirable feature of piperidine analogue

21d, reducing our interest in further studies with this compound.

Diethylamine analogue 21b suffered from low stability in mouse and

moderate stability in human microsomes in addition to significant

CYP3A4 inhibition. Although the CYP inhibition for 21b and 22b

was moderate, it could not be excluded as these plasma concentra-

tions could be reached in human subjects, and the compounds were

therefore not further tested. However, 21c and 22f were much less

active against CYP3A4 (20% inhibition at 5 μM and IC₅₀>10 μM,

respectively), and both compounds had good selectivity and

microsomal stability. Compound 22f, although possessing excellent

enzyme selectivity, was less active against FLT3. Methyl pyrimidine

Pharmacokinetics of 21c in Multiple Species. The PK

properties of 21c are summarized in Table 6. Compound 21c

showed rapid absorption in mice (t_max=1.0 h), a mean C_maxand

AUC of 292 ng/mL and 399 ng h/mL, respectively, with a

3

mean terminal half-life of 0.8 h following a single oral dose of

30 mg/kg. The promising efficacy of 21c in several preclinical

pharmacology models (see below) was consistent with this

exposure. In rats, 21c showed moderately fast absorption (t_max=4h),

with a peak concentration of 114 ng/mL, AUC of 599 ng h/mL, and

3

a terminal half-life of ∼6 h following a single oral dose of 10 mg/kg. In

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Figure 6. Compound 21c reduces splenomegaly and hepatomegaly in a Ba/F3-JAK2^V617Fmodel of leukemia. Ba/F3-JAK2^V617F-bearing nude mice

were dosed p.o., b.i.d., with 75 and 150 mg/kg 21c (HCl salt) for 13 consecutive days. On day 13, mice were sacriﬁced and spleen and liver weights

determined (n = 8; * p < 0.05, **p < 0.01 ANOVA, Dunnett’s post test). The dotted line represents baseline weights.

Figure 7. Compound 21c is eﬃcacious in a xenograft derived from a cell line harboring FLT3-ITD. MV4ꢀ11 tumor bearing nude mice were

randomized into 4 groups of 8ꢀ10 animals each. Mice were treated orally (p.o.) once daily at doses of 25, 50, or 100 mg/kg of 21c for 21 consecutive

days, and median time to end point (= median survival) was determined on day 55. The ﬁgures given indicate doses calculated using the molecular weight

of the free base. The Log-rank (Mantel-Cox) test was used for statistical analysis.

dogs, 21c was rapidly absorbed (t_max= 2.0 h), with a peak

Efficacy of 21c in Mouse Models. On the basis of its efficacy

on JAK2 and FLT3 dependent tumor cell lines¹⁹and good oral

bioavailability, 21c was selected for evaluation in mouse tumor

concentration of ∼12 ng/mL, AUC of 53 ng h/mL, and a terminal

3

half-life of 3.4 h following a single oral dose of 3 mg/kg.

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models. Two models were selected on the basis of their relevance

to the molecular targets: Ba/F3-JAK2^V617Fand MV4ꢀ11 allo-

graft and xenograft studies representing cell lines dependent on

mutant JAK2 and FLT3 signaling, respectively.

cell lines driven by mutant JAK2 and FLT3, and target inhibition

in the cell has been demonstrated. The favorable oral pharma-

cokinetic properties of 21c prompted us to explore its pharma-

codynamic eﬀects in relevant mouse models in a dose-dependent

manner. An eﬃcacy study in a model of JAK2^V617F-driven disease

demonstrated dose-dependent tumor growth inhibition¹⁹and

normalization of splenomegaly and hepatomegaly at well-toler-

ated doses. In a second study in a model of FLT3-ITD driven

leukemia, 21c demonstrated signiﬁcant survival beneﬁts at very

well-tolerated doses. Extensive cellular proﬁling of 21c on

The Ba/F3-JAK2^V617Fmouse allograft represents a model for

a JAK2-driven disease exhibiting hallmark symptoms of myelo-

proliferative diseases such as splenomegaly and hepatomegaly. In

this model, murine Ba/F3-JAK2^V617Fcells were engrafted by tail

vein injection. Treatment with 21c at doses of 75 and 150 mg/kg

p.o. b.i.d. was started 4 days after cell inoculation for 13

consecutive days. At study termination, vehicle control mice

exhibited splenomegaly and hepatomegaly (∼7-fold and 1.6-fold

respectively); 21c treatment at 150 mg/kg p.o. b.i.d. signiﬁcantly

ameliorated the disease symptoms, with 42% normalization of

spleen weight and 99% normalization of liver weight (Figure 6).

For evaluation of the in vivo eﬃcacy of 21c on FLT3-ITD

driven tumors, MV4ꢀ11 xenografts were established in nude

mice. To identify eﬀects on tumor growth delay, MV4ꢀ11 tumor

bearing mice (average starting tumor size of 130 mm³) were

treated orally once daily at doses of 25, 50, or 100 mg/kg for 21

consecutive days. After termination of the treatment, tumor

growth was measured up to day 55, and median time-to-end

point (= median survival) was calculated. The end point of the

experiment for an individual mouse was reached on day 55 or

when the tumor reached a size of 1000 mm³, whichever occurred

earlier. Figure 7 shows the KaplanꢀMeier survival curves for the

diﬀerent groups. The median survival was 33.0 days for the

vehicle group. The lowest dose-treated group (25 mg/kg 21c)

did not show statistically signiﬁcant activity with survival of

35.0 days. However, the 50 mg/kg and 100 mg/kg 21c treatment

groups showed a signiﬁcantly increased median survival of 55 days

(p < 0.01) with no signiﬁcant body weight loss in the treated

animals at the termination of the study. Clearly, 21c is eﬀective at

lower doses in the MV4ꢀ11 model than in the more aggressive

Ba/F3 JAK2^V617Fmodel. This is partly explained by diﬀerences

in the degree of oncogene addiction in the two models, reﬂected

in the cell viability IC₅₀of both cell lines: the MV4ꢀ11 cell line is

more sensitive than the Ba/F3 JAK2^V617Fcell line (MV4ꢀ11

showed an approximately 4-fold diﬀerence in sensitivity com-

pared to that of Ba/F3 JAK2^V617F); this may account for the

diﬀerence in dose required in vivo. Both 75 mg/kg and 150 mg/kg

bid doses are well tolerated in the Ba/F3 inoculated animals with

no signiﬁcant body weight loss.

myeloid and lymphoid cells and its activity in the JAK2^V617F

-

driven SET-2 model have been reported elsewhere.¹⁹Its attrac-

tive preclinical proﬁle led to the selection of 21c for preclinical

development, which in turn resulted in progression into the clinic

where it has shown clinical beneﬁt in myeloﬁbrosis^13,14and

lymphoma patients in two completed phase 2 trials.⁴⁸

’ EXPERIMENTAL SECTION

Chemistry. Workup for chemical reactions was typically done by

diluting the reaction mixture or residue with the reaction solvent or

extraction solvent and then washing with the indicated aqueous

solution(s). Product solutions were dried over anhydrous sodium sulfate

prior to filtration, and the solvents were removed under reduced

pressure using a rotary evaporator. All compounds were purified by

either flash or reversed phase chromatography. Flash column chroma-

tography was conducted using Silica gel 60 (Merck KGaA, 0.040ꢀ0.063

mm, 230ꢀ400 mesh ASTM). Reverse-phase preparative high perfor-

mance liquid chromatography (rpHPLC) was operated on a Phenom-

enex column (Luna 5 μm C18 (2) 100A 150 ꢁ 21.2 mm) with

adjustable solvent gradients, usually 5 to 95% acetonitrile in water þ

0.1% trifluoroacetic acid (TFA), with a run time of 18 min, at a flow rate

of 20 mL/min were used for routine purification. Fractions containing

the desired product were lyophilized or evaporated to dryness under

vacuum to provide the dry compound, or evaporated to remove volatile

organic solvents, then extracted with a suitable organic solvent (ethyl

acetate or dichloromethane were commonly used; if necessary, the pH of

the aqueous solution was adjusted in order to get free base, acid, or the

neutral compound).

The preliminary purity and identity of all compounds was assessed

post-puriﬁcation by tandem HPLC/mass spectral (LC/MS) analyses on

a Waters Micromass ZQ mass spectrometer in electrospray ionization

(ESI) positive mode after separation on Waters 2795 Separations

Module. The HPLC separations were performed on a Phenomenex

column (Luna 5 μm C18 (2) 100A 50 ꢁ 2.00 mm) with a ﬂow rate of

0.8 mL/min and a 4 min gradient of xꢀ100% acetonitrile in water þ

0.05% TFA (x = 5, or 30, or 50), using a Waters 2996 photodiode array

detector. Purity and identity were assessed on the integrated UV

chromatogram (220ꢀ400 nm) and the mass spectrum (homogeneity

of the product peak and its fragmentation(s)). The ﬁnal purity was

determined using Waters 2695 Separations Module on a Waters Xterra

RP18 3.5 mm 4.6 ꢁ 20 mm IS column with a ﬂow rate of 2.0 mL/min,

gradient 5ꢀ65% B over 4 min, then 65ꢀ95% B over 1 min, and 95% B

for an additional 0.1 min (solvent A, H₂O with 0.1% TFA; solvent B,

acetonitrile with 0.1% TFA), and a Waters 2996 photodiode array

detector. For compounds not suitable for the above HPLC methods

either due to polarity or poor peak separation, a longer column

(Phenomenex Gemini 5 μm C18, 110A, 4.6 ꢁ 150 mm) together with

a ﬂow rate of 1.0ꢀ1.2 mL/min and a 15 min gradient of 5ꢀ95%

acetonitrile in water þ 0.1% TFA was used for purity determination.

Purity was >95% for all test compounds except those indicated in their

respective synthesis descriptions.

’ CONCLUSIONS

We have described the discovery of a series of small molecule

macrocycles as potent inhibitors of JAK2 and FLT3 kinases. SAR

studies using data from biochemical and ADME assays led to

potent JAK2 inhibitors with an appropriate balance of physico-

chemical and ADME properties suitable for daily oral dosing.

Our original hypothesis of using a macrocyclic structure with

limited conformational options by constraining the binding

moieties at speciﬁc points in space has produced a novel series

of JAK2/FLT3 selective inhibitors able to achieve interactions in

the active sites of the target enzymes not easily accomplished

with an acyclic structure. A subset of diverse compounds showed

good potency and selectivity for JAK2 within the JAK family,

good potency for FLT3, and also retaining potency for TYK2.

Importantly, selectivity for CDK2 was assured in this series,

which is not the case with at least one reported clinical-stage

JAK1/2 inhibitor.⁴⁷Cell proliferation was potently inhibited in

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1

All the 1D and 2D NMR experiments for H (400.13 MHz), ¹³C

(100.61 MHz), ¹⁵N (40.55 MHz), and ¹⁹F (376.47 MHz) nuclei were

performed on a Bruker AVANCE-400 digital NMR spectrometer.

3-(2-Chloro-pyrimidin-4-yl)-4-methoxy-benzaldehyde (5h). The ti-

tle compound was synthesized from 3a and 5-formyl-2-methoxyphenyl

boronic acid (4f) (yield, 84%). LC-MS (ESI positive mode) m/z 249

([M þ H]^þ).

¹Hꢀ H, ¹Hꢀ C, and ¹Hꢀ N 2D experiments (COSY, HMQC,

HSQC, and HMBC) were run with Z-gradient selection. NMR spectra

are reported in ppm with reference to an internal tetramethylsilane

standard (0.00 ppm for ¹H and ¹³C) or solvent peak(s) of CDCl₃(7.26

and 77.1 ppm) or CD₃OD (3.31 and 49.0 ppm), or DMSO-d₆(2.50 and

39.5 ppm). Other NMR solvents were used as needed. When peak

multiplicitiesare reported, thefollowing abbreviationsare used: s=singlet,

d = doublet, t = triplet, q = quartet, m = multiplet, br = broadened, dd =

doublet of doublets, dt = doublet of triplets, and bs = broadened singlet.

Coupling constants, when given, are reported in Hertz.

1

13

15

[3-(2-Chloro-5-methyl-pyrimidin-4-yl)-phenyl]-methanol (5j). The

title compound was synthesized from 2,4-dichloro-5-methylpyrimidine

(3b) and 4b (yield, 56%). LC-MS (ESI positive mode) m/z 235 ([M þ

H]^þ).

[3-(2-Chloro-5-fluoro-pyrimidin-4-yl)-phenyl]-methanol (5k). The

title compound was synthesized from 2,4-dichloro-5-fluoropyrimidine

(3c) and 4b (yield, 74%). LC-MS (ESI positive mode) m/z 239 ([M þ

H]^þ).

[5-(2-Chloro-pyrimidin-4-yl)-2-methoxy-phenyl]-methanol (5e).

To a solution of 5d (5 g, 20.1 mmol) in THF (25 mL, 5 vol) at 0 °C

was added NaBH₄(0.76 g, 20.1 mmol), and the resulting mixture was

stirred for 30 min before warming to rt. The reaction mixture was

quenched with water. The product was extracted with CH₂Cl₂thrice,

and the combined organic extracts were washed with H₂O followed by

brine, dried over Na₂SO₄, and concentrated under reduced pressure to

furnish without purification 5 g of compound 5e (yield, quantitative).

LC-MS (ESI positive mode) m/z 251 ([M þ H]^þ).

All melting points are uncorrected. Elemental analyses of CHN were

performed on Perkin-Elmer 2400 CHN/CHNS Elemental Analyzers.

HRMS was obtained from a Bruker microTOF-Q II with direct

injection.

3-(2-Chloro-pyrimidin-4-yl)-phenol (5a). To a degassed (nitrogen)

solution of 2,4-dichoropyrimidine (3a) (1.0 g, 6.71 mmol) and 3-hydro-

xylphenyl boronic acid (4a) (1.1 g, 8.05 mmol) in 1,2 dimethoxyethane

(10 mL) was sequentially added aqueous Na₂CO₃(1.06 g, 10.06 mmol)

and Pd(PPh₃)₄(0.387 g, 0.335 mmol). The resultant mixture was stirred

at 80ꢀ85 °C for 4 h, cooled to 0 °C, and quenched with saturated

NH₄Cl. The product was extracted with CH₂Cl₂thrice and the

combined organic extracts were washed with brine, dried over Na₂SO₄,

and concentrated under reduced pressure. The crude mixture was

column purified (EtOAc/Hexane) to furnish 0.550 g of 5a (yield,

[3-(2-Chloro-pyrimidin-4-yl)-5-fluoro-phenyl]-methanol (5g). To a

cooled mixture of 5f (560 mg, 2.0 mmol) in CH₂Cl₂(6 mL, 10 vol) at

0 °C, 1.0 M DIBAL solution (4 mL, 4.00 mmol) was added dropwise.

The resulting mixture was stirred for 1 h and allowed to warm to rt.

Water was added, and the product was extracted with CH₂Cl₂(3 ꢁ

10 mL). The combined organic extracts were washed with brine, dried

(Na₂SO₄), and concentrated under reduced pressure to obtain the crude

residue, which was purified by column chromatography (using 35%

ethyl acetate in hexane) to furnish 5g (230 mg; yield, 48%) as a colorless

oil. LC-MS (ESI positive mode) m/z 239 ([M þ H]^þ).

40%). LC-MS (ESI positive mode) m/z 207 ([M þ H]^þ). H NMR

1

(400 MHz, CDCl₃): δ 9.74 (s, 1H), 9.23 (d, 1H), 8.83 (d, 1H), 8.01 (dd,

1H), 7.60ꢀ7.65 (m, 1H), 7.35 (t, 1H), 6.94ꢀ6.99 (m, 1H).

[3-(2-Chloro pyrimidine-4-yl) phenyl] Methanol (5b). The synthesis

for this intermediate has been improved for scale-up and was prepared

using a different Suzuki coupling procedure as compared to that of 5a.

To a degassed (nitrogen) solution of 3a (50 g, 335 mmol) and

3-(hydroxymethyl)phenylboronic acid (4b) (48.5 g, 318 mmol) in

THF (500 mL, 10 vol) was added saturated Na₂CO₃(88.6 g, 838

mmol), palladium acetate (0.15 g, 0.67 mmol), and triphenylphosphine

(0.35 g, 1.34 mmol). The resultant mixture was stirred at 70 °C for 6 h,

and filtered. Water was added to the filtrate and extracted with ethyl

acetate (3 ꢁ 250 mL). The combined organic extracts were washed with

brine, dried (Na₂SO₄), and filtered. The filtrate was treated with

activated charcoal, and the mixture was filtered and the filtrate concen-

trated under reduced pressure, and the crude product was slurried in

isopropyl alcohol (300 mL). The mixture was filtered, and the solid was

slurried in cold n-heptane and filtered to furnish 5b (42.8 g; yield, 61%).

This material was taken up for the next step without any purification.

LC-MS (ESI positive mode) m/z 221 ([M þ H]^þ); ¹H NMR (DMSO-

d₆) δ 8.81 (d, 1H), 8.15 (br, 1H), 8.10 (d, 1H), 8.04 (dt, 1H), 7.57ꢀ7.54

(m, 1H), 4.63 (s, 2H); ¹³C NMR (DMSO-d₆) δ 166.7, 161.4, 160.8,

143.9, 134.7, 130.5, 129.4, 126.1, 125.4, 116.4, 62.9.

[3-(2-Chloro-pyrimidin-4-yl)-4-methoxy-phenyl]-methanol (5i).

The title compound was synthesized following a procedure similar to

that of 5e, from 5h (yield, quantitative). LC-MS (ESI positive mode)

m/z 251 ([M þ H]^þ).

4-(3-Allyloxymethyl phenyl)-2-chloro Pyrimidine (6b). A mixture of

5b (20 g, 90.6 mmol), KOH (23.8 g, 172 mmol), and tetrabutyl

ammonium hydrogen sulfate (1.5 g, 4.53 mmol) in allyl bromide

(80 mL, 4 vol) was stirred at rt for 12 h. The reaction mixture was

quenched with water and extracted with CH₂Cl₂(3 ꢁ 25 mL). The

combined organic extracts were washed with water, brine, dried

(Na₂SO₄), and concentrated under reduced pressure to obtain the

crude residue, which was purified by column chromatography (using

15% ethyl acetate in hexane) to furnish 6b (13 g; yield, 55%) as a pale

yellow oil. LC-MS (ESI positive mode) m/z 261 ([M þ H]^þ); ¹H NMR

(CDCl₃) δ 8.62 (d, 1H), 8.06 (br, 1H), 8.00 (dt, 1H), 7.66 (d, 1H),

7.54ꢀ7.46 (m, 2H), 6.02ꢀ5.93 (m, 1H), 5.27 (ddq, 2H), 4.60 (s, 2H),

4.08 (dq, 2H); ¹³C NMR (CDCl₃) δ 167.1, 161.9, 159.9, 139.7, 135.3,

134.6, 131.2, 129.7, 129.3, 126.6, 117.5, 115.4, 71.7, 71.6; IR (KBr

pellet): 1568, 1535, 1344, 1186, 1079 cm^ꢀ1

.

Following a procedure similar to that of 5a, the following intermedi-

ates were synthesized:

Following a procedure similar to that of 6b, the following intermedi-

ates were synthesized:

[5-(2-Chloro-pyrimidin-4-yl)-2-fluoro-phenyl]-methanol (5c). The title

compound was synthesized from 3a and [4-fluoro-3-(hydroxy-

methyl)phenyl] boronic acid (4c) (yield, 66%). LC-MS (ESI positive

mode) m/z 239 ([M þ H]^þ).

4-(3-Allyloxy-phenyl)-2-chloro-pyrimidine (6a). The title com-

pound was synthesized from 5a and allyl bromide (yield, 59%). LC-

MS (ESI positive mode) m/z 247 ([M þ H]^þ).

4-(3-Allyloxymethyl-4-fluoro-phenyl)-2-chloro-pyrimidine (6c).

The title compound was synthesized from 5c and allyl bromide (yield,

63%). LC-MS (ESI positive mode) m/z 279 ([M þ H]^þ).

4-(3-Allyloxymethyl-4-methoxy-phenyl)-2-chloro-pyrimidine (6d).

The title compound was synthesized from 5e and allyl bromide (yield,

51%). LC-MS (ESI positive mode) m/z 291 ([M þ H]^þ).

4-(3-Allyloxymethyl-5-fluoro-phenyl)-2-chloro-pyrimidine (6e).

The title compound was synthesized from 5g and allyl bromide (yield,

71%). LC-MS (ESI positive mode) m/z 279 ([M þ H]^þ).

5-(2-Chloro-pyrimidin-4-yl)-2-methoxy-benzaldehyde (5d). The ti-

tle compound was synthesized from 3a and 3-formyl-4-methoxyphe-

nylboronic acid (4d) (yield, 58%). LC-MS (ESI positive mode) m/z 249

([M þ H]^þ).

3-(2-Chloro-pyrimidin-4-yl)-5-fluoro-benzoic Acid Ethyl Ester (5f).

The title compound was synthesized from 3a and (3-fluoro-5-ethoxy-

carbonylphenyl) boronic acid (4e) (yield, 89%). LC-MS (ESI positive

mode) m/z 281 ([M þ H]^þ).

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4-(5-Allyloxymethyl-2-methoxy-phenyl)-2-chloro-pyrimidine (6f).

The title compound was synthesized from 5i and allyl bromide (yield,

55%). LC-MS (ESI positive mode) m/z 291 ([M þ H]^þ).

3.96 (t, 2H); ¹³C NMR (CDCl₃) δ 187.3, 164.2, 142.1, 130.6, 125.0,

124.6, 113.2, 69.4, 41.3.

Following a procedure similar to that of 9e, the following intermedi-

ates were synthesized:

4-(3-Allyloxymethyl-phenyl)-2-chloro-5-methyl-pyrimidine (6g).

The title compound was synthesized from 5j and allyl bromide (yield,

42%). LC-MS (ESI positive mode) m/z 275 ([M þ H]^þ).

3-(2-Chloro-ethoxy)-5-nitro-benzaldehyde (9f). The title compound

was synthesized from 3-hydroxy-5-nitrobenzaldehyde (8d) and 1,2-dichloro-

ethane (yield, 66%). LC-MS (ESI positive mode) m/z 230 ([M þ H]^þ).

2-(2-Chloro-ethoxy)-3-methoxy-5-nitro-benzaldehyde (9g). The title

compound was synthesized from 2-hydroxy-3-methoxy-5-nitro-benzalde-

hyde (8e) and 1,2-dichloroethane (yield, 64%). LC-MS (ESI positive

mode) m/z 260 ([M þ H]^þ).

4-(3-Allyloxymethyl-phenyl)-2-chloro-5-fluoro-pyrimidine (6h).

The title compound was synthesized from 5k and allyl bromide (yield,

67%). LC-MS (ESI positive mode) m/z 279 ([M þ H]^þ).

4-(3-But-3-enyloxy-phenyl)-2-chloro-pyrimidine (7). To a mixture of

5a (2.0 g, 9.68 mmol) and 4-bromo-but-1-ene (7.8 g, 5.80 mmol) in dry

DMF (10 mL) at ambient temperature was added cesium carbonate

(14.19 g, 43.55 mmol), and the resulting mixture was stirred at 40 °C for

6 h. The reaction mixture was cooled to 0 °C and quenched with water.

The product was extracted with CH₂Cl₂thrice, and the combined organic

extracts were washed with water followed by brine, dried over Na₂SO₄,

and concentrated under reduced pressure to furnish an oil, which was

purified by column (EtOAc/hexane) to obtain 1.61 g of 7 (yield, 64%).

LC-MS (ESI positive mode) m/z 261 ([M þ H]^þ); ¹H NMR (400 MHz,

DMSO-d₆): δ 8.82 (d, 1H), 8.12 (d, 1H), 7.77 (d, 1H), 7.70 (br s, 1H),

7.48 (t, 1H), 7.18 (dd, 1H), 5.86ꢀ5.98 (m, 1H), 5.16ꢀ5.24 (m, 1H),

5.09ꢀ5.13 (m, 1H), 4.13 (t, 2H), 2.49ꢀ2.56 (m, 2H).

Following a procedure similar to that of 5e, the following intermedi-

ates were synthesized:

(2-Methoxy-5-nitro-phenyl)-methanol (10b). The title compound

was synthesized from 2-methoxy-3-nitrobenzaldehyde 8a (yield,

quantitative). LC-MS (ESI positive mode) m/z 184 ([M þ H]^þ).

(2-Morpholin-4-yl-5-nitro-phenyl)-methanol (10c). The title com-

pound was synthesized from 9a (yield, quantitative). LC-MS (ESI

positive mode) m/z 239 ([M þ H]^þ).

4-(2-Hydroxymethyl-4-nitro-phenyl)-piperazine-1-carboxylic Acid

tert-Butyl Ester (10d). The title compound was synthesized from 9b

(yield, quantitative). LC-MS (ESI positive mode) m/z 338 ([M þ H]^þ).

[2-(4-Methyl-piperazin-1-yl)-5-nitro-phenyl]-methanol (10e). The

title compound was synthesized from 9c (yield, quantitative). LC-MS

(ESI positive mode) m/z 252 ([M þ H]^þ).

2-(4-Methyl-piperazin-1-yl)-5-nitro-benzaldehyde (9c). To a mix-

ture of 2-chloro-5-nitrobenzaldehyde 8b (3 g, 16.2 mmol) and 1-methyl-

piperazine (3.5 mL, 31.6 mmol) in DMF (30 mL, 10 vol) was added

K₂CO₃(4 g, 28.9 mmol), and the resulting mixture was stirred at 90 °C

for 4 h. The reaction mixture was cooled to rt, and water (60 mL) was

added to quench. The aqueous layer was extracted thrice with EtOAc

(40 mL each), and the combined organic extract was washed with brine,

dried (Na₂SO₄), and concentrated under reduced pressure. Product 9c

(3.6 g; yield, 90%) was taken forward for the next step without any

{2-[(2-Dimethylamino-ethyl)-methyl-amino]-5-nitro-phenyl}-

methanol (10f). The title compound was synthesized from 9d (yield,

quantitative). LC-MS (ESI positive mode) m/z 254 ([M þ H]^þ).

[2-(2-Chloro ethoxy)-5-nitro-phenyl-methanol (10g). The title

compound was synthesized from 9e (yield, quantitative). LC-MS (ESI

positive mode) m/z 232 ([M þ H]^þ); ¹H NMR (DMSO-d₆) δ

8.27ꢀ8.26 (m, 1H), 8.17 (dd, 1H), 7.19 (d, 1H), 4.60 (s, 2H), 4.44

(t, 2H), 4.00 (t, 2H); ¹³C NMR (DMSO-d₆) δ 160.0, 141.4, 132.6,

124.4, 122.0, 111.8, 69.2, 57.5, 43.1.

1

purification. LC-MS (ESI positive mode) m/z 250 ([M þ H]^þ). H

NMR (CDCl₃): δ 10.10 (s, 1H), 8.62 (d, 1H), 8.30 (dd, 1H), 7.09

(d, 1H), 3.35ꢀ3.32 (m, 4H), 2.64ꢀ2.63 (m, 4H), 2.39 (s, 3H).

Following a procedure similar to that of 9c, the following intermedi-

ates were synthesized:

[3-(2-Chloro-ethoxy)-5-nitro-phenyl]-methanol (10h). The title

compound was synthesized from 9f (yield, quantitative). LC-MS (ESI

positive mode) m/z 232 ([M þ H]^þ);

2-Morpholin-4-yl-5-nitro-benzaldehyde (9a). The title compound

was synthesized from 8b and morpholine (yield, quantitative). LC-MS

(ESI positive mode) m/z 237 ([M þ H]^þ).

[2-(2-Chloro-ethoxy)-3-methoxy-5-nitro-phenyl]-methanol (10i).

The title compound was synthesized from 9g (yield, quantitative).

LC-MS (ESI positive mode) m/z 262 ([M þ H]^þ).

4-(2-Formyl-4-nitro-phenyl)-piperazine-1-carboxylic Acid tert-Butyl

Ester (9b). The title compound was synthesized from 8b and 1-Boc-

piperazine (yield, quantitative). LC-MS (ESI positive mode) m/z 336

([M þ H]^þ).

Following a procedure similar to that of 6a, the following intermedi-

ates were synthesized:

1-Allyloxy-3-nitro-benzene (11a). The title compound was synthe-

sized from 10a and allyl bromide (yield, 62%). LC-MS (ESI positive

mode) m/z 180 ([M þ H]^þ).

2-[(2-Dimethylamino-ethyl)-methyl-amino]-5-nitro-benzaldehyde

(9d). The title compound was synthesized from 8b and N,N,N⁰-trimethyl-

ethane-1,2-diamine (yield, 96%). LC-MS (ESI positive mode) m/z 252

([M þ H]^þ).

2-Allyloxymethyl-1-methoxy-4-nitro-benzene (11b). The title com-

pound was synthesized from 10b and allyl bromide (yield, 68%). LC-MS

(ESI positive mode) m/z 224 ([M þ H]^þ).

2-(2-Chloro ethoxy)-5-nitro-benzaldehyde (9e). To a mixture of

2-hydroxy-4-nitrobenzaldehyde (8c) (50 g, 299 mmol) and 1,2-dichlor-

oethane (300 mL, 6 vol) in DMF (600 mL, 12 vol)) was added K₂CO₃

(62.5 g, 450 mmol), and the resulting mixture was stirred at 100ꢀ105 °C

for 6 h. The reaction mixture was quenched with water (500 mL), and

the product was extracted four times with CH₂Cl₂(200 mL each). The

combined organic extracts were washed with water (500 mL), brine,

dried (Na₂SO₄), and concentrated under reduced pressure, and n-

hexane (500 mL) was added to the crude product. The resulting mixture

was stirred for 30 min and evaporated to dryness under reduced

pressure, and one more hexane washing was repeated. A third aliquot

of cold n-hexane (500 mL) was added, and the yellow solid was collected

by filtration and washing with cold n-hexane. Product 9e (49.3 g; yield,

72%) was taken forward for the next step without any purification. LC-

4-(2-Allyloxymethyl-4-nitro-phenyl)-morpholine (11c). The title

compound was synthesized from 10c and allyl bromide (yield, 55%).

LC-MS (ESI positive mode) m/z 279 ([M þ H]^þ).

4-(2-Allyloxymethyl-4-nitro-phenyl)-piperazine-1-carboxylic Acid

tert-Butyl Ester (11d). The title compound was synthesized from 10d

and allyl bromide (yield, 60%). LC-MS (ESI positive mode) m/z 378

([M þ H]^þ).

1-(2-Allyloxymethyl-4-nitro-phenyl)-4-methyl-piperazine (11e).

The title compound was synthesized from 10e and allyl bromide

(yield, 48%). LC-MS (ESI positive mode) m/z 292 ([M þ H]^þ).

N-(2-Allyloxymethyl-4-nitro-phenyl)-N,N⁰,N⁰-trimethyl-ethane-1,2-

diamine (11f). The title compound was synthesized from 10f and allyl

bromide (yield, 42%). LC-MS (ESI positive mode) m/z 294 ([M þ

H]^þ); ¹H NMR (CDCl₃) δ 8.35 (d, 1H), 8.12 (dd, 1H), 7.09 (d, 1H),

6.01ꢀ5.94 (m, 1H), 5.37ꢀ5.24 (m, 2H), 4.53 (s, 2H), 4.13ꢀ4.11 (m,

2H), 3.63ꢀ3.59 (m, 2H), 2.90ꢀ2.84 (m, 2H), 2.88 (s, 3H), 2.60 (s, 6H).

1

MS (ESI positive mode) m/z 230 ([M þ H]^þ); H NMR (CDCl₃)

δ 10.52 (s, 1H), 8.71 (d, 1H), 8.45 (dd, 1H), 7.14 (d, 1H), 4.51 (t, 2H),

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ARTICLE

2-Allyloxymethyl-1-(2-chloro ethoxy)-4-nitro Benzene (11g). The

title compound was synthesized from 10g and allyl bromide (yield,

3-Allyloxymethyl-4-(4-methyl-piperazin-1-yl)-phenylamine (12e).

The title compound was synthesized from 11e (yield, 76%). LC-MS

(ESI positive mode) m/z 262 ([M þ H]^þ).

1

63%). LC-MS (ESI positive mode) m/z 272 ([M þ H]^þ); H NMR

(CDCl₃) δ 8.37 (t, 1H), 8.17 (dd, 1H), 6.88 (dd, 1H), 6.04ꢀ5.91 (m,

1H), 5.43ꢀ5.21 (m, 2H), 4.35 (t, 2H), 4.15ꢀ4.13 (m, 2H), 3.87 (t, 2H);

¹³C NMR (CDCl₃) δ 160.2, 142.0, 134.5, 129.1, 124.8, 124.2, 117.5,

110.7, 72.1, 68.9, 66.0, 41.7. Anal. Calcd. for C₁₂H₁₄ClNO₄: C, 53.05; H,

5.19; N, 5.16; Cl: 13.05. Found: C, 53.09; H, 5.30; N, 5.14; Cl, 13.37. IR

2-Allyloxymethyl-N1-(2-dimethylamino-ethyl)-N1-methyl-benzene-

1,4-diamine (12f). The title compound was synthesized from 11f (yield,

1

71%). LC-MS (ESI positive mode) m/z 264 ([M þ H]^þ); H NMR

(CDCl₃) δ 6.99 (d, 1H), 6.81 (d, 1H), 6.61 (dd, 1H), 6.00ꢀ5.93

(m, 1H), 5.34ꢀ5.19 (m, 2H), 4.51 (s, 2H), 4.07ꢀ4.05 (m, 2H), 3.58 (br,

2H), 3.28ꢀ3.24 (m, 2H), 2.81ꢀ2.60 (m, 2H), 2.60 (s, 3H), 2.51 (s, 6H).

3-Allyloxymethyl-4-(2-chloro-ethoxy)-phenylamine (12g). The title

compound was synthesized from 11g (yield, 86%). LC-MS (ESI positive

mode) m/z 242 ([M þ H]^þ).

(KBr pellet): 1593, 1519, 1341, 1270, 1077 cm^ꢀ1

.

1-Allyloxymethyl-3-(2-chloro-ethoxy)-5-nitro-benzene (11h). The

title compound was synthesized from 10h and allyl bromide (yield, 59%).

LC-MS (ESI positive mode) m/z 272 ([M þ H]^þ).

1-Allyloxymethyl-2-(2-chloro-ethoxy)-3-methoxy-5-nitro-benzene (11i).

The title compound was synthesized from 10i and allyl bromide (yield, 38%).

LC-MS (ESI positive mode) m/z 302 ([M þ H]^þ).

3-Allyloxymethyl-5-(2-chloro-ethoxy)-phenylamine (12h). The title

compound was synthesized from 11h (yield, 89%). LC-MS (ESI positive

mode) m/z 242 ([M þ H]^þ).

1-Allyloxymethyl-3,5-dinitro-benzene (19). The title compound was

synthesized from 3,5-dinitrobenzyl alcohol 18 and allyl bromide (yield,

41%). LC-MS (ESI positive mode) m/z 239 ([M þ H]^þ).

1-[2-(2-Allyloxymethyl-4-nitro-phenoxy] Pyrrolidine (11j). To a solu-

tion of 11g (10 g, 36.8 mmol) in N,N-dimethylacetamide (DMA, 50 mL)

wasadded pyrrolidine(20mL, 2 vol), and theresulting mixture wasstirred

at 90 °C for 20 h. The reaction mixture was brought to rt and quenched

with water (100 mL) and extracted in ethyl acetate (3 ꢁ 50 mL). The

combined organic extracts were washed with water, brine, dried

(Na₂SO₄), and concentrated under reduced pressure to afford the crude

product 11m (8.5 g; yield, 76%) as a pale yellow oil. LC-MS (ESI positive

mode) m/z 307 ([M þ H]^þ); ¹H NMR (CDCl₃) δ 8.33

(d, 1H), 8.17 (dd, 1H), 6.91 (d, 1H), 6.03ꢀ5.93 (m, 1H), 5.37ꢀ5.23

(m, 2H), 4.56 (s, 2H), 4.26 (t, 2H), 4.12 (dt, 2H), 2.99 (t, 2H), 2.71ꢀ2.68

(m, 4H), 1.90ꢀ1.86 (m, 4H); ¹³C NMR (CDCl₃) δ 161.0, 141.7, 134.6,

128.6, 125.0, 124.3, 117.6, 110.7, 72.2, 68.5, 66.3, 55.1, 54.7, 23.8.

1-[2-(2-Allyloxymethyl-6-methoxy-4-nitro-phenoxy)-ethyl]-pyrroli-

dine (11k). Following a similar procedure to that of 11i, the title

compound was synthesized from 11h and pyrrolidine (yield, 66%). LC-

MS (ESI positive mode) m/z 337 ([M þ H]^þ).

3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenylamine (12j).

The title compound was synthesized from 11j (yield, 90%). LC-MS (ESI

positive mode) m/z 277 ([M þ H]^þ); ¹H NMR (MeOD-d₄) δ 6.82ꢀ6.78

(m, 2H), 6.69 (dd, 1H), 5.93ꢀ5.83 (m, 1H), 5.26ꢀ5.11 (m, 2H), 4.45 (s,

2H), 4.20 (t, 2H), 3.97 (dt, 2H), 3.56 (t, 2H); ¹³C NMR (MeOD-d₄) δ

149.4, 140.2, 134.7, 127.6, 118.2, 116.5, 116.2, 113.9, 70.8, 67.3, 64.6, 54.5,

54.1, 22.6. IR (KBr pellet): 1626, 1502, 1225, 1084, 798 cm^ꢀ1

.

3-Allyloxymethyl-5-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyla-

mine (12k). The title compound was synthesized from 11k (yield,

74%). LC-MS (ESI positive mode) m/z 307 ([M þ H]^þ).

3-But-3-enyloxy-phenylamine (14).The title compound was synthesized

from 13 (yield, 89%). LC-MS (ESI positive mode) m/z 164 ([M þ H]^þ).

5-Allyloxymethyl-benzene-1,3-diamine (20). The title compound

was synthesized from 19, using twice the amount of Fe powder and

NH₄Cl solution (yield, 73%). LC-MS (ESI positive mode) m/z 179

([M þ H]^þ).

(3-Allyloxymethyl-5-amino-phenyl)-carbamic acid 9H-fluoren-9-

ylmethyl Ester (12i). To a mixture of 20 (290 mg, 1.63 mmol) and

Fmoc-Cl (350 mg, 1.35 mmol) in 1,2-dioxane was added di-isopropy-

lethylamine (0.5 mL), and the resulting mixture was stirred at rt for 2 h.

The reaction mixture was concentrated under reduced pressure to

furnish an oil, which was purified by column (40% EtOAc/n-hexane)

to obtain 370 mg of 12k (yield, 95%). LC-MS (ESI positive mode) m/z

401 ([M þ H]^þ).

1-But-3-enyloxy-3-nitro-benzene (13). Following a similar proce-

dure to that of 7, the title compound was synthesized from 10a and

4-bromo-but-1-ene (yield, 52%). LC-MS (ESI positive mode) m/z 194

([M þ H]^þ).

3-Allyloxy-phenylamine (12a). Compound 11a (1.0 g, 5.58 mmol)

was taken in EtOH (10 mL, 10 vol), and fine Fe powder (0.93 g, 16.7

mmol) was added at 50ꢀ55 °C followed by NH₄Cl solution (1.76 g,

32 mmol in 2 mL water). The reaction mixture was refluxed for 4 h, and

EtOH was removed from the reaction mixture under reduced pressure.

The residue was basified with NaHCO₃solution (pH 7ꢀ8) and

extracted with CH₂Cl₂(3 ꢁ 25 mL). The combined organic extracts

were washed with brine, dried (Na₂SO₄), and concentrated under

reduced pressure to furnish 12a (0.75 g; yield, 90%) as a brown oil.

This material was taken up for the next step without any purification.

LC-MS (ESI positive mode) m/z 150 ([M þ H]^þ).

(3-But-3-enyloxy-phenyl)-[4-(3-but-3-enyloxy-phenyl)-pyrimidin-

2-yl]-amine (15a). To a mixture of 7 (100 mg, 0.38 mmol) and 14

(75 mg, 0.46 mmol) in n-butanol (2 mL) at ambient temperature was

added 4 M HCl (0.5 mL), and the resulting mixture was stirred at 80 °C

for 4 h. The reaction mixture was cooled to 0 °C and quenched with water.

The product was extracted with CH₂Cl₂thrice, and the combined organic

extracts were washed with saturated NaHCO₃followed by brine, dried

over Na₂SO₄, and concentrated under reduced pressure to furnish an oil,

which was purified by column (EtOAc/n-hexane) to obtain 71 mg of 15a

(yield, 48%). LC-MS (ESI positive mode) m/z 388 ([M þ H]^þ).

Following a procedure similar to that of 15a, the following inter-

mediates were synthesized:

Following a procedure similar to that of 12a, the following inter-

mediates were synthesized:

(3-Allyloxy-phenyl)-[4-(3-but-3-enyloxy-phenyl)-pyrimidin-2-yl]-amine

(15b). The title compound was synthesized from 7 and 12a (yield, 21%).

LC-MS (ESI positive mode) m/z 374 ([M þ H]^þ).

3-Allyloxymethyl-4-methoxy-phenylamine (12b). The title com-

pound was synthesized from 11b (yield, 88%). LC-MS (ESI positive

mode) m/z 194 ([M þ H]^þ).

(3-Allyloxymethyl-4-methoxy-phenyl)-[4-(3-but-3-enyloxy-phenyl)-

pyrimidin-2-yl]-amine (15c). The title compound was synthesized

from 7 and 12b (yield, 26%). LC-MS (ESI positive mode) m/z 418

([M þ H]^þ).

3-Allyloxymethyl-4-morpholin-4-yl-phenylamine (12c). The title

compound was synthesized from 11c (yield, 83%). LC-MS (ESI positive

mode) m/z 249 ([M þ H]^þ).

4-(2-Allyloxymethyl-4-amino-phenyl)-piperazine-1-carboxylic Acid tert-

Butyl Ester (12d). The title compound was synthesized from 11d (yield,

(3-Allyloxymethyl-4-methoxy-phenyl)-[4-(3-allyloxy-phenyl)-pyrimi-

din-2-yl]-amine (15d). The title compound was synthesized from 6a and

12b (yield, 51%). LC-MS (ESI positive mode) m/z 404 ([M þ H]^þ).

(3-Allyloxymethyl-4-methoxy-phenyl)-[4-(3-allyloxymethyl-phenyl)-

pyrimidin-2-yl]-amine (15e). The title compound was synthesized from 6b

and 12b (yield, 56%). LC-MS (ESI positive mode) m/z 418 ([M þ H]^þ).

1

80%). LC-MS (ESI positive mode) m/z 348 ([M þ H]^þ). H NMR

(CDCl₃) δ 6.90 (d, 1H), 6.81 (d, 1H), 6.59 (dd, 1H), 6.00ꢀ5.93 (m, 1H),

5.34ꢀ5.29 (m, 2H), 4.55 (s, 2H), 4.07ꢀ4.05 (m, 2H), 3.55ꢀ3.50 (m, 4H),

2.78ꢀ2.72 (m, 4H), 1.48 (s, 9H).

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ARTICLE

(3-Allyloxymethyl-4-morpholin-4-yl-phenyl)-[4-(3-allyloxymethyl-

phenyl)-pyrimidin-2-yl]-amine (15f). The title compound was synthe-

sized from 6b and 12c (yield, 78%). LC-MS (ESI positive mode) m/z 473

([M þ H]^þ).

360 ([M þ H]^þ); ¹H NMR (DMSO-d₆) δ9.77 (s, 1H), 8.57 (d, 1H), 8.48

(t, 1H), 8.09 (t, 1H), 7.65 (d, 1H), 7.46 (d, 1H), 7.44 (t, 1H), 7.17 (t, 1H),

7.10 (dd, 1H), 6.84 (dd, 1H), 6.54 (dd, 1H), 5.60ꢀ5.68 (m, 2H), 4.12

(t, 2H), 4.06 (t, 2H), 2.56ꢀ2.61 (m, 4H).

4-{2-Allyloxymethyl-4-[4-(3-allyloxymethyl-phenyl)-pyrimidin-2-

ylamino]-phenyl}-piperazine-1-carboxylic Acid tert-Butyl Ester (15g).

The title compound was synthesized from 6b and 12d (yield, 86%). LC-

MS (ESI positive mode) m/z 572 ([M þ H]^þ).

Following a procedure similar to that of 16a, the following com-

pounds were synthesized:

(16b). The title compound was synthesized from 15b (yield, 51%;

>95% trans by NMR). LC-MS (ESI positive mode) m/z 346 ([M þ

H]^þ). ¹H NMR (CDCl₃): δ 11.30 (s, 1H), 8.29 (d, 1H), 8.21 (t, 1H),

8.11 (t, 1H), 7.57 (d, 1H), 7.48 (t, 1H), 7.32ꢀ7.35 (m, 2H), 7.22ꢀ7.25

(m, 1H), 6.95 (dd, 1H), 6.82 (dd, 1H), 6.02ꢀ6.08 (m, 1H), 5.87ꢀ5.93

(m, 1H), 4.78 (d, 2H), 4.29 (t, 2H), 2.63ꢀ2.68 (m, 2H).

[3-Allyloxymethyl-4-(4-methyl-piperazin-1-yl)-phenyl]-[4-(3-ally-

loxymethyl-phenyl)-pyrimidin-2-yl]-amine (15h). The title compound

was synthesized from 6b and 12e (yield, 88%). LC-MS (ESI positive

mode) m/z 486 ([M þ H]^þ).

2-Allyloxymethyl-N4-[4-(3-allyloxymethyl-phenyl)-pyrimidin-2-yl]-

N1-(2-dimethylamino-ethyl)-N1-methyl-benzene-1,4-diamine (15i).

The title compound was synthesized from 6b and 12f (yield, 71%).

LC-MS (ESI positive mode) m/z 488 ([M þ H]^þ).

(16c). The title compound was synthesized from 15c (yield, 45%;

>95% trans by NMR). LC-MS (ESI positive mode) m/z 390 ([M þ

H]^þ); ¹H NMR.

(16d). The title compound was synthesized from 15d (yield, 45%;

>95% trans by NMR). LC-MS (ESI positive mode) m/z 502 ([M þ

H]^þ). ¹H NMR (CDCl₃): δ 11.76 (s, 1H), 8.16 (d, 1H), 8.14 (d, 1H),

7.66 (s, 1H), 7.40ꢀ7.42 (m, 2H), 7.28 (dd, 1H), 7.19ꢀ7.21 (m, 1H),

7.17 (d, 1H), 6.90 (d, 1H), 6.07ꢀ6.14 (m, 1H), 5.82ꢀ5.88(m, 1H), 5.67

(d, 2H), 4.60 (s, 2H), 4.14 (dd, 2H), 3.86 (s, 3H).

[3-Allyloxymethyl-4-(2-chloro-ethoxy)-phenyl]-[4-(3-allyloxymethyl-

phenyl)-pyrimidin-2-yl]-amine (15j). The title compound was synthe-

sized from 6b and 12g (yield, 92%). LC-MS (ESI positive mode) m/z 466

([M þ H]^þ).

[3-Allyloxymethyl-5-(2-chloro-ethoxy)-phenyl]-[4-(3-allyloxymethyl-

phenyl)-pyrimidin-2-yl]-amine (15k). The title compound was synthe-

sized from 6b and 12h (yield, 82%). LC-MS (ESI positive mode) m/z 466

([M þ H]^þ).

(16e). The title compound was synthesized from 15e (yield, 48%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

390 ([M þ H]^þ). ¹H NMR (DMSO-d₆): δ 9.55 (s, 1H), 8.54ꢀ8.52 (m,

2H), 8.19 (s, 1H), 8.02 (d, 1H), 7.56 (dd, 1H), 7.40 (d, 1H), 7.15 (d,

1H), 6.98 (d, 1H), 5.84ꢀ5.72 (m, 2H), 4.57 (s, 2H), 4.49 (s, 2H), 4.06

(d, 2H), 4.01 (d, 2H), 3.78 (s, 3H). ¹³C NMR (DMSO-d₆): δ 63.4,

160.7, 159.8, 152.5, 139.1, 137.3, 134.2, 131.5, 131.2, 130.3, 129.6, 127.0,

126.9, 126.2, 121.2, 120.1, 111.9, 107.8, 69.6, 69.3, 67.5, 65.7, 56.1.

(17a). The title compound was synthesized from 15f (yield, 43%;

mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive mode) m/z

502 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 11.56 (s, 1H), 8.45 (d, 1H),

8.29ꢀ8.27 (m, 1H), 8.22 (d, 1H), 7.90 (d, 1H), 7.76 (d, 1H), 7.59 (t,

1H), 7.31 (d, 1H), 7.26ꢀ7.21 (m, 1H), 7.10 (d, 1H), 5.91ꢀ5.69 (m,

2H), 4.66 (s, 2H), 4.64 (s, 2H), 4.17ꢀ4.15 (m, 2H), 4.06ꢀ4.04 (m, 2H),

3.89ꢀ3.82 (m, 4H), 2.30ꢀ2.96 (m, 4H).

{3-Allyloxymethyl-5-[4-(3-allyloxymethyl-phenyl)-pyrimidin-2-

ylamino]-phenyl}-carbamic Acid 9H-Fluoren-9-yl-methyl Ester (15l).

The title compound was synthesized from 6b and 12i (yield, 90%). LC-

MS (ESI positive mode) m/z 625 ([M þ H]^þ).

[3-Allyloxymethyl-5-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-

[4-(3-allyloxymethyl-phenyl)-pyrimidin-2-yl]-amine (15m). The title

compound was synthesized from 6b and 12k (yield, 21%) LC-MS (ESI

positive mode) m/z 531 ([M þ H]^þ).

[4-(3-Allyloxymethyl-phenyl)-5-methyl-pyrimidin-2-yl]-[3-allyloxy-

methyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15n). The title

compound was synthesized from 6g and 12j (yield, 65%). LC-MS

(ESI positive mode) m/z 515 ([M þ H]^þ).

[4-(3-Allyloxymethyl-phenyl)-5-fluoro-pyrimidin-2-yl]-[3-allylox-

ymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15o). The title

compound was synthesized from 6h and 12j (yield, 65%). LC-MS (ESI

positive mode) m/z 519 ([M þ H]^þ).

(17b). The title compound was synthesized from 15g using 10 mol %

Zhan catalyst-1B instead of the Grubbs second generation catalyst

(yield, 96%, isomers ration not determined). LC-MS (ESI positive

mode) m/z 502 ([M þ H]^þ).

[4-(5-Allyloxymethyl-2-methoxy-phenyl)-pyrimidin-2-yl]-[3-ally-

loxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15p). The ti-

tle compound was synthesized from 6f and 12j (yield, 91%). LC-MS

(ESI positive mode) m/z 531 ([M þ H]^þ).

(17d). The title compound was synthesized from 15h (yield, 52%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

502 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 10.62 (s, 1H), 8.59 (d, 1H),

8.29ꢀ8.24 (m, 2H), 7.88 (d, 1H), 7.72 (d, 1H), 7.59ꢀ7.56 (m, 1H),

7.30 (d, 1H), 7.18ꢀ7.10 (m, 2H), 5.90ꢀ5.69 (m, 2H), 4.65 (s, 2H), 4.61

(s, 2H), 4.17ꢀ4.15 (m, 2H), 4.08ꢀ4.06 (m, 2H), 3.68ꢀ3.66 (m, 2H),

3.37ꢀ3.25 (m, 4H), 2.93ꢀ2.91 (m, 2H), 2.90 (s, 3H).

[4-(3-Allyloxymethyl-5-fluoro-phenyl)-pyrimidin-2-yl]-[3-allylox-

ymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15q). The title

compound was synthesized from 6e and 12j (yield, 47%). LC-MS (ESI

positive mode) m/z 519 ([M þ H]^þ).

(17h). The title compound was synthesized from 15i (yield, 56%;

mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive mode) m/z

502 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.87 (s, 1H), 8.48 (s, 1H),

8.29 (s, 1H), 7.98 (d, 1H), 7.64 (d, 1H), 7.54 (d, 1H), 7.40 (d, 1H), 7.37

(d, 1H), 7.20 (dd, 1H), 6.00ꢀ5.78 (m, 2H), 4.67 (s, 2H), 4.27 (d, 2H),

4.06 (d, 2H), 3.40 (t, 2H), 3.15 (t, 2H), 2.89 (s, 6H), 2.76 (s, 3H).

(21a). The title compound was synthesized from 15j (yield, 74%;

mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive mode) m/z

438 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 8.70 (d, 1H), 8.43 (d, 1H), 8.30

(s, 1H), 7.81 (d, 1H), 7.61ꢀ7.59 (m, 1H), 7.50 (t, 1H), 7.17ꢀ7.15 (m,

2H), 6.85ꢀ6.82 (m, 2H), 5.91ꢀ5.79 (m, 2H), 4.66 (s, 2H), 4.65 (s, 2H),

4.27 (t, 2H), 4.17 (d, 2H), 4.07 (d, 2H), 3.83 (t, 2H). ¹³C NMR

(DMSO-d₆): δ 163.4, 160.7, 159.9, 151.1, 139.1, 137.2, 134.9, 131.5,

131.3, 130.4, 129.6, 127.4, 127.0 (duplicate), 121.0, 120.1, 114.4, 107.9,

69.8, 69.7, 69.4, 67.5, 65.5, 43.8.

[4-(3-Allyloxymethyl-4-methoxy-phenyl)-pyrimidin-2-yl]-[3-ally-

loxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15r). The ti-

tle compound was synthesized from 6d and 12j (yield, 81%). LC-MS

(ESI positive mode) m/z 531 ([M þ H]^þ).

[4-(3-Allyloxymethyl-4-fluoro-phenyl)-pyrimidin-2-yl]-[3-allylox-

ymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (15s). The title

compound was synthesized from 6c and 12j (yield, 39%). LC-MS

(ESI positive mode) m/z 519 ([M þ H]^þ).

(16a). To 15a (20 mg, 0.052 mmol) in CH₂Cl₂(50 mL) at ambient

temperature was added 4 M HCl until the pH reached 2.0ꢀ2.2. The

solution was degassed (N₂), and Grubbs second generation catalyst

(7 mg, 0.005 mmol) was added in two portions at 1 h intervals. The

resulting mixture was stirred at 40ꢀ45 °C for 4 h. The reaction

mixture was cooled and concentrated under reduced pressure to

furnish an oil, which was purified by preparative HPLC to obtain 9 mg

of 16a (yield, 48%, >95% trans by NMR). LC-MS (ESI positive mode) m/z

(21g). The title compound was synthesized from 15k (yield, 56%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

4653

dx.doi.org/10.1021/jm200326p |J. Med. Chem. 2011, 54, 4638–4658

Journal of Medicinal Chemistry

ARTICLE

438 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 8.46 (d, 1H), 8.43 (s, 1H), 8.39

(s, 1H), 7.83 (d, 1H), 7.58ꢀ7.54 (m, 2H), 7.51ꢀ7.49 (m, 1H), 7.21 (d,

1H), 6.70 (s, 1H), 6.49 (t, 1H), 5.92ꢀ5.81 (m 2H), 4.63 (s, 2H), 4.56

(s, 2H), 4.27 (t, 2H), 4.15 (d, 2H), 4.09 (d, 2H), 3.83 (t, 2H).

(21j). The title compound was synthesized from 15l using 10 mol %

Zhan catalyst-1B instead of the Grubbs second generation catalyst

(yield, 73%; mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive

mode) m/z 597 ([M þ H]^þ).

(dd, 1H), 6.76 (d, 1H), 5.85ꢀ5.73 (m, 2H), 4.56 (s, 2H), 4.47 (s, 2H),

4.06 (s, 2H), 4.01 (d, 2H). ¹³C NMR (DMSO-d₆): δ 163.3, 160.7, 159.8,

150.6, 139.1, 137.3, 132.8, 131.6, 131.2, 130.3, 129.6, 127.1, 126.9, 124.3,

121.3, 120.3, 115.8, 107.5, 169.5, 69.4, 67.6, 66.0.

(17c). To a mixture of 17b (131 mg, 0.241 mmol) in CH₂Cl₂(5 mL)

was added TFA (0.5 mL), and the resulting mixture was stirred at 40 °C

for 2 h. It was cooled and concentrated under reduced pressure. The

crude product was further purified by preparative HPLC to furnish 17c

(70 mg; yield, 73%; mixture of trans/cis 80:20 by NMR) as a pale yellow

(22a). The title compound was synthesized from 15m (yield, 43%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

503 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 8.33ꢀ8.28 (m, 2H), 8.10 (d,

1H), 7.93 (d, 1H), 7.79 (d, 1H), 7.63 (t, 1H), 7.40 (d, 1H), 6.93 (d, 1H),

5.94ꢀ5.82 (m, 2H), 4.68 (s, 1H), 4.60 (s, 2H), 4.36 (m, 2H), 4.18 (d,

2H), 4.11 (d, 2H), 4.12ꢀ4.03 (m, 2H), 3.94 (s, 3H), 3.63(m, 2H), 3.19

(m, 2H), 2.34ꢀ2.22 (m, 4H).

1

solid. LC-MS (ESI positive mode) m/z 444 ([M þ H]^þ). H NMR

(MeOD-d₄): δ 8.77 (d, 1H), 8.46 (d, 1H), 8.31 (s, 1H), 8.00 (d, 1H),

7.65ꢀ7.63 (m, 1H), 7.56 (t, 1H), 7.41 (d, 1H), 7.19 (d, 1H), 7.11 (dd,

1H), 5.95ꢀ5.76 (m, 2H), 4.68 (s, 2H), 4.67 (s, 2H), 4.17 (d, 2H), 4.07

(d, 2H), 3.42ꢀ3.38 (m, 4H), 3.25ꢀ3.21 (m, 4H).

(17e). To a mixture of 17c (16 mg, 0.036 mmol) and 2-bromoethanol

(5 μL, 0.072 mmol) in CH₂Cl₂(1 mL) was added Et₃N (14 μL, 0.10

mmol), and the resulting mixture was stirred at rt for 2 h. It was

concentrated under reduced pressure and further purified by preparative

HPLC to furnish 17e (6 mg; yield, 34%; mixture of trans/cis 80:20 by

NMR) as a pale yellow solid. LC-MS (ESI positive mode) m/z 488

([M þ H]^þ). ¹H NMR (CDCl₃): δ 11.76 (s, 1H), 8.51 (d, 1H), 8.26 (s,

1H), 8.23 (d, 1H), 7.90 (d, 1H), 7.76 (d, 1H), 7.62 (t, 1H), 7.34 (d, 1H),

7.30ꢀ7.27 (m, 1H), 7.15 (d, 1H), 5.90ꢀ5.73 (m, 2H), 4.65 (s, 2H), 4.60

(s, 2H), 4.14 (d, 2H), 4.07 (d, 4H), 3.83ꢀ3.80 (m, 2H), 3.40ꢀ3.38 (m,

2H), 3.30ꢀ3.27 (m, 4H), 3.17ꢀ3.13 (m, 2H).

(22b). The title compound was synthesized from 15n (yield, 79%;

mixture of trans/cis 88:12 by NMR). LC-MS (ESI positive mode) m/z

487 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 10.4 (s, 1H), 8.40 (s, 1H), 8.12

(s, 1H), 7.78 (s, 1H), 7.65ꢀ7.36 (m, 3H), 7.07 (d, 1H), 6.78 (d, 1H),

5.84ꢀ5.64 (m, 2H), 4.56 (s, 2H), 4.42 (s, 2H), 4.31 (br s, 2H), 4.10 (d,

2H), 3.99 (d, 2H), 3.85 (m, 2H), 3.50 (m, 2H), 2.97 (m, 2H), 2.26 (s,

3H), 2.05 (m, 4H).

(22c). The title compound was synthesized from 15o (yield, 56%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/

z 491 ([M þ H]^þ). ¹H NMR (CDCl₃): δ 8.23 (m, 1H), 8.12 (s, 1H),

7.95 (d, 1H), 7.52 (d, 1H), 7.42 (m, 1H), 7.12 (s, 1H), 6.76 (d, 2H),

5.75 (m, 2H), 4.59 (s, 2H), 4.47 (m, 2H), 4.14 (m, 2H), 4.05 (m, 2H),

4.01 (m, 2H), 2.96 (m, 2H), 2.71 (m, 4H), 1.79 (m, 4H).

(22d). The title compound was synthesized from 15p (yield, 32%;

mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive mode) m/z

503 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.61 (d, 1H), 8.36 (d, 1H),

8.07 (s, 1H), 7.59 (d, 1H), 7.53 (dd, 1H), 7.19 (d, 1 H), 7.13 (dd, 1H),

7.04 (d, 1H), 5.90 (dt, 1H), 5.79 (dt, 1H), 4.61 (s, 2H), 4.59 (s, 2H),

4.37 (t, 2H), 4.11 (d, 2H), 4.08 (d, 2H), 3.95 (s, 3H), 3.80 (m, 2H); 3.68

(t, 2H), 2.17 (m, 2H), 2.08 (m, 2H), 1.30 (m, 2H).

(22e). The title compound was synthesized from 15q (yield, 42%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

491 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.72 (d, 1H), 8.48 (m, 1H),

8.11 (s, 1H), 7.72 (m, 1H), 7.34 (m, 2H), 7.05ꢀ7.15 (m, 2H),

5.82ꢀ5.90 (m, 2H), 4.65 (m, 2H), 4.39 (m, 2H), 4.16 (m, 2H), 4.09

(m, 2H), 3.80 (m, 2H), 3.71 (m, 2H), 3.27 (m, 4H), 2.08ꢀ2.24 (m, 4H).

(22f). The title compound was synthesized from 15r (yield, 48%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

503 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.50ꢀ8.48 (m, 1H), 8.37 (d,

1H), 8.27 (d, 1H), 8.07 (dd, 1H), 7.38 (d, 1H), 7.17ꢀ7.15 (m, 2H),

7.08ꢀ7.06 (m, 1H), 5.98ꢀ5.86 (m, 2H), 4.69 (s, 2H), 4.64 (s, 2H), 4.39

(t, 2H), 4.17 (d, 2H), 4.08 (d, 2H), 3.88ꢀ3.82 (m, 2H), 3.70 (t, 2H),

2.23ꢀ2.21 (m, 2H), 2.10ꢀ2.07 (m, 2H).

(17f). To a mixture of 17c (9 mg, 0.020 mmol) and acetyl chloride (3 μL,

0.041 mmol) in CH₂Cl₂(1 mL) was added Et₃N (9 μL, 0.060 mmol), and

the resulting mixture was stirred at rt for 2 h. It was concentrated under

reduced pressure and further purified by preparative HPLC to furnish 17f (6

mg; yield, 63%; mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive

mode) m/z486 ([M þ H]^þ). ¹HNMR(CDCl₃):δ12.00 (s, 1H), 8.46 (d,

1H), 8.28 (s, 1H), 8.25 (d, 1H), 7.92 (d, 1H), 7.78 (d, 1H), 7.59 (t, 1H),

7.36 (d, 1H), 7.31ꢀ7.28 (m, 1H), 7.09 (d, 1H), 5.91ꢀ5.75 (m, 2H), 4.67

(s, 2H), 4.65 (s, 2H), 4.17 (d, 2H), 4.07 (d, 2H), 3.83ꢀ3.67 (m, 4H),

3.06ꢀ2.93 (m, 4H), 2.22 (s, 3H).

(17g). To a mixture of 17c (14 mg, 0.032 mmol) and ethanesulfonyl

chloride (6 μL, 0.063 mmol) in THF (1 mL) was added K₂CO₃(10 mg,

0.072 mmol), and the resulting mixture was stirred at 70 °C for 4 h. It was

cooled and concentrated under reduced pressure. The crude product was

further purified by preparative HPLC to furnish 17g (7 mg; yield, 41%;

mixture of trans/cis 80:20 by NMR) as a pale yellow solid. LC-MS (ESI

1

positive mode) m/z 536 ([M þ H]^þ). H NMR (CDCl₃): δ 8.50 (d,

1H), 8.29 (s, 1H), 8.22 (d, 1H), 7.90 (d, 1H), 7.75 (d, 1H), 7.57 (t, 1H),

7.32 (d, 1H), 7.25ꢀ7.21 (m, 1H), 7.10 (d, 1H), 5.90ꢀ5.73 (m, 2H), 4.65

(s, 2H), 4.64 (s, 2H), 4.15 (d, 2H), 4.06 (d, 2H), 3.07ꢀ2.99 (m, 4H), 3.02

(d, 2H), 3.25ꢀ3.21 (m, 4H), 1.45 (t, 3H).

(21b). To a microwave glass vial was added 21a (20 mg, 0.046 mmol),

diethylamine (10 μL, 0.11 mmol), and DMA (1 mL), and the resulting

mixture was stirred under microwave conditions at 80 °C for 30 min.

The mixture was diluted with MeOH and purified directly by preparative

HPLC to furnish 21b (18 mg; yield, 82%; mixture of trans/cis 85:15 by

NMR) as a pale yellow solid. LC-MS (ESI positive mode) m/z 475

(22g). The title compound was synthesized from 15s (yield, 49%;

mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive mode) m/z

491 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.45 (m, 2H), 8.30 (m, 1H),

8.05 (m, 1H), 7.38 (m, 1H), 7.26 (m, 1H), 7.16 (m, 1H), 7.08 (m, 1H),

5.90ꢀ5.92 (m, 2H), 4.65 (m, 4H), 4.39 (m, 2H), 4.16 (m, 2H), 4.09 (m,

2H), 3.85 (m, 2H), 3.71 (m, 2H), 3.32 (m, 2H), 2.08ꢀ2.25 (m, 4H).

(16f). To a mixture of 16e (20 mg, 0.051 mmol) in DMF (2 mL) was

added sodium ethanethiolate (5 mg, 0.061 mmol), and the resulting

mixture was stirred at 120 °C for 20 h. It was cooled, and saturated NH₄Cl

was added. The product was extracted with CH₂Cl₂thrice, and the

combined organic extracts were dried over Na₂SO₄and concentrated

under reduced pressure to furnish an oil which was further purified by

preparative HPLC to furnish 16f (6 mg; yield, 30%; mixture of trans/cis

80:20 by NMR). LC-MS (ESI positive mode) m/z 376 ([M þ H]^þ). ¹H

NMR (DMSO-d₆): δ 9.43 (s, 1H), 9.11 (br, 1H), 8.50 (d, 1H), 8.44 (d,

1H), 8.20 (s, 1H), 8.01 (dd, 1H), 7.56ꢀ7.54 (m, 2H), 7.37 (d, 1H), 6.99

1

([M þ H]^þ). H NMR (MeOD-d₄): δ 8.49 (s, 1H), 7.98ꢀ7.97 (m,

1H), 7.77 (s, 1H), 7.63ꢀ7.61 (m, 1H), 7.57ꢀ7.55 (m, 1H), 7.42ꢀ7.38

(m, 4H), 7.14ꢀ7.11 (m, 1H), 5.83ꢀ5.76 (m, 1H), 5.42ꢀ5.34 (m, 1H),

4.29ꢀ4.27 (m, 1H), 4.13ꢀ4.10 (m, 2H), 3.83ꢀ3.72 (m, 2H), 3.24 (s,

2H), 3.25ꢀ2.97 (m, 4H), 2.29ꢀ2.24 (m, 2H), 2.11ꢀ1.92 (m, 4H).

Following a procedure similar to that of 21b, the following com-

pounds were synthesized:

(21c). The title compound was synthesized from 21a and pyrrolidine

(yield, 83%; mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive

mode) m/z 473 ([M þ H]^þ). HRMS: theoretical C₂₈H₃₂N₄O₃MW,

472.2474; found, 473.2547. ¹HNMR(MeOD-d₄):δ8.79(d, 1H), 8.46(d,

1H), 8.34ꢀ8.31 (m, 1H, CH), 7.98ꢀ7.96 (m, 1H), 7.62ꢀ7.49 (m, 2H),

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7.35 (d, 1H), 7.15ꢀ7.10 (m, 1H), 7.07ꢀ7.02 (m, 1H), 5.98ꢀ5.75 (m,

2H), 4.67 (s, 2H), 4.67 (s, 2H), 4.39ꢀ4.36 (m, 2H), 4.17 (d, 2H), 4.08 (d,

2H), 3.88ꢀ3.82 (m, 2H), 3.70 (t, 2H), 2.23ꢀ2.21 (m, 2H), 2.10ꢀ2.07

(m, 2H); chloride content (titration) 7.7% (1.18 equivs); water content

Enzyme Assays. The recombinant enzymes (CDK2/CyclinA,

JAK1, JAK2, JAK3 and FLT3) were prepared by the S*BIO biochemistry

group according to published procedures. All assays were carried out in

384-well white microtiter plates using the PKLight assay system from

Cambrex. This assay platform is essentially a luminometric assay for the

detection of ATP in the reaction using a luciferase-coupled reaction. The

compounds were tested at 8 concentrations prepared from 3- or 4-fold

serial dilution starting at 10 μM. For the CDK2/cyclin A assay, the

reaction mixture consisted of the following components in 25 μL assay

buffer (50 mM Hepes, pH 7.5, 10 mM MgCl₂, 5 mM MnCl₂, 5 mM

BGP, 1 mM DTT, and 0.1 mM sodium orthovanadate), 1.4 μg/mL of

CDK2/cyclin A complex, 0.5 μM of RbING substrate (Invitrogen, cat #

PV2939), and 0.5 μM of ATP. The reaction was incubated at room

temperature for 2 h. Thirteen microliters of PKLight ATP detection

reagent was added, and the reaction was incubated for 10 min.

Luminescence signals were detected on a multilabel plate reader

(Victor²V 1420, Perkin-Elmer). The other kinase assays were similar,

with the following differences in reagents. For FLT3 assays, the reaction

contained 2.0 μg/mL FLT3 enzyme, 5 μM of poly(Glu,Tyr) substrate

(Sigma, cat # P0275), and 4 μM of ATP. For JAK1 assays, the reaction

contained 2.5 μg/mL of JAK1 enzyme, 10 μM of poly(Glu,Ala,Tyr)

substrate (Sigma, cat # P3899), and 1.0 μM of ATP. For JAK2 assays, the

reaction contained 0.35 μg/mL of JAK2 enzyme, 10 μM of poly(Glu,

Ala,Tyr) substrate (Sigma, cat # P3899), and 0.15 μM of ATP. For JAK3

assays, the reaction contained 3.5 μg/mL of JAK3 enzyme, 10 μM of

poly(Glu,Ala,Tyr) substrate (Sigma, cat # P3899), and 6.0 μM of ATP.

The analytical software, Prism 5.0 (GraphPad Software Pte Ltd.) was

used to generate IC₅₀values from the data.

(Karl Fischer) 6.1% (1.85 equivs); Anal. Calcd. for C₂₈H₃₂N₄O₃1.18

3

HCl 1.85H₂O: C, 61.46; H, 6.46; N, 10.24; Cl, 7.65. Found: C, 61.99; H,

6.91; N, 10.25; Cl, 7.45.

3

(21d). The title compound was synthesized from 21a and piperidine

(yield, 84%; mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive

mode) m/z 487 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ 8.65 (d, 1H),

8.33 (d, 1H), 8.15 (s, 1H), 7.84 (d, 1H), 7.44 (m, 2H), 7.22 (d, 1H), 7.00

(m, 1H), 6.93 (d, 1H), 5.82ꢀ5.66 (m, 2H), 4.52 (s, 2H), 4.50 (s, 2H),

4.30 (t, 2H), 4.02 (d, 2H), 3.95 (d, 2H), 3.61 (br, 2H), 3.49 (t, 2H), 3.03

(m, 2H), 1.98 (m, 2H), 1.77 (m, 4H). ¹³C NMR (MeOD-d₄): δ 159.6,

152.8, 140.2, 138.6, 132.5, 132.4, 132.1, 130.4, 128.7, 128.0, 127.7, 123.0,

121.2, 114.6, 109.0, 70.8, 70.6, 68.9, 67.7, 64.7, 57.6, 55.3, 24.4, 22.7.

(21e).The title compound was synthesized from 21a and 2-pyrrolidinone

(yield, 72%; mixture of trans/cis 85:15 by NMR). LC-MS (ESI positive

mode) m/z 487 ([M þ H]^þ). ¹H NMR (DMSO-d₆): δ 9.55 (s, 1H), 8.57

(s, 1H), 8.52 (d, 1H), 8.19 (s, 1H), 8.01 (d, 1H), 7.56 (m, 2H), 7.39 (d, 1H),

7.13 (dd, 1H), 6.96 (d, 1H), 5.88ꢀ5.69 (m, 2H), 4.57 (s, 2H), 4.47 (s, 2H),

4.09 (m, 6H), 3.58 (t, 2H), 3.51 (t, 2H), 2.24 (t, 2H), 1.95 (t, 2H).

(21f). The title compound was synthesized from 21a and 3-pyrroli-

dinone (yield, 75%; mixture of trans/cis 85:15 by NMR). LC-MS (ESI

positive mode) m/z 487 ([M þ H]^þ). ¹H NMR (DMSO-d₆): δ 9.55 (s,

1H), 8.57 (s, 1H), 8.52 (d, 1H), 8.19 (s, 1H), 8.01 (d, 1H), 7.56 (m, 2H),

7.39 (d, 1H), 7.13 (dd, 1H), 6.97 (d, 1H), 5.87ꢀ5.69 (m, 2H), 4.56 (s,

2H), 4.48 (s, 2H), 4.11 (t, 2H), 4.03 (m, 4H), 3.08 (s, 2H), 2.99 (t, 2H),

2.92 (t, 2H), 2.34 (t, 2H).

Cell Proliferation Assays. Ba/F3-JAK2^V617F-GFP-Luc cells were

generous gifts from Dr. Martin Sattler (Dana-Farber Cancer Institute,

Boston, MA). The generation and culture of these cells has been

described previously {Walz, 2006 #2}. MV4ꢀ11 cells was obtained

from the American Type Culture Collection (ATCC, Manassas, VA)

and cultured according to the recommended guidelines. For prolifera-

tion assays in 96-well plates, 20.000 cells were seeded in 100 μL and

treated the following day with compounds (in triplicates) at concentra-

tions up to 10 μM for 48 h. Cell viability was monitored using the

CellTiter-Glo cell proliferation assay (Promega, Madison, WI). Doseꢀ

response curves were plotted to determine IC₅₀values for the com-

pounds using the XL-fit software (IDBS Ltd., Alameda, CA).

(21h). The title compound was synthesized from 21g and pyrrolidine

(yield, 90%; mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive

mode) m/z 473 ([M þ H]^þ). ¹H NMR(MeOD-d₄): δ8.47 (d, 1H), 8.35

(d, 2H), 8.00 (d, 1H), 7.62ꢀ7.59 (m, 1H), 7.56ꢀ7.52 (m, 1H), 7.43 (d,

1H), 6.80ꢀ6.79 (m, 1H), 6.75ꢀ6.74 (m, 1H), 5.92ꢀ5.79 (m, 2H), 4.61

(s, 2H), 4.57 (s, 2H), 4.37 (t, 2H), 4.15 (d, 2H), 4.08 (d, 2H), 3.77ꢀ3.72

(m, 2H), 3.68 (t, 2H), 3.26ꢀ3.21 (m, 2H), 2.23ꢀ2.16 (m, 2H),

2.11ꢀ2.03 (m, 2H).

(21i). The title compound was synthesized from 21g and morpholine

(yield, 79%; mixture of trans/cis 80:20 by NMR). LC-MS (ESI positive

mode) m/z 489 ([M þ H]^þ). ¹H NMR(MeOD-d₄): δ8.47 (d, 1H), 8.36

(d, 2H), 8.00 (d, 1H), 7.62ꢀ7.60 (m, 1H), 7.55ꢀ7.51 (m, 1H), 7.42 (d,

1H), 6.80ꢀ6.79 (m, 1H), 6.75ꢀ6.73 (m, 1H), 5.92ꢀ5.79 (m, 2H), 4.61

(s, 2H), 4.56 (s, 2H), 4.43 (t, 2H), 4.15 (d, 2H), 4.08 (d, 2H), 4.08ꢀ4.04

(m, 2H), 3.90ꢀ3.79 (m, 2H), 3.66 (t, 2H), 3.65ꢀ3.58 (m, 2H).

(21k). To a mixture of 21j (156 mg, 0.261 mmol) in CH₂Cl₂(5 mL)

was added piperidine (1 mL, 20% v/v), and the resulting mixture was

stirred at rt for 30 min. It was concentrated under reduced pressure and

purified by preparative HPLC to furnish 21k (80 mg; yield, 82%;

mixture of trans/cis 85:15 by NMR) as a pale yellow solid. LC-MS

(ESI positive mode) m/z 375 ([M þ H]^þ). ¹H NMR (MeOD-d₄): δ

8.83 (s, 1H), 8.53 (d, 1H), 8.34 (s, 1H), 7.99 (dd, 1H), 7.61ꢀ7.56 (m,

1H), 7.54ꢀ7.51 (m, 1H), 7.45 (d, 1H), 7.08ꢀ7.06 (m, 2H), 5.94ꢀ5.81

(m, 2H), 4.63 (s, 4H), 4.18 (d, 2H), 4.10 (d, 2H).

(21l). To a mixture of 21k (20 mg, 0.053 mmol) and 3-(die-

thylamino)propionic acid hydrochloride (17 mg, 0.096 mmol) in

CH₂Cl₂(1 mL) was added HOBt (14 mg, 0.11 mmol) and EDCI (21

mg, 0.11 mmol), and the resulting mixture was stirred at rt for 4 h. It was

concentrated under reduced pressure and purified by preparative HPLC

to furnish 21l (15 mg; yield, 55%; mixture of trans/cis 85:15 by NMR) as

a pale yellow solid. LC-MS (ESI positive mode) m/z 502 ([M þ H]^þ).

¹H NMR (MeOD-d₄): δ 8.50ꢀ8.49 (m, 2H), 8.37 (s, 1H), 8.01 (d, 1H),

7.62ꢀ7.61 (m, 1H), 7.56 (t, 1H), 7.48ꢀ7.47 (m, 1H), 7.42 (d, 1H), 7.28

(s, 1H), 5.95ꢀ5.81 (m, 2H), 4.64 (s, 2H), 4.59 (s, 2H), 4.17 (d, 2H),

4.10 (d, 2H), 3.55 (t, 2H), 3.34 (q, 4H), 2.94 (t, 2H), 1.40 (t, 6H).

Allograft Studies with Ba/F3-JAK2^V617FCells. All mice were

obtained from the Biological Resource Centre (Biopolis, Singapore).

Female athymic BALB/c nude mice (BALB/cOlaHsd-Foxn1^nu) were 12

weeks of age; female SCID Beige mice (CB17.Cg-Prkdc^scidLyst ^bg/Crl)

werr 9ꢀ10 weeks of age. Standard protocols were followed, in com-

pliance with the NIH and National Advisory Committee for Laboratory

Animal Research guidelines (IACUC approval #0800371).

For the mouse model of MPD, 2 ꢁ 10⁶Ba/F3-JAK2^V617Fcells were

resuspended in 100 μL serum-free culture medium and injected

intravenously into the tail vein of BALB/c nude mice. Treatment was

started on day 4 for 13 consecutive days for three groups of 8 mice each.

€

Four naive mice served to establish baseline values. Animals were

sacriﬁced at the end of study, and the spleens and livers were harvested

and weighed.

Xenograft Studies with MV4ꢀ11 Cells. Female BALB/c mice

and female athymic BALB/c nude mice (BALB/cOlaHsd-Foxn1^nu)

were obtained from the Biological Resource Centre (BRC, Biopolis,

Singapore) and were 9ꢀ16 weeks of age at the time of tumor implanta-

tion. Standard protocols were followed, in compliance with the National

Institutes of Health and National Advisory Committee for Laboratory

Animal Research guidelines (IACUC approval #0800371). Female

BALB/c nude mice were implanted subcutaneously in the right flank

with 1 ꢁ 10⁷MV4ꢀ11 human AML cells. Cells were resuspended

in 50 μL of serum-free growth medium, mixed 1:1 with Matrigel

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ARTICLE

(Cat. No:354248; BD Bioscience), and injected in a total volume of

100 μL, using a 23-gauge needle. MV4ꢀ11 tumor-bearing mice (average

starting tumor size of 130 mm³) were treated orally (p.o.) once daily at

doses of 25, 50, or 100 mg/kg for 21 consecutive days. After termination

of the treatment, tumor growth was measured up to day 55, and the

median time to end point (TTE) (median survival) was calculated. End

point of experiment for an individual mouse was reached either on day

55 or when a tumor reached a size of 1000 mm³. The tumor volumes

were calculated using the formula: Tumor volume (mm³) = (w²ꢁ l)/2

(w = width, and l = length in mm of the tumor xenograft). All doses for

in vivo experiments used in this article are given as free base equivalent.

Metabolic Stability in Liver Microsomes. Test compounds

(5 μM) were incubated with MLM, RLM, DLM, and HLM (final micro-

somal concentration of ∼0.87 mg/mL), in a reaction mix containing

50 mM potassium phosphate buffer (pH 7.4), and a NADPH regenera-

tion system, at 37 °C, in a total reaction volume of 1 mL. Reactions were

terminated at 0, 15, 30, 45, and 60 min of incubation with a chilled

mixture of acetonitrile and DMSO (80:20), vortexed for 5 min, and

centrifuged at 13200 rpm for 15 min at 4 °C, and the supernatants were

analyzed by LC/MS/MS. Stability was assessed by plotting the percent

of parent compound remaining against time on a logꢀlinear scale and

estimating the half-life from the linear portion of the logꢀlinear curve

using the first order equation t_1/2= 0.693/k, where k = slope of the curve

(equal to the first order elimination rate constant).

Human in vitro CYP450 Inhibition Assay. Test compounds

were incubated (at concentrations of 0.05, 0.25, 0.5, 2.5, 5, and

25 μM in DMSO; final DMSO concentration = 0.35%) with human liver

microsomes (0.25 mg/mL for CYP1A and CYP3A4, 0.5 mg/mL for

CYP2C19 and CYP2D6, 1 mg/mL for CYP2C9) and NADPH (1 mM)

in the presence of the probe substrate ethoxyresorufin (0.5 μM) for

5 min (CYP1A), tolbutamide (120 μM) for 60 min (CYP2C9), mephe-

nytoin (25 μM) for 60 min (CYP2C19), dextromethorphan (5 μM) for

30 min (CYP2D6), and midazolam (2.5 μM) for 5 min (CYP3A4) at

37 °C. The selective inhibitors R-naphthoflavone, sulphaphenazole,

tranylcypromine, quinidine, and ketoconazole were used as positive

controls for CYP1A, CYP2C9, CYP2C19, CYP2D6, and CYP3A4

inhibitors, respectively. For CYP1A, the reactions were terminated by

the addition of methanol, and the formation of the metabolite, resorufin,

was monitored by fluorescence (excitation wavelength = 535 nm, and

emission wavelength = 595 nm). For the CYP2C9, CYP2C19, CYP2D6,

and CYP3A4 incubations, the reactions were terminated by the addition

of methanol containing an internal standard. The samples were cen-

trifuged, and the supernatants were combined, for the simultaneous

analysis of 4-hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan,

1-hydroxymidazolam, and the internal standard by LC-MS/MS. Formic

acid in deionized water (final concentration = 0.1%) was added to the

final sample prior to analysis. A decrease in the formation of the

metabolites compared to vehicle control was used to calculate an IC₅₀

value (test compound concentration which produces 50% inhibition).

Caco-2 Bidirectional Permeability Assay. Compound 21c at

5 μM in Hank’s balanced salt solution (HBSS), final DMSO concentration

less than 1%, was placed in 21ꢀ28 day confluent monolayer cells in

Transwell assay plates. Both apical and basolateral sides were maintained

at pH 7.4. When dosed on the apical side, the permeability in the AfB

direction was assessed and when dosed on the basolateral side, the BfA

direction was assessed. Both apical and basolateral sides were sampled at

2 h. The concentration of 21c was determined by LC/MS using a

4-point calibration curve. Atenolol (P_app<0.5 ꢁ 10^ꢀ6cm/s), propra-

nolol (15 < P_app< 25 ꢁ 10^ꢀ6cm/s), Lucifer Yellow (P_app> 0.4 ꢁ

10^ꢀ6cm/s), and digoxin (efflux ratio >3) were used in the quality

control of the monolayer batch. The integrity of the monolayer was

determined by measuring the pre-experiment TEER (between 450 and

650 Ωcm²) and using Lucifer Yellow (% efflux e0.5). The efflux ratio

In Vitro Plasma Protein Binding. Equilibrium dialysis was

performed in Micro-Equilibrium Dialyzer (Harvard Apparatus) with a

chamber volume of 500 μL (each compartment with a volume of 250 μL).

The semipermeable membrane used was rinsed with Milli-Q-water

and soaked for 10 min in PBS. Compound 21c was added to plasma

(from mouse, dog and humans) to obtain a final concentration of

1000 ng/mL. The spiked plasma was vortexed, and 250 μL was aliquoted

into one chamber of the dialyzer cell. The other chamber was filled with

250 μL of PBS buffer. The assembled cell was placed into a water-bath at

37 °C, and dialysis was performed for 4 h. Following dialysis, 50 μL of

PBS dialyzed samples containing free 21c was transferred into 2 mL

Eppendorf tubes in triplicate for extraction. Samples were extracted with

1500 μL of MTBE (methyl tertiary-butyl ether) for 30 min using a mixer

at motor speed setting 60 with pulsing. After 30 min, the sample tubes

were centrifuged at 4 °C for 10 min at 13,000 rpm in a microcentrifuge.

The supernatant (1400 μL) was transferred into fresh 2 mL Eppendorf

tubes and dried in SpeedVac at 43 °C for 35 min. The dried samples were

reconstituted with 100 μL of methanol/Milli-Q-H₂O (60:40) and

analyzed by LC/MS/MS.

Pharmacokinetics. Male BALB/c mice (aged ∼8 weeks and

weighing 20ꢀ23 g), male Beagle dogs (∼ 6 to7 months of age, weighing

9ꢀ11 kg), and male Wistar rats (aged 6ꢀ8 weeks, weighing 270 to 325 g)

were used in this study. All of the animal studies were performed as

per approved internal protocols for animal care and use. The oral doses

for mice, dogs, and rats were 30, 3, and 10 mg/kg, respectively. The

doses were administered, by gavage, as suspensions (0.5 % methylcellu-

lose and 0.1%tween 80) to mice and rats, and as gelatin capsules (12

Torpac) to dogs. Following oral dosing, serial blood samples were

collected (retroorbital plexus in mice, jugular vein in dogs, and superior

vena cava in rats) at different time points (0 to 24 h) in tubes containing

K₃EDTA as anticoagulant, and centrifuged, the plasma was separated

and stored at ꢀ70 °C until analysis. Plasma samples were processed and

analyzed by LC/MS/MS. Pharmacokinetic parameters were estimated

by noncompartmental methods using WinNonlin (ver 5.2, Pharsight,

CA)

PAMPA (Parallel Artificial Membrane Permeability Assay).

This assay measures the permeability of compounds in a high

throughput format. The assay was performed in a 96 well format

(Millipore MultiScreen 96 well acceptor plate, Teflon tray, Millipore

MultiScreen 0.4 μm PCTE membrane clear base plate, 96 well,

surface area = 0.24 cm², porosity ∼20%). The final concentration of

the compounds in the assay was 1 μM. The artificial membrane in the

donor plate was prepared by mixing 5% hexadecane in hexane. The

donor plate contained 0.15 mL of 5% DMSO in PBS, and the

compound (1 μM) and the acceptor plate had 0.3 mL of 5% DMSO

in PBS. The assembled plates were placed in a Ziploc bag with wet

C-fold towels to prevent evaporation and incubated for 5 h at room

temperature. Following incubation, the donor and acceptor solutions

were analyzed by LC/MS/MS, and the apparent permeability coeffi-

cient (P_app) was estimated.

High Throughput solubility Assay. This assay measures the

solubility of a compound in PBS in high throughput mode. The assay was

done using 96-well semitransparent PP microplates with V-shaped

bottoms (Greiner Bio-one) and 96-well UV transparent microplates

(Greiner Bio-One). Compound solutions (250 μM) were prepared in

10 mM phosphate buffer (pH 7.0) containing 20% DMSO in a total

volume of 0.2 mL. Plates were placed on a shaker set at 600 rpm for 1.5 h,

following which the plates were allowed to stand for 2 h at room

temperature. The plates were centrifuged at 1500g for 15 min. The

supernatants were transferred to a UV transparent microplate and

analyzed by UV-spectrophotometry at the appropriate absorption max-

ima. The concentration of the compound in the supernatant was

quantified using a calibration curve. For calculated solubilities of 250 (

30 μM, solubilities are reported as >250 μM (>150 μg/mL).

was defined as the ratio of P_app,BfAto P_app,AfB

.

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ARTICLE

Computational. The molecules were built using Maestro 8.0.308 or

converted to 3D structures from the 2D structure using LigPrep version

2.1.207.⁴⁹Basic amines were protonated as in the aqueous solution at

physiological pH. The conformational space was searched using the

Monte Carlo (MCMM) method as implemented in MacroModel version

9.5.207.⁴⁹All heavy atoms and hydrogens on heteroatoms were included

in the test for duplicate conformations. All rotatable single bonds were

included in the conformational search, and all aliphatic rings were ring-

opened, and quaternary atoms were allowed to invert. Each search was

continued until the global energy minima were found at least 3 times. The

energy minimizations were carried out using the truncated Newton

conjugate gradient algorithm (TNCG) and the OPLS-AA force field as

implemented in MacroModel.⁵⁰Default parameters were used. The

conformational searches were done for aqueous solution using the

generalized Born/solvent accessible surface (GB/SA) continuum solva-

tion model.⁵¹

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’ ASSOCIATED CONTENT

S

Supporting Information. Explanation for CDK2 potency

b

of 17h, Western blots showing intracellular target inhibition by

21c, and structures of the RCM catalysts employed. This material

is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

(11) Verstovsek, S. Therapeutic potential of JAK2 inhibitors. Hema-

tol. Am. Soc. Hematol. Educ. Prog. 2009, 636–642.

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therapeutic potential? Blood Rev. 2011, 25, 53–63.

Corresponding Author

*Tel: (0065) 6827-5021. Fax: (0065) 6827-5005. E-mail: anthony_

william@sbio.com.

(13) Verstovsek, S.; Odenike, O.; Scott, B.; Estrov, Z.; Cortes,

J.; Thomas, D. A.; Wood, J.; Ethirajulu, K.; Lowe, A.; Zhu, H. J.;

Kantarjian, H.; Deeg, H. J. Phase I dose escalation trial of SB1518, a

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SB1518, a novel JAK2/FLT3 inhibitor, in the treatment of primary

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’ ACKNOWLEDGMENT

We thank Changyong Hu, Bee Kheng Ng, and Yong Cheng

Tan for the generation of screening data, Venkatesh Reddy for

PK sample analysis, Zahid Bonday and Miah Kiat for protein

preparation work, Sam Ramanujulu Murugappan for NMR

related work, and Dr. Simon Campbell for helpful discussions.

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Sun, E. T.; Tan, E. Y.-L.; William, A. D. Oxygen linked pyrimidine

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JAK2, Janus kinase 2; FLT3, fms-like receptor tyrosine

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MPN, myeloproliferative neoplasms; PV, polycythemia vera; ET,

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