Isoquinoline derivatives as potent, selective, and orally active CRTH2 antagonists

Article abstract of DOI:10.1248/cpb.c13-00980

Synthesis and structure-activity relationship of a novel series of isoquinoline CRTH2 antagonists bearing a methylene linker between the isoquinoline and benzamide moieties were described. Optimization focusing on the substituents of the benzamide portion in the right hand part of the molecule led to the identification of TASP0412098 (9l), which is a potent, selective CRTH2 antagonist (binding affinity: IC₅₀=2.1 nM, functional activity: IC₅₀=12 nM). Compound 9l, which was orally bioavailable in mice and guinea pigs, showed in vivo efficacy after oral administration in a bronchial asthma model of guinea pigs.

Full text of DOI:10.1248/cpb.c13-00980

528
Regular Article
Chem. Pharm. Bull. 62(6) 528–537 (2014)
Vol. 62, No. 6
Isoquinoline Derivatives as Potent, Selective, and Orally Active CRTH2
Antagonists
,a
Rie Nishikawa-Shimono,* Yoshinori Sekiguchi,a Madoka Kawamura,a Daisuke Wakasugi,a
Masahumi Kawanishi,b Kazuhito Watanabe,b Yumiko Asakura,b Akiko Takaoka,b and
Tetsuo Takayamaa
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd.; and b Molecular Function and Pharmacology
Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama 331–9530, Japan.
Received December 18, 2013; accepted March 14, 2014
Synthesis and structure–activity relationship of a novel series of isoquinoline CRTH2 antagonists bear-
ing a methylene linker between the isoquinoline and benzamide moieties were described. Optimization focus-
ing on the substituents of the benzamide portion in the right hand part of the molecule led to the identifica-
tion of TASP0412098 (9l), which is a potent, selective CRTH2 antagonist (binding affinity: IC50=2.1nM, func-
tional activity: IC50=12nM). Compound 9l, which was orally bioavailable in mice and guinea pigs, showed in
vivo efficacy after oral administration in a bronchial asthma model of guinea pigs.
Key words CRTH2 antagonist; allergic diseases; isoquinoline; prostaglandin D2
The chemoattractant receptor–homologous molecule ex- chemotype by changing its physicochemical properties. In the
pressed on Th2 cells (CRTH2) was reported in 2001 as a present study, we described the synthesis and SAR of isoquin-
G protein-coupled receptor for prostaglandin D2 (PGD2). oline CRTH2 antagonists containing a methylene linker, and
CRTH2 is expressed on eosinophils, basophils, and Th2 theꢀpreliminaryꢀpharmacokineticꢀandꢀpharmacologicalꢀprofilesꢀ
cells, and plays an important role in allergic diseases, driv- of the potent, selective, and orally active CRTH2 antagonist
ing the immunoglobulin E (IgE) response, eosinophilia, and 9l, which is the most potent compound of this class.
theꢀ releaseꢀ ofꢀ proinflammatoryꢀ cytokines.1–3) Activation of
Chemistry Preparation of isoquinoline derivatives 9a–9u
CRTH2 promotes the release of histamine from basophils and is outlined in Chart 1. The ylide 3, derived from commer-
degranulation of eosinophiles.4–6) Thus, a CRTH2 antagonist cially available benzyl bromide 2, was treated with methyl
mightꢀ beꢀ beneficialꢀ forꢀ aꢀ varietyꢀ ofꢀ inflammatoryꢀ diseases.7,8) 1-chloroisoquinoline-4-carboxylate in the presence of sodium
Research designed to identify selective CRTH2 antagonists hexamethyldisilazane, followed by hydrolysis, to give 4 in
dramatically increased after the discovery that the thrombox- 58% yield.11) The carboxylic acid 5, prepared by the basic
ane receptor antagonist, ramatroban, which is approved for the hydrolysis of 4, was converted into the corresponding acid
treatmentꢀofꢀallergicꢀrhinitis,ꢀandꢀtheꢀanti-inflammatoryꢀdrug,ꢀ chloride, which was treated with trimethylsilyldiazomethane
indomethacin, were found to be CRTH2 antagonists. A large to afford the diazoketone intermediate. Wolff rearrangement
number of structure–activity relationship (SAR) information of the diazoketone intermediate followed by methylation of the
around the indoleacetic acids have been disclosed.
resulting carboxylic acid moiety with trimethylsilyldiazometh-
In our previous paper, we reported that the lead generation ane provided 6 in 35% yield from 5.12) Deprotection of the
process and initial SAR study of a novel isoquinoline che- t-butoxycarbonyl group of 6ꢀunderꢀanꢀacidicꢀconditionꢀ(trifluo-
motypeꢀ ofꢀ CRTH2ꢀ antagonists,ꢀ ledꢀ toꢀ theꢀ identificationꢀ ofꢀ roacetic acid, TFA), and subsequent acylation of the resulting
compound 1ꢀ(bindingꢀaffinity:ꢀIC50=19 nM,ꢀfunctionalꢀactivity:ꢀ acid chloride of 7 with appropriate amines gave 8a, 8l, 8o, and
IC50=13 nM) as a potent and selective CRTH2 antagonist.9,10) 8s. Compound 8b–8k, 8m, 8n, 8p–8v, 8t, and 8u were ob-
In our continuous research focused on identifying an alterna- tained by coupling the acid 7 with appropriate amines using a
tive lead class, we took particular note of the carbonyl linker standard protocol (WSC·HCl, HOBT·H2O). Finally, hydrolysis
of 1,ꢀbecauseꢀtheꢀmodificationꢀofꢀthisꢀlinkerꢀwouldꢀimpactꢀtheꢀ of the ester moieties of 8a–8u afforded the target isoquinoline
physicochemical properties of 1. We designed novel CRTH2 derivatives 9a–9u.
antagonists bearing a methylene linker in place of the car-
Compounds 9v, 9w, dl-9x, and 9y, in which the acetic acid
bonylꢀlinker,ꢀexpectingꢀtoꢀchangeꢀtheꢀbiologicalꢀprofileꢀofꢀthisꢀ moietyꢀatꢀ theꢀ 4-positionꢀofꢀtheꢀ isoquinolineꢀ coreꢀ isꢀ modified,ꢀ
Fig. 1. Ligands for the CRTH2 Receptor
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
© 2014 The Pharmaceutical Society of Japan
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀr-nishikawa@so.taisho.co.jp

June 2014
529
(a) n-Bu3P, toluene, 60°C, 93%; (b) methyl 1-chloroisoquinoline-4-carboxylate, NaHMDS, THF, −30–50°C, then Na2CO3 aq., 60°C, 58%; (c) 1N NaOH aq., THF, 50°C,
quant; (d) (COCl)2, CHCl3, rt, TMSCHN2, THF/MeCN, rt, silver acetone, H2O/dioxane, 60°C then TMSCHN2, MeOH, rt, 35% (4 steps); (e) TFA, CHCl3, 0°C-rt, quant;
(f) for 8a, 8l, 8o, 8s, (COCl)2, DMF, CHCl3 then R2NH2, pyridine, 0°C-rt, 23–93%, for 8b–k, 8m, 8n, 8p–v, 8t, 8u, R2NH2, WSC·HCl, HOBT·H2O, Et3N, CHCl3, rt,
36–80%; (g) 1N NaOH aq., THF, 0°C, 19–90%.
Chart 1
(a) CDI then NH3 aq., THF, rt, 96%; (b) POCl3, DMF, rt, 74%; (c) NaN3, Et3N–HCl, 14%.
Chart 2
(a) t-BuOK, MeI, THF, 0°C, 61%; (b) TFA, CHCl3, 0°C-rt; (c) 2-(4-chlorophenyl)ethane amine, WSC·HCl, HOBT·H2O, Et3N, CHCl3, rt, 58% (2 steps); (d) 1N NaOH aq.,
THF, 0°C, 71%.
Chart 3
were synthesized as shown in Charts 2, 3, and 4. Carbamo- to give compound 9w. Methylation of 6 using t-BuOK and io-
ylation of compound 9l afforded compound 9v in 96% yield. domethane produced dl-11 in 61% yield. Deprotection of the t-
Following the reaction of 9v with phosphorus oxychloride, butylꢀesterꢀmoietyꢀusingꢀtrifluoroaceticꢀacidꢀ(TFA),ꢀfollowedꢀbyꢀ
cyclization to the tetrazole was achieved using sodium azide the condensation of dl-12 with 2-(4-chlorophenyl)ethanamine

530
Vol. 62, No. 6
(a) BH3-THF, THF, 0°C-rt, 39%; (b) Dess–Martin periodinane, CHCl3, 0°C, 85%; (c) ethyl 2-(dithoxyphosphoryl)acetate, NaH, 0°C, 82%; (d) NaBH4, CuCl2, MeOH, rt,
0°C, 60%; (e) TFA, CHCl3, 0°C-rt, quant; (f) 2-(4-chlorophenyl)ethaneamine, WSC·HCl, HOBT·H2O, THF, rt, 82%; (g) 1 n NaOH aq., THF, 0°C-rt, 83%.
Chart 4
Table 1. In VitroꢀDataꢀofꢀIsoquinolineꢀDerivativesꢀwithꢀLinkerꢀModifica-
tions
(WSC·HCl, HOBT·H2O) gave dl-13 in 58% yield. Finally,
hydrolysis of the ester moiety afforded the target derivative
dl-9x. Compound 9y was synthesized from the intermediate
5, which was reduced by borane–tetrahydrofuran complex to
give alcohol 14. Subsequent oxidation with Dess–Martin peri-
odinane,ꢀfollowedꢀbyꢀolefinationꢀofꢀtheꢀaldehyde,ꢀaffordedꢀ16.
Compound 16 was reduced by sodium borohydride to give 17,
which was converted to 9y using the following conventional
method:ꢀ (1)ꢀ deprotectionꢀ ofꢀ theꢀ t-butoxycarbonyl group of 17
using TFA; (2) condensation reaction with 2-(4-chlorophenyl)-
ethanamine (WSC·HCl, HOBT·H2O); and (3) hydrolysis of the
ethyl ester moiety.
IC50 (nM)a)
Compound
W
Binding
Ca2+
1
CO
19
13
15
9a
CH2
8.3
a) Mean values from at two or more independent experiments. IC50 values were
determined from full ten-point, half-log concentration–response curves.
Results and Discussion
Compounds were tested in a radioligand binding assay
3
using H-PGD2 and human CRTH2 stably transfected in CHO
cells. Their functional activities were assessed in PGD2 driven
Ca2+ꢀfluxꢀassayꢀinꢀKB8ꢀcellsꢀexpressingꢀhumanꢀCRTH2.
Initially,ꢀ theꢀ bindingꢀ affinityꢀ andꢀ theꢀ functionalꢀ activityꢀ ofꢀ
the methylene linker compound 9a were compared with those
of the original carbonyl linker compound 1 (Table 1). These
results suggested that both compounds were potent CRTH2
antagonists. And compound 1 and compound 9a shows dif-
ferent physicochemical properties. For example, with regard
to the aqueous solubility (20mM phosphate buffer), 9a showed
much lower solubility than 1 (9a:ꢀ 1.2ꢀmg/mL,ꢀ 1:ꢀ 540ꢀmg/mL).ꢀ
In addition, comparison of the logP values at pH 6.8 revealed
that 9a is more hydrophobic than 1 (9a:ꢀ3.1,ꢀ1:ꢀ2.7).ꢀTheseꢀdataꢀ
suggested that the change of this linker part would have an
impact on the physicochemical properties as we had expected.
With these data in hand, we conducted the SAR study of
the methylene linker compounds, focusing on the effect of the
R2 substituent of the benzamide moiety on the CRTH2 bind-
ingꢀaffinityꢀandꢀfunctionalꢀactivityꢀ(Tableꢀ2).ꢀWhenꢀtheꢀmethylꢀ
group in 9b, which showed moderate potency (IC50=360nM)
in the binding assay, was replaced with a cycloalkylethyl
moiety such as a cyclopropylethyl (9c), cyclohexylethyl (9d),
Table 2. In Vitro Data of Isoquinoline Derivatives with R2 Substitution
Modifications
IC50 (nM)a)
Compound
R2
Binding
Ca2+
9b
9c
9d
9e
9f
Methyl
360
16
>1000
141
Cyclopropylethyl
Cyclohexylethyl
Adamantylethyl
3.7
14
29
6.6
2-(Tetrahydro-2H-pyran-
4-yl)ethyl
35
>1000
9g
9h
9i
2-(Morpholin-4-yl)ethyl
Phenethyl
>1000
4.6
>1000
110
2-(Pyridin=4-yl)ethyl
320
>1000
a) Mean values from at two or more independent experiments. IC50 values were
determined from full ten-point, half-log conentration–response curves.

June 2014
531
Table 3. In Vitro Data of Isoquinoline Derivatives with R2 Substitution Table 4. In Vitro Data of Isoquinoline Derivatives with R3 Substitution
Modifications
Modifications
IC50 (nM)a)
Binding
IC50 (nM)a)
R3
Compound
R2
Compound
Ca2+
Binding
2.1
Ca2+
12
9h
9j
Phenethyl
4.6
2.4
3.8
2.1
4.8
110
28
9l
2-Cl-phenythyl
9k
9l
3-Cl-phenethyl
21
4-Cl-phenethyl
12
9v
>1000
>1000
>1000
790
9m
9n
9o
9p
9q
9r
9s
4-Me-Phenethyl
4-NMe2-phenethyl
4-OMe-phenethyl
4-OH-phenethyl
4-SO2Me-phenethyl
4-CO2H-phenethyl
4-Cl-benzyl
17
10
19
9.0
210
52
9w
dl-9x
9y
590
34
510
>1000
>1000
400
42
180
900
27
9t
3-(4-Cl-phenyl)propyl
2-(4-Cl-phenoxy)ethyl
6.5
7.7
9u
250
28
54
a) Mean values from at two or more independent experiments. IC50 values were
determined from full ten-point, half-log concentration–response curves.
a) Mean values from two or more independent experiments. IC50 values were de-
termined from full ten-point, half-log concentration–response curves.
or adamantylethyl (9e), these moieties contributed greatly to
enhancingꢀnotꢀonlyꢀtheꢀbindingꢀaffinityꢀbutꢀalsoꢀtheꢀfunctionalꢀ
activity. The 2-(tetrahydro-2H-pyran-4-yl)ethyl group (9f)
showedꢀ onlyꢀ moderateꢀ bindingꢀ affinityꢀ (IC50=35 nM), while
the 2-(morpholin-4-yl)ethyl group (9g) resulted in the com-
plete loss of potency. These data suggested that the R2 group
does not accept hydrophilic substituents. Interestingly, the
phenethyl (9h)ꢀgroupꢀledꢀtoꢀgreatlyꢀenhancedꢀbindingꢀaffinity,ꢀ
(IC50=4.6nM), although the functional activity was not suf-
ficientꢀ(IC50=110 nM).
Table 5. Pharmacokinetic Parameters of 1 and 9l Following Oral Admin-
istration to Preclinical Species
Compound
Species
1
9l
Mouse
25
Mouse
10
Guinea pig
30
Dose (mg/kg)
Cmax (nm)
1600
0.50
1.7
2200
0.50
2.0
540
Tmax (h)
0.75
T1/2 (h)
6.5
AUC0–24h (ngh/mL)
510
1400
1500
Further SAR study on 9h was conducted to examine the
effect of substituents of the terminal phenyl ring on the representative CRTH2 antagonists (Table 4). Replacement of
CRTH2 binding and functional activities (Table 3). Incorpo- the carboxylic acid with its isosteres, such as a carbamoyl and
ration of a chlorine atom into the 2-, 3-, or 4-position on the tetrazole revealed that the carbamoyl led to the loss of binding
phenyl group led to an increase not only in the binding poten- activity (9v:ꢀ IC50=>1000nM, 9w:ꢀ IC50=590 nM). Methylation
cy (9j:ꢀIC50=2.4nM, 9k:ꢀIC50=3.8 nM, 9l:ꢀIC50=2.1 nM) but also of the methylene moiety next to the carboxylic acid of 9l re-
in the functional assay (9j:ꢀ IC50=28nM, 9k:ꢀ IC50=21 nM, 9l:ꢀ sultedꢀinꢀaꢀ17-foldꢀdropꢀinꢀtheꢀbindingꢀaffinityꢀcomparedꢀwithꢀ
IC50=12nM). With regard to the substituent at the 4-position, the original compound. And the carboxymethyl moiety in 9l
methyl (9m), dimethylamino (9n), methoxy (9o), and hydroxyl was replaced with the corresponding carboxyethyl group, the
(9p)ꢀ groupsꢀ wereꢀ foundꢀ toꢀ beꢀ toleratedꢀ inꢀ theꢀ bindingꢀ affin- bindingꢀ affinityꢀ ofꢀ theꢀ resultingꢀ compoundꢀ 9y was decreased
ity (9m:ꢀ IC50=4.8nM, 9n:ꢀ IC50=10 nM, 9o:ꢀ IC50=19 nM, 9p:ꢀ by a factor of 13, although both compounds were tolerable (dl-
IC50=9.0 nM), while dimethylamino (9n) and hydroxyl (9p) 9x:ꢀ IC50=34nM, 9y:ꢀ IC50=28nM). These data suggest that the
resulted in a decrease in the functional activity compared to carboxylic acid moiety is essential for CRTH2 activity and the
9h. Installation of a methylsulfonyl (9q) or a carboxylic acid binding space, where the acid moiety of the antagonists inter-
(9r)ꢀ atꢀ thisꢀ positionꢀ resultedꢀ inꢀ aꢀ significantꢀ lossꢀ inꢀ bothꢀ theꢀ acts with the CRTH2, is limited.
bindingꢀ affinityꢀ andꢀ functionalꢀ activity.ꢀ Regardingꢀ theꢀ linkerꢀ
Thus, compound 9l was selected as one of the most potent
part between the amide and the terminal 4-chlorophenyl group antagonists of this chemotype. The preliminary physicochemi-
of 9l, replacement of the ethylene linker with a methylene (9s), cal study indicated that 9l had moderate aqueous solubility
propylene (9t), or oxyethylene (9u) linker led to slight losses (14 µg/mL in 20mM phosphate buffer at pH 6.8) and its logP
inꢀbothꢀtheꢀbindingꢀaffinityꢀandꢀfunctionalꢀactivity,ꢀcomparedꢀ value (pH 6.8) was 2.4. The selectivity for the binding of 9l
to those of 9l. These data suggest that a two-carbon linker is to CRTH2 over not only DP1, TP, EP1, FP, and IP (prostanoid
the most favorable.
receptors), but also BLT1, cysLT1, and cysLT2 (leukotriene
Following this, we examined the rough SAR of the car- receptors) was tested (IC50:ꢀ>10 µM),13) revealing that this com-
boxylic acid moiety, which is commonly shared among the pound is a highly selective CRTH2 antagonist. Furthermore,

532
Vol. 62, No. 6
Fig. 2. Effect of TASP0412098 (9l) on Antigen-Induced 1AR (A) and LAR (B) in Guinea Pigs
sRaw was measured before, 1min, 2, 4, 5, 6 and 7h after OVA challenge. The results are expressed as IAR (1min after the challenge) (a) and the LAR (AUC of 4–7h
after the challenge; AUC4–7h) (B). Values represent means S.E.M. (n=17–20). Dex, dexamethasone.
inhibitory effects on LAR were observed at 100mg/kg of 9l
(Fig. 2B). The discrepancy between the in vitro functional ac-
tivity (IC50:ꢀ12ꢀnM) and in vivo effects (100mg/kg, oral admin-
istration) may be due to the high protein binding ratio of 9I
(>99.4%ꢀinꢀhumans).ꢀMoreꢀspecificallyꢀtheꢀfreeꢀfractionꢀofꢀ9I
in the plasma at 30mg/kg (Cmax:ꢀ540ꢀnM) might not rise to the
level needed for eliciting the in vivo effects. At 24h after OVA
challenge,ꢀ aꢀ significantꢀ increaseꢀ inꢀ theꢀ totalꢀ numberꢀ ofꢀ cellsꢀ
and the number of macrophages, eosinophils, and neutrophils
wasꢀ observedꢀ inꢀ theꢀ bronchoalveolarꢀ lavageꢀ fluidꢀ (BALF).ꢀ
Compound 9lꢀ significantlyꢀ inhibitedꢀ theꢀ migrationꢀ ofꢀ eosino-
phils into the BALF in a dose-dependent manner (30, 100mg/
kg),ꢀ andꢀ significantꢀ inhibitoryꢀ effectsꢀ wereꢀ observedꢀ afterꢀ
oral administration at 100mg/kg (Fig. 3). These data suggest
that CRTH2 has an important role in bronchial asthma, and
compound 9l may be useful as therapeutic drug for bronchial
asthma.
Conclusion
In our continuing research focused on identifying an alter-
Fig. 3. Effect of TASP0412098 (9l) on Antigen-Induced Increase in native lead class, novel CRTH2 antagonists bearing a methy-
Eosinophil Counts in the BALF of Guinea-Pig
lene linker in place of the carbonyl linker were designed and
Values represent means S.E.M. (n=17–20). Dex, dexamethasone.
synthesized. These compounds were expected to change the
biologicalꢀ profileꢀ ofꢀ thisꢀ class.ꢀ Asꢀ aꢀ resultꢀ ofꢀ SARꢀ studiesꢀ ofꢀ
9l was effective in chemotaxis assay (IC50=23nM). A mouse the methylene linker isoquinoline CRTH2 antagonists, we
pharmacokinetic (PK) study of 1 and 9l indicated that 9l had identifiedꢀ theꢀ potent,ꢀ selective,ꢀ andꢀ orallyꢀ activeꢀ CRTH2ꢀ an-
a longer half-life and higher exposure than 1. In addition, a tagonist 9l. A pharmacological study revealed that 9l showed
guinea pig PK study suggested that 9l gave acceptable plasma anti-asthma effect in the bronchial asthma model using guinea
exposure for an in vivo pharmacological study (Table 5).
pigs at 100mg/kg, suggesting that CRTH2 has an important
Given the favorable in vitroꢀprofileꢀofꢀ9l and acceptable PK role in bronchial asthma. Based on these results, compound 9l
profile,ꢀ compoundꢀ 9l was assessed in the ovalbumin (OVA)- (TASP0412098) is a promising lead compound that warrants
induced asthmatic model. Inhalational OVA challenge led to further preclinical development.
a two-phase airway response at 1min [immediate airway re-
sponse (IAR)] and at 4–7h [late airway response (LAR)] after
the OVA challenge in OVA-sensitized guinea pigs, compared
Experimental
Chemistry All starting materials and reagents were com-
with the airway response obtained from OVA-sensitized and mercialꢀ productsꢀ thatꢀ wereꢀ usedꢀ withoutꢀ furtherꢀ purification.ꢀ
saline-challenged guinea pigs. Inhibitory effects on IAR were The reaction progresses were usually monitored by TLC
not observed at any dose of 9l (Fig. 2A), while the remarkable using Merck silica gel 60F254ꢀ platesꢀ orꢀ Fujiꢀ Silysiaꢀ chroma-

June 2014
533
torex NH plates. Column chromatography was performed 2.0M of TMSCHN2 in CH2Cl2 (29.5mL, 59.0mmol) at 0°C,
using silica gel Wako Pure Chemical Industries, Ltd. C-200 and the resulting mixture was stirred for 3h. Then the reac-
andꢀ NH-silicaꢀ gelꢀ Fujiꢀ Silysiaꢀ chromatorexꢀ DM1020.ꢀ Meltingꢀ tion mixture was concentrated under reduced pressure. To a
points were determined on a Mettler FP-61 or a Yanaco MP- solution of the residue in a mixture of 1,4-dioxane (133mL)
1
500D melting point apparatus and were uncorrected. HNMR and H2O (133mL) was added silver acetate (2.95g, 17.6mmol),
spectra were recorded on a Varian Gemini-600 instrument at and the mixture was stirred at 60°C for 45min. After cooling,
600MHz. Chemical shifts were reported in parts per million the mixture was extracted with EtOAc, and the organic layer
as δ units relative to tetramethylsilane as an internal reference. was washed with brine, dried over MgSO4 and concentrated
Multiplicityꢀ wasꢀ definedꢀ asꢀ sꢀ (singlet),ꢀ dꢀ (doublet),ꢀ tꢀ (triplet),ꢀ under reduced pressure. The resulting residue was dissolved
q (quartet), dd (double doublet), m (multiplet), or brs (broad in MeOH (265mL), and a 2.0M of TMSCHN2 in CH2Cl2
singlet). Mass spectra (MS) were recorded on a Shimadzu (44.2mL, 88.4mmol) was added to this mixture. After being
LCMS-2010EV mass spectrometer with electrospray ioniza- stirred for 20min, the reaction was quenched by adding a few
tion (ESI)/atmospheric pressure chemical ionization (APCI) drops of AcOH. The mixture was diluted with EtOAc and
dual source. Elemental analyses were performed on a Yanaco the organic layer was washed with brine, dried over MgSO4
MT-6 elemental analyzer, and the results were within 0.4% and concentrated under reduced pressure. The residue was
of the calculated values.
Tributyl-[[4-[(2-methylpropan-2-yl)oxycarbonyl]phenyl]- with 10–40% EtOAc/hexane to give 6 (4.07g, 35% from 5)
methyl]phosphanium Bromide (3) To
solution of asꢀ brownꢀ oil:ꢀ 1H-NMR (CDCl3) δ:ꢀ 1.50ꢀ (s,ꢀ 9H),ꢀ 4.76ꢀ (s,ꢀ 2H),ꢀ
tert-butyl 4-(bromomethyl)benzoate (22.1g, 81.6mmol) in 7.38–8.99 (m, 9H); MS (ESI/APCI Dual) m/z 392 (M+H)+.
toluene (440mL) was added tri-n-butyl phosphine (30.5mL,
4-[[4-(2-Methoxy-2-oxoethyl)isoquinolin-1-yl]methyl]ben-
purifiedꢀ byꢀ silicaꢀ gelꢀ columnꢀ chromatographyꢀ (60N)ꢀ elutingꢀ
a
121mmol), and the mixture was stirred at 60°C for 70min. zoic Acid (7) To a solution of 6 (4.07g, 10.4mmol) in CHCl3
The reaction mixture was evaporated and the residue was (40mL) was added TFA (20mL) at 0°C, and the mixture was
added n-hexane. The mixture was stirred and the result- stirred at room temperature for 3.5h. The reaction mixture
ingꢀ precipitateꢀ wasꢀ collectedꢀ byꢀ filtration,ꢀ andꢀ driedꢀ toꢀ yieldꢀ wasꢀ basifiedꢀ withꢀ 1ꢀN NaOH and extracted with EtOAc. The
3ꢀ (36.0ꢀg,ꢀ 93%)ꢀ asꢀ aꢀ colorlessꢀ powder:ꢀ 1H-NMR (CDCl3) δ:ꢀ organic layer was washed with brine, dried over MgSO4 and
0.93–0.98 (m, 9H), 1.44–1.52 (m, 12H), 1.60 (s, 9H), 2.37–2.46 concentrated under reduced pressure to give 7 (3.42g, 98%)
(m, 6H), 4.34–4.40 (m, 2H), 7.50–8.02 (m, 4H); MS (ESI/ as a pale brown powder. This compound was used for the next
APCI Dual) m/z 393 (M+H)+.
reactionꢀwithoutꢀfurtherꢀpurification:ꢀ1H-NMR (CDCl3) δ:ꢀ3.62ꢀ
Methyl 1-[[4-[(2-Methylpropan-2-yl)oxycarbonyl]phenyl]- (s, 3H), 4.13 (s, 2H), 4.71 (s, 2H), 7.39–8.36 (m, 8H), 8.37 (s,
methyl]isoquinoline-4-carboxylate (4) A 1.9M of NaHMDS 1H), 12.79 (brs, 1H); MS (ESI/APCI Dual) m/z 336 (M+H)+.
in THF (2.60mL, 2.60mmol) was added to a solution of 3
(33.8g, 71.4mmol) and methyl 1-chlorosioquinoline-4-car- yl]methyl]isoquinolin-4-yl]acetate (8l) To
Methyl 2-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phen-
suspension
a
boxylate (11.3g, 60.0mmol) in THF (235mL) at −30°C, and of 7 (2.69g, 8.02mmol) in CHCl3 (40mL) was added oxalyl
the mixture was stirred for 15min and at room temperature chloride (1.07mL, 12.0mmol) and DMF (3 drop) at 0°C,
for 45min. After stirring 50°C for 75min, to the mixture was and the mixture was stirred for 1h. After evaporation of the
added a sodium carbonate solution (10.8g in 120mL) and the volatile in the reaction mixture, the residue was dissolved in
mixture was stirred at 60°C for 2h. The reaction was acidi- CHCl3 (40mL). To this mixture was added 2-(4-chlorophenyl)-
fiedꢀ(pH=5) by adding 1N HCl, and the mixture was extracted ethylamine (1.68mL, 12.0mmol) and pyridine (0.973mL,
with EtOAc. The organic layer was dried over MgSO4, and 12.0mmol) at 0°C, and the resulting mixture was stirred for
concentrated under reduced pressure. The residue was puri- 1h. Then the reaction was quenched by adding a saturated am-
fiedꢀ byꢀ silicaꢀ gelꢀ columnꢀ chromatographyꢀ (OH)ꢀ elutingꢀ withꢀ monium chloride solution, and the mixture was extracted with
10–30% EtOAc/hexane to give 4ꢀ (11.1ꢀg,ꢀ 58%)ꢀ asꢀ orangeꢀ oil:ꢀ EtOAc. The organic layer was dried over MgSO4, and con-
1H-NMR (CDCl3) δ:ꢀ 1.55ꢀ (s,ꢀ 9H),ꢀ 4.02ꢀ (s,ꢀ 3H),ꢀ 4.76ꢀ (s,ꢀ 2H),ꢀ centratedꢀunderꢀreducedꢀpressure.ꢀTheꢀresidueꢀwasꢀpurifiedꢀbyꢀ
7.27–8.98 (m, 8H), 9.14 (s, 1H); MS (ESI/APCI Dual) m/z 378 silica gel column chromatography (NH) eluting with 10–100%
(M+H)+.
EtOAc/hexane to give 8lꢀ(1.31ꢀg,ꢀ35%)ꢀasꢀaꢀpaleꢀyellowꢀpowder:ꢀ
1-[[4-[(2-Methylpropan-2-yl)oxycarbonyl]phenyl]methyl]- 1H-NMR (CDCl3) δ:ꢀ2.87ꢀ(t,ꢀJ=6.9Hz, 2H), 3.61–3.67 (m, 2H),
isoquinoline-4-carboxylic Acid (5) To a solution of 4 3.70 (s, 3H), 4.01 (s, 2H), 4.68 (s, 2H), 6.04 (t, J=5.3Hz, 1H),
(11.8g, 29.5mmol) in THF (150mL) was added 1N NaOH 7.11–8.13 (m, 12H), 8.40 (s, 1H); MS (ESI/APCI Dual) m/z 473
(150mL), and the mixture was stirred at 50°C for 2.5h. The (M+H)+.
mixtureꢀwasꢀacidifiedꢀwithꢀ1ꢀN HCl, and extracted with EtOAc.
2-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phenyl]-
The organic layer was dried over MgSO4, and concentrated methyl]isoquinolin-4-yl]acetic Acid (9l) To a solution of
to give 5ꢀ (11.1ꢀg,ꢀ quant)ꢀ asꢀ aꢀ paleꢀ orangeꢀ powder:ꢀ 1H-NMR 8l (1.31g, 2.77mmol) in THF (55mL) was added 1N NaOH
(CDCl3) δ:ꢀ 1.50ꢀ (s,ꢀ 9H),ꢀ 4.76ꢀ (s,ꢀ 2H),ꢀ 7.38–8.99ꢀ (m,ꢀ 9H);ꢀ MSꢀ (14mL) at 0°C, and the mixture was stirred for 2.5h at room
(ESI/APCI Dual) m/z 364 (M+H)+.
temperature.ꢀ Theꢀ mixtureꢀ wasꢀ acidifiedꢀ withꢀ dilutedꢀ hydro-
tert-Butyl 4-[[4-(2-methoxy-2-oxoethyl)isoquinolin-1-yl]- chloric acid and extracted with EtOAc. The organic layer was
methyl]benzoate (6) To a suspension of 5 (11.1g, 29.5mmol) dried over MgSO4 and concentrated under reduced pressure.
in CHCl3 (265mL) was added oxalyl chloride (5.1mL, Theꢀresidueꢀwasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ
59.0mmol) and DMF (1 drop) at 0°C, and the mixture was (OH) eluting with 0–15% CHCl3/MeOH. The resulting pow-
stirred for 70min. After evaporation of the volatile in the re- der was added ethanol (250mL) and the mixture was stirred
action mixture, the residue was dissolved in a mixture of THF at room temperature for 17h. Then the resulting precipitate
(133mL) and CH3CN (133mL). To this mixture was added a wasꢀ collectedꢀ byꢀ filtration,ꢀ andꢀ driedꢀ toꢀ yieldꢀ 9l (944mg,

534
Vol. 62, No. 6
74%)ꢀ asꢀ aꢀ paleꢀ yellowꢀ powder:ꢀ mpꢀ 184.0–185.0°C;ꢀ 1H-NMR 197.0–199.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.80ꢀ (t,ꢀ J=7.3Hz,
(DMSO-d6) δ:ꢀ 2.79ꢀ (t,ꢀ J=7.3Hz, 2H), 3.39–3.46 (m, 2H), 2H), 3.41–3.46 (m, 2H), 4.02 (s, 2H), 4.68 (s, 2H), 7.16–8.45
4.00 (s, 2H), 4.67 (s, 2H), 7.20–8.43 (m, 14H), 12.59 (brs, (m, 15H), 12.52 (brs, 1H); MS (ESI/APCI Dual) m/z 425 (M+
1H); MS (ESI/APCI Dual) m/z 459 (M+H)+; Anal. Calcd for H)+; Anal. Calcd for C27H24N2O3·0.5H2O:ꢀC,ꢀ74.81;ꢀH,ꢀ5.81;ꢀN,ꢀ
C27H23ClN2O3·0.6H2O:ꢀ C,ꢀ 69.04;ꢀ H,ꢀ 5.19;ꢀ N,ꢀ 5.96.ꢀ Found:ꢀ C,ꢀ 6.46.ꢀFound:ꢀC,ꢀ75.10;ꢀH,ꢀ5.80;ꢀN,ꢀ6.36.
69.01; H, 5.17; N, 5.97.
2-[1-[[4-(2-Pyridin-4-ylethylcarbamoyl)phenyl]meth-
Compound 9a–9u were obtained according to the procedure yl]isoquinolin-4-yl]acetic Acid (9i) Colorless powder;
of 9l.
1H-NMR (DMSO-d6) δ:ꢀ 2.80–2.86ꢀ (m,ꢀ 2H),ꢀ 3.45–3.51ꢀ (m,ꢀ
2-[1-[[4-[(3,4-Dichlorobenzoyl)amino]phenyl]methyl]- 2H), 3.69 (brs, 2H), 4.63 (s, 2H), 7.19–8.48 (m, 14H); MS
isoquinolin-4-yl]acetic Acid (9a) Colorless powder; mp (ESI/APCI Dual) m/z 426 (M+H)+; HR-MS m/z Calcd for
145.0–152.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 4.00ꢀ (s,ꢀ 2H),ꢀ 4.60ꢀ C26H23N3O3 (M+H)+,ꢀ426.1812.ꢀFound:ꢀ426.1782.
(s, 2H), 7.24–7.32 (m, 2H), 7.58–7.67 (m, 3H), 7.73–7.83 (m,
2-[1-[[4-[2-(2-Chlorophenyl)ethylcarbamoyl]phenyl]-
2H), 7.87–7.92 (m, 1H), 7.94–8.00 (m, 1H), 8.14–8.19 (m, methyl]isoquinolin-4-yl]acetic Acid (9j) Pale yellow pow-
1
1H), 8.29–8.37 (m, 2H), 10.31 (brs, 1H), 12.59 (brs, 1H); der; mp 181.0–183.0°C; H-NMR (DMSO-d6) δ:ꢀ2.91–2.97ꢀ(m,ꢀ
MS (ESI/APCI Dual) m/z 465 (M+H)+; Anal. Calcd for 2H), 3.44–3.50 (m, 2H), 4.01 (s, 2H), 4.67 (s, 2H), 7.21–8.49
C25H18Cl2N2O3·1.0H2O:ꢀ C,ꢀ 62.12;ꢀ H,ꢀ 4.17;ꢀ N,ꢀ 5.80.ꢀ Found:ꢀ C,ꢀ (m, 14H), 12.52 (brs, 1H); MS (ESI/APCI Dual) m/z 459 (M+
61.17; H, 4.12; N, 5.67.
2-[1-[[4-(Methylcarbamoyl)phenyl]methyl]isoquinolin- N,ꢀ5.81.ꢀFound:ꢀC,ꢀ69.15;ꢀH,ꢀ5.33;ꢀN,ꢀ5.81.
H)+; Anal. Calcd for C27H23ClN2O3·0.5H2O:ꢀC,ꢀ69.30;ꢀH,ꢀ5.33;ꢀ
4-yl]acetic Acid (9b) Pale yellow powder; mp 219.0–221.0°C;
2-[1-[[4-[2-(3-Chlorophenyl)ethylcarbamoyl]phenyl]-
1H-NMR (DMSO-d6) δ:ꢀ2.73ꢀ (d,ꢀ J=4.6Hz, 3H), 4.00 (s, 2H), methyl]isoquinolin-4-yl]acetic Acid (9k) Pale yellow pow-
4.67 (s, 2H), 7.32–8.38 (m, 10H), 12.55 (brs, 1H); MS (ESI/ der; mp 121.0–123.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.82ꢀ (t,ꢀ
APCI Dual) m/z 335 (M+H)+; high resolution (HR)-MS m/z J=7.1Hz, 2H), 3.42–3.48 (m, 2H), 4.01 (s, 2H), 4.67 (s, 2H),
Calcd for C20H18N2O3 (M+Na)+,ꢀ357.1210.ꢀFound:ꢀ357.1199.
7.14–8.45 (m, 14H), 12.54 (brs, 1H); MS (ESI/APCI Dual) m/z
2-[1-[[4-(2-Cyclopropylethylcarbamoyl)phenyl]methyl]- 459 (M+H)+; Anal. Calcd for C27H23ClN2O3·0.4H2O:ꢀC,ꢀ69.57;ꢀ
isoquinolin-4-yl]acetic Acid (9c) Pale yellow powder; mp H,ꢀ5.15;ꢀN,ꢀ6.01.ꢀFound:ꢀC,ꢀ69.66;ꢀH,ꢀ5.27;ꢀN,ꢀ5.81.
188.5–189.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 0.00–0.06ꢀ (m,ꢀ 2H),ꢀ
2-[1-[[4-[2-(4-Methylphenyl)ethylcarbamoyl]phenyl]meth-
0.34–0.42 (m, 2H), 0.64–0.74 (m, 1H), 1.33–1.44 (m, 2H), yl]isoquinolin-4-yl]acetic Acid (9m) Pale yellow powder;
1
3.23–3.32 (m, 2H), 4.00 (s, 2H), 4.67 (s, 2H), 7.32–8.40 (m, mp 121.0–124.0°C; H-NMR (DMSO-d6) δ:ꢀ2.24ꢀ(s,ꢀ3H),ꢀ2.75ꢀ
10H), 12.57 (brs, 1H); MS (ESI/APCI Dual) m/z 389 (M+H)+; (t, J=7.6Hz, 2H), 3.36–3.43 (m, 2H), 4.19 (s, 2H), 4.90 (s, 2H),
Anal. Calcd for C24H24N2O3·0.3H2O:ꢀC,ꢀ73.19;ꢀH,ꢀ6.30;ꢀN,ꢀ7.11.ꢀ 7.05–8.58 (m, 14H); MS (ESI/APCI Dual) m/z 439 (M+H)+;
Found:ꢀC,ꢀ73.03;ꢀH,ꢀ6.13;ꢀN,ꢀ6.98.
2-[1-[[4-(2-Cyclohexylethylcarbamoyl)phenyl]methyl]iso- 439.1974.
HR-MS m/z Calcd for C28H26N2O3 (M+H)+,ꢀ439.2016.ꢀFound:ꢀ
quinolin-4-yl]acetic Acid (9d) Pale yellow amorphous; mp
2-[1-[[4-[2-[4-(Dimethylamino)phenyl]ethylcarbamoyl]-
1
176.5.0–177.0°C; H-NMR (DMSO-d6) δ:ꢀ 0.82–1.72ꢀ (m,ꢀ 13H),ꢀ phenyl]methyl]isoquinolin-4-yl]acetic Acid (9n) Pale yel-
3.20–3.26 (m, 2H), 4.01 (s, 2H), 4.67 (s, 2H), 7.34–8.39 (m, low powder; mp 187.0–188.0°C; 1H-NMR (DMSO-d6) δ:ꢀ
10H), 12.52 (brs, 1H); MS (ESI/APCI Dual) m/z 431 (M+H)+; 2.67 (t, J=7.6Hz, 2H), 2.83 (s, 6H), 3.32–3.38 (m, 2H), 4.00
HR-MS m/z Calcd for C27H30N2O3 (M+H)+,ꢀ431.2329.ꢀFound:ꢀ (s, 2H), 4.67 (s, 2H), 6.62–8.40 (m, 14H), 12.57 (brs, 1H);
431.2309.
MS (ESI/APCI Dual) m/z 468 (M+H)+; Anal. Calcd for
2-[1-[[4-(2-Cyclohexylethylcarbamoyl)phenyl]methyl]- C29H29N3O3·0.5H2O:ꢀ C,ꢀ 73.09;ꢀ H,ꢀ 6.34;ꢀ N,ꢀ 8.82.ꢀ Found:ꢀ C,ꢀ
isoquinolin-4-yl]acetic Acid (9e) Pale yellow powder; mp 73.00; H, 6.19; N, 8.76.
1
140.0–142.0°C; H-NMR (DMSO-d6) δ:ꢀ 1.24–1.70ꢀ (m,ꢀ 14H),ꢀ
2-[1-[[4-[2-(4-Methoxyphenyl)ethylcarbamoyl]phenyl]-
1.87–1.95 (m, 3H), 3.18–3.26 (m, 2H), 4.00 (s, 2H), 4.67 (s, methyl]isoquinolin-4-yl]acetic Acid (9o) Colorless powder;
2H), 7.32–8.37 (m, 10H), 12.57 (brs, 1H); MS (ESI/APCI mp 172.0–173.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.70–2.76ꢀ (m,ꢀ
Dual) m/z 483 (M+H)+; Anal. Calcd for C31H34N2O3:ꢀC,ꢀ77.15;ꢀ 2H), 3.37–3.42 (m, 2H), 3.70 (s, 3H), 4.01 (s, 2H), 4.67 (s, 2H),
H,ꢀ7.10;ꢀN,ꢀ5.80.ꢀFound:ꢀC,ꢀ76.17;ꢀH,ꢀ7.04;ꢀN,ꢀ5.72.
6.79–8.42 (m, 14H), 12.53 (brs, 1H); MS (ESI/APCI Dual) m/z
2-[1-[[4-[2-(Oxan-4-yl)ethylcarbamoyl]phenyl]methyl]- 477 (M+H)+; Anal. Calcd for C28H26N2O4·0.4H2O:ꢀ C,ꢀ 72.84;ꢀ
isoquinolin-4-yl]acetic Acid (9f) Pale yellow powder; mp H,ꢀ5.85;ꢀN,ꢀ6.07.ꢀFound:ꢀC,ꢀ72.88;ꢀH,ꢀ5.67;ꢀN,ꢀ6.01.
197.5–198.5°C; 1H-NMR (DMSO-d6) δ:ꢀ 1.07–1.61ꢀ (m,ꢀ 7H),ꢀ
2-[1-[[4-[2-(4-Hydroxyphenyl)ethylcarbamoyl]phenyl]-
3.20–3.28 (m, 4H), 3.77–3.83 (m, 2H), 4.01 (s, 2H), 4.67 (s, methyl]isoquinolin-4-yl]acetic Acid (9p) Pale yellow pow-
2H), 7.35–8.37 (m, 10H), 12.57 (brs, 1H); MS (ESI/APCI der; mp 145.0–150.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.67ꢀ (t,ꢀ
Dual) m/z 433 (M+H)+; Anal. Calcd for C26H28N2O4 ·0.5H2O:ꢀ J=7.6Hz, 2H), 3.34–3.39 (m, 2H), 4.00 (s, 2H), 4.67 (s,
C,ꢀ70.73;ꢀH,ꢀ6.62;ꢀN,ꢀ6.34.ꢀFound:ꢀC,ꢀ70.61;ꢀH,ꢀ6.47;ꢀN,ꢀ6.13.
2H), 6.63–8.41 (m, 14H), 9.15 (brs, 1H), 12.54 (brs, 1H);
2-[1-[[4-(2-Morpholin-4-ylethylcarbamoyl)phenyl]meth- MS (ESI/APCI Dual) m/z 441 (M+H)+; Anal. Calcd for
yl]isoquinolin-4-yl]acetic Acid (9g) Colorless powder; mp C27H24N2O4·0.8H2O·0.1Et2O:ꢀ C,ꢀ 71.18;ꢀ H,ꢀ 5.80;ꢀ N,ꢀ 6.06.ꢀ
130.0–131.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.36–2.43ꢀ (m,ꢀ 6H),ꢀ Found:ꢀC,ꢀ71.54;ꢀH,ꢀ5.86;ꢀN,ꢀ5.69.
2.60–2.62 (m, 2H), 3.54 (s, 4H), 3.95 (brs, 2H), 4.67 (s, 2H),
2-[1-[[4-[2-(4-Methylsulphonylphenyl)ethylcarbamoyl]-
7.32–8.36 (m, 10H), 12.55 (brs, 1H); MS (ESI/APCI Dual) phenyl]methyl]isoquinolin-4-yl]acetic Acid (9q) Pale yel-
m/z 434 (M+H)+; HR-MS m/z Calcd for C25H27N3O4 (M+H)+, low powder; mp 121.5–122.5°C; 1H-NMR (DMSO-d6) δ:ꢀ
434.2074.ꢀFound:ꢀ434.2065.
2.89–2.95 (m, 2H), 3.17 (s, 3H), 3.45–3.51 (m, 2H), 4.00
2-[1-[[4-(2-Phenylethylcarbamoyl)phenyl]methyl]iso- (s, 2H), 4.67 (s, 2H), 7.35–8.47 (m, 14H), 12.56 (brs, 1H);
quinolin-4-yl]acetic Acid (9h) Pale yellow powder; mp MS (ESI/APCI Dual) m/z 503 (M+H)+; Anal. Calcd for

June 2014
535
C28H26N2O5S·1.2H2O:ꢀ C,ꢀ 64.16;ꢀ H,ꢀ 5.46;ꢀ N,ꢀ 5.34.ꢀ Found:ꢀ C,ꢀ diluted with CHCl3 and MeOH, washed with H2O, dried over
64.17; H, 5.33; N, 5.21. MgSO4 and concentrated under reduced pressure. The residue
4-[2-[[4-[[4-(Carboxymethyl)isoquinolin-1-yl]methyl]ben- wasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(60N)ꢀelut-
zoyl]amino]ethyl]benzoic Acid (9r) Pale yellow powder; ing with 10–20% CHCl3/MeOH to give triethylamine salt of
1
mp 198.0–200.5°C; H-NMR (DMSO-d6) δ:ꢀ2.84ꢀ(t,ꢀJ=7.1Hz, 9w. The salt was dissolved with CHCl3 and MeOH, and the
2H), 3.41–3.46 (m, 2H), 3.98 (s, 2H), 4.64 (s, 2H), 7.28–8.43 solution was washed with 0.5N HCl aq and H2O, dried over
(m, 14H), 12.62 (brs, 1H); MS (ESI/APCI Dual) m/z 469 (M+ MgSO4 and concentrated under reduced pressure to give
H)+; Anal. Calcd for C28H24N2O5·0.8H2O:ꢀ C,ꢀ 69.64;ꢀ H,ꢀ 5.34;ꢀ 9wꢀ (3.1ꢀmg,ꢀ 14%)ꢀ asꢀ aꢀ colorlessꢀ powder:ꢀ mpꢀ 237.0–239.0°C;ꢀ
N,ꢀ5.80.ꢀFound:ꢀC,ꢀ69.71;ꢀH,ꢀ5.29;ꢀN,ꢀ5.71.
1H-NMR (DMSO-d6) δ:ꢀ2.77–2.82ꢀ(m,ꢀ2H),ꢀ3.40–3.46ꢀ(m,ꢀ2H),ꢀ
2-[1-[[4-[(4-Chlorophenyl)methylcarbamoyl]phenyl]meth- 4.69 (s, 2H), 4.68 (s, 2H), 7.21–8.45 (m, 14H); MS (ESI/APCI
yl]isoquinolin-4-yl]acetic Acid (9s) Colorless powder; mp Dual) m/z 483 (M+H)+; HR-MS m/z Calcd for C27H23ClN6O
1
187.5–188.5°C; H-NMR (DMSO-d6) δ:ꢀ3.98ꢀ(s,ꢀ2H),ꢀ4.37–4.41ꢀ (M+H)+,ꢀ483.1695.ꢀFound:ꢀ483.1694.
(m, 2H), 4.65 (s, 2H), 7.24–8.94 (m, 14H), 12.33 (brs, 1H);
MS (ESI/APCI Dual) m/z 445 (M+H)+; HR-MS m/z Calcd for lin-1-yl]methyl]benzoate (dl-11) To a solution of 6 (500mg,
C26H21ClN2O3 (M+Na)+,ꢀ467.1133.ꢀFound:ꢀ467.1125.
1.28mmol) in THF (5mL) was added potassium tert-butoxide
tert-Butyl 4-[[4-(1-Methoxy-1-oxopropan-2-yl)isoquino-
2-[1-[[4-[3-(4-Chlorophenyl)propylcarbamoyl]phenyl]- (150mg, 1.34mmol) at −30°C and stirred for 5min. Then the
methyl]isoquinolin-4-yl]acetic Acid (9t) Pale yellow pow- mixture was added iodomethane (0.318mL, 5.10mmol) and
der; mp 124.0–127.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 1.74–1.80ꢀ stirred for 3h. The reaction was quenched by adding a satu-
(m, 2H), 2.56–2.61 (m, 2H), 3.19–3.25 (m, 2H), 4.01 (s, rated ammonium chloride solution, and the mixture was ex-
2H), 4.67 (s, 2H), 7.21–8.37 (m, 14H), 12.54 (brs, 1H); tracted with EtOAc. The organic layer was dried over MgSO4
MS (ESI/APCI Dual) m/z 473 (M+H)+; Anal. Calcd for and concentrated under reduced pressure. The residue was pu-
C28H25ClN2O3·0.2H2O:ꢀ C,ꢀ 70.57;ꢀ H,ꢀ 5.37;ꢀ N,ꢀ 5.88.ꢀ Found:ꢀ C,ꢀ rifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(OH)ꢀelutingꢀwithꢀ
70.41; H, 5.43; N, 5.78.
10–20% EtOAc/hexane to give dl-11 (319mg, 61%) as a yellow
2-[1-[[4-[2-(4-Chlorophenoxy)ethylcarbamoyl]phenyl]- oil:ꢀ 1H-NMR (CDCl3) δ:ꢀ 1.55ꢀ (s,ꢀ 9H),ꢀ 1.70ꢀ (d,ꢀ J=7.0Hz, 3H),
methyl]isoquinolin-4-yl]acetic Acid (9u) Pale yellow pow- 3.67 (s, 3H), 4.36 (q, J=7.0Hz, 1H), 4.70 (s, 2H), 7.30–8.14 (m,
der; mp 116.0–118.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 3.52–3.57ꢀ 8H), 8.45 (s, 1H); MS (ESI/APCI Dual) m/z 406 (M+H)+.
(m, 2H), 3.97 (s, 2H), 4.03 (t, J=6.0Hz, 2H), 4.64 (s, 2H),
4-[[4-(1-Methoxy-1-oxopropan-2-yl)isoquinolin-1-yl]-
6.90–8.56 (m, 14H), 12.54 (brs, 1H); MS (ESI/APCI Dual) methyl]benzoic Acid (dl-12) To a solution of dl-11 (319mg,
m/z 475 (M+H)+; Anal. Calcd for C27H23ClN2O4·0.4H2O:ꢀ C,ꢀ 0.787mmol) in CHCl3 (3mL) was added TFA (1.5mL) at 0°C,
67.26;ꢀH,ꢀ4.98;ꢀN,ꢀ5.81.ꢀFound:ꢀC,ꢀ67.26;ꢀH,ꢀ5.04;ꢀN,ꢀ5.71.
and the mixture was stirred at room temperature for 2h. The
4-[[4-(2-Amino-2-oxoethyl)isoquinolin-1-yl]methyl]- reactionꢀ mixtureꢀ wasꢀ basifiedꢀ withꢀ 1ꢀN NaOH and extracted
N-[2-(4-chlorophenyl)ethyl]benzamide (9v) To a suspen- with EtOAc. The organic layer was washed with brine, dried
sion of 9l (400mg, 0.872mmol) in THF (9mL) was added over MgSO4 and concentrated under reduced pressure to
carbonyldiimidazole (293mg, 1.74mmol), and the mixture give dl-12 (254mg) as a pale yellow powder. This compound
was stirred for 2h at room temperature. Then the mixture was wasꢀ usedꢀ forꢀ theꢀ nextꢀ reactionꢀ withoutꢀ furtherꢀ purification:ꢀ
added 28% ammonia solution (0.45mL, 6.75mmol) and stirred 1H-NMR (CDCl3) δ:ꢀ1.55ꢀ(s,ꢀ9H),ꢀ1.70ꢀ(d,ꢀJ=7.0Hz, 3H), 3.67
forꢀ 1ꢀh.ꢀ Theꢀ resultingꢀ precipitateꢀ wasꢀ collectedꢀ byꢀ filtration,ꢀ (s, 3H), 4.36 (q, J=7.0Hz, 1H), 4.70 (s, 2H), 7.30–8.14 (m,
and dried to yield 9vꢀ(384ꢀmg,ꢀ96%)ꢀasꢀaꢀcolorlessꢀpowder:ꢀmpꢀ 8H), 8.45 (s, 1H); MS (ESI/APCI Dual) m/z 350 (M+H)+.
265.0–267.0°C; 1H-NMR (DMSO-d6) δ:ꢀ 2.77–2.82ꢀ (m,ꢀ 2H),ꢀ
Methyl 2-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phen-
3.40–3.45 (m, 2H), 3.81 (s, 2H), 4.66 (s, 2H), 7.00–8.43 (m, yl]methyl]isoquinolin-4-yl]propanoate (dl-13) A mixture of
16H); MS (ESI/APCI Dual) m/z 480 (M+H)+; Anal. Calcd for dl-12 (254mg), 2-(4-chlorophenyl)amine (0.132mL, 0.994mmol),
C27H24ClN3O2·0.4H2O:ꢀ C,ꢀ 69.72;ꢀ H,ꢀ 5.37;ꢀ N,ꢀ 9.03.ꢀ Found:ꢀ C,ꢀ WSC (266mg, 0.1.18mmol), HOBT (181mg, 1.18mmol), and
69.41; H, 5.42; N, 9.33.
triethylamine (0.331mL, 2.36) in CHCl3 (5mL) was stirred at
N-[2-(4-Chlorophenyl)ethyl]-4-[[4-(cyanomethyl)iso- room temperature for 12h. The reaction was quenched by add-
quinolin-1-yl]methyl]benzamide (10) To a suspension of 9v ing a saturated ammonium chloride solution, and the mixture
(362mg, 0.790mmol) in DMF (8mL) was added phosphoryl was extracted with EtOAc. The organic layer was dried over
chloride (0.088mL, 0.949mmol), and the mixture was stirred MgSO4, and concentrated under reduced pressure. The residue
for 1h at room temperature. The mixture was poured into ice wasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(OH)ꢀelutingꢀ
water, extracted with EtOAc, dried over MgSO4 and concen- with 10–50% EtOAc/hexane to give dl-13 (289mg, 58%) as a
tratedꢀ underꢀ reducedꢀ pressure.ꢀ Theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ paleꢀyellowꢀamorphous:ꢀ1H-NMR (CDCl3) δ:ꢀ1.70ꢀ(d,ꢀJ=7.2Hz,
silica gel column chromatography (60N) eluting with 2–10% 3H), 2.87 (t, J=6.9Hz, 2H), 3.63–3.66 (m, 2H), 3.67 (s, 3H),
CHCl3/MeOH to give 10 (256mg, 74%) as a pale yellow pow- 4.36 (q, J=7.2Hz, 1H), 4.68 (s, 2H), 6.03 (brs, 1H), 7.12–8.14
der:ꢀ1H-NMR (DMSO-d6) δ:ꢀ2.80ꢀ(t,ꢀJ=7.3Hz, 2H), 3.40–3.47 (m, 12H), 8.45 (s, 1H); MS (ESI/APCI Dual) m/z 487 (M+H)+.
(m, 2H), 4.45 (s, 2H), 4.70 (s, 2H), 7.20–8.44 (m, 13H), 8.49 (s,
2-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phenyl]meth-
yl]isoquinolin-4-yl]propanoic Acid (dl-9x) To a solution
1H); MS (ESI/APCI Dual) m/z 462 (M+Na)+.
N-[2-(4-Chlorophenyl)ethyl]-4-[[4-(1H-tetrazol-5-yl- of dl-13 (110mg, 0.226mmol) in THF (1.5mL) was added 1N
methyl)isoquinolin-1-yl]methyl]benzamide (9w) To a sus- NaOH (1mL), and the mixture was stirred for 17h at room
pension of 10 (20mg, 0.045mmol) in toluene (0.45mL) was temperature.ꢀ Theꢀ mixtureꢀ wasꢀ acidifiedꢀ withꢀ 1ꢀN HCl aq.
added sodium azide (9mg, 0.136mmol) and triethylamine hy- and extracted with AcOEt. The organic layer was dried over
drochlorideꢀ(19ꢀmg,ꢀ0.136ꢀmmol),ꢀandꢀtheꢀmixtureꢀwasꢀrefluxedꢀ MgSO4 and concentrated under reduced pressure. The residue
for 72h. After cooling to room temperature, the mixture was wasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(OH)ꢀelut-

536
Vol. 62, No. 6
ing with 30–75% EtOAc/hexane to give dl-9x (76mg, 71%) as 7.85–7.89 (m, 2H), 7.98–8.02 (m, 1H), 8.10 (d, J=8.3Hz, 1H),
aꢀ colorlessꢀ powder:ꢀ mpꢀ 189.0–190.0°C;ꢀ 1H-NMR (DMSO-d6) 8.37 (s, 1H); MS (ESI/APCI Dual) m/z 420 (M+H)+.
δ:ꢀ 1.55ꢀ (d,ꢀ J=7.3Hz, 3H), 2.77–2.83 (m, 2H), 3.40–3.46 (m,
4-[[4-(3-Ethoxy-3-oxopropyl)isoquinolin-1-yl]methyl]ben-
2H), 4.39 (q, J=7.3Hz, 1H), 4.67 (s, 2H), 7.21–8.44 (m, 14H), zoic Acid (18) To a solution of 17 (110mg, 0.262mmol) in
12.49 (brs, 1H); Anal. Calcd for C28H25ClN2O3 ·0.4H2O:ꢀ C,ꢀ CHCl3 (2mL) was added TFA (1mL) at 0°C, and the mixture
70.04;ꢀH,ꢀ5.42;ꢀN,ꢀ5.83.ꢀFound:ꢀC,ꢀ69.98;ꢀH,ꢀ5.48;ꢀN,ꢀ5.78.
was stirred at room temperature for 1h. The reaction mixture
tert-Butyl 4-[[4-(Hydroxymethyl)isoquinolin-1-yl]methyl]- wasꢀ basifiedꢀ withꢀ 1Nꢀ NaOHꢀ andꢀ extractedꢀ withꢀ EtOAc.ꢀ Theꢀ
benzoate (14) To a solution of 5 (600mg, 1.65mmol) in THF organic layer was washed with brine, dried over MgSO4 and
(17mL) was added BH3-THF (3.30mL, 3.30mmol) at 0°C, concentrated under reduced pressure to give 18 (95mg) as a
and the mixture was stirred at room temperature for 1h. The yellow powder. This compound was used for the next reaction
1
reaction mixture was added MeOH at 0°C and evaporated. withoutꢀ furtherꢀ purification:ꢀ H-NMR (CDCl3) δ:ꢀ 2.70–2.74ꢀ
Theꢀresidueꢀwasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ (m, 3H), 3.25–3.30 (m, 2H), 4.02–4.07 (m, 2H), 4.68 (s, 2H),
(OH)ꢀelutingꢀwithꢀ30–70%ꢀEtOAc/hexaneꢀandꢀthenꢀpurifiedꢀbyꢀ 7.38 (d, J=8.3Hz, 2H), 7.62–7.67 (m, 1H), 7.77–7.84 (m, 3H),
silica gel column chromatography (NH) eluting with 50–70% 8.10 (d, J=8.3Hz, 1H), 8.30–8.33 (m, 2H); MS (ESI/APCI
EtOAc/hexane to give 14 (235mg, 39%) as a pale yellow pow- Dual) m/z 364 (M+H)+.
der:ꢀ 1H-NMR (CDCl3) δ:ꢀ 1.55ꢀ (s,ꢀ 9H),ꢀ 4.69ꢀ (s,ꢀ 2H),ꢀ 5.10ꢀ (s,ꢀ
Ethyl 3-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phen-
2H), 7.27–7.32 (m, 2H), 7.50–7.58 (m, 1H), 7.68–7.76 (m, 1H), yl]methyl]isoquinolin-4-yl]propanoate (19) A mixture of
7.83–7.89 (m, 2H), 8.08–8.20 (m, 2H), 8.47 (s, 1H); MS (ESI/ 18 (95mg), 2-(4-chlorophenyl)amine (0.055mL, 0.393mmol),
APCI Dual) m/z 350 (M+H)+.
WSC (75mg, 0.393mmol), and HOBT (75mg, 0.393mmol) in
tert-Butyl 4-[(4-Formylisoquinolin-1-yl)methyl]benzoate THF (3mL) was stirred at room temperature for 3h. The reac-
(15) To a solution of 14 (235mg, 0.674mmol) in CHCl3 tion mixture was diluted with EtOAc, and washed with H2O
(7mL) was added Dess–Martin periodinane (344mg,, and brine. The organic layer was dried over MgSO4, and con-
0.812mmol) at 0°C, and the mixture was stirred for 1h. The centratedꢀunderꢀreducedꢀpressure.ꢀTheꢀresidueꢀwasꢀpurifiedꢀbyꢀ
reaction mixture was washed with H2O and brine, dried over silica gel column chromatography (OH) eluting with 10–50%
MgSO4 and concentrated under reduced pressure. The residue CHCl3/AcOEt to give 19 (107mg, 82%) as a pale yellow pow-
wasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(OH)ꢀelut- der:ꢀ1H-NMR (CDCl3) δ:ꢀ1.25ꢀ(t,ꢀJ=7.1Hz, 3H), 2.73–2.78 (m,
ing with 20–30% EtOAc/hexane to give 15 (200mg, 85%) as a 2H), 2.87 (t, J=6.9Hz, 2H), 3.34–3.39 (m, 2H), 3.62–3.68 (m,
yellowꢀamorphous:ꢀ1H-NMR (CDCl3) δ:ꢀ1.55ꢀ(9H,ꢀs),ꢀ4.79ꢀ(2H,ꢀ 2H), 4.14–4.19 (m, 2H), 4.67 (s, 2H), 6.02 (brs, 1H), 7.11–7.32
s), 7.32 (d, 2H, J=8.3Hz), 7.62–7.68 (m, 1H), 7.82–7.92 (m, (m, 6H), 7.52–7.59 (m, 3H), 7.68–7.73 (m, 1H), 8.01–8.03 (m,
3H), 8.23 (d, J=8.3Hz, 1H), 8.90 (s, 1H), 9.24–9.28 (m, 1H), 1H), 8.09–8.12 (m, 1H), 8.37 (s, 1H).
10.36 (s, 1H); MS (ESI/APCI Dual) m/z 380 (M+Na)+.
tert-Butyl 4-[[4-[(E)-3-Ethoxy-3-oxoprop-1-enyl]iso- methyl]isoquinolin-4-yl]propanoic Acid (9y) To a solution
3-[1-[[4-[2-(4-Chlorophenyl)ethylcarbamoyl]phenyl]-
quinolin-1-yl]methyl]benzoate (16) To a solution of ethyl of 19 (106mg, 0.214mmol) in THF (4mL) was added 1N
2-(diethoxyphosphoryl)acetate (0.161mL, 0.806mmol) in THF NaOH (2mL), and the mixture was stirred for 6h at room tem-
(4mL) was added sodium hydride (38mg, 0.950mmol) at 0°C, perature.ꢀTheꢀmixtureꢀwasꢀacidifiedꢀwithꢀ1ꢀN HCl aq. and ex-
and the mixture was stirred for 30min. The reaction mixture tracted with EtOAc. The organic layer was dried over MgSO4
was added a solution of 15 (200mg, 0.576mmol) in THF and concentrated under reduced pressure to give 9y (84mg,
1
(2mL) and stirred for 1h. The reaction was quenched by add- 83%)ꢀ asꢀ aꢀ colorlessꢀ powder:ꢀ mpꢀ 208.0–210.0°C;ꢀ H-NMR
ing a saturated ammonium chloride solution, and the mixture (DMSO-d6) δ:ꢀ 2.65ꢀ (t,ꢀ J=7.7Hz, 2H), 2.80 (t, J=7.1Hz,
was extracted with EtOAc. The organic layer was dried over 2H), 3.24 (t, J=7.7Hz, 2H), 3.40–3.46 (m, 2H), 4.65 (s, 2H),
MgSO4, and concentrated under reduced pressure. The residue 7.20–8.43 (m, 14H), 12.23 (brs, 1H); MS (ESI/APCI Dual) m/z
wasꢀpurifiedꢀbyꢀsilicaꢀgelꢀcolumnꢀchromatographyꢀ(NH)ꢀelut- 473 (M+H)+; Anal. Calcd for C28H25ClN2O3·0.2H2O:ꢀC,ꢀ70.57;ꢀ
ing with 20–30% EtOAc/hexane to give 16 (198mg, 82%) as H,ꢀ5.37;ꢀN,ꢀ5.88.ꢀFound:ꢀC,ꢀ70.35;ꢀH,ꢀ5.22;ꢀN,ꢀ5.81.
aꢀpaleꢀyellowꢀoil:ꢀ1H-NMR (CDCl3) δ:ꢀ1.36–1.41ꢀ(m,ꢀ3H),ꢀ1.55ꢀ
In Vitro Pharmacological Studies. Binding Assay
(s, 9H), 4.29–4.36 (m, 2H), 4.73 (s, 2H), 6.56–6.61 (m, 2H), CRTH2 transfected cells were washed and suspended in
7.31 (d, 2 H, J=8.7Hz), 7.55–7.61 (m, 1H), 7.72–7.77 (m, 1H), HBSS containing 10mM HEPES, pH 7.3 (2×105 cells/100µL).
3
7.86–7.91 (m, 2H), 8.12–8.18 (m, 1H), 8.34–8.39 (m, 1H), 8.72 The cells were incubated with 5nM H-PGD2 (GE Healthcare,
(s, 1H); MS (ESI/APCI Dual) m/z 418 (M+H)+.
Little Chalfont, United Kingdom) for 60min on ice. The cells
tert-Butyl 4-[[4-(3-Ethoxy-3-oxopropyl)isoquinolin-1-yl]- inꢀ suspensionꢀ wereꢀ rapidlyꢀ filteredꢀ underꢀ vacuumꢀ throughꢀ
methyl]benzoate (17) To solution of 16 (181mg, aꢀ glassꢀ fiberꢀ filterꢀ plateꢀ GF/Cꢀ (Whatmanꢀ Inc.,ꢀ Clifton,ꢀ NJ,ꢀ
a
0.434mmol) in MeOH (9mL) was added CuCl2 (44mg, U.S.A.) using a cell harvester Filtermate (Packard, Meriden,
0.326mmol) and sodium borohydride (91mg, 2.17mmol) at CT,ꢀ U.S.A.).ꢀ Theꢀ filtersꢀ wereꢀ thenꢀ washedꢀ fourꢀ timesꢀ withꢀ
0°C, and the mixture was stirred for 1h. The reaction mixture ice-cold phosphate buffered saline containing 0.1% bovine
was diluted with EtOAc, and washed with H2O and brine. serum albumin. Bound radio activity was measured with a
The organic layer was dried over MgSO4, and concentrated liquid scintillation counter Top Count NXT (PerkinElmer,
underꢀreducedꢀpressure.ꢀTheꢀresidueꢀwasꢀpurifiedꢀbyꢀsilicaꢀgelꢀ Inc., Boston, MA, U.S.A.) using a liquid scintillation cocktail
column chromatography (OH) eluting with 20–40% EtOAc/ Micro-scinti-O (Packard, Meriden, CT, U.S.A.).
hexane to give 17ꢀ (110ꢀmg,ꢀ 60%)ꢀ asꢀ aꢀ paleꢀ yellowꢀ powder:ꢀ
Intracellular Ca2+ Influx Assay Human CRTH2 trans-
1H-NMR (CDCl3) δ:ꢀ1.23–1.28ꢀ(m,ꢀ3H),ꢀ1.55ꢀ(s,ꢀ9H),ꢀ2.73–2.77ꢀ fectant KB8 cells (BML Co. Kawagoe, Japan) were incu-
(m, 2H), 3.34–3.39 (m, 2H), 4.14–4.20 (m, 2H), 4.68 (s, 2H), bated with 1µM Fluo-4 AM for 30min at 37°C in the dark.
7.30 (d, J=8.3Hz, 2H), 7.50–7.55 (m, 1H), 7.67–7.72 (m, 1H), After incubation, the cells were washed and suspended

June 2014
537
in HBSS containing 10mM HEPES, pH 7.3, 1mM CaCl2
In Vivo Pharmacological Studies. Antigen-Induced
(2×105 cells/100µL). The compound and 100nM of PGD2 were Bronchoconstriction in Guinea Pigs Guinea pigs were
added, and the increase of intracellular Ca2+ concentration sensitized by aerosolized 1% OVA for 10min once daily for
([Ca2+]i) was measured using the functional drug screening 8ꢀ consecutiveꢀ days.ꢀ Oneꢀ weekꢀ afterꢀ theꢀ finalꢀ sensitization,ꢀ
system FDSS6000 (Hamamatsu Photonics, Shizuoka, Japan).
the animals were challenged for 5min with aerosolized 2%
Th2 Cells Migration Assay Human Th2 cells were pre- OVA. TASP0412098 was administered 30min before and 2h
paredꢀ asꢀ follows:ꢀ theꢀ CD4+T lymphocytes separated from after the OVA challenge, and dexamethasone was adminis-
PBMCs using anti-CD4 mAb were stimulated with anti-CD3 teredꢀ 3ꢀhꢀ beforeꢀ theꢀ OVAꢀ challenge.ꢀ Oneꢀ weekꢀ afterꢀ theꢀ firstꢀ
mAb and anti-CD28 mAb in the presence of IL-4 and neu- OVA challenge, animals were challenged OVA again with the
tralizing anti-IFNγ mAb for 3d and, then, expanded by IL-2 same protocol and asthmatic responses were measured using a
and IL-4 for 7d. Th2 cells highly expressing CRTH2 were two-chambered,ꢀdouble-flowꢀplethysmographꢀsystem.ꢀAntigen-
separated with anti-CRTH2 mAb, and 2d after the separation, induced bronchoconstriction was evaluated as the change in
these cells were used in the cell migration assay. Compound- sRaw at 1min after the antigen challenge for the immediate
treated Th2 cells and 100nM solution of PGD2 were applied airway response (IAR), and the area under the change in the
to top and bottom wells of the 5µm-poreꢀfilterꢀChemoꢀTx-96ꢀ sRaw curve (AUC) between 4 and 7h (AUC4–7h) after the anti-
chamber (Neuroprobe, Gaithersburg, MD, U.S.A.), respective- gen challenge for the late airway response (LAR).
ly. After incubation at 37°C for 1h, the number of the cells in
Cell Counts in Bronchoalveolar Lavage Fluid of Guinea
the bottom wells was counted by Burker–Turk hemocytometer. Pigs Twenty-four hours after the last antigen challenge, the
Pharmacokinetics. Animals Male NC/Nga mice (SLC, guineaꢀpigsꢀwereꢀsacrificedꢀbyꢀanesthesiaꢀandꢀrecoveredꢀbron-
Japan) 11–13 weeks old were used in the experiments. All choalveolarꢀ lavageꢀ fluidꢀ (BALF).ꢀ Theꢀ BALFꢀ wasꢀ hemolyzedꢀ
animals were used after acclimation for at least 4d. The mice and cell count per 1mL was taken as the total cell count. The
were given free access to water and atandard laboratory diet slides were prepared and stained using May–Grunwald stain
(MF, Oriental Yeast Co., Japan) during the acclimation period. and Giemza stain. Approximately 300 cells were counted and
Environmental conditions were controlled at a relative humid- the ratios of neutrophils, eosinophils, macrophages and lym-
ity of 50 20% and temperature of 23 3°C.
phocytes relative to the total were calculated.
Male guinea pigs (SLC, Japan) 9–11 weeks old were used in
the experiments after acclimation that described above.
All the studies were reviewed by the Taisho Pharmaceutical
Co., Ltd. Animal Care Committee.
References and Notes
1) Nagata K., Tanaka K., Ogawa K., Kenmotsu K., Imai T., Yoshie
O., Abe H., Tada K., Nakamura M., Sugamura K., Takano S.,
J. Immunol., 162, 1278–1286 (1999).
Plasma Concentrations of 1 in Mice Compound 1 was
orally administered to male NC/Nga mice. The animals were
anesthetizedꢀwithꢀisoflurane,ꢀandꢀbloodꢀsamplesꢀwereꢀcollectedꢀ
from the postcava and stored at −80°C until the bioanalysis.
The plasma concentration of 1 was determined by liquid chro-
matography-tandem mass spectrometry (LC-MS/MS) with
electrospray ionization (ESI). A 50µL aliquot of plasma was
added with 200µL of organic solvent (acetonitrile–methanol
(9ꢀ:ꢀ1,ꢀ v/v))ꢀ containingꢀ anꢀ internalꢀ standardꢀ andꢀ vortex-mixed.ꢀ
After centrifugation, the supernatant (5µL) obtained was
directlyꢀ injectedꢀ intoꢀ aꢀ LC-MS/MSꢀ systemꢀ composedꢀ ofꢀ aꢀ
CTC HTS-PAL autosampler (CTC Analytics AG, Zwingen,
Switzerland), HP1100 binary pump system (Agilent Technolo-
gies, CA, U.S.A.), and an API 3000 tandem mass spectrometer
equipped with a Turbo Ionspray interface (Applied Biosys-
tems, CA, U.S.A.). Chromatographic separation with linear
gradient elution was performed on a Shim-pack XR-ODS
(2.2 µM, 3.0×30mm, Shimadzu) using a 0.1% aqueous solu-
2) Nagata K., Hirai H., Tanaka K., Ogawa K., Aso T., Sugamura K.,
Nakamura M., Takano S., FEBS Lett., 459, 195–199 (1999).
3) Pettipher R., Hansel T. T., Armer R., Nat. Rev. Drug Discov., 6,
313–325 (2007).
4) Hirai H., Tanaka K., Yoshie O., Ogawa K., Kenmotsu K., Takamori
Y., Ichimasa M., Sugamura K., Nakamura M., Takano S., Nagata
K., J. Exp. Med., 193, 255–261 (2001).
5) Monneret G., Gravel S., Diamond M., Rokach J., Powell W. S.,
Blood, 98, 1942–1948 (2001).
6) Xue L., Gyles S. L., Wettey F. R., Gazi L., Townsend E., Hunter M.
G., Pettipher R., J. Immunol., 175, 6531–6536 (2005).
7) Woodward D. F., Jones R. L., Narumiya S., Pharmacol. Rev., 63,
471–538 (2011).
ꢀ 8)ꢀ SchuligoiꢀR.,ꢀ Sturmꢀ E.,ꢀ Luschnigꢀ P.,ꢀ Konyaꢀ V.,ꢀ Philiposeꢀ S.,ꢀ Sedejꢀ
M., Waldhoer M., Peskar B. A., Heinemann A., Pharmacology, 85,
372–382 (2010).
9) Nishikawa-Shimono R., Sekiguchi Y., Koami T., Kawamura M.,
Wakasugi D., Watanabe K., Wakahara S., Matsumoto K., Takayama
T., Bioorg. Med. Chem. Lett., 22, 3305–3310 (2012).
tion of formic acid and acetonitrile. The column temperature 10) Nishikawa-Shimono R., Sekiguchi Y., Koami T., Kawamura M.,
Wakasugi D., Watanabe K., Wakahara S., Kimura K., Yamanobe S.,
Takayama T., Bioorg. Med. Chem., 21, 7674–7685 (2013).
11) Copp F. C., Franzmann K. W., Grundy J., Whalley W. B., J. Chem.
Soc., Perkin Trans. 1, 11, 2455–2462 (1985).
12) Kirmse W., Eur. J. Org. Chem., 14, 2193–2256 (2002).
13) The binding assays for TP, EP1, FP, IP, BLT1, cysLT1, and cysLT2,
andꢀmobileꢀphaseꢀflowꢀrateꢀwereꢀsetꢀatꢀ50°Cꢀandꢀ1.2ꢀmL/min,ꢀ
respectively.ꢀ Approximatelyꢀ 2/7ꢀ ofꢀ theꢀ columnꢀ effluentꢀ wasꢀ
directed to the ion source, and multiple reaction monitoring
(MRM) with negative ion detection was performed. The lower
limitꢀ ofꢀ quantificationꢀ (LLOQ)ꢀ forꢀ mouseꢀ plasmaꢀ wasꢀ 1ꢀng/
mL.
Plasma Concentrations of 9l in Mice and Guinea Pigs
Plasma concentrations of 9l in mice and guinea pigs were
and the enzyme inhibition assays for COX-1 and COX-2 were run
byꢀCerepꢀ(Paris,ꢀFrance)ꢀusingꢀtheꢀprofilerꢀserviceꢀaccordingꢀtoꢀtheꢀ
manufacturer’s procedures.
measured by almost the same method as above.