Title 熱帯熱マラリア原虫チミジル酸キナーゼ阻害活性を有する炭素環ヌクレオシド誘導体の合成( 本文（Fulltext) ) Author(s) 野口, 義紘 Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第22号 Issue Date 2013-06-30 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/47881 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Synthesis of carbocyclic nucleosides possessing inhibitory activities
against Plasmodium falciparum thymidylate kinase
i
Synthesis of carbocyclic nucleosides possessing inhibitory activities against Plasmodium falciparum thymidylate kinase
1 1 2 3 4 5 S -Adenosyl-L-homocysteine hydrolase 6 2 1 2 (1) 3 (2) 4 (1) 5 (2) 6 7 3 1 2 3 4 6- -2-5 6 4
ii 2 2 2 3 2 4 2 5 2 6 2 7 3 2 3 3 3 4 3 5 3 6
1
1 “mal aria” (Plasmodium spp.) (Anopheles) (P. falciparum) (P. vivax) (P. ovale) (P. malariae) 4 4 2004 (P. Knowlesi) P. Knowlesi 5 (Table 1) 1, 2) Table 1. * (P. falciparum) 7 14 / A R D S D I C (P. vivax) 12 17 48 (P. ovale) 11 18 48 (P. malariae) 18 40 72 ( ) (P. Knowlesi) 10 12 24 * 2 3/ARDA DIC 5 6 1) 1910 3) (Figure 1.) 4) Figure 1. ( (WHO) 2011 7 )
5) (Aedes polynesiensis) (Anopheles punctulatus) (An. farauti) 2100 m 6) (AIM ) 100 1 3 7) (HYV) HYV 5) 8)
2 3500 9) (Figure 2.) 10) Figure 2. 1500 (Atharva-Veda) 1400
( ) 11) 5 (Figure 3.) 12) 9000 22000 1930 5000 9000 1946 28,200 13) 2 Fugure 3. 5
1999 4 ( ) 50 80 1999 4 112 2000 154 2001 109 100 2002 83 2003 78 2007 50 (Figure 4.) 14) 2001 2003 SARS Figure 4. . (1999 4 2009 ) n = 902 (IDWR 2010 38 ) 2010 6 1 1 (70 )
3
Cinchona ( )
17
Cinchona Cardinal Juan de Lugo
1765 James Lind Cinchona
1820 Pelletier Caventou Cinchona
(Quinine) (Figure 5.) Cinchona Cinchona 1 1910 (Chloroquine) (Figure 6.) 1934 1946 Figure 6. Chloroquine. Figure 5. Quinine.
(Figure 7.) 15)
Fp9 (ferriprotoporphyrin IX) hemozoin Fp9
Fp9 hemozoin
1960
(Figure 8.) 16)
(Mefloquine) (Halofantrine) (Figure 9.)
Figure 8. ( : CDC Travelers’ Health, 2010) Figure 7.
(Artemisia annua)
(artemisinin) 1970
(Figure 10.)
(Artemisia annua)
( ( ) )
Figure 10. (Artemisia annua) Artemisinin.
(artemisinin-based combination therapy ACT) Figure 9. Mefloquine ( ) Halofantrine ( )
(Figure 11.) 17) 45 1 3 ( ) ( ) ( ) ( ) 16 24 Figure 11. : Vol. 81, No. 7, 1999
4
purine HIV ddI ara-A
pyrimidine HIV AZT
IDU (Figure 12.) Figure 12. 18-22) Sterptomuces citricolor (aristeromycin) (Figure 13.)
23) (Table 2.) Table 2.
H
F
2.2 4.0 1.14 Å 1.29 Å CH3-X (X H, F) 105 kcal/mol 110 kcal/mol - -P450 1 1 (Figure 14. )-F
⇔
-OH
⇔
-NH
2⇔
-CH
3Figure 14. 24)
5 S -Adenosyl-L-homocysteine hydrolase
DNA mRNA
mRNA 5’
mRNA (Figure 15.) DNA
DNA DNA
X
S - a d e n o s y l
-L-methionine (SAM) SAM SAM-dependent methyltransferase
S -Adenosyl-L-homocysteine (SAH)
SAH SAM SAM-dependent methyl-transferase
SAH SAH
hydrolase (SAHH) SAHH SAH adenosine L-homocysteine
SAH (Figure 16.)18, 24 - 26)
Figure 16. SAH
Palmer Abeles SAHH 27) 1976
NAD+ NAD+
SAHH Figure 17. SAHH
432
1 mol NAD+ NAD+
Open form
SAH Closed form
NAD+ NADH SAH 3’
3’-keto 4’
4’ homocysteine β
3’-keto-4’,5’-didehydro-5’-deoxyadenosine
H2O Michael 3’-keto-adenosine NADH
adenosine Open form L- homocysteine
Figure 17. SAH
in vitro SAH (Keq = 106 M)
in vivo adenosine adenosine monophosphanate inosine
L-homocysteine L-cysteine L-methionine
28, 29)
SAHH in vitro SAH
adenosine L-homocysteine SAH
in vivo SAH SAM
SAH adenosine L-homocysteine
SAH adenosine adenosine deaminase
adenosine kinase adenosine monophosphate
adenosine DNA ATP
adenosine
cystathionine-β-synthase
betaine-methionine methyltransferase
L-methionine SAM 30)
SAHH
SAHH
SAHH SAH mRNA
mRNA
SAHH
SAHH SAHH
S-Adenosyl-L-homocysteine (Pf SAHH)
SAHH Type I mechanism-based inhibitor
Type II mechanism-based inhibitor Type III mechanism-based inhibitor
Type I 3’ NAD+ 3’
Type II 5’
(Figure 18.) 31) Type III 3’ 5’
Open form
(aristeromycin) Type I
SAHH adenosine
Figure 18. Type I Type II
SAHH 4
(noraristeromycin)
2
SAHH (Hs SAHH) Pf SAHH
(Table 3.) 32 - 34)
Table 3. 2-substituted noraristeromycinst
Compound -X IC50 (μM) Selective index Hs SAHH Pf SAHH 1a H 1.1 3.1 0.35 1b F 63 13 4.8 1c NH2 60 18 3.3 2 (Table 4.)
Table 4. 2-substituted aristeromycins Compound -X IC50 (μM) Selective index Hs SAHH Pf SAHH 2a H 4.85 57 0.085 2b F 47.2 1.98 24 2c NH2 90.7 4.51 20 Pf SAHH Hs SAHH Pf SAHH Hs SAHH adenosine adenosine 2 Hs SAHH Thr60 Pf SAHH Cys59 (Figure 19., 20A., 20B.) 35) 2 SAHH Pf SAHH Figure.20. Hs SAHH Pf SAHH adenosine 4’ (Table 5.) 36) Figure 19.
Table 5. Carbocyclic fluoro nucleosides .
N.A. :No inhibitory activity showed at 1000 µM.
Hs SAHH (3a) Pf SAHH (4b)
DHCaA 10 1 Compound IC50 (μM) Selective index Hs SAHH Pf SAHH DHCaA 9 18 0.5 3a 200 N.A. < 0.2 3b N.A. N.A. - 4a N.A. N.A. - 4b N.A. 220 > 4.5
6
(Thymidylate kinase; TMK) Nucleoside monophosphate
kinase (NMPK) de novo Mg2+ ATP TMP TDP (TPP) Figure 21. (Hs TMK) ATP TMP Figure 21. Hs TMK ATP TMP Salvage de novo Salvage de novo TDP DNA (Figure 22.)
Figure 22. Pf TMK
(Pf TMK) (Hs TMK)
18)
Pf TMK Hs TMK NMP
(nucleoside monophosphate)
Pf TMK Saccharomyces cerevisiae E. coli
27-41% Hs TMK
39 % (Figure 23.) 37)
Hs TMK Pf TMK Pf TMK (Figure 24.) 38) Figure 24. Hs TMK Pf TMK Pf TMK Hs TMK Pf TMK α-2‒α3 α-5‒α-6
α-8 Hs TMK His66 Lys109 Phe156
Pf TMK Glu68 Ala111 Thr158 Pf TMK TMK Pf TMK TMP dUMP Hs TMK dGMP Pf TMK (Table 6., Figure 25.) 38)
Table 6. nucleosides monophosphate . Nucleoside monophosphate Concentration
(mM) Activity (%) TMP 1 100 dGMP 1 86 dUMP 10 43 dIMP 10 15 GMP 10 1.5 AMP/dAMP/CMP/dCMP/UMP Up to 5a N.D.b a
Substrate concentrations are 0.02, 0.1, 0.5, 1, 2 and 5 mM. b
Activity was not detectable.
TMP TMP dGMP
Figure 25. Hs TMK Pf TMK nucleosides monophosphate
Pf TMK
(Table 7.) 39)
Table 7. Nucleoside analogues .
Cpd. R1 R2 R3 R4 KiTMP(μM) KidGMP(μM) 5a NH2 H H OH N.D. N.D. 5b NH2 H OH OH N.D. N.D. 5c OH NH2 OH OH N.D. N.D. 5d OH NH2 H OH 270 80 5e OH H H OH N.D. N.D. Cpd. X1 X2 X3 Ki TMP(μM) KidGMP(μM) 6a H OH OH N.D. N.D. 6b CH3 OH OH 22 18 6c H NH2 OH N.D. N.D. 6d Br OH OH 68 60 6e I OH OH 175 170 6f CH3 OH N3 470 56
The Ki values are KiTMP and KidGMP when the substrate is TMP and dGMP, respectively. N.D.: Activity was not detectable.
Pf TMK (dG) Pf TMK dG dGMP kinase TMK TMP Ki 22 µM dGMP 18 µM 5 5 38) (Table 8.) 40, 41)
Table 8. Pf TMK carbocyclic thymidine analogues . Compound Thymidine 7a 7b 7c Ki (μM) TMP 22 355 246 206 dGMP 18 103 89 36 Compound 7d 7e 7f 7g Ki (μM) TMP 20 N.A. 2500 208 dGMP 7 1540 67 55 Compound 7h 7i Ki (μM) TMP 86 N.A. dGMP 47 N.A.
N.A. : No inhibitory activity was detectible at 10 μM concentration of the compound.
TMP dGMP (7d) TMP dGMP Ki 20 μM 7 μM (7e) (7f) (7g) 2’ 3’ PfTMK 2’ 3’ 2’ 3’ (7d) (7h)
5’ 5’
Pf TMK Hs TMK
2
1 1 PfTMK 1 6 Table 8. 5 4’ (Figure 26.) Figure 26. .2 (1)
(2-acetoxycyclopent-3-en-1-yl)methyl acetate (13)
( diacetate (13)) 42 - 45) (Scheme 1, 2)
Dicyclopentadiene 180-190 oC cyclopentadiene (8) Glyoxylic acid
4 2-oxabicyclo[3,3,0]oct-7en-3-one (9) ( hydroxylactone (9))
33% (Scheme 1.)
Scheme 1.
Hydroxylactone (9) LiAlH4 triol (10) 32%
NaIO4 Malaprade Glycol NaBH4
diol (12) 62%
diacetate (13) 78%
(Scheme 2.) diacetate (13)
3 (2)
2 hydroxylactone (9)
endo-hydroxylactone (9A) exo- (9B) 2
endo-(1R, 4S, 5S) (9Aa) endo-(1S, 4R, 5R) (9Ab) exo-(1S, 4S,
5R) (9Ba) exo- (1R, 4S, 5S) (9Bb) 4 (Figure
27.)
Figure 27. hydroxylactone (9) 4
hydroxylactone (9) LiAlH4 triol (10) 4
4 (Figure 28.)
Figure 28.
Figure 28.
5’
endo-(1R, 4S, 5S)-hydroxylactone (9Aa)
endo-hydroxylactone (9A) exo- (9B)
46) (9Aa) endo- (9A) 47) (Scheme 3.) 44) (Scheme 4.) 44) (Scheme 5.) (Scheme 3.) Scheme 3. (14) (15) chiral derivatizing
agent endo-hydroxylactone (9A) (16A) (16B)
30 g (16A) (16B)
Scheme 4.
endo-hydroxylactone (9A) butyryl chloride (13A)
SD (1R,4S,5S) endo-hydroxylactone (9Aa) 28% 44) (9Aa) (18Aa) HPLC 95%ee (Scheme 5.) Scheme 5. endo-Hydroxylactone (9A) SD (1R,4S,5S) (9Aa) acetoxylactone (19Aa) 42% (Scheme 4.) (Scheme 5.)
(1R,4S,5S)-hydroxylactone (9Aa) (1R,4S,5S)-acetoxylactone (19Aa) 48)
Triol (10Aa) LiAlH4
EtOH 68% (Scheme 6.)
Scheme 6.
48)
triol (10Aa) triacetate (20)
65% triacetate (20) TLC
tert-butyldiphenylsilyl
(TBDPS) (21)
(22) 49, 50) (Scheme 7.)
4 (1)
Diacetate (17) thymine (Scheme 7, 8.)
Thymine DMSO NaH Pd[(C6H5)3P]4
2 diacetate (17) -allylpalladium(0)
(7c) 48) (Scheme 8.)
Scheme 8.
(7d) N,N-diethylaminosulfur trifluoride (DAST)
(23) 48%
(Scheme 9.)
5 (2)
Triacetate (20) thymine (Scheme 9, 10.)
4 diacetate (17) thymine
2
thymine N-benzoyl
(Scheme 10.)
Scheme 10.
3-benzoylthymine (24) DMSO NaH
Pd[(C6H5)3P]4 3 triacetate (20) -allylpalladium(0) (25) 47% (Scheme 11.) Scheme 11. benzoyl acetyl (26) 96% N,N-diethylaminosulfur trifluoride (DAST) (27) 21% 0 oC 30 (26) quench (Scheme 12.)
6
Triacetate (20) uracil (Scheme 13 - 15.)
5 uracil
N-benzoyl (Scheme 13.)
Scheme 13.
3-benzoyluracil (28) DMSO NaH
Pd[(C6H5)3P]4 2 2 triacetate (20) -allylpalladium(0) (29) 41% (Scheme 14.) Scheme 14. benzoyl acetyl (30) 89% N,N-diethylaminosulfur trifluoride (DAST) (31) 25% 5 0 oC 30 (30) quench (Scheme 15.)
7 PfTMK 1 mL (Pf TMK) 30 oC 38) (Figure 29.)
Figure 29. Enzyme assay .
PfTMK NADH NAD+ NADH 335nm 335nm Ki (Figure 30.) Figure 30. Table 9. (23) (±) (25) (26) (27) (29) (30) (31) v = V max[S] / (Km + [S])･････････････(1) 1/v = (K m / Vmax)(1 / [S]) + 1 / Vmax･･･(2) K` m = K``m(1 + [I] / Ki)･･････････････(3)
Table 9. Pf TMK carbocyclic pyrimidine nucleoside analogues . Compound 7d 7h 23 Ki (μM) TMP 20 86 50 dGMP 7 47 21 Compound 25 26 27 Ki (μM) TMP 295 142 14 dGMP 183 260 20 Compound 29 30 31 Ki (μM) TMP N.A. 1238 N.A. dGMP 525 1259 45
N.A. : No inhibitory activity was detectible at 10 μM concentration of the compound.
(7d) 5’ (23) Ki TMP 50 μM dGMP 21 μM (7d) (23) (4.0) (3.49) 51) (23) 1 7
(7d), (7h) 5’ (7d; CH2OH) (23; CH2F) (7h; OH) 4’ (27) Ki TMP 14 μM dGMP 20 μM TMP (7d) 5’ (27) (26) 5’ (26) 6’ (29) (30) (31) TMP Ki N.A. 1238μM N.A. 5- Pf TMK TMP Pf TMK (Figure 31A, 31B, 31C.) Figure 31.
Ligand group in Pf TMK (A, B). TMP is shown in green (A) and included in the binding cavity (B, C) in the surface representation.
TMP
dGMP deoxyguanylate
(31) Ki 45μM (27)
3
1 Pf TMK Hs TMK TMP dGMP Pf TMK 2 7 Table 9. Pf TMK (Figure 32.) Figure 32. .2
2-Aamino-6-chloropurine DMSO NaH
Pd[(C6H5)3P]4 2 3 triacetate (20) -allylpalladium(0) 2 4 (32) (Scheme 16.) Scheme 16. 2-Amino-6-chloro (32) 0.2N NaOH 6 (33) 50 v/v% CF3CO2H (Scheme 17.) Scheme 17. 50 v/v% CF3CO2H (33) 2 6 (33) 77%
3
2 6-chloropurine DMSO NaH
Pd[(C6H5)3P]4 Triacetate (20) -allylpalladium(0) (34) 6-chloro (34) 50 v/v% CF3CO2H 2 6 (35) 78% (Scheme 18.) Scheme 18.
4 6-
-2-6-
52, 53)
4-amino-5-imidazolecarboxamide hydrochloride trifluoroacetamide 180
o
C 5 ether
2-trifluoromethyl-6-hydroxypurine (36) 87% (36)
dry CH3CN N,N’-dimethylaniline POCl3
95 oC 5
15 hexane
2-trifluoromethyl-6-chloropurine (37) 89% (Scheme 19.)
Scheme 19.
2 2-trifluoromethyl-6-chloropurine (37)
DMSO NaH Pd[(C6H5)3P]4 triacetate (20)
-allylpalladium(0) (38) (Scheme
20.)
5 54, 55) 2,4-dihydro-3-nitropyridine POCl3 2 2,4-dichloro-3-nitropyridine (39) 77% (39) DMF Et3N p-methoxybenzylamine (PMB-NH2) 4 PMB-NH (40) (40) Raney-Ni HCl (41) 52% (Scheme 21.) Scheme 21. (41) HC(OEt)3 Ac2O deazapurine (42) 82% trifluoroacetic acid (42) PMB 6-chloro-3-deazapurine (43) 60% (Scheme 22.) Scheme 22.
2 6-chloro-3-deazapurine (43) DMSO NaH Pd[(C6H5)3P]4 triacetate (20) -allylpalladium(0) (44) 50 v/v% CF3CO2H 2 6 (45) 66% (Scheme 23.) Scheme 23.
6 Pf TMK 2 7 dGMP ATP Figure 33A. ATP TMP dGMP (
Competitive inhibition) Figure 33B. ATP ( Uncompetitive inhibition) Figure 33C. ATP TMP dGMP ( Liner mixed inhibition) ATP TMP dGMP (Non detectable inhibition) Figure 33.
A
B
C
Table 10., Table 11.
Table 10. Pf TMK carbocyclic purine nucleoside analogues (1).
Substrate : TMP
I. (Liner mixed inhibition)
Cpd. 34
Ki (μM) 568
II. (Uncompetitive inhibition)
Cpd. 35 32 33 Ki (μM) 590 87 440 Cpd 44 45 38 Ki (μM) 550 412 635 TMP (Competitive inhibition) (Figure 34.) Pf TMK ATP TMP ( dGMP) Figure 34.
TMP ATP (Uncompetitive inhibition)
(34) (Liner mixed inhibition) ATP dGMP
Table 11. Pf TMK carbocyclic purine nucleoside analogues (2).
Substrate : dGMP
I. (Competitive inhibition) II. (Liner mixed inhibition)
Cpd. 33 Cpd. 34
Ki (μM) 93 Ki (μM) 162
III. Uncompetitive inhibition
Cpd. 35 32 44
Ki (μM) 235 250 314
IV. Non detectable inhibition
Cpd 45 38
Ki (μM) N.A. N.A.
dGMP (33) Ki 93 μM (Figure 35.) TMP 5 TMP dGMP Figure 35.
4
100 100 (Pf TMK) 5 4’ Pf TMK Pf TMK I. (2-acetoxycyclopent-3-en-1-yl)methyl acetate (13) thymine (7d) (23) (S)-1-((1S,2R)-2-acetoxycyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) N-benzoylthymine (24) N-benzoyluracil (28) (25) (29) (26) (30) (27) (31) Pf TMK (27) TMP Ki 14 μM dGMP Ki 20.4 μM II. (20) 2-amino-6-chloropurine6-chloropurine 2-trifluoromethyl-6-chloropurine (37) 6-chloro-
3-deazapurine (43) (32) (34) (38) (44)
(33) (35) (45)
(33) Ki 93 μM TMP (Competitive inhibition) Pf TMK Pf TMK Hs TMK dGMP Pf TMK X Pf TMK Hs TMK Pf TMK
( )
Mahmoud Kandeel
Pyridine CaH2 4 Å
DMF 4 Å
THF
1
H, 13C NMR JEOL JNM ECS-400 spectrometer (400 MHz for 1H-NMR,
100 MHz for 13C-NMR) 1H (δ)
(TMS) 0.00 ppm CDCl3 7.26 ppm, DMSO-d6 2.49
ppm, CD3OD 3.31 ppm 13C
CDCl3 77.0 ppm, DMSO-d6 39.5 ppm, CD3OD 49.0 ppm
JEOL The AccuTOF LC-plus JMS-T100LP
Yanaco MP-J3
60N ( )
HITACHI U-2001 spectrophotometer SHIMADZU RF-5300PC
2
2
1,3-Cyclopentadiene (8)
Dicyclopentadiene (197.2 g, 200 mL, 1.49 mol) 180-190 oC
5 1,3-cyclopentadiene (8) (149.5 g, 2.26 mol, 76 %)
2-Oxabicyclo[3,3,0]oct-7en-3-one (9)
1,3-Cyclopentadiene (8) (56.7 g, 0.86 mol) 50 % glyoxylic acid (61 mL, 1.12 mol )
4 TLC (hexane : EtOAc = 1 : 2 )
EtOAc Na2SO4
(hexane : EtOAc = 1 : 1 ) 2-oxabicyclo[3,3,0]oct-7en-3-one (9) (39.8 g, 0.28 mol, 33 %)
1-(2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10)
2-Oxabicyclo[3,3,0]oct-7en-3-one (9) (2.67 g, 19.1 mmol) dry THF (37.5 mL) LiAlH4 (0.85 g, 22.4 mmol) 70°C 2 H2O (0.85 mL) 15% NaOH (0.85 mL) H2O (2.6 mL) 10 1-(2-hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10) (0.874 g, 6.07 mmol, 32%) 5-(Hydroxymethyl)cyclopent-2-enol (12)
1-(2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10) (10.5 g, 72.8 mmol, 1.0 eq.) diethyl ether (400 mL) H2O (400 mL)
NaIO4 (19.6 g, 91.6 mmol, 1.25 eq.) 1 1
ethylene glycol (1.73 mL, 31.1 mmol, 0.43 eq.) 1 1
H2O EtOH (150 mL)
NaBH4 (3.47 g, 91.6 mmol, 1.25 eq.) 1
1 ethylene glycol
sat NaCl EtOAc
(CHCl3 : MeOH = 9 : 1 → 1 : 1 ) 5-(hydroxymethyl)cyclopent-2-enol (12) (3.26 g, 28.6 mmol, 39%) 1 H-NMR (CDCl3, 400 MHz) δ 6.02 (m, 1H, H-4 or H-5), 5.84 (m, 1H, H-4 or H-5), 4.93 (m, 1H, H-1), 3.81 (m, 2H, C2-CH2), 2.38‒2.73 (m, 3H, H-2 and H-3), 2.24 (m, 2H, OH) 2-Acetoxycyclopent-3-en-1-yl)methyl acetate (13)
5-(Hydroxymethyl)cyclopent-2-enol (12) (687 mg, 6.02 mmol, 1.0 eq.) CH2Cl2 (10 mL) Et3N (3.8 mL, 27.2 mmol, 4.5 eq.)
Ac2O (2.5 mL, 27.1 mmol, 4.5 eq.) 3 TLC
(hexane : EtOAc = 1 : 2 ) 24
24 5% HClaq. 2,3 CHCl3
sat.NaHCO3aq., sat.NaClaq. Na2SO4
(hexane EtOAc = 5 : 1 → 1 : 1 ) 2-acetoxycyclopent-3-en-1-yl)methyl acetate (13) (930 mg, 4.69 mmol, 78%) 1 H-NMR (CDCl3, 400 MHz) δ 6.10 (m, 1H, H-4 or H-5), 5.79 (m, 2H, H-4 or H-5 and H-1), 4.18 (m, 2H, C2-CH2), 2.70 - 2.49 (m, 2H, H-3), 2.25 (m, 1H, H-2), 2.07 (6H, dd, CH3).
2
3
endo-2-Oxabicyclo[3,3,0]oct-7en-3-one(9A)
1,3-Cyclopentadiene (8) (60 g, 0.91 mol) 50 % glyoxylic acid (95 mL, 2.95 mol )
40 oC 8
hexane (50mL × 4) 1,3-cyclopentadiene
NaCl EtOAc (50 mL × 10) EtOAc
CO2 sat.NaHCO3 aq. Na2SO4
NaHCO3 NaCl EtOAc Na2SO4
EtOAc Ether
Ether-hexane (9:1) Ether endo-2-oxabicyclo[3,3,0]oct-
7en-3-one (9A) (14.7 g, 0.10 mol, 11.5%)
endo-4-Butyryloxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (17A)
endo-2-Oxabicyclo[3,3,0]oct-7en-3-one (9A) (8.363 g, 59.7 mmol) dry CH2Cl2 (60
mL) dry pyridine (7.08 g [7.2 mL], 89.6 mmol, 1.5 equiv.)
0 oC butyryl chloride (6.99 g, 89.6 mmol) dry CH2Cl2
(6mL) 1 3 sat.NaCl aq. 1 sat.NaCl aq. 10%HCl (5mL) 2 1 sat.NaHCO3 aq. 2 Na2SO4 (CHCl3 → CHCl3 : acetone = 100 : 1 ) endo-4-butyryloxy-2-oxabicyclo-[3,3,0]oct-7-en-3- one (17Aa) (12.5 g) (1R,4S,5R)-4-Hydroxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (9Aa)
endo-4-Butyryloxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (17A) (3.0 g, 14.3 mmol) lipase PS Amano SD (70 mg) phosphate buffer (0.1M KH2PO4 pH 7.0, 29 mL)
35 oC 30 pH pH 6.5-7.0
7 pH overnight brine
AcOEt Na2SO4
CHCl3 :
acetone = 100 : 1 ester (17Ab) CHCl3 : acetone = 10 : 1
(1R,4S,5R)-4-hydroxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (9Aa) (552 mg, 28%, 95%ee) (18Aa)
(3S,3aS,6aR)-2-Oxo-3,3a,4,6a-tetrahydro-2H-cyclopenta[b]furan-3-yl 4-methyl- benzoate (18Aa)
(1R,4S,5R)-4-Hydroxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (9Aa) (70 mg, 0.5 mmol) dry pyridine (61 μL, 0.75 mmol) dichloromethane (0.5 mL) 0 oC
p-toluoyl chloride (93 mg, 0.6 mmol)
sat.NaCl aq. dichloromethane sat.NaHCO3 aq. Na2SO4
hexane AcOEt-hexane (3S,3aS,6aR)-2-oxo-
3,3a,4,6a-tetrahydro-2H-cyclopenta[b]furan-3-yl 4-methyl-benzoate (18Aa) (93 mg, 72%, 95%ee)
Colorless prisms, mp 142 oC. Anal. Calcd for C15H14O4 : C, 69.76; H, 5.46 Fonud: C, 69.48;
H, 5.36.
(18Aa) 1A CHCl3: hexane =
3 : 7 endo-2-oxabicyclo[3,3,0]oct-7en-3-one (9A) (18A)
(3S,3aS,6aR)-2-Oxo-3,3a,4,6a-tetrahydro-2H-cyclopenta[b]furan-3-yl acetate (19Aa)
endo-2-Oxabicyclo[3,3,0]oct-7en-3-one (9A) (1.4 g, 1.0 mmol) vinyl acetate (50 mL) triethylamine (1.0 mL) lipase PS Amano SD (1.0 g)
(CHCl3 : acetone = 10 : 1) (3S,3aS,6aR)-2-oxo-3,3a,4,6a-tetrahydro-2H-
cyclopenta[b]furan-3-yl acetate (19Aa) (0.76 g, 42 %)
1
H-NMR (CDCl3, 400 MHz) δ 6.24 - 6.18 (m, 1H), 5.98 - 5.92 (m, 1H), 5.62 (dd, 1H, J =
9.6 Hz, 3.6 Hz), 5.41 - 5.35 (m, 1H), 3.41 - 3.31 (m, 1H), 2.60 - 2.37 (m, 2H), 2.19 (d, 3.6 Hz).
(S)-1-((1S,2R)-2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10Aa)
LiAlH4 (1.52 g, 40 mmol, 2 eq.) dry THF (30 mL)
(1R,4S,5R)-4-hydroxy-2-oxabicyclo[3,3,0]oct-7-en-3-one (9Aa) (2.80 g, 20 mmol) dry
(1.5 mL) 15% NaOHaq. (1.5 mL) (4.5mL)
30 EtOH
THF EtOH Na2SO4
toluene ((1S,2R)-2-hydroxycyclo
-pent-3-en-1-yl)ethane-1,2-diol (10Aa) (1.50 g, 52%)
LiAlH4 (866 mg, 23 mmol) dry THF (20 mL)
(3S,3aS,6aR)-2-oxo-3,3a,4,6a-tetrahydro-2H-cyclopenta[b]furan-3-yl acetate (19Aa) (2.60 g,
14 mmol) dry THF (30 mL) 10 2 16 (0.9 mL) 15% NaOHaq. (0.9 mL) (2.7mL) 30 MeOH THF MeOH Na2SO4 toluene (S)-1-((1S,2R)-2-hydroxycyclopent-3-en-1-yl) ethane-1,2-diol (10Aa) (1.40 g, 68%) (1R,5S)-5-((S)-2-((tert-Butyldiphenylsilyl)oxy)-1-hydroxyethyl)cyclopent-2-enol (21)
((1S,2R)-2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10Aa) (1.136 g, 7.9 mmol) imidazole (1.076 g, 15.8 mmol, 2 eq.) dry DMF (16 mL)
tert-butyl(chloro)diphenylsilane (TBDPSCl) (2.382 g, 8.7 mmol, 1.1 eq.)
sat.NaCl aq. ether
Ether Na2SO4
CHCl3 :
hexane = 1 : 3 silane CHCl3 : acetone = 100 : 1
(1R,5S)-5-((S)-1-((tert-butyldiphenylsilyl)oxy)-2-hydroxyethyl)cyclopent-2-enol (21) (2.20 g, 73%)
((1S,2R)-2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10Aa) (1.416 g, 9.8 mmol) dry pyridine (35 mL) tert-butyl(chloro)diphenylsilane (TBDPSCl)
(2.97 g, 10.8 mmol, 1.1 eq.)
Na2SO4
CHCl3 : hexane = 1 : 3 silane CHCl3 : acetone
= 100 : 1 (1R,5S)-5-((S)-1-((tert-butyldiphenylsilyl)oxy)-2-hydroxyethyl) -cyclopent-2-enol (21) (2.95 g, 82%)
(1R,5R)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)-1-hydroxyethyl)cyclopent-2-en-1-yl acetate (22)
(1R,5S)-5-((S)-2-((tert-Butyldiphenylsilyl)oxy)-1-hydroxyethyl)cyclopent-2-enol (21) (2.14
g, 5.6 mmol) dry pyridine (12 mL) DMAP (DMAP 21
mg) acetic anhydride (2.86 g, 28.0 mmol, 5 eq.)
CH2Cl2 5%
Na2SO4 hexane
(1R,5R)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)-1-hydroxyethyl)cyclo -pent-2-en-1-yl acetate (22) (2.229 g, 85%)
(S)-1-((1S,2R)-2-Acetoxycyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20)
(S)-1-((1S,2R)-2-Hydroxycyclopent-3-en-1-yl)ethane-1,2-diol (10Aa) (688 mg, 4.86 mmol)
dry pyridine (10 mL) (10mL) 1
CH2Cl2 CH2Cl2
sat.NaCl aq., 5%HCl, sat.NaHCO3 aq. Na2SO4
hexane (S)-1-((1S,2R)-2-acetoxy-cyclo-pent-3-en-1 -yl)ethane-1,2-diyl diacetate (20) (841 mg, 65%)
(1R,5R)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)-1-hydroxyethyl)cyclopent-2-en-1-yl acetate (22) (1.62 g, 3.45 mmol) dry THF (2.5 mL) BuN4F-THF
1 THF CHCl3 sat.NaCl
aq., sat.NaHCO3 aq. Na2SO4
dry pyridine (8mL) DMAP (13mg) (1.76g, 17.2
mmol) 1
pyridine CHCl3 CHCl3 sat.NaCl
aq., 5%HCl, sat.NaHCO3 aq. Na2SO4
(S)-1-((1S,2R)-2-acetoxycyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) (741 mg, 79%) 1 H-NMR (CDCl3, 400 MHz) δ 6.15 - 6.11 (m, 1H), 5.92 - 5.88 (m, 1H ), 5.68 (ddd, J = 6.4 Hz, 1.6 Hz, 1.6 Hz, 1H), 5.31 - 5.23 (m, 1H), 4.44 (dd, J = 2.6 Hz, 1.1 Hz, 1H), 4.01 (dd,12.2 Hz, 5.4 Hz, 1H), 2.64 - 2.53 (m, 1H), 2.42 – 2.25 (m, 2H), 2.62 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H).
2
4
4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopent-2-en-1-yl)methyl acetate (7c)
Thymine (636 mg, 5.04 mmol, 2.0 eq.) dry DMSO (20 mL) Ar NaH (60% in mineral oil) (151 mg, 3.78 mmol, 1.5 eq.) thymine Na
2 Ar 2-acetoxycyclopent-3-en-1-yl)methyl
acetate (13) (500 mg, 2.52 mmol, 1.0 eq.) Pd(PPh3)4 (291 mg, 0.252 mmol, 0.1 eq.), PPh3
(66 mg, 0.252 mmol, 0.1eq.) in dry THF (20 mL) π-60 oC 24 TLC (CHCl3 : MeOH = 10 : 1) CHCl3 (CHCl3 : MeOH = 60 : 1 → 30 : 1) × 2 4-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopent-2-en-1-yl)methyl acetate (23) (0.491 g, 1.86 mmol, 74%) 1 H-NMR (CDCl3, 400 MHz) δ 8.13 (s, 1H, NH-3), 7.05 (s, 1H, H-6), 6.10 (dt, 1H, H-2), 5.74 - 5.69 (m, 2H, H-1, H-3), 4.17, 4.08 (2dd, 2H, H-6α, 6β), 3.09 (m, 1H, H-4), 2.78 (dt, 1H, H-5α), 2.08 (s, 3H, CH3), 1.92 (s, 3H, CH3), 1.37 (dt, 1H, H-5β); 13C-NMR (CDCl3, 100 MHz) δ 170.89, 163.51, 150.82, 138.13, 136.38, 130.60, 111.05, 66.40, 60.88, 44.03, 33.97, 20.90, 12.63; DIFNOE: H-4 (H-1; 2.8 %, H-2; 6.1 %, H-5α; 10.1%, H-6α; and H-6β; 4.9% and 5.2% ), H-5β (H-1; 4.6%, H-4; 2.7%, H-5β; 18.5%); MS (DART) m/z 265
(M++H), HRMS (DART) Calcd for C13H16N2O4 (M++H): 265.1188 Fonud: 265.1224
1-(4-(Hydroxymethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7d)
4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopent-2-en-1-yl)methyl acetate
(23) (262 mg, 0.991 mmol) MeOH 28% CH3ONa pH 9 1
2 18 TLC (CHCl3 : MeOH = 10 : 1) AcOH 1 2 (CHCl3 : MeOH = 10 : 1) 1-(4-(hydroxymethyl)cyclopent-2-en -1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7d) (212mg, 0.954 mmol, 96%) 1 H-NMR (CDCl3, 400 MHz) δ 7.98 (s, 1H, NH-3), 6.12 (dt, 1H, H-2), 5.71 (m, 2H, H-1, H-3), 3.82, 3.67 (2dd, 2H, H-6α, 6β), 2.97 (br, 1H, H-4), 2.71 (dt, 1H, H-5α), 1.90 (s, 3H,
CH3), 1.24 (dt, 1H, H-5β); MS (DART) m/z 223 (M++H), HRMS (DART) Calcd for
C13H14N2O3 (M++H): 223.1083 Fonud: 223.1105
1-(4-(Fluoromethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (23) 1,4-(Hydroxymethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7d)(23.6
mg, 0.106 mmol) CH2Cl2 (4.6 mL) DAST (27.8
µL, 0.212 mmol, 2 eq.) TLC (CHCl3 : MeOH = 10 : 1)
2 sat.NaHCO3aq. CHCl3 Na2SO4 (CHCl3 : MeOH = 50 : 1) 1-(4-(fluoromethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (23) (11.3 mg, 0.0504 mmol, 48%) 1 H-NMR (CDCl3, 400 MHz) δ 8.46 (s, 1H, NH-3), 7.20 - 7.06 (m, 1H, H-Ar), 6.12 - 6.11 (m, 1H, H-2’), 5.74 - 5.66 (m, 2H, H-1 and H-3), 4.65 - 4.05 (m, 2H, H-6), 3.26 - 3.02 (m, 1H, H-4), 2.80 - 2.69 (m, 1H, H-5α), 1.96 - 1.90 (m, 3H, CH3), 1.55 - 1.43 (m, 1H, H-5β); MS (DART) m/z 225 (M++H)
2
5
3-Benzoylthymine (24)
Thymine (1.21 g, 9.59 mmol) MeCN (10 mL) pyridine (4 mL) benzoyl chloride (2.55
mL, 22.1 mmol, 2.2 eq.) Ar 20 TLC (CHCl3 : MeOH
= 15 : 1) CHCl3
0.5 M K2CO3 aq. (20 mL) 1,4-dioxane (20 mL) 1 pH 5
AcOH sat.NaHCO3aq. (50 mL)
1 acetone 13 (0.843g, 3.66 mmol, 52%) 1 H-NMR (CDCl3, 400MHz) δ 9.94 (s, 1H, N-H), 7.95 - 7.01 (m, 6H, Ar-H), 1.91 (s, 3H, -CH3) (S)-1-((1S,4R)-4-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclope nt-2-en-1-yl)ethane-1,2-diyl diacetate (25)
3-Benzoylthymine (24) (0.230 g, 1.00 mmol, 2.0 eq.) dry DMSO (7 mL) Ar NaH (60% in mineral oil) (18.0 mg, 0.75 mmol, 1.5 eq.)
3-Benzoylthymine Na 2 Ar
(S)-1-((1S,2R)-2-acetoxycyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) (0.140 g, 0.52 mmol, 1.0 eq.), Pd(PPh3)4 (62 mg, 0.0535 mmol, 0.1 eq..), PPh3 (13.9 mg, 0.053 mmol, 0.1
eq.) in dry THF (5 mL) π- 60 oC
24 TLC (CH3Cl : MeOH = 20 : 1) THF
DMSO CHCl3
(CHCl3 )
(S)-1-((1S,4R)-4-(3-benzoyl-5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H) -yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (25) (108 mg, 0.246 mmol, 47%)
1-((1R,4S)-4-((S)-1,2-Dihydroxyethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3 H)-dione (26)
(S)-1-((1S,4R)-4-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclo -pent-2-en-1-yl)ethane-1,2-diyl diacetate (25) (98 mg, 0.223 mmol) NH3/MeOH (15 mL)
TLC (CHCl3 : MeOH = 10 : 1) 1-((1R,4S)-4-((S)-1,2-dihydroxyethyl)cyclopent-2-en-1-yl)-5-methyl- pyrimidine-2,4(1H,3H)-dione (26) (54.1 mg, 0.388 mmol, 96 %.) 1 H-NMR (CD3OD, 400MHz) δ 8.02 (s, 1H, NH-3), 7.47 (d, J =4.0 1H, H-Ar), 6.27 - 6.13 (m, 1H, H-2), 5.73 - 5.66 (m, 1H, H-1), 5.64 - 5.52(m, 1H, H-3), 3.63 - 3.46 (m, 3H, H-6 and H-7), 3.01 - 2.80 (m, 1H, H-4), 2.73 - 2.49 (m, 1H, H-5α), 1.87 - 1.76 (m, 3H, CH3), 1.54 -
1.36 (m, 1H, H-5β); MS (DRAT) m/z 253 [M+H]+, HRMS (DART) Calcd for C12H17N2O4
[M+H]+: 253.1188. Found: 253.1180. 1-((1R,4S)-4-((S)-2-Fluoro-1-hydroxyethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4 (1H,3H)-dione (27) 1-((1R,4S)-4-((S)-1,2-Dihydroxyethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H )-dione (26) (30.2 mg, 0.217 mmol) CH2Cl2 (5mL) DAST (15.6μL) 30 TLC
sat.NaHCO3aq. CHCl3 TLC (CHCl3 : MeOH = 10 : 1)
1-((1R,4S)-4-((S)-2-fluoro-1-hydroxyethyl)cyclopent-2-en-1-yl) -5-methylpyrimidine-2,4(1H,3H)-dione (27) (11.3mg, 0.044mmol, 21 %.) 1 H-NMR (CDCl3, 400MHz) δ 7.06 (dd, J = 1.2 Hz, 20.8 Hz 1H, ), 6.30 - 6.10 (m, 1H, H-2), 5.89 - 5.64 (m, 2H, H-1, and H-3), 4.96 - 4.75 (m, 1H), 4.62 - 4.42 (m, 1H), 3.24 - 2.96 (m, 1H), 2.94 - 2.68 (m, 1H, H-5α), 1.98 - 1.86 (m, 3H, CH3), 1.60 - 1.38 (m, 1H,), 1.37 - 1.16
(m, 1H,); MS (DRAT) m/z 255 [M+H]+, HRMS (DART) Calcd for C12H16FN2O3 [M+H]+:
2
6
3-Benzoyluracil (28)
Uracil (1.13 g, 10.0 mmol) MeCN (10 mL) pyridine (4 mL) benzoyl chloride (2.6 mL,
22.4 mmol, 2.2 eq.) Ar 68 TLC (CHCl3 : MeOH =
10 : 1) CHCl3
0.5 M K2CO3aq. (10 mL) 1,4-dioxane (20 mL) 1 pH 5
AcOH sat.NaHCO3aq. (50 mL)
1 acetone 3-benzoyluracil (18) (0.864 g, 4.00 mmol, 40%) 1H-NMR (DMSO-d6, 400 MHz) δ 11.5 (s, 1H, H-N), 7.95 - 7.57 (m, 7H, H-Ar) (S)-1-((1S,4R)-4-(3-Benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (29)
3-Benzoyluracil (28) (0.303 g, 1.4 mmol, 2.0 eq.) dry DMSO (7 mL) Ar NaH (60% pure in mineral oil) (44 mg, 1.1mmol) 3-benzoyl uracil
Na 2 Ar (S)-1-((1S,2R)-2-acetoxy
-cyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) (0.190 g, 0.71 mmol, 1.0 eq.), Pd(PPh3)4
(82 mg, 0.071 mmol, 0.1 eq.), PPh3 (18.6 mg, 0.071 mmol, 0.1 eq.) in dry THF (7 mL)
π- 60 oC 24 TLC (CH3Cl : MeOH = 20 : 1) THF DMSO CHCl3 (CHCl3 ) (124.6 mg, 0.29 mmol, 41%) (S)-1-((1S,4R)-4-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H) -yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (29) 1-((1R,4S)-4-((S)-1,2-Dihydroxyethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (30) (S)-1-((1S,4R)-4-(3-Benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopent-2-en-1-yl )ethane-1,2-diyl diacetate (29) (116 mg, 0.272 mmol) NH3/MeOH (10 mL)
120°C 14
TLC (CHCl3 : MeOH = 10 : 1)
1-((1R,4S)-4-((S)-1,2-dihydroxyethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (30) (57.7 mg, 0.242 mmol, 89 %.)
MS (DRAT) m/z 239 [M+H]+, HRMS (DART) Calcd for C11H15N2O4 [M+H]+: 239.1032. Found: 239.1034. 1-((1R,4S)-4-((S)-2-Fluoro-1-hydroxyethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (31) 1-((1R,4S)-4-((S)-1,2-Dihydroxyethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (30) (37.6 mg, 0.158 mmol) CH2Cl2 (5mL) DAST (11.4μL) 30 TLC
sat.NaHCO3aq. CHCl3 TLC (CHCl3 : MeOH = 10 : 1)
1-((1R,4S)-4-((S)-2-fluoro-1-hydroxyethyl)cyclopent-2-en-1-yl)- pyrimidine-2,4(1H,3H)-dione (31) (9.6mg, 0.040mmol, 25 %.)
MS (DRAT) m/z 241 [M+H]+, HRMS (DART) Calcd for C11H14FN2O3 [M+H]+:241.0989.
2
7
N Pf TMK 50 µg/mL LB 10 mL 37 °C 50 µg/mL 1 L LB 37 °C OD600 0.60 1 mM IPTG 37 °C 4 6,000 × g 15 4 °C -20 °C 40 mL (25 mMTris-HCl buffer pH 7.2 150 mM NaCl) 40
3 3 12,000 rpm 15 4 °C
Talon
(25 mM Tris-HCl buffer pH7.2 150 mM NaCl)
30 (25 mM Tris-HCl buffer pH7.2 150
mM NaCl 10 mM imidazole)
(25 mM Tris-HCl buffer pH7.2 150 mM NaCl 200 mM imidazole)
(50 mM Tris-HCl pH 7.2 0.5 mM phosphoenolpyruvate 40 mM KCl 2 mM MgCl2 2 mM ATP 0.15 mM NADH 2 unit pyruvate kinase 2 unit lactate
dehydrogenase TMP and dGMP ) 1 mL (Pf TMK) 30°C 335 nm Ki 1 unit 1 1 μmol v = V max[S] / (Km + [S])･････････････(1) 1/v = (K m / Vmax)(1 / [S]) + 1 / Vmax･･･(2) K` m = K``m(1 + [I] / Ki)･･････････････(3)
3
2
(1’R,4’S,5’S)-2-Amino-6-chloro-9-[4’-(1”,2”-diacetoxyethyl)cyclopent-2’-en-1-yl]purine (32)
2-Amino-6-chloropurine (339 mg, 2.0 mmol) dry DMSO (8 mL) Ar NaH (60% pure in mineral oil) (80 mg, 2mmol) 2-amino-6-chloropurine
Na 2 Ar (S)-1-((1S,2R)-2-acetoxy-
cyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) (541 mg, 2 mmol), Pd(PPh3)4 (231 mg,
0.2 mmol), PPh3 (52 mg, 0.2 mmol) in dry THF (8 mL)
π-60 oC 24 CHCl3
CHCl3 mineral oil PPh3
AcOEt : hexane = 1 : 2 (20) acetone :
hexane = 1 : 1 (1’R,4’S,5’S)-2-amino-6-chloro-9-[4’-(1”,2”-diacetoxy- ethyl)cyclopent-2’-en-1-yl]purine (32) (301 mg, 40%) (1S)-1-((1S,4R)-4-(2-Amino-6-hydroxy-1H-purin-9(6H)-yl)cyclopent-2-en-1-yl)ethane-1, 2-diol (33) (1’R,4’S,5’S)-2-Amino-6-chloro-9-[4’-(1”,2”-diacetoxyethyl)cyclopent-2’-en-1-yl]purine (32) (75 mg) 50v/v% CF3CO2H (5 mL) 2 Toluene CF3CO2H MeOH (1 mL) 1M NH4OH (1 mL) 2 (CHCl3 ; MeOH = 4 : 1) (1S)-1-((1S,4R)-4-(2-amino-6-hydroxy-1H-purin-9(6H)- yl)cyclopent-2-en-1-yl)ethane-1,2-diol (33) 42 mg, 77% 1 H NMR (CD3OD) δ 1.67 - 1.74 (1H, m, 5’-CH2), 2.71 - 2.76 (1H, m, 5’-CH2), 2.90 - 3.03 (1H, m, 4’-CH), 3.50 - 3.54 (1H, m, 2’’-CH2-O), 3.54 - 3.62 (1H, m, 2’’-CH2-O), 4.57 (1H, br s, 1’-CH-N), 5.45 - 5.52 (1H, m, 1’’-CH-O), 5.85 - 5.88 (1H, m, =CH), 6.20 - 6.26 (1H, m, =CH), 7.75 (1H, s, 8-H). ; Anal. Calcd. for C12H15N5O3 1/2H2O : C, 50.34; H, 5.63; N, 24.46.
3
3
(1’R,4’S,5’S)- 6-Chloro-9-[4’-(1”,2”-diacetoxyethyl)cyclopent-2’-en-1-yl]purine (34)
6-Chloropurine (309 mg, 2.0 mmol) dry DMSO (8 mL) Ar NaH
(60% pure in mineral oil) (80 mg, 2mmol) 6-chloropurine Na
2 Ar (S)-1-((1S,2R)-2-acetoxycyclopent-3-en-1-yl)- ethane-1,2-diyl diacetate (20) (541 mg, 2 mmol), Pd(PPh3)4 (231 mg, 0.2 mmol), PPh3 (52
mg, 0.2 mmol) in dry THF (8 mL)
π-60 oC 24 CHCl3
CHCl3 mineral oil PPh3 AcOEt : hexane
= 1 : 2 (20) (145mg) acetone : hexane =1 : 1 (1’R,4’S,5’S)-6-chloro-9-[4’-(1”,2”-diacetoxyethyl)cyclopent-2’-en-1- yl]purine (34) (34) ether (108 mg, 15% 1 H NMR (CDCl3) δ 1.63 - 1.74 (1H, m, 5’-CH2), 2.05 (3H, s, COCH3), 2.08 (3H, s, COCH3), 2.88 - 2.98 (1H, m, 5’-CH2), 3.17 - 3.25 (1H, m, 4’-CH), 4.05 - 4.12 (1H, m, 2’’-CH2-O), 4.33 - 4.38 (1H, m, 2’’-CH2-O), 5.06 - 5.12 (1H, m, 1’-CH-N), 5.77 - 5.84 (1H, m, 1’’-CH-O), 5.97 - 6.02 (1H, m, =CH), 6.23 - 6.26 (1H, m, =CH), 8.18 (1H, s, 8-H), 8.74 (1H, s, 4-H). (1S)-1-((1S,4R)-4-(6-Hydroxy-1H-purin-9(6H)-yl)cyclopent-2-en-1-yl)ethane-1,2-diol (35) (1’R,4’S,5’S)- 6-Chloro-9-[4’-(1”,2”-diacetoxyethyl)cyclopent-2’-en-1-yl]purine (34) (80 mg) 50v/v% CF3CO2H (5 mL) 2 toluene CF3CO2H MeOH (1 mL) 1M NH4OH (1 mL) 2 (CHCl3 : MeOH = 5 : 1) MeOH (1S)-1-((1S,4R)-4-(6- hydroxy-1H-purin-9(6H)-yl)cyclopent-2-en-1-yl)ethane-1,2-diol(35) (45 mg, 78%) mp 280 oC. (dec.) 1H NMR (CD3OD) δ 1.76 (1H, ddd, J =13.7Hz, 6.4Hz, 6.4Hz 5’-CH2), 2.80 (1H, ddd, J =13.7Hz, 8.8Hz, 8.8Hz Hz, 5’-CH2), 3.00 - 3.08 (1H, m, 4’-CH), 3.51 -
3.64 (2H, m, 2’’-CH2-O), 4.82 - 4.90 (1’-CH-N) Overlapped with the H2O Peak , 5.65 -
8-H), 8.12 (1H, br s, 2-H).; Anal. Calcd. for C12H14N4O3 2/3H2O : C, 52.55; H, 5.63; N,
3
4
2-(Trifluoromethyl)-9H-purin-6-ol (36)
4-Amino-5-imidazolecarboxamide hydrochloride (2.0 g, 12 mmol) trifluoroacetamide
(13.6 g, 120 mmol) 180 oC 5 2 ether ether EtOH MeOH EtOH 2-(trifluoromethyl)-9H-purin-6-ol (36) (2.14 g, 87%) 2-Trifluoromethyl-6-chloropurine (37)
2-(Trifluoromethyl)-9H-purin-6-ol (36) (1.52 g, 7.4 mmol) dry CH3CN (22 mL)
N,N’-dimethylaniline (1.35 g, 11.1 mmol, 1.5 eq) POCl3 (3.43 g, 22.2
mmol, 3 eq.) 95 oC 5 (10 mL) 15 hexane 2-trifluoromethyl-6-chloropurine (37) (1.47 g, 89%) (S)-1-((1S,4R)-4-(6-Chloro-2-(trifluoromethyl)-9H-purin-9-yl)cyclopent-2-en-1-yl)ethan e-1,2-diyl diacetate (38)
2-Trifluoromethyl-6-chloropurine (37) (222 mg, 1.0 mmol) dry DMSO (5 mL)
Ar NaH (60% pure in mineral oil) (40 mg, 1 mmol) 6-chloro-2-
trifluoromethylpurine Na 2 Ar
1-acetoxy-5-(1,2-diacetoxyethyl)cyclopent-2-ene (270 mg, 1 mmol), Pd(PPh3)4 (116 mg, 0.1
mmol), PPh3 (26 mg, 0.1 mmol) in dry THF (5 mL)
π-Purine Na DMSO 60 oC 24
CHCl3
CHCl3
mineral oil PPh3 AcOEt hexane=1:2
triacetate acetone:hexane=1:1 (S)-1-((1S,4R)-4-(6-chloro-2- (trifluoromethyl)-9H-purin-9-yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (38)
1
H NMR (CDCl3) δ 1.63 - 1.72 (1H, m, 5’-CH2), 2.05 (3H, s, COCH3), 2.08 (3H, s, COCH3),
4.32 - 4.38 (1H, m, 2’’-CH2-O), 5.06 (1H, m, 1’-CH-N), 5.88 (1H, m, 1’’-CH-O), 5.98 (1H,
3
5
2,4-Dichloro-3-nitropyridine (39)
2,4-Dihydroxy-3-nitropyridine (4.73g, 30.2 mmol) POCl3 29 mL 95 oC
17 50 g 30 22 mL 4.51 g, 77% 2-Chloro-4-(p-methoxybenzylamino)-3-nitropyridine (40) 2,4-Dichloro-3-nitropyridine (39) (4.51 g, 23.4 mmol) DMF (40 mL) triethylamine (2.08 g, 20.6 mmol) p-Methoxybenzylamine (3.3 g, 24.0 mmol) 1 2 200 mL 50 mL 2-Chloro-4-(p-methoxybenzyl amino)-3-nitropyridine (40) (6.24 g, 91%) 3-Amino-2-chloro-4-(p-methoxybenzylamino)pyridine (41)
2-Chloro-4-(p-methoxybenzylamino)-3-nitropyridine (40) (6.24 g, 21.2 mmol) EtOH (115 mL) Iron powder (6.51 g) H2O (21 mL) HCl (4.6 mL) 95 oC
17
(CH2Cl2:MeOH = 9:1)
3-amino-2-chloro-4-(p-methoxybenzylamino)pyridine (41) (2.93 g, 52.4%)
4-Chloro-1-(p-methoxybenzyl)-1H-imidazo[4,5-c]pyridine (42)
3-Amino-2-chloro-4-(p-methoxybenzylamino)pyridine (41) (2.93 g, 11.1 mmol) triethyl orthoformate (25 mL) acetic anhydride (25 mL) 160 oC 2
(CH2Cl2 : acetone = 20:1 → 10:1 → 5:1) 4-chloro-1-(p-methoxybenzyl)-1H-imidazo[4,5-c] pyridine (42) (2.48 g, 82%) 4-Chloro-1H-imidazo[4,5-c]pyridine (43) 4-Chloro-1-(p-methoxybenzyl)-1H-imidazo[4,5-c]pyridine (42) (2.4 g, 8.8 mmol) trifluoroacetic acid (11.0 mL) 80 oC 2 CHCl3
CHCl3 acetone AcOEt CHCl3 : MeOH = 5 : 1 5 CHCl3 PMB CHCl3 : MeOH = 10 : 1 4-chloro-1H- imidazo[4,5-c]pyridine (43) (0.811 g, 60%) (S)-1-((1S,4R)-4-(4-Chloro-1H-imidazo[4,5-c]pyridin-1-yl)cyclopent-2-en-1-yl)ethane-1, 2-diyl diacetate (44)
4-Chloro-1H-imidazo[4,5-c]pyridine (43) (307 mg, 2 mmol) dry DMSO (8 mL) NaH (60% pure in mineral oil) (136 mg, 3.4 mmol, 1.7 eq.) Ar
2 Na Ar (S)-1-((1S,2R)-2-acetoxy-
cyclopent-3-en-1-yl)ethane-1,2-diyl diacetate (20) (541 mg, 2 mmol), PPh3 (73 mg, 0.28
mmol, 0.14 eq.), Pd(PPh3)4 (324 mg, 0.28 mmol, 0.14 eq.) in dry THF (8 mL)
π- Deazapurine Na DMSO 60 oC
24
AcOEt:hexane=1:2 Ph3P acetone : hexane = 1 : 1
9- coupling product TLC (44) (S)-1-((1S,4R)-4-(4-chloro-1H-imidazo[4,5-c]pyridin- 1-yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (44) (39 mg, 47%) (S)-1-((1S,4R)-4-(4-Chloro-1H-imidazo[4,5-c]pyridin-1-yl)cyclopent-2-en-1-yl)ethane-1, 2-diol (45) (S)-1-((1S,4R)-4-(4-chloro-1H-imidazo[4,5-c]pyridin-1-yl)cyclopent-2-en-1-yl)ethane-1,2-diyl diacetate (44) (80 mg) 50v/v%CF3CO2H (3 mL) 2 Toluene CF3CO2H MeOH (1 mL), 1M NH4OH (1 mL) 2 TLC (CHCl3 : MeOH = 10 : 1) (S)-1-((1S,4R)-4-(4-chloro-1H-imidazo[4,5-c]pyridin-1-yl)cyclopent-2 -en-1-yl)ethane-1,2-diol (45) (42mg 66%) MS (m/z): 279 (M+), 281 ([M+2]+).
3
6
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