Discovery of the macrocycle (9 E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25- trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9, 14(26),15,17,20,22-nonaene (SB1578), a potent inhibitor of Janus kinase 2/Fms-liketyrosine kinase-3 (J

Article abstract of DOI:10.1021/jm201454n

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (

Full text of DOI:10.1021/jm201454n

Article
pubs.acs.org/jmc
Discovery of the Macrocycle (9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-
trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,-
4,9,14(26),15,17,20,22-nonaene (SB1578), a Potent Inhibitor of Janus
Kinase 2/Fms-LikeTyrosine Kinase-3 (JAK2/FLT3) for the Treatment of
Rheumatoid Arthritis
Anthony D. William,* Angeline C.-H. Lee, Anders Poulsen, Kee Chuan Goh, Babita Madan, Stefan Hart,
Evelyn Tan, Haishan Wang, Harish Nagaraj, Dizhong Chen, Chai Ping Lee, Eric T. Sun,
Ramesh Jayaraman, Mohammad 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
ABSTRACT: Herein, we describe the synthesis and SAR of a
series of small molecule macrocycles that selectively inhibit JAK2
kinase within the JAK family and FLT3 kinase. Following a
multiparameter optimization of a key aryl ring of the previously
described SB1518 (pacritinib), the highly soluble 14l was selected
as the optimal compound. Oral efficacy in the murine collagen-
induced arthritis (CIA) model for rheumatoid arthritis (RA)
supported 14l as a potential treatment for autoimmune diseases
and inflammatory disorders such as psoriasis and RA. Compound
14l (SB1578) was progressed into development and is currently
undergoing phase 1 clinical trials in healthy volunteers.
Inhibition of protein kinases implicated in cytokine signaling
networks has emerged as a potential new treatment paradigm
for RA.5 The hematopoietin family of cytokines, which includes
several postulated to have roles in RA (e.g., interferons, IL-2,
IL-6, IL-7, IL-12, and IL-15), bind to Type I and II cytokine
receptors and signal through the janus kinase-signal transducer
and activator of the transcription (JAK-STAT) pathway.6 There
are 4 members of the JAK kinase family (JAK1, JAK2, JAK3,
and TYK2 (tyrosine kinase 2)) which play essential roles in
several important physiological processes such as hematopoiesis
and lipid homeostasis.7 JAK inhibitors with a range of selectivity
profiles have been described with many now in clinical trials for
treatment of various cancers (JAK1/2) and for immunosup-
pressive disorders (JAK3).8−17 However, the immunosuppres-
sive effects of inhibiting JAK3 may cause severe combined
immunodeficiency syndrome (SCID); hence, targeting selected
JAK proteins is thought to be desirable. JAK2 plays an essential
role in the signaling of pro-inflammatory cytokines involved
in the pathogenesis of RA.18 Hence, JAK2 specific inhibitors
may be differentiated from other JAK inhibitors and produce
therapeutic benefits in inflammatory autoimmune diseases without
immunosuppression. TYK2 is involved in Type I IFN signaling,
INTRODUCTION
■
An estimated 1% of the world’s population is afflicted by
rheumatoid arthritis (RA), a chronic, systemic inflammatory
disorder leading to the destruction of articular cartilage and
ankylosis of the joints.1 RA is a disabling and painful condition,
which leads to substantial loss of functioning and mobility if not
adequately treated. Nonpharmacological treatment includes
physical, occupational, and nutritional therapy, but these do not
stop the progression of joint destruction. Analgesic and anti-
inflammatory drugs, including steroids, are used to suppress the
symptoms, while disease-modifying antirheumatic drugs
(DMARDs), such as methotrexate, hydroxychloroquin, and
sulfasalazine, modify the underlying immune process in an
effort to prevent long-term damage.2 Although many patients
derive benefit from DMARDs, few achieve remission, and there
are many adverse events related to the liver, bone marrow, and
renal toxicity, allergic skin reactions, autoimmunity and infections.
More recently, the advent of biological agents targeted at pro-
inflammatory cytokines, such as the anti-TNFα drugs etanercept,
infliximab, and adalimumab, have successfully suppressed inflam-
mation in many individuals with rheumatoid arthritis (RA).3
However, these and other biologics are expensive drugs with
significant side effects, and the majority of patients relapse when
treatment is withdrawn.4
Received: October 27, 2011
Published: February 17, 2012
© 2012 American Chemical Society
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Figure 1. Modulating the macrocycle phenyl ring system.
and mice with a mutation in TYK2 have reduced susceptibility
to collagen-induced arthritis.19 FLT3 (fms-like tyrosine kinase 3), a
class III receptor tyrosine kinase, has been implicated in RA through
high levels of expression of FLT3-ligand at the site of inflammation
in RA patients.20 In a mouse antigen-induced arthritis (AIA) model
treatment with the FLT3 inhibitor sunitinib reduced the intensity of
synovitis and the incidence of bone destruction.21
Given the above evidence for therapeutic intervention via
blockade of kinase signaling pathways implicated in RA, a specific
JAK2/FLT3/TYK2 antagonist may have therapeutic potential.
We have recently reported the discovery of pacritinib
(SB1518, (2, where R2 = N-pyrrolidinylethoxy, Figure 1), a
unique macrocyclic JAK2/FLT3/TYK2 inhibitor currently in
clinical development for myelofibrosis, lymphoma, and other
cancers,22 and SB1317/TG02 (3, R1 = Me, R2 = H), a CDK/
JAK2/FLT3 inhibitor for the treatment of advanced leukemias
and multiple myeloma.23 Herein, we describe the discovery of a
further series of macrocycles targeting JAK2, FLT3, and TYK2
kinases, a compelling profile for the treatment of rheumatoid
arthritis (RA).
Chemistry. We were inspired to continue to evolve the
macrocycle theme aiming to develop a compound with higher
selectivity, particularly against JAK3, a higher therapeutic
window for nononcology applications, and improved solubility
(lower cLogP), hence improving and differentiating from the
first generation series.22 We set ourselves the challenge of fine-
tuning various aspects of both series described above, searching
for a higher selectivity for JAK2 within the janus kinase family,
while achieving our solubility goals. We set out to explore a
wide range of heterocyclic rings, both saturated and
unsaturated, as well as, monocyclic and bicyclic, in the biaryl-
pyrimidine system (Figure 1).
Synthesis of the macrocycles via the Ring Closing Metathesis
(RCM) reaction pioneered by Grubbs was carried out as
previously reported employing either Grubbs second gener-
ation catalyst ((C46H65Cl2N2PRu), [1,3-bis(2,4,6-trimethyl-
phenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)-
(tricyclohexylphosphine)ruthenium), or Zhan-1B catalyst
((RuCl2[C21H26N2][C12H17NO3S]), 1,3-Bis(2,4,6-trimethyl-
phenyl)-4,5-dihydroimidazol-2-ylidene[2-(isopropoxy)-5-(N,N-
dimethylaminosulfonyl)phenyl]methylene ruthenium(II) di-
chloride).24,25 Most products were isolated as predominately trans
isomers, with a minority as inseparable mixtures of geometrical
isomers. Schemes 1 and 2 illustrate the synthetic routes for con-
struction of the left-hand and right-hand fragments, respectively.
Herein, we have introduced both saturated and unsaturated
ring systems to the left-hand fragments: commercially available
2,4-dichloropyrimidine (1a) reacted readily with 4-piperidine-
methanol under mild basic conditions to afford primary alcohol
4a. Sodium hydride-assisted displacement with allyl bromide
furnished allylether 5a (Scheme 1). Suzuki coupling of 1a with
heterocyclic boronic acids or esters 3a−h gave rise to a diverse
range of intermediates. Indolylpyrimidines 4b and 4q were
subsequently alkylated with either 4-bromo-1-butene, giving 5b,
or allyl bromide to give 5c and 5j. Thiophenes 4h and 4j, furans
4c, 4l, and 4n, and 3-pyridyl 4f aldehyde Suzuki products were
readily reduced to alcohols by sodium borohydride, and alkylation
of the alcohols was achieved under phase transfer conditions with
allyl bromide, giving allylethers 5d−i in moderate to good yield
(Scheme 1).
Methods for preparing right-hand fragments 10a−e have
been previously reported for analogous compounds.22,23 Reduc-
tive amination of 3-nitrobenzaldehye (6a) with N-methyl-
allylamine followed by tin chloride reduction of the nitro group
gave aniline 10a. In a similar reduction/allylation/reduction
sequence, 10b was formed from 6b following an initial alkyla-
tion with bromoethylmethyl ether. Alternatively, alkylation of
6b with 1,2-dichloroethane gives versatile intermediate 6d,
which was reductively aminated, as for 8a, then aminated with
pyrrolidine before nitro reduction to furnish 10c. Allylation of
7a gave 9a in moderate to good yield, followed by nitro reduction
to give anline 10d. Facile reduction of 6d to 7d followed by
allylation, pyrrolidine displacement, and nitro reduction furnished
aniline 10e (Scheme 2).
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a
Scheme 1
a
Reactions and conditions: (a) 3-piperidinemethanol, Et3N, CH2Cl2, rt, 93%. (b) Allyl bromide, NaH (60% in mineral oil), −78 °C→rt, 52%. (c)
Pd(dppf)Cl2·CH2Cl2 (1:1), Na2CO3, 1,2-dimethoxyethane, water, 80 °C, 58−92%. (d) 4-Bromobut-1-ene/allyl bromide, NaH (60% in mineral oil),
DMF, rt, 62−77%. (e) NaBH4, THF, MeOH, 0 °C, 70−94%. (f) Allyl bromide, KOH, TBAHSO4, CH2Cl2, rt, 46−84%.
Terminal alkenes 10a−e were coupled with the left-hand
fragments 5a−j in a high yielding acidic displacement using
hydrochloric acid (Scheme 3). Ruthenium catalyzed RCM
directly formed macrocycles 13a−c, 13e−h, 14a−d, and 14f−n
(Tables 1 and 2). Following coupling of 2-fluoropyridyl biaryls
4e and 4p with 10a and 10c, fluoride displacement with 3-
buten-1-ol, under basic conditions, produced the desired dienes
in moderate yields. Macrocyclisation with Grubbs second genera-
tion catalyst, using hydrochloric acid as an addictive, to neutralize
the basic center, provided macrocyclic 2-pyridyl ethers 13d, 14d,
and 14e.
series of phenyl-pyrimidine compounds.22 A selection of those
linkers combined with (i) saturated; (ii) 5- or 6-membered; and
(iii) bicyclic heterocycles were installed in some initial exploratory
compounds. We rapidly lost interest in saturated compounds
given the complete lack of activity of 13a. However, other
saturated series, with shorter linkers, were more active and will
be reported in a future publication. Focusing on heteroaromatic
rings, we decided to maintain the macrocycle ring size at either
17 or 18 atoms, which was found to be optimal for JAK2
activity in the phenyl series.22 Incorporation of a bicyclic indole
into the structure was possible with these restrictions giving
13b and 13c. Both compounds were potent against FLT3 and
selective against JAK3, two key aspects of our target profile.
However, only 13c was potent against JAK2, the observed 10-
fold JAK2 potency difference may be explained by the larger
ring size of 13b leading to some unfavorable clashes with the
RESULTS AND DISCUSSION
■
Preliminary Study of Heterocycle−Linker Combina-
tions. Linkers of 8 atoms in length between the meta positions
of both aryl groups were previously found to be optimal in a
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a
Scheme 2
a
Reactions and conditions: (a) N-Allylmethyl amine, Na(OAc)3BH, CH2Cl2, rt, 75−95%. (b) SnCl2·2H2O, CH2Cl2/MeOH (1:1), rt, 64−98%. (c)
Allyl bromide, KOH, TBAHSO4, CH2Cl2, rt, 55−78%. (d) 2-Bromoethyl methyl ether, K2CO3, DMF, 80 °C, 86%. (e) 1,2-Dichloroethane, K2CO3,
DMF, 100 °C, 72%. (f) NaBH4, THF, water, 0 °C, 94%. (g) Pyrrolidine, DMA, 90 °C, 75−78%.
protein (Figure 2). More importantly, both compounds were
active against CDK2 in the same concentration range as FLT3,
a critical selectivity issue. Given this negative aspect and the
generally greater challenges of synthesizing the indole macro-
cycles, we focused our attention on monocyclic rings. Pyridyl
rings with the basic linker, such as 13d, retained their high
potency for CDK2 as expected but unfortunately lost JAK2
potency as compared to the corresponding phenyl ring (CDK2
IC50 0.013 μM and JAK2 IC50 0.073 μM).23 CDK2 selectivity
can be increased by exchanging the basic center in the linker for
an ether oxygen, as in 13e, but surprisingly, JAK2 potency was
completely abolished. This appears to be due to the placement
of the pyridyl nitrogen, which is in a highly unfavorable location
with regard to Asp994. Since the pyridyl is not protonated, the
opportunity to interact with Asp994 is lost, and instead, a
repulsive interaction forces the ligand out of the binding site
(Figure 3). This is significantly reversed on changing the nature
of the linker to a neutral ether group (13f), although selectivity
for JAK1 and 3 has been achieved, CDK2 potency is high,
whereas JAK2 and FLT3 activity is weak. The smaller furan ring,
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a
Scheme 3
a
Reactions and conditions: (a) 4 M HCl, n-butanol, 80−100 °C, 81−86%. (b) 3-Buten-1-ol, NaH (60% in mineral oil), THF, 0 °C, 36−48%. (c).
Grubbs second generation or Zhan catalyst, HCl, CH2Cl2, 45 °C, 36−76%.
with its hydrogens projecting externally from the macrocycle,
was then explored with the same two linker types for com-
parison. Basic 13g was a generally uninteresting moderately
potent CDK2 inhibitor, but 13h was much more interesting
with submicromolar JAK2, TYK2, and FLT3 potency, as well as
selectivity for JAK1 and 3. CDK2 potency is still a major issue
(see Figure S2, Supporting Information), but we felt this series
was worthy of further work to attempt to improve potency
against the key targets and eliminate CDK2 activity.
Study of Heterocycle−Linker Combinations with Solu-
bilizing Side Chains. From our earlier work,22 we had
identified a robust solubilizing side chain which conferred good
JAK2 and FLT3 potency as well as physicochemical properties.
Given the similarities between this reported series and the
heterocyclic series discussed herein, we decided to install the
same side chain in a selection of heterocyclic macrocycles from
Table 1 to understand if the same benefits were transferred.
Adding the preferred side chain to 13c gave 14a, which did
experience a slight improvement in JAK2 potency; however, its
FLT3 potency was increased by an order of magnitude giving a
single digit nanomolar IC50 (Table 2). Although CDK2 potency
of 14a dropped by 3-fold, JAK3 potency had become
significant. Addition of a 5′-methyl group to the pyrimidine
ring has been previously found to boost JAK2 (and,
undesirably, JAK3) potency in the phenyl series.22 However,
when the same change is installed in the indole, giving 14b,
JAK2 potency is actually reduced, and surprisingly, JAK3 acti-
vity is abolished. CDK2 potency of 14b dropped by 10-fold
compared to that of 14a, but the high cLogP and disappointing
JAK2 activity prompted us to move our focus to monocyclic rings.
Comparing 3-pyridyl 14c with 13f, we observed a trend
similar to that of the indole series: a modest improvement in
JAK2 potency, good FLT3 activity, and much improved
CDK2/JAK2 selectivity. However, JAK3/JAK2 selectivity had
dropped significantly. In general, 3-pyridyl compounds do not
benefit fully from the addition of the pyrrolidinylethoxy side
chain most likely due to repulsive forces between the unpro-
tonated lone pair and amino acid residues (Figure 3). However,
this is not the case with the 4-pyridyl compounds typified by
14d, which has an IC50 of 18 nM against JAK2. In addition, 14d
has good activity against TYK2 and FLT3 and over 10-fold
selectivity for CDK2 and over 50-fold selectivity for JAK1 and
JAK3. The direct comparator without the side chain was not
prepared, but clearly this profile is much improved over 13d
and moving toward our target profile. As discussed above for
14b, previous studies had shown that a 5′-methyl pyrimidine
group could boost JAK2 potency. When 14e was prepared and
tested, we found that JAK2 potency remained unchanged, but
we were pleasantly surprised to see much increased selectivity
for CDK2 (CDK2/JAK2 = 278-fold for 14e); however, the
extra methyl had increased cLogP to 4.8. For this reason, we
did not extend the study of the 5′-pyrimidyl substituents.
Such good potency of pyridyl analogues 14d and 14e may be
explained by a potential water-mediated H-bond to Asp981
(Figure 4).
5-Membered heterocycles were also investigated with the
preferred pyrrolidinyl-ethyloxy side chain. Furan and thiophene
analogues 14f and 14g with the basic N-methyl linker were
moderately potent against JAK2 but the latter exhibited good
activity for TYK2 and FLT3. An alternative side chain,
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Table 1. SAR of Macrocycles with Hetero-Atomic Ring Bi-Aryl to Pyrimidine
a
b
All compounds were isolated as c.95:5 trans/cis mixtures. All IC50 values are the mean SD of at least two independent experiments.
methoxyethoxy (compounds 14h and 14i), showed that the
pyrrolidine enhances JAK2 potency by 3−5-fold (compare 14f
with 14h and 14g with 14i). Again, as for 14f and 14g, the
thiophene was more active than furan. This may be explained
by the highly lipophilic nature of the binding site where the
thiophene is interacting (Figure S1, Supporting Information).
Significant JAK2 potency gains were achieved with the com-
bination of thiophene, diether linker, and pyrroidine side chain:
14j exhibited a JAK2 IC50 of 7 nM with 32 and 24 nM for
TYK2 and FLT3, respectively. Furthermore, this compound
was 400-, 57-, and 89-fold selective for CDK2, JAK1, and JAK3,
respectively. Although cLogP was a little high, we were encouraged
to progress the profiling of 14j due to its excellent potency and
selectivity profile.
When the thiophene sulfur is moved to a regioisomeric
position at the exterior of the macrocycle (14k), the in vitro
potency profile was quite similar to that of 14j, but cLogP rises
significantly. Constraints on the molecular conformation of the
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macrocycles clearly have the potential to result in significantly
different physicochemistry lending another dimension to the
SAR and opportunities to tune the structure to a preferred
profile. The analogous furan to 14j was less active against JAK2
(see Figure S1, Supporting Information) but did have a very
positive selectivity profile: 14l has a JAK2 IC50 of 46 nM and
selectivity of 152-, 58-, and 93-fold for CDK2, JAK1, and JAK3,
respectively. Its potency against FLT3 is in the same con-
centration range as JAK2 with 5-fold selectivity against TYK2.
cLogP is lower for 14l than for 14j, but both compounds were
selected for further assessment along with 14k. Another com-
pound selected for further profiling was 14m, the furan com-
parator of 14k. In this case, the cLogP is reduced to 3.6 with
the polar oxygen atom more exposed to solvent. The overall
potency profile of 14m is similar to that of 14l but with improved
CDK2 selectivity (14m CDK2 IC50 >10 μM). Moving the oxygen
atom in the furan to the remaining ortho position (14n), with
respect to the pyrimidine, results in a further drop in JAK2 and
Table 2. SAR Study of Aromatic Ring Substitutions with Small Groups for Fine Tuning Selectivity and Solubility
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Table 2. continued
a
b
All compounds were isolated as c.95:5 trans/cis mixtures. All IC50 values are the mean SD of at least two independent experiments.
Figure 2. (A) Compound 13c docked into JAK2. Hydrogen bonds are
shown in magenta dashed lines and close contacts in orange dashed
lines. The only close contact between 13c and JAK2 is between the
backbone carbonyl of Glu930 and the free hydrogen next to the
pyrimidine nitrogen of 13c. This is an attractive interaction and may
be described as a weak hydrogen bond. (B) Compound 13b docked
into JAK2. The macrocyclic linker of 13b is more bulky than that of
13c, and consequently 13b has several close contacts to the protein.
The reduced JAK2 activity of 13b may be due to slight clashes with the
protein.
Figure 3. Sitemap analysis of JAK2 with 13e docked into the ATP-
binding site. White dots indicate positions where it is favorable to
place ligand atoms. The yellow, blue, and red grids are hydrophobic
and hydrogen bond acceptor and donor areas of the binding site,
respectively. The pyridine nitrogen falls into the hydrogen bond donor
FLT3 activity below 100 nM, although the excellent CDK2 selec-
area around the carboxylic acid group of Asp994. The interaction with
tivity was retained.
Asp994 will be repulsive (distance 3.7 Å) unless the pyridine is
protonated. The pyridine pKa is calculated by Epik to be 3.6, which
indicates it is not protonated; hence, the negative interaction of the
pyridine forces the compound out of the binding site leading to a
lower IC50.
Further In Vitro Profiling of Selected Compounds.
Selected inhibitors which met our criteria of JAK2 and FLT3
potency with selectivity against CDK2, JAK1, and JAK3 were
further profiled in vitro (Table 3). A few potent compounds
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Figure 4. Compound 14d docked into JAK2. There is room for a
water molecule which may mediate a hydrogen bond between the
pyridine nitrogen and the side chain of Asp981. This may explain the
increased JAK2 activity of pyrimidine analogues like 14d and 14e.
with higher cLogPs (14e and 14k) are compared for
information purposes. Even the higher cLogP compounds
had acceptable solubility, but 14l and 14m were superior in this
important respect. CYP3A4 and 2D6 were not an issue with the
tested compounds with the exception of the high cLogP 14e,
which inhibited CYP3A4 with an IC50 of 0.97 μM. Human
microsomal stabilities were remarkably good, as has been
previously noted with these macrocyclic compounds. Stability
was lower in mouse microsomes but still acceptable for further
preclinical evaluation in mouse models. On-target cellular
potency was evaluated in a cell proliferation assay using two
cancer cells lines: Ba/F3 (driven by the JAK2 V617F mutant)
and HL60 (driven by FLT3). Cell proliferation data (GI50) vary
about 5-fold between the selected compounds (Table 3). As a
measure of cell inhibition efficiency, GI50/IC50 shows that 14k
and 14l were the most efficient compounds in Ba/F3 cells
(JAK2 driven) and that 14j was the best performing compound,
relative to its enzyme IC50, in HL60 cells. Compounds 14k, 14l,
and 14m were similarly efficient in HL60 cells. Apart from
solubility, there did not appear to be any significant differences
in vitro between high and low cLogP compounds. Of the
preferred lower cLogP compounds 14j, 14l, and 14m, we were
concerned about the metabolites of furan compounds. Furans
can be metabolized to 1,4-cis-butendials, which are toxic.26,27
However, it is reported that 2,5-disubstitued furans, such as
ranitidine, are quite stable in vivo;28 hence, we felt that 14l,
being 2−5-disubstituted, posed less of a risk than 14m. Highly
potent 14j was an attractive compound from most respects but
only moderately soluble. Selection of a preferred single
candidate was aided by hERG screening (binding assay). In
general, the pyridyl derivatives were moderately potent with
IC50s in the 1−5 μM range. We were aiming to eliminate hERG
activity as a concern; hence, 14l was preferred over 14j (>10
μM (14% at 10 μM) and 7.2 μM, respectively). Taken together,
this complex set of data supported 14l as the preferred
candidate. Although 14l was not always the best compound in
each test category, it did, however, meet our minimum target
profile in all areas with a balanced compromise between the
criteria. Hence, 14l was selected as the preferred drug candidate
and went forward into more advanced profiling studies.
In silico, 14l meets all accepted drug-like guidelines for an
orally absorbed small molecule with molecular weight less than
500, a cLogP less than 5, numbers of H-bond donors and
acceptors less than 5 and 10, respectively, and a polar surface
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area of less than 120 Å2. Compound 14l is highly soluble,
>250 μg/mL, in the kinetic experiment and 0.88−1.43 mg/mL
in the thermodynamic experiment buffered to pH 7.0. This
solubility compares very well to that of 2 (where R2 = N-
pyrrolidinylethoxy, Figure 1), which is 150 μg/mL at pH 7.0. In
the Caco-2 bidirectional permeability assay, 14l showed high
permeability with an efflux ratio of 1.1, which indicated that 14l
is probably not a substrate for p-glycoprotein in humans and
thus may show high intestinal absorption. Compound 14l was
found to be stable in liver microsomal stability studies with HLM,
MLM, RLM, and DLM t1/2 > 60 min and was moderately stable
in MoLM with t1/2 of 23 min (Table 4). Compound 14l did not
Table 5. Oral Pharmacokinetic Parameters (Mean SD) of
14l in Mouse, Rat, Dog, and Rhesus Monkey
dog
(n = 3)
a
parameter
mouse
rat (n = 3)
monkey(n = 3)
dose (mg/kg)
Cmax (ng/mL)
tmax (h)
50
50
5
20
1227
0.5
231 85
2.3 1.5
3.6 1.4
1499 192
6
27
7
1976 575
2.0 1.0
1.1 0.3
7590 2958
32
3.0 1.0
1.5 0.5
96 33
6
t1/2 (h)
0.8
AUC0‑∞(ng·h/mL)
2020
34
oral bioavailability
b
(F%)
a
Estimated from mean concentration time data (n = 3 mice per time
b
point). Mean.
Table 4. Physicochemical Properties and in Vitro ADME
of 14l
The terminal t1/2 was moderate in mice, dogs, and monkeys
(ranged between 0.8 and 1.5 h) and longer in rats (3.6 h). The
oral bioavailability was moderate in mice and monkeys (32−
34%) and poor in rats and dogs (6%). Exposures achieved in
mice at 50 mg/kg exceeded the pharmacological concentrations
(JAK2 IC50 = 46 nM and FLT3 IC50 = 62 nM).
Modulation of JAK2 Signaling Pathways. The effects of
14l on JAK2 signaling were investigated in 4 different cellular
contexts: a cell line expressing constitutively activated mutant
JAK2 (SET2, Figure 5A);31 a cell line expressing constitutively
activated wild-type JAK2 (MDA-MB-231, Figure 5B); a cell
line requiring exogenous ligand to activate wild-type JAK2
(HEK293, Figure 5C); and primary human T cells (Figure
5D). JAK2 signaling was dose-dependently inhibited by 14l
treatment in all four cell systems. Note that 14l inhibited cell
proliferation in SET2 cells with an IC50 of 0.246 nM.
property
value
mol.wt
463
no. of HDB
no. of HBA
cLogP
1
7
3.9
log D (pH 7.4)
PSA (Å2)
1.58
82
a
thermodynamic solubility pH 7.0
(mg/mL)
0.88−1.43
b
permeability (Papp, A→B, × 10−6 cm/
24.8, efflux ratio = 1.1 (low)
sec)
c
liver microsomes (t1/2, min)
MoLM (t1/2, min)
>60
d
23
e
f
human CYP inhibition IC50 (μM)
CYP450 phenotyping
>10 ; 1.4
cleared by 3A4
Following cell incubation with 14l, 30 μg of cell lysates were
resolved on SDS−PAGE, transferred onto PVDF membranes,
and probed with the respective antibodies. (A) SET2 cells were
treated with various concentrations of 14l for 4 h. (B) MDA-
MB-231 cells were treated with various concentrations of 14l
for 4 h. (C) HEK293 cells were incubated with various con-
centrations of 14l for 3 h followed by treatment with 1000
U/mL IFN-γ. (D) Human CD4+ T cells were incubated with
various concentrations of 14l for 3 h followed by treatment
with 10 ng/mL interleukin-12.
Treatment with 14l is Efficacious in the Murine
Collagen Induced Arthritis Model. The murine CIA
model recapitulates the clinical and histological development
of human rheumatoid arthritis and has been useful in evaluating
the effects of clinically used agents.32,33 After the onset of
collagen induced arthritis, mice were administered twice daily
with doses of 105 mg/kg or 210 mg/kg (free base equivalent)
of 14l, and the treatment was continued for 10 days. In the
vehicle group, the clinical arthritic score of mice increased
rapidly from day 0 to disease day 11 (Figure 6). Treatment with
14l led to a dose-dependent inhibition of arthritis, with a 23%
and 59% reduction in the AUC in the mice administered
105 mg/kg and 210 mg/kg 14l, respectively. Treatment effects
at the higher dose were comparable to the mice treated with
dexamethasone with a 63% reduction in the AUC. This
reduction in the clinical arthritis scores was also accompanied
by substantial improvement in the histopathology of the drug-
treated mice (data not shown). This data shows that inhibition
of JAK2 signaling, which is involved in the pathological
responses of inflammatory and autoimmune diseases, could be
an efficacious new paradigm for the treatment of rheumatoid
arthritis.
CYP450 induction
negative for 3A4 and 1A2
g
plasma protein binding (%)
99.0 (H); 98.7(Mo), 87.3−87.5
(M, R, D)
a
b
Two determinations of two different batches of 14l. Caco-2
c
bidirectional permeability assay. Human, dog, rat, and mouse.
d
e
Rhesus monkey microsomes. Human CYP3A4, 1A2, 2D6,
f
g
2C9·CYP3A4, and 2D6; fluorescence detection. 2C19. Equilibrium
dialysis assay at 1000 ng/mL. H = human, Mo = monkey, M = mouse,
R = rat, and D = dog.
inhibit the major drug metabolizing human CYP450s 1A2, 3A4,
2D6, and 2C9 at below 10 μM but did inhibit 2C19 (IC50 ∼1.4
μM), suggesting that it may not have the potential to cause
drug−drug interactions in vivo. In vitro plasma protein binding
of 14l was high and similar in humans and monkeys and was
significantly lower in mouse, dog, and rat plasma.
Testing against a panel of kinases revealed that 14l is
approximately 20-fold selective against a cross-section of other
kinases with the exception of c-fms receptor kinase (IC50 69 nM)
and CDK9 (IC50 36 nM). Elevated levels of c-fms ligand,
macrophage colony stimulating factor (M-CSF), are observed
in the joints of RA patients; hence, due to its pivotal role in the
pathogenesis of RA, the blockade of the M-CSF/c-fms axis has
been shown to inhibit the progression of arthritis.29 CDK9
promotes RNA synthesis for cell growth and differentiation and
has been identified as a potential target for therapeutic inter-
vention in oncology, virology, and cardiology.30
Pharmacokinetics of 14l in Multiple Species. The
pharmacokinetic properties of 14l are sumarized in Table 5.
Compound 14l showed rapid to moderately fast absorption
(tmax ranged between 0.5 to ∼2.3 h) in mice, rats, and monkeys
and was relatively slower in dogs.
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Journal of Medicinal Chemistry
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Figure 5. Compound 14l blocks JAK2 signaling pathways in multiple cellular contexts.
selectivity is, in fact, achievable starting from rather unpro-
mising N-aryl pyrimidines. Ring-closing metathesis chemistry
has been successfully employed to construct relatively rigid
ATP-competitive heterobiaryl macrocycles, with significantly
reduced degrees of conformational freedom over typical non-
macrocyclic kinase inhibitors. In addition to being potent
inhibitors of JAK2, FLT3, and TYK2, this series has good
physicochemical properties explaining the good translation of
potency to the cell. Exchange of the previously described
phenyl system22 for heteroaryls was found to be fruitful with
SAR studies leading to the 5-membered thiophene and furan
rings as preferred motifs. Following extensive in vitro and in
vivo profiling, highly soluble 14l, with negligible hERG binding
inhibition, was shown to inhibit its targets in cells and in vivo.
Tolerable twice daily oral doses of 14l in the mouse CIA model
led to efficacy similar to that of dexamethasone; hence, 14l was
therefore selected for further development. Compound 14l
(SB1578) is currently being evaluated in phase 1 clinical trials
in healthy volunteers.
Figure 6. Compound 14l attenuates clinical symptoms of arthritis in a
mouse CIA model.
EXPERIMENTAL SECTION
Arthritis was induced in mice by immunization with type II
collagen emulsified in Freund’s Complete Adjuvant. Following
the disease onset (day 0), the mice were enrolled into
treatment groups of 10 and were orally administered with 14l
(105 or 210 mg/kg b.i.d, free base equivalent) or dexametha-
■
Chemistry. Purity was >95% for all tested compounds except those
indicated in their respective synthesis descriptions. For detailed HPLC
analytical methods and general chemistry, refer to our earlier
publication.22
[1-(2-Chloro-pyrimidin-4-yl)-piperidin-3-yl]-methanol (4a). To a
solution of 2,4-dichloropyrimidine 1a (1.86 g, 12.5 mmol), 3-
piperidinemethanol (1.59 g, 13.8 mmol) in CH2Cl2 (80 mL) was
added Et3N (2.0 mL, 14.4 mmol), and the resulting mixture was
stirred at room temp for 2 h. Aqueous Na2CO3 was added, and the
product was extracted with CH2Cl2 thrice. After workup, a solid (2.67
g; yield, 94%) was obtained as a mixture of title compound and isomer
(i.e., [1-(4-chloro-pyrimidin-2-yl)-piperidin-4-yl]-methanol) in a ratio
̈
sone (0.1 mg/kg); naive group had 4 mice. Clinical arthritis
scores were evaluated daily for 10 days. The graph represents
the analysis of clinical arthritis scores with area under the
dosing curve calculation, *p ≤ 0.05, Mann−Whitney U test to
vehicle.
CONCLUSIONS
■
1
of 87:13 as determined by H NMR. The crude mixture was taken
This article provides a further demonstration that conforma-
tionally constrained macrocycles can be potent and selective
inhibitors of kinases. This research has general applicability to
kinase inhibitors since the macrocyclic linkage is distinct from
the hinge binding region of the inhibitor. Using this simple
paradigm, we demonstrate that a significant degree of kinase
forward for the next step without any further purification. The title
compound (the major product): LC-MS (ESI positive mode) m/z 228
([M + H]+). 1H NMR (DMSO-d6) selected peaks: δ 8.00 (d, 1H, J =
6.2 Hz), 6.76 (d, 1H, J = 6.3 Hz), 4.67 (t, 2H, J = 5.2 Hz); 13C NMR
(DMSO-d6) δ 162.0, 159.6, 157.1, 102.1, 63.3, 47.0, 44.1, 38.3, 26.7,
23.9.
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6-(2-Chloro-pyrimidin-4-yl)-1H-indole (4b). To a prestirred
mxiture of 2,4-dichloropyrimidine 1a (0.83 g, 5.5 mmol), 1H-indol-
6-yl boronic acid (0.83 g, 5.0 mmol) in 1,2-dimethoxyethane (40 mL)
and saturated aqueous solution of NaHCO3 (4 mL) was added
Pd(PPh3)4 (0.30 g, 0.26 mmol). The solution was heated to 85 °C for
6 to 8 h. After the reaction was completed, the solution was cooled
down and filtered through Celite. The filtrate was added to ethyl
acetate (40 mL) and water (20 mL), and the organic layer was
separated, washed with water (3×) and brine (3×), and concentrated.
The residue was purified by flash chromatography (silica, EtOAc/
hexanes from 1:5 to 1:2) to afford the title compound (0.76 g, yield
66%). 1H NMR (CDCl3) δ 9.74 (s, 1H), 9.23 (d, 1H, J = 1.2 Hz), 8.83
(d, 1H, J = 5.3 Hz), 8.01 (dd, 1H, J = 5.3, 1.2 Hz), 7.60−7.65 (m, 1H),
7.35 (t, 1H, J = 8.2 Hz), 6.94−6.99 (m, 1H). LC-MS (ESI positive
mode) m/z 230 ([M + H])+ .
5-(2-Chloro-pyrimidin-4-yl)-furan-2-carbaldehyde (4c). To a
degassed (nitrogen) solution of 2,4-dichloropyrimidine 1a (3.8 g,
25.51 mmol) and 5-formyl-2-furan boronic acid (3 g, 21.44 mmol) in
1,2-dimethoxy ethane [DME] (100 mL) were added aq. Na2CO3 (5.5
g in 10 mL of H2O, 51.0 mmol) and Pd(dppf)Cl2 CH2Cl2 [1:1] (0.87
g, 1.07 mmol). The reaction mixture was stirred to 80 °C for 4 h.
It was cooled to rt and poured into water and extracted with CH2Cl2
(3 × 40 mL). The combined organic extracts were washed with brine,
dried over Na2SO4, and concentrated under reduced pressure to
obtain the crude residue. The crude material was purified by column
chromatography (EtOAc/Hexane 1:2) to afford 4c (3.01 g; yield,
67%) as an off-white solid. LC-MS (ESI positive mode) m/z 209
([M + H]+).
furnish without purification 2.14 g of compound 4d (yield, 94%). LC-
MS (ESI positive mode) m/z 211 ([M + H]+).
Following a procedure similar to that of 4d, the following
intermediates were synthesized.
[5-(2-Chloro-pyrimidin-4-yl)-pyridin-3-yl]-methanol (4g). The
title compound was synthesized from 4f (yield, 75%). LC-MS (ESI
positive mode) m/z 222 ([M + H]+).
[5-(2-Chloro-pyrimidin-4-yl)-thiophen-2-yl]-methanol (4i). The
title compound was synthesized from 4h (yield, 70%). LC-MS (ESI
1
positive mode) m/z 227 ([M + H]+); H NMR (CDCl3) δ 8.62 (d,
1H), 7.55 (m, 2H), 7.25 (d, 1H), 4.83 (s, 2H), 4.68 (bs, 1H).
[4-(2-Chloro-pyrimidin-4-yl)-thiophen-2-yl]-methanol (4k). The
title compound was synthesized from 4j (yield, 80%). LC-MS (ESI
positive mode) m/z 227 ([M + H]+).
[4-(2-Chloro-pyrimidin-4-yl)-furan-2-yl]-methanol (4m). The title
compound was synthesized from 4l (yield, 93%). LC-MS (ESI positive
mode) m/z 211 ([M + H]+).
[5-(2-Chloro-pyrimidin-4-yl)-furan-3-yl]-methanol (4o). The title
compound was synthesized from 4n (yield, 78%). LC-MS (ESI
positive mode) m/z 211 ([M + H]+).
4-(3-Allyloxymethyl-piperidin-1-yl)-2-chloro-pyrimidine (5a). A
solution of 4a (1.12 g, 4.91 mmol) in anhydrous THF (40 mL) was
added allyl bromide (1.5 mL, 17.3 mmol) and cooled in an acetone/
dry ice bath. Sodium hydride (60% in mineral oil, 0.910 g, 22.7 mmol)
was added to the mixture under N2, after 10 min, the dry ice bath was
removed, and the resulting mixture was stirred at room temp
overnight. The reaction was quenched by the addition of dry ice
powder and MeOH, and then concentrated under reduced pressure.
The residue was diluted with aqueous NaHCO3 and extracted with
CH2Cl2 thrice. After workup, the crude product was purified by flash
chromatography (silica, 30−60% EtOAc in hexanes) to afford the title
compound as a colorless oil (0.68 g; yield, 52%). LC-MS (ESI positive
Following a procedure similar to that of 4c, the following
intermediates were synthesized.
2-Chloro-4-(2-fluoro-pyridin-4-yl)-pyrimidine (4e). The title com-
pound was synthesized from 1a and 2-fluoro-4-pyridine boronic acid
3c (yield, 61%). LC-MS (ESI positive mode) m/z 210 ([M + H]+);
1H NMR (CDCl3): δ 8.85 (d, 1H), 8.47 (d, 1H), 7.87 (dt, 1H), 7.75
(d, 1H), 7.67−7.68 (m, 1H).
5-(2-Chloro-pyrimidin-4-yl)-pyridine-3-carbaldehyde (4f). The
title compound was synthesized from 1a and 5-formyl-3-pyridine
boronic acid pinacol ester 3a (yield, 80%). LC-MS (ESI positive
mode) m/z 220 ([M + H]+).
1
mode) m/z 268/270 ([M + H]+); H NMR (CDCl3) δ 7.89 (d, 1H,
J = 6.2 Hz), 6.35 (d, 1H, J = 6.2 Hz), 5.82 (ddt, 1H, J = 17.2, 10.4, 5.6
Hz), 5.20 (ddt, 1H, J = 17.2, 1.7, 1.7 Hz), 5.10 (ddt, 1H, J = 10.4, 1.6,
1.5 Hz), 4.08 (b s, 2H), 3.88 (dm, 2H, J = 7.1 Hz), 3.29 (dd, 1H, J =
9.4, 5.0 Hz), 3.22 (dd, 1H, J = 9.3, 7.7 Hz), 3.05 (ddd, 1H, J = 13.1,
10.6, 3.0 Hz), 2.86 (dd, 1H, J = 13.0, 9.7 Hz), 1.86−1.74 (m, 2H),
1.72−1.64 (m, 1H), 1.51−1.39 (m, 1H), 1.36−1.13 (m, 1H); 13C
NMR (CDCl3) δ 162.4, 160.6, 156.9, 134.7, 116.9, 101.3, 72.1, 72.0,
47.8, 44.8, 36.2, 27.3, 24.1.
5-(2-Chloro-pyrimidin-4-yl)-thiophene-2-carbaldehyde (4h). The
title compound was synthesized from 1a and 5-formyl-2-thiophene
boronic acid 3e (yield, 84%). LC-MS (ESI positive mode) m/z 225
1-Allyl-6-(2-chloro-pyrimidin-4-yl)-1H-indole (5c). A solution of
4b (115 mg, 0.5 mmol) and NaH (73 mg, 60% in mineral oil, 0.9
mmol) in 2 mL of dried DMF was stirred at room temperature for
30 min. Allyl bromide (121 mg, 1.0 mmol) in 1 mL of dried DMF was
added to the solution. The mixture was stirred at room temperature
overnight. The reaction was quenched by adding water slowly. The
mixture was extracted with EtOAc thrice, and the combined extracts
were washed with water 3 times and then washed with brine once. The
product 5c (112 mg; yield, 83%) was obtained by flash column
chromatography. (EtOAc/hexanes from 1:10 to 1:4) LC-MS (ESI
1
([M + H]+); H NMR (CDCl3) δ 10.64 (s, 1H), 8.68 (d, 1H), 7.66
(m, 2H), 7.57 (d, 1H).
4-(2-Chloro-pyrimidin-4-yl)-thiophene-2-carbaldehyde (4j). The
title compound was synthesized from 1a and 2-formyl-4-thiophene
boronic acid 3f (yield, 92%). LC-MS (ESI positive mode) m/z 225
([M + H]+).
4-(2-Chloro-pyrimidin-4-yl)-furan-2-carbaldehyde (4l). The title
compound was synthesized from 1a and 2-formyl-4-furan boronic acid
3g (yield, 61%). LC-MS (ESI positive mode) m/z 209 ([M + H]+).
5-(2-Chloro-pyrimidin-4-yl)-furan-3-carbaldehyde (4n). The title
compound was synthesized from 1a and 4-formyl-2-furan boronic acid
3h (yield, 70%). LC-MS (ESI positive mode) m/z 209 ([M + H]+).
2-Chloro-4-(2-fluoro-pyridin-4-yl)-5-methyl-pyrimidine (4p). The
title compound was synthesized from 2,4-dichloro-5-methylpyrimidine
1b and 2-fluoro-4-pyridine boronic acid 3c (yield, 58%). LC-MS (ESI
positive mode) m/z 224 ([M + H]+).
1
positive mode) m/z 270 ([M + H]+); H NMR (CDCl3) δ 8.54 (d,
1H), 8.21 (s, 1H), 7.75 (d, 1H), 7.71 (d, 1H), 7.66 (d, 1H), 7.25 (d, 1H),
6.56 (d, 1H), 6.02 (m, 1H), 5.25 (d, 1H), 5.12 (d, 1H), 4.83 (d, 2H).
Following a procedure similar to that of 5c, the following
intermediates were synthesized.
1-But-3-enyl-6-(2-chloro-pyrimidin-4-yl)-1H-indole (5b). The title
compound was synthesized from 4b and 4-bromo-1-butene (yield,
62%). LC-MS (ESI positive mode) m/z 284 ([M + H]+).
1-Allyl-6-(2-chloro-5-methyl-pyrimidin-4-yl)-1H-indole (5j). The
title compound was synthesized from 4q and allyl bromide (yield,
77%). LC-MS (ESI positive mode) m/z 284 ([M + H]+).
6-(2-Chloro-5-methyl-pyrimidin-4-yl)-1H-indole (4q). The title
compound was synthesized from 2,4-dichloro-5-methylpyrimidine 1b
and 6-indole boronic acid 3a (yield, 77%). LC-MS (ESI positive
mode) m/z 244 ([M + H]+).
[5-(2-Chloro-pyrimidin-4-yl)-furan-2-yl]-methanol (4d). To a
solution of 4c (2.25 g, 10.79 mmol) in THF (40 mL), cooled at
0 °C, was added NaBH4 (0.82 g, 21.58 mmol), followed by MeOH
(10 mL) dropwise, maintaining the temperature at 0 °C. The resulting
mixture was brought to rt and stirred for 30 min. The reaction mixture
was quenched with H2O and extracted with EtOAc (3 × 25 mL). The
combined organic extracts were washed with H2O, followed by brine,
dried over Na2SO4, and concentrated under reduced pressure to
4-(5-Allyloxymethyl-furan-2-yl)-2-chloro-pyrimidine (5d). A mix-
ture of 4d (10 g, 47.5 mmol), KOH (5.3 g, 94.6 mmol), and tetrabutyl
ammonium hydrogen sulfate [TBAHSO4] (0.79 g, 2.38 mmol) in allyl
bromide (40 mL, 4 vol) was stirred at rt for 12 h. The reaction mixture
was quenched with water and extracted with CH2Cl2 (3 × 20 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
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15% EtOAc/hexanes) to furnish 5d (10.0 g; yield, 84%) as a pale
yellow oil. LC-MS (ESI positive mode) m/z 251 ([M + H]+).
Following a procedure similar to that of 5d, the following
intermediates were synthesized.
Allyl-[2-(2-chloro-ethoxy)-5-nitro-benzyl]-methyl-amine (8c). The
title compound was synthesized from 6d and N-allyl methyl amine
(yield, 85%). LC-MS (ESI positive mode) m/z 285 ([M + H]+).
[5-(2-Chloro-pyrimidin-4-yl)-furan-3-yl]-methanol (8d). To a
solution of 8c (2 g, 7.02 mmol) in N,N-dimethylacetamide (DMA,
10 mL) was added pyrrolidine (4 mL, 2 vol), and the resulting mixture
was stirred at 90 °C for 20 h. The reaction mixture was brought to rt
and quenched with water (20 mL) and extracted in ethyl acetate (3 ×
20 mL). The combined organic extracts were washed with water,
brine, dried (Na2SO4), and concentrated under reduced pressure to
afford the crude 8d (1.68 g; yield, 75%) as thick syrup. This
intermediate was taken forward for the next step without any
purification. LC-MS (ESI positive mode) m/z 320 ([M + H]+).
1-[2-(2-Allyloxymethyl-4-nitro phenoxy] pyrrolidine (9c). Follow-
ing a procedure similar to that of 8d, the title compound was
synthesized from 9b and pyrrolidine (yield, 78%). LC-MS (ESI
positive mode) m/z 307 ([M + H]+). Theoretical HRMS ([M + H]+)
4-(5-Allyloxymethyl-pyridin-3-yl)-2-chloro-pyrimidine (5e). The
title compound was synthesized from 4g and allyl bromide (yield,
46%). LC-MS (ESI positive mode) m/z 262 ([M + H]+).
4-(5-Allyloxymethyl-thiophen-2-yl)-2-chloro-pyrimidine (5f). The
title compound was synthesized from 4i and allyl bromide (yield,
80%). LC-MS (ESI positive mode) m/z 267 ([M + H]+).
4-(5-Allyloxymethyl-thiophen-3-yl)-2-chloro-pyrimidine (5g). The
title compound was synthesized from 4k and allyl bromide (yield,
80%). LC-MS (ESI positive mode) m/z 267 ([M + H]+).
4-(5-Allyloxymethyl-furan-3-yl)-2-chloro-pyrimidine (5h). The
title compound was synthesized from 4m and allyl bromide (yield,
82%). LC-MS (ESI positive mode) m/z 251 ([M + H]+).
4-(4-Allyloxymethyl-furan-2-yl)-2-chloro-pyrimidine (5i). The title
compound was synthesized from 4o and allyl bromide (yield, 74%).
LC-MS (ESI positive mode) m/z 251 ([M + H]+).
1
307.1652, found 307.1653; H NMR (CDCl3) δ 8.33 (d, 1H), 8.17
(dd, 1H), 6.91 (d, 1H), 6.03−5.93 (m, 1H), 5.37−5.23 (m, 2H), 4.56
(s, 2H), 4.26 (t, 2H), 4.12 (dt, 2H), 2.99 (t, 2H), 2.71−2.68 (m, 4H),
1.90−1.86 (m, 4H); 13C NMR (CDCl3) δ 23.8, 54.7, 55.1, 66.3, 68.5,
72.2, 110.7, 117.6, 124.3, 125.0, 128.6, 134.6, 141.7, 161.0.
1-Allyloxymethyl-3-nitro-benzene (9a). The title compound was
synthesized from 3-nitrobenzyl alcohol 7a and allyl bromide (yield,
1
78%). LC-MS (ESI positive mode) m/z 194 ([M + H]+); H NMR
(CDCl3): δ 8.27 (s, 1H), 8.18 (dd, 1H), 7.73 (dd, 1H), 7.57 (t, 1H),
6.01 (m, 1H), 5.38 (m, 1H), 5.29 (m, 1H), 4.65 (s, 2H), 4.13 (dt, 2H).
4-(3-Allyloxymethyl phenyl)-2-chloro pyrimidine (9b). The title
compound was synthesized from 7b and allyl bromide (yield, 55%).
LC-MS (ESI positive mode) m/z 261 ([M + H]+); 1H NMR (CDCl3)
δ 8.62 (d, 1H), 8.06 (br, 1H), 8.00 (dt, 1H), 7.66 (d, 1H), 7.54−7.46
(m, 2H), 6.02 − 5.93 (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. 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 (KBr pellet): 1568, 1535, 1344, 1186,
1079 cm−1.
2-(2-Methoxy−ethoxy)-5-nitro-benzaldehyde (6c). To a mixture
of 2-hydroxy-5-nitrobenzaldehyde 6b (5 g, 29.9 mmol) and 1-bromo-
2-methoxyethane (5.6 mL, 59.8 mmol) in DMF (50 mL, 10 vol)) was
added K2CO3 (6.25 g, 45.0 mmol), and the resulting mixture was
stirred at 100−105 °C for 6 h. The reaction mixture was cooled to
0 °C and quenched with water. The product was extracted with
CH2Cl2 thrice, and the combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure to furnish an oil,
which was purified by column chromatography (EtOAc/Hexane) to
obtain 5.8 g of 6c (yield, 86%). LC-MS (ESI positive mode) m/z 226
([M + H]+).
3-[(Allylmethylamino) methyl] Phenyl Amine (10a). To a solution
of 8a (6.9 g, 33.5 mmol) in CH2Cl2/MeOH (1:1, 100 mL), cooled to
0 °C, SnCl2·2H2O (25.4 g, 134 mmol) was added. The reaction
mixture was gradually raised to rt and stirred for 12 h. After
completion of the reaction, the solvent from the reaction mixture was
removed under reduced pressure, and the residue was taken into
aqueous Na2CO3 and CH2Cl2 (100 mL). The reaction mixture was
filtered through filter paper. The organic layer was separated out from
the filtrate, and the aqueous layer was extracted with CH2Cl2 (3 × 50
mL). The combined organic extracts were dried (Na2SO4) and
concentrated under reduced pressure to afford 7 (5.8 g; yield, 98%) as
a thick syrup, which was used for the next step without further
1
purification. LC-MS (ESI positive mode) m/z 177 ([M + H]+). H
NMR (CDCl3) δ 7.09 (t, 1H), 6.57−6.69 (m, 2H), 6.55 (dd, 1H),
5.85−5.95 (m, 1H), 5.12−5.17 (m, 2H), 3.67 (br, 1H), 3.39 (s, 2H),
3.02 (d, 2H), 2.18 (s, 3H); 13C NMR (CDCl3) δ 146.4, 140.3, 136.0,
129.1, 119.4, 117.4, 115.7, 113.8, 61.7, 60.6, 42.2. Anal. Calcd. for
C11H16N2: C, 74.96; H, 9.15; N, 15.89. Found: 74.34; H, 9.04; N,
15.68. IR (KBr pellet): 3346, 3210, 2782, 1605, 1460, 1294, 921, 785,
696 cm−1.
Following a procedure similar to that of 10a, the following
intermediates were synthesized.
3-[(Allyl-methyl-amino)-methyl]-4-(2-methoxy−ethoxy)-phenyl-
amine (10b). The title compound was synthesized from 8b (yield,
64%). LC-MS (ESI positive mode) m/z 251 ([M + H]+).
3-[(Allyl-methyl-amino)-methyl]-4-(2-pyrrolidin-1-yl-ethoxy)-phe-
nylamine (10c). The title compound was synthesized from 8d (yield,
88%). LC-MS (ESI positive mode) m/z 290 ([M + H]+).
3-Allyloxymethyl-phenylamine (10d). The title compound was
synthesized from 9a (yield, 91%). LC-MS (ESI positive mode) m/z
164 ([M + H]+).
2-(2-Chloro ethoxy)-5-nitro-benzaldehyde (6d) and [2-(2-Chloro-
ethoxy)-5-nitro-phenyl]-methanol (7b). The title compounds were
synthesized according to a literature procedure.22
Allyl Methyl-(3 nitro benzyl) Amine (8a). To a solution of 3-
nitrobenzaldehyde 6a (20 g, 132.4 mmol) in CH2Cl2 (500 mL) was
added N-allyl methyl amine (18.8 mL, 198.6 mmol), and the resulting
mixture was stirred at rt for 12 h, after which Na(OAc)3BH (56.13 g,
265 mmol) was added portion wise and stirred for additional 4 h. The
reaction mixture was then quenched with sat. NH4Cl solution and
extracted with CH2Cl2 (3 × 150 mL). The combined organic extracts
were dried over Na2SO4 and concentrated under reduced pressure to
give the crude material, which was purified by column chromatography
(EtOAc/hexane 1:19) to afford 8a (25.9 g; yield, 95%) as a pale yellow
3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)phenylamine (10e).
The title compound was synthesized from 9c (yield, 89%). LC-MS
(ESI positive mode) m/z 277 ([M + H]+). Theoretical HRMS [M +
1
H]+ 277.1911, found 277.1916; H NMR (MeOD-d4) δ 6.82−6.78
1
syrup. LC-MS (ESI positive mode) m/z 207 ([M + H]+); H NMR
(m, 2H), 6.69 (dd, 1H), 5.93−5.83 (m, 1H), 5.26−5.11 (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.
(CDCl3) δ 8.20 (s, 1H), 8.11 (dd, 1H), 7.68 (dd, 1H), 7.48 (t, 1H),
5.85−5.94 (m, 1H), 5.20 (t, 1H), 3.60 (s, 2H), 3.07 (dd, 1H), 2.22 (s,
3H); 13C NMR (CDCl3) δ 148.4, 141.5, 135.20, 134.9, 129.1, 123.7,
122.1, 118.1, 60.52, 60.50. Anal. Calcd. for C11H14N2O2: C, 64.06; H,
6.84; N, 13.58. Found: C, 62.31; H, 6.69; N, 13.25. IR (KBr pellet):
2787, 1528, 1349, 924, 733 cm−1.
Following a procedure similar to that of 8a, the following
intermediates were synthesized:
Allyl-[2-(2-methoxy−ethoxy)-5-nitro-benzyl]-methyl-amine (8b).
The title compound was synthesized from 6c and N-allyl methyl amine
(yield, 92%). LC-MS (ESI positive mode) m/z 281 ([M + H]+).
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(5-allyloxymethyl-
furan-2-yl)-pyrimidin-2-yl]-amine (11c). To a solution of 5d (80 mg,
0.319 mmol) and 10a (60 mg, 0.340 mmol) in n-butanol (2 mL) was
added 4 M HCl (0.2 mL), and the resulting mixture was heated to
80−100 °C for 12 h. The reaction mixture was brought to rt, and the
solvent was removed under reduced pressure. The residue was
dissolved in minimum amount of MeOH and purified by preparative
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rpHPLC to furnish 11c as a TFA salt (112 mg; yield, 90%). LC-MS
(ESI positive mode) m/z 391 ([M + H]+).
Following a procedure similar to that of 11c, the following
intermediates were synthesized.
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(3-allyloxymethyl-pi-
peridin-1-yl)-pyrimidin-2-yl]-amine (11a). The title compound was
synthesized from 5a and 10a and was obtained as a crude yellow
residue (HPLC purity = 94% at 254 nm), which was used for the next
step of the reaction without further purification. LC-MS (ESI positive
mode) m/z 408 ([M + H]+).
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(1-but-3-enyl-1H-
indol-6-yl)-pyrimidin-2-yl]-amine (11b). The title compound was
synthesized from 5b and 10a (yield, 94%). LC-MS (ESI positive
mode) m/z 424 ([M + H]+).
was synthesized from 5d and 10e (yield, 96%). LC-MS (ESI positive
mode) m/z 491 ([M + H]+).
[4-(4-Allyloxymethyl-furan-2-yl)-pyrimidin-2-yl]-[3-allyloxymeth-
yl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12j). The title com-
pound was synthesized from 5i and 10e (yield, 63%). LC-MS (ESI
positive mode) m/z 491 ([M + H]+).
[4-(5-Allyloxymethyl-pyridin-3-yl)-pyrimidin-2-yl]-[3-allyloxy-
methyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12k). The title
compound was synthesized from 5e and 10e (yield, 68%). LC-MS
(ESI positive mode) m/z 502 ([M + H]+).
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(2-fluoro-
pyridin-4-yl)-pyrimidin-2-yl]-amine (12l). The title compound was
synthesized from 4e and 10e (yield, 81%). LC-MS (ESI positive
1
mode) m/z 450 ([M + H]+); H NMR (CDCl3) δ 8.47−8.35 (m,
3H), 7.81 (d, 1H), 7.62 (dd, 1H), 7.50 (dd, 1H), 7.39 (s, 1H), 6.93 (d,
1H), 5.90−6.00 (dq, 1H), 5.33 (dd, 1H), 5.17 (dd, 1H), 4.58 (s, 2H),
4.45 (s, 2H), 4.10 (d, 2H), 4.04 (s, 2H), 3.65 (br s, 2H), 3.10 (m, 2H),
2.21−2.10 (m, 4H).
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(2-fluoro-pyridin-4-
yl)-pyrimidin-2-yl]-amine (11d). The title compound was synthesized
from 4e and 10a (yield, 86%). LC-MS (ESI positive mode) m/z 350
([M + H]+).
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(2-fluoro-
pyridin-4-yl)-5-methyl-pyrimidin-2-yl]-amine; compound with methane
(12n). The title compound was synthesized from 4p and 10e (yield,
83%). LC-MS (ESI positive mode) m/z 479 ([M + H]+).
[4-(1-Allyl-1H-indol-6-yl)-5-methyl-pyrimidin-2-yl]-[3-allyloxy-
methyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12p). The title
compound was synthesized from 5c and 10e (yield, 86%). LC-MS
(ESI positive mode) m/z 524 ([M + H]+).
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(5-allyloxymethyl-
pyridin-3-yl)-pyrimidin-2-yl]-amine (11f). The title compound was
synthesized from 5e and 10a (yield, 84%). LC-MS (ESI positive
mode) m/z 402 ([M + H]+).
[4-(5-Allyloxymethyl-furan-2-yl)-pyrimidin-2-yl]-(3-allyloxymeth-
yl-phenyl)-amine (11g). The title compound was synthesized from 5d
and 10d (yield, 93%). LC-MS (ESI positive mode) m/z 478 ([M +
H]+).
{3-[(Allyl-methyl-amino)-methyl]-phenyl}-[4-(2-but-3-enyloxy-
pyridin-4-yl)-pyrimidin-2-yl]-amine (11d). To a solution of 3-buten-
1-ol (30 μL, 0.349 mmol), in anhydrous THF (2 mL), was added 60%
sodium hydride in mineral oil (16 mg, 0.400 mmol) at 0 °C. The
solution was stirred at 0 °C was 0.5 h and then slowly allowed to warm
to rt. To this solution of sodium salt of 3-buten-1-ol was added a
solution of 11c (TFA salt, 120 mg, 0.269 mmol) in anhydrous THF
(2 mL). The resulting mixture was heated at 70 °C for 12 h, quenched
with water, and extracted with CH2Cl2. The combined organic layers
were dried over Na2SO4. Evaporation and purification by preparative
HPLC afforded the title compound in 40% yield. LC-MS (ESI positive
mode) m/z 402 ([M + H]+).
Following a procedure similar to that of 11d, the following
intermediates were synthesized.
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(2-but-
3-enyloxy-pyridin-4-yl)-pyrimidin-2-yl]-amine (12m). The title com-
pound was synthesized from 12l and 3-buten-1-ol (yield, 48%). LC-
MS (ESI positive mode) m/z 502([M + H]+).
[4-(1-Allyl-1H-indol-6-yl)-pyrimidin-2-yl]-(3-allyloxymethyl-phe-
nyl)-amine (11h). The title compound was synthesized from 5c and
10d (yield, 79%). LC-MS (ESI positive mode) m/z 397 ([M + H]+).
(3-Allyloxymethyl-phenyl)-[4-(5-allyloxymethyl-pyridin-3-yl)-pyri-
midin-2-yl]-amine (11i). The title compound was synthesized from 5e
and 10d (yield, 92%). LC-MS (ESI positive mode) m/z 389 ([M +
H]+).
[3-[(Allyl-methyl-amino)-methyl]-4-(2-pyrrolidin-1-yl-ethoxy)-
phenyl]-[4-(5-allyloxymethyl-furan-2-yl)-pyrimidin-2-yl]-amine
(12a). The title compound was synthesized from 5d and 10c (yield,
72%). LC-MS (ESI positive mode) m/z 504 ([M + H]+).
[3-[(Allyl-methyl-amino)-methyl]-4-(2-pyrrolidin-1-yl-ethoxy)-
phenyl]-[4-(5-allyloxymethyl-thiophen-2-yl)-pyrimidin-2-yl]-amine
(12b). The title compound was synthesized from 5f and 10c (yield,
62%). LC-MS (ESI positive mode) m/z 520 ([M + H]+).
[3-[(Allyl-methyl-amino)-methyl]-4-(2-methoxy−ethoxy)-phe-
nyl]-[4-(5-allyloxymethyl-thiophen-2-yl)-pyrimidin-2-yl]-amine
(12c). The title compound was synthesized from 5f and 10b (yield,
72%). LC-MS (ESI positive mode) m/z 481 ([M + H]+).
[3-[(Allyl-methyl-amino)-methyl]-4-(2-methoxy−ethoxy)-phe-
nyl]-[4-(5-allyloxymethyl-furan-2-yl)-pyrimidin-2-yl]-amine (12d).
The title compound was synthesized from 5d and 10b (yield, 68%).
LC-MS (ESI positive mode) m/z 465 ([M + H]+).
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(2-but-
3-enyloxy-pyridin-4-yl)-5-methyl-pyrimidin-2-yl]-amine (12o). The
title compound was synthesized from 12n and 3-buten-1-ol (yield,
36%). LC-MS (ESI positive mode) m/z 516 ([M + H]+).
15-(2-Methoxyethoxy)-12-methyl-7-oxa-25-thia-12,19,21,24-tet-
raaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14-
(26),15,17,20,22-nonaene (14i). To 12i (TFA salt, 100 mg, 0.170
mmol) in CH2Cl2 (50 mL) at ambient temperature was added 4 M
HCl until the pH reached 2.0−2.2. The solution was degassed (N2),
and Grubbs second generation catalyst (14 mg, 0.017 mmol) was
added in two portions (7 mg each) at 1 h intervals. The resulting
mixture was stirred at 40−45 °C for 4 h. The reaction mixture was
cooled and concentrated under reduced pressure to furnish an oil,
which was purified by preparative rpHPLC to obtain 63 mg of 14i as a
TFA salt (yield, 66%; >95% trans by 1H NMR). LC-MS (ESI positive
[4-(1-Allyl-1H-indol-6-yl)-pyrimidin-2-yl]-[3-allyloxymethyl-4-(2-
pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12e). The title compound
was synthesized from 5c and 10e (yield, 71%). LC-MS (ESI positive
1
mode) m/z 510 ([M + H]+); H NMR (MeOD-d4) δ 8.20 (s, 1H),
8.15 (d, 1H), 7.81 (d, 1H), 7.64 (d, 1H), 7.43 (d, 1H), 7.33 (m, 1H),
7.02 (d, 1H), 6.43 (d, 1H), 5.85 (m, 2H), 5.15 (d, 1H), 5.06 (m, 2H),
4.95 (d, 1H), 4.79 (m, 2H), 4.55 (s, 2H), 4.30 (m, 2H), 4.00 (m, 2H),
3.68 (m, 2H), 3.60 (m, 2H), 3.18 (m, 2H), 2.10 (m, 2H), 1.91 (m, 2H).
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(5-allyl-
oxymethyl-thiophen-2-yl)-pyrimidin-2-yl]-amine (12f). The title
compound was synthesized from 5f and 10e (yield, 87%). LC-MS
(ESI positive mode) m/z 507 ([M + H]+).
[3-Allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-[4-(5-allyl-
oxymethyl-thiophen-3-yl)-pyrimidin-2-yl]-amine (12g). The title
compound was synthesized from 5g and 10e (yield, 88%). LC-MS
(ESI positive mode) m/z 507 ([M + H]+).
[4-(5-Allyloxymethyl-furan-3-yl)-pyrimidin-2-yl]-[3-allyloxymeth-
yl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12h). The title com-
pound was synthesized from 5h and 10e (yield, 92%). LC-MS (ESI
positive mode) m/z 491 ([M + H]+).
[4-(5-Allyloxymethyl-furan-2-yl)-pyrimidin-2-yl]-[3-allyloxymethyl-4-
(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (12i). The title compound
1
mode) m/z 463 ([M + H]+); H NMR (MeOD-d4): δ 8.56 (d, 1H),
8.38 (d, 1H), 8.29 (br s, 1H), 7.17 (d, 1H), 7.11−7.06 (m, 2H), 7.03−
7.01 (m, 1H), 5.99 (dt, 1H), 5.84 (dt, 1H), 4.66 (s, 2H2), 4.57 (s, 2H),
4.37 (t, 2H), 4.18 (d, 2H), 4.09 (d, 2H), 3.79 (br s, 2H), 3.69 (t, 2H),
3.35−3.34 (m, 2H), 2.21−2.07 (m, 4H).
Following a procedure similar to that of 14i, the following
compounds were synthesized.
14-Methyl-19-oxa-1,5,7,14,26-pentaaza-tetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-2(26),3,5,8(27), 9,11,16-heptaene (13a).
The title compound was synthesized from 11a and isolated as
freebase (33 mg, 15% from 5a in two steps; mixture of cis/trans 1:9 by
1H NMR). LC-MS (ESI positive mode) m/z 380 ([M + H]+). For the
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1
major trans isomer: 1H NMR (CDCl3) δ 8.33 (t, 1H, J = 1.6 Hz), 7.92
(d, 1H, J = 6.1), 7.57 (b s, 1H), 7.16 (t, 1H, J = 7.7 Hz), 6.85 (d, 1H,
J = 7.5 Hz), 6.75 (dd, 1H, J = 7.9, 1.3 Hz), 5.98 (d, 1H, J = 6.2 Hz),
5.92 (dm, 1H, J = 15.6 Hz), 5.61 (ddd, 1H, J = 15.7, 4.5, 3.7 Hz), 4.88
(dm, 1H, J = 13.0 Hz), 4.14 (dd, 1H, J = 15.1, 1.3 Hz), 3.91 (dd, 1H,
J = 14.9, 4.6 Hz), 3.80 (d, 1H, J = 13.3 Hz), 3.58 (d, 1H, J = 12.4 Hz),
3.47 (m, 2H), 3.22 (d, 1H, J = 12.4 Hz), 3.20 (t, 1H, J = 5.4 Hz), 3.05
(dd, 1H, J = 13.1, 7.4 Hz), 2.93 (m, 1H), 2.87 (m, 1H), 2.16 (s, 3H),
1.95 (m, 1H), 1.83−1.75 (m, 2H), 1.65−1.49 (m, 2H); 13C NMR
(CDCl3) δ 161.9, 159.6, 156.5, 140.6, 140.1, 129.9, 129.3, 128.1, 122.9,
119.6, 117.9, 94.2, 71.2, 68.5, 60.43, 60.38, 46.7, 45.4, 42.9, 36.1, 27.9,
25.2.
from 12a (yield, 65%; >95% trans by H NMR). LC-MS (ESI positive
mode) m/z 476 ([M + H]+); H NMR (MeOD-d4) δ 9.02 (d, 1H),
1
8.40 (d, 1H), 7.31−7.25 (m, 2H), 7.22−7.14 (m, 2H), 6.69 (d, 1H, CH),
6.28 (dt, 1H), 6.20−6.11 (m, 1H), 4.66 (s, 2H), 4.49−4.39 (m, 4H),
4.25−4.15 (m, 2H), 3.88−3.76 (m, 6H), 2.89−2.84 (m, 4H), 2.31−2.04
(m, 5H).
19,21-Etheno-3-methyl-11-(2-(pyrrolidin-1-yl)ethoxy)-14-oxa-
5,7,19,25-tetraaza-tetracyclo[18.3.1.1(2,6).1(8,12)]hexacosa-1-
(24),2(25),3,5,8(26),9,11,16,20,22-decaene (14b). The title com-
pound was synthesized from 12b (yield, 44%; >95% trans by 1H
NMR). LC-MS (ESI positive mode) m/z 492 ([M + H]+); H NMR
1
(MeOD-d4) δ 8.80 (d, 1H), 8.32 (d, 1H), 7.83 (d, 1H), 7.31−7.28 (m,
2H), 7.18 (d, 1H), 7.07 (d, 1H), 6.31−6.17 (m, 2H), 4.56−4.47 (m,
4H), 4.36−4.24 (m, 4H), 4.04−3.95 (m, 2H), 3.82−3.77 (m, 5H),
2.71 (s, 3H), 2.21−2.13 (m, 5H).
11-(2-(Pyrrolidin-1-yl)ethoxy)-14,19-dioxa-5,7,23,26-tetraaza-
tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (14c). The title compound was synthe-
20,22-Etheno-14-methyl-5,7,14,20,26-pentaaza-tetracyclo-
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-
decaene (13b). The title compound was synthesized from 11b (yield,
1
69%; mixture of cis/trans 13:87 by H NMR). LC-MS (ESI positive
1
mode) m/z 396 ([M + H]+); H NMR (MeOD-d4) δ 8.27 (m, 1H),
8.10−8.16 (m, 2H), 7.53−7.59 (m, 2H), 7.25−7.40 (m, 3H), 7.03 (m,
2H), 6.42 (m, 1H), 5.50−5.80 (m, 2H), 5.05 (m, 1H), 4.80 (m, 1H),
4.59 (m, 1H), 4.42 (m, 1H), 4.15 (m, 1H), 3.95 (m, 1H).
1
sized from 12c (yield, 48%; >95% trans by H NMR). LC-MS (ESI
positive mode) m/z 453 ([M + H]+); 1H NMR (DMSO-d6) δ 9.78 (s,
1H), 9.54 (br s 1H), 8.73 (d, 1H), 8.48 (d, 1H), 7.90 (d, 1H), 7.35 (d,
1H), 7.15 (s, 1H), 7.10−7.14 (m, 1H), 6.23 (dt, 1H), 6.10 (dt, 1H),
4.90 (d, 2H), 4.11−4.35 (m, 11H), 3.70−3.74 (m, 2H), 2.63
(brs, 3H).
19,21-Etheno-14-oxa-5,7,19,25-tetraaza-tetracyclo[18.3.1.1-
(2,6).1(8,12)]hexacosa-1(24),2(25),3,5,8(26),9,11,16,20,22-decaene
(13c). The title compound was synthesized from 11c (yield, 76%;
1
>95% trans by H NMR). LC-MS (ESI positive mode) m/z 363 ([M
1
+ H]+); H NMR (MeOD-d4) δ 9.04−9.03 (m, 1H), 8.42 (d, 1H)
11-(2-(Pyrrolidin-1-yl)ethoxy)-14,20-dioxa-5,7,22,26-tetraaza-
tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8-
(27),9,11,16,21,23-decaene (14d). The title compound was synthe-
7.33 (d, 1H) 7.23−7.15 (m, 4H), 6.69 (d, 1H), 6.28 (dt, 1H), 6.20−
6.09 (m, 1H), 4.66 (s, 2H), 4.52−4.47 (m, 1H), 4.20 (br d, 2H),
4.11−4.04 (m, 1H), 3.92−3.81 (m, 2H), 2.90−2.84 (m, 3H).
14-Methyl-20-oxa-5,7,14,22,26-pentaaza-tetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene
(13d). The title compound was synthesized from 11e (yield, 52%;
1
sized from 12d (yield, 39%; >95% trans by H NMR). LC-MS (ESI
positive mode) m/z 437 ([M + H]+); 1H NMR (DMSO-d6) δ 9.40 (s,
1H), 8.48 (d, 1H), 8.40 (d, 1H), 7.46 (d, 1H), 7.16 (d, 1H), 7.0 (dd,
1H), 6.90 (d, 1H), 6.67 (d, 1H), 5.93 (dt, 1H,), 5.69 (dt, 1H), 4.52 (s,
2H), 4.04−4.07 (m, 2H), 3.97 (d, 2H), 3.62−3.90 (m, 2H), 3.44 (s,
2H), 3.33 (s, 3H), 3.06 (d, 2H), 2.08 (s, 3H).
1
>95% trans by H NMR). LC-MS (ESI positive mode) m/z 374 ([M
+ H]+); 1H NMR (MeOD-d4) δ 9.08 (m, 1H), 8.64 (d, 1H), 8.31 (m,
1H), 7.84 (m, 1H), 7.53−7.52 (dd, 1H), 7.48−7.43 (m, 2H), 7.29−
7.27 (m, 1H), 7.18 (m,1H), 6.29−6.21 (m, 1H), 5.90−5.82 (m, 1H),
4.39−3.72 (m, 6H), 2.68 (s, 5H).
3-Methyl-11-(2-(pyrrolidin-1-yl)ethoxy)-14,20-dioxa-5,7,22,26-
tetraaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2-
(26),3,5,8(27),9,11,16,21,23-decaene (14e). The title compound was
1
14-Methyl-19-oxa-5,7,14,23,26-pentaaza-tetracyclo[19.3.1.1-
(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene
(13e). The title compound was synthesized from 11f (yield, 44%;
synthesized from 12e (yield, 65%; >95% trans by H NMR). LC-MS
(ESI positive mode) m/z 482 ([M + H]+); H NMR (DMSO-d6) δ
1
9.58 (s, 1H), 8.69 (m, 1H), 8.47 (m, 1H), 8.17 (s, 1H), 7.68 (m, 1H),
7.49−7.53 (m, 2H), 7.20 (m, 2H), 7.01 (m, 1H), 6.52 (m, 1H), 6.17
(m, 1H), 5.66 (m, 1H), 4.97 (m, 2H), 4.49 (m, 2H), 4.26 (m, 2H),
3.97 (m, 2H), 3.59−3.67 (m, 4H), 3.18 (m, 2H), 2.05 (m, 2H), 1.90
(m, 2H).
1
>95% trans by H NMR). LC-MS (ESI positive mode) m/z 374 ([M
1
+ H]+); H NMR (DMSO-d6) δ 10.04 (s, 1H), 9.30 (s, 1H), 8.78 (s,
1H), 8.72 (s, 1H), 8.67 (d, 1H), 8.61 (s, 1H), 7.67 (d, 1H), 7.39 (t,
1H), 7.31 (d, 1H), 7.15 (d, 1H), 6.24−6.18 (m, 1H), 5.96−5.88 (m,
1H), 4.75−4.62 (m, 2H), 4.50 (d, 1H), 4.15 (d, 2H), 4.05−3.93 (m,
2H), 3.84−3.76 (m, 1H), 2.68 (s, 1H).
12-Methyl-15-(2-(pyrrolidin-1-yl)ethoxy)-7,25-dioxa-12,19,21,24-
tetraaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14-
(26),15,17,20,22-nonaene (14f). The title compound was synthesized
from 12f (yield, 55%; >95% trans by 1H NMR). LC-MS (ESI positive
14,19-Dioxa-5,7,23,26-tetraaza-tetracyclo[19.3.1.1(2,6).1(8,12)]-
heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (13f). The
title compound was synthesized from 11g (yield, 73%; >95% trans
mode) m/z 479 ([M + H]+); H NMR (MeOD-d4) δ 8.66 (d, 1H),
1
1
by 1H NMR). LC-MS (ESI positive mode) m/z 350 ([M + H]+); H
8.32 (d, 1H), 7.81 (d, 1H), 7.27 (d, 1H), 7.12 (dd, 1H), 7.07−7.02
(m, 2H), 6.08 (dt, 1H), 5.98 (dt, 1H), 4.61 (s, 2H), 4.38 (t, 2H), 4.18
(d, 4H), 3.81 (br s, 2H), 3.69 (t, 2H), 3.37−3.35 (m, 2H), 2.22−2.08
(m, 6H).
NMR (CDCl3) δ 10.91(s, 1H), 8.67−8.66 (m, 1H), 8.17−8.14 (m,
1H), 7.37−7.32 (m, 1H), 7.28−7.27 (m, 1H), 7.18−7.16 (m, 2H),
6.97 (d, 1H), 6.65 (d, 1H), 6.00 (dt, 1H), 5.89 (dt, 1H), 4.61 (s, 2H),
4.58 (s, 2H), 4.15 (d, 2H), 4.08 (d, 2H).
12-Methyl-15-(2-(pyrrolidin-1-yl)ethoxy)-7-oxa-25-thia-
12,19,21,24-tetraaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1-
(24),2,4,9,14(26),15,17,20,22-nonaene (14g). The title compound
was synthesized from 12g (yield, 70%; >95% trans by 1H NMR). LC-
MS (ESI positive mode) m/z 479 ([M + H]+); 1H NMR (MeOD-d4)
δ 9.03 (d, 1H), 8.86 (d, 1H), 8.81 (d, 1H), 8.26 (s, 1H), 7.81 (d, 1H),
7.59 (dd, 1H), 7.56−7.51 (m, 1H), 6.38 (dt, 1H), 6.31 (dt, 1H), 5.24
(s, 2H), 5.14 (s, 2H), 4.86 (t, 2H), 4.65 (d, 2H), 4.54 (d, 2H), 4.29 (br
s, 2H), 4.18 (t, 2H), 3.84−3.83 (m, 1H), 2.80−2.48 (m, 5H).
15-(2-Methoxyethoxy)-12-methyl-7,25-dioxa-12,19,21,24-tetra-
aza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14-
(26),15,17,20,22-nonaene (14h). The title compound was synthesized
from 12h (yield, 54%; >95% trans by 1H NMR). LC-MS (ESI positive
12-Methyl-7,25-dioxa-12,19,21,24-tetraaza-tetracyclo[18.3.1.1-
(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26),15,17,20,22-nonaene
(13g). The title compound was synthesized from 11h (yield, 51%;
mixture of cis/trans 15:85 by 1H NMR). LC-MS (ESI positive mode)
m/z 369 ([M + H]+); 1H NMR (MeOD-d4) δ 8.68 (m, 1H), 8.37 (m,
1H), 8.40 (m, 1H), 8.21 (m, 1H), 7.71 (m, 2H), 7.48 (m, 2H), 7.30
(m, 2H), 6.62 (m, 1H), 5.75 (m, 2H), 4.46 (m, 2H), 4.25 (m, 2H),
3.75−3.95 (m, 2H), 2.87 (s, 3H), 2.75 (m, 2H).
7,12,25-trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]-
hexacosa-1(24),2,4,9,14(26),15,17,20,22-nonaene (13h). The title
1
compound was synthesized from 11i (yield, 46%; >95% trans by H
1
NMR). LC-MS (ESI positive mode) m/z 361 ([M + H]+); H NMR
(DMSO-d6) δ 9.29 (s, 1H), 8.78 (s, 1H), 8.63 (d, 1H), 8.57 (s, 1H),
8.52 (s, 1H), 7.62 (d, 1H), 7.28 (t, 1H), 7.14 (d, 1H), 6.98 (d, 1H),
5.90−5.81 (m, 2H), 4.62 (s, 2H), 4.49 (s, 2H), 4.09−4.05 (m, 4H).
19,21-Etheno-11-(2-(pyrrolidin-1-yl)ethoxy)-14-oxa-5,7,19,25-tet-
raaza-tetracyclo[18.3.1.1(2,6).1(8,12)]hexacosa-1(24),2(25),3,5,8-
(26),9,11,16,20,22-decaene (14a). The title compound was synthesized
1
mode) m/z 463 ([M + H]+); H NMR (MeOD-d4) δ 8.56 (d, 1H),
8.38 (d, 1H), 8.29 (br s, 1H), 7.17 (d, 1H), 7.11−7.06 (m, 2H), 7.03−
7.01 (m, 1H), 5.99 (dt, 1H), 5.84 (dt, 1H), 4.66 (s, 2H2), 4.57 (s, 2H),
4.37 (t, 2H), 4.18 (d, 2H), 4.09 (d, 2H), 3.79 (brs, 2H), 3.69 (t, 2H),
3.35−3.34 (m, 2H), 2.21−2.07 (m, 4H).
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15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12-dioxa-25-thia-19,21,24-triaza-
tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14-
(26),15,17,20,22-nonaene (14j). The title compound was synthesized
from 12j (yield, 52%; >95% trans by 1H NMR). LC-MS (ESI positive
0.5 mg/mL for CYP1A2, CYP2C9, CYP2C19, and CYP2D6) and
NADPH (1 mM) in the presence of the probe substrates testosterone
for CYP3A4, phenacetin (CYP1A2), diclofenac (CYP2C9), omepra-
zole (CYP2C19), and dextromethorphan (CYP2D6) at 37 °C. The
selective inhibitors ketoconazole, furafylline, sulphaphenazole, ticlopi-
dine, and quinidine were used as positive controls for CYP3A4, 1A2,
CYP2C9, CYP2C19, and CYP2D6, respectively. For all CYP enzymes,
the reactions were terminated by the addition of a mixture of acetonitrile
and DMSO (8:2). The samples were centrifuged, and the supernatants
were analyzed for respective metabolites 6-β-hydroxytestosterone
(CYP3A4), acetaminophen (CYP1A2), 4-hydroxydiclofenac (CYP2C9),
5-hydroxyomeprazole (CYP2C19), and dextrorphan (CYP2D6) by
LC-MS/MS. Formic A decrease in the formation of the metabolites
compared to the vehicle control was used to calculate an IC50 value (test
compound concentration which produces 50% inhibition).
Caco-2 Bidirectional Permeability Assay. Caco-2 cells obtained
from ATCC were used between passage numbers 40−60. Cells were
seeded on to Millipore Multiscreen Caco-2 plates at 1 × 105 cells/cm2.
The permeability study was performed on day 20. The incubation
medium used was Hanks Balanced Salt Solution (HBSS) at pH 7.4,
with 25 mM HEPES and 4.45 mM glucose at 37 °C. Incubations were
carried out for 120 min in an atmosphere of 5% CO2 with a relative
humidity of 95% at 37 °C. The compound solutions were made by
diluting 10 mM 14l in DMSO with HBSS to give a final concentration
of 10 μM (final DMSO concentration 1%). The apical compartment
inserts were placed into companion plates containing fresh HBSS. For
basolateral to apical (B→A) permeability determination, the experi-
ment was initiated by replacing the buffer in the inserts, then placing
them in companion plates containing dosing solutions. After 120 min,
the companion plate was removed, and apical and basolateral samples
were diluted and analyzed by LC-MS/MS. The permeability of 14l was
assessed in duplicate. On each plate, reference compounds of known
permeability characteristics were run as controls. The control compound
and 14l were quantified by LC-MS/MS. The integrity of the monolayers
throughout the experiment was checked by monitoring lucifer yellow
permeation using fluorimetric analysis.
1
mode) m/z 463 ([M + H]+); H NMR (MeOD-d4) δ 8.51 (bs, 1H),
8.34 (bd, 1H), 7.75 (s, 1H), 7.49 (s, 1H), 7.11−7.06 (m, 2H), 7.00 (d,
1H), 6.07 (dt, 1H), 5.92 (dt, 1H), 4.63 (s, 2H), 4.47 (s, 2H), 4.36 (t,
2H), 4.19 (d, 2H), 4.08 (d, 2H), 3.79 (bs, 2H), 3.69 (t, 2H), 3.27−
3.25 (m, 2H), 2.21−2.08 (m, 4H).
15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12-dioxa-4-thia-19,21,24-triaza-
tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,5(25),9,14-
(26),15,17,20,22-nonaene (14k). The title compound was synthesized
from 12k (yield, 34%; >95% trans by 1H NMR). LC-MS (ESI positive
mode) m/z 474 ([M + H]+); 1H NMR (MeOD-d4) δ 9.26 (brs, 1H),
8.90−8.87 (m, 2H), 8.61−8.58 (m, 2H), 7.44 (d, 1H), 7.15−7.12 (m,
1H), 7.06 (d, 1H), 5.96 (dt, 1H), 5.88 (dt, 1H), 4.77 (s, 2H), 4.64 (s,
2H), 4.38 (t, 2H), 4.15 (q, 4H), 3.81 (brs, 2H), 3.70 (t, 2H), 3.27−
3.25 (m, 2H), 2.23−1.99 (m, 5H).
(9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza-
tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26),
15,17,20,22-nonaene (14l). The title compound was synthesized from
1
12n (yield, 46%; mixture of cis/trans 33:67 by H NMR). LC-MS
1
(ESI positive mode) m/z 474 ([M + H]+); H NMR (MeOD-d4) δ
8.91 (d, 1H), 8.57−8.54 (m, 1H), 8.28 (d, 1H), 7.70 (s, 1H), 7.51−
7.46 (m, 1H), 7.38−7.32 (m, 1H), 7.14−7.12 (m, 1H), 7.05 (s, 1H),
5.93−5.85 (m, 1H), 5.68−5.62 (m, 1H), 4.46 (s, 2H), 4.58 (m, 2H),
4.46−4.34 (m, 2H), 4.12 (d, 2H), 3.82 (m, 2H), 3.72 (m, 2H), 3.37
(m, 2H), 2.52 (m, 2H), 2.25 (m, 2H), 2.10 (m, 2H).
15-(2-(Pyrrolidin-1-yl)ethoxy)-4,7,12-trioxa-19,21,24-triaza-
tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,5(25),9,14-
(26),15,17,20,22-nonaene (14m). The title compound was synthe-
1
sized from 12p (yield, 58%; >95% trans by H NMR). LC-MS (ESI
positive mode) m/z 488 ([M + H]+); 1H NMR (DMSO-d6) δ 8.82 (d,
1H), 8.38 (s, 1H), 8.25 (d, 1H); 7.24−7.30 (m, 2H); 7.07−7.11 (m,
2H); 6.99−7.03 (m, 2H), 5.81−5.92 (m, 1H); 5.54−5.64 (m, 1H),
4.55−4.60 (m, 2H); 4.33−4.36 (m, 2H); 4.07 (d, 2H); 3.75−3.85 (m,
2H); 3.65−3.71 (m, 2H); 3.22−3.29 (m, 4H); 2.54−2.60 (m, 2H);
2.35 (s, 3H); 2.18−2.28 (m, 2H); 2.04−2.12 (m, 2H).
In Vitro Plasma Protein Binding. Equilibrium dialysis was
performed in a 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 14l was added to
plasma (from mouse, rat, dog, monkey, 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 14l
was transferred into 2 mL Eppendorf tubes in triplicate for extraction.
Samples were extracted with 1250 μL of MTBE (methyl tertiary-butyl
ether) for 30 min and centrifuged at 4 °C for 10 min at 13,000 rpm in
a microcentrifuge. The supernatants (1000 μL) were transferred into
fresh Eppendorf tubes and dried in a SpeedVac at 43 °C for 35 min.
The dried samples were reconstituted with 100 μL of acetonitrile/
Milli-Q-water (60:40), vortexed, centrifuged for 2 min at 13,000 rpm,
and analyzed by LC-MS/MS.
Pharmacokinetics. Female BALB/c nude mice (aged 8−10 weeks
and weighing 18−22 g), male Wistar rats (aged 8−10 weeks, weighing
230−260 g), male Beagle dogs (∼5−6 months of age, weighing
∼10 kg), and male rhesus monkeys (∼4 years of age and weighing
6−7 kg) were used in the studies. All the animal studies were
performed as per approved internal protocols for animal care and use.
The oral doses for mice, rats, dogs, and monkeys were 50, 50, 5, and
20 mg/kg, respectively. The doses were administered, by gavage, as
suspensions (0.5% methylcellulose and 0.1% Tween 80) to mice, rats,
and monkeys and as gelatin capsules (SNAP-FIT, Capsugel) to dogs.
Following oral dosing, serial blood samples were collected at different
time points (0 to 24 h) in tubes containing K3EDTA as anticoagulant
and centrifuged, and plasma was separated and stored at −70 °C until
analysis. Plasma samples were processed and analyzed by LC/MS/MS.
15-(2-(Pyrrolidin-1-yl)ethoxy)-3,7,12-trioxa-19,21,24-triaza-
tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2(25),4,9,14-
(26),15,17,20,22-nonaene (14n). The title compound was synthesized
from 12q (yield, 49%; >95% trans by 1H NMR). LC-MS (ESI positive
mode) m/z 496 ([M + H]+); 1H NMR (MeOD-d4) δ 8.54 (brs, 1H),
8.24 (s, 1H), 7.72 (s, 1H), 7.55 (d, 1H), 7.26 (d, 1H), 7.15 (d, 1H),
6.98 (dd, 1H), 6.88 (d, 1H), 6.44 (d, 1H), 5.96−6.01 (m, 1H), 5.37−
5.43 (m, 1H), 4.85 (s, 2H), 4.39 (s, 2H), 4.20 (d, 2H), 3.90 (d, 2H),
3.65 (brs, 2H), 3.51 (d, 2H), 3.03−3.15 (m, 2H), 2.19 (s, 3H), 2.07
(brs, 2H), 1.92 (brs, 2H).
Biology. Enzyme assays (JAK1, JAK2, JAK3, TYK2, CDK2, and
FLT3), cell proliferation, and computational algorithms were carried
out as per the previously reported methods.22,34 hERG activity was
determined by MDS Pharma Services (www.mdsps.com). CYP induction
and phenotyping was carried out at Cyprotex (www.cyprotex.com).
Metabolic Stability in Liver Microsomes. Reactions were
carried out in a 96 well microtiter plate. Compound 14l (5 μM)
was incubated with HLM/MLM/DLM (at 0.87 mg/mL) or RLM and
MoLM (at 1 mg/mL) in a reaction mix containing 50 mM potassium
phosphate buffer (pH 7.4) and an NADPH regeneration system. The
plate was incubated in an incubator shaker set at 37 °C. At
predetermined time points, 0 min, 5,15, 30, 45, and 60 min, reactions
(50 μL aliquots) were terminated by the addition of 100 μL of an ice-
cold mixture of acetonitrile and DMSO (80:20, v/v).The terminated
reaction mixtures were centrifuged and the supernatants analyzed by
LC-MS. Stability was assessed by plotting the percent of parent
compound remaining against time on a log−linear scale and estimating
the half-life from the linear portion of the log−linear curve using the
first order equation t1/2 = 0.693/k, where k = slope of the curve (equal
to the first order elimination rate constant).
Human in Vitro CYP450 Inhibition Assay. Compounds
were incubated (at concentrations of 0.001, 0.01, 0.1, 1.0, 5, 10, and
25 μM) with human liver microsomes (0.25 mg/mL for CYP3A4 and
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Article
Pharmacokinetic parameters were estimated by noncompartmental
methods using WinNonlin (ver 5.2, Pharsight, CA).
area under the dosing curve (AUC). The daily mouse scores were
obtained, and the area under the curve between the treatment days and
the final day was computed. Means for each group were determined, and
percentage inhibition from arthritis controls was calculated by comparing
values for treated and normal animals.
High Throughput Solubility Assay. This assay measures the
solubility of a compound in PBS in a high throughput mode. The assay
was done using 96-well semitransparent PP microplates with a V-
shaped bottom (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 maxima. The concentration of the compound in the
supernatant was quantified using a calibration curve.
Thermodynamic Solubility Assay. This assay measures the
solubility of a compound in pH buffered solutions in a low throughput
mode with HPLC quantification. Approximately 5 mg of 14l (citrate)
was weighed into individual 4 mL glass vials. Appropriate volumes of
buffers of pH 1, 3, 5, 7, and 9 were added to corresponding vials
containing the compound to make 5 mg/mL (w/v) stock of 14l. Each
sample was made in triplicate. Prior to the addition of buffers, their
initial pH was recorded. Following addition of the buffers, the vials
were capped tightly with screw caps, transferred to the shaking
platform, and allowed to shake for 24 h at 600 rpm at room
temperature. After 24 h of shaking, each sample was filtered using a
syringe filter (0.4 μm) into clean Eppendorf tubes, and final pH of the
filtrate was measured. The filtrates were appropriately diluted using
acetonitrile/water (1:1), vortexed for 3 min, and centrifuged at 13200
rpm for 3 min. The resultant supernatant was transferred into HPLC
vials and analyzed by HPLC-UV.
Cell Lines. SET2 cells were obtained from DSMZ (Braunschweig,
Germany), while HL60, MDA-MB-231, HEK293, and 32D cells were
obtained from the American Type Culture Collection (ATCC,
Manassas, VA). These cells were cultured as described previously.34
Human CD4+ T cells, isolated from peripheral blood mononuclear
cells with >95% purity, were purchased from AllCells (Emeryville, CA)
and maintained in X-VIVO 20 media (Lonza, Basel, Switzerland).
Western Blot Analyses. Cells were lysed and proteins
immunoprecipitated as previously described.34 Following SDS−
polyacrylamide gel electrophoresis, proteins were transferred to
PVDF membranes. Western blots were performed according to
standard methods. pJAK2 (Y1007/8) (Cat #3776), pSTAT3 (Y705),
antimouse IgG (Cat #7074), and antirabbit IgG, HRP-linked (Cat
#7076) antibodies were purchased from Cell Signaling Technology
(Beverly, MA). pSTAT4 (Y693) (Cat# AF4319) was from R&D
systems (Minneapolis, MN). pSTAT5 (Y694, Cat #611965) was
obtained from BD Biosciences (San Jose, CA), tubulin (Cat #65−829)
from Millipore (Billerica, MA), and actin (Cat #A2066) from Sigma
(St Louis, MO). After the final wash, membranes were incubated for
5 min in ECL from GE-Healthcare (Singapore). The images were
captured digitally using the LAS-3000 Life Science Imager from
Fujifilm (Tokyo, Japan). Densitometric analysis was performed using
MultiGauge software (v3.1) from Fujifilm.
ASSOCIATED CONTENT
* Supporting Information
■
S
Explanation for CDK2 selectivity of 14i versus 14h and explanation
for the CDK2 potency of 13 h. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +65 62195443. E-mail: wanthony11@yahoo.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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, and
Dr. Simon Campbell for helpful discussions.
ABBREVIATIONS USED
■
CIA, collagen induced arthritis; CDKs, cyclin-dependent kinases;
CYP, cytochrome P450; FLT3, fms-like tyrosine kinase 3; JAK2,
janus kinase 2; RA, rheumatoid arthritis; RCM, ring closing
metathesis; STAT, signal transducer and activator of transcription;
TYK2, tyrosine kinase 2
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Collagen-Induced Arthritis (CIA).32,33 The mouse CIA study
was performed at Bolder BioPATH (Boulder, CO) and was approved
by the Bolder BioPATH’s Institutional Animal Care and Use
Committee (IACUC). Six to eight week old male B10/RIII mice
were obtained from Jackson Laboratories (Bar Harbor, ME). The mice
were immunized by injecting 300 μg of bovine type II collagen (Elastin
Products, Owensville, MO) emulsified in Freund’s Complete Adjuvant
(Sigma) at the base of the tail on day 0 and again on day 15. The mice
were randomized into treatment groups, and oral treatment with 14l
was initiated after the onset of arthritis (usually between days 18−22)
identified by establishment of swelling in at least one paw. Treatment
was continued twice daily (b.i.d) for 10 days. Arthritis severity was
evaluated on a scale of 0−5 based on the number of joints affected,
swelling of the joints, and the severity of erythema. The sum of scores
for all four paws from each mouse was used as the total clinical score. The
data for paw scores (mean for animal) was analyzed by determining the
2639
dx.doi.org/10.1021/jm201454n | J. Med. Chem. 2012, 55, 2623−2640

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