Mendeleev Commun., 2012, 22, 15–17
Table 1 New PARP1 inhibitors.
O
IC50a/mm
K /μM
b
NH
Substance
i
1
2
3
4
5
6
16 (6; 39)
160 (141; 181)
47 (no data)
27 (18; 42)
26 (8; 92)
1.21
12.3
3.6
2.1
2.0
n
N
S
CN
1 n = 1
n = 2
2
COOEt
n = 1 (92%)
n = 2 (73%)
HC(O)NH2
12 (6; 24)
0.9
NH3 100%
aExperimental IC50 values (the numbers in parentheses show the confidence
CONH2
CN
interval for 95% probability). Experimental K values, calculated from IC .
b
S8, morpholine
i
50
n
NH2
CONH2
S
(Figure 2) reveals that the new compounds are comparable to
O
n
benzamide and quinazoline base fragments in binding affinity,
which confirms the efficiency of a preliminary simulation in the
organic synthesis of bioactive compounds.
n = 1 (53%)
n = 2 (67%)
2
ClCH CN, n = 1
HCl
62%
O
O
3
NaN3,
DMF
NH
NH
N3
Cl
9
1%
N
S
N
S
5
i, PPh3
ii, H2O, H+
NaOH,
EtOH
7
7%
61%
O
O
NH
NH
NH2
OH
N
N
S
S
6
4
Figure 2
Scheme 1 Synthesis of compounds 1–6.
‡
Virtual screening and docking procedure. Ligand docking was carried
out using published procedures.1 The procedures used for syn-
0,11
19
out using the Lead Finder software, version 1.1.15, with default con-
thesizing compounds 3–6 were described elsewhere.1
2–15
Table 1
figuration parameters. The full-atomic model of PARP1 was prepared
using the Model Builder software supplied in the Lead Finder distribution
package. The starting structure of PARP1 was obtained from Protein
Database, PDB ID: 1UK0. The binding energies of the ligands were
assessed using the dG-scoring function available in Lead Finder. Fragment
screening was based on an STK library provided by VitasM, http://www.
vitasmlab.com/. Compounds from the STK library were broken into
fragments according to a published procedure.20 Calculated inhibition
constants for compounds 1–6 are 80, 27, 11, 20, 23 and 20 mm, respec-
tively.
summarizes the inhibiting effects of the synthesized compounds.
According to IC50 measurements, the efficiency of compound 2
(
IC50 160 mM, 95% confidence interval of 141–181 mM) is con-
siderably lower than that of reference compound 1 (IC50 17 mM,
5% confidence interval of 6–39 mM). IC measurements (Table 1)
have shown that the displacement of conservative water apparently
does not result in significant changes in the binding energy.
9
50
§
A comparison of the inhibition constants of the compounds
synthesized with the well-known data for PARP1 inhibitors
Experimental measurement of inhibition constants. Recombinant human
enzyme PARP1 was purified according to a reported procedure. PARP1
2
1
†
inhibition was measured as follows: samples (15 ml) containing 200 nm
3
,5,6,7-Tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-one
1
of purified PARP1 protein, 2 OU cm–3 DNAse I-activated calf thymus
1
: mp 162–163°C. H NMR (500 MHz, DMSO-d ) d: 2.38 (q, 2H, CH ,
6
2
+
3
+
J 7.1 Hz), 2.91 (t, 4H, CH , J 7.1 Hz), 8.00 (s, 1H, CH), 12.39 (br.s, 1H, NH).
DNA, 600 mm NAD , 0.5 mCi [ H]NAD , 10% DMSO and compounds
2
5
,6,7,8-Tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one
2,
of interest in various concentrations were incubated for 1 min at 37°C in
1
a buffer solution (50 mm Tris, pH 8.0, 20 mm MgCl , 150 mm NaCl,
mp 164–165°C. H NMR (500 MHz, DMSO-d ) d: 1.77 (m, 4H, CH ),
2
6
2
2
1
.73 (br.s, 2H, CH ), 2.87 (br.s, 2H, CH ), 7.99 (s, 1H, CH), 12.29 (br.s,
7 mm b-mercaptoethanol). The reaction rate was linear during the first
20 min under these conditions. The reaction was terminated by trans-
ferring a 12 ml aliquot portion on Whatman 1 paper filters soaked in 5%
trichloroacetic acid. The filters were washed three times with 150 ml of
5% trichloroacetic acid, which was then washed off with 90% ethanol,
2
2
H, NH).
-(Chloromethyl)-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]-
2
1
pyrimidin-4-one 3: mp 159–161°C. H NMR (500 MHz, DMSO-d ) d:
6
2.30 (q, 2H, CH , J 7.1 Hz), 2.68 (t, 4H, CH , J 7.1 Hz), 3.66 (s, 2H, CH ).
2
2
2
3
2
-(Hydroxymethyl)-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno-
and the filters were dried. The degree of inclusion of [ H]ADP-ribose into
1
an acid-insoluble material was measured with a QuantaSmart scintillation
counter in a toluene scintillator. IC50 values were calculated using the
Origin Pro 8.0 software by means of nonlinear regression analysis. All
[2,3-d]pyrimidin-4-one 4: mp 129–133°C. H NMR (500 MHz, DMSO-d )
6
d: 2.38 (q, 2H, CH , J 7.2 Hz), 2.90 (t, 4H, CH , J 7.2 Hz), 4.36 (s, 2H,
CH ), 5.43 (br.s, 1H, OH), 12.02 (s, 1H, NH).
2
2
2
2
-(Azidomethyl)-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]-
experiments were carried out in duplicate.
1
§ Calculation of inhibition constants from IC . IC values were con-
pyrimidin-4-one 5: mp 140–143°C. H NMR (500 MHz, DMSO-d ) d:
2
50
50
6
.37 (q, 2H, CH , J 7.1 Hz), 2.90 (t, 4H, CH , J 7.1 Hz), 3.41 (s, 2H, CH ),
verted to Ki using the competitive inhibition equation
2
2
2
1
2.77 (s, 1H, NH).
+
K = IC /(1 + [NAD ]/Km),
i
50
2-(Aminomethyl)-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]-
1
+
+
pyrimidin-4-one 6: mp 132–135°C. H NMR (500 MHz, DMSO-d ) d:
where [NAD ] is the NAD concentration used in the experiments, and
Km is the Michaelis constant of the reaction (according to published
data,22 Km = 50 mm).
6
2
.38 (q, 2H, CH , J 7.2 Hz), 2.90 (t, 4H, CH , J 7.2 Hz), 3.61 (s, 2H,
2
2
CH ), 5.51 (br.s, 2H, NH ), 12.41 (s, 1H, NH).
2
2
–
16 –