D. Branca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4042–4045
4045
O
Acknowledgments
O
NH
a
The authors thank Monica Bisbocci and Claudia Giomini for bio-
logical testing.
NH
HN
NH2
F
26
27
References and notes
O
1. Schreiber, V.; Dantzer, F.; Amé, J.-C.; De Murcia, G. Nat. Rev. Mol. Cell Biology
2006, 7, 517.
2. Ogata, N.; Ueda, K.; Kawaichi, M.; Hayaishi, O. J. Biol. Chem. 1981, 256, 4135.
3. Jagtap, P.; Szabò, C. Nat. Rev. Drug Discov. 2005, 4, 421.
4. (a) Moroni, F. Curr. Opin. Pharmacol. 2008, 8, 96; (b) Pacher, P.; Szabò, C.
Cardiovasc. Drug Rev. 2007, 25, 235.
O
Cl
O
H2N
5. Rodon, J.; Inisesta, M. D.; Papadopoulos, K. Expert Opin. Inv. Drugs 2005, 18, 31.
6. Lindahl, T.; Satoh, M. S.; Poirier, G. G.; Klungland, A. TIBS 1995, 20, 405.
7. Cantoni, O.; Cattabeni, F.; Stocchi, V.; Meyn, R. E.; Cerutti, P.; Murray, D.
Biochem. Biophys. Acta 1989, 1014, 1.
8. Purnell, M. R.; Whish, W. J. D. Biochem. J 1980, 185, 775.
9. For reviews, see (a) Cosi, C. Expert Opin. Ther. Patents 2002, 12, 1047; (b)
Southan, G. J.; Szabo, C. Curr. Med. Chem 2003, 10, 321; (c) Peukert, S.; Schwahn,
U. Exp. Opin. Ther. Pat. 2004, 14, 1531.
NH
O
HN
+
NH
N
H
NH2
1
10. Plewczynski, D.; Spieser, S. A.; Koch, U. Comb Chem High Throughput Screen
2009, 12, 358.
11. Plewczynski, D.; Spieser, S. A.; Koch, U. J Chem Inf Model. 2006, 46, 1098.
12. MDL Information Systems Inc., San Leandro, CA. MACCS Drug Data Report
(MDDR).
c or d
or e
13. Carhart, R. E.; Smith, D. H.; Venkataraghavan, R. J. Chem. Inf. Comput. Sci. 1985,
25, 64.
14. Vapnik, V. Statistical Learning Theory 1998.
O
NH
15. The ability of compounds to inhibit PARP1 activity was tested using a modified
literature procedures (Cheung A., Zhang J. Analytical Biochemistry 2000, 282,
24), using PARP1 enzyme isolated from HeLa cells. Enzyme assay was
conducted in buffer containing 25 mM Tris pH 8.0, 1 mM DTT, 1 mM
Spermine, 50 mM KCl, 0.01% Nonidet P-40 and 1 mM MgCl2. PARP reactions
HN
contained 0.1
NAD+, 1
g/mL activated calf thymus, and 1–5 nM PARP-1. Auto reactions
utilizing SPA bead-based detection were carried out in 50 L volumes in white
96-well plates. Compounds were prepared in 11-point serial dilution in 96 well
plate, 5
L/well in 5% DMSO/H2O (10ꢀ concentrated). Reactions were initiated
by adding first 35 L of PARP-1 enzyme in buffer and incubating for 5 min at rt,
then 10
L of NAD+ and DNA substrate mixture. After 3 h at rt these reactions
were terminated by the addition of 50 L Streptavidin-SPA beads (2.5 mg/ml in
200 mM EDTA pH 8). After 5 min, they were counted using TopCount
l l
Ci [3H]-NAD (200,000 DPM), 1.5 M NAD+, 150 nM biotinylated
NH
l
R: -Alkyl; -COR; -CONR1R2
l
R
l
Scheme 1. Reagents and conditions: (a) NH2NH2ꢁH2O,
EtOH/H2O, rt, 24 h; (c) when R = alkyl: aldehyde or ketone, MP-triacetoxyborohy-
dride, DMF, W (80 °C, 10 min); (d) when R = COR: RCOOH, PL-Mukaiyama, PS-
dimethylaminopyridine (PS-DMAP), DCM/DMF, rt, 24 h; (e) when R = CONR1R2: (i)
CDI, DIPEA, DMF, W (80 °C, 5 min).
W (80 °C, 5 min); (ii) HNR1R2, DIPEA,
lW (115 °C, 15 min); (b)
l
l
l
l
a
l
l
microplate scintillation counter. IC50 data was determined from inhibition
curves at various substrate concentrations.
16. The PARP isoforms were assayed using a TCA protocol using [3H]-NAD and
activated calf thymus (PARPs 2 and 3 only). hPARP-2 was expressed using a
baculovirus, hPARP-3 was available from Alexis Biochemical ALX-201-170,
while the catalytic domains of the human vPARP (aa. 209-661) and human
Tankyrase 1 (aa. 1013-132) were expressed in Escherichia Coli.
by addition of hydrogen peroxide, unfortunately the compounds
were inactive at 5 M.
The synthetic route for the synthesis of the pyrrolo-
dihydroisoquinolone scaffold is outlined in Scheme 1.
l
17. Kelly, M. G.; Kang, Y. H. US 4,432,974, 2003.
The treatment of 2617 with hydrazine, upon microwave irradia-
tion at 115 °C for 15 min, provided compound 27 (yield: 85%) that,
after reaction with 2-(3-chloropropyl)-1,3-dioxolane, gave rise to a
mixture of two regioisomers (1:1 ratio, by HPLC). Their separation
by HPLC provided the desired pyrrolo-tetrahydroisoquinolinone 1
(yield: 32%).18 All the derivatives in Tables 1–3 were prepared by
functionalization of 1.
18. Synthesis of compound 1. Step 1:
A
suspension of 6-fluoro-3,4-
dihydroisoquinolin-1(2H)-one (1 equiv) (obtained as described in Ref. 12)
and hydrazine monohydrate (25 equiv) was irradiated in a microwave oven at
110 °C for 15 min. The reaction mixture was cooled to rt, water was added and
the resulting precipitate was filtered and washed with diethyl ether. MS (EI+)
C9H11N3O calcd: 177, found: 178 (M+H)+. Step 2: A solution of 6-hydrazino-
3,4-dihydroisoquinolin-1(2H)-one (1 equiv) and 2-(3-chloropropyl)-1,3-
dioxolane (1 equiv) in EtOH/H2O (5:1) was stirred at rt for 24 h. After
aqueous work up, a crude mixture of the two isomers 1-(2-aminoethyl)-
3,7,8,9-tetrahydro-6H-pyrrolo[3,2-f]isoquinolin-6-one and 3-(2-aminoethyl)-
1,6,7,8-tetrahydro-5H-pyrrolo[2,3-g]isoquinolin-5-one was obtained. This
Alkyl derivatives (Table 1) were obtained by reductive amina-
tion of 1 with the appropriate aldehyde or ketone.
mixture was purified by RP-HPLC (column Symmetry C18 7 lm, gradient A:
Amide compounds in Table 2 were prepared by amide coupling
reaction of 1 with the appropriate carboxylic acid. Activation of 1
H2O + 0.1% TFA; B: MeCN + 0.1% TFA) to afford the desired product (first eluted
isomer), as its trifluoroacetate salt. 1H NMR (400 MHz, DMSO-d6, 300 K): d
11.27 (br s, 1H), 7.81 (br s, 3H), 7.66 (d, J = 8.7 Hz, 1H), 7.62 (br s, 1H), 7.27 (d,
J = 8.7 Hz, 1H), 7.25 (s, 1H), 3.48-3.38 (m, 2H), 3.30–3.20 (m, 2H), 3.17–3.00 (m,
4H). MS m/z (EI+) C13H15N3O calcd: 229, found: 230 (M+H)+.
with 1,10-carbonyldiimidazole (
lW, 80 °C, 5 min), followed by
addition of the appropriate amine upon microwave irradiation,
afforded urea analogs (Table 3).19
19. As an example, synthesis of 21: A 0.3 M solution of 1 (1 equiv), DIPEA (1.1 equiv)
and CDI (1 equiv) in DMF was heated in a microwave oven at 80 °C for 5 min.
This mixture was added to a 0.4 M solution of tert-butyl 1,4-diazepane-1-
carboxylate and DIPEA (1.1 equiv) in DMF and heated in a microwave at 80 °C
for 5 min. The crude mixture was purified by RP-HPLC (column Waters X-
TERRA MS C18, gradient A: H2O + 0.1% TFA; B: MeCN + 0.1% TFA). During
evaporation of the HPLC fractions Boc-deprotection occurred, providing 21 as
its trifluoroacetate salt (yield: 65%). 1H NMR (300 MHz, DMSO-d6+ 2% TFA,
300 K): d 11.11 (s, lH), 8.68 (br s, 2H), 7.63 (d, J = 8.6 Hz, lH), 7.59 (br s, lH), 7.24
(d, J = 8.6 Hz, lH), 7.20–7.16 (m, lH), 6.93–6.83 (m, lH), 3.53–3.46 (m, 4H), 3.44–
3.38 (m, 2H), 3.34–3.27 (m, 4H), 3.10–3.01 (m, 4H), 2.99–2.92 (m, 2H).
C18H23N5O2 calcd: 341, found: 342 (M+H)+.
In conclusion, the synthesis and preliminary biological evalua-
tion of a novel class of pyrrolo-isoquinolinones as PARP1 inhibitors
have been described. After a virtual screening of the proprietary
collection, compound 1 was identified as potent PARP1 inhibitor
(IC50 = 40 nM). A variety of functionalizations on the terminal
nitrogen of the pendant 2-aminoethyl chain were evaluated. The
introduction of alkyl and acyl groups resulted in compounds with
low nanomolar activity in the PARP1 enzymatic assay (Tables 1 and
2). Urea derivatives, such as compounds 21 and 22, proved to be
the most promising PARP1 inhibitors in the series displaying an
IC50 = 25 and 27 nM, respectively (Table 3).
20. Halgren, T. A. Curr. Opin. Struct. Biol. 1995, 5, 205.
21. MacroModel Version 7.0: Schroedinger Inc., Portland OR 97201. http://