Synthesis and antiviral activity evaluation of some novel acyclic C-nucleosides

Article abstract of DOI:10.1248/cpb.56.775

The preparation of novel 5-amino or 7-hydroxy substituted pyrazolo[4,3-b]pyridine and pyrazolo[3,4-c]pyridine acyclic C-nucleosides is described. Their synthesis was carried out by condensation of suitably substituted lithiated picolines with 2-benzyloxyethoxymethylchloride followed by pyrazole ring annulation. The compounds were evaluated for their antiviral activity against a wide panel of viruses, but were found inactive at subtoxic concentrations.

Full text of DOI:10.1248/cpb.56.775

June 2008
Chem. Pharm. Bull. 56(6) 775—780 (2008)
775
Synthesis and Antiviral Activity Evaluation of some Novel Acyclic
C-Nucleosides
b
Nikolaos LOUGIAKIS,a Panagiotis MARAKOS,a Nicole POUL,*,a and Jan BALZARINI
a Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens; Panepistimiopolis Zografou
15771, Athens, Greece: and b Rega Institute for Medical Research; K.U.Leuven, B-3000 Leuven, Belgium.
Received November 2, 2007; accepted March 10, 2008; published online March 19, 2008
The preparation of novel 5-amino or 7-hydroxy substituted pyrazolo[4,3-b]pyridine and pyrazolo[3,4-c]pyri-
dine acyclic C-nucleosides is described. Their synthesis was carried out by condensation of suitably substituted
lithiated picolines with 2-benzyloxyethoxymethylchloride followed by pyrazole ring annulation. The compounds
were evaluated for their antiviral activity against a wide panel of viruses, but were found inactive at subtoxic con-
centrations.
Key words acyclonucleoside; pyrazolopyridine; antiviral activity
The discovery of 9-[(2-hydroxyethoxy)methyl]guanine reported for acyclic nucleosides, we were interested in deter-
(acyclovir, Fig. 1)1) as the first potent and selective inhibitor mining whether antiviral activity is retained when the (2-hy-
of a human herpes virus2) has stimulated continuing research droxyethoxy)methyl group of acyclovir is employed in com-
into the preparation and evaluation of novel acyclic ana- bination with a base structurally related to 3-deazaadenine.
logues of nucleosides as potential substrates of virus-specific Within this context we report here the synthesis and the
thymidine kinase (TK) and/or inhibition of DNA polymerase in vitro antiviral evaluation of some new acyclic C-nucleo-
after activation by the viral TK and subsequent phosphoryla- side analogues, bearing the 8-aza-1,9-dideazapurine or the
tion by cellular enzymes to the 5ꢀ-triphosphates.3,4) Structure– 8-aza-3,9-dideazapurine skeleton.
activity relationship studies have been explored extensively
and revealed that both the nature of the heterocyclic base and Results and Discussion
the chemical structure of the aliphatic substituent have a pro-
Chemistry For the preparation of the pyrazolopyridi-
nounced effect on the antiviral activity of these molecules. none 13 (Chart 1) we used commercial 2-picoline N-oxide
Purine nucleosides structurally related to acyclovir that are (1) which was converted to the picoline 4 through known
currently used in the clinic are ganciclovir,5,6) which is useful procedures.12) Subsequent nitration of 4 provided a mixture
against cytomegalovirus (HCMV) infections and the penci- of the isomeric nitroderivatives 5 and 6 and from this mixture
clovir prodrug famciclovir,7) which is marketed for shingles. only the desired compound 6 could be isolated in pure form
The antiviral spectrum of these agents is limited to the her- by column chromatography, whereas 5 was contaminated
pes virus genus. On the other hand, 3-deazaadenosine as well with traces of 6. The isomer 6 was reduced to the amino-
as a number of acyclic and carbocyclic adenosine analogues, derivative 7, which has been previously reported as a side-
such as the S enantiomer of 9-(2,3-dihydroxypropyl)-adenine product of the ammonolysis of 3-methoxy-2-acylfuran.13)
(S-DHPA), aristeromycin, neplanocin A and 3-deazane-
planocin A (Fig. 1) were found to act as reversible inhibitors
of S-adenosyl-L-homocysteine (SAH) hydrolase.8) These
SAH hydrolase inhibitors exhibit potent antiviral activity
against a number of DNA and RNA viruses, which heavily
depend on methylation reactions for viral replication. More
interestingly, acyclic nucleoside phosphonates have been
developed, which retain marked activity against thymidine
kinase deficient viral strains and some of them are useful for
the treatment of HCMV, retrovirus (HIV) and human hepati-
tis B virus (HBV) infections.9)
In the course of our studies, concerning the preparation of
a number of C-nucleosides structurally related to inosine and
adenosine10,11) and in conjunction with the stimulating results
Reagents and conditions: a) H2SO4, HNO3, 160 °C; b) CH3ONa, CH3OH, 80 °C; c)
Fe, CH3CO2H, 130 °C; d) H2SO4, HNO3, 65 °C; e) H2, Pd–C, EtOH; f) Ac2O, CH2Cl2,
rt; g) (1) n-BuLi (2.5 eq), THF, ꢁ78 °C, (2) 2-benzyloxyethoxymethylchloride, THF; h)
AcOK, Ac2O, isoamyle nitrite, C6H6, reflux; i) BCl3, CH2Cl2, ꢁ70 °C; j) HCl (36%),
EtOH, 80 °C.
Chart 1
Fig. 1. Structures of Acyclovir and Adenosine Analogues
∗ To whom correspondence should be addressed. e-mail: pouli@pharm.uoa.gr
© 2008 Pharmaceutical Society of Japan

776
Vol. 56, No. 6
Compound 7 was then acetylated to give the acetamide 8, first converted to the Boc-protected analogue 25 and then,
which was lithiated using n-butyllithium in THF solution. upon reduction, to the acetamide 27 (Chart 3). The Boc-
The resulting anion was reasonably stable to be trapped protecting group is suitable in terms of its stability to anionic
by 2-benzyloxyethoxymethylchloride,14) thus allowing the reaction conditions. Thus, lithiation of 27 with 3.3 eq
preparation of the substituted pyridine 9. From this reaction of n-butyllithium followed by treatment with 2-benzyl-
we have also isolated a significant amount (32%) of a oxyethoxymethylchloride provided the intermediate pyridine
by-product the structure of which was unambiguously 28, which was subjected to ring-closure according to the
assigned as 10 (Fig. 2), on the basis of 1-D and 2-D NMR afore-mentioned conditions to result in the pyrazolopyridine
data. The formation of 10 is due to a competitive anion for- 29. The Boc-group was then easily cleaved by the use of
mation on the acetamide’s methyl group.
trifluoracetic acid and the resulting pyrazolopyridine 30 was
Compound 9 was then heated at reflux in benzene with converted to the target compound 31 upon treatment with
isoamyl nitrite, in the presence of acetic anhydride.15) This boron trichloride in dichloromethane solution.
furnished the substituted 1-acetylpyrazolo[4,3-b]pyridine 11
The application of this strategy using the isomeric picoline
through a rearrangement of the intermediate N-nitroso com- 33 (Chart 4) which was prepared from the previously
pound. Both the N-acetyl and benzyl groups were easily reported amino-derivative 32,18) resulted in six steps in the
cleaved upon treatment of 11 with a 1 M solution of BCl3 in 3-substituted 5-aminopyrazolopyridine 39.
hexane at low temperature. This provided the 7-methoxy-
derivative 12 which was then converted to the target pyra- and antiviral activity of compounds against the replication of
zolopyridinone by heating into an ethanolic HCl solution. Herpes simplex virus-1 (KOS), Herpes simplex virus-2 (G),
Antiviral and Cytostatic Evaluations The cytotoxicity
Following an analogous procedure, using the readily pre- Vaccinia virus, Vesicular stomatitis virus and Herpes simplex
pared acetamide 19 (Chart 2),16) we prepared the isomeric virus-1 ACVr in HEL cell cultures, against the replication of
pyrazolo[3,4-c]pyridinone 23 in reasonable overall yield.
Vesicular stomatitis virus, Coxsackie virus B4, Respiratory
In order to prepare the corresponding 5-aminosubstituted syncytial virus in HeLa cell cultures, against the replication
derivatives, we used the aminonitropicoline 2417) which was of Para-influenza-3 virus, Reovirus-1, Sindbis virus, Cox-
sackie virus B4, Punta Toro virus in Vero cell cultures,
against the replication of Feline Corona virus and Human
Corona (SARS) virus in feline kidney Crandel cell cultures
and against HIV-1- and HIV-2-induced cytopathicity in CEM
cell cultures were evaluated. The compounds were antivirally
inactive at subtoxic concentrations.
The inhibitory effects of the compounds on the prolifera-
Fig. 2. Structure of the By-product 10
tion of murine leukemia cells (L1210) and human T-lympho-
Reagents and conditions: a) (1) NaH, THF, 0 °C, (2) Boc2O, THF; b) H2, Pd–C,
EtOH; c) Ac2O, CH2Cl2, r.t.; d) (1) n-BuLi (3.3 eq), THF, ꢁ78 °C, (2) 2-benzyl-
oxyethoxymethylchloride, THF; e) AcOK, Ac2O, isoamyle nitrite, benzene, reflux; f)
CF3CO2H, CH2Cl2, rt; g) BCl3, CH2Cl2, ꢁ70 °C.
Reagents and conditions: a) NaNO2, H2SO4, H2O; b) PCl5, POCl3, 130 °C; c)
CH3ONa, CH3OH, 80 °C; d) H2, Pd–C, EtOH; e) Ac2O, CH2Cl2, rt; f) (1) n-BuLi (2.5
eq), THF, ꢁ78 °C, (2) 2-benzyloxyethoxymethylchloride, THF; g) AcOK, Ac2O,
isoamyle nitrite, C6H6, reflux; h) BCl3, CH2Cl2, ꢁ70 °C; i) HCl/CH3OH, rt.
Chart 4
Chart 2
Reagents and conditions: a) (1) NaH, THF, 0 °C, (2) Boc2O, THF; b) H2, Pd–C, EtOH; c) Ac2O, CH2Cl2, rt; d) (1) n-BuLi (3.3 eq), THF, ꢁ78 °C, (2) 2-benzy-
loxyethoxymethylchloride, THF; e) AcOK, Ac2O, isoamyle nitrite, benzene, reflux; f) CF3CO2H, CH2Cl2, rt; g) BCl3, CH2Cl2, ꢁ70 °C.
Chart 3

June 2008
777
cyte cells (Molt4/C8, CEM) were also determined. Only OCH2CH2O), 3.81 (2H, t, Jꢃ5.5 Hz, PyrCH2CH2O), 3.84 (3H, s, OCH3),
4.51 (2H, s, CH2Ph), 6.74 (1H, d, Jꢃ5.7 Hz, H-5), 7.27—7.33 (5H, m, Ph-
compound 13 exhibited a slight antiproliferative activity with
IC50 values within the range of 126—220 mM against the
tested cell lines.
H), 8.16 (1H, br s, D2O exchang., NH), 8.31 (1H, d, Jꢃ5.7 Hz, H-6). 13C-
NMR (50 MHz, CDCl3) d: 23.10 (CH3CO), 35.06 (PyrCH2CH2O), 55.86
(CH3O), 69.48 (OCH2CH2O), 69.94 (OCH2CH2O), 71.59 (PyrCH2CH2O),
The inactivity of the new compounds is likely due to their
inability to be phosphorylated to a potentially active metabo-
lite and to the lack of being efficient substrates for viral
enzymes. It seems that a closer similarity of the purine-like
nucleus to guanine should be taken under consideration,
since the replacement of the imidazole ring of the purine
skeleton by the isosteric pyrazole nucleus and the lack of the
suitable second substituent on the pyridine ring, are not in
favour of the antiviral activity of this class of compounds.
73.41 (CH2Ph), 105.72 (C-5), 122.23 (C-3), 127.62 (C-4ꢀ), 127.97 (2ꢄ
Phenyl C), 128.52 (2ꢄPhenyl C), 137.69 (C-1ꢀ), 149.05 (C-6), 157.34 (C-2),
161.41 (C-4), 169.18 (CH3CO). Anal. Calcd for C19H24N2O4: C, 66.22; H,
7.02; N, 8.13. Found: C, 66.03; H, 6.91; N, 8.07.
Data for 3-[3-(2-Benzyloxyethoxy)-1-oxopropanamine]-4-methoxy-2-
1
methylpyridine (10): Yield: 32%. Oil. H-NMR (400 MHz, CDCl3) d: 2.38
(3H, s, CH3), 2.67 (2H, t, Jꢃ5.7 Hz, NH(Oꢃ)CCH2CH2O), 3.64—3.66 (2H,
m, OCH2CH2O), 3.71—3.73 (2H, m, OCH2CH2O), 3.76 (3H, s, OCH3), 3.85
(2H, t, Jꢃ5.7 Hz, NH(Oꢃ)CCH2CH2O), 4.47 (2H, s, CH2Ph), 6.63 (1H, d,
Jꢃ5.7 Hz, H-5), 7.17—7.24 (5H, m, Ph-H), 8.05 (1H, br s, D2O exchang.,
NH), 8.26 (1H, d, Jꢃ5.7 Hz, H-6). 13C-NMR (50 MHz, CDCl3) d: 21.19
(NHCOCH2), 37.13 (PyrCH3), 55.72 (CH3O), 67.31 (COCH2CH2O), 69.15
(OCH2CH2O), 70.40 (OCH2CH2O), 73.35 (CH2Ph), 104.69 (C-5), 120.79
(C-3), 127.77 (C-4ꢀ), 127.93 (2ꢄPhenyl C), 128.41 (2ꢄPhenyl C), 137.76
(C-1ꢀ), 148.57 (C-6), 156.78 (C-2), 160.54 (C-4), 170.59 (CH3CO). Anal.
Calcd for C19H24N2O4: C, 66.22; H, 7.02; N, 8.13. Found: C, 65.94; H, 7.17;
N, 8.32.
Experimental
All chemicals were purchased from Aldrich Chemical Co. Melting points
were determined on a Büchi apparatus and are uncorrected. Flash chro-
matography was performed on Merck silica gel 60 (0.040—0.063 mm).
Analytical thin layer chromatography (TLC) was carried out on precoated
1
(0.25 mm) Merck silica gel F-254 plates. H-NMR spectra and 2D spectra
1-Acetyl-3-(2-benzyloxyethoxy)methyl-7-methoxy-1H-pyrazolo[4,3-
b]pyridine (11) Potassium acetate (23 mg, 0.233 mmol) and acetic anhy-
dride (0.07 ml, 0.74 mmol) were added to a solution of 9 (70 mg, 0.20 mmol)
in dry benzene (20 ml) under Ar. The reaction mixture was heated at reflux,
isoamyl nitrite (0.06 ml, 0.44 mmol) was added and the resulting mixture
was refluxed for 7 h. The insoluble material was then filtered off, the solvent
was vacuum-evaporated and the residue was purified by column chromatog-
raphy (silica gel) using a mixture of cyclohexane/EtOAc (7/3, v/v) as the
eluent, to give 11 (65 mg, 87%) as an oil. 1H-NMR (400 MHz, CDCl3) d:
2.81 (3H, s, COCH3), 3.70 (2H, t, Jꢃ4.7 Hz, OCH2CH2OBn), 3.87 (2H, t,
Jꢃ4.7 Hz, OCH2CH2OBn), 4.05 (3H, s, OCH3), 4.57 (2H, s, CH2Ph), 5.02
(2H, s, pyrazol-CH2O), 6.88 (1H, d, Jꢃ5.3 Hz, H-6), 7.29—7.35 (5H, m, Ph-
H), 8.56 (1H, d, Jꢃ5.3 Hz, H-5). 13C-NMR (50 MHz, CDCl3) d: 23.72
(COCH3), 56.36 (OCH3), 64.33 (pyrazol-CH2O), 69.41 (OCH2CH2OBn),
70.73 (OCH2CH2OBn), 73.34 (CH2Ph), 104.98 (C-6), 124.83 (C-7a),
127.66 (C-4ꢀ), 127.84 (2ꢄPhenyl C), 128.43 (2ꢄPhenyl C), 138.28 (C-1ꢀ),
145.26 (C-3a), 148.06 (C-3), 150.04 (C-5), 154.31 (C-7), 169.23 (COCH3).
Anal. Calcd for C19H21N3O4: C, 64.21; H, 5.96; N, 11.82. Found: C, 63.89;
H, 5.90; N, 11.73.
3-(2-Hydroxyethoxy)methyl-7-methoxy-1H-pyrazolo[4,3-b]pyridine
(12) A solution of BCl3 (1 M in hexane, 0.84 ml, 0.84 mmol) was added
dropwise under argon to a solution of 11 (150 mg, 0.42 mmol) in dry CH2Cl2
(25 ml) at ꢁ70 °C. The reaction mixture was stirred at ꢁ70 °C for 1 h, the
temperature was then raised to ꢁ25 °C and a cold solution of CH2Cl2/MeOH
(4 ml, 1/1 v/v) was added. The solvents were evaporated and the residue
was purified by flash chromatography (silica gel) using a mixture of
CH2Cl2/MeOH (95/5, v/v) as the eluent to give pure 12 (79.6 mg, 85%) as a
white solid. mp: 139—140 °C (EtOH). 1H-NMR (400 MHz, CDCl3) d:
3.80—3.85 (4H, m, OCH2CH2O), 4.00 (3H, s, OCH3), 5.09 (2H, s, pyrazol-
CH2O), 6.66 (1H, d, Jꢃ5.4 Hz, H-6), 8.42 (1H, d, Jꢃ5.4 Hz, H-5), 11.60
(1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, CDCl3) d: 56.03 (OCH3),
61.54 (OCH2CH2OH), 65.10 (pyrazol-CH2O), 72.82 (OCH2CH2OH), 101.31
(C-6), 127.18 (C-7a), 140.30 (C-3a), 143.01 (C-3), 147.18 (C-5), 152.80
(C-7). Anal. Calcd for C10H13N3O3: C, 53.81; H, 5.87; N, 18.82. Found: C,
53.57; H, 5.93; N, 18.61.
were recorded on a Bruker Avanche 400 instrument, whereas 13C-NMR
spectra were recorded on a Bruker AC 200 spectrometer in deuterated sol-
vents and were referenced to TMS (d scale). The signals of 1H and 13C spec-
tra were unambiguously assigned by using 2D NMR techniques: 1H–1H
COSY, NOESY HMQC and HMBC. Elemental analyses were performed on
a Perkin-Elmer PE 240C Elemental Analyzer (Norwalk, CT, U.S.A.) and
were within ꢂ0.4% of the theoretical values. The oily analytical samples,
upon the appropriate chromatographic purification were dried in vacuo
(vacuum pump) at 90 °C in the presence of phosphorous pentoxide for 12 h.
4-Methoxy-2-methyl-3-nitropyridine (6) A mixture of sulfuric acid
(98%, 1.6 ml) and nitric acid (65%, 1.6 ml) was added at 0 °C to a solution
of 412) (2.4 g, 19.51 mmol) in sulfuric acid (98%, 11.1 ml) and the resulting
solution was heated at 65 °C for 12 h. The mixture was then poured into
ice-water, neutralized with a 40% NaOH solution and the precipitate was
filtered and air-dried. Flash chromatography of the residue, on silica gel,
using a mixture of CH2Cl2/EtOAc (85/15, v/v) as the eluent, provided pure
only the title nitrocompound 6 in 48% yield, whereas the isomeric
nitroderivative 5 was eluted in mixture with 6. mp: 55—56°C (EtOAc–
1
n-hexane). H-NMR (400 MHz, CDCl3) d: 2.52 (3H, s, CH3), 3.95 (3H, s,
OCH3), 6.85 (1H, d, Jꢃ5.8 Hz, H-5), 8.45 (1H, d, Jꢃ5.8 Hz, H-6). 13C-NMR
(50 MHz, CDCl3) d: 20.37 (CH3), 56.64 (CH3O), 105.93 (C-5), 138.80 (C-
3), 151.42 (C-2), 151.70 (C-6), 157.30 (C-4).
N-(4-Methoxy-2-methylpyridin-3-yl)acetamide (8) To a solution of
the amine 7 (1.1 g, 7.97 mmol) in dry CH2Cl2 (25 ml) was added acetic anhy-
dride (0.95 ml, 10 mmol) and the mixture was stirred at room temperature
for 10 h. The solvent was then vacuum-evaporated and the residue was puri-
fied by column chromatography (silica gel) using a mixture of CH2Cl2/
CH3OH (98/2, v/v) as the eluent, to give pure 8 (1.32 g, 92%). mp: 136 °C
1
(EtOH). H-NMR (400 MHz, CDCl3) d: 2.15 (3H, s, COCH3), 2.37 (3H, s,
CH3), 3.79 (3H, s, OCH3), 6.65 (1H, d, Jꢃ5.7 Hz, H-5), 7.47 (1H, br s, D2O
exchang., NH), 8.23 (1H, d, Jꢃ5.7 Hz, H-6). 13C-NMR (50 MHz, CDCl3) d:
21.10 (CH3), 23.34 (CH3CO), 55.78 (CH3O), 104.67 (C-5), 120.83 (C-3),
148.52 (C-6), 156.88 (C-2), 160.54 (C-4), 169.12 (CH3CO). Anal. Calcd for
C9H12N2O2: C, 59.99; H, 6.71; N, 15.55. Found: C, 60.12; H, 6.55; N, 15.43.
N-[2-(2-Benzyloxyethoxy)ethyl-4-methoxypyridin-3-yl]acetamide (9)
To a solution of the picoline 8 (0.6 g, 3.33 mmol) in dry THF (30 ml) at
ꢁ78 °C was added under argon n-BuLi (5.2 ml, 8.32 mmol, 1.6 M solution in
hexanes). The resulting light yellow solution was stirred at ꢁ78 °C for
15 min and the temperature then raised to ꢁ50 °C for 50 min. The orange-
colored solution was cooled to ꢁ78 °C and a solution of 2-benzyl-
oxyethoxymethylchloride14) (0.86 g, 4.29 mmol) in dry THF (5 ml) was
added dropwise. The resulting mixture was stirred at ꢁ78 °C for 15 min and
the temperature was then allowed to rise to the ambient one. A saturated am-
monium chloride solution was then added to the reaction mixture to quench
the excess n-BuLi. The solvent was vacuum-evaporated, water was added to
the residue and this was extracted with CH2Cl2. The organic extracts were
dried (Na2SO4) and concentrated to dryness to give an oil which was purified
by flash chromatography (silica gel) using EtOAc as the eluent, to give com-
pounds 9 and 10.
1,4-Dihydro-3-(2-hydroxyethoxy)methyl-7H-pyrazolo[4,3-b]pyridin-7-
one (13) A mixture of HCl (36%, 1 ml) in ethanol (2 ml) was added to a
solution of 12 (50 mg, 0.22 mmol) in ethanol (15 ml) and the resulting solu-
tion was heated at reflux for 36 h. The mixture was then neutralized with a
saturated NaHCO3 solution, the inorganic material was filtered off, the sol-
vents were evaporated and the residue was purified by column chromatogra-
phy (silica gel) using a mixture of CH2Cl2/CH3OH (9/1, v/v) as the eluent, to
1
give 13 (25 mg, 54%) as a white solid. mp: 200—201 °C (EtOH). H-NMR
(400 MHz, DMSO-d6) d: 3.45—3.55 (4H, m, OCH2CH2O), 4.69 (2H, s,
pyrazol-CH2O), 5.94 (1H, d, Jꢃ7.43 Hz, H-6), 7.69 (1H, m, H-5), 11.82
(1H, br s, D2O exchang., NH-4), 13.86 (1H, br s, D2O exchang., NH-1). 13C-
NMR (50 MHz, DMSO-d6) d: 60.14 (OCH2CH2OH), 64.21 (pyrazol-CH2O),
71.62 (OCH2CH2OH), 109.12 (C-6), 127.54 (C-3a), 133.45 (C-7a), 136.09
(C-3), 137.23 (C-5), 168.55 (C-7). Anal. Calcd for C9H11N3O3: C, 51.67; H,
5.30; N, 20.09. Found: C, 51.36; H, 5.12; N, 19.84.
1
Data for 9: Yield: 40%. Oil. H-NMR (400 MHz, CDCl3) d: 2.01 (3H, s,
N-[4-(2-Benzyloxyethoxy)ethyl-2-methoxypyridin-3-yl]acetamide (20)
This compound was prepared by a procedure analogous to that of 9, starting
COCH3), 3.01 (2H, t, Jꢃ5.5 Hz, PyrCH2CH2O), 3.58—3.60 (4H, m,

778
Vol. 56, No. 6
from 1916) (700 mg, 3.89 mmol), using 2.5 eq of n-BuLi. The product was
purified by column chromatography (silica gel) using a mixture of cyclo-
hexane/EtOAc (6/4, v/v) as the eluent to provide 20 (575 mg, 43%) as an oil.
1H-NMR (400 MHz, CDCl3) d: 2.04 (3H, s, COCH3), 2.82 (2H, t, Jꢃ5.9 Hz,
PyrCH2CH2O), 3.57—3.60 (4H, m, OCH2CH2O), 3.71 (2H, t, Jꢃ5.9 Hz,
PyrCH2CH2O), 3.96 (3H, s, OCH3), 4.53 (2H, s, CH2Ph), 6.78 (1H, d, Jꢃ
5.1 Hz, H-5), 7.28—7.35 (5H, m, Ph-H), 7.78 (1H, br s, D2O exchang., NH),
7.97 (1H, d, Jꢃ5.1 Hz, H-6). 13C-NMR (50 MHz, CDCl3) d: 23.24
(CH3CO), 32.05 (PyrCH2CH2O), 54.05 (CH3O), 69.47 (OCH2CH2O), 70.09
(OCH2CH2O), 71.41 (PyrCH2CH2O), 73.44 (CH2Ph), 118.15 (C-5), 120.36
(C-3), 127.96 (3ꢄPhenyl C), 128.54 (2ꢄPhenyl C), 137.80 (C-1ꢀ), 144.33
(C-6), 147.34 (C-4), 159.67 (C-2), 169.20 (CH3CO). Anal. Calcd for
C19H24N2O4: C, 66.22; H, 7.02; N, 8.13. Found: C, 65.97; H, 7.19; N, 8.28.
1-Acetyl-3-(2-benzyloxyethoxy)methyl-7-methoxy-1H-pyrazolo[3,4-
c]pyridine (21) This compound was prepared by a procedure analogous to
that of 11, starting from 20 (500 mg, 1.45 mmol). The product was purified
by column chromatography (silica gel) using a mixture of cyclohexane/
tion of 25 (2.5 g, 9.88 mmol) in dry ethanol (40 ml) was hydrogenated in the
presence of 10% Pd/C (130 mg) under a pressure of 50 psi at rt for 5 h. The
solution was filtered through a celite pad to remove the catalyst and the
filtrate was evaporated to dryness to give pure 9 (2.18 g, 99%). mp: 122—
123 °C (Et2O) 1H-NMR (400 MHz, CDCl3) d: 1.47 [9H, s, (CH3)3], 2.29
(3H, s, CH3), 3.40 (2H, br s, D2O exchang., NH2), 6.95 (1H, d, Jꢃ8.61 Hz,
H-4), 7.30 (1H, br s, D2O exchang., NH), 7.54 (1H, d, Jꢃ8.61 Hz, H-3). 13C-
NMR (50 MHz, CDCl3) d: 19.94 (CH3), 28.42 [(CH3)3], 80.30 [(CH3)3C],
111.07 (C-3), 124.74 (C-4), 136.18 (C-5), 141.57 (C-6), 143.45 (C-2),
152.88 (OCONH). Anal. Calcd for C11H17N3O2: C, 59.17; H, 7.67; N, 18.82.
Found: C, 58.84; H, 7.79; N, 18.97.
tert-Butyl-N-(5-acetamido-6-methylpyridin-2-yl) Carbamate (27) To
a solution of 26 (2 g, 8.97 mmol) in dry dichloromethane (20 ml) acetic an-
hydride (0.95 ml, 10 mmol) was added and the resulting solution was stirred
at rt for 10 h. The solvent was vacuum-evaporated and the residue was puri-
fied by column chromatography (silica gel) using a a mixture of cyclo-
hexane/EtOAc (6/4, v/v) as the eluent, to give 27 (2.15 g, 91%). mp: 131 °C
(Et2O). 1H-NMR (400 MHz, CDCl3) d: 1.50 [9H, s, (CH3)3], 2.18 (3H, s,
COCH3), 2.35 (3H, s, CH3), 7.00 (1H, br s, D2O exchang., NHAc), 7.42 (1H,
br s, D2O exchang., NHBoc), 7.73 (1H, d, Jꢃ8.97 Hz, H-3), 7.88 (1H, d, Jꢃ
8.97 Hz, H-4). 13C-NMR (50 MHz, CDCl3) d: 20.25 (CH3), 23.56 (CH3CO),
28.25 [(CH3)3], 80.90 [(CH3)3C], 109.97 (C-3), 126.93 (C-5), 135.06 (C-4),
148.68 (C-2), 150.51 (C-6), 152.54 (OCONH), 169.40 (COCH3). Anal.
Calcd for C13H19N3O3: C, 58.85; H, 7.22; N, 15.84. Found: C, 58.61; H,
7.09; N, 15.68.
tert-Butyl-N-[5-acetamido-6-(2-benzyloxyethoxy)ethylpyridin-2-yl]
Carbamate (28) This compound was prepared by a procedure analogous
to that of 9, starting from 27 (650 mg, 2.45 mmol), using 3.3 eq of n-BuLi.
The anion formation was complete at ꢁ20 °C. The product was purified by
column chromatography (silica gel) using a mixture of cyclohexane/EtOAc
(7/3, v/v) as the eluent to provide 28 (420 mg, 40%) as an oil. 1H-NMR
(400 MHz, CDCl3) d: 1.51 [9H, s, (CH3)3], 2.01 (3H, s, COCH3), 2.93 (2H,
t, Jꢃ5.3 Hz, PyrCH2CH2O), 3.60—3.63 (4H, m, OCH2CH2O), 3.80 (2H, t,
Jꢃ5.3 Hz, PyrCH2CH2O), 4.50 (2H, s, CH2Ph), 7.09 (1H, br s, D2O
exchang., NHBoc), 7.27—7.36 (5H, m, Ph-H), 7.77 (1H, d, Jꢃ8.6 Hz, H-3),
8.03 (1H, d, Jꢃ8.6 Hz, H-4), 8.86 (1H, br s, D2O exchang., NHAc).
13C-NMR (50 MHz, CDCl3) d: 23.94 (CH3CO), 28.42 [(CH3)3], 35.76
(PyrCH2CH2O), 69.29 (OCH2CH2O), 70.40 (OCH2CH2O), 72.09
(PyrCH2CH2O), 73.30 (CH2Ph), 80.98 [(CH3)3C], 110.72 (C-3), 127.84
(2ꢄPhenyl C), 128.06 (C-4ꢀ), 128.62 (2ꢄPhenyl C), 128.88 (C-5), 134.61
(C-4), 137.73 (C-1ꢀ), 147.95 (C-2), 150.37 (C-6), 152.36 (OCONH), 169.04
(CH3CO). Anal. Calcd for C23H31N3O5: C, 64.32; H, 7.27; N, 9.78. Found:
C, 64.21; H, 7.23; N, 9.60.
1
EtOAc (8/2, v/v) as the eluent to provide 21 (475 mg, 92%) as an oil. H-
NMR (400 MHz, CDCl3) d: 2.78 (3H, s, COCH3), 3.65—3.69 (2H, m,
OCH2CH2O), 3.71—3.75 (2H, m, OCH2CH2O), 4.13 (3H, s, OCH3), 4.56
(2H, s, CH2Ph), 4.89 (2H, s, pyrazol-CH2O), 7.30—7.34 (5H, m, Ph-H),
7.36 (1H, d, Jꢃ5.5 Hz, H-4), 7.99 (1H, d, Jꢃ5.5 Hz, H-5). 13C-NMR
(50 MHz, CDCl3) d: 24.12 (COCH3), 54.34 (OCH3), 66.32 (pyrazol-CH2O),
69.48 (OCH2CH2O), 70.32 (OCH2CH2O), 73.45 (CH2Ph), 108.51 (C-4),
127.84 (2ꢄPhenyl C), 128.50 (2ꢄPhenyl C), 129.79 (C-4ꢀ), 133.28 (C-3a),
136.22 (C-7a), 138.17 (C-1ꢀ), 140.45 (C-5), 147.58 (C-3), 151.77 (C-7),
168.75 (COCH3). Anal. Calcd for C19H21N3O4: C, 64.21; H, 5.96; N, 11.82.
Found: C, 64.33; H, 5.86; N, 11.67.
3-(2-Hydroxyethoxy)methyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine
(22) This compound was prepared by a procedure analogous to that of 12,
starting from 21 (420 mg, 1.18 mmol). The product was purified by column
chromatography (silica gel) using a mixture of EtOAc/CH3OH (95/5, v/v) as
the eluent to provide 22 (220 mg, 85%) as an amorphous solid. 1H-NMR
(400 MHz, CDCl3) d: 2.75 (1H, br s, D2O exchang., OH), 3.69 (2H, t, Jꢃ
4.8 Hz, OCH2CH2OH), 3.78 (2H, t, Jꢃ4.8 Hz, OCH2CH2OH), 4.15 (3H, s,
OCH3), 4.94 (2H, s, pyrazol-CH2O), 7.27 (1H, d, Jꢃ5.9 Hz, H-4), 7.81 (1H,
d, Jꢃ5.9 Hz, H-5), 10.85 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz,
CDCl3) d: 53.68 (OCH3), 61.95 (OCH2CH2OH), 66.01 (pyrazol-CH2O),
72.12 (OCH2CH2OH), 108.38 (C-4), 127.35 (C-3a), 128.27 (C-7a), 136.54
(C-5), 143.84 (C-3), 150.76 (C-7). Anal. Calcd for C10H13N3O3: C, 53.81; H,
5.87; N, 18.82. Found: C, 53.62; H, 5.79; N, 18.97.
1,6-Dihydro-3-(2-hydroxyethoxy)methyl-7H-pyrazolo[3,4-c]pyridin-7-
one (23) Compound 22 (50 mg, 0.22 mmol) was added at 0 °C to a satu-
rated solution of HCl in dry methanol (10 ml) and the resulting solution was
stirred at room temperature for 18 h. The solvent was vacuum-evaporated
and the residue was purified by column chromatography (silica gel) using a
mixture of CH2Cl2/CH3OH (95/5, v/v) as the eluent to give 23 (40 mg, 85%).
mp: 235 °C (EtOH). 1H-NMR (400 MHz, DMSO-d6) d: 3.45 (2H, t, Jꢃ
4.9 Hz, OCH2CH2OH), 3.50 (2H, t, Jꢃ4.9 Hz, OCH2CH2OH), 4.68 (2H, s,
pyrazol-CH2O), 6.58 (1H, d, Jꢃ6.6 Hz, H-4), 6.92 (1H, m, H-5), 11.25 (1H,
br s, D2O exchang., NH-6), 13.88 (1H, br s, D2O exchang., NH-1). 13C-NMR
(50 MHz, DMSO-d6) d: 60.20 (OCH2CH2OH), 64.99 (pyrazol-CH2O), 71.62
(OCH2CH2OH), 98.44 (C-4), 124.03 (C-3a), 125.59 (C-5), 132.79 (C-7a),
143.32 (C-3), 154.18 (C-7). Anal. Calcd for C9H11N3O3: C, 51.67; H, 5.30;
N, 20.09. Found: C, 51.41; H, 5.24; N, 19.94.
tert-Butyl-N-(6-methyl-5-nitropyridin-2-yl) Carbamate (25) To a so-
lution of the aminopicoline 2417) (2 g, 13.07 mmol) in dry THF (40 ml)
sodium hydride (0.78 g, 19.4 mmol, 60% suspension in paraffin oil) was
added under argon at 0 °C and the resulting mixture was stirred at rt for 2 h.
It was then cooled at 0 °C, a solution of di-tert-butyl dicarbonate (3.3 ml,
14.38 mmol) in dry THF (10 ml) was added dropwise and the mixture was
stirred at rt for an additional 4 h. The solvent was vacuum-evaporated, a
solution of HCl (0.5 N, 20 ml) was added to the residue and the product was
extracted with ethyl acetate. The organic extracts were dried (Na2SO4) and
concentrated to dryness and the residue was purified by column chromatog-
raphy (silica gel) using a a mixture of cyclohexane/EtOAc (98/2, v/v) as the
eluent, to give 25 (3.15 g, 95%). mp: 127—128 °C (EtOAc). 1H-NMR
(400 MHz, CDCl3) d: 1.47 [9H, s, (CH3)3], 2.73 (3H, s, CH3), 7.93 (1H, d,
Jꢃ9.12 Hz, H-3), 8.32 (1H, d, Jꢃ9.12 Hz, H-4), 8.37 (1H, br s, D2O
exchang., NH). 13C-NMR (50 MHz, CDCl3) d: 24.29 (CH3), 28.11 [(CH3)3],
82.27 [(CH3)3C], 109.63 (C-3), 135.85 (C-4), 140.43 (C-5), 151.76
(OCONH), 154.28 (C-6), 154.34 (C-2). Anal. Calcd for C11H15N3O4: C,
52.17; H, 5.97; N, 16.59. Found: C, 52.34; H, 5.81; N, 16.38.
tert-Butyl-N-[1-acetyl-3-(2-benzyloxyethoxy)methyl-1H-pyrazolo[4,3-
b]pyridin-5-yl] Carbamate (29) This compound was prepared by a proce-
dure analogous to that of 11, starting from 28 (400 mg, 0.93 mmol). The
product was purified by column chromatography (silica gel) using a mixture
of cyclohexane/EtOAc (7/3, v/v) as the eluent to provide 29 (370 mg, 90%)
1
as an oil. H-NMR (400 MHz, CDCl3) d: 1.53 [9H, s, (CH3)3], 2.76 (3H, s,
COCH3), 3.68 (2H, t, Jꢃ4.7 Hz, OCH2CH2O), 3.83 (2H, t, Jꢃ4.7 Hz,
OCH2CH2O), 4.56 (2H, s, CH2Ph), 4.93 (2H, s, pyrazol-CH2O), 7.27—7.34
(5H, m, Ph-H), 7.49 (1H, br s, D2O exchang., NH), 8.23 (1H, d, Jꢃ9.2 Hz,
H-6), 8.61 (1H, d, Jꢃ9.2 Hz, H-7). 13C-NMR (50 MHz, CDCl3) d: 22.43
(COCH3), 28.35 [(CH3)3C], 64.30 (pyrazol-CH2O), 69.48 (OCH2CH2O),
70.62 (OCH2CH2O), 73.37 (CH2Ph), 81.50 [(CH3)3C], 114.02 (C-6), 125.60
(C-7), 127.81 (3ꢄPhenyl C), 128.47 (2ꢄPhenyl C), 130.82 (C-7a), 138.28
(C-1ꢀ), 140.67 (C-3a), 147.69 (C-3), 150.74 (C-5), 152.43 (OCONH),
170.95 (COCH3). Anal. Calcd for C23H28N4O5: C, 62.71; H, 6.41; N, 12.72.
Found: C, 62.58; H, 6.36; N, 12.57.
1-Acetyl-3-(2-benzyloxyethoxy)methyl-1H-pyrazolo[4,3-b]pyridin-5-
yl-amine (30) Trifluoroacetic acid (0.1 ml, 1.28 mmol) was added at 0 °C
to a solution of 29 (60 mg, 0.14 mmol) in dry dichloromethane (10 ml) and
the mixture was stirred at rt for 10 h. The solvent was vacuum-evaporated
and the residue was treated with a saturated NaHCO3 solution and
dichloromethane. The organic layer was dried (Na2SO4) and concentrated to
dryness. The residue was purified by flash chromatography (silica gel) using
a mixture of cyclohexane/EtOAc (1/1, v/v) as the eluent to give 30 (45 mg,
97%). mp: 103 °C (EtOAc). 1H-NMR (400 MHz, CDCl3) d: 2.72 (3H, s,
COCH3), 3.71 (2H, t, Jꢃ4.7 Hz, OCH2CH2O), 3.83 (2H, t, Jꢃ4.7 Hz,
OCH2CH2O), 4.57 (2H, s, CH2Ph), 4.87 (2H, s, pyrazol-CH2O), 5.55 (2H,
br s, D2O exchang., NH2), 6.45 (1H, d, Jꢃ9 Hz, H-6), 7.28—7.36 (5H, m,
Ph-H), 8.28 (1H, d, Jꢃ9 Hz, H-7). 13C-NMR (50 MHz, CDCl3) d: 22.36
(COCH3), 64.41 (pyrazol-CH2O), 69.63 (OCH2CH2O), 70.51 (OCH2CH2O),
73.48 (CH2Ph), 112.11 (C-6), 125.78 (C-7), 127.81 (C-4ꢀ), 127.99 (2ꢄ
tert-Butyl-N-(5-amino-6-methylpyridin-2-yl) Carbamate (26) A solu-

June 2008
779
of cyclohexane/EtOAc (95/5, v/v) as the eluent to provide 37 (315 mg, 88%)
as an amorphous solid. 1H-NMR (400 MHz, CDCl3) d: 1.55 [9H, s, (CH3)3],
2.75 (3H, s, COCH3), 3.71 (2H, t, Jꢃ4.7 Hz, OCH2CH2OBn), 3.79 (2H, t,
Jꢃ4.7 Hz, OCH2CH2OBn), 4.59 (2H, s, CH2Ph), 4.92 (2H, s, pyrazol-
CH2O), 7.31—7.35 (5H, m, Ph-H), 8.25 (1H, br s, D2O exchang., NH), 8.37
(1H, s, H-4), 9.41 (1H, s, H-7). 13C-NMR (50 MHz, CDCl3) d: 22.61
(COCH3), 28.48 [(CH3)3C], 65.95 (pyrazol-CH2O), 69.52 (OCH2CH2OBn),
70.65 (OCH2CH2OBn), 73.47 (CH2Ph), 81.19 [(CH3)3C], 101.87 (C-4),
127.73 (C-4ꢀ), 127.87 (2ꢄPhenyl C), 128.50 (2ꢄPhenyl C), 133.05 (C-3a),
133.33 (C-7a), 136.42 (C-7), 138.31 (C-1ꢀ), 147.65 (C-5), 148.59 (C-3),
152.73 (OCONH), 170.23 (COCH3). Anal. Calcd for C23H28N4O5: C, 62.71;
H, 6.41; N, 12.72. Found: C, 62.80; H, 6.44; N, 12.77.
1-Acetyl-3-(2-benzyloxyethoxy)methyl-1H-pyrazolo[3,4-c]pyridin-5-
yl-amine (38) This compound was prepared by a procedure analogous to
that of 30, starting from 37 (260 mg, 0.59 mmol). The product was purified
by column chromatography (silica gel) using a mixture of cyclohexane/
EtOAc (4/6, v/v) as the eluent to provide 38 (175 mg, 87%). mp: 98—100 °C
(dec.) (EtOAc). 1H-NMR (400 MHz, CDCl3) d: 2.69 (3H, s, COCH3),
3.67—3.70 (2H, m, OCH2CH2OBn), 3.72—3.75 (2H, m, OCH2CH2OBn),
4.36 (2H, br s, D2O exchang., NH2), 4.57 (2H, s, CH2Ph), 4.86 (2H, s, pyra-
zol-CH2O), 6.85 (1H, s, H-4), 7.30—7.36 (5H, m, Ph-H), 9.22 (1H, s, H-7).
13C-NMR (50 MHz, CDCl3) d: 22.51 (COCH3), 66.58 (pyrazol-CH2O),
69.59 (OCH2CH2OBn), 70.32 (OCH2CH2OBn), 73.41 (CH2Ph), 96.60 (C-
4), 127.84 (3ꢄPhenyl C), 128.61 (2ꢄPhenyl C), 131.56 (C-7a), 133.72 (C-
3a), 136.63 (C-7), 138.24 (C-1ꢀ), 147.95 (C-3), 154.71 (C-5), 169.93
(COCH3). Anal. Calcd for C18H20N4O3: C, 63.52; H, 5.92; N, 16.46. Found:
C, 63.66; H, 5.71; N,16.65.
3-(2-Hydroxyethoxy)methyl-1H-pyrazolo[3,4-c]pyridin-5-yl-amine
(39) This compound was prepared by a procedure analogous to that of 12,
starting from 38 (120 mg, 0.35 mmol) using 5 eq of BCl3. The product was
purified by column chromatography (silica gel) using a mixture of CH2Cl2/
CH3OH (9/1, v/v) as the eluent to provide 39 (60 mg, 83%) as an amor-
phous solid. 1H-NMR (400 MHz, DMSO-d6) d: 3.44 (2H, t, Jꢃ4.9 Hz,
OCH2CH2OH), 3.50 (2H, t, Jꢃ4.9 Hz, OCH2CH2OH), 4.69 (2H, s, pyrazol-
CH2O), 5.40 (2H, br s, D2O exchang., NH2), 6.65 (1H, s, H-4), 8.47 (1H, s,
H-7), 12.98 (1H, br s, D2O exchang., NH-1). 13C-NMR (50 MHz, DMSO-d6)
d: 60.16 (OCH2CH2OH), 65.15 (pyrazol-CH2O), 71.49 (OCH2CH2OH),
92.56 (C-4), 128.99 (C-3a), 131.97 (C-7), 133.59 (C-7a), 140.01 (C-3),
152.90 (C-5). Anal. Calcd. for C9H12N4O2: C, 51.92; H, 5.81; N, 26.91.
Found: C, 51.77; H, 5.60; N, 27.18.
Phenyl C), 128.54 (2ꢄPhenyl C), 128.91 (C-7a), 138.17 (C-1ꢀ), 141.04 (C-
3a), 147.03 (C-3), 157.80 (C-5), 170.92 (COCH3). Anal. Calcd for
C18H20N4O3: C, 63.52; H, 5.92; N, 16.46. Found: C, 63.34; H, 6.15; N,
16.63.
3-(2-Hydroxyethoxy)methyl-1H-pyrazolo[4,3-b]pyridin-5-yl-amine
(31) This compound was prepared by a procedure analogous to that of 12,
starting from 30 (40 mg, 0.12 mmol) using 5 eq of BCl3. The product was
purified by column chromatography (silica gel) using a mixture of CH2Cl2/
CH3OH (95/5, v/v) as the eluent to provide 31 (20 mg, 82%). mp:
146—148 °C (EtOH). 1H-NMR (400 MHz, DMSO-d6) d: 3.48 (4H, m,
OCH2CH2O), 4.63 (2H, s, pyrazol-CH2O), 4.72 (1H, br s, D2O exchang.,
OH), 5.95 (2H, br s, D2O exchang., NH2), 6.60 (1H, d, Jꢃ8.9 Hz, H-6), 7.62
(1H, d, Jꢃ8.9 Hz, H-7), 12.78 (1H, br s, D2O exchang., NH-1). 13C-NMR
(DMSO-d6) d: 60.09 (OCH2CH2-OH), 63.24 (pyrazol-CH2O), 71.40
(OCH2CH2OH), 110.99 (C-6), 120.77 (C-7), 129.07 (C-7a), 136.91 (C-3a),
138.71 (C-3), 156.31 (C-5). Anal. Calcd for C9H12N4O2: C, 51.92; H, 5.81;
N, 26.91. Found: C, 52.17; H, 5.76; N, 26.82.
tert-Butyl-N-(4-methyl-5-nitropyridin-2-yl) Carbamate (33) This
compound was prepared by a procedure analogous to that of 25, starting
from 3218) (3 g, 19.6 mmol). The product was purified by column chromatog-
raphy (silica gel) using a mixture of cyclohexane/EtOAc (4 : 6, v/v) as the
eluent, to give pure 33 (4.71 g, 95%). mp: 186 °C (Et2O/n-pentane); 1H-
NMR (400 MHz, CDCl3) d: 1.59 [9H, s, (CH3)3], 2.69 (3H, s, CH3), 8.10
(1H, s, H-3), 9.12 (1H, s, H-6), 10.43 (1H, br s, D2O exchang., NH). 13C-
NMR (50 MHz, CDCl3) d: 21.59 (CH3), 28.42 [(CH3)3CO], 82.74
[(CH3)3C], 114.39 (C-3), 140.71 (C-5), 146.00 (C-6), 147.02 (C-4), 152.35
(OCONH), 155.87 (C-2). Anal. Calcd for C11H15N3O4: C, 52.17; H, 5.97; N,
16.59. Found: C, 52.34; H, 5.81; N, 16.38.
tert-Butyl-N-(5-amino-4-methylpyridin-2-yl) Carbamate (34) A solu-
tion of 33 (1 g, 3.95 mmol) in dry ethanol (40 ml) was hydrogenated in the
presence of 10% Pd/C (130 mg) under a pressure of 50 psi at rt for 5 h. The
solution was filtered through a celite pad to remove the catalyst and the
filtrate was evaporated to dryness to give pure 34 (880 mg, 98%). mp:
1
167 °C (EtOAc); H-NMR (400 MHz, CDCl3) d: 1.50 [9H, s, (CH3)3], 2.14
(3H, s, CH3), 3.34 (2H, br s, D2O exchang., NH2), 7.69 (1H, s, H-3), 7.78
(1H, s, H-6), 9.28 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz,
CDCl3) d: 17.38 (CH3), 28.47 [(CH3)3C], 80.08 [(CH3)3C], 114.01 (C-3),
133.86 (C-6), 134.49 (C-5), 136.96 (C-4), 145.25 (C-2), 153.26 (OCONH).
Anal. Calcd for C11H17N3O2: C, 59.17; H, 7.67; N, 18.82. Found: C, 58.84;
H, 7.79; N, 18.97.
Antiviral and Cytostatic Assays Antiviral activity against vesicular
stomatitis virus, HSV-1, HSV-2, Vaccinia virus, VZV and CMV in HEL cell
cultures, Coxsackie B4 virus, respiratory syncytial virus, reovirus-1, Sindbis
virus and Punta Toro virus in Vero and HeLa cell cultures was determined
by adding virus (100 CCID50) to confluent human cell cultures in 96-well
microtiter plates. After a 1—2 h incubation period, residual virus was
removed and the infected cells were further incubated with the medium con-
taining different concentrations of the compounds. After incubation at 37 °C,
virus-induced cytopathogenicity was monitored microscopically when the
infected control cell cultures reached full cytopathicity. For the anti-HIV
assays, the viruses were administered to CEM cell cultures in the presence
of different dilutions of the test compounds, and virus-induced cytopathicity
(syncytia formation) was microscopically recorded. Antiviral activity was
expressed as the EC50 or compound concentration required to reduce virus-
induced cytopathogenicity by 50%. EC50 values were calculated from
graphic plots of the percentage of cytopathogenicity as a function of concen-
tration of the compounds.
Cytostatic activity against L1210 (murine leukemia), Molt4/C8 and CEM
(human T-lymphocytes) cells were measured by adding ca. 50000 to 75000
cells per 200-ml well in 100-ml growth medium. Then, medium (100 ml) con-
taining different concentrations of the test compounds were added. After 2—
3 d of incubation at 37 °C, the cell number was determined with a Coulter
counter. The cytostatic concentration was calculated as the CC50 or the com-
pound concentration required to reduce cell growth by 50% relative to the
number of cells in the untreated controls. CC50 values were estimated from
graphic plots of the number of cells (percentage of control) as a function of
the concentration of the test compounds.
tert-Butyl-N-(5-acetamido-4-methylpyridin-2-yl) Carbamate (35) To
a solution of 34 (880 mg, 3.95 mmol) in dry dichloromethane (20 ml) acetic
anhydride (0.56 ml, 5.93 mmol) was added and the resulting solution was
stirred at rt for 12 h. The solvent was vacuum-evaporated and the residue
was triturated with diethyl ether to give pure 35 (0.98 g, 93%). mp: 208—
210 °C (EtOAc); 1H-NMR (400 MHz, DMSO-d6) d: 1.46 [9H, s, (CH3)3],
2.05 (3H, s, COCH3), 2.19 (3H, s, CH3), 7.69 (1H, s, H-3), 8.15 (1H, s, H-
6), 9.43 (1H, br s, D2O exchang., NHAc), 9.83 (1H, br s, D2O exchang.,
NHBoc). 13C-NMR (50 MHz, DMSO-d6) d: 17.95 (CH3), 23.02 (CH3CO),
28.09 [(CH3)3C], 79.65 [(CH3)3C], 113.22 (C-3), 128.70 (C-5), 143.78 (C-
4), 144.55 (C-6), 149.62 (C-2), 152.80 (OCONH), 168.85 (COCH3). Anal.
Calcd for C13H19N3O3: C, 58.85; H, 7.22; N, 15.84. Found: C, 58.61; H,
7.09; N, 15.68.
3-tert-Butyl-N-[5-acetamido-4-(2-benzyloxyethoxy)ethylpyridin-2-yl]
Carbamate (36) This compound was prepared by a procedure analogous
to that of 9, starting from 35 (600 mg, 2.26 mmol), using 3.3 eq of n-BuLi.
The anion formation was complete at ꢁ20 °C. The product was purified by
column chromatography (silica gel) using a mixture of cyclohexane/EtOAc
(1/1, v/v) as the eluent to provide 36 (390 mg, 40%) as an oil. 1H-NMR
(400 MHz, CDCl3) d: 1.51 [9H, s, (CH3)3], 1.99 (3H, s, COCH3), 2.82 (2H,
t, Jꢃ5.2 Hz, PyrCH2CH2O), 3.56—3.59 (4H, m, OCH2CH2O), 3.73 (2H, t,
Jꢃ5.2 Hz, PyrCH2CH2O), 4.49 (2H, s, CH2Ph), 7.23—7.36 (5H, m, Ph-H),
7.81 (1H, s, H-3), 8.41 (1H, br s, D2O exchang., NHBoc), 8.58 (1H, s, H-6),
8.75 (1H, br s, D2O exchang., NHAc). 13C-NMR (50 MHz, CDCl3) d: 23.78
(CH3CO), 28.43 [(CH3)3C], 33.37 (PyrCH2CH2O), 69.21 (OCH2CH2O),
70.39 (OCH2CH2O), 72.60 (PyrCH2CH2O), 73.32 (CH2Ph), 81.04
[(CH3)3C], 112.97 (C-3), 127.87 (2ꢄPhenyl C), 128.08 (C-4ꢀ), 128.63 (2ꢄ
Phenyl C), 129.39 (C-5), 137.68 (C-1ꢀ), 144.07 (C-4), 144.54 (C-6), 148.95
(C-2), 152.78 (OCONH), 169.08 (CH3CO). Anal. Calcd for C23H31N3O5: C,
64.32; H, 7.27; N, 9.78. Found: C, 64.41; H, 7.19; N, 9.93.
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