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Syntheses and Antiviral Activities of Some 5 В╨Ж-O-Acyl Derivatives.

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Prodrugs of 5-Ethyl-2'-deoxyuridine
317/84
867
Mo1.-Masse 364 (ms). - IR (KBr): 3070,3030,2940,2890,2870,2840 (CH), 1710,1655,1598 (C=C)
cm-';- 'H-NMR(CDC13): 6 (ppm) = 8.00-8.40(m, 4H), 7.27 (s, 5H), 3.82 (d, l H , J = 2Hz), 3.57 (s,
2H), 2.63-2.13 (m, 2H), 2.00-1.43 (m, 6H).
7,7a,8,9,10,I1-Hexahydro-7-phenyl-6H,1laH-[l]-benzothiopyrano[4,3-b]chromen-lIa-o1-5,5dioxid (13)
Darstellung analog 8a aus 1.35 g (3 mmol) l l b in 15 ml Dioxan und 15 ml2N-HCVWasser (1 : 2).
Reaktionszeit 5 1/2 h. Nach Waschen des Riickstandes mit Petrolether wurde aus Ethanol
umkristallisiert. Farblose Kristalle, Schmp. 156-158", Ausb. 0.81 g (71 %). C,H,,O,S
Ber. C 69.1 H
5.80S8.4Mol.-Masse382.48;Gef. C68.9H5.82S8.1Mol.-Masse382(ms).-IR(KBr):3465(OH),
3070,3030,2940,2900,2860(CH), 1635,1595(C=C)cm-';-'H-NMR(CDC13):
G(ppm) = 8.00-7.00
(m, 9H), 3.50 (s, 2H), 3.37 (d, l H , J = lOHz), 2.90 (s, breit, 1WD-Tausch), 2.20-1.00 (m, 9H).
Literatur
104. Mitt. iiber Untersuchungen an Pyran-Derivaten; 103. Mitt. F. Eiden und G. Felbermeir,
Arch. Pharm. (Weinheim) 317, 675 (1984).
P. Pfeiffer und J. Grimmer, Ber. Dtsch. Chem. Ges. 50, 911 (1917); W. H. Perkin, I. N. Ray und
R. Robinson, J. Chem. SOC.1926,941; F. Arndt und G. Kallner, Ber. Dtsch. Chem. Ges. 57,202
(1924); 0.Dann und H. Hofmann, Chem. Ber. 95, 1446 (1962); P. Pfeiffer, H. Oberlin und
E. Konermann, Ber. Dtsch. Chem. Ges. 58, 1946 (1925); G. Wagner, C. Garbe, H. Vieweg,
M. Briinn und A. Dittrich, Pharmazie 34, 55 (1979).
L. Farkas, A. Gottsegen und M. NogrBdi, Tetrahedron Lett. 1968, 4099.
A. LBvai und J. B. SchBg, Pharmazie 34,749 (1979).
D. Mulvagh, M. J. Meegen und D. Donnelly, J. Chem. Res. 1979, 137; A. LCvai, Z. Dinya,
J. B. SchBg, G.Tbth und A. Szollosy, Pharmazie 36, 465 (1981).
Dissertation W. Winkier, Munchen 1981; W. Winkler und F. Eiden, Dtsch. Apoth. Ztg. 123,Nr.
15 (Sonderbeilage) (1983).
[Ph 8241
Arch. Pharm. (Weinheim) 317, 867-873 (1984)
Prodrugs of 5-Ethyl-Z'-deoxyuridine, I.
Syntheses and Antiviral Activities of Some 5 '-0-Acyl Derivatives
Klaus Keppeler*)+,Gebhard Kiefer+ and Erik De Clercq++
Research Laboratories of Robugen GmbH, PoBox266, 7300 Esslingen, West Germany and the
Katholieke Universiteit Leuven, Rega Instituut, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
+
++
Eingegangen am 19. Juli 1983
A series of 5'-0- acyl derivatives (2a-e) of 5-ethyl-2'-deoxyuridine(EDU, EtUdR) were prepared by
(DMF). These
direct acylation of the parent nucleoside 1 in pyridine/N,N-dimethylformamide
compounds, designed as prodrugs of 1, offer a wider range of solubilities and lipophilicitiesthan 1.
03654233/84/101&0867 $ 02.50/0
0 Verlag Chemie GmbH, Weinheim 1984
868
Arch. Pharm.
Keppeler, Kiefer and De Clercq
The antiviral activities (against herpes simplex virus types 1and 2) of all compounds were determined
in primary rabbit kidney (PRK) cell cultures.
Prodrugs von 5-Ethyl-2'-desoxyuridin,
1. Mitt.: Synthese und antivirale Aktivitiit einiger 5'-0(Acy1)-Derivate
Eine Sene von 5'-0-(Acy1)-Derivaten (2e-e)von 5-Ethyl-2'-desoxyuridin (EDU, EtUdR) wurde
durch direkte Acylierung des Ausgangsnukleosides 1 in PyrididN,N-Dimethylformamid (DMF)
hergestellt. Diese Substanzen, Prodrugs von 1,bieten ein groBeres Spektrum an Loslichkeit und
Lipophilie gegenuber 1.Die antivirale Aktivitat (gegen Herpes Simplex Virus Typ 1 und 2) aller
Verbindungen wurde in primaren Kaninchennieren-Zellkulturen bestimmt.
The nucleoside analog 5-ethyl-2'-deoxyuridine (1)(EDU, EtUdR) has shown promising activity
against different strains of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type2
(HSV-2) in cell culture'-3). Compound 1has proven to be useful as an effective, relatively nontoxic,
antiviral agent to treat experimental HSV-1 keratitis in rabbits4', herpes labialis as well as herpes
genitalid) and herpes virus infections of the cornea in humans6"). Despite its efficacy, 1does suffer
from a number of limitations: (a) a high aqueous solubility of 70mg/ml at 20°C that causes a rapid
elimination after parenteral administration of the drug, (b) a rapid enzymatic degradation to give the
nonvirostatic metabolites 5-ethyluracil') and 5-(l-hydroxyethyl)uracil9.'0),(c) a low lipophilicity that
hampers the passage of 1 through the blood brain barrier.
In an attempt to prevent these problems of drug formulation, delivery and enzymatic
deactivation, several 5'monoesters of 1have been synthesized"). In addition we have also
synthesized a series of 5'monoesters (2a-e) of 1, based on dicarboxylic acids. Similar
procedures have been followed in prodrug design for a variety of pharmaceutical^'^^'^).
Chemistry
The monoalkylester chlorides were prepared according to literature methods or are
commercially available. Direct acylation of the free nucleoside 1 was effected by adding
the acid chlorides, dissolved in N,N-dimethylformamide (DMF), in a molecular ratio of
1:1to a solution of 1 in pyridine at ice-bath temperature, in order to prevent side reactions
at the 3'-position of 1 (Scheme 1). The 5'-acylated nucleosides 2a-e were obtained in yields
of 13-57 % after column chromatography over silica gel to separate minor, peracylated,
I
0
0
H
H
1
Scheme 1: General Procedure for the Acylation of Compound 1
2a-e
31 7/84
869
Prodrugs of 5-Ethyl-2'-deoxyuridine
contaminants and unreacted 1. The low-yieldingreactions contained much unreacted 1 (as
monitored by TLC) that could not be recovered. The structures of the compounds were
confirmed by NMR spectra. In contrast with other worker~'~)
we found that acylation of a
nucleoside, in casu 1, decreased the aqueous solubility. Further details are presented in
table 1.
Physical Properties
Compounds 2a-e show an about 100-folddecrease in aqueous solubility, as compared to
the parent compound 1 (Table 1).The melting points of 2a-e drop by about 15-20 "C(m. p.
of 1: 152-153 O C ) " ) . Only 2a shows an increase of the melting point (by 25 "C).
Table 1: S-O-(Acyl)-Esters2a-2e of 5-Ethyl-2'-deoxyuridine
Compd.
Yield m.p.
% oc
0
II
R-C-
hmaxH20 Formula
nm (1% E) (MW)
Analyses
Calc.:
Found
C
Solubilitya
(mgiml)
H
N
267 (3.97) ClsHlsNzO8 49.1
(342.3)
49.0
5.29
5.26
8.2 3.47c
8.1
267 (3.97) C 1 7 H ~ N 2 0 8 53.1
(384.4)
53.0
6.29
6.28
7.3 1.14
7.1
138-139
267 (3.97) ClsHxNzOs 54.3
(398.4)
54.1
6.57
6.47
7.0
6.8
13.3 136-138
267 (3.89) C16NzzNzO8 51.9
(370.4)
51.5
5.99
5.87
7.6 0.60
7.7
137.5-138.5 268(3.98) C18H26N~O8 54.3
(398.4)
54.2
6.57
6.56
7.0 0.87
7.1
0
2a
I
CH3O-d-C=O
N.Db 1785
0
II
2b
CH30-C-(CH2)3-C=O
57.2 144-145
2c
0
II
CH30-C -(CH2)4-C=O50.0
2d
CH3CH20-C -CH2-C=O
1.36
0
II
0
2e
II
CH3CH20-C -CH-C=O
$HZ
34.7
CH 3
a)
Water at 2 0 T , for methodology see the Experimental Section. Solubility of 1: 70mgiml.
b,
Not done.
')
After 24h, there is a great amount of EtUdR in solution (indicated by TLC).
870
Keppeler, Kiefer and De Clercq
Arch. Pharrn.
Antiviral Activity
The antiviral activity of the compounds was determined in primary rabbit kidney (PRK)
cell cultures (Tables 2 and 3). For a detailed description of the virus strains and procedures
used to evaluate antiviral activity in PRK cell cultures,
The esters 2a-e function as
effective antivirals. They inhibit viral cytopathogenicity at a concentration which is only
slightly higher than the concentration at which 1inhibits viral cytopathogenicity (Table 2)
(with the exception of 2d in its activity against vaccinia virus). These data suggest that the
5’-0-(acy1)-estersof EtUdR are readily hydrolyzed under our in vitro conditions to release
the parent compound. Supporting evidence for this notion stems from the finding that
5’-derivatives of 1,which failed to release 1are uneffective as antiviral agent~’~).
That the
inhibitory effects of the 5’-0-(acy1)-estersof EtUdR on virusinduced cytopathogenicity
(Table 2) truly reflect an inhibition of virus multiplication was ascertained by measuring
the effects of the compounds on virus growth (Table 3). When added to the PRK cells at
10&ml immediately after the cells have been infected with HSV-1, the EtUdR esters 2a-e
cause a reduction in virus yield (circa 3.3-3.61og10, as determined 24h after virus
infection) which is identical to that achieved by the free nucleoside.
Table2 Antiviral Activity of 5’-0-(Acy1)-Estersof 1 in PRK Cell Cultures
Minimum inhibitory concentration (MIC) (Mg/ml)a
Compd. HSV-1
HSV-2‘
Vaccinia virus
1
0.33
(0.1-0.7)
0.41
(0.1-0.7)
2a
0.7
0.3
{0.2-0.7)
10
2b
2 .o
2.6
(2-4)
10
2c
1.6
(0.7 -2.0)
1.8
(0.4-4 .O)
10
3 .O
2.6
(2.O-4.0)
150
(2 .O -7 .O)
2.6
(2.0-4.0
(0.7-1.0)
2d
2e
0.8
a)
Required to reduce virus-induced cytopathogenicity by 50 %.
b,
Average values for three HSV-1 strains (KOS, F and McIntyre).
2.7
1
Average values for three HSV-2 strains (Lyons, G and 196). The range of individual values is
indicated in parentheses.
871
Prodrugs of 5-Ethyl-2’-deoxyuridine
31 7/84
Table3 Inhibitory Effects of 5’-0-(Acy1)-Estersof 1 on the Multiplication of HSV-1 (strain KOS) in
PRK Cell Cultures
Compd.
1
2a
2b
Virus yield (loglo PFU/mBa
Compd.
Virus yield (loglo PFU/ml)a
2c
2d
2e
3.8
3.9
3.9
Control
3.7
4 .O
3.8
7.3
a) The cell cultures were inoculated with HSV-1 (strain KOS)at lo4.’ PFW per petri dish (lo6 cells).
The compounds were added at a concentration of lOpg/ml (which is well above their MIC for
inhibition of virus-induced cytopathogenicity:see Table 2). Virus yield was determined at 24 h after
virus infection by plaque formation in Vero cell cultures. Average values for two separate
experiments.
Antimetabolic Activity
Like EtUdR (1)itself, all 5’-0-(acy1)-esters2a-e can be considered as rather specific in
their activity against HSV-1 and HSV-2, since they inhibit the replication of these viruses at
concentrations well below those that affect normal cell metabolism (Table 4). Normal cell
metabolism was monitored by the incorporation of 2’-deoxythymidine (dThd) or
2’-deoxyuridine (dUrd) into DNA of uninfected PRK cell cultures. A relatively greater
inhibition of dUrd than of dThd incorporation can thereby be interpreted as evidence for a
specific action at the thymidylate synthetase level”). EtUdR and its esters inhibit dUrd
incorporation at a consistently lower concentration than dThd incorporation, the
difference varying from 2.2- to 13.2-fold. These data indicate that a concentration higher
than that required for inhibition of virus replication EtUdR and its esters may interfere
with the host cell thymidylate synthetase.
Table4 Antimetabolic Activity of 5‘-O-(Acyl)-Estersof 1 in PRK Cell Cultures
Compd.
1
2a
2b
2c
2d
2e
(3H-methy1)dThd
incorporation
46
83
17
31
123
43
(3H-l ‘,Z‘)dUrd
incorporation
4.6
6.3
7 .I
9 .o
30
8
a) Required to inhibit (3H-metvl)dThd or (3H-l’,2’)dUrd incorporation by 50%. Input of the
radiolabeled precursor (per lo5 PRK cells) : 10 pmoles (0.38pCi) of (3H-methyl)dThd and 6 pmoles
(0.25 pCi) of (3H-1’,2’)dUrd. Average values for three separate determinations.
872
Keppeler, Kiefer and De Clercq
Arch. Pharm.
Discussion
All 5'-0-(acy1)-esters of EtUdR exhibit an antiviral activity in vitro that is comparable to
EtUdR, in both potency and selectivity. These esters should therefore be further pursued
for their antiviral potentials in vivo. One may postulate that in vivo the 5'-O-(acyI)-esters
of EtUdR may be degraded less effectively than the parent compound to the inactive
metabolites 5-ethyluracil and 5-(l-hydroxyethyl)uracil.
Experimental Part
M.p. : (uncorr.) Dr. Tottoli apparatus (Biichi, Switzerland). Microanalyses: Fa. Pascher, Bonn, West
Germany. TLC: Silica gel plates 6OFz5,, (E. Merck, Darmstadt, West Germany). Column
chromatography: Silica gel 60 (E. Merck), chloroform/i-propano19/1(v/v). 'H-NMR:Bruker XL-90,
DMSO-d6. UV-spectra: Zeiss Opton M 4 Q spectrophotometer.
General Procedure for Acylation
To a solution of 2,568 (0,Ol mol) 5-ethyl-2'-deoxyuridine in 20ml dry pyridine cooled to 0°C in an
ice-bath, dropwise and while stirring, 0,Ol mol of the corresponding acyl chloride was added,
dissolvedin DMF. After stirring overnight at room temp., the mixture was partitioned between 150ml
water and 50 ml dichloromethane. The aqueous layer was extracted with 2 X 50 ml dichloromethane.
The combined organic layers (1501111) were washed with 3 X 30ml 3 N-H,SO, and 2 x 30ml water,
dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude 5'-monoesters of
1were purified by column chromatography, using chlorofordi-propano19:l (vh) as eluent, or were
recrystallized with the aid of activated carbon (Darco G 60).
Determination of Water Solubility: 200mg of each compound were agitated for 24h in lOml
volumetric flask with distilled water at 20 "C. To remove undissolved material, the solutions were
filtered through 40-100 pm sintered glass funnels, and the concentration of dissolved compound was
determined by UV absorbance.
References
1 K. K. Gauri and G. Malorny, Naunyn-Schmiedebergs Arch. Pharmacol. Exp. Pathol. 257, 21
(1967).
2 E. De Clercq and D. Shugar, Biochem. Pharmacol. 24, 1073 (1975).
3 E. De Clercq, J. Descamps, G. Verhelst, R. T. Walker, A. S. Jones, P. F. Torrence and D.
Shugar, J. Infect. Dis. 141, 563 (1980).
4 K. K. Gauri, Klin. Monatsbl. Augenheilkd. 153, 837 (1968).
5 S. W. Wassilew, Z. Hautkrankh. 54, 251 (1979).
6 K. K. Elze, Adv. Ophthalmol. 38, 134 (1979).
8 R. Kaul, G. Kiefer, S. Erhardt and B. Hempel, J. Pharm. Sci. 69, 531 (1980).
9 R. Kaul, K. Keppeler, G. Kiefer, B. Hempel and P. Fischer, Chemosphere 11,539 (1982).
10 K. Keppeler, G. Kiefer and E. De Clercq, paper in preparation.
11 G. Kiefer, K. Keppeler and E. De Clercq, paper in preparation.
12 T. Higuchi and V. Stella, Eds., Pro-Drugs as Novel Drug Delivery Systems, Am. Chem. SOC.,
Washington D. C. 1975.
13 E. B. Roche, Ed., Design of Biopharmaceutical Properties through Prodrugs and Analogs, Am.
Pharm. Assoc., Washington D. C. 1977.
317/84
873
MS von a-Ethoxy-Pyridinen und Pyrimidinen
14 D.C. Baker, T. H. Haskell and S. R. Putt, J. Med. Chem. 21, 1218 (1978).
15 M. Swierkowski and D. Shugar, J. Med. Chem. 12, 533 (1969).
16 E. De Clercq, J. Descamps, P. De Somer, P. J. Barr, A. S. Jones and R. T. Walker, Proc. Natl.
Acad. Sci. USA 76, 2947 (1979).
17 E. De Clercq, K. Keppeler and G. Kiefer, unpublished data.
18 E. De Clercq, J. Descamps, G.-F. Huang and P.F. Torrence, Mol. Pharmacol. 14, 422
(1978).
[Ph 8251
Arch. Pharm. (Weinheim) 317, 873-877 (1984)
Massenspektrometrie einiger substituierter a-Ethoxy-Pyridine und
-Pyrimidine
Ivo C . Ivanov* und Piroschka B. Sulay
Pharmazeutische Fakultat der Medizinischen Akademie, Duvav 2, BG-1000 Sofia, Bulgarien
Eingegangen am 21. Juli 1983
Die Molekulionen der a-Ethoxyheterocyclen 1-8 fragmentieren unter Abspaltung von * CH3
und/oder C,H, bzw. *C2H5aus der a-Ethoxygruppe. Die Fragmentierung der Ester 2b, 3b, 4-8
erfolgt auch durch den Zerfall der Ethoxycarbonylgruppe.
Mass Spectrometry of Substituted a-Ethoxypyridines and -Pyrimidines
The molecular ions of the a-ethoxy heterocycles 1-8 lose CH, and CZH40r' C2H5from the a-ethoxy
group. Also, fragmentation of the esters 2b, 3b, 4-8 occurs by degradation of the ethoxycarbonyl
group.
Unlangst wurde von uns eine Reihe von Pyridin- und Pyrimidinderivaten, darunter die aethoxysubstituierten Verbindungen 2b, 3b, 4 und 5, durch Selbstkondensation des 3-Amino3-ethoxypropensaure-ethylestersgewonnen'). Aus derselben Ausgangsverbindung wurden auherdem 3a, 6 und 7 dargestellt'.'). Das 2-Pyridinon 2a ist ein Verseifungs- und Decarboxylierungsprodukt
von 2b.
Die vorliegende Arbeit bezweckt einen Vergleich der Massenspektren dieser neuen
Derivate, wobei auch das in Lit.3)beschriebene MS-Spektrum des 2-Ethoxypyridins 1 mit
betrachtet wird (Tab. 1). Die Verbindungen zeichnen sich durch drei Hauptfragmentierungswege des Molekiilions aus, das nur in den Spektren von 2a, 3b und 5 einen base peak
bildet. Nach einem dieser Wege spaltet sich das Methylradikal der a-Ethoxygruppe ab,
und die relative Intensitat des entsprechenden Peaks [M-'CH3]+ iibersteigt 70 % in den
fur eine Reihe
Spektren von 1, 2a, 3b und 4. Dieser Vorgang ist laut
a-substituierter Pyridine vom Typ a kennzeichnend und wird offenbar von der Bildung
eines Vierringes im Ion b hervorgerufen.
03654?33/84/10100873 $ 02.50/0
0 Verlag Chemie GmbH. Weinheim 1984
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