2-Alkynyl-8-aryladenines possessing an amide moiety: Their synthesis and structure-activity relationships of effects on hepatic glucose production induced via agonism of the A2B adenosine receptor

Bioorganic& Medicinal Chemistry 9 (2001) 2709–2726

2-Alkynyl-8-aryladenines Possessing an Amide Moiety: Their

Synthesis and Structure–Activity Relationships of Eﬀects on

Hepatic Glucose Production Induced Via Agonism of the

A_2BAdenosine Receptor

Hitoshi Harada,* Osamu Asano, Tsutomu Kawata, Takashi Inoue, Tatsuo Horizoe,

Nobuyuki Yasuda, Kaya Nagata, Manabu Murakami, Junsaku Nagaoka,

Seiichi Kobayashi, Isao Tanaka and Shinya Abe

Tsukuba Research Laboratories, Eisai Company, Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan

Received 6 April 2001; accepted 10 June 2001

Abstract—A series of 2-alkynyl-8-aryladenine derivatives bearing an amide moiety at the 9-position of adenine was synthesized.

These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in

primary cultured rat hepatocytes. The m-primary benzamide derivative 15f was the most potent compound (IC₅₀=0.017 mM), being

15-fold more active than the corresponding 9-methyl derivative (1). Compound 15f showed 72- and 5.2-fold selectivity for human

A_2Breceptor versus human A₁and A_2Areceptors, respectively. Structure–activity relationship (SAR) studies of the synthesized

compounds indicated that a three-carbon linker, ﬁxed in the form of a benzene ring, between the adenine core and the amide moiety

is important for both A_2Bantagonistic activity and selectivity. The IC₅₀values in rat hepatocyte glucose assay correlated well with

the IC₅₀values in cAMP assay using Chinese hamster ovary cells stably transfected with human A_2Breceptors (r²=0.94). The A₁

and A_2Aaﬃnities showed no correlation with the potency to inhibit NECA-induced glucose production. These results strongly

support our previous conclusion that adenosine agonist-induced hepatic glucose production in rat hepatocytes is mediated through

Introduction

hepatocytes, and we suggested that these hepatic eﬀects

are mediated through the A_2Breceptor.⁶We have also

shown that one of these compounds has hypoglycemic

activity in genetically diabetic KK-A^ymice.⁶

The purine nucleoside, adenosine, is produced in many

organs and tissues, and has a variety of biological

actions via adenosine receptors.¹So far, four subtypes

of adenosine receptors (i.e., A₁, A_2A, A_2B, and A₃) have

been deﬁned on the basis of pharmacological and

molecular cloning characterization.^2ꢀ5Activation of A₁

and A₃receptors can lead to inhibition of adenylate

cyclase activity, while this activity is stimulated by the

activation of A_2Aand A_2Breceptors.¹

In our previously reported SAR studies, we had ﬁxed

the 9-substituent of the adenine ring as a methyl group,⁶

so we could not examine the role of the substituent at

this position in A_2Bantagonisticpotency and selectivity.

The nonselective adenosine agonist, NECA,⁷is a potent

Recently, we have discovered novel adenosine antago-

nists, 2-alkynyl-8-aryl-9-methyladenine derivatives (1

and its analogues, Chart 1), as candidate antidiabetic

agents by means of structure–activity relationship

(SAR) studies on inhibitory activity towards NECA-

induced glucose production in primary cultured rat

*Corresponding author. Tel.: +81-298-47-5874; fax: +81-298-47-

4012; e-mail: h-harada@hhc.eisai.co.jp

Chart 1. Structure of 2-alkynyl-8-aryl-9-methyladenine derivatives.

PII: S0968-0896(01)00201-2

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

toward NECA-induced glucose production in rat

hepatocytes. The A_2Bantagonisticatcivities of these

compounds were also measured in terms of the inhibi-

tory activities on NECA-induced cyclic AMP accumu-

lation in CHO.K1 cells stably transfected with human

adenosine A_2Breceptor cDNA.^4bMoreover, to examine

the selectivity of our compounds, we tested their aﬃ-

nities for human A₁and A_2Areceptors using a radi-

oligand binding technique.

Results and Discussion

Chemistry

Chart 2. Structure of 2-alkynyl-8-aryladenines possessing an amide

moiety.

A_2Breceptor ligand,^8,9and its 5⁰-amide group has been

found to be favorable for high-aﬃnity binding to the

A_2Breceptor.⁹Therefore, it is of interest to introduce

various substituents bearing an amide moiety at the

9-position of 2-alkynyl-8-aryladenines (Chart 2).

The 2-alkynyl-8-aryl-9-amidoalkyladenine derivatives

(15a and 15b) were synthesized by using 6-chloro-2-

iodo-9-hydroxyalkylpurine derivatives (9a and 9b,

respectively) as key intermediates, as previously repor-

ted in the synthesis of 2-alkynyl derivatives of

NECA.^10,11These intermediates were prepared starting

from N1-(4,6-dichloro-5-nitropyrimidin-2-yl)acetamide

(4),¹²and 4-amino-1-butanol or 3-amino-1-propanol

according to our established method for the synthesis of

2-alkynyl-8-aryl-9-methyladenine derivatives (Scheme 1).⁶

In the present study, as a continuation of our previous

work,⁶we synthesized a series of 2-alkynyl-8-aryladenine

derivatives bearing an amide moiety at the 9-position of

the adenine core, and evaluated their inhibitory activities

Scheme 1. Preparation of 8-aryl-6-chloro-2-iodopurine intermediates. Reagents: (a) R¹NH₂, AcOH/THF(–MeOH); (b) POCl₃, N,N-dimethylani-

.

line, Et₄NCl/CH₃CN; (c) H₂, Raney Ni/MeOH; (d) NaBH₄, SnCl₂2H₂O/EtOH; (e) 3-ﬂuorobenzaldehyde, AcOH/MeOH; (f) FeCl₃/EtOH; (g) HCl/

H₂O–THF; (h) CH₂I₂, isoamyl nitrite, CuI/THF.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2711

Regioselective amination¹³of the 6-position of 9 with

saturated ammonia in MeOH in a sealed tube at 60 ^ꢁC

yielded the 6-amino compound (10). Oxidation of the

hydroxyl group in 10 with RuO₄(prepared from

(Scheme 2).⁶The carbamate and sulfonamide deriva-

tives (15d and 15e) were similarly synthesized starting

from readily available 2-aminoethyl N-ethylcarbamate

(24) and N-ethyl-3-aminopropanesulfonamide (29),

respectively (Schemes 2 and 4).

.

RuO₂H₂O with NaIO₄in a mixture of CH₃CN, CHCl₃,

and H₂O),^11,14followed by esteriﬁcation¹¹with thionyl

chloride in methanol aﬀorded the methyl ester (12).

Palladium-catalyzed cross-coupling reaction^11,15of 12

with terminal alkyne yielded the 2-alkynyl compound

(13). Reaction¹¹of 9 with ethylamine in EtOH–H₂O in a

sealed tube at 80 ^ꢁC followed by treatment with 20%

HCl/EtOH gave the desired compounds (15a and 15b)

as the HCl salts (Scheme 2).

Primary benzamide derivatives (15f and 15g) were

obtained by oxidative hydrolysis¹⁶of the cyano group in

16f and 16g, respectively (Scheme 3). In the synthesis of

these compounds, however, the reaction of 4 with amino-

benzonitrile provided the diarylamino compound, as

well as the desired monoarylamino compound (5f and

5g). Monoarylamino pyrimidines (5f and 5g) were

obtained by replacement of the chlorine atom of N1-(6-

chloro-3,4-dihydro-5-nitro-4-oxopyrimidine-2-yl)aceta-

mide (2)¹²by aminobenzonitrile, followed by chlorina-

tion of the 4-oxo group. Additionally, because of the

sensitivity of the cyano group to Raney nickel hydrogena-

tion, reduction of the nitro groups of 5f and 5g was

achieved by using the sodium borohydride-stannous chlo-

ride system,¹⁷along with deacetylation. Protection of the

2-amino group of pyrimidine with an acetyl group was not

indispensable for ring closure to purine (Scheme 1).

In the synthesis of 15c, since oxidation of the primary

alcohol was unsuccessful, we examined an alternative

route in which the ethylamide moiety was introduced

beforehand. That is, glycine ethylamide (20), readily

prepared from Z-glycine (18) in two steps (Scheme 4),

was reacted with 4 to give 5c in the ﬁrst step (Scheme 1).

With the exception of the palladium-catalyzed cross-

coupling reaction prior to ammonolysis, the following

steps were basically the same as described above

.

Scheme 2. Synthesis of 9-amidoalkylademine derivatives. Reagents: (a) NH₃/MeOH; (b) RuO₂H₂O, NaIO₄/CH₃CN–CHCl₃–H₂O; (c) SOCl₂/

MeOH; (d) 1-ethynyl-1-cyclohexanol, (PPh₃)₂PdCl₂, CuI, Et₃N/DMF; (e) EtNH₂/EtOH–H₂O; (f) HCl/MeOH; (g) 1-ethynyl-1-cyclohexanol,

(PPh₃)₂PdCl₂, CuI, Et₃N/THF; (h) NH₃/EtOH.

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

Scheme 3. Synthesis of 9-(N-ethyl)benzamidoadenine derivatives. Reagents: (a) 1-ethynyl-1-cyclohexanol, (PPh₃)₂PdCl₂, CuI, Et₃N/THF; (b) NH₃/

.

EtOH; (c) NaOH, H₂O₂/CHCl₃–MeOH–H₂O; (d) HCl/MeOH or EtOH; (e) NaOH/CHCl₃–MeOH–H₂O; (f) EtNH₂HCl, Et₃N, WSC, HOBt/DMF.

.

Scheme 4. Preparation of alkylamines possessing an amide moiety. Reagents: (a) EtNH₂HCl, Et₃N, WSC HCl, HOBt/THF; (b) H₂, 10% Pd/C/

.

MeOH; (c) Z-Cl, K₂CO₃/H₂O; (d) EtNCO, pyridine/CH₂Cl₂; (e) EtNH₂HCl, Et₃N/CH₂Cl₂; (f) NaI/2-butanone; (g) NaN₃/MeOH–H₂O.

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2713

The synthesis of N-ethylbenzamide derivatives (15h–j)

was achieved by condensation¹⁸of the carboxyl group

in 17h–j with ethylamine (Scheme 3). Compounds 17h–j

were obtained by alkaline hydrolysis of carboxylic ester

derivatives (16h–j) prepared in a similar manner to that

described for 16f and 16g.

the adenine backbone and the amide group was

decreased (15b, IC₅₀=1.6 mM; 15c, IC₅₀=23 mM). The

optimum length of the alkyl linker for activity appeared

to be three methylene groups. The carbamate derivative

(15d) and sulfonamide derivative (15e) showed signiﬁcant

decreases in activity (IC₅₀=14 and 8.2 mM, respectively).

Our syntheticmethod allowed the introdutcion of a

variety of substituents (substituted alkyl and aromatic

groups) at the 9-position of adenine.

N-Ethylbenzamide derivatives (15h–j) were also synthe-

sized and evaluated in the rat hepatocyte glucose assay.

Among the three regioisomers, the meta-derivative (15i),

with three carbons between the adenine backbone and

the amide group as in 15a, was the most potent

(IC₅₀=0.044 mM). Moving the amide group of 15i from

the meta- to the para- or the ortho-position markedly

decreased the activity (15j, IC₅₀=0.52 mM; 15h,

IC₅₀>30 mM). The primary benzamide analogue of 15i

(15f) displayed more potent inhibitory activity than its

parent compound (15f, IC₅₀=0.017 mM; 15i,

IC₅₀=0.044 mM). Movement of the amide group from

the meta- to the para-position diminished the potency

(15g, IC₅₀=0.098 mM; 15f, IC₅₀=0.017 mM), but this

tendency was not as marked as that observed in the N-

ethylbenzamide derivatives. This may be attributed to

the existence of steric limitation at the para-position of

the 9-phenyl ring.

Pharmacology

All the synthesized compounds were tested in rat hepa-

tocyte glucose assay. The methods for the preparation

of hepatocytes and for the inhibition assay of glucose

production are described in the Experimental. The 9-

methyladenine compound (1)⁶was used as an active

control in each screening assay. The inhibitory activities

of the title compounds toward NECA-induced glucose

production in rat hepatocytes are summarized in Table 1.

The 9-(N-ethylamidopropyl)adenine compound (15a)

showed about 3.5-fold more potent inhibitory activity

than the 9-methyl compound (1)⁶(15a, IC₅₀=0.071 mM;

1, IC₅₀=0.25 mM). The potency was signiﬁcantly atte-

nuated when the number of methylene groups between

The A_2Bantagonisticactivities of the newly synthesized

compounds were also measured in terms of inhibitory

Table 1. Inhibitory activities on NECA-induced glucose production in primary cultured rat hepatocytes and on NECA-induced cyclic AMP

accumulation in CHO.K1 cells, and adenosine A₁and A_2areceptor binding aﬃnities

Compd

R⁹

IC₅₀(mM)^a

K_i(mM)^b

Hepatocyte

A_2B-CHO.K1 cell

A₁

A_2A

15a

15b

15c

15d

15e

15f

15g

15h

15i

15j

1^d

FK453^d

KF17837^d

(CH₂)₃CONHEt

(CH₂)₂CONHEt

CH₂CONHEt

0.071ꢂ0.020

1.6

23

0.0080

0.23

1.8

0.96

0.0025

0.0040

>10^c

0.024

0.0074

0.078

1.4

0.90

0.081

0.18

0.086

>10^c

0.36

0.0080

0.014

0.025

0.18

(CH₂)₂OCONHEt

(CH₂)₃SO₂NHEt

3-(CONH₂)C₆H₄

4-(CONH₂)C₆H₄

2-(CONHEt)C₆H₄

3-(CONHEt)C₆H₄

4-(CONHEt)C₆H₄

Me

14

8.2

0.028

0.013

0.0098

0.017ꢂ0.002

0.098ꢂ0.027

>30^c

>10^c

0.044ꢂ0.014

0.019

0.045

0.52

0.063

0.064

0.25ꢂ0.04

8.1ꢂ1.6

>10 (>10, >10)^c

0.023ꢂ0.003

0.98 (0.92–1.0)

1.5 (1.4–1.7)

0.014ꢂ0.003

0.018ꢂ0.002

>10^c

0.016ꢂ0.001

1.3ꢂ0.4

0.071ꢂ0.014

^aIC₅₀values were determined from the logarithmic concentration–inhibition curve (at least three points). In cases where the number of independent

experiments (n)>2, data are shown as mean IC₅₀values (mM)ꢂSEM. In cases where n=2, IC₅₀values are the means of two independent experi-

ments (values of individual measurements in parentheses). In other cases where n=1, the values are the results of one experiment performed at least

in duplicate and in which individual determinations varied by less than 10%.

^bK_ivalues were determined in radioligand binding assays for recombinant human A₁and A_2Areceptors expressed in CHO.K1 cells versus

[³H]CCPA and HEK-293 cells versus [³H]CGS21680, respectively. Concentration-inhibition curves were obtained using three or more concentra-

tions of each test agent, and IC₅₀values were determined from the logarithmic concentration-inhibition curve (at least three points). IC₅₀values were

converted to K_ivalues using the Cheng-Prusoﬀ equation.²¹In cases where n>2, data are shown as mean K_ivalues (mM)ꢂSEM. In cases where n=1,

the values are the results of one experiment performed at least in duplicate and in which individual determinations varied by less than 10%.

^c>10 and >30 mean that the IC₅₀values were greater than 10 and 30 mM, respectively.

^dData from our previous report.⁶

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

activities on NECA-induced cyclic AMP accumulation

in CHO.K1 cells stably transfected with human adeno-

sine A_2Breceptor cDNA. The compounds antagonized

NECA-induced stimulation of cyclic AMP production

in a dose-dependent manner, and the IC₅₀values are

listed in Table 1.

Our previously reported 9-methyl derivative (1) was a

nonselective adenosine antagonist.⁶Among the 9-ali-

phaticamide derivatives ( 15a–e), there was no A_2B

-

selective compound. Fixing the amide moiety of 15a

with a benzene ring increased the selectivity for A_2B

receptor versus A₁, but not A_2A(15i, 15-fold vs A₁).

However, the para-isomer of 15i (15j) lost this selectiv-

ity. The primary benzamide derivatives tended to be

more A_2B-selective than the corresponding N-ethylbenza-

mide compounds (15f vs 15i, 15g vs 15j). The m-primary

benzamide compound (15f) showed 72- and 5.2-fold

selectivity for human A_2Breceptor versus human A₁

and A_2Areceptors, respectively. Movement of the amide

moiety of 15f from the meta- to the para-position of the

9-phenyl ring decreased the selectivity (15g, 22- and 2.5-

fold vs A₁and A_2Areceptors, respectively). These results

indicate that ﬁxing the amide moiety at the meta-position

To assess the selectivity of our compounds for the A_2B

receptor, their aﬃnities for human A₁and A_2Arecep-

tors were evaluated using a radioligand binding techni-

que. An A₁selective antagonist, (E)-(2R)-1-[3-(2-

phenylpyrazolo[1,5-a]pyridin-3-yl)acryloyl]-2-piperidine-

ethanol (FK453)^8,19and an A_2Aselective antagonist,

(E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxan-

thine (KF17837),^8,20were used as positive controls for

A₁and A_2Abinding assay, respectively. The results are

summarized in Table 1.

Figure 1. Correlation between inhibitory activities on NECA-induced glucose production in primary cultured rat hepatocytes and (a) inhibitory

activities on NECA-induced cyclic AMP accumulation in CHO.K1 cells, (b) A₁binding aﬃnities, and (c) A_2Abinding aﬃnities (least-squares

regression analysis).

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2715

of a benzene ring is favorable for not only A_2B-antag-

onisticactivity, but also A selectivity. Furthermore, it

is worthy of note that the N-ethylacetamide derivative

(15c) was highly selective for the A_2Areceptor (56- and

72-fold vs A₁and A_2Breceptors, respectively).

N1-[4-Chloro-6-[(4-hydroxybutyl)amino]-5-nitro-2-pyri-

midinyl]acetamide (5a). General procedure. Glacial ace-

tic acid (2.6 mL, 45.4 mmol) was carefully added with

cooling to 4-amino-1-butanol (4.1 g, 46.0 mmol) in

MeOH (10 mL). This solution was added dropwise to a

stirred solution of N1-(4,6-dichloro-5-nitro-2-pyri-

midinyl)acetamide (4) (5.00 g, 19.9 mmol) in THF (50

mL), previously cooled to 0 ^ꢁC over 1 h. The mixture

was stirred at this temperature for an additional 1.5 h,

acidiﬁed with 1 N HCl to pH 2, and then diluted with

EtOAc. The insoluble material was removed by ﬁltra-

tion. The organiclayer of the ﬁltrate was washed with

1 N HCl, saturated aqueous NaHCO₃, and saturated

aqueous NH₄Cl, dried over Na₂SO₄, and then con-

centrated. The residue was suspended in MeOH. The

precipitated material was collected by ﬁltration and

washed with MeOH to give 5a (2.98 g, 49%) as a pale

2B

The IC₅₀values in rat hepatocyte glucose assay were

found to be well correlated with IC₅₀values in the A_2B

cAMP assay [r²=0.94, Fig. 1(a)]. The A₁and A_2Aaﬃ-

nities showed no correlation with the potency to inhibit

NECA-induced glucose production [r²=0.10 for A₁

receptor K_iand hepatocyte IC₅₀, Fig. 1(b); r²=0.37 for

A_2Areceptor K_iand hepatocyte IC₅₀, Fig. 1(c)]. These

results are consistent with our previous conclusion that

adenosine agonist-induced hepatic glucose production

in rat hepatocytes results from the activation of the A_2B

receptor.

1

yellow solid. H NMR (DMSO-d₆) d 1.39–1.47 (2H, m,

CH₂), 1.56–1.64 (2H, m, CH₂), 2.25 (3H, s, Ac), 3.40

(2H, t, J=6.4 Hz, CH₂O), 3.48 (2H, dt, J=6.0 and 7.2

Hz, NCH₂), 4.41 (1H, br, OH), 8.55 (1H, t, J=6.0 Hz,

NHCH₂), 10.78 (1H, s, NHAc).

Conclusion

A series of 2-alkynyl-8-aryladenine derivatives bearing

an amide moiety at the 9-position of adenine was syn-

thesized. Using the screening system of adenosine ago-

nist-induced hepatic glucose production, compounds

15a, 15f, and 15i were found to be about 3.5–15-fold

more potent than our previously reported compound

(1). The most potent compound in hepatocyte glucose

assay, 15f (IC₅₀=0.017 mM), showed 72- and 5.2-fold

selectivity for human A_2Breceptor versus human A₁

and A_2Areceptors, respectively. SAR studies of our

N1-[5-Amino-4-chloro-6-[(4-hydroxybutyl)amino]-2-pyri-

midinyl]acetamide (6a). General procedure. A suspen-

sion of 5a (2.8 g, 9.22 mmol) in MeOH (60 mL) was

hydrogenated in the presence of Raney nickel (2.8 g wet,

washed with H₂O and MeOH) at room temperature and

atmosphericpressure. The theoretical amount of H ₂was

absorbed within 17 h. The reaction mixture was ﬁltered

through a Celite pad, which was then washed with

MeOH, and the ﬁltrate was evaporated. The residue was

suspended in EtOH, and the resulting precipitate was

collected by ﬁltration and washed with EtOH to aﬀord

6a (1.43 g, 57%) as a pale yellow solid. ¹H NMR

(DMSO-d₆) d 1.40–1.50 (2H, m, CH₂), 1.52–1.62 (2H,

m, CH₂), 2.09 (3H, br s, Ac), 3.30–3.43 (4H, m, CH₂O

and NCH₂), 4.38 (1H, t, J=5.2 Hz, OH), 4.67 (2H, br s,

NH₂), 6.85 (1H, br, NHCH₂), 9.70 (1H, br s, NHAc).

compounds indicated that

a three-carbon linker

between the adenine core and the amide moiety was

important for A_2Bantagonisticactivity, and ﬁxing the

conformation of the amide group at the meta-position

of a benzene ring imparted A_2Bselectivity. Functional

assay using recombinant human A_2Breceptors con-

ﬁrmed that our compounds show A_2Bantagonistic

activity. The correlation between the IC₅₀values in rat

hepatocyte glucose assay and in A_2BcAMP assay

strongly support our previous conclusion that adenosine

agonist-induced hepatic glucose production in rat

hepatocytes is mediated through the A_2Breceptor.

4-[2-Amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-purinyl]-1-

butanol (8a). General procedure. A mixture of 6a (5.0 g,

18.3 mmol), 3-ﬂuorobenzaldehyde (2.7 g, 21.8 mmol),

and acetic acid (1.2 mL) in MeOH (100 mL) was stirred

at room temperature for 28.5 h. The reaction mixture

was concentrated to dryness under reduced pressure.

The residue was azeotroped with toluene (50 mLꢃ2),

and used directly in the next step. To a suspension of

the crude imine in EtOH (100 mL) was added a solution

of anhydrous FeCl₃(3.0 g, 18.5 mmol) in EtOH (20 mL)

at room temperature. The mixture was heated under

reﬂux for 1 h and then allowed to cool to room tem-

perature. The solvent was removed under reduced pres-

sure, and the residue was suspended in EtOH and H₂O.

The precipitated product was ﬁltered oﬀ and washed

with H₂O to give crude 8a (3.60 g) as a pale yellow solid.

Experimental

Chemistry

Column chromatography was performed on silica gel

(Merck, particle size 0.063–0.200 mm). TLC analyses

were done on silica gel plates (Merck, Art 5715). All H

1

NMR spectra were measured on a Varian Unity 400

(400 MHz) spectrometer, and chemical shifts are

expressed in d units from tetramethylsilane (TMS) as an

internal standard; coupling constants (J) are reported in

hertz. Abbreviations are as follows: s, singlet; d, doub-

let; t, triplet; q, quartet; quint, quintet; m, multiplet; br,

broad peak; Hz, hertz. Mass spectra (FAB-MS) were

obtained on a JEOL SX102 mass spectrometer. Mass

spectra and elemental analyses were performed at the

Analytical Chemistry Section of Eisai Research

Laboratories.

1

Examination of the H NMR spectrum indicated that a

small amount of the N-acetyl purine derivative

remained in the crude material. A solution of crude 8a

(3.60 g) in 1 N HCl (25 mL)–THF (100 mL) was heated

under reﬂux for 1 h and then allowed to cool to room

temperature. The mixture was ﬁltered through a Celite

pad, which was then washed with MeOH, and the

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H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

ﬁltrate was evaporated. The residue was suspended in

H₂O. The precipitated product was ﬁltered oﬀ and

washed with H₂O to give 8a (2.86 g, 47%) as a white

Methyl 4-[6-amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-puri-

nyl]butanoate (12a). General procedure. SOCl₂(0.66

mL, 9.05 mmol) was added dropwise to a suspension of

11a (800 mg, 1.81 mmol) in MeOH (20 mL) over 1 h at

2–5 ^ꢁC. The mixture was stirred at room temperature

for 1 h and concentrated to dryness under reduced

pressure. The residue was partitioned between EtOAc

and saturated aqueous NaHCO₃. The separated organic

phase was washed with saturated aqueous NaHCO₃and

saturated aqueous NH₄Cl, dried over Na₂SO₄, and

concentrated. The residue was suspended in MeOH, and

the precipitated product was collected by ﬁltration and

washed with MeOH to give 12a (720 mg, 87%) as a

1

solid. H NMR (DMSO-d₆) d 1.21–1.28 (2H, m, CH₂),

1.60–1.69 (2H, m, CH₂), 3.26 (2H, t, J=6.4 Hz, CH₂O),

4.19 (2H, t, J=7.2 Hz, NCH₂), 7.02 (2H, br s, NH₂),

7.40–7.47 (1H, m, phenyl), 7.60–7.66 (3H, m, phenyl).

4-[6-Chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]-1-bu-

tanol (9a). General procedure. Isoamyl nitrite (3.0 mL,

22.3 mmol) was added to a mixture of 8a (2.50 g, 7.45

mmol), CH₂I₂(3.0 mL, 37.2 mmol), and CuI (1.42 mg,

7.46 mmol) in THF (50 mL). The mixture was heated

under reﬂux for 1 h and then allowed to cool to room

temperature. The reaction mixture was partitioned

between EtOAcand 1 N HCl. The separated organic

phase was washed with concentrated aqueous ammonia,

and the aqueous ammonia layer was extracted with

EtOAc. The combined organic layer was washed with

saturated aqueous NH₄Cl, dried over Na₂SO₄, and

concentrated. The residue was puriﬁed by silica gel col-

umn chromatography (eluent hexane, hexane/

EtOAc=4:1, 2:1, 3:2) to give a crude material. This

crude product was suspended in Et₂O, and the resulting

precipitate was collected by ﬁltration to aﬀord 9a (1.78

1

white solid. H NMR (DMSO-d₆) d 1.87 (2H, quint,

J=7.2 Hz, NCH₂CH₂), 2.22 (2H, t, J=7.2 Hz,

CH₂CO), 3.48 (3H, s, CH₃), 4.24 (2H, t, J=7.2 Hz,

NCH₂), 7.39–7.46 (1H, m, phenyl), 7.59–7.66 (3H, m,

phenyl), 7.76 (2H, br s, NH₂).

Methyl 4-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycy-

clohexyl)-1-ethynyl]-9H-9-purinyl]butanoate (13a). Gen-

eral procedure. Triethylamine (0.27 mL, 1.93 mmol)

was added dropwise to a mixture of 12a (450 mg, 0.989

mmol), CuI (19 mg, 0.0997 mmol), dichlorobis(tri-

phenylphosphine)palladium(II) (69 mg, 0.0983 mmol),

and 1-ethynyl-1-cyclohexanol (246 mg, 1.98 mmol) in

N,N-dimethylformamide (5 mL). The mixture was stir-

red under an atmosphere of N₂at room temperature for

13.5 h and then partitioned between EtOAcand satu-

rated aqueous NH₄Cl. The separated organicphase was

washed with concentrated aqueous ammonia/saturated

aqueous NH₄Cl (1:1) and saturated aqueous NH₄Cl,

dried over Na₂SO₄, and concentrated. The residue was

suspended in MeOH. The precipitated product was ﬁl-

tered oﬀ and washed with MeOH to give 13a (286 mg,

64%) as a white solid. ¹H NMR (DMSO-d₆) d 1.21–1.34

(1H, m, c-C₆H₁₀), 1.42–1.70 (7H, m, c-C₆H₁₀), 1.80–1.88

(2H, m, c-C₆H₁₀), 1.89 (2H, quint, J=7.2 Hz,

NCH₂CH₂), 2.22 (2H, t, J=7.2 Hz, CH₂CO), 3.48 (3H,

s, CH₃), 4.29 (2H, t, J=7.2 Hz, NCH₂), 5.55 (1H, s,

OH), 7.39–7.46 (1H, m, phenyl), 7.50 (2H, br s, NH₂),

7.59–7.66 (3H, m, phenyl).

1

g, 54%) as a white solid. H NMR (DMSO-d₆) d 1.23–

1.31 (2H, m, CH₂), 1.65–1.74 (2H, m, CH₂), 3.28 (2H,

dt, J=5.6 and 6.0 Hz, CH₂O), 4.36 (2H, t, J=7.2 Hz,

NCH₂) 4.38 (1H, t, J=5.6 Hz, OH), 7.50–7.56 (1H, m,

phenyl), 7.66–7.74 (3H, m, phenyl).

4-[6-Amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]-1-bu-

tanol (10a). General procedure. A solution of 9a (2.51 g,

5.60 mmol) in methanolicammonia (saturated at 0 ^ꢁC)

(250 mL) in a sealed steel tube was heated at 60 ^ꢁC for

48 h and then allowed to cool to room temperature. The

solvent was removed in vacuo, and EtOH and H₂O were

added. The resulting precipitate was ﬁltered oﬀ and

washed with EtOH and Et₂O to give 10a (1.50 g, 63%)

1

as a white solid. H NMR (DMSO-d₆) d 1.18–1.26 (2H,

m, CH₂), 1.58–1.67 (2H, m, CH₂), 3.26 (2H, dt, J=5.2

and 5.6 Hz, CH₂O), 4.22 (2H, t, J=7.2 Hz, NCH₂), 4.36

(1H, t, J=5.2 Hz, OH), 7.39–7.46 (1H, m, phenyl),

7.59–7.66 (3H, m, phenyl), 7.75 (2H, br s, NH₂).

N1-Ethyl-4-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]butanamide hydro-

chloride (15a). General procedure. A solution of 13a

(663 mg, 1.47 mmol) in a 70% aqueous solution of

ethylamine (20 mL) and MeOH (40 mL) in a sealed steel

tube was heated at 80 ^ꢁC for 20 h and then allowed to

cool to room temperature. The solvent was removed in

vacuo and the residue was puriﬁed by silica gel column

4-[6-Amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]buta-

noic Acid (11a). General procedure. Ruthenium(IV)

oxide hydrate (61 mg, 0.458 mmol) was added to a sus-

pension of 10a (1.30 g, 3.04 mmol) and NaIO₄(3.30 g,

15.4 mmol) in a mixture of CH₃CN (26 mL), CHCl₃(26

mL), and H₂O (39 mL). The mixture was vigorously

stirred at room temperature for 4 h. The reaction was

quenched by dropwise addition of 2-propanol (2.0 mL),

and the mixture was adjusted with 1 N NaOH to pH 12–

13. The insoluble material was removed by ﬁltration

and washed with MeOH. The organicsolvent of the ﬁl-

trate was evaporated and acidiﬁed with 1 N HCl to pH

2–3. The resulting precipitate was collected by ﬁltration

and washed with H₂O to give 11a (1.21 g, 90%) as a

chromatography

(eluent

CH₂Cl₂,

CH₂Cl₂/

MeOH=100:1, 50:1, 40:1, 30:1, 20:1) to give a crude

material. The crude product was suspended in EtOAc,

and the resulting precipitate was collected by ﬁltration

to aﬀord the title compound (396 mg) as the free form.

This was suspended in MeOH, 20% HCl/EtOH, and the

solvent was evaporated. The residue was diluted with

Et₂O, and the precipitated product was ﬁltered oﬀ and

washed with Et₂O to give 15a (400 mg, 54%) as a white

1

grayish solid. H NMR (DMSO-d₆) d 1.86 (2H, quint,

1

J=7.2 Hz, NCH₂CH₂), 2.16 (2H, t, J=7.2 Hz, CH₂CO),

4.23 (2H, t, J=7.2 Hz, NCH₂), 7.39–7.46 (1H, m,

phenyl), 7.57–7.66 (3H, m, phenyl), 7.76 (2H, br s, NH₂).

solid. H NMR (DMSO-d₆) d 0.91 (3H, t, J=7.2 Hz,

CH₂CH₃), 1.20–1.32 (1H, m, c-C₆H₁₀), 1.40–1.67 (7H,

m, c-C₆H₁₀), 1.78–1.87 (4H, m, c-C₆H₁₀and

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2717

NCH₂CH₂), 1.93 (2H, t, J=7.1 Hz, CH₂CO), 2.94 (2H,

dq, J=5.5 and 7.2 Hz, CH₂CH₃), 4.25 (2H, t, J=7.1

Hz, NCH₂CH₂), 7.40–7.45 (1H, m, phenyl), 7.58–7.65

(3H, m, phenyl), 7.72 (1H, t, J=5.5 Hz, NHEt); MS m/e

Methyl 3-[6-amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-puri-

nyl]propanoate (12b). This compound (white solid, 77%

yield) was prepared in a manner similar to that descri-

bed for 12a, except that 11b was used instead of 11a. ¹H

NMR (DMSO-d₆) d 2.82 (2H, t, J=7.0 Hz, CH₂CO),

3.49 (3H, s, CH₃), 4.43 (2H, t, J=7.0 Hz, NCH₂), 7.40–

7.46 (1H, m, phenyl), 7.58–7.66 (3H, m, phenyl), 7.77

(2H, br s, NH₂).

(FAB)

465

.

(MH⁺).

Anal.

calcd

for

1

.

C₂₅H₂₉FN₆O₂HCl /₂H₂O: C, 58.88, H, 6.13, N, 16.48;

found: C, 58.80, H, 6.18, N, 16.85.

N1-[4-Chloro-6-[(3-hydroxypropyl)amino]-5-nitro-2-pyri-

midinyl]acetamide (5b). This compound (pale yellow

solid, 44% yield) was prepared in a manner similar to

that described for 5a, except that 3-amino-1-propanol

was used instead of 4-amino-1-butanol. ¹H NMR

Methyl 3-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]propanoate (13b).

This compound (white solid, 76% yield) was prepared

in a manner similar to that described for 13a, except

1

(DMSO-d₆)

d

1.72 (2H, quint, J=6.4 Hz,

that 12b was used instead of 12a. H NMR (DMSO-d₆)

CH₂CH₂CH₂), 2.25 (3H, s, Ac), 3.47 (2H, t, J=6.4 Hz,

CH₂O), 3.55 (2H, dt, J=5.6 and 6.4 Hz, NCH₂), 4.56

(1H, br, OH), 8.63 (1H, t, J=5.6 Hz, NHCH₂), 10.82

(1H, s, NHAc).

d 1.21–1.33 (1H, m, c-C₆H₁₀), 1.42–1.70 (7H, m, c-

C₆H₁₀), 1.80–1.90 (2H, m, c-C₆H₁₀), 2.85 (2H, t, J=7.2

Hz, CH₂CO), 3.49 (3H, s, CH₃), 4.47 (2H, t, J=7.2 Hz,

NCH₂), 5.55 (1H, s, OH), 7.40–7.46 (1H, m, phenyl),

7.51 (2H, br s, NH₂), 7.61–7.67 (3H, m, phenyl).

N1-[5-Amino-4-chloro-6-[(3-hydroxypropyl)amino]-2-pyr-

imidinyl]acetamide (6b). This compound (pale brown

solid, 59% yield) was prepared in a manner similar to

that described for 6a, except that 5b was used instead of

N1-Ethyl-3-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]propanamide hydro-

chloride (15b). This compound (pale yellow solid, 40%

yield) was prepared in a manner similar to that descri-

bed for 15a, except that 13b was used instead of 13a. ¹H

NMR (DMSO-d₆) d 0.90 (3H, t, J=7.2 Hz, CH₂CH₃),

1.18–1.33 (1H, m, c-C₆H₁₀), 1.42–1.68 (7H, m, c-C₆H₁₀),

1.80–1.88 (2H, m, c-C₆H₁₀), 2.55 (2H, t, J=7.5 Hz,

CH₂CO), 2.93 (2H, dq, J=5.5 and 7.2 Hz, CH₂CH₃),

4.43 (2H, t, J=7.5 Hz, NCH₂CH₂), 7.39–7.45 (1H, m,

phenyl), 7.58–7.66 (3H, m, phenyl), 7.90 (1H, t, J=5.5

Hz, NHEt); MS m/e (FAB) 451 (MH⁺). Anal. calcd for

1

5a. H NMR (DMSO-d₆) d 1.69 (2H, quint, J=6.4 Hz,

CH₂CH₂CH₂), 2.08 (3H, br s, Ac), 3.37–3.47 (4H, m,

CH₂O and NCH₂), 4.47 (1H, t, J=5.4 Hz, OH), 4.67

(2H, s, NH₂), 6.86 (1H, t, J=5.2 Hz, NHCH₂), 9.76

(1H, br s, NHAc).

3-[2-Amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-purinyl]-1-

propanol (8b). This compound (white solid, 46% yield)

was prepared in a manner similar to that described for

1

.

8a, except that 6b was used instead of 6a. H NMR

C₂₄H₂₇FN₆O₂HCl H₂O: C, 57.08, H, 5.99, N, 16.64;

found: C, 57.26, H, 5.72, N, 16.80.

(DMSO-d₆) d 1.78–1.87 (2H, m, CH₂CH₂CH₂), 3.34

(2H, t, J=5.8 Hz, CH₂O), 4.24 (2H, t, J=7.6 Hz,

NCH₂), 7.02 (2H, br, NH₂), 7.40–7.46 (1H, m, phenyl),

7.59–7.69 (3H, m, phenyl).

Benzyl N-[2-(Ethylamino)-2-oxoethyl]carbamate (19). To

a mixture of 2-[(benzyloxycarbonyl)amino]acetic acid

(18) (15.0 g, 71.7 mmol), ethylamine hydrochloride (17.5

g, 215 mmol), 1-hydroxybenzotriazole (13.1 g, 85.6

mmol), and 1-ethyl-3-(3⁰-dimethylaminopropyl)carbo-

diimide hydrochloride (16.5 g, 86.1 mmol) in THF (300

mL) was added dropwise triethylamine (30.0 mL, 215

mmol) over 10 min at 5–10 ^ꢁC. The mixture was stirred

at room temperature for 13 h. The reaction mixture was

partitioned between EtOAcand saturated aqueous

NH₄Cl. The separated organicphase was washed with

1 N NaOH and saturated aqueous NH₄Cl, dried over

Na₂SO₄, and concentrated. The residue was suspended

in Et₂O, and the precipitated product was collected by

ﬁltration and washed with Et₂O to give 19 (10.4 g, 61%)

3-[6-Chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]-1-

propanol (9b). This compound (white solid, 62% yield)

was prepared in a manner similar to that described for

1

9a, except that 8b was used instead of 8a. H NMR

(DMSO-d₆) d 1.82–1.92 (2H, m, CH₂CH₂CH₂), 3.36

(2H, dt, J=4.8 and 5.6 Hz, CH₂O), 4.40 (2H, t, J=7.6

Hz, NCH₂) 4.56 (1H, t, J=4.8 Hz, OH), 7.49–7.55 (1H,

m, phenyl), 7.65–7.76 (3H, m, phenyl).

3-[6-Amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]-1-

propanol (10b). This compound (white solid, 72% yield)

was prepared in a manner similar to that described for

1

10a, except that 9b was used instead of 9a. H NMR

as a white powder. H NMR (CDCl₃) d 1.13 (3H, t,

(DMSO-d₆) d 1.77–1.86 (2H, m, CH₂CH₂CH₂), 3.30–

3.37 (2H, m, CH₂O), 4.25 (2H, t, J=7.4 Hz, NCH₂),

4.58 (1H, t, J=4.8 Hz, OH), 7.39–7.45 (1H, m, phenyl),

7.58–7.66 (3H, m, phenyl), 7.75 (2H, br s, NH₂).

J=7.2 Hz, CH₂CH₃), 3.31 (2H, dq, J=6.6 and 7.2 Hz,

CH₂CH₃), 3.84 (2H, d, J=6.0 Hz, CH₂CO), 5.13 (2H, s,

CH₂Ph), 5.42 (1H, br, NH), 5.96 (1H, br, NH), 7.31–

7.38 (5H, m, Ph).

3-[6-Amino-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]pro-

panoic acid (11b). This compound (grayish solid, 85%

yield) was prepared in a manner similar to that descri-

bed for 11a, except that 10b was used instead of 10a. ¹H

NMR (DMSO-d₆) d 2.75 (2H, t, J=7.2 Hz, CH₂CO),

4.38 (2H, t, J=7.2 Hz, NCH₂), 7.39–7.46 (1H, m,

phenyl), 7.58–7.66 (3H, m, phenyl), 7.76 (2H, br s, NH₂).

N1-Ethyl-2-aminoacetamide (20). A solution of 19 (9.0

g, 38.1 mmol) in MeOH (400 mL) was hydrogenated in

the presence of 10% Pd/C (1.0 g) at room temperature

and atmosphericpressure. The theoretical amount of H

2

was absorbed within 12.5 h. The reaction mixture was

ﬁltered through a Celite pad, which was then washed

with MeOH, and the ﬁltrate was evaporated to give 20

2718

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

1

(3.86 g, 99%) as a colorless oil. H NMR (DMSO-d₆) d

1.02 (3H, t, J=7.2 Hz, CH₂CH₃), 3.04 (2H, s, CH₂CO),

3.07 (2H, br, NH₂), 3.10 (2H, dq, J=5.8 and 7.2 Hz,

CH₂CH₃), 7.79 (1H, br, NHEt).

removed in vacuo, and the residue was puriﬁed by silica

gel column chromatography (eluent CH₂Cl₂, CH₂Cl₂/

MeOH=100:1, 40:1, 30:1, 20:1) to give the title com-

pound (90 mg) as the free form. This was suspended in

MeOH, 20% HCl/EtOH was added, and the solvent

was evaporated. The residue was diluted with Et₂O, and

the precipitated product was ﬁltered oﬀ and washed

with Et₂O to give 15c (86 mg, 53%) as a white solid. ¹H

NMR (DMSO-d₆) d 0.96 (3H, t, J=7.2 Hz, CH₂CH₃),

1.18–1.31 (1H, m, c-C₆H₁₀), 1.41–1.67 (7H, m, c-C₆H₁₀),

1.78–1.90 (2H, m, c-C₆H₁₀), 3.06 (2H, dq, J=5.5 and

7.2 Hz, CH₂CH₃), 4.84 (2H, s, CH₂CO), 7.38–7.44 (1H,

m, phenyl), 7.52–7.63 (3H, m, phenyl), 8.40 (1H, t,

J=5.5 Hz, NHEt); MS m/e (FAB) 437 (MH⁺). Anal.

N1-Ethyl-2-[2-(acetylamino)-6-chloro-5-nitro-4-pyrimidi-

nyl]amino]acetamide (5c). This compound (pale yellow

solid, 44% yield) was prepared in a manner similar to

that described for 5a, except that 20 was used instead of

1

4-amino-1-butanol. H NMR (DMSO-d₆) d 1.00 (3H, t,

J=7.4 Hz, CH₂CH₃), 2.24 (3H, s, Ac), 3.08 (2H, dq,

J=5.6 and 7.4 Hz, CH₂CH₃), 4.06 (2H, d, J=5.6 Hz,

CH₂CO), 8.04 (1H, t, J=5.6 Hz, NH), 8.79 (1H, t,

J=5.6 Hz, NH), 10.82 (1H, s, NHAc).

.

calcd for C₂₃H₂₅FN₆O₂HCl H₂O: C, 56.27, H, 5.75, N,

17.12; found: C, 55.97, H, 5.72, N, 16.93.

N1-Ethyl-2-[2-(acetylamino)-5-amino-6-chloro-4-pyrimi-

dinyl]amino]acetamide (6c). This compound (brown

solid, 75% yield) was prepared in a manner similar to

that described for 6a, except that 5c was used instead of

5a. ¹H NMR (DMSO-d₆) d 0.96 (3H, t, J=7.2 Hz,

CH₂CH₃), 2.06 (3H, s, Ac), 3.05 (2H, dq, J=5.2 and 7.2

Hz, CH₂CH₃), 3.89 (2H, d, J=5.2 Hz, CH₂CO), 4.73

(1H, br, NH), 7.34 (1H, t, J=5.2 Hz, NH), 8.09 (2H, br

s, NH₂), 9.87 (1H, br s, NHAc).

Benzyl N-(2-hydroxyethyl)carbamate (22). To a solution

of ethanolamine (21) (15.0 g, 245 mmol) in H₂O (150

mL) were simultaneously added dropwise benzyl chlor-

oformate (35.0 mL, 245 mmol) and aqueous 2 N

Na₂CO₃(125 mL, 250 mmol) over 2 h at 0–8 ^ꢁC. After

the addition was complete, the mixture was stirred at

0 ^ꢁC for an additional 1 h and extracted with EtOAc.

The organiclayer was washed with H ₂O, saturated

aqueous NaHCO₃, and brine, dried over Na₂SO₄, and

concentrated. The residue was diluted with hexane, and

the resulting precipitate was collected by ﬁltration and

washed with hexane to aﬀord 22 (40.0 g, 83%) as a

N1-Ethyl-2-[2-amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-

purinyl]acetamide (8c). This compound (white solid,

27% yield) was prepared in a manner similar to that

described for 8a, except that 6c was used instead of 6a.

¹H NMR (DMSO-d₆) d 0.97 (3H, t, J=7.2 Hz,

CH₂CH₃), 3.07 (2H, dq, J=5.2 and 7.2 Hz, CH₂CH₃),

4.73 (2H, s, CH₂CO), 7.03 (1H, br s, NH₂), 7.38–7.45

(1H, m, phenyl), 7.50–7.63 (3H, m, phenyl), 8.30 (1H, t,

J=5.2 Hz, NHEt).

1

white solid. H NMR (CDCl₃) d 2.76 (1H, br s, OH),

3.32 (2H, q, J=5.2 Hz, NCH₂), 3.67 (2H, t, J=5.2 Hz,

CH₂CH₂O), 5.09 (2H, s, CH₂Ph), 5.36 (1H, br, NH),

7.26–7.41 (5H, m, phenyl).

2-[(Benzyloxy)carbonyl)]amino]ethyl N-ethylcarbamate

(23). To a solution of 22 (10.0 g, 51.2 mmol) and pyri-

dine (1 drop) in CH₂Cl₂(100 mL) was added dropwise

ethyl isocyanate (10.6 mL, 134 mmol) at 0 ^ꢁC, and the

mixture was stirred at this temperature for 62 h. The

solvent was removed under reduced pressure. The resi-

due was diluted with Et₂O, and the resulting precipitate

was collected by ﬁltration and washed with Et₂O to aﬀord

23 (9.0 g, 66%) as a white solid. ¹H NMR (DMSO-d₆) d

0.98 (3H, t, J=7.2 Hz, CH₂CH₃), 2.97 (2H, dq, J=6.0

and 7.2 Hz, CH₂CH₃), 3.13–3.23 (2H, m, NCH₂CH₂),

3.93 (2H, t, J=6.0 Hz, CH₂CH₂O), 5.01 (2H, s, CH₂Ph),

7.08 (1H, br, NH), 7.25–7.45 (6H, m, NH and phenyl).

N1-Ethyl-2-[6-chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-pur-

inyl]acetamide (9c). This compound (pale brown solid,

68% yield) was prepared in a manner similar to that

described for 9a, except that 8c was used instead of 8a.

¹H NMR (DMSO-d₆) d 0.97 (3H, t, J=7.2 Hz,

CH₂CH₃), 3.07 (2H, dq, J=5.6 and 7.2 Hz, CH₂CH₃),

4.95 (2H, s, CH₂CO), 7.48–7.54 (1H, m, phenyl), 7.61–

7.70 (3H, m, phenyl), 6.15 (1H, t, J=5.6 Hz, NHEt).

N1-Ethyl-2-[6-chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]acetamide

(14c).

This compound (white solid, 88% yield) was prepared

from 9c in a manner similar to that described for 13a,

except that THF was used instead of N,N-dimethylfor-

2-Aminoethyl N-ethylcarbamate (24). A solution of 23

(8.50 g, 31.9 mmol) in MeOH (400 mL) was hydro-

genated in the presence of 10% Pd/C (1.0 g) at room

temperature and atmosphericpressure. The theoretical

amount of H₂was absorbed within 13 h. The reaction

mixture was ﬁltered through a Celite pad, which was

then washed with MeOH, and the ﬁltrate was evapo-

1

mamide as a reaction solvent. H NMR (DMSO-d₆) d

0.98 (3H, t, J=7.2 Hz, CH₂CH₃), 1.22–1.37 (1H, m, c-

C₆H₁₀), 1.44–1.74 (7H, m, c-C₆H₁₀), 1.84–1.96 (2H, m,

c-C₆H₁₀), 3.08 (2H, dq, J=6.0 and 7.2 Hz, CH₂CH₃),

4.98 (2H, s, CH₂CO), 5.72 (1H, s, OH), 7.48–7.56 (1H,

m, phenyl), 7.61–7.72 (3H, m, phenyl), 8.41 (1H, t,

J=6.0 Hz, NHEt).

1

rated to give 24 (4.38 g, quant) as a colorless oil. H

NMR (DMSO-d₆) d 1.00 (3H, t, J=7.2 Hz, CH₂CH₃),

2.69 (2H, t, J=6.0 Hz, NCH₂CH₂), 2.99 (2H, dq, J=5.8

and 7.2 Hz, CH₂CH₃), 3.10 (2H, br, NH₂), 3.87 (2H, t,

J=6.0 Hz, CH₂O), 7.06 (1H, br, NH).

N1-Ethyl-2-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]acetamide Hydro-

chloride (15c). A solution of 14c (157 mg, 0.344 mmol)

in ethanolicammonia (saturated at 0 ^ꢁC) (40 mL) in a

sealed steel tube was heated at 100 ^ꢁC for 12 h and then

allowed to cool to room temperature. The solvent was

2-[2-(Acetylamino)-6-chloro-5-nitro-4-pyrimidinyl]amino]-

ethyl N-ethylcarbamate (5d). This compound (pale

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2719

yellow solid, 57% yield) was prepared in a manner

similar to that described for 5a, except that 24 was used

instead of 4-amino-1-butanol. H NMR (DMSO-d₆) d

0.98 (3H, t, J=7.2 Hz, CH₂CH₃), 2.25 (3H, s, Ac), 2.97

(2H, dq, J=5.2 and 7.2 Hz, CH₂CH₃), 3.69 (2H, dt,

J=5.2 and 5.8 Hz, NCH₂CH₂), 4.15 (2H, t, J=5.8 Hz,

CH₂O), 7.11 (1H, t, J=5.2 Hz, NH), 8.56 (1H, t, J=5.2

Hz, NH), 10.83 (1H, s, NHAc).

4.52 (2H, m, NCH₂CH₂), 6.96 (1H, t, J=5.5 Hz,

NHEt), 7.40–7.48 (1H, m, phenyl), 7.58–7.66 (3H, m,

phenyl); MS m/e (FAB) 467 (MH⁺). Anal. calcd for

C₂₄H₂₇FN₆O₃HCl: C, 57.31, H, 5.61, N, 16.71; found:

C, 57.12, H, 5.76, N, 16.61.

1

.

N1-Ethyl-3-chloro-1-propanesulfonamide (26). Triethyla-

mine (31.2 mL, 224 mmol) was added dropwise to a

suspension of ethylamine hydrochloride (18.2 g, 223

mmol) in CH₂Cl₂(150 mL) over 20 min with stirring at

2–5 ^ꢁC, and 3-chloropropanesulfonyl chloride (25) (13.2

g, 74.6 mmol) was added dropwise at 3–10 ^ꢁC for 1 h.

The mixture was stirred at room temperature for a fur-

ther 19 h. The reaction mixture was poured into H₂O,

and the organicphase was washed with 1 N HCl and

saturated aqueous NaHCO₃, dried over anhydrous

Na₂SO₄, and concentrated under reduced pressure to

give 26 (10.4 g, 75%) as a colorless oil. ¹H NMR

(CDCl₃) d 1.23 (3H, t, J=7.2 Hz, CH₂CH₃), 2.25–2.32

(2H, m, CH₂CH₂CH₂), 3.19 (2H, dq, J=6.4 and 7.2 Hz,

CH₂CH₃), 3.19 (2H, dd, J=6.0 and 7.2 Hz, CH₂S), 3.69

(2H, t, J=6.0 Hz, CH₂Cl), 4.26 (1H, br, NH).

2-[2-(Acetylamino)-5-amino-6-chloro-4-pyrimidinyl]amino]-

ethyl N-ethylcarbamate (6d). This compound (pale yel-

low solid, 73% yield) was prepared in a manner similar

to that described for 6a, except that 5d was used instead

1

of 5a. H NMR (DMSO-d₆) d 1.00 (3H, t, J=7.2 Hz,

CH₂CH₃), 2.09 (3H, br s, Ac), 3.00 (2H, dq, J=5.2 and

7.2 Hz, CH₂CH₃), 3.53–3.63 (2H, m, NCH₂CH₂), 4.13

(2H, t, J=5.8 Hz, CH₂O), 4.72 (2H, br s, NH₂), 7.04

(1H, t, J=5.2 Hz, NH), 7.12 (1H, t, J=5.2 Hz, NH),

9.80 (1H, s, NHAc).

2-[2-Amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-purinyl]-

ethyl N-ethylcarbamate (8d). This compound (white

solid, 51% yield) was prepared in a manner similar to

that described for 8a, except that 6d was used instead of

6a. ¹H NMR (DMSO-d₆) d 0.89 (3H, t, J=7.2 Hz,

CH₂CH₃), 2.84 (2H, dq, J=5.2 and 7.2 Hz, CH₂CH₃),

4.23 (2H, t, J=5.0 Hz, CH₂O), 4.36 (2H, t, J=5.0 Hz,

NCH₂CH₂), 6.96 (1H, t, J=5.2 Hz, NHEt), 7.03 (2H,

br s, NH₂), 7.39–7.46 (1H, m, phenyl), 7.56–7.64 (3H,

m, phenyl).

N1-Ethyl-3-iodo-1-propanesulfonamide (27). A mixture

of 26 (10.0 g, 53.9 mmol) and NaI (24.2 g, 161 mmol) in

2-butanone (200 mL) was stirred under reﬂux for 6.5 h.

The mixture was cooled, the solvent was evaporated,

and the residue was partitioned between EtOAcand

H₂O. The separated organicphase was washed with

brine, dried over Na₂SO₄, and concentrated to give 27

1

(14.0 g, 84%) as a brown oil. H NMR (CDCl₃) d 1.24

2-[6-Chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]ethyl

N-ethylcarbamate (9d). This compound (white solid,

60% yield) was prepared in a manner similar to that

described for 9a, except that 8d was used instead of 8a.

¹H NMR (DMSO-d₆) d 0.86 (3H, t, J=7.2 Hz,

CH₂CH₃), 2.81 (2H, dq, J=5.0 and 7.2 Hz, CH₂CH₃),

4.22 (2H, t, J=4.0 Hz, CH₂O), 4.56 (2H, t, J=4.0 Hz,

NCH₂CH₂), 6.95 (1H, t, J=5.0 Hz, NHEt), 7.48–7.56

(1H, m, phenyl), 7.63–7.76 (3H, m, phenyl).

(3H, t, J=7.2 Hz, CH₂CH₃), 2.29–2.36 (2H, m,

CH₂CH₂CH₂), 3.07–3.26 (4H, m, CH₂CH₃and CH₂S),

3.31 (2H, t, J=6.4 Hz, CH₂I), 4.23 (1H, br, NH).

1-[3-(Ethylamino)sulfonyl]propyl]-1,2-triazadien-2-ium

(28). A mixture of 27 (14.0 g, 45.3 mmol) and NaN₃

(7.6 g, 115 mmol) in H₂O (43 mL)–MeOH (15 mL) was

stirred under reﬂux for 3 h. After having been cooled,

the reaction mixture was diluted with EtOAc, washed

with H₂O (ꢃ2) and brine, then dried over Na₂SO₄, and

1

2-[6-Chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]ethyl N-ethylcarbamate (14d).

This compound (colorless foam, 92% yield) was pre-

pared from 9d in a manner similar to that described for

13a, except that THF was used instead of N,N-dime-

thylformamide as a reaction solvent. ¹H NMR (DMSO-

d₆) d 0.85 (3H, t, J=7.2 Hz, CH₂CH₃), 1.22–1.36 (1H,

m, c-C₆H₁₀), 1.44–1.74 (7H, m, c-C₆H₁₀), 1.86–1.95 (2H,

m, c-C₆H₁₀), 2.80 (2H, dq, J=5.6 and 7.2 Hz,

CH₂CH₃), 4.24 (2H, t, J=4.8 Hz, CH₂O), 4.60 (2H, t,

J=4.8 Hz, NCH₂CH₂), 6.94 (1H, t, J=5.6 Hz, NHEt),

7.48–7.58 (1H, m, phenyl), 7.59–7.78 (3H, m, phenyl).

concentrated to give 28 (6.4 g, 74%) as a brown oil. H

NMR (CDCl₃) d 1.23 (3H, t, J=7.2 Hz, CH₂CH₃),

2.04–2.11 (2H, m, CH₂CH₂CH₂), 3.10 (2H, dd, J=6.0

and 7.2 Hz, CH₂S), 3.19 (2H, dq, J=6.0 and 7.2 Hz,

CH₂CH₃), 3.50 (2H, t, J=6.4 Hz, CH₂N₃), 4.21 (1H, br,

NH).

N1-Ethyl-3-amino-1-propanesulfonamide (29). A solu-

tion of 28 (6.0 g, 31.2 mmol) in MeOH (250 mL) was

hydrogenated in the presence of 10% Pd/C (3.0 g) at

room temperature and atmosphericpressure. The theo-

retical amount of H₂was absorbed within 10 h. The

reaction mixture was ﬁltered through a Celite pad,

which was then washed with MeOH, and the ﬁltrate was

evaporated to give 29 (4.92 g, 95%) as pale yellow nee-

2-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]ethyl N-ethylcarbamate hydro-

chloride (15d). This compound (white solid, 67% yield)

was prepared in a manner similar to that described for

1

dles. H NMR (DMSO-d₆) d 1.07 (3H, t, J=7.2 Hz,

CH₂CH₃), 1.65–1.72 (2H, m, CH₂CH₂CH₂), 2.60 (2H, t,

J=6.4 Hz, CH₂NH₂), 2.94 (2H, q, J=7.2 Hz, CH₂CH₃),

2.98–3.03 (2H, m, CH₂S), 3.12 (2H, br, NH₂).

1

15c, except that 14d was used instead of 14c. H NMR

(DMSO-d₆) d 0.86 (3H, t, J=7.2 Hz, CH₂CH₃), 1.19–

1.32 (1H, m, c-C₆H₁₀), 1.41–1.70 (7H, m, c-C₆H₁₀),

1.80–1.92 (2H, m, c-C₆H₁₀), 2.80 (2H, dq, J=5.5 and

7.2 Hz, CH₂CH₃), 4.19 (2H, t, J=5.7 Hz, CH₂O), 4.44–

N1-[4-Chloro-6-[3-[(ethylamino)sulfonyl]propyl]amino]-5-

nitro-2-pyrimidinyl]acetamide (5e). This compound

2720

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

(yellow powder, 54% yield) was prepared in a manner

similar to that described for 5a, except that 29 was used

instead of 4-amino-1-butanol. H NMR (DMSO-d₆) d

1.05 (3H, t, J=7.2 Hz, CH₂CH₃), 1.90–2.00 (2H, m,

CH₂CH₂CH₂), 2.23 (3H, s, Ac), 2.93 (2H, dq, J=5.6

and 7.2 Hz, CH₂CH₃), 3.00–3.08 (2H, m, CH₂S), 3.58

(2H, dt, J=5.6 and 6.4 Hz, NCH₂CH₂), 6.98 (1H, t,

J=5.6 Hz, NH), 8.63 (1H, t, J=5.6 Hz, NH), 10.85

(1H, s, NHAc).

that described for 15c, except that 14e was used instead

of 14c. ¹H NMR (DMSO-d₆) d 0.93–1.00 (3H, m,

CH₂CH₃), 1.19–1.32 (1H, m, c-C₆H₁₀), 1.40–1.68 (7H,

m, c-C₆H₁₀), 1.77–1.90 (2H, m, c-C₆H₁₀), 1.95–2.14 (2H,

m, CH₂CH₂CH₂), 2.75–2.87 (2H, m, CH₂CH₃), 2.90–

2.98 (2H, m, CH₂S), 4.35–4.42 (2H, m, NCH₂CH₂),

6.94–7.02 (1H, m, NHEt), 7.40–7.48 (1H, m, phenyl),

1

7.58–7.78 (3H, m, phenyl); MS m/e (FAB) 501 (MH⁺).

1

.

Anal. calcd for C₂₄H₂₉FN₆O₃S HCl /₂H₂O: C, 52.79,

H, 5.72, N, 15.39; found: C, 52.83, H, 5.62, N, 15.40.

N1-[5-Amino-4-chloro-6-[3-[(ethylamino)sulfonyl]propyl]-

amino]-2-pyrimidinyl]acetamide (6e). This compound

(pale yellow solid, 57% yield) was prepared in a manner

similar to that described for 6a, except that 5e was used

instead of 5a. ¹H NMR (DMSO-d₆) d 1.05 (3H, t,

J=7.2 Hz, CH₂CH₃), 1.90–2.00 (2H, m, CH₂CH₂CH₂),

2.10 (3H, br s, Ac), 2.95 (2H, dq, J=6.0 and 7.2 Hz,

CH₂CH₃), 3.02–3.10 (2H, m, CH₂S), 3.48 (2H, q, J=6.0

Hz, NCH₂CH₂), 4.70 (2H, br s, NH₂), 7.00 (2H, two t,

J=6.0 Hz, two NH), 9.79 (1H, br s, NHAc).

N1-[4-(3-Cyanoanilino)-5-nitro-6-oxo-1,6-dihydro-2-pyri-

midinyl]acetamide (3f). General procedure. N1-(6-

Chloro-3,4-dihydro-5-nitro-4-oxopyrimidine-2-yl)aceta-

mide (2) (15.0 g, 64.5 mmol) was added to a mixture of

3-aminobenzonitrile (19.0 g, 161 mmol) and glacial ace-

ticacid (9.2 mL, 161 mmol) in THF (150 mL) at 5 ^ꢁC,

and the mixture was stirred at room temperature for 4

h. The precipitated solid was collected by ﬁltration,

washed with THF, H₂O, MeOH, and diethyl ether, and

dried to give 3f (19.0 g, 94%) as a white solid. ¹H NMR

(DMSO-d₆) d 2.20 (3H, s, Ac), 7.60 (1H, t, J=8.0 Hz,

phenyl), 7.68–6.72 (1H, m, phenyl), 7.85–7.90 (1H, m,

phenyl), 8.09 (1H, t, J=2.0 Hz, phenyl), 11.07 (1H, s,

NHPh), 11.70 (2H, br, N1-H and NHAc).

N1-Ethyl-3-[2-amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-

purinyl]-1-propanesulfonamide (8e). This compound

(pale yellow solid, 32% yield) was prepared in a manner

similar to that described for 8a, except that 6e was used

instead of 6a. ¹H NMR (DMSO-d₆) d 0.98 (3H, t,

J=7.2 Hz, CH₂CH₃), 2.03 (2H, quint, J=7.2 Hz,

CH₂CH₂CH₂), 2.85 (2H, dq, J=5.6 and 7.2 Hz,

CH₂CH₃), 2.92 (2H, t, J=7.2 Hz, CH₂S), 4.28 (2H, t,

J=7.2 Hz, NCH₂CH₂), 6.96 (1H, t, J=5.6 Hz, NHEt),

7.03 (2H, br s, NH₂), 7.40–7.48 (1H, m, phenyl), 7.58–

7.78 (3H, m, phenyl).

N1-[4-Chloro-6-(3-cyanoanilino)-5-nitro-2-pyrimidinyl]-

acetamide (5f). General procedure. A mixture of 3f (5.0

g, 15.9 mmol), Et₄NCl (5.3 g, 32.0 mmol), N,N-dime-

thylaniline (4.0 mL, 31.6 mmol), and POCl₃(15.7 mL,

191 mmol) in CH₃CN (100 mL) was heated under reﬂux

for 10 h. After having been cooled, the mixture was

poured into crushed ice (200 g) and vigorously stirred

for 2 h. The resulting precipitate was collected by ﬁltra-

tion, washed with H₂O, MeOH, and Et₂O, and dried to

give 5f (4.27 g, 81%) as a yellow solid. ¹H NMR

(DMSO-d₆) d 2.14 (3H, s, Ac), 7.52–7.52 (2H, m,

phenyl), 8.03–8.09 (1H, m, phenyl), 8.64 (1H, s, phenyl),

10.10 (1H, s, NHPh), 11.14 (1H, s, NHAc).

N1-Ethyl-3-[6-chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-pur-

inyl]-1-propanesulfonamide (9e). This compound (white

solid, 29% yield) was prepared in a manner similar to

that described for 9a, except that 8e was used instead of

8a. ¹H NMR (DMSO-d₆) d 1.01 (3H, t, J=7.2 Hz,

CH₂CH₃), 2.08 (2H, quint, J=7.2 Hz, CH₂CH₂CH₂),

2.88 (2H, dq, J=5.6 and 7.2 Hz, CH₂CH₃), 3.02 (2H, t,

J=7.2 Hz, CH₂S), 4.46 (2H, t, J=7.2 Hz, NCH₂CH₂),

6.98 (1H, t, J=5.6 Hz, NHEt), 7.50–7.57 (1H, m,

phenyl), 7.65–7.73 (3H, m, phenyl).

3-[(2,5-Diamino-6-chloro-4-pyrimidinyl)amino]benzonitrile

(7f). General procedure. A stirred mixture of 5f (3.80 g,

.

11.4 mmol) and SnCl₂2H₂O (12.9 g, 57.2 mmol) in

EtOH (380 mL) was heated to 60 ^ꢁC, and NaBH₄(216

mg, 5.71 mmol) in EtOH (38 mL) was added dropwise.

The mixture was stirred at this temperature for 5.5 h.

After having been cooled, the reaction mixture was

concentrated to dryness, and the residue was diluted

with H₂O. The mixture was neutralized with 1 N

NaOH. The insoluble material was removed by ﬁltra-

tion and washed thoroughly with THF and EtOAc. The

ﬁltrate was diluted with saturated aqueous NH₄Cl. The

separated organicphase was washed with saturated

aqueous NH₄Cl, dried over Na₂SO₄, and evaporated to

give 7f (2.10 g, 71%) as a pinkish solid. ¹H NMR

(DMSO-d₆) d 4.25 (2H, br s, NH₂), 6.09 (2H, br s,

NH₂), 7.39–7.44 (1H, m, phenyl), 7.49 (1H, t, J=8.0

Hz, phenyl), 7.99–8.04 (1H, m, phenyl), 8.35 (1H, t,

J=2.0 Hz, phenyl), 8.63 (1H, s, NHPh).

N1-Ethyl-3-[6-chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]-1-propanesulfona-

mide (14e). This compound (colorless foam, 81% yield)

was prepared from 9e in a manner similar to that

described for 13a, except that THF was used instead of

N,N-dimethylformamide as a reaction solvent. ¹H

NMR (DMSO-d₆) d 1.00 (3H, t, J=7.2 Hz, CH₂CH₃),

1.25–1.37 (1H, m, c-C₆H₁₀), 1.45–1.74 (7H, m, c-C₆H₁₀),

1.86–1.95 (2H, m, c-C₆H₁₀), 2.09 (2H, quint, J=7.6 Hz,

CH₂CH₂CH₂), 2.86 (2H, dq, J=5.6 and 7.2 Hz,

CH₂CH₃), 3.02 (2H, t, J=7.6 Hz, CH₂S), 4.50 (2H, t,

J=7.6 Hz, NCH₂CH₂), 5.71 (1H, s, OH), 6.97 (1H, t,

J=5.6 Hz, NHEt), 7.50–7.58 (1H, m, phenyl), 7.59–7.75

(3H, m, phenyl).

N1-Ethyl-3-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]-1-propanesulfona-

mide hydrochloride (15e). This compound (pale yellow

solid, 53% yield) was prepared in a manner similar to

3-[2-Amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-purinyl]ben-

zonitrile (8f). General procedure. A mixture of crude

2,5-diaminopyrimidine (7f) (8.0 g, 30.7 mmol), 3-

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2721

ﬂuorobenzaldehyde (5.0 mL, 47.5 mmol), and acetic

acid (14.0 mL) in MeOH (450 mL) was stirred at room

temperature for 4.5 h. The reaction mixture was con-

centrated to dryness under reduced pressure. The resi-

due was azeotropically distilled with toluene (ꢃ2), and

used directly in the next step. To a suspension of the

crude imine in EtOH (450 mL) was added a solution of

anhydrous FeCl₃(6.5 g, 40.1 mmol) in EtOH (50 mL) at

room temperature, and the mixture was stirred for 1 h.

The solvent was removed under reduced pressure, and

the residue was suspended in a small amount of EtOH.

The resulting precipitate was ﬁltered oﬀ, washed with

EtOH, and dried to give 8f (7.1 g, 63%) as a slightly

brown solid. The ﬁltrate was evaporated to dryness. The

residue was dissolved in EtOAc, washed with H₂O,

dried over Na₂SO₄, and evaporated. The residue was

suspended in a small amount of EtOH, and the resulting

precipitate was ﬁltered oﬀ, washed with EtOH, and

dried to give additional 8f (2.1 g, 19%) as a slightly

brown solid. ¹H NMR (DMSO-d₆) d 7.12 (2H, s, NH₂),

7.21–7.34 (3H, m, 8-phenyl), 7.40–7.46 (1H, m, 8-

phenyl), 7.72–7.81 (2H, m, 9-phenyl), 7.98–8.03 (1H, m,

9-phenyl), 8.09–8.12 (1H, m, 9-phenyl).

stirred at room temperature for 4 h. The precipitated

solid was collected by ﬁltration, washed with H₂O, and

dried to give the desired compound (1.14 g) as the free

form. This was suspended in EtOH, 6 N HCl was

added, and the solvent was evaporated to give 15f (1.14

1

g, 67%) as a white solid. H NMR (DMSO-d₆) d 1.15–

1.28 (1H, m, c-C₆H₁₀), 1.25–1.63 (7H, m, c-C₆H₁₀),

1.72–1.80 (2H, m, c-C₆H₁₀), 7.22–7.30 (3H, m, 8-

phenyl), 7.37–7.43 (1H, m, 8-phenyl), 7.54–7.58 (1H, m,

9-phenyl), 7.60–7.64 (1H, m, 9-phenyl), 7.77 (1H, br,

CONH₂), 7.89–7.91 (1H, m, 9-phenyl), 8.01–8.04 (1H,

m, 9-phenyl), 8.08 (1H, br s, CONH₂); MS m/e (FAB)

+

3

.

471 (MH ). Anal. calcd for C₂₆H₂₃FN₆O₂HCl /₄H₂O:

C, 60.00, H, 4.94, N, 16.15; found: C, 60.19, H, 4.72, N,

16.38.

N1-[4-(4-Cyanoanilino)-5-nitro-6-oxo-1,6-dihydro-2-pyri-

midinyl]acetamide (3g). This compound (pale yellow

solid, quantitative yield) was prepared in a manner

similar to that described for 3f, except that 4-amino-

benzonitrile was used instead of 3-aminobenzonitrile.

¹H NMR (DMSO-d₆) d 2.19 (3H, s, Ac), 7.83 (4H, s,

phenyl), 11.09 (1H, br s, NHPh), 11.70 (2H, br, N1-H

and NHAc).

3-[6-Chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]ben-

zonitrile (9f). This compound (white solid, 50% yield)

was prepared in a manner similar to that described for

9a, except that 8f was used instead of 8a. ¹H NMR

(CDCl₃) d 7.19–7.27 (2H, m, 8-phenyl), 7.31–7.40 (2H,

m, 8-phenyl), 7.57–7.60 (1H, m, 9-phenyl), 7.64–7.66

(1H, m, 9-phenyl), 7.68–7.72 (1H, m, 9-phenyl), 7.84–

7.87 (1H, m, 9-phenyl).

N1-[4-Chloro-6-(4-cyanoanilino)-5-nitro-2-pyrimidinyl]a-

cetamide (5g). This compound (yellow powder, 86%

yield) was prepared in a manner similar to that descri-

1

bed for 5f, except that 3g was used instead of 3f. H

NMR (DMSO-d₆) d 2.15 (3H, s, Ac), 7.77–7.81 (2H, m,

phenyl), 8.16–8.21 (2H, m, phenyl), 10.17 (1H, br,

NHPh), 11.16 (1H, br s, NHAc).

3-[6-Chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzonitrile (14f). This com-

pound (pale yellow solid, 70% yield) was prepared from

9f in a manner similar to that described for 13a, except

that THF was used instead of N,N-dimethylformamide

4-[(2,5-Diamino-6-chloro-4-pyrimidinyl)amino]benzonitrile

(7g). This compound (brown powder, 88% yield) was

prepared in a manner similar to that described for 7f,

except that 5g was used instead of 5f. ¹H NMR

(DMSO-d₆) d 4.32 (2H, br s, NH₂), 6.09 (2H, br s,

NH₂), 7.71 (2H, d, J=8.8 Hz, phenyl), 8.03 (2H, d,

J=8.8 Hz, phenyl), 8.76 (1H, br, NHPh).

1

as a reaction solvent. H NMR (CDCl₃) d 1.27–1.40

(1H, m, c-C₆H₁₀), 1.57–1.78 (7H, m, c-C₆H₁₀), 2.01–

2.08 (2H, m, c-C₆H₁₀), 2.13 (1H, s, OH), 7.18–7.24

(1H, m, 8-phenyl), 7.25–7.28 (1H, m, 8-phenyl), 7.32–

7.40 (2H, m, 8-phenyl), 7.55–7.58 (1H, m, 9-phenyl),

7.66–7.72 (2H, m, 9-phenyl), 7.83–7.86 (1H, m, 9-

phenyl).

4-[2-Amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-purinyl]ben-

zonitrile (8g). This compound (ocher powder, 76%

yield) was prepared in a manner similar to that descri-

1

bed for 8f, except that 7g was used instead of 7f. H

NMR (DMSO-d₆) d 7.11 (2H, br s, NH₂), 7.18–7.23

(1H, m, 8-phenyl), 7.24–7.34 (2H, m, 8-phenyl), 7.39–

7.46 (1H, m, 8-phenyl), 7.68 (2H, d, J=8.4 Hz, 9-

phenyl), 8.05 (2H, d, J=8.4 Hz, 9-phenyl).

3-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzonitrile (16f). This com-

pound (pale yellow solid, 73% yield) was prepared in a

manner similar to that described for 15c, except that 14f

1

was used instead of 14c. H NMR (DMSO-d₆) d 1.19–

4-[6-Chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-purinyl]ben-

zonitrile (9g). This compound (white solid, 70% yield)

was prepared in a manner similar to that described for

1.31 (1H, m, c-C₆H₁₀), 1.38–1.66 (7H, m, c-C₆H₁₀),

1.75–1.83 (2H, m, c-C₆H₁₀), 5.50 (1H, s, OH), 7.20–7.24

(1H, m, 8-phenyl), 7.27–7.34 (2H, m, 8-phenyl), 7.40–

7.47 (1H, m, 8-phenyl), 7.69 (2H, br s, NH₂), 7.73–7.81

(2H, m, 9-phenyl), 8.02 (1H, dt, J=2.0 and 7.2 Hz, 9-

phenyl), 8.04–8.06 (1H, m, 9-phenyl).

1

9a, except that 8g was used instead of 8a. H NMR

(DMSO-d₆) d 7.26–7.30 (1H, m, 8-phenyl), 7.33–7.44

(2H, m, 8-phenyl), 7.46–7.53 (1H, m, 8-phenyl), 7.72–

7.76 (2H, m, 9-phenyl), 8.09–8.14 (2H, m, 9-phenyl).

3-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzamide hydrochloride (15f).

General procedure. A mixture of 16f (1.40 g, 3.10

mmol), 1 N NaOH (1.55 mL, 1.55 mmol), and 30%

H₂O₂(1.55 mL, 13.66 mmol) in MeOH (70 mL) was

4-[6-Chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzonitrile (14g). This com-

pound (pale yellow solid, 67% yield) was prepared from

9g in a manner similar to that described for 13a, except

that THF was used instead of N,N-dimethylformamide

2722

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

1

as a reaction solvent. H NMR (DMSO-d₆) d 1.22–1.34

(1H, m, c-C₆H₁₀), 1.39–1.52 (3H, m, c-C₆H₁₀), 1.54–1.70

(4H, m, c-C₆H₁₀), 1.80–1.89 (2H, m, c-C₆H₁₀), 5.67 (2H,

s, OH), 7.28–7.32 (1H, m, 8-phenyl), 7.35–7.44 (2H, m,

8-phenyl), 7.47–7.54 (1H, m, 8-phenyl), 7.73–7.78 (2H,

m, 9-phenyl), 8.10–8.14 (2H, m, 9-phenyl).

Ethyl 3-[2-amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-puri-

nyl]benzoate (8i). This compound (pale brown solid,

79% yield) was prepared in a manner similar to that

described for 8f, except that 7i was used instead of 7f.

¹H NMR (DMSO-d₆) d 1.31 (3H, t, J=7.2 Hz,

CH₂CH₃), 4.33 (2H, q, J=7.2 Hz, CH₂CH₃), 7.09 (2H,

br s, NH₂), 7.22–7.32 (3H, m, 8-phenyl), 7.36–7.45 (1H,

m, 8-phenyl), 7.65–7.74 (2H, m, 9-phenyl), 8.06–8.12

(2H, m, 9-phenyl).

4-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzonitrile (16g). This com-

pound (pale yellow solid, 79% yield) was prepared in a

manner similar to that described for 15c, except that 14g

Ethyl 3-[6-chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-puri-

nyl]benzoate (9i). This compound (white solid, 66%

1

was used instead of 14c. H NMR (DMSO-d₆) d 1.22–

1.34 (1H, m, c-C₆H₁₀), 1.38–1.68 (7H, m, c-C₆H₁₀),

1.74–1.84 (2H, m, c-C₆H₁₀), 5.50 (1H, s, OH), 7.19–7.24

(1H, m, 8-phenyl), 7.27–7.34 (2H, m, 8-phenyl), 7.40–

7.48 (1H, m, 8-phenyl), 7.66 (2H, d, J=8.4 Hz, phenyl),

7.70 (2H, br s, NH₂), 8.05 (2H, d, J=8.4 Hz, phenyl).

yield) was prepared in a manner similar to that descri-

1

bed for 9a, except that 8i was used instead of 8a. H

NMR (DMSO-d₆) d 1.31 (3H, t, J=7.2 Hz, CH₂CH₃),

4.34 (2H, q, J=7.2 Hz, CH₂CH₃), 7.30–7.42 (3H, m, 8-

phenyl), 7.44–7.51 (1H, m, 8-phenyl), 7.70–7.82 (2H, m,

9-phenyl), 8.14–8.19 (2H, m, 9-phenyl).

4-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzamide hydrochloride (15g).

This compound (white solid, 85% yield) was prepared

in a manner similar to that described for 15f, except that

Ethyl 3-[6-chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclo-

hexyl)-1-ethynyl]-9H-9-purinyl]benzoate (14i). This com-

pound (white solid, 74% yield) was prepared from 9i in

a manner similar to that described for 13a, except that

THF was used instead of N,N-dimethylformamide as a

1

16g was used instead of 16f. H NMR (DMSO-d₆) d

1.16–1.28 (1H, m, c-C₆H₁₀), 1.35–1.64 (7H, m, c-C₆H₁₀),

1.72–1.81 (2H, m, c-C₆H₁₀), 7.22–7.29 (3H, m, 8-

phenyl), 7.39–7.44 (1H, m, 8-phenyl), 7.50 (2H, d,

J=8.2 Hz, 9-phenyl), 7.55 (1H, s, CONH₂), 7.99 (2H, d,

J=8.2 Hz, 9-phenyl), 8.13 (1H, s, CONH₂); MS m/e

1

reaction solvent. H NMR (DMSO-d₆) d 1.31 (3H, t,

J=7.2 Hz, CH₂CH₃), 1.24–1.35 (1H, m, c-C₆H₁₀), 1.40–

1.52 (3H, m, c-C₆H₁₀), 1.52–1.70 (4H, m, c-C₆H₁₀),

1.80–1.88 (2H, m, c-C₆H₁₀), 4.33 (2H, q, J=7.2 Hz,

CH₂CH₃), 5.65 (1H, s, OH), 7.32–7.42 (3H, m, 8-

phenyl), 7.44–7.52 (1H, m, 8-phenyl), 7.73–7.82 (2H, m,

9-phenyl), 8.14–8.18 (2H, m, 9-phenyl).

(FAB)

471

.

(MH⁺).

Anal.

calcd

for

3

.

C₂₆H₂₃FN₆O₂HCl /₄H₂O: C, 60.00, H, 4.94, N, 16.15;

found: C, 60.00, H, 4.74, N, 16.10.

Ethyl

3-[2-(acetylamino)-5-nitro-6-oxo-1,6-dihydro-4-

Ethyl

3-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

pyrimidinyl]amino]benzoate (3i). This compound (pale

yellow solid, 94% yield) was prepared in a manner

similar to that described for 3f, except that ethyl 3-ami-

nobenzoate was used instead of 3-aminobenzonitrile. ¹H

NMR (DMSO-d₆) d 1.33 (3H, t, J=7.2 Hz, CH₂CH₃),

2.17 (3H, s, Ac), 4.34 (2H, q, J=7.2 Hz, CH₂CH₃), 7.56

(1H, t, J=8.0 Hz, phenyl), 7.82–7.86 (1H, m, phenyl),

7.88–7.91 (1H, m, phenyl), 7.96 (1H, t, J=2.0 Hz,

phenyl), 11.06 (1H, s, NHPh), 11.63 (2H, br, N1-H and

NHAc).

cyclohexyl)-1-ethynyl]-9H-9-purinyl]benzoate (16i). This

compound (white solid, 90% yield) was prepared in a

manner similar to that described for 15c, except that 14i

1

was used instead of 14c. H NMR (DMSO-d₆) d 1.18–

1.28 (1H, m, c-C₆H₁₀), 1.31 (3H, t, J=7.2 Hz,

CH₂CH₃), 1.38–1.66 (7H, m, c-C₆H₁₀), 1.74–1.83 (2H,

m, c-C₆H₁₀), 4.33 (2H, q, J=7.2 Hz, CH₂CH₃), 5.49

(1H, s, OH), 7.22–7.33 (3H, m, 8-phenyl), 7.38–7.45

(1H, m, 8-phenyl), 7.66 (2H, br s, NH₂), 7.68–7.72 (2H,

m, 9-phenyl), 8.00–8.03 (1H, m, 9-phenyl), 8.07–8.13

(1H, m, 9-phenyl).

Ethyl 3-[(2-amino-6-chloro-5-nitro-4-pyrimidinyl)amino]-

benzoate (5i). This compound (yellow solid, 78% yield)

was prepared in a manner similar to that described for

5f, except that 3i was used instead of 3f. ¹H NMR

(DMSO-d₆) d 1.30 (3H, t, J=7.2 Hz, CH₂CH₃), 2.07

(3H, s, Ac), 4.30 (2H, q, J=7.2 Hz, CH₂CH₃), 7.49 (1H,

t, J=8.0 Hz, phenyl), 7.70–7.75 (1H, m, phenyl), 8.07

(1H, t, J=2.0 Hz, phenyl), 8.39–8.44 (1H, m, phenyl),

10.10 (1H, s, NHPh), 10.95 (1H, s, NHAc).

3-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzoic acid (17i). General pro-

cedure. A mixture of 16i (798 mg, 1.60 mmol) and 1 N

NaOH (30 mL) in CHCl₃–MeOH (2:1) (90 mL) was

stirred at room temperature for 5 h. The mixture was

acidiﬁed with 1 N HCl, and the precipitated solid was

collected by ﬁltration, washed with H₂O, and dried to

give 17i (644 mg, 86%) as a white solid. ¹H NMR

(DMSO-d₆) d 1.16–1.27 (1H, m, c-C₆H₁₀), 1.37–1.65

(7H, m, c-C₆H₁₀), 1.72–1.81 (2H, m, c-C₆H₁₀), 5.49 (1H,

br s, OH), 7.20–7.30 (3H, m, 8-phenyl), 7.36–7.43 (1H,

m, 8-phenyl), 7.59–7.71 (4H, m, 9-phenyl and NH₂),

7.93–7.96 (1H, m, 9-phenyl), 8.03–8.09 (1H, m, 9-

phenyl).

Ethyl

3-[(2,5-diamino-6-chloro-4-pyrimidinyl)amino]-

benzoate (7i). This compound (brown solid, 82% yield)

was prepared in a manner similar to that described for

7f, except that 5i was used instead of 5f. ¹H NMR

(DMSO-d₆) d 1.34 (3H, t, J=7.2 Hz, CH₂CH₃), 4.28

(2H, br, NH₂), 4.32 (2H, q, J=7.2 Hz, CH₂CH₃), 5.92

(2H, br s, NH₂), 7.43 (1H, t, J=8.0 Hz, phenyl), 7.54–

7.60 (1H, m, phenyl), 8.10 (1H, t, J=2.0 Hz, phenyl),

8.04–8.40 (1H, m, phenyl), 8.59 (1H, br, NHPh).

N1-Ethyl-3-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]benzamide hydro-

chloride (15i). General procedure. A mixture of 17i (200

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2723

1

mg, 0.424 mmol), 1-hydroxybenzotriazole (200 mg, 1.31

mmol), and 1-ethyl-3-(3⁰-dimethylaminopropyl)carbo-

diimide (200 mg, 1.29 mmol) in DMF (5.0 mL) was

stirred at room temperature for 4 h. Ethylamine hydro-

chloride (110 mg, 1.35 mmol) and triethylamine (0.18

mL, 1.29 mmol) were added, and the mixture was stir-

red for an additional 13 h. The solvent was removed in

vacuo, and the residue was puriﬁed by silica gel column

chromatography (eluent CH₂Cl₂, CH₂Cl₂/MeOH=40:1,

20:1) to give the title compound (136 mg) as the free

form. This was suspended in MeOH, 20% HCl/EtOH

was added, and the solvent was evaporated. The residue

was diluted with Et₂O, and the precipitated product was

ﬁltered oﬀ and washed with Et₂O to give 15i (126 mg,

56%) as a white solid. ¹H NMR (DMSO-d₆) d 1.10 (3H,

t, J=7.2 Hz, CH₂CH₃), 1.14–1.28 (1H, m, c-C₆H₁₀),

1.34–1.64 (7H, m, c-C₆H₁₀), 1.71–1.81 (2H, m, c-C₆H₁₀),

3.26 (2H, dq, J=5.6 and 7.2 Hz, CH₂CH₃), 7.22–7.30

(3H, m, 8-phenyl), 7.37–7.44 (1H, m, 8-phenyl), 7.53–

7.57 (1H, m, 9-phenyl), 7.62 (1H, t, J=8.0 Hz, 9-

phenyl), 7.89 (1H, m, 9-phenyl), 8.00 (1H, m, 9-phenyl),

8.61 (1H, t, J=5.6 Hz, NHEt); MS m/e (FAB) 499

for 8f, except that 7h was used instead of 7f. H NMR

(DMSO-d₆) d 0.87 (3H, t, J=7.2 Hz, CH₂CH₃), 3.88–

4.01 (2H, m, CH₂CH₃), 7.03 (2H, br s, NH₂), 7.16–7.30

(3H, m, 8-phenyl), 7.36–7.42 (1H, m, 8-phenyl), 7.68–

7.74 (2H, m, 9-phenyl), 7.83 (1H, dt, J=1.6 and 8.0 Hz,

9-phenyl), 8.01 (1H, dd, J=1.6 and 8.0 Hz, 9-phenyl).

Ethyl 2-[6-chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-puri-

nyl]benzoate (9h). This compound (white powder, 65%

yield) was prepared in a manner similar to that descri-

1

bed for 9a, except that 8h was used instead of 8a. H

NMR (DMSO-d₆) d 0.85 (3H, t, J=7.2 Hz, CH₂CH₃),

3.88–4.00 (2H, m, CH₂CH₃), 7.26–7.32 (2H, m, 8-

phenyl), 7.35–7.41 (1H, m, 8-phenyl), 7.43–7.50 (1H, m,

8-phenyl), 7.78–7.83 (2H, m, 9-phenyl), 7.91 (1H, dt,

J=1.6 and 8.0 Hz, 9-phenyl), 8.08 (1H, dd, J=1.6 and

8.0 Hz, 9-phenyl).

Ethyl 2-[6-chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycy-

clohexyl)-1-ethynyl]-9H-9-purinyl]benzoate (14h). This

compound (white solid, 48% yield) was prepared from

9h in a manner similar to that described for 13a, except

that THF was used instead of N,N-dimethylformamide

+

5

.

(MH ). Anal. calcd for C₂₈H₂₇FN₆O₂HCl /₄H₂O: C,

60.32, H, 5.51, N, 15.07; found: C, 60.03, H, 5.68, N,

15.33.

1

as a reaction solvent. H NMR (DMSO-d₆) d 0.83 (3H,

t, J=7.2 Hz, CH₂CH₃), 1.20–1.32 (1H, m, c-C₆H₁₀),

1.37–1.51 (3H, m, c-C₆H₁₀), 1.51–1.69 (4H, m, c-C₆H₁₀),

1.78–1.88 (2H, m, c-C₆H₁₀), 3.92 (2H, q, J=7.2 Hz,

CH₂CH₃), 5.65 (1H, s, OH), 7.26–7.34 (2H, m, 8-

phenyl), 7.34–7.41 (1H, m, 8-phenyl), 7.44–7.50 (1H, m,

8-phenyl), 7.78–7.86 (2H, m, 9-phenyl), 7.93 (1H, dt,

J=1.6 and 8.0 Hz, 9-phenyl), 8.08 (1H, dd, J=1.6 and

8.0 Hz, 9-phenyl).

Ethyl

2-[2-(acetylamino)-5-nitro-6-oxo-1,6-dihydro-4-

pyrimidinyl]amino]benzoate (3h). This compound (pale

yellow solid, 80% yield) was prepared in a manner

similar to that described for 3f, except that ethyl 2-ami-

nobenzoate was used instead of 3-aminobenzonitrile. ¹H

NMR (DMSO-d₆) d 1.30 (3H, t, J=7.2 Hz, CH₂CH₃),

2.18 (3H, s, Ac), 4.32 (2H, q, J=7.2 Hz, CH₂CH₃), 7.36

(1H, t, J=8.0 Hz, phenyl), 7.66 (1H, dt, J=1.6 and 8.0

Hz, phenyl), 7.97 (1H, dd, J=1.6 and 8.0 Hz, phenyl),

8.02 (1H, d, J=8.0 Hz, phenyl), 11.67 (2H, br, two

NH), 12.08 (1H, br s, NH).

Ethyl 2-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclo-

hexyl)-1-ethynyl]-9H-9-purinyl]benzoate (16h). This

compound (white powder, 95% yield) was prepared in a

manner similar to that described for 15c, except that 14h

1

was used instead of 14c. H NMR (DMSO-d₆) d 0.86

Ethyl 2-[(2-amino-6-chloro-5-nitro-4-pyrimidinyl)amino]-

benzoate (5h). This compound (ocher powder, 91%

yield) was prepared in a manner similar to that descri-

(3H, t, J=7.2 Hz, CH₂CH₃), 1.20–1.30 (1H, m, c-

C₆H₁₀), 1.38–1.66 (7H, m, c-C₆H₁₀), 1.73–1.82 (2H, m,

c-C₆H₁₀), 3.87–3.96 (2H, m, CH₂CH₃), 5.47 (1H, s,

OH), 7.16–7.29 (3H, m, 8-phenyl), 7.36–7.43 (1H, m, 8-

phenyl), 7.61 (2H, br s, NH₂), 7.66 (1H, dd, J=1.2 and

8.0 Hz, 9-phenyl), 7.72 (1H, dt, J=1.2 and 8.0 Hz, 9-

phenyl), 7.83 (1H, dt, J=1.6 and 8.0 Hz, 9-phenyl), 8.00

(1H, dd, J=1.6 and 8.0 Hz, 9-phenyl).

1

bed for 5f, except that 3h was used instead of 3f. H

NMR (DMSO-d₆) d 1.32 (3H, t, J=7.2 Hz, CH₂CH₃),

2.12 (3H, s, Ac), 4.35 (2H, q, J=7.2 Hz, CH₂CH₃), 7.29

(1H, t, J=8.0 Hz, phenyl), 7.65 (1H, dt, J=1.6 and 8.0

Hz, phenyl), 8.01 (1H, dd, J=1.6 and 8.0 Hz, phenyl),

8.93 (1H, d, J=8.0 Hz, phenyl), 11.15 (1H, br s,

NHPh), 11.67 (1H, br s, NHAc).

2-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzoic acid (17h). This com-

pound (white solid, 85% yield) was prepared in a man-

ner similar to that described for 17i, except that 16h was

Ethyl

2-[(2,5-diamino-6-chloro-4-pyrimidinyl)amino]-

benzoate (7h). This compound (yellow solid, 93% yield)

was prepared in a manner similar to that described for

7f, except that 5h was used instead of 5f. ¹H NMR

(DMSO-d₆) d 1.34 (3H, t, J=7.2 Hz, CH₂CH₃), 3.88

(2H, br s, NH₂), 4.34 (2H, q, J=7.2 Hz, CH₂CH₃), 6.22

(2H, br s, NH₂), 7.07 (1H, t, J=8.0 Hz, phenyl), 7.57

(1H, dt, J=1.6 and 8.0 Hz, phenyl), 7.98 (1H, dd,

J=1.6 and 8.0 Hz, phenyl), 8.92 (1H, d, J=8.0 Hz,

phenyl), 10.72 (1H, br s, NHPh).

1

used instead of 16i. H NMR (DMSO-d₆) d 1.15–1.28

(1H, m, c-C₆H₁₀), 1.33–1.62 (7H, m, c-C₆H₁₀), 1.70–1.80

(2H, m, c-C₆H₁₀), 7.16–7.26 (3H, m, 8-phenyl), 7.33–

7.40 (1H, m, 8-phenyl), 7.57 (1H, dd, J=1.2 and 8.0 Hz,

9-phenyl), 7.60 (2H, br s, NH₂), 7.68 (1H, dt, J=1.2 and

8.0 Hz, 9-phenyl), 7.88 (1H, dt, J=1.6 and 8.0 Hz, 9-

phenyl), 8.02 (1H, dd, J=1.6 and 8.0 Hz, 9-phenyl).

N1-Ethyl-2-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

Ethyl 2-[2-amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-puri-

nyl]benzoate (8h). This compound (ocher solid, 81%

yield) was prepared in a manner similar to that described

cyclohexyl)-1-ethynyl]-9H-9-purinyl]benzamide hydro-

chloride (15h). This compound (pale yellow solid, 42%

yield) was prepared in a manner similar to that described

2724

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

for 15i, except that 17h was used instead of 17i. ¹H

NMR (DMSO-d₆) d 0.80 (3H, t, J=7.2 Hz, CH₂CH₃),

1.16–1.27 (1H, m, c-C₆H₁₀), 1.35–1.63 (7H, m, c-C₆H₁₀),

1.70–1.81 (2H, m, c-C₆H₁₀), 2.80–2.91 (1H, m,

CH₂CH₃), 2.92–3.03 (1H, m, CH₂CH₃), 7.17–7.25 (2H,

m, 8-phenyl), 7.26–7.30 (1H, m, 8-phenyl), 7.32–7.41

(1H, m, 8-phenyl), 7.44–7.49 (1H, m, 9-phenyl), 7.58–

7.66 (3H, m, 9-phenyl), 8.28–8.34 (1H, m, NHEt); MS

that THF was used instead of N,N-dimethylformamide

1

as a reaction solvent. H NMR (DMSO-d₆) d 1.35 (3H,

t, J=7.2 Hz, CH₂CH₃), 1.23–1.32 (1H, m, c-C₆H₁₀),

1.40–1.53 (3H, m, c-C₆H₁₀), 1.53–1.70 (4H, m, c-C₆H₁₀),

1.79–1.89 (2H, m, c-C₆H₁₀), 4.37 (2H, q, J=7.2 Hz,

CH₂CH₃), 5.65 (1H, s, OH), 7.31–7.42 (3H, m, 8-

phenyl), 7.45–7.53 (1H, m, 8-phenyl), 7.68 (2H, d,

J=8.2 Hz, 9-phenyl), 8.16 (2H, d, J=8.2 Hz, 9-phenyl).

m/e (FAB) 499 (MH⁺). Anal. calcd for

1

.

C₂₈H₂₇FN₆O₂HCl /₂H₂O: C, 61.82, H, 5.37, N, 15.45;

found: C, 61.50, H, 5.53, N, 15.42.

Ethyl

4-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]benzoate (16j). This

compound (white solid, 77% yield) was prepared in a

manner similar to that described for 15c, except that 14j

Ethyl

4-[2-(acetylamino)-5-nitro-6-oxo-1,6-dihydro-4-

1

pyrimidinyl]amino]benzoate (3j). This compound (yellow

solid, 89% yield) was prepared in a manner similar to

that described for 3f, except that ethyl 4-aminobenzoate

was used instead of 3-aminobenzonitrile. ¹H NMR

(DMSO-d₆) d 1.33 (3H, t, J=7.2 Hz, CH₂CH₃), 2.21

(3H, s, Ac), 4.32 (2H, q, J=7.2 Hz, CH₂CH₃), 7.77–7.82

(2H, m, phenyl), 7.94–7.99 (2H, m, phenyl), 11.17 (1H,

br s, NHPh), 11.68 (1H, br, NH), 11.73 (1H, br s, NH).

was used instead of 14c. H NMR (DMSO-d₆) d 1.20–

1.30 (1H, m, c-C₆H₁₀), 1.35 (3H, t, J=7.2 Hz,

CH₂CH₃), 1.38–1.66 (7H, m, c-C₆H₁₀), 1.75–1.83 (2H,

m, c-C₆H₁₀), 4.36 (2H, q, J=7.2 Hz, CH₂CH₃), 5.49

(1H, s, OH), 7.20–7.33 (3H, m, 8-phenyl), 7.39–7.46

(1H, m, 8-phenyl), 7.56–7.60 (2H, m, 9-phenyl), 7.68

(2H, br s, NH₂), 8.08–8.13 (2H, m, 9-phenyl).

4-[6-Amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclohexyl)-

1-ethynyl]-9H-9-purinyl]benzoic acid (17j). This com-

pound (white solid, 93% yield) was prepared in a man-

ner similar to that described for 17i, except that 16j was

Ethyl 4-[(2-amino-6-chloro-5-nitro-4-pyrimidinyl)amino]-

benzoate (5j). This compound (yellow solid, 91% yield)

was prepared in a manner similar to that described for

5f, except that 3j was used instead of 3f. ¹H NMR

(DMSO-d₆) d 1.33 (3H, t, J=7.2 Hz, CH₂CH₃), 2.15

(3H, s, Ac), 4.30 (2H, q, J=7.2 Hz, CH₂CH₃), 7.93 (2H,

d, J=8.6 Hz, phenyl), 8.12 (2H, d, J=8.6 Hz, phenyl),

10.15 (1H, br s, NHPh), 11.11 (1H, br s, NHAc).

1

used instead of 16i. H NMR (DMSO-d₆) d 1.18–1.30

(1H, m, c-C₆H₁₀), 1.38–1.66 (7H, m, c-C₆H₁₀), 1.74–1.84

(2H, m, c-C₆H₁₀), 5.53 (1H, br s, OH), 7.22–7.32 (3H,

m, 8-phenyl), 7.39–7.46 (1H, m, 8-phenyl), 7.52–7.57

(2H, m, 9-phenyl), 7.66 (2H, br s, NH₂), 8.06–8.12 (2H,

m, 9-phenyl).

Ethyl

4-[(2,5-diamino-6-chloro-4-pyrimidinyl)amino]-

benzoate (7j). This compound (brown solid, 82% yield)

was prepared in a manner similar to that described for

7f, except that 5j was used instead of 5f. ¹H NMR

(DMSO-d₆) d 1.32 (3H, t, J=7.2 Hz, CH₂CH₃), 4.28

(2H, q, J=7.2 Hz, CH₂CH₃), 4.33 (2H, br s, NH₂), 6.03

(2H, br s, NH₂), 7.86–7.92 (2H, m, phenyl), 7.94–8.00

(2H, m, phenyl), 8.68 (1H, br s, NHPh).

N1-Ethyl-4-[6-amino-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxy-

cyclohexyl)-1-ethynyl]-9H-9-purinyl]benzamide hydro-

chloride (15j). This compound (white solid, 49% yield)

was prepared in a manner similar to that described for

1

15i, except that 17j was used instead of 17i. H NMR

(DMSO-d₆) d 1.12 (3H, t, J=7.2 Hz, CH₂CH₃), 1.16–

1.28 (1H, m, c-C₆H₁₀), 1.36–1.64 (7H, m, c-C₆H₁₀),

1.72–1.82 (2H, m, c-C₆H₁₀), 3.29 (2H, dq, J=5.6 and

7.2 Hz, CH₂CH₃), 7.21–7.31 (3H, m, 8-phenyl), 7.38–

7.45 (1H, m, 8-phenyl), 7.50 (2H, d, J=8.0 Hz, 9-

phenyl), 7.95 (2H, d, J=8.0 Hz, 9-phenyl), 8.64 (1H, t,

Ethyl 4-[2-amino-6-chloro-8-(3-ﬂuorophenyl)-9H-9-puri-

nyl]benzoate (8j). This compound (white powder, 63%

yield) was prepared in a manner similar to that descri-

1

bed for 8f, except that 7j was used instead of 7f. H

J=5.6 Hz, NHEt); MS m/e (FAB) 499 (MH⁺). Anal.

1

.

NMR (DMSO-d₆) d 1.34 (3H, t, J=7.2 Hz, CH₂CH₃),

4.35 (2H, q, J=7.2 Hz, CH₂CH₃), 7.08 (2H, br s, NH₂),

7.21–7.32 (3H, m, 8-phenyl), 7.38–7.45 (1H, m, 8-

phenyl), 7.60 (2H, d, J=8.4 Hz, 9-phenyl), 8.09 (2H, d,

J=8.4 Hz, 9-phenyl).

calcd for C₂₈H₂₇FN₆O₂HCl /₄H₂O: C, 62.33, H, 5.32,

N, 15.58; found: C, 62.35, H, 5.40, N, 15.52.

Materials. NECA was purchased from Sigma (St.

Louis, MO, USA). Compounds 1,⁶FK453,¹⁹and

KF17837^20cwere synthesized as reported. [³H]-2-

Chloro-N⁶-cyclopentyladenosine ([³H]CCPA) and [³H]-

2-[4-(2-carboxyethyl)phenyl]ethylamino]-5⁰-N-ethylcar-

boxamidoadenosine ([³H]CGS21680) were obtained

from NEN Life Science, Inc. (Boston, MA, USA).

Ethyl

4-[6-chloro-8-(3-ﬂuorophenyl)-2-iodo-9H-9-puri-

nyl]benzoate (9j). This compound (pale yellow powder,

46% yield) was prepared in a manner similar to that

described for 9a, except that 8j was used instead of 8a.

¹H NMR (DMSO-d₆) d 1.35 (3H, t, J=7.2 Hz, CH₂CH₃),

4.37 (2H, q, J=7.2 Hz, CH₂CH₃), 7.28–7.42 (3H, m, 8-

phenyl), 7.44–7.52 (1H, m, 8-phenyl), 7.66 (2H, d, J=8.4

Hz, 9-phenyl), 8.16 (2H, d, J=8.4 Hz, 9-phenyl).

Pharmacology

inhibitory activity on neca-induced glucose production in

primary cultured rat hepatocytes. Hepatocytes were iso-

lated by collagenase type I (Gibco BRL Products,

Tokyo, Japan) digestion from livers of 5-week-old male

Wistar rats (Charles River Japan Inc., Atsugi, Japan).

Isolated hepatocytes were cultured in William’s E

Ethyl 4-[6-chloro-8-(3-ﬂuorophenyl)-2-[2-(1-hydroxycyclo-

hexyl)-1-ethynyl]-9H-9-purinyl]benzoate (14j). This com-

pound (white powder, 85% yield) was prepared from 9j

in a manner similar to that described for 13a, except

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

2725

medium. After having been cultured for 24 h, hepato-

cytes were washed and then incubated in 500 mL of

Krebs Ringer bicarbonate buﬀer containing 0.1%

bovine serum albumin for 30 min. To each well was

added 100 mL of solution containing 0.6 mM NECA and

the test antagonist (appropriate concentration). One h

thereafter, glucose released from the hepatocytes was

assayed using a commercial kit (Glucose CII-test

WAKO, Wako Pure Chemical Industries, Ltd., Osaka,

Japan).

with human adenosine A_2Areceptor cDNA^3bwere used

in this assay. The A_2Areceptor binding assay was per-

formed in a manner similar to that described for A₁

binding assay, except that 500 nM [³H]CGS21680 was

used as the radioligand.

Acknowledgement

Inhibitory activity on NECA-induced cyclic AMP pro-

duction in CHO.K1 cells expressing human adenosine

A_2breceptor. A Chinese hamster ovary (CHO.K1) cell

stably transfected with human adenosine A_2Breceptor

cDNA^4bwas used in this assay. Cells were cultured

under a 5% CO₂/95% O₂atmosphere at 37 ^ꢁC in D-

MEM/F-12 (1:1 mixture) medium (Gibco BRL Pro-

ducts, Tokyo, Japan) with 10% fetal calf serum (Gibco

BRL Products, Tokyo, Japan), 100 U/mL penicillin

(Gibco BRL Products, Tokyo, Japan), 100 U/mL

streptomycin (Gibco BRL Products, Tokyo, Japan),

and 1 mg/mL G418 (Gibco BRL Products, Tokyo,

Japan). Experimental cultures were grown overnight as

a monolayer in 24-well tissue culture plates (1.5ꢃ10⁵

cells/well). Each well was washed twice with 2 mL of

Krebs buﬀer and then incubated in 0.5 mL of this buﬀer

for 30 min. To each well was added 100 mL of a solution

containing 600 mM phosphodiesterase inhibitor Ro20-

1724 (Research Biochemicals Inc., Natick, MA, USA),

180 nM NECA, 6 U/mL adenosine deaminase, and the

test compound (appropriate concentration). After incu-

bation for 15 min, the reaction was terminated by

removing the medium and adding 0.1 N HCl (300 mL/

well). The intracellular cyclic AMP contents were mea-

sured using a commercial radioimmunoassay (cyclic

AMP EIA Kit, Amersham, Chicago, IL, USA).

The authors thank the staﬀ of Eisai Analytical Chemistry

Section for mass spectra and elemental analysis.

References and Notes

1. Poulsen, S.-A.; Quinn, R. J. Bioorg. Med. Chem. 1998, 6,

619.

2. (a) Libert, F.; Schiﬀmann, S. N.; Lefort, A.; Parmentier,

M.; Gerard, C.; Dumont, J. E.; Vanderhaeghen, J. J.; Vassart,

G. EMBO J. 1991, 10, 1677. (b) Libert, F.; van Sande, J.;

Lefort, A.; Czernilofsky, A.; Dumont, J. E.; Vassart, G.;

Ensinger, H. A.; Mendla, K. D. Biochem. Biophys. Res. Com-

mun. 1992, 187, 919.

3. (a) Maenhaut, C.; Sande, J. V.; Libert, F.; Abramowicz,

M.; Parmentier, M.; Vanderhaeghen, J. J.; Dumont, J. E.;

Vassart, G.; Schiﬀmann, S. Biochem. Biophys. Res. Commun.

1990, 173, 1169. (b) Furlong, T. J.; Pierce, K. D.; Selbie, L. A.;

Shine, J. Mol. Brain Res. 1992, 15, 62.

4. (a) Stehle, J. H.; Rivkees, S. A.; Lee, J. J.; Weaver, D. R.;

Deeds, J. D.; Reppert, S. M. Mol. Endocrinol. 1992, 6, 384.

5. (b) Pierce, K. D.; Furlong, T. J.; Selbie, L. A.; Shine, J.

Biochem. Biophys. Res. Commun. 1992, 187, 86. (a) Zhou,

Q. Y.; Li, C. Y.; Olah, M. E.; Johnson, R. A.; Stiles, G. L.;

Civelli, O. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7432. (b)

Salvatore, C. A.; Jacobson, M. A.; Tailor, H. E.; Linden, J.;

Johnson, R. G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10365.

6. Harada, H.; Asano, O.; Hoshino, Y.; Yoshikawa, S.; Mat-

sukura, M.; Kabasawa, Y.; Niijima, J.; Kotake, Y.; Wata-

nabe, N.; Kawata, T.; Inoue, T.; Horizoe, T.; Yasuda, N.;

Minami, H.; Nagata, K.; Murakami, M.; Nagaoka, J.;

Kobayashi, S.; Tanaka, I.; Abe, S. J. Med. Chem. 2001, 44,

170.

Human adenosine A₁receptor binding assay. CHO.K1

cells stably transfected with human adenosine A₁recep-

tor cDNA^2bwere used in this assay. The transfected

cells were maintained under a 5% CO₂/95% O₂atmo-

sphere at 37 ^ꢁC in D-MEM/F-12 (1:1 mixture) medium

with 10% fetal calf serum, 100 U/mL penicillin, 100 U/

mL streptomycin, and 1 mg/mL G418. Conﬂuent

monolayers of the cells were detached by 1 mM EDTA

in phosphate-buﬀered saline, then sonicated for about

15 s on ice, and centrifuged for 15 min at 50,000g and

4 ^ꢁC. The membrane pellet was resuspended in HEPES

buﬀer (20 mM HEPES, 10 mM MgCl₂, 100 mM NaCl,

pH 7.4). The binding assay was performed in a total

volume of 500 mL containing membrane suspension, 60

nM [³H]CCPA (25 mL), the appropriate concentration

of test antagonist (25 mL), and 1 U/mL adenosine dea-

minase. The mixture was incubated for 2 h at 25 ^ꢁC, and

ﬁltered, and the collected membranes were washed twice

with 5 mL of ice-cold buﬀer containing 50 mM Tris–

HCl (pH 7.4) using a Brandel cell harvester. The radio-

activity of the bound ligand was measured using a liquid

scintillation counter.

7. Prasad, R. N.; Bariana, D. S.; Fung, A.; Savic, M.; Tietje,

K.; Stein, H. H.; Brondyk, H.; Egan, R. S. J. Med. Chem.

1980, 23, 313.

8. Muller, C. E.; Stein, B. Curr. Pharm. Des 1996, 2, 501.

¨

9. de Zwart, M.; Link, R.; von Frijtag Drabbe Kunzel, J. K.;

¨

Cristalli, G.; Jacobson, K. A.; Townsend-Nicholson, A.;

Ijzerman, A. P. Nucleosides Nucleotides 1998, 17, 969.

10. Cristalli, G.; Eleuteri, A.; Vittori, S.; Volpini, R.; Lohse,

M. J.; Klotz, K.-N. J. Med. Chem. 1992, 35, 2363.

11. Homma, H.; Watanabe, Y.; Abiru, T.; Murayama, T.;

Momura, Y.; Matsuda, A. J. Med. Chem. 1992, 35, 2881.

12. Temple, C., Jr.; Smith, B. H.; Montgomery, J. A. J. Org.

Chem. 1975, 40, 3141.

13. Nair, V.; Young, D. A. J. Org. Chem. 1985, 50, 406.

14. Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless,

K. B. J. Org. Chem. 1981, 46, 3936.

15. Matsuda, A.; Shinozaki, M.; Yamaguchi, T.; Homma, H.;

Nomoto, R.; Miyasaka, T.; Watanabe, Y.; Abiru, T. J. Med.

Chem. 1992, 35, 241.

16. (a) Buck, J. S.; Ide, W. S. Org. Synth. II, 44. (b) Noller, C.

R. Org. Synth. II, 586.

Human adenosine A_2Areceptor binding assay. Human

embryonic kidney (HEK-293) cells stably transfected

17. Satoh, T.; Mitsuo, N.; Nishiki, M.; Inoue, Y.; Ooi, Y.

Chem. Pharm. Bull. 1981, 29, 1443.

2726

H. Harada et al. / Bioorg. Med. Chem. 9 (2001) 2709–2726

18. Sheehan, J. C.; Hlavka, J. J. J. Org. Chem. 1956, 21, 439.

19. Akahane, A.; Katayama, H.; Mitsunaga, T.; Kita, Y.;

Kusunoki, T.; Terai, T.; Yoshida, K.; Shinokawa, Y. Bioorg.

Med. Chem. Lett. 1996, 7, 2059.

20. (a) Shimada, J.; Suzuki, F.; Nonaka, H.; Ishii, A.; Ichi-

kawa, S. J. Med. Chem. 1992, 35, 2342. (b) Nonaka, Y.; Shi-

mada, J.; Nonaka, H.; Koike, N.; Aoki, N.; Kobayashi, H.;

Kase, H.; Yamaguchi, K.; Suzuki, F. J. Med. Chem. 1993, 36,

3731. (c) Suzuki, F.; Shimada, J.; Ishii, A.; Nonaka, H.;

Kosaka, N.; Ichikawa, S. PCT Patent WO, 92/06976, 1992.

21. Cheng, Y. C.; Prusoﬀ, W. H. Biochem. Pharmacol. 1973,

22, 3099.

Article Doi

2-Alkynyl-8-aryladenines possessing an amide moiety: Their synthesis and structure-activity relationships of effects on hepatic glucose production induced via agonism of the A2B adenosine receptor

DOI: 10.1016/S0968-0896(01)00201-2

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