Novel 7H-pyrrolo[2,3-d]pyrimidine derivatives as potent and orally active STAT6 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.021

Signal transducers and activators of transcription 6 (STAT6) is an important transcription factor in interleukin (IL)-4 signaling pathway and a key regulator of the type 2 helper T (Th2) cell immune response. Therefore, STAT6 may be an excellent therapeut

Full text of DOI:10.1016/j.bmc.2009.08.021

Bioorganic & Medicinal Chemistry 17 (2009) 6926–6936
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 7H-pyrrolo[2,3-d]pyrimidine derivatives as potent and orally active
STAT6 inhibitors
a
a
a
a
a
Shinya Nagashima a, , Takeshi Hondo , Hiroshi Nagata , Takashi Ogiyama , Jun Maeda , Hiroaki Hoshii ,
Toru Kontani a, Sadao Kuromitsu a, Keiko Ohga a, Masaya Orita a, Kazuki Ohno a, Ayako Moritomo a,
Koichi Shiozuka b, Masako Furutani a, Makoto Takeuchi a, Mitsuaki Ohta a, Shin-ichi Tsukamoto a
*
a Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Astellas Research Technologies Co. Ltd, 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Signal transducers and activators of transcription 6 (STAT6) is an important transcription factor in inter-
leukin (IL)-4 signaling pathway and a key regulator of the type 2 helper T (Th2) cell immune response.
Therefore, STAT6 may be an excellent therapeutic target for allergic conditions, including asthma and ato-
pic diseases. Previously, we reported 4-aminopyrimidine-5-carboxamide derivatives as STAT6 inhibitors.
To search for novel STAT6 inhibitors, we synthesized fused bicyclic pyrimidine derivatives and identified
a 7H-pyrrolo[2,3-d]pyrimidine derivative as a STAT6 inhibitor. Optimization of the pyrrolopyrimidine
derivatives led to identification of 2-[4-(4-{[7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
amino}phenyl)piperazin-1-yl]acetamide (24, AS1810722) which showed potent STAT6 inhibition and a
good CYP3A4 inhibition profile. Compound 24 also inhibited in vitro Th2 differentiation without affecting
type 1 helper T (Th1) cell differentiation and eosinophil infiltration in an antigen-induced mouse asth-
matic model after oral administration.
Received 16 July 2009
Accepted 12 August 2009
Available online 15 August 2009
Keywords:
Signal transducers and activators of
transcription 6
Allergic disease
Interleukin-4
Th2 differentiation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
that STAT6 might be an excellent therapeutic target for allergic dis-
eases, such as asthma and atopic diseases.
Peripheral T helper (Th) cells are classified into two subsets,
type 1 helper T (Th1) and type 2 helper T (Th2), based on cytokine
production and functional ability.1 Th1 cells produce Th1 cytokines
Previously, we reported several 4-benzylaminopyrimidine-5-
carboxamide derivatives as STAT6 inhibitors (Fig. 1).6 Among these
compounds, 1b (YM-341619, AS161672) inhibited Th2 differentia-
tion and suppressed antigen-induced eosinophil accumulation in
the lungs of mice and rats after oral administration.7 In the further
studies of STAT6 inhibitors, we explored new lead compounds
for identification of a novel scaffold (based on 1a), since this is
such as interferon-c (IFN-c), interleukin (IL)-2, and tumor necrosis
factor-b for enhancement of cellular immunity for elimination of
intracellular pathogens. In contrast, Th2 cells produce Th2 cyto-
kines such as IL-4, IL-5, IL-10 and IL-13 for modulation of humoral
immunity for protection against parasites and allergens. The im-
mune system maintains a proper balance between Th1 and Th2
responses, and Th1/Th2 polarization is thought to play an impor-
tant role in the pathogenesis of allergic and autoimmune diseases.2
Differentiation of naive Th cells into Th2 cells is induced by
antigen stimulation of T cell receptors in the presence of IL-4. Sig-
R
R
R
nal transducers and activators of transcription
6 (STAT6) is
O
NH
O
strongly associated with the IL-4 signaling pathway and plays an
important role in Th2 differentiation.3 In STAT6-deficient mice,
Th cells are unable to differentiate into Th2 cells, and B cells cannot
switch immunoglobulin (Ig) production to IgG1 and IgE.4 In addi-
tion, antigen-induced airway hyperresponsiveness and eosinophil
infiltration are significantly decreased.5 These findings indicate
N
N
NH2
N
H
N
1a R=H
1b (YM-341619, AS1617612) R=F
* Corresponding author. Tel.: +81 (0)29 829 6176; fax: +81 (0)29 852 5378.
E-mail address: shinya.nagashima@jp.astellas.com (S. Nagashima).
Figure 1. Structures of 4-benzylamino-2-[(4-morpholin-4-ylphenyl)amino]pyrim-
idine-5-carboxamide derivatives.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.021

S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
6927
important in drug discovery. In these studies, we focused on the
carboxamide group and the amino group of the benzylamine moi-
ety, which could form a pseudo-six-membered ring through an
intramolecular hydrogen bond between the oxygen of the carbox-
amide and the hydrogen of the amine. We hypothesized that the
bicyclic system formed by pyrimidine and the pseudo ring might
be an important factor for activity. Therefore, we designed fused
bicyclic pyrimidine derivatives that may mimic the 4-aminopyrim-
idine-5-carboxamide system (Fig. 2).
In the discovery and development of new drugs, examination of
the inhibitory activity against cytochrome P450 (CYP) is also an
important issue, since CYP inactivation may produce unfavorable
drug–drug interactions (DDIs) and toxicity. In particular, time-
dependent inhibition (TDI) of CYP has recently become a concern
in clinical practice with regard to the duration of the CYP inhibitory
effect.8 CYP3A4 is a major CYP isoform and is thought to be in-
volved in the metabolism of approximately 50% of drugs used in
humans. Since our previous STAT6 inhibitor 1b showed TDI of
CYP3A4, we assessed potential DDIs and evaluated CYP3A4 TDI
in the novel series of STAT6 inhibitors. In this paper, we report
the synthesis of novel fused bicyclic pyrimidine derivatives,
together with their structure–activity relationships (SARs) and
CYP3A4 profiles. We also describe the in vitro and in vivo pharma-
cological profiles of one derivative.
of the Z and E isomers (Z/E > 20/1).13 Cyclization of compound 12
with trifluoroacetic acid gave 2-chloro-7H-pyrrolo[2,3-d]pyrimi-
dine 13 in good yield. Alkylation of the 7-position of compound
13 with 3-fluorobenzyl bromide and 3,5-difluorobenzyl bromide
readily proceeded in the presence of potassium carbonate. A palla-
dium catalyzed cross-coupling reaction of compounds 14a–b with
4-morpholinoaniline and 4-(1-tert-butoxycarbonylpiperazin-4-
yl)aniline provided compounds 9e and 15, respectively. Deprotec-
tion of the tert-butyldimethylsilyl (TBDMS) group of 18 and the Boc
group of compound 20 gave the 2-hydroxyethyl derivative 19 and
the 2-aminoethyl derivative 21, respectively. Hydrolysis of 22 gave
the acetic acid derivative 23, followed by condensation with aque-
ous ammonia and methylamine in the presence of 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCꢀHCl) and
1-hydroxybenzotriazole (HOBt) to afford the amide derivatives
24 and 25, respectively.
3. Results and discussion
The ability of the compounds to inhibit STAT6 activation was
measured in an IL-4-stimulated STAT6-dependent promoter repor-
ter assay.6 In this assay, the 7H-pyrrolo[2,3-d]pyrimidine deriva-
tive 9a was identified as a new lead compound with moderate
activity (Table 1). Reduction of the double bond in the pyrrole
moiety (10) led to a decrease in activity. To understand the rela-
tionship between the molecular features and the activity, a molec-
ular modeling study was performed. Superimposition of 9a and 10
with the lowest-energy conformation of 1a showed good overlap,
except for the amino group of the carbamoyl moiety (Fig. 3). In
contrast, the atomic charge distribution of the 4-aminopyrimi-
dine-5-carboxamide moiety of 1a and the pyrrolopyrimidine ring
of 9a differed from that of the dihydropyrrolopyrimidine ring of
10 (Fig. 4). For 1a and 9a, the negative charge (red) was distributed
on the right side of the molecule, whereas 10 had a positive charge
(blue) in the corresponding region. These results suggest that the
7H-pyrrolo[2,3-d]pyrimidine may mimic the 4-aminopyrimidine-
5-carboxamide moiety electrostatically. The negative potential
surface may interact with a positive region. Therefore, we hypoth-
esized that the pyrrole moiety of 9a and the 4-amino-5-carbamoyl
moiety of 1a might interact with a protonated site on the target
protein.
The inhibitory activities of 7H-pyrrolo[2,3-d]pyrimidine deriv-
atives with modification of the benzyl and morpholine moieties
are shown in Table 2. In our previous work, we found that intro-
ducing a fluorine atom at the 2- or 3-position of the benzene
ring in the benzyl moiety enhanced the activity, whereas the
4-fluoro derivative had diminished activity.6 Similar SARs were
observed in the 7H-pyrrolo[2,3-d]pyrimidine derivatives: the
2- and 3-fluoro derivatives (9b, 9d) were about two- and
threefold more potent than 9a, respectively, and the 4-fluoro
derivative 9c was 10-fold less potent than 9a. Interestingly, the
3,5-difluoro derivative 9e was 10-fold more potent than 9a.
2. Chemistry
The syntheses of 7H-pyrrolo[2,3-d]pyrimidine derivatives and a
6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine derivative are shown in
Scheme 1. A substitution reaction of methyl 1,6-dihydro-2-methyl-
thio-6-oxo-5-pyrimidinecarboxylate (2)9 with 4-morpholinoani-
line gave the 2-phenylamino derivative 3.10 Chlorination of 3
with phosphorus trichloride in the presence of diethylaniline affor-
ded the 4-chloro derivative 4. Displacement of the chloro group of
4 with the appropriate benzylamine furnished 4-benzylamino
derivatives 5a–d. Hydrolysis of the compounds 5a–d afforded car-
boxylic acids 6a–d, followed by condensation with N,O-dim-
ethylhydroxylamine to give N-methoxy-N-methylcarboxamide
derivatives 7a–d, respectively. Treatment of 7a–d with lithium
aluminum hydride afforded the corresponding aldehyde deriva-
tives 8a–d in 53–82% yields. A Wittig reaction of 8a–d with
(methoxymethyl)triphenylphosphonium chloride and subsequent
treatment with concentrated HCl in methanol gave the desired
7H-pyrrolo[2,3-d]pyrimidine derivatives 9a–d. Conversion of 9a
into 6,7-dihydoropyrrolopyrimidine derivative 10 was achieved
by hydrogenation in the presence of palladium on carbon.11
Scheme 2 shows an alternative synthetic route for the 7H-pyr-
rolo[2,3-d]pyrimidine derivatives. Stille coupling of 4-amino-5-
bromo-2-chloropyrimidine (11)12 with (Z)-1-ethoxy-2-(tributhyl-
stannyl)ethene using a catalytic amount of dichlorobis(triphenyl-
phosphine) palladium in the presence of n-tetrabutylammonium
chloride afforded alkenylpyrimidine 12 in 57% yield as a mixture
H
N
N
O
O
O
N
N
N
N
NH2
N
N
N
H
N
H
1a
Figure 2. Design of novel fused pyrimidine derivatives.

6928
S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
O
O
N
O
Cl
N
O
CO2Me
N
CO2Me
CO2Me
N
HN
a
b
N
HN
N
N
N
H
MeS
H
3
2
4
1
R1
R
c
d
O
N
NH
N
O
NH
N
CO2Me
CO2H
N
N
N
N
N
H
H
5a—d
6a—d
No.
5a
5b
R1
H
No.
5c
5d
R1
No.
6a
6b
R1
H
No.
6c
6d
R1
4-F
4-F
2-F
3,5-F
2
2-F
3,5-F
2
1
1
R
R
e
f
O
O
NH
O
NH
N
OMe
CHO
N
N
N
N
N
Me
N
N
H
N
H
7a—d
8a—d
No.
R1
H
No.
7c
7d
R1
No.
8a
8b
R1
No.
8c
8d
R1
4-F
7a
7b
4-F
H
2-F
3,5-F
2
2-F
3,5-F
2
1
R
O
N
g
O
N
N
h
N
N
N
N
N
N
H
N
H
9a—d
10
No.
9a
9b
R1
H
No.
9c
9d
R1
4-F
2-F
3,5-F
2
i
Scheme 1. Reagents and conditions: (a) 4-(morpholin-4-yl)aniline/EtOH, reflux; (b) POCl3, diethylaniline/MeCN, reflux; (c) benzylamine derivatives, Pr2NEt/dimethylacet-
amide, rt; (d) 1 M NaOH/MeOH–THF, reflux; (e) N,O-dimethylhydroxylamine hydrochloride, Et3N, EDCꢀHCl, HOBt/DMF, rt; (f) LiAlH4/THF, ꢁ78 to 0 °C; (g) (i)
(methoxymethyl)triphenylphosphonium chloride, nBuLi/THF, (ii) concd HCl/MeOH, reflux; (h) H2, 10% Pd–C/AcOEt–AcOH, rt.
Replacement of the morpholine group of 9e with a piperazine
group led to improved activity (16), and methylation of the ter-
minal nitrogen of the piperazine gave further improvement in
the activity (17). Replacing the methyl substituents with 2-
hydroxyethyl (19), acetamide (24) and N-methylacetamide (25)
retained the activity, whereas the 2-aminoethyl derivative (21)
and acetic acid derivative (23) had decreased activity. These re-
sults indicate that neutral substituents at the terminal nitrogen
of the piperazine moiety might give favorable activity over basic
and acidic substituents.
To evaluate the inhibitory effects of the 7H-pyrrolo[2,3-d]-
pyrimidine derivatives against CYP3A4, the metabolic activity of
human liver microsomes (HLMs) for midazolam, a substrate for
CYP3A4, was measured at 0 and 30 min after preincubation with
the compounds. The residual activities of the HLMs in the presence
of the pyrrolopyrimidine derivatives are shown in Table 2. The
metabolic activity at 0 min after preincubation with compound
9a was 88% compared to the activity without 9a, but the activity
at 30 min after preincubation was only 34% based on the activity
at 0 min. These results show that 9a exhibits TDI of CYP3A4. The

S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
6929
R1
NH2
NH2
HN
N
Br
CH=CHOEt
a
N
N
b
c
N
N
N
Cl
N
12
Cl
N
13
N
Cl
Cl
11
14a―b
No.
14a
R1
3-F
14b 3,5-F2
F
F
R1
F
F
R2
N
d
f or
N
e
HN
N
N
R
N
N
N
N
N
N
N
H
N
N
N
H
N
H
17―25
16
9e, 15
No.
17
R2
No.
R1
R
Me
N
O
9e
3-F
18
TBDMS-O-(CH2)2-
HO-(CH2)2-
h
i
19
20
21
22
23
24
25
N
Boc
N
15 3,5-F2
BocHN-(CH2)2-
H2N-(CH2)2-
EtO2CCH2-
j
HO2CCH2-
k
H2NOCCH2-
MeHNOCCH2-
Scheme 2. Reagents and conditions: (Z)-1-ethoxy-2-(tributylstannyl)ethane, PdCl2(PPh3)2, nBu4NCl/DMF, 140 °C; (b) trifluoroacetic acid/Cl(CH2)2Cl, 80 °C; (c) 3-fluorobenzyl
bromide or 3,5-difluorobenzyl bromide, K2CO3/DMSO, 60 °C; (d) 4-(morphplin-4-yl)aniline and 4-(1-tert-butoxycarbonylpiperazin-4-yl)aniline, Pd(OAc)2, rac-BINAP, Cs2CO3/
dioxane, 80 °C; (e) 4 M HCl–dioxane/MeOH–THF, 50 °C; (f) CH3I or BrCH2CO2Et, K2CO3/DMF, rt; (g) TBDMSO(CH2)2Br or BocHN(CH2)2Br, NaI, Na2CO3/DMF, 100 °C; (h) nBu4NF/
THF, rt; (i) 4 M HCl–AcOEt/MeOH, rt; (j) 1 M NaOH/MeOH–THF, 50 °C; (k) NH4OH (28%) or aqueous methylamine (25%), EDCꢀHCl, HOBt/DMF.
morpholine derivatives 9b–e also produced time-dependent
decreases of CYP3A4 activity. In the piperazine series, compound
16 did not show TDI of CYP3A4, but did produce moderate inhibi-
tion of CYP3A4 at 0 min after preincubation. Introduction of a
methyl group at the terminal nitrogen of the piperazine moiety
in 16 led to decreased CYP3A4 inhibition at 0 min (compound
17). The N-(2-hydroxy)ethyl and N-methylacetamide derivatives
(19, 25) showed similar CYP3A4 profiles to 17. The 2-aminoethyl
derivative (21) showed no TDI, but the residual activity at 0 min
after preincubation was only 65%. These findings suggest that pri-
mary and secondary amino groups (16, 21) are important in
CYP3A4 inhibition. Further improvement of the CYP profile was
found for the acetic acid and acetamide derivatives (23, 24), which
showed no CYP inhibition and weak TDI. These data suggest that
TDI of CYP by the 7H-pyrrolo[2,3-d]pyrimidine derivatives might
be related to the lipophilicity of the compounds, since the calcu-
lated log D values of the piperazine derivatives at pH 7.4
(c Log D7.4) are <4, whereas those for the morpholine derivatives
are >4 (Table 2). TDI is often caused by generation of reactive
metabolites8 and we speculate that the less lipophilic piperazine
derivatives might be metabolically more stable than the morpho-
line derivatives.
The results in Table 2 suggest that 24 is the most promising
compound from the perspectives of STAT6 inhibition and the
CYP3A4 profile. Therefore, the effect of 24 on differentiation of T
cells to Th subsets was examined using a previously reported
method.15 The extent of Th1 and Th2 differentiation was deter-
mined based on production of IFN-
shown in Figure 5, 24 inhibited production of IL-4 with an IC50 of
2.4 nM, but showed no effect on production of IFN- . These
c and IL-4, respectively. As
c
observations indicate that 24 inhibits Th2 differentiation without
influencing Th1 differentiation. To evaluate its potency as an
anti-asthmatic drug, 24 was tested in an antigen-induced mouse
asthmatic model, and was found to suppress eosinophil infiltration
in the lung in a dose-dependent manner after oral administration
(0.03–0.3 mg/kg, po, Fig. 6). Collectively, our findings indicate that
compound 24 (AS1810722) is a potent and orally active STAT6
inhibitor, and we suggest that this compound may be useful for
treatment of allergic diseases such as asthma and atopic diseases.
4. Conclusion
In an attempt to find novel STAT6 inhibitors, we synthesized
fused bicyclic pyrimidine derivatives and evaluated their activities.

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S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
Table 1
description: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; and br, broad peak). Mass spectra were recorded with a Hit-
achi M-80 or a JEOL JMS-DX300 spectrometer. Organic solutions
were dried over anhydrous MgSO4 during work-up. Column chro-
matography was carried out on silica gel (Kieselgel 60). Unless
otherwise noted, all commercial reagents and solvents were used
without further purification.
STAT6 inhibitory activity of 4-benzylaminopyrimidine-5-carboxamide derivative 1a
and fused bicyclic pyrimidine derivatives 9a and 10
O
N
scaffold
N
H
5.1.1. Methyl 2-[(4-morpholin-4-ylphenyl)amino]-6-oxo-1,6-
dihydropyrimidine-5-carboxylate (3)
a
Compound
Scaffold
IC50 (nM)
4-Morpholinoaniline (32.7 g, 187 mmol) was added to a mix-
ture of 29 (35.0 g, 174.8 mmol) and EtOH (350 mL) and the mixture
was refluxed for 10 h. The mixture was cooled to room tempera-
ture and resulting precipitate was collected by filtration and
washed with EtOH (100 mL), MeOH (100 mL) and hexane
(100 mL). A mixture of the crude solid and DMF (250 mL) was stir-
red for 1 h at 130 °C. The reaction mixture was cooled to room tem-
perature, and MeOH (250 mL) was added. The resulting solid was
collected by filtration and washed with MeOH (100 mL) to give 3
(48.6 g, 84%) as a pale green solid. 1H NMR (DMSO-d6) d 3.06–
3.09 (4H, m), 3.69 (3H, s), 3.72–3.75 (4H, m), 6.93 (2H, d,
J = 9.2 Hz), 7.37 (2H, d, J = 9.2 Hz), 8.41 (1H, s), 9.14 (1H, br),
10.95 (1H, br); FAB MS m/e [M+H]+ 331.
*
NH
O
1a
6.5
NH2
N
N
*
*
N
9a
10
400
N
N
*
*
*
N
8300
N
N
a
IC50 values are averages from two or three experiments.
5.1.2. Methyl 4-chloro-2-[(4-morpholin-4-ylphenyl)amino]-
pyrimidine-5-carboxylate (4)
Among these compounds, the 7H-pyrrolo[2,3-d]pyrimidine deriva-
tive 9a showed moderate activity. The atomic charge distribution
of 9a suggested that the 7H-pyrrolo[2,3-d]pyrimidine moiety could
mimic a 4-aminopyrimidine-5-carboxamide moiety. Optimization
of thebenzylgroupand morpholinemoietyof 9a led toidentification
of a piperazinylacetamide derivative (24, AS1810722) that showed
potent STAT6 inhibitory activity and a good CYP3A4 profile. Com-
pound 24 (AS1810722) inhibited in vitro Th2 differentiation with
an IC50 of 2.4 nM without affecting Th1 cell differentiation, and also
suppressed eosinophil infiltration in an antigen-induced mouse
asthmatic model after oral administration. These results suggest
that 24 is a novel potent and orally active STAT6 inhibitor that could
be useful for treatment of allergic diseases such as asthma and atopic
diseases.
Diethylaniline (5.8 mL, 36.3 mmol) was added to a mixture of 3
(10.0 g, 30.3 mmol) and phosphorus trichloride (5.6 mL, 60.5
mmol) and the mixture was refluxed for 3.5 h at 90 °C. The reaction
mixture was cooled to room temperature and concentrated in va-
cuo. AcOEt (200 mL) was added to the residue and resulting solid
was collected by filtration. The solid was dissolved in AcOEt–THF
and the organic layer was washed successively with H2O and sat-
urated aqueous NaCl. The organic layer was dried and concentrated
in vacuo. The resulting solid was triturated with MeOH and col-
lected by filtration to give 4 (5.18 g, 49%) as a pale green solid,
which was used in the next reaction without further purification.
1H NMR (DMSO-d6) d 3.14–3.16 (4H, m), 3.86–3.89 (4H, m), 3.91
(3H, s), 6.93 (2H, d, J = 8.8 Hz), 7.41 (1H, br), 7.47 (2H, d,
J = 8.8 Hz), 8.89 (1H, s); FAB MS m/e [M+H]+ 349, 351.
5. Experimental
5.1. Chemistry
5.1.3. Methyl 4-(benzylamino)-2-[(4-morpholin-4-ylphenyl)-
amino]pyrimidine-5-carboxylate (5a)
Benzylamine (344 mg, 3.21 mmol) was added to a solution of 4
(800 mg, 2.29 mmol) and diisopropylethylamine (0.56 mL,
3.21 mmol) in dimethylacetamide (8 mL) and the mixture was stir-
red for 3 h at room temperature. The reaction mixture was then di-
luted with H2O and the resulting solid was collected by filtration
and washed successively with MeOH and Et2O to give 5a
(920 mg, 96%) as a colorless solid, which was used in the next reac-
tion without further purification. 1H NMR (DMSO-d6) d 3.01–3.08
(4H, m), 3.71–3.74 (4H, m), 3.78 (3H, s), 4.70 (2H, d, J = 5.9 Hz),
6.81 (2H, d, J = 8.8 Hz), 7.23–7.27 (1H, m), 7.33–7.35 (4H, m),
7.47 (2H, d, J = 8.8 Hz), 8.55 (1H, s), 8.65 (1H, br), 9.60 (1H, s);
FAB MS m/e [M+H]+ 420.
1H NMR spectra were measured with a JEOL EX400 (400 MHz)
or GX500 (500 MHz) spectrometer; chemical shifts are expressed
in d units using tetramethylsilane as the standard (NMR peak
5.1.4. Methyl 4-[(2-fluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carboxylate (5b)
Compound 5b was prepared from compound 4 and 2-fluorob-
enzylamine in 93% yield as a colorless solid, using a similar ap-
proach to that described for 5a, and used in the next reaction
without further purification. 1H NMR (DMSO-d6) d 3.00–3.03 (4H,
m), 3.72–3.74 (4H, m), 3.79 (3H, s), 4.75 (2H, d, J = 5.8 Hz), 6.78
(2H, d, J = 8.8 Hz), 7.14–7.16 (1H, m), 7.25–7.32 (3H, m), 7.42
(2H, d, J = 8.8 Hz), 8.55 (1H, s), 8.65 (1H, br), 9.58 (1H, s); FAB MS
m/e [M+H]+ 438.
Figure 3. Superposition of 1a (gray), 9a (orange) and 10 (green).

S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
6931
Figure 4. Atomic charge distributions of the 4-aminopyrimidine-5-carboxamide moiety, pyrrolopyrimidine ring, and dihydropyrrolopyrimidine ring of 1a, 9a, and 10,
respectively. Compounds are colored blue for nitrogen, red for oxygen and green for carbon. Connolly surfaces around the core scaffolds are colored red for negative charge
and blue for positive charge. See Section 5.
5.1.5. Methyl 4-[(4-fluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carboxylate (5c)
NMR (DMSO-d6) d 3.01–3.04 (4H, m), 3.72–3.77 (4H, m), 4.67
(2H, d, J = 5.6 Hz), 6.82 (2H, d, J = 8.8 Hz), 7.13–7.18 (2H, m),
7.35–7.39 (2H, m), 7.48 (2H, d, J = 8.8 Hz), 8.51 (1H, s), 8.77 (1H,
br), 9.47 (1H, s), 12.55 (1H, br); FAB MS m/e [M+H]+ 424.
Compound 5c was prepared from compound 4 and 4-fluoroben-
zylamine in 96% yield as a pale brown solid, using a similar ap-
proach to that described for 5a, and used in the next reaction
without further purification. 1H NMR (DMSO-d6) d 3.02–3.04 (4H,
m), 3.72–3.74 (4H, m), 3.78 (3H, s), 4.67 (2H, d, J = 5.6 Hz), 6.82
(2H, d, J = 8.8 Hz), 7.13–7.17 (2H, m), 7.35–7.37 (2H, m), 7.47
(2H, d, J = 8.8 Hz), 8.54 (1H, s), 8.65 (1H, br), 9.57 (1H, s); FAB MS
m/e [M+H]+ 438.
5.1.10. 4-[(3,5-Difluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carboxylic acid (6d)
Compound 6d was prepared from compound 5d in 95% yield as
a pale yellow solid, using a similar approach to that described for
6a, and used in the next reaction without further purification. 1H
NMR (DMSO-d6) d 3.01–3.03 (4H, m), 3.72–3.74 (4H, m), 4.68
(2H, d, J = 5.2 Hz), 6.79 (2H, d, J = 8.8 Hz), 7.02–7.10 (3H, m), 7.39
(2H, d, J = 8.8 Hz), 8.52 (1H, s), 8.85 (1H, br), 9.50 (1H, br), 12.62
(1H, br); FAB MS m/e [MꢁH]ꢁ 440.
5.1.6. Methyl 4-[(3,5-difluorobenzyl)amino]-2-[(4-morpholin-
4-ylphenyl)amino]pyrimidine-5-carboxylate (5d)
Compound 5d was prepared from compound 4 and 3,5-difluo-
robenzylamine in 92% yield as a pale yellow solid, using a similar
approach to that described for 5a, and used in the next reaction
without further purification. 1H NMR (DMSO-d6) d 3.01–3.03 (4H,
m), 3.72–3.74 (4H, m), 3.80 (3H, s), 4.68 (2H, d, J = 6.0 Hz), 6.79
(2H, d, J = 8.3 Hz), 7.02–7.10 (3H, m), 7.39 (2H, br), 8.55 (1H, s),
8.73 (1H, s), 9.58 (1H, br); FAB MS m/e [M+H]+ 455.
5.1.11. 4-(Benzylamino)-N-methoxy-N-methyl-2-[(4-
morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide (7a)
EDCꢀHCl (1.02 g, 5.30 mmol) and HOBt (718 mg, 5.30 mmol)
were added to a mixture of 6a (1.43 g, 3.53 mmol) in DMF
(20 mL). After stirring for 30 min at room temperature, N,O-dim-
ethylhydroxylamine hydrochloride (516 mg, 5.30 mmol) and tri-
ethylamine (1.72 mL, 12.4 mmol) were added and the mixture
was stirred for 24 h at room temperature. The reaction mixture
was then diluted with H2O and extracted with AcOEt–THF. The
organic layer was washed successively with H2O and saturated
aqueous NaCl. The organic layer was dried and concentrated in va-
cuo. The resulting pale yellow solid was recrystallized from THF–
MeOH to give 7a (1.15 g, 73%) as a pale pink solid.1H NMR
(DMSO-d6) d 3.00–3.03 (4H, m), 3.23 (3H, s), 3.61 (3H, s), 3.71–
3.74 (4H, m), 4.64 (2H, d, J = 5.9 Hz), 6.81 (2H, d, J = 8.8 Hz),
7.22–7.27 (1H, m), 7.33–7.40 (4H, m), 7.49 (2H, d, J = 8.8 Hz),
8.38 (1H, s), 8.45 (1H, br), 9.28 (1H, br); FAB MS m/e [M+H]+ 449.
5.1.7. 4-(Benzylamino)-2-[(4-morpholin-4-ylphenyl)amino]-
pyrimidine-5-carboxylic acid (6a)
1 M NaOH (12.9 mL) was added to a solution of 5a (2.00 g,
4.61 mmol) in MeOH (20 mL) and THF (20 mL) and the mixture
was stirred for 10 h at 60 °C. 1 M HCl (14 mL) was added and the
resulting solid was collected by filtration and washed with H2O to
give 6a (1.83 g, 98%) as a colorless solid, which was used in the next
reaction without further purification. 1H NMR (DMSO-d6) d 3.01–
3.03 (4H, m), 3.71–3.74 (4H, m), 4.70 (2H, d, J = 5.9 Hz), 6.81 (2H, d,
J = 8.8 Hz), 7.25–7.27 (1H, m), 7.33–7.35 (4H, m), 7.49 (2H, d,
J = 8.8 Hz), 8.51 (1H, s), 8.75 (1H, br), 9.48 (1H, s), 12.56 (1H, br);
FAB MS m/e [M+H]+ 406.
5.1.12. 4-[(2-Fluorobenzyl)amino]-N-methoxy-N-methyl-2-[(4-
morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide (7b)
Compound 7d was prepared from compound 6b in 51% yield as
a dark red solid, using a similar approach to that described for 7a,
and used in the next reaction without recrystallization. 1H NMR
(DMSO-d6) d 2.99–3.02 (4H, m), 3.24 (3H, s), 3.63 (3H, s), 3.71–
3.75 (4H, m), 4.68 (2H, d, J = 5.8 Hz), 6.77 (2H, d, J = 8.8 Hz),
7.12–7.17 (1H, m), 7.21–7.32 (3H, m), 7.43 (2H, d, J = 8.8 Hz),
8.32 (1H, br), 8.39 (1H, br), 9.30 (1H, s); FAB MS m/e [M+H]+ 467.
5.1.8. 4-[(2-Fluorobenzyl)amino]-2-[(4-morpholin-4-ylphenyl)-
amino]pyrimidine-5-carboxylic acid (6b)
Compound 6b was prepared from compound 5b in 96% yield as
a colorless solid, using a similar approach to that described for 6a,
and used in the next reaction without further purification. colorless
solid. 1H NMR (DMSO-d6) d 3.01–3.03 (4H, m), 3.71–3.75 (4H, m),
4.74 (2H, d, J = 5.3 Hz), 6.78 (2H, d, J = 8.8 Hz), 7.12–7.42 (4H, m),
7.43 (2H, d, J = 8.8 Hz), 8.52 (1H, s), 8.74 (1H, br), 9.50 (1H, br),
12.61 (1H, br); FAB MS m/e [M+H]+ 424.
5.1.13. 4-[(4-Fluorobenzyl)amino]-N-methoxy-N-methyl-2-[(4-
morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide (7c)
Compound 7c was prepared from compound 6c in 71% yield
as a pale brown solid, using a similar approach to that described
for 7a, and used in the next reaction without recrystallization. 1H
NMR (DMSO-d6) d 3.00–3.03 (4H, m), 3.23 (3H, s), 3.61 (3H, s),
5.1.9. 4-[(4-Fluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carboxylic acid (6c)
Compound 6c was prepared from compound 5c in 96% yield as
a pale brown solid, using a similar approach to that described for
6a, and used in the next reaction without further purification. 1H

6932
S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
Table 2
STAT6 inhibitory activity, residual CYP3A4 activities and c Log D7.4 of 7H-pyrrolo[2,3-d]pyrimidine derivatives 9a–e, 16, 17, 19, 21 and 23–25
R1
N
R2
N
N
N
H
R1
R2
IC50 a(nM)
CYP3A4b
c Log D7.4
c
Compound
Preincubation time
0 min
100
30 min
1b
9a
0.7
31
3.44
4.03
H
400
88
78
80
82
97
74
34
O
N
N
N
N
N
*
*
*
*
*
9b
9d
9c
9e
16
2-F
220
120
4100
37
32
37
43
56
95
4.19
4.29
4.19
4.53
1.36
O
3-F
O
4-F
O
3,5-F2
3,5-F2
O
12
HN
N
*
17
19
21
23
3,5-F2
3,5-F2
3,5-F2
3,5-F2
3.2
5.6
34
90
86
88
86
3.85
3.21
1.19
0.73
Me
HO
N
N
*
N
N
*
*
H2N
HO
65
105
98
N
N
O
26
100
N
N
*
O
H2N
Me
24
25
3,5-F2
3,5-F2
1.9
4.7
104
85
85
77
2.92
3.12
N
N
*
O
N
H
N
N
*
a
IC50 values are averages from two or three experiments.
Residual activities of HLM for metabolism of midazolam in the presence of 7H-pyrrolo[2,3-d]pyrimidine derivatives (5 lM) were determined following preincubation for
b
0 and 30 min. See Section 5.
c
c Log D7.4 values were calculated using Pallas PrologD (ver.3.0, CompuDrug).14
3.72–3.74 (4H, m), 4.61 (2H, d, J = 6.0 Hz), 6.81 (2H, d, J = 8.8 Hz),
7.13–7.17 (2H, m), 7.35–7.38 (2H, m), 7.47 (2H, d, J = 8.8 Hz),
8.30 (1H, br), 8.37 (1H, br), 9.27 (1H, br); FAB MS m/e [M+H]+
467.
5.1.15. 4-(Benzylamino)-2-[(4-morpholin-4-ylphenyl)amino]
pyrimidine-5-carbaldehyde (8a)
A solution of 7a (1.87 g, 4.17 mmol) in THF (20 mL) was added
dropwise to a suspension of lithium aluminum hydride (174 mg,
4.49 mmol) and THF (20 mL) at ꢁ78 °C. The mixture was stirred
for 2.5 h below 0 °C. H2O (0.17 g), 15% aqueous NaOH solution
(0.17 g) and H2O (0.51 g) were successively added to the mixture
at 0 °C. The resulting precipitate was filtered and the filtrate was
concentrated in vacuo, and the residue was chromatographed on
silica gel with elution using CHCl3–MeOH (50:1–25:1) to give 8a
(1.50 g, 81%) as a yellow solid, which was used in the next reaction
without further purification.1H NMR (DMSO-d6) d 3.02–3.05 (4H,
m), 3.71–3.74 (4H, m), 4.71 (2H, d, J = 5.7 Hz), 6.83 (2H, d,
J = 8.6 Hz), 7.25–7.28 (1H, m), 7.33–7.35 (4H, m), 7.50 (2H, d,
5.1.14. 4-[(3,5-Difluorobenzyl)amino]-N-methoxy-N-methyl-2-
[(4-morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide (7d)
Compound 7d was prepared from compound 6d in 66% yield as
a pale brown solid, using a similar approach to that described for
7a, and used in the next reaction without recrystallization. 1H
NMR (DMSO-d6) d 3.00–3.02 (4H, m), 3.25 (3H, s), 3.62 (3H, s),
3.72–3.74 (4H, m), 4.62 (2H, d, J = 6.0 Hz), 6.79 (2H, d, J = 8.8 Hz),
7.02–7.09 (3H, m), 7.40 (2H, d, J = 8.8 Hz), 8.32 (1H, br), 8.35 (1H,
s), 9.27 (1H, br); FAB MS m/e [M+H]+ 485.

S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
6933
1.4
1.2
1
140
120
100
80
IFN-g
IL-4
0.8
0.6
0.4
#
p=0.059
60
40
0.2
##
0
20
(mg/kg)
Normal Control
0.03
0.1
24
0.3
3
0
Prednisolone
0.1
1
10
n=10
24 (nM)
#<0.05: significant difference from control as determined by Student’s t-test
Figure 6. Effects of compound 24 and prednisolone on accumulation of eosinophils
in bronchoalveolar lavage fluid (BALF) in ovalbumin (OVA)-sensitized mice.
Figure 5. The effect of compound 24 on cytokine production of spleen T cells from
mice. Squares indicate the amount of IFN- produced in a culture in the presence of
c
IL-12 to induce Th1 cells. Circles indicate the amount of IL-4 produced from Th2
cells differentiated in a culture with IL-4. The cytokine levels in the DMSO control
were 1280 60 ng/mL for IFN-c and 3.9 0.5 ng/mL for IL-4. Data are means SEM
on silica gel with elution using CHCl3–MeOH (50:1–30:1) to give
the crude methoxyvinyl derivative (1.0 g) as a yellow solid. A mix-
ture of the crude methoxyvinyl derivative, concentrated HCl (5 mL)
and MeOH (50 mL) was refluxed for 16 h. The reaction mixture was
cooled to room temperature and concentrated in vacuo. The resi-
due was diluted with CHCl3 and basicified with saturated aqueous
NaHCO3. The organic layer was separated from the aqueous phase,
washed with saturated aqueous NaCl and then dried and concen-
trated in vacuo. The resulting solid was recrystallized from THF–
MeOH to give 9a (725 mg, 51% for 2 steps) as a colorless solid.
Mp 153–155 °C; 1H NMR (DMSO-d6) d 3.01–3.03 (4H, m), 3.72–
3.75 (4H, m), 5.34 (2H, s), 6.43 (1H, d, J = 3.4 Hz), 6.87 (2H, d,
J = 8.8 Hz), 7.24–7.36 (6H, m), 7.69 (2H, d, J = 8.8 Hz), 8.65 (1H, s),
9.16 (1H, s); FAB MS m/e [M+H]+ 386. Anal. Calcd for C23H23N5O:
C, 71.67; H, 6.01; N, 18.17. Found: C, 71.63; H, 6.04; N, 18.26.
expressed as percentages relative to the DMSO control (n = 3).
J = 8.6 Hz), 8.46 (1H, s), 9.05 (1H, br), 9.54 (1H, s), 9.84 (1H, br); FAB
MS m/e [M+H]+ 390.
5.1.16. 4-[(2-Fluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carbaldehyde (8b)
Compound 8b was prepared from compound 7b in 53% yield as
a yellow solid, using a similar approach to that described for 8a,
and used in the next reaction without further purification. 1H
NMR (DMSO-d6) d 3.02–3.05 (4H, m), 3.72–3.75 (4H, m), 4.75
(2H, d, J = 5.7 Hz), 6.80 (2H, d, J = 8.8 Hz), 7.13–7.17 (1H, m),
7.23–7.34 (3H, m), 7.45 (2H, d, J = 8.8 Hz), 8.47 (1H, s), 9.08 (1H,
br), 9.56 (1H, s), 9.86 (1H, br); FAB MS m/e [M+H]+ 408.
5.1.20. 7-(2-Fluorobenzyl)-N-(4-morpholin-4-ylphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (9b)
5.1.17. 4-[(4-Fluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carbaldehyde (8c)
Compound 9b was prepared from compound 8b in 28% yield as
a pale brown solid, using a similar approach to that described for
9a: mp 162–164 °C (MeOH–THF); 1H NMR (DMSO-d6) d 3.01–
3.03 (4H, m), 3.73–3.75 (4H, m), 5.40 (2H, s), 6.45 (1H, d,
J = 3.9 Hz), 6.87 (2H, d, J = 8.8 Hz), 7.12–7.18 (2H, m), 7.23–7.27
(2H, m), 7.31–7.37 (1H, m), 7.67 (2H, d, J = 8.8 Hz), 8.65 (1H, s),
9.16 (1H, s); FAB MS m/e [M+H]+ 404. Anal. Calcd for C23H22N5OF:
C, 68.47; H, 5.50; N, 17.36; F, 4.71. Found: C, 68.23; H, 5.57; N,
17.52; F, 4.63.
Compound 8c was prepared from compound 7c in 82% yield as
a yellow solid, using a similar approach to that described for 8a,
and used in the next reaction without further purification.1H
NMR (DMSO-d6) d 3.03–3.06 (4H, m), 3.72–3.75 (4H, m), 3.78
(3H, s), 4.68 (2H, d, J = 5.6 Hz), 6.84 (2H, d, J = 8.8 Hz), 7.13–7.18
(2H, m), 7.35–7.38 (2H, m), 7.50 (2H, d, J = 8.8 Hz), 8.45 (1H, s),
9.10 (1H, br), 9.53 (1H, s), 9.83 (1H, br); FAB MS m/e [M+H]+ 408.
5.1.18. 4-[(3,5-Difluorobenzyl)amino]-2-[(4-morpholin-4-
ylphenyl)amino]pyrimidine-5-carbaldehyde (8d)
5.1.21. 7-(4-Fluorobenzyl)-N-(4-morpholin-4-ylphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (9c)
Compound 8d was prepared from compound 7d in 75% yield as
a yellow solid, using a similar approach to that described for 8a,
and used in the next reaction without further purification. 1H
NMR (DMSO-d6) d 3.02–3.04 (4H, m), 3.72–3.75 (4H, m), 4.68
(2H, d, J = 5.6 Hz), 6.81 (2H, d, J = 8.3 Hz), 7.02–7.11 (3H, m), 7.41
(2H, d, J = 8.3 Hz), 8.47 (1H, s), 9.12 (1H, br), 9.56 (1H, s), 9.83
(1H, br); FAB MS m/e [M+H]+ 426.
Compound 9c was prepared from compound 8c in 29% yield as
a pale yellow solid, using a similar approach to that described for
9a: mp 170–172 °C (MeOH–THF); 1H NMR (DMSO-d6) d 3.02–
3.04 (4H, m), 3.73–3.75 (4H, m), 5.32 (2H, s), 6.43 (1H, d,
J = 3.4 Hz), 6.88 (2H, d, J = 8.8 Hz), 7.15–7.20 (2H, m), 7.28 (1H, d,
J = 3.4 Hz), 7.33–7.36 (2H, m), 7.68 (2H, d, J = 8.8 Hz), 8.65 (1H, s),
9.16 (1H, s); FAB MS m/e [M+H]+ 404. Anal. Calcd for C23H22N5OF:
C, 68.47; H, 5.50; N, 17.36; F, 4.71. Found: C, 68.32; H, 5.65; N,
17.36; F, 4.67.
5.1.19. 7-Benzyl-N-(4-morpholin-4-ylphenyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-amine (9a)
n-BuLi (1.58 M) solution in hexane (4.7 mL, 7.45 mmol) was
added dropwise to a mixture of (methoxymethyl)triphenylphos-
phonium chloride (2.55 g, 7.45 mmol) at 0–5 °C and the mixture
was stirred for 10 min. To the resulting red solution, a solution of
8a (1.45 g, 3.72 mmol) in THF (50 mL) was added dropwise at
0 °C and the mixture was stirred for 2 h at room temperature.
The mixture was then diluted with AcOEt and washed successively
with H2O and saturated aqueous NaCl. The organic layer was dried
and concentrated in vacuo, and the residue was chromatographed
5.1.22. 7-(3,5-Difluorobenzyl)-N-(4-morpholin-4-ylphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (9d)
Compound 9d was prepared from compound 8d in 57% yield as
a colorless solid, using a similar approach to that described for 9a:
mp 203–206 °C (MeOH–THF); 1H NMR (DMSO-d6) d 3.01–3.04 (4H,
m), 3.73–3.75 (4H, m), 5.33 (2H, s), 6.45 (1H, d, J = 3.4 Hz), 6.88
(2H, d, J = 9.3 Hz), 7.09–7.13 (1H, m), 7.31 (1H, d, J = 3.4 Hz),
7.38–7.45 (2H, m), 7.67 (2H, d, J = 9.3 Hz), 8.66 (1H, s), 9.16 (1H,

6934
S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
s); FAB MS m/e [M+H]+ 422. Anal. Calcd for C23H21N5OF2: C, 65.55;
H, 5.02; N, 16.62; F, 9.02. Found: C, 65.54; H, 5.08; N, 16.83; F, 9.08.
(CDCl3) d 5.43 (2H, s), 6.60 (1H, d, J = 3.7 Hz), 6.86–6.91 (1H, m),
6.97–7.03 (2H, m), 7.16 (1H, d, J = 3.7 Hz), 7.28–7.34 (1H, m),
8.32 (1H, s); FAB MS m/e [M+H]+ 262.
5.1.23. 7-Benzyl-N-(4-morpholin-4-ylphenyl)-6,7-dihydro-5H-
pyrrolo[2,3-d]pyrimidin-2-amine (10)
5.1.27. 2-Chloro-7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidine (14b)
Palladium (10%) on carbon (150 mg) was added to a solution of
9a (500 mg, 130 mmol) in AcOEt (7 mL) and acetic acid (0.7 mL).
The mixture was stirred for 6 h at room temperature under a
hydrogen atmosphere. The mixture was then filtered with Celite
and the filtrate was concentrated in vacuo. The residue was chro-
matographed on silica gel with elution using CHCl3–MeOH–
NH4OH(28%) (200:10:1) to give a colorless solid, which was recrys-
tallized from AcOEt to give 10 (149 mg, 36%) as a colorless solid.
Mp 175–177 °C; 1H NMR (DMSO-d6) d 2.28 (2H, t, J = 8.2 Hz),
2.86–2.90 (4H, m), 3.43 (2H, t, J = 8.2 Hz), 3.70–3.73 (4H, m), 4.56
(2H, s), 6.82 (2H, d, J = 9.1 Hz), 7.26–7.38 (5H, m), 7.61 (2H, d,
J = 9.1 Hz), 7.65 (1H, s), 8.74 (1H, s); FAB MS m/e [M+H]+ 388. Anal.
Calcd for C23H25N5O: C, 71.29; H, 6.50; N, 18.07. Found: C, 71.07; H,
6.45; N, 18.15.
Compound 14b was prepared from compound 13 and 3,5-diflu-
orobenzyl bromide in 99% yield as a pale brown solid, using a sim-
ilar approach to that described for 14a: 1H NMR (CDCl3) d 5.41 (2H,
s), 6.63 (1H, d, J = 3.6 Hz), 6.68–6.78 (3H, m), 7.16 (1H, d, J = 3.6 Hz),
8.84 (1H, s); FAB MS m/e [M+H]+ 280.
5.1.28. 7-(3-Fluorobenzyl)-N-(4-morpholin-4-ylphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-amine (9e)
Palladium diacetate (14 mg, 0.061 mmol), 2’-bis(diphenylphos-
phino)-1,10-binaphthyl (BINAP, 57 mg, 0.092 mmol) and cesium
carbonate (239 mg, 0.73 mmol) were added to a mixture of 14a
(160 mg, 0.61 mmol), 4-morpholinoaniline (120 mg, 0.67 mmol)
and 1,4-dioxane (3.2 mL), and the mixture was heated at 80 °C
for 3 h under an argon atmosphere. The reaction mixture was
cooled to room temperature and concentrated in vacuo. The resi-
due was chromatographed on silica gel with elution using hex-
ane–AcOEt–THF (2:1:1) to give a crude solid (147 mg, 60%),
which was recrystallized from EtOH to give 9e (56 mg, 23%) as
an ivory solid. Mp 183–184 °C; 1H NMR (DMSO-d6) d 3.01–3.03
(4H, m), 3.72–3.75 (4H, m), 5.35 (2H, s), 6.45 (1H, d, J = 3.4 Hz),
6.87 (2H, d, J = 9.3 Hz), 7.08–7.13 (1H, m), 7.32 (1H, d, J = 3.4 Hz),
7.36–7.41 (1H, m), 7.65 (2H, d, J = 9.2 Hz), 8.66 (1H, s), 9.16 (1H,
s); FAB MS m/e [M+H]+ 404. Anal. Calcd for C23H22N5OF: C, 68.47;
H, 5.50; N, 17.36; F, 4.71. Found: C, 68.43; H, 5.64; N, 17.33; F, 4.69.
5.1.24. 2-Chloro-5-(2-ethoxyvinyl)pyrimidin-4-amine (12)
Dichlorobis(triphenylphosphine)palladium (2.74 g, 3.91 mmol)
and n-tetrabutylammonium chloride (21.7 g, 78.1 mmol) were
added to a mixture of 1112 (16.3 g, 78.1 mmol), (Z)-1-ethoxy-2-
(tributylstannyl)ethane (42.3 g, 117 mmol) and DMF (150 mL),
and the mixture was heated at 150 °C for 1 h under an argon ato-
mosphere. The reaction mixture was cooled to room temperature
and diluted with AcOEt (300 mL) and H2O (300 mL), and then fil-
tered with Celite. The organic layer was separated from the aque-
ous layer and the aqueous layer was extracted with AcOEt. The
combined organic layers were washed with saturated aqueous
NaCl, dried and concentrated in vacuo. The residue was chromato-
graphed on silica gel with elution using hexane–AcOEt (3:1–1:1) to
give 12 (8.84 g, 57%) as a colorless solid, which was used in the
next reaction without further purification. 1H NMR (CDCl3) d 1.34
(3H, t, J = 7.1 Hz), 4.02 (2H, q, J = 7.1 Hz), 4.95 (1H, d, J = 7.1 Hz),
5.64 (2H, br), 6.31 (1H, d, J = 7.1 Hz), 8.25 (1H, s); FAB MS m/e
[M+H]+ 200.
5.1.29. tert-Butyl 4-(4-{[7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]amino}phenyl)piperazine-1-carboxylate (15)
Compound 15 was prepared from compound 14b and tert-butyl
4-(4-aminophenyl)piperazine-1-carboxylate in 84% yield as a pale
brown solid, using a similar approach to that described for 9e: 1H
NMR (DMSO-d6) d 1.42 (9H, s), 2.98–3.00 (4H, m), 3.45–3.47 (4H,
m), 5.36 (2H, s), 6.47 (1H, d, J = 3.6 Hz), 6.88 (2H, d, J = 9.2 Hz),
6.97–6.98 (2H, m), 7.14–7.18 (2H, m), 7.33 (1H, d, J = 3.6 Hz),
7.64 (2H, d, J = 9.2 Hz), 8.67 (1H, br), 9.18 (1H, s); FAB MS m/e
[M+H]+ 521.
5.1.25. 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (13)
Trifluoroacetic acid (10 mL) was added to a solution of 12
(9.34 g, 46.8 mmol) in dichloroethane (100 mL) and the mixture
was stirred at 100 °C for 1 h. The reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue was di-
luted with AcOEt–THF and basicified with saturated aqueous NaH-
CO3. The organic layer was separated from the aqueous phase and
the aqueous layer was extracted with AcOEt–THF. The combined
organic layers were washed with saturated aqueous NaCl, dried
and concentrated in vacuo. The residue was chromatographed on
silica gel with elution using hexane–AcOEt (2:1), followed by hex-
ane–AcOEt–THF (7:7:6) to give 13 (6.61 g, 92%) as an ivory solid,
which was used in the next reaction without further purification.
1H NMR (CDCl3) d 6.64–6.66 (1H, m), 7.41–7.43 (1H, m), 8.89
(1H, s), 10.74 (1H, br); FAB MS m/e [M+H]+ 154.
5.1.30. 7-(3,5-Difluorobenzyl)-N-(4-piperazin-1-ylphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-amine dihydrochloride (16)
4 M HCl–AcOEt (47 mL) was added to a mixture of 15 (24.4 g,
46.9 mmol) and MeOH–THF (1:1, 400 mL), and the mixture was
stirred at 80 °C for 13 h. The reaction mixture was cooled to room
temperature and concentrated in vacuo to give 16 (23.0 g, 99%) as a
yellow solid recrystallized from aqueous EtOH. Mp 243–249 °C
(dec.); 1H NMR (DMSO-d6) d 3.23 (4H, br), 3.33–3.35 (4H, m),
5.37 (2H, s), 6.67 (1H, d, J = 3.7 Hz), 6.99–7.04 (4H, m), 7.17–7.22
(1H, m), 7.55 (2H, d, J = 9.0 Hz), 7.60 (1H, d, J = 3.7 Hz), 8.85 (1H,
s), 9.23 (1H, br), 10.18 (1H, s); FAB MS m/e [M+H]+ 421. Anal. Calcd
for C23H22N6OF2ꢀ2HClꢀ4H2O: C, 48.86; H, 5.70; N, 14.86; F, 6.72; Cl,
12.54. Found: C, 48.92; H, 5.49; N, 14.70; F, 6.79; Cl, 12.79.
5.1.26. 2-Chloro-7-(3-fluorobenzyl)-7H-pyrrolo[2,3-d
]pyrimidine (14a)
5.1.31. 7-(3,5-Difluorobenzyl)-N-[4-(4-methylpiperazin-1-
yl)phenyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (17)
3-Fluorobenzyl bromide (257 mg, 1.36 mmol) was added to a
mixture of 13 (173 mg, 1.13 mmol) and potassium carbonate
(187 mg, 1.35 mmol) in DMSO (4 mL) and the mixture was stirred
at 60 °C for 2 h. The mixture was then diluted with AcOEt and ex-
tracted with H2O. The organic layer was washed with saturated
aqueous NaCl, dried and concentrated in vacuo. The residue was
chromatographed on silica gel with elution using hexane–AcOEt
(3:1–2:1) to give 14a (334 mg, 94%) as an ivory solid, which was
used in the next reaction without further purification.1H NMR
Dipotassium carbonate (840 mg, 6.08 mmol) and iodomethane
(0.13 mL, 2.09 mmol) were added to a DMF (9.3 mL) solution of
16 (926 mg, 1.88 mmol) and the mixture was stirred for 2 h at
room temperature. The mixture was then diluted with AcOEt–
THF and washed successively with H2O and saturated aqueous
NaCl. The organic layer was dried and concentrated in vacuo. The
resultant solid was recrystallized from AcOEt–MeOH to give 17
(150, 15%) as a pale yellow solid. Mp 181–184 °C; 1H NMR

S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
6935
(DMSO-d6) d 2.42–2.48 (4H, m), 2.50 (3H, s), 3.03–3.06 (4H, m),
5.35 (2H, s), 6.46 (1H, d, J = 3.6 Hz), 6.85 (2H, d, J = 9.1 Hz), 6.97–
7.01 (2H, m), 7.13–7.19 (1H, m), 7.33 (1H, d, J = 3.6 Hz), 7.61 (2H,
d, J = 9.1 Hz), 8.66 (1H, s), 9.16 (1H, s); FAB MS m/e [M+H]+ 435.
Anal. Calcd for C24H24N6F2ꢀ0.2H2O: C, 65.80; H, 5.61; N, 19.18; F,
8.67. Found: C, 65.66; H, 5.56; N, 19.25; F, 8.53.
7.17–7.23 (1H, m), 7.56–7.59 (3H, m), 8.42 (3H, br), 8.84 (1H, s),
10.09 (1H, br), 11.38 (1H, br); FAB MS m/e [M+H]+ 464. Anal. Calcd
for C25H27N7F2ꢀ3.1HClꢀ3H2O: C, 47.62; H, 5.77; N, 15.55; F, 6.03; Cl,
17.43. Found: C, 47.33; H, 5.71; N, 15.35; F, 6.39; Cl, 17.32.
5.1.36. Ethyl [4-(4-{[7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetate (22)
Ethyl bromoacetate (0.2 mL) was added to a mixture of 16
(780 mg, 1.58 mmol), dipotassium carbonate (813 mg, 5.88 mmol)
and DMF (20 mL) and the mixture was stirred for 4 h at 100 °C. The
mixture was then diluted with AcOEt and washed successively
with H2O and saturated aqueous NaCl. The organic layer was dried
and concentrated in vacuo. The residue was chromatographed on
silica gel with elution using CHCl3–MeOH (100:1 to 50:1) to give
a crude solid, which was recrystallized from EtOH to give 22
(561 mg, 75%) as a pale brown solid. 1H NMR (DMSO-d6) d 1.21
(3H, t, J = 7.3 Hz), 2.65–2.62 (4H, m), 3.04–3.06 (4H, m), 3.27 (2H,
s), 4.11 (2H, q, J = 7.3 Hz), 5.35 (2H, s), 6.46 (1H, d, J = 3.9 Hz),
6.86 (2H, d, J = 9.3 Hz), 6.96–7.00 (2H, m), 7.13–7.18 (1H, m),
7.33 (1H, d, J = 3.9 Hz), 7.62 (2H, d, J = 9.3 Hz), 8.66 (1H, s), 9.15
(1H, s); FAB MS m/e [M+H]+ 507.
5.1.32. N-{4-[4-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)pip-
erazin-1-yl]phenyl}-7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-amine (18)
(2-Bromoethoxy)-tert-butyldimethylsilane (630 mg, 2.64 mmol)
and sodium iodide (394 mg, 2.64 mmol) were added to a mixture
of 16 (926 mg, 1.88 mmol), disodium carbonate (751 mg,
7.10 mmol) and DMF (9 mL). The mixture was stirred for 4 h at
100 °C. The mixture was then diluted with AcOEt–THF and washed
successively with H2O and saturated aqueous NaCl. The organic
layer was dried and concentrated in vacuo to give 18 (676 mg,
62%) as an ivory solid, which was used in the next reaction without
further purification. 1H NMR (DMSO-d6) d 0.06 (6H, s), 0.89 (9H, s),
2.47 (2H, t, J = 6.0 Hz), 2.57–2.59 (4H, m), 3.01–3.03 (4H, m), 3.72
(2H, t, J = 6.0 Hz), 5.35 (2H, s), 6.46 (1H, d, J = 3.6 Hz), 6.85 (2H, d,
J = 9.2 Hz), 6.97–6.99 (2H, m), 7.14–7.19 (1H, m), 7.31 (1H, d,
J = 3.6 Hz), 7.61 (2H, d, J = 9.2 Hz), 8.66 (1H, s), 9.15 (1H, s); FAB
MS m/e [M+H]+ 579.
5.1.37. [4-(4-{[7-(3,5-Difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetic acid (23)
1 M NaOH (1.9 mL) was added to a solution of 22 (490 mg,
0.97 mmol) and MeOH–THF (1:1, 20 mL), and the mixture was stir-
red at 50 °C for 15 h.1 M HCl (2.0 mL) was added to the mixture
and resulting solid was collected by filtration and washed with
H2O. The crude solid was recrystallized from EtOH–THF to give
23 (284 mg, 61%) as a colorless solid. Mp 107–110 °C; 1H NMR
(DMSO-d6) d 2.73–2.76 (4H, m), 3.08–3.10 (4H, m), 3.22 (2H, s),
5.35 (2H, s), 6.46 (1H, d, J = 3.4 Hz), 6.86 (2H, d, J = 9.3 Hz), 6.96–
7.01 (2H, m), 7.13–7.19 (1H, m), 7.33 (1H, d, J = 3.4 Hz), 7.62 (2H,
d, J = 9.3 Hz), 8.66 (1H, s), 9.16 (1H, s); FAB MS m/e [MꢁH]ꢁ 477.
Anal. Calcd for C25H24N6F2O2ꢀ0.6H2O: C, 61.37; H, 5.19; N, 17.18;
F, 7.77. Found: C, 61.20; H, 5.23; N, 17.23; F, 7.90.
5.1.33. 2-[4-(4-{[7-(3,5-Difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]ethanol (19)
A 1 M solution of n-tetrabuthylammonium fluoride in THF
(5.7 mL) was added to a solution of 18 (655 mg, 1.13 mmol) and
THF (10 mL), and the mixture was stirred for 16 h at room temper-
ature. The mixture was then diluted with AcOEt and washed suc-
cessively with H2O and saturated aqueous NaCl. The organic
layer was dried and concentrated in vacuo. The resultant solid
was recrystallized from AcOEt to give 19 (305 mg, 58%) as an ivory
solid. Mp 160–163 °C; 1H NMR (DMSO-d6) d 2.43–2.46 (2H, m),
2.56–2.58 (4H, m), 3.02–3.05 (4H, m), 3.54 (2H, dt, J = 6.0,
6.8 Hz), 4.43 (1H, br), 5.35 (2H, s), 6.46 (1H, d, J = 3.6 Hz), 6.85
(2H, d, J = 9.2 Hz), 6.97–6.99 (2H, m), 7.13–7.19 (1H, m), 7.33
(1H, d, J = 3.6 Hz), 7.61 (2H, d, J = 9.2 Hz), 8.66 (1H, s), 9.15 (1H,
s); FAB MS m/e [M+H]+ 465. Anal. Calcd for C25H26N6F2Oꢀ0.3H2O:
C, 63.90; H, 5.71; N, 17.88; F, 8.09. Found: C, 63.70; H, 5.62; N,
17.83; F, 8.18.
5.1.38. 2-[4-(4-{[7-(3,5-Difluorobenzyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetamide (24)
EDCꢀHCl (75 mg, 0.39 mmol) and HOBt (53 mg, 0.39 mmol)
were added to a mixture of 23 (157 mg, 0.33 mmol) in DMF
(5 mL). After stirring for 30 min at room temperature, 28% aqueous
ammonia (1.0 mL) was added, and the mixture was stirred for 16 h
at room temperature. The reaction mixture was then diluted with
H2O and extracted with AcOEt–THF. The organic layer was washed
successively with H2O and saturated aqueous NaCl and then dried
and concentrated in vacuo. The residue was chromatographed on
silica gel with elution using CHCl3–MeOH (30:1 to 20:1) to give
crude solid, which was recrystallized from EtOH to give 24
(95 mg, 60%) as a colorless solid. Mp 201–203 °C; 1H NMR
(DMSO-d6) d 2.58–2.60 (4H, m), 2.92 (2H, s), 3.08–3.10 (4H, m),
5.35 (2H, s), 6.46 (1H, d, J = 3.2 Hz), 6.83 (2H, d, J = 9.2 Hz), 6.96–
7.01 (2H, m), 7.13–7.22 (1H, m), 7.33 (1H, d, J = 3.2 Hz), 7.62 (2H,
d, J = 9.2 Hz), 8.66 (1H, s), 9.16 (1H, s); FAB MS m/e [M+H]+ 478.
Anal. Calcd for C25H25N7F2O: C, 62.88; H, 5.28; N, 20.53; F, 7.96.
Found: C, 62.72; H, 5.32; N, 20.50; F, 8.10.
5.1.34. tert-Butyl {2-[4-(4-{[7-(3,5-difluorobenzyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]-
ethyl}carbamate (20)
Compound 20 was prepared from compound 16 and 2-(tert-
butoxycarbonylamino)ethyl bromide in 59% yield as a pale yellow
solid, using a similar approach to that described for 18, and used in
the next reaction without further purification. 1H NMR (DMSO-d6)
d 1.38 (9H, s), 2.37 (2H, t, J = 6.4 Hz), 2.53 (4H, br), 3.02–3.08 (6H,
m), 5.35 (2H, s), 6.45 (1H, d, J = 3.6 Hz), 6.67 (1H, br), 6.85 (2H, d,
J = 9.2 Hz), 6.97–6.99 (2H, m), 7.14–7.18 (1H, m), 7.34 (1H, d,
J = 3.6 Hz), 7.61 (2H, d, J = 9.2 Hz), 8.31 (1H, s), 9.15 (1H, s); FAB
MS m/e [M+H]+ 564.
5.1.35. N-{4-[4-(2-Aminoethyl)piperazin-1-yl]phenyl}-7-(3,5-
difluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine
hydrochloride (21)
4 M HCl–AcOEt (7.2 mL) was added to a solution of 20 (651 mg,
1.16 mmol) in MeOH (20 mL), and the mixture was stirred for 16 h
at room temperature. The resulting solid was collected by filtration
and washed with AcOEt. The solid was recrystallized from AcOEt–
MeOH to give 21 (240 mg, 36%) as a yellow solid. Mp 169–171 °C;
1H NMR (DMSO-d6) d 3.17–3.26 (4H, m), 3.36–3.43 (4H, m), 3.68–
3.76 (4H, m), 5.37 (2H, s), 6.54 (1H, d, J = 3.6 Hz), 7.01–7.04 (3H, m),
5.1.39. 2-[4-(4-{[7-(3,5-Difluorobenzyl)-7H-pyrrolo[2,3-d]pyr-
imidin-2-yl]amino}phenyl)piperazin-1-yl]-N-methylacetamide
hydrochloride (25)
EDCꢀHCl (78 mg, 0.41 mmol) and HOBt (56 mg, 0.41 mmol)
were added to a mixture of 23 (150 mg, 0.31 mmol) in DMF
(5 mL). After stirring for 30 min at room temperature, 40%
methylamine in MeOH solution (0.1 mL) was added and the mix-
ture was stirred for 16 h at room temperature. The reaction mix-
ture was then diluted with H2O and extracted with AcOEt. The

6936
S. Nagashima et al. / Bioorg. Med. Chem. 17 (2009) 6926–6936
organic layer was washed successively with H2O and saturated
aqueous NaCl, and was then dried and concentrated in vacuo.
The residue was chromatographed on silica gel with elution
using CHCl3–MeOH (50:1 to 30:1). The collected fraction was
concentrated to give an amorphous solid. 4 M HCl–AcOEt
(1 mL) was added to the amorphous solid, and the mixture
was concentrated. The residue was triturated with EtOH–AcOEt
to give 25 (100 mg, 29%) as a pale yellow solid. Mp 154–
156 °C; 1H NMR (DMSO-d6) d 2.68 (3H, d, J = 4.4 Hz), 3.16 (2H,
br), 3.33 (2H, br), 3.56 (2H, br), 3.74 (2H, br), 4.02 (2H, s),
5.38 (2H, s), 6.72 (1H, d, J = 3.4 Hz), 7.02 (2H, d, J = 9.3 Hz),
7.03–7.07 (2H, m), 7.18–7.24 (1H, m), 7.53 (2H, d, J = 9.3 Hz),
7.67 (1H, d, J = 3.4 Hz), 8.69 (1H, br q, J = 4.4 Hz), 8.93 (1H, s),
10.55 (1H, s); FAB MS m/e [M+H]+ 492. Anal. Calcd for
C26H27N7F2Oꢀ2.8HClꢀ0.7H2O: C, 51.51; H, 5.19; N, 16.17; F, 6.27;
Cl, 16.37. Found: C, 51.51; H, 5.21; N, 16.15; F, 6.34; Cl, 16.07.
5.5. In vitro T cell differentiation
The effect of compound 24 on Th1 and Th2 differentiation from
naive Th cells in mice were determined as described previously.15
5.6. OVA-induced pulmonary eosinophilia in actively sensitized
rats
Female Balb/c mice were actively sensitized by intraperitoneal
injection of OVA-containing aluminum hydroxide gel. This was re-
peated twice at four-day intervals. At 12 days after the first sensi-
tization, the mice were exposed to an aerosolized solution of OVA
for 1 h. At 72 h after OVA exposure, mice were sacrificed and BAL
was performed. Compound 24 was administered orally 30 min be-
fore, and 24 and 48 h after OVA exposure. Prednisolne was admin-
istered orally 2 h before, and 24 and 48 h after OVA exposure. The
number of eosinophils was determined as described previously.7b
5.2. Molecular modeling
Acknowledgments
The geometry of 1a was optimized using the Conformation Im-
port function in the Molecular Operating Environment (MOE) pro-
gram.16 Structure alignment of 1a, 9a and 10 was performed
with the Flexible Alignment tool in MOE, using the geometry of 1a
as a template and the MMFF94x force field.
The authors are grateful to the staff of the Division of Analytical
Science Laboratories for elemental analysis and spectral measure-
ments. We thank Dr. Tadashi Terasaka for valuable comments
and help in preparation of the manuscript.
5.3. Calculation of atomic charge distribution
References and notes
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MOE (revision 2007.0902) was used to build the ligand structures
of 1a, 9a, and 10. The molecular conformations were optimized at
the semiempirical AM1 level and electrostatic potential-fitted
atomic partial charges were calculated using the MOPAC 7.1 mod-
ule in MOE. Connolly surfaces17 that surround the van der Waals
surfaces of the core scaffolds were generated and colored with
red for negative charge and blue for positive charge, using the
MOE program.
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5.4. Measurement of residual activities of HLMs for metabolism
of midazolam
The test compounds (5 lM) were pre-incubated with HLM
(0.1 mg/mL) in the presence of NADPH (1 mM) and ethylenedi-
aminetetraacetic acid (EDTA, 0.1 mM) in potassium sodium phos-
phate buffer (100 mM, pH 7.4). Midazolam was added to the
reaction mixture following pre-incubation for 0 and 30 min at
37 °C. The reaction was stopped by addition of acetonitrile after
20 min incubation. The metabolite of midazolam (10-hydroxymi-
dazolam) was analyzed by LC–MS, comprising an HPLC system
(Waters 2795) to separate the peaks and atmospheric pressure
electrospray ionization MS (Waters ZQ). The residual midazolam
12. Cheung, M.; Harris, P. A.; Lackey, K. E. Tetrahedron Lett. 2001, 42, 999.
13. Sakamoto, T.; Kondo, Y.; Yasuhara, Y.; Yanamaka, H. Heterocycles 1990, 31, 219.
14. 115 Morgan Drive, Sedona, AZ 86351, USA.
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Tsukamoto, S.; Ohta, M. Bioorg. Med. Chem. 2007, 15, 1044.
10-hydroxylase activity in the presence of 5
lM of the test com-
pound was expressed as the percentage activity of that in a control
incubation performed without compound. Verapamil was used as a
positive control CYP inactivator.
16. MOE 200810; Chemical Computing Group: Montreal, Canada.
17. Connolly, M. L. J. Appl. Crystallogr. 1983, 16, 548.