J. Med. Chem. 1993,36, 3834-3042
3834
Synthesis, Antiproliferative, and Antiviral Activity of 4-Amino-l-(~-~-ribofuranosyl)pyrrolo[2,3-dJpyr~daz~n-7(6~-one and Related Derivatives Eric A. Meade, Linda L. Wotring, John C. Drach, and Leroy B. Townsend’ Departments of Medicinal Chemistry and Pharmaceutical Chemistry, College of Pharmacy, Department of Chemistry, College of Literature, Sciences, and Arts, and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1065 Received March 15, 1993.
The synthesis of 4-amino-l-~-~-ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one (3) from the reaction of ethyl 3-cyano-1-8-~-ribofuranosylpyrrole-2-carboxylate (10) and hydrazine is described. The 5:6 pyrrolo[2,3-d]pyridazin-7(6H)-one structure of 3 was established via a three-step conversion of 3 into l-~-~-ribofuranosylpyrrolo[2,3-dlpyridazin-4,7(5H,6H)-dione (14). 4-Amino-3-chlorol-~-~-ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one (16) and 4-amino-3-bromo-l-8-~-ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one(18) were prepared uia N-chlorosuccinimide or N-bromosuccinimide treatment of 4-amino-1-(2,3,5-tri-0-benzyl-~-~-ribofuranosyl)pyrrolo[2,3d]pyridazin-7(6H)-one (7) followed by a removal of the benzyl groups with boron trichloride. Direct treatment of 3 with N-iodosuccinimide furnished 4-amino-3-iodo-l-~-~-ribofuranosylpyrolo[2,3-d]pyridazin-7(6H)-one(19). The antiproliferative activity of the compounds was determined in L1210, H. Ep. 2 and several additional human tumor cell lines. In L1210 cells, the 3-hdosubstituted compounds 16, 18, and 19 exhibited significant cytotoxicity (ICs0 = 0.2,0.1,0.08 p M , respectively), in contrast to the 3-unsubstituted compound 3, which had only slight activity. The greater antiproliferative activity of 18 and 19 in contrast to 3 was confirmed in H. Ep. 2 cells and KB cells. The antiviral evaluation of these compounds revealed that compounds 16, 18, and 19 were active against human cytomegalovirus in both plaque- and yield-reduction assays. However, this activity was only partially separated from cytotoxicity in human cell lines. Introduction Significantbiological activity has been observed for the nucleoside antibiotic tubercidin (la)(7-deazaadenosine)
How
R &
now no
OH
1 1.R H b, R-Br
this analog showed no antineoplastic or antiviral acti~ity.~ During our attempts to establish a facile preparation of 2a,we prepared another novel adenosineanalog, 4-aminol-~-~-ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one (3). This prompted us to investigate its biological activity and to synthesize related derivatives. The %halo (Cl, Br, I) H analogsof 3were of particular interest because the 3-bromo derivative (2b)of 2a had shown modest antiproliferative and antiviral activity while 2a itself was completely ina~tive.~ Also, the corresponding &halo analogs in the pyrrolo[2,3-d]pyrimidineseries demonstrated significant antiproliferative and antiviral a c t i ~ i t i e s . ~ ~ ~
~
N
O
HO
OH
2
a. R-H
OH
HO
3
b, R-Br
Results and Discussion Chemistry. Our initial synthetic efforts were focused and certain structurally related analogs which have been on the preparation of 4-amino-l-&~-ribofuranosylpyrroloisolated from naturally occurring sources or prepared by [2,3-dlpyridazin-7(6H)-one(3). After a comprehensive synthetic methods. Tubercidin and especially certain literature search, we chose to use ethyl 3-cyanopyrrole5-substituted analogs have shown antineoplastic activity 2-carboxylate (41’0 as our starting material. This pyrrole in a variety of assays both in vitro and in viu0.l The had substituents in the proper juxtaposition and in 5-carboxamidoderivativeof tubercidin, sangivamycin,has addition these substituents had different chemical reacundergone phase I clinicaltrials as an antineoplasticagente2 tivities which could be exploited at a later stage of the Although the 7-deazaadenosineanalogsshowed significant synthesis. We elected to use 2,3,5-tri-O-benzyl-~-riboantiproliferative activity, their anticancer potential was furanosyl chloride (5)11as the sugar for the glycosylation tempered by the fact that these analogsalsohad significant procedure with the pyrrole 4. Even though 5 had not been host toxicity. Therefore, there is a continuing need to used previously in the sodium salt mediated coupling of synthesize other structurally related nucleoside analogs pyrroles, the benzyl protecting groups were considered which possess less host toxicity. necessary for further transformations in the synthetic As part of an ongoing program to investigate other basesequence. The sodium salt mediated coupling of 4 and 5 modified adenosine analogs in which the 7-nitrogen atom was initially attempted in acetonitrile, the usual solvent has been replaced by a carbon atom,- we have prepared for the pyrrole coupling reaction.12 However the reaction the novel 4-amino-l-~-~-ribofuranosylp~rolo[2,3-dlpywas quite sluggish in this solvent. Substitution of aceridazine (2a),an isomer of tubercidin (la). Surprisingly tonitrile by dimethylformamide overcame this problem and DMF became our solvent of choice for this particular Abstract publiihed in Advance ACS Abstracts, September 1,1993. 0022-2623/93/1836-3834$04.00/00 1993 American Chemical Society
Pyrrolo[2,3-d]pyridazin- 7(6H)-ones as Antiviral Agents
Journal of Medicinal Chemistry, 1993, Vol. 36,No.24 388s
Scheme I
Scheme I1 CN
CN 1. NaH
fiCO2E1 N H
2 ' B n o o a BnO OBn 5
3
M
c
Pd(OH)d
'
""U O
I
Bzo*a BzO 8
NH
NH
0 BnO OBn 7
OBz
BzodBzO OBz
9.
I
H2WH2
~
&02Et
2.
4
BnO OBn
ecyclohexene
3
QCGEt
CN
1. NaH
H
4
HO OH
CN
& Q E t
w
HO OH
3
NaOE"EtOH
CN
(&&Et
Hod HO OH 10
Scheme I11 reaction (Scheme I). The coupling reaction in dimethylformamide afforded 3640% yields of the desired ethyl 3-cyano-l-(2,3,5-tri-O-benzyl-&~-ribofuranosyl)pyrrole-2carboxylate (6), after column chromatography. That the pyrrole had been alkylated at N-1 and not at a carbon atom was evident from the lH NMR of 6, which revealed HO OH the presence of two doublets corresponding to the pyrrole 3 H's. The anomeric confiiation of 6 could be assigned from the 1H NMR spectrum since the peak at 6.70 ppm correspondingto the anomeric proton appeared as a singlet. The coupling constant for the H-1' and H-2' protons was HO OH 10 therefore less than 1 Hz, and this corresponds to a j3 configuration for 6.15 The pyridazine portion of the desired heterocyclic 11 moiety was obtained via a ring annulation reaction of 6 using anhydrous hydrazine in refluxing EtOH (Scheme Scheme IV I). From the IR spectrum which revealed the loss of the NY absorbances at 2200 and 1708 cm-l for the nitrile ester, respectively, it was determined that ring closure had ~ N H occurred to form 4-amino-l-(2,3,5-tri-O-benzyl-j3-~-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7(6H)-one (7). In the HO OH O FAB+ mass spectrum of 7 a peak at mlz 553 was observed which corresponded to the M + 1 ion expected for a 3 molecular formula of CszH3zNrOs. Finally as further NaNWOAc structure proof, 7 was deprotected using transfer hydrogenation conditions (Pearlman's catalystlcyclohexene)to afford 4-amino-l-~-~-ribofuranosylpyrrolo[2,3-dlpyridazin7(6H)-one (3) in 23% yield. In addition to the DzONaOMeNK4-I NH exchangeablepeaks corresponding to the sugar hydroxyls in the lH NMR spectrum of 3, there were DzO-exchangee3 able singlets at 6 11.23 and 5.69. The downfield singlet HO OH l 4 Aco OAC which integrated for one proton was assigned to the NH-6 The elemental analyses and the electron impact mass proton and the upfield signal, which integrated for two spectrum of 3 suggested that compound 3 possessed a protons, was assigned to the 4-amino protons. The rather low overall yield for this particular synthetic molecular formula of CllH14N405. These data were sequence prompted an investigation for a higher yielding consistent with the proposed 5 6 pyrrolo[2,3-dlpyridazin7(6H)-one structure. Also consistent with this structure procedure for the preparation of 3. Compound 3 was prepared more efficiently by a slightlymodified procedure were DzO-exchangeable singlets at S 11.23 integrating for one proton and at S 5.69 integrating for two protons. (Scheme 11). Ethyl 3-cyano-1-(2,3,5-tri-0-benzoyl-8-Dribofuranosyl)pyrrole-2-carboxylate (9) was readily obHowever, the data did not completely rule out the tained by the sodium salt glycosylation of 4 with l-chloropossibility of the isomeric 5 5 structure dhydrazino-l-& 2,3,5-tri-O-benzoyl-~-ribofuranose (81.7 Treatment of 9 ~ n b o ~ a n ~ l p y r r o l o [ 3 , 4 ~ l p y r(1r1)o(Scheme l~~~~ 111). In order to establish the 5 6 pyrrolo[2,3-d3pyridazinwith a solution of sodium ethoxide in ethanol effected debenzoylation and provided ethyl 3-cyano-l-p-~-ribo- 7(6H)-one structure unequivocally, 3 was further derivatized (Scheme IV). Compound 3 was first acetylated with furanosylpyrrole-2-carboxylate(10). A ring annulation exactly 3 equiv of acetic anhydride to afford 4amino-lof 10 with hydrazine then furnished 3 in 62.5% yield. The (2,3,S-tri-0-acetyl-~-~-r~bofuranosyl)pyrrolo[2,3-d1compound obtained via this procedure had the same pyridazin-7(6H)-one (12). Thfs modification furnished a physical properties as those of the compound (3)obtained by the earlier procedure. compound which was soluble in organic solvente and
HovY
1
- -e
3836 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 24
Meade et al.
(centeredat 6 124.35) as a doubletof doublets. Thispattern of peaks was expected for C-2, as it was coupled to both H-2 (‘J) and H-1’ ( 3 4 . Upon irradiation of 16 with the frequency corresponding to the anomeric proton, these same peaks collapsed to a clean doublet. This observation was in agreement with the fact that chlorination of the heterocycle had occurred at the 3 position. The spectra of the analogous 3-bromo (18) and 3-iOdO (19) compounds showed similar patterns in their I3C NMR spectra. Biology. In Vitro Antiproliferative Activity. The pyrrolo[2,3-dlpyridazin-7(6H)-one analogs of adefour BnO OBn nosine (3,16,18, and 19) and the inosine analog 14 were 7 evaluated for their antiproliferative activity in L1210 L murine leukemia cells (Table I). Data for tubercidin (la) 17 and its bromo analog l b (both pyrrolopyrimidines),plus Scheme VI two pyrrolopyridazine analogs described previouslyg(2a and 2b), also are included in the table for comparison. The well-establishedantiproliferativeactivity of tubercidin NH was readily detected while 5-bromotubercidin was 100H O U 0 % H O U 0 fold less active. In contrast to these pyrrolo[2,3-d]pyrimidine nucleosides, halogenation of the new pyrroloHO OH HO OH [2,3-d]pyridazinenucleosides at the 3-position (analogous 3 1s to the 5-position of the pyrrolo[2,3-dlpyrimidinenucleosides) conferred increased cytotoxic activity. The 4-amifacilitated the workup of the next reaction. Upon diazno-3-bromopyrrolo[2,3-dlpyridazinenucleoside 2b was otization of 12 using sodium nitrite and acetic acid, the moderately cytotoxic, whereas the corresponding 3-untetraacetylated compound 13 was isolated. Four singlet substituted adenosineanalog 2a was not growth inhibitory peaks each integrating for three protons assigned to the at 100 pM, the highest concentration tested. The new, methyl protons of the acetyl groups were evident in the 3-halogenated4-aminopyrrolo[2,3-dlppidazin-7(6H)-one 1H NMR of 13. The electron impact mass spectrum of 13 nucleosides 16,18, and 19 were highly cytotoxic, while the (with a molecular ion of 451) and elemental analyses were corresponding3-unsubstitutedadenosineanalog3 was only in agreement with a compound with a molecular formula marginallygrowth inhibitory. It is of considerableinterest of CuHzlNsOlo. Deprotection of 13 using a sodium that the 3-bromo (18) and 3-iodo (19) analogs appear to methoxide/methanol solution yielded 1-8-D-ribofuranobe almost as potent as the naturally occurring pyrrolosylpyrrolo[2,3-d]pyridazin-4,7(5H,GH)-dione (14). The [2,3-dlpyrimidine nucleoside tubercidin. mass spectrum of 14 showed the presence of a peak The new compounds also were evaluated in H. Ep. 2 corresponding to the molecular ion at 283 and an intense human epidermoid carcinoma cells, and selected compeak at 151correspondingto the B + 1. Elementalanalyses pounds from this series also were evaluated in nine were also consistent with a molecular formula of (211additional human tumor cell lines (Table 11) for their H13N306. Further proof was derived from the observation ability to inhibit cell growth (Table I). The structureof a bathochromic shift in the UV spectrum at pH 11, activity relationships were consistent with the pattern which was consistent with the inosine-like structure for observed for L1210 cells. Introduction of either a %halo 14. Had the structure of 3 been the isomeric 11, the (2b)or 7-0ne (3) substituent provided moderate cytotoxic expected product from the diazotization would have in contrast to the unsubstituted adenosineanalog activity, contained an azido f~ncti0n.l~ The observed product (14) (2a), which was completely inactive at 100 pM. The of this sequence was therefore in agreement with a 5:6 compounds with both %halo and 7-one substituents (16, pyrrolo[2,3-d]pyridazin-7(6H)-one structure for 3. 18, 19) were by far the most active. Their ICm’s were With the free nucleoside 3 and a protected analog, lower than that of compound 2b by a factor of 100 or 4-amino-1-(2,3,5-tri-O-benzyl-~-~-ribofuranosyl)pyrrolomore. [2,3-d]pyridazin-7(6H)-one(7), availableas intermediates, In VitroAntiviral Activity. All compoundsalso were the preparation of the 3-halogenatedanalogswas initiated. tested for activity against two herpes viruses, human The chloro and bromo analogs were prepared by the cytomegalovirus (HCMV) and herpes simplex virus type treatment of 7 with N-chlorosuccinimide or N-bromo1 (HSV-l), because halogenated analogs in the pyrrolosuccinimide,respectively,in dichloromethane(SchemeV). pyrimidine series were very active against these viruse~.~*~ The presumed intermediates 16 and 17 were then deproAs a means of determining the selectivity of this activity, tected using boron trichlorideto furnish 4-amino-3-chlorothe compounds also were evaluated for cytotoxicity to l-~-~ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one (16) and 4-amino-3-bromo-l-/3-~-ribofuranosylpyrrolo[2,3-d] - uninfected cells used to propagate HCMV and HSV-1, human foreskin fibroblasts (HFF cells) and KB cells, pyridazin-7(6H)-one (181, respectively. 4-Amino-3-iodorespectively (Table 111). The structural requirements for 1-~-~ribofuranosylpyrrolo[2,3-dlpyridazin-7(6H)-one (19) antiviral activity in this series of compoundswere similar was obtained by a direct treatment of 3 with N-iodosucto those discussedabove for antiproliferativeactivity. Only cinimide in glacial acetic acid (Scheme VI). That the those analogs which contained both 3-hdo and 7-one halogen had added at the 3 position was established by substituents (16, 18, 19) exhibited significant activity the lH NMR and lSCNMR spectra. In the fully coupled against HCMV in the plaque-reduction assay (Table 111). lsC NMR spectrum of 16 the peaks corresponding to the These three compounds were more active than the pyrrole carbon attached directly to a hydrogen appeared Scheme V
f&
~
Pyrrol0[2~3-d]pyridazin-7(6iY)-onesas Antiviral Agents
Journal of Medicinal Chemistry, 1993, Vol. 36]No.24 3837
Table I. In Vitro Cell Growth Inhibition by 4-Aminopyrrolo[2,3-d]pyridazin-7-oneand Rslated Nucleosides
HO
OH
le,lb
2a-19
ICw (rM)" R R1 & L1210 H.Ep.2 UMSCC-1OA CALU-1 NCI-H1437 NCLH460 NCI-H146 NCLN417 DLD-1 HT-29 SW-620 lab H NH2 0.043 0.06 lbb Br NH2 4 2ac H NH2 H -d 2be Br NH2 H 24 5.2 11 11 3 H NH2 OHe 74 14 H OH OH 16 C1 NH2 OH 0.23 18 Br NH2 OH 0.10 0.20 1.3 0.23 0.50 0.50 19 I NH2 OH 0.08 0.076 2.5 0.07 0.13 0.20 0.42 0.28 0.07 0.50 0.23 0 Concentration required to decrease growth rate to 50%of control. See Experimental Section for complete definition. b Results for theee pyrrolopyrimidineshave been reported previously'. Results for these pyrrolopyridazines have been reported previously.@d A dash indicah that 100 pM, the highest concentration tested, had no significant effect on cell growth. * Shown as OH for convenience in presenting the structures. no.
Table 11. Human Tumor Cell Linea Used for in Vitro Evaluation of Antiproliferative Activity plating Yname tumor site histologicaltype source cells/well day medium squamous cell carcinoma Carep larynx 2000 5 MEM-6' UMSCC-1OA squamous cell carcinoma ATCCC lung 2000 5 R-5' CALU-1 Gazde 2000 1l.W adenocarcinoma 5 R-5 NCI-H1437 large cell carcinoma Gazdarf 1000 4 R-5 lw! NCI-H460 classic small cell carcinoma Gazd& 2 m 7 R-5 lung NCI-H146 GazdM 2 m 1l-W variant small cell carcinoma 7 R-5 NCI-N417 DLD-1 colon adenocarcinoma ATCC 1000 5 R-5 HT-29 colon adenocarcinoma ATCC 2000 5 R-5 SW-620 colon adenocarcinoma ATCC 7500 4 R-5 a Thomas E. Carey, University of Michigan, Ann Arbor, MLl6 MEM-5, Minimal Essential Medium with Earle's salts and 5% fetal bovine serum. ATCC, American Type Culture Collection, Rockville, MD. d R-5, RPMI 1640 with 5% fetal bovine serum, 2 mM L-glutamine, 50 units/mL penicillin,50 pg/mLstreptomycin, and 100pg/mLneomycin. e Adi F. Gazdar, SimmonsCancer Center, Dallas, TX.16 f Adi F. Gazdar, Simmons Cancer Center, Dallas, TX.17 8 Adi F. Gazdar, Simmons Cancer Center, Dallas, TX.l*
marketed antiviral drug ganciclovir (Table 111). Furthermore, they were highly active against HCMV in a yieldreduction assay, which is a more rigorous measure of antiviral activity. This assay also showed that the brominated pyrrolopyridazine 2b was active against HCMV, even though this activity was not apparent in the plaque assay.g Compounds 16,18,19,and2b alsoappeared to have some activity against HSV-1, but this activity was not separated from cytotoxicity in uninfected HFF and KB cells (Table 111). Because this apparent activity occurred at cytotoxicconcentrations,the activity observed in the HSV-1 assay probably was a consequence of cytotoxicity and not specific antiviral activity. Likewise, activity against HCMV of tubercidin and possibly bromotubercidin most likely was a consequence of cytotoxicity. In contrast, activity of 16,18,19, and 2b against HCMV was separated to some extent from cytotoxicity. A comparison of ICw's in the HCMV yield assay to ICw's for cytotoxicity in HFF, KB and H. Ep. 2 cells (Table IV) illustrates that the bromo and iodo analogs 18 and 19 were more selective than the C1 analog 16, due to their greater potency against HCMV. Depending upon which cell line and assay was used for determination of cytotoxicity, differencesin antiviral selectivitywere noted. In general, compounds appeared to be more selective on the basis of
their cytotoxicity in HFF cells than in KB or H. Ep. 2 cells. This is probably a consequence of using visual inspectionas a measure of cytotoxicityin nonproliferating HFF cells compared to measurement of growth in KB and H. Ep. 2 cells. Because growingcells are more susceptible to effects of antimetabolites than are stationary cells, determinations of cytotoxicity and antiviral selectivityare most meaningful using results obtained in proliferating cells. Apparent differences in cytotoxicity and antiviral selectivity between the KB and H. Ep. 2 cells shown in Table IV are more difficult to understand but may reflect the more rigorous assay used to determine the growth rate of H. Ep. 2 cells. Regardless of which of these two cell lines is used, the results support the conclusion that real but not extensive separation exists between anti-HCMV activity and cytotoxicity for all of the halogenated compounds. The mechanism for this selectivity has not been explored nor has the mechanism by which the compounds act. By analogy to the pyrrolopyrimidine analog of compounds 2b and 18, we speculate that the halogenated compounds are converted to the respectivetriphosphates which inhibit viral and cellular DNA polymerases. However, we have no data to substantiate these speculations. In summary, the effect of placing a carbonyl group at C-7 of 2 to afford 3 has led to an increase in the biological activity of the latter nucleoside. As was the case in the
3838 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 24
Meade et al.
Table 111. Antiviral Activity and Cytotoxicity of Pyrrolo[2,3-d]pyrid~n-7(6H)-oneand Related Nuclewides
HO
OH
ICWor ICW(rM) antiviral activity substituent HCMV cytotoxicity no. R Ri Rz plaque yield HSV-1:* EIA HFF KB la (tubercidin)d 0.5e 0.4e 1* l b (5-bromotubercidin)d 0.6e 3.5 4 1.2' 3' 2af H 2" H >looB >lo0 >100 2w Br NHz H 121e 1.4e 3oc 1W 12a 3 H 2" OH >lo0 90 >lo0 16 c1 2" OH 0.2e 0.7e 6.8e 2.18 &le 18 Br 2" OH 0.1 0.04e 2.0. 2.5'
