Synthesis and biological evaluation of novel quinazoline-derived human Pin1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.03.058

A series of novel 2,4-disubstituted quinazoline derivatives were prepared and their inhibitory activities on hPin1 were evaluated. Of all the synthesized compounds, eight compounds displayed inhibitory activities with IC₅₀ value at the level of 10^-6 mol/L. Preliminary structure-activity relationships were analyzed in details and the binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule Pin1 inhibitors will be guided by the results of this report.

Full text of DOI:10.1016/j.bmc.2011.03.058

Bioorganic & Medicinal Chemistry 19 (2011) 2797–2807
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel quinazoline-derived human
Pin1 inhibitors
Lina Zhu a,b, , Jing Jin a, , Chang Liu a,b, Chongjing Zhang a, Yan Sun a, Yanshen Guo a, Decai Fu b,
Xiaoguang Chen a, Bailing Xu a,
⇑
a Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
b Hebei University of Science and Technology, Shijiazhuang 050018, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2,4-disubstituted quinazoline derivatives were prepared and their inhibitory activities
on hPin1 were evaluated. Of all the synthesized compounds, eight compounds displayed inhibitory activ-
ities with IC50 value at the level of 10À6 mol/L. Preliminary structure–activity relationships were analyzed
in details and the binding mode of the titled compounds was predicted using FlexX algorithm. The design
and optimization of novel small molecule Pin1 inhibitors will be guided by the results of this report.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 22 February 2011
Revised 23 March 2011
Accepted 23 March 2011
Available online 29 March 2011
Keywords:
Quinazoline
Pin1
Pin1 inhibitor
PPIase
Anti-cancer agents
1. Introduction
with the aminophenylpropanol scaffold.12–15 However, the
reported highly potent Pin1 inhibitors have lacked the whole cell
Pin1 (Protein interaction with NIMA1), a phosphorylation-
dependent peptidyl-prolyl cis/trans isomerase (PPIase),1,2 can cat-
alyze the rotation of pSer-Pro/pThr-Pro amide bond in its substrate
protein.3 Thus, Pin1 works as a conformational switcher to regulate
the function of many phosphoproteins. The over-expression of Pin1
could lead to oncogenesis by increasing cyclin D1 transcription and
stability in multiple oncogenic signaling pathways.4 It has been
demonstrated that Pin1 is prevalently over-expressed in some
commonly encountered cancers such as prostate, breast, brain,
lung and colon cancer,5 and its over-expression level correlates
with tumor grades and clinical outcomes.6 The depletion of Pin1
in various cancer cells may cause apoptosis and suppress the trans-
formed phenotypes and tumorigenicity in nude mice.7,8 Therefore,
inhibition of Pin1 is becoming a potential therapeutic strategy for
anticancer treatment and has attracted many efforts on the devel-
opment of Pin1 inhibitors.
activity because of their poor permeability.12 In view of the fact,
the development of small molecule as Pin1 inhibitors is still highly
desirable and challenging with respect to improving the binding
affinity and physiochemical properties.
The crystal structure of Pin1 has shown that the active site of
Pin1 consists of two hydrophobic domains and one basic cluster
formed by the side chains of Lys63, Arg68 and Arg69 residues.12–
16 One of hydrophobic binding site defined as prolyl pocket formed
by His59, His157, Met130 and Phe134 residues, the other one is a
slightly shallow hydrophobic shelf and includes His59, Ala118 and
Leu122 residues. As a part of our program to discover novel Pin1
inhibitors, quinazoline compound 6a was identified as a hit with
an IC50 of 4.87 lM by a random screening. In order to investigate
the structure–activity relationships (SAR), a series of quinazoline
derivatives were designed by diversifying the substituents at
2- and 4-position of the parent quinazolines. We envisioned that
the introduced carboxyl group at 4-position of quinazoline allowed
to bind with the charged side chains of the basic cluster. The 2-sub-
stituents on the quinazoline scaffold may produce a hydrophobic
interaction with the shallow surface, and the prolyl pocket of
Pin1 is able to be occupied by the present quinazoline fragment.
In this work, the chemical synthesis of these new quinazoline
derivatives is described in details. The inhibitory activities on
Pin1 of these synthetic compounds are presented along with their
SAR analysis as follows.
In this regard, a number of small molecules have been investi-
gated as inhibitors of Pin1 by several groups.9–11 Lately, the design
of Pin1 inhibitors has been carried out using structure-based strat-
egy and the enzymatic inhibition was achieved in nanomolar level
⇑
Corresponding author. Tel./fax: +86 10 63166764.
E-mail address: xubl@imm.ac.cn (B. Xu).
The first two authors contributed equally.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.058

2798
L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
the presence of 1H-tetrazole and hydrogen peroxide, compounds
2. Chemistry
8a–8c were reacted with dibenzyl diisopropylphosphoramidite to
offer the phosphorylated products 9a–9c in 74–90% yields, respec-
tively. The target compounds 10a–10c were provided in high
yields when compounds 9a–9c were deprotected with TFA.
Compounds 13a–13h featuring a urea motif were synthesized
according to Scheme 3. Under microwave irradiation, compounds
11a and 11b were obtained in 98% and 69% yields by the reaction
of 4a and 4c with saturated NH3 in ethanol, respectively. Com-
pounds 11a and 11b were converted into the desired products
12a–12h in moderate to good yields upon treatment with a variety
of isocyanate derivatives under microwave irradiation. Finally, un-
der basic condition the hydrolysis of compounds 12a–12h fur-
nished the corresponding target compounds 13a–13h.
The synthesis of a variety of 2,4-disubstituted quinazolines
(6a–6h, 10a–10c and 13a–13h) was depicted in Schemes 1–3. Tar-
get compounds 6a–6h were achieved in five steps using o-amino-
benzoic acid derivatives as starting materials as shown in Scheme
1. Starting from 2-aminobenzoic acid or 5-nitro-2-aminobenzoic
acid, the desired compounds 2a and 2b were obtained, respec-
tively, (2a: 80% yield; 2b: 65% yield) upon treatment with urea un-
der microwave irradiation. Chlorination of compounds 2a and 2b
was accomplished in 48% yield by employing POCl3 or the mixture
of POCl3 and PCl5. In the presence of DIEA, the regioselective sub-
stitution reaction at 4-position of compounds 3a or 3b was per-
formed with an array of primary amines, thus furnishing
compounds 4a–4f in 63–96% yields. The subsequent substitution
reaction of compounds 4a–4f with several amines was carried
out under the different reaction conditions. Under catalysis of con-
centrated HCl(aq), the reaction of compounds 4a–4f with 4-phen-
oxyphenylamine or methyl 4-aminobenzate underwent readily,
thus furnishing the desired products 5a, 5c, 5d, 5g and 5h in mod-
erate to high yields. In contrast, in the absence of any catalyst the
amination reaction of 4c and 4d with 3,5-bis(trifluoromethyl)ben-
zylamine proceeded smoothly to give compounds 5e and 5f in 75%
and 67% yields, respectively. The conversion of compound 5a to 5b
was successfully completed in quantitative yield under catalytic
hydrogenation in hexafluoroisopropanol (HFIP) at room tempera-
ture. Upon treatment with basic reaction conditions, compounds
5a–5h were hydrolyzed into the corresponding carboxylic acid
derivatives 6a–6h in a moderate to excellent yield.
3. Biological results and discussion
The inhibitory activities on Pin1 of all target compounds (6a–
6h, 10a–10c and 13a–13h) were evaluated by a protease-coupled
enzyme assay with Suc-Ala-Glu-Pro-Phe-pNA as the substrate.17,18
The corresponding results are expressed as IC50 values and pre-
sented in Tables 1–3.
As shown in Table 1, the SAR was investigated initially on the
series of compounds 6a–6h. Compound 6a with an aminobenzoic
acid as R4 group was found to have an IC50 value of 4.87
lM on
the enzymatic inhibition. When the aminobenzoate substituent
in compound 6a was replaced by a carboxylmethylene (6c, IC50
16.4 lM) or carboxylethylene (6d, IC50 31.4 lM), the inhibitory
activities were decreased to about 1/3 or 1/6, presumably due to
the more flexible alkyl linker in compounds 6c and 6d resulting
in additional entropy penalty. In comparison with compound 6d,
the inhibitory potency of compound 6e (IC50 16.4 lM) was in-
creased to about twofold by changing the 4-phenoxyphenyl to
The synthesis of the phosphoric acid derivatives 10a–10c was
outlined in Scheme 2. The key intermediate 7 was obtained in
97% yield via the reaction of compound 3a with 2-aminoethanol
in the presence of hünig’s base. The access to compounds 8a and
8c was achieved in 99% and 80% yields, respectively, via the reac-
tion of compound 7 with 4-phenoxyphenylamine or 3-fluorophe-
nylamine under catalysis of concentrated hydrochloric acid.
Compound 8b was easily accessed via the reaction of compound
7 with an excessive amount of benzylamine in isopropanol. In
3,5-bis(trifluoromethyl)benzyl group. The potency of compound
6f (IC50 33.2 lM) is about half of that of compound 6e when an
additional CH2 was introduced into the R4 group. Based on the
above the results, it is reasonable to hypothesize that the rigid lin-
ker can make a beneficial contribution to the binding affinity. In
R2
HN
O
Cl
R1
R1
COOH
NH2
R1
R1
N
NH
N
b
c
a
N
Cl
N
H
O
N
Cl
4
3
1
2
4a: R1 = NO2, R2 = 4-COOCH3-Ph
4b: R1 = NO2, R2 = CH2COOCH3
4c: R1 = NO2, R2 = (CH2)2COOCH3
4d: R1 = NO2, R2 = (CH2)3COOCH3
4e: R1 = NO2, R2 = 4-PhOPh
1a: R1 = NO2
3a: R1 = NO2
2a: R1 = NO2
1b: R1 = H
3b: R1 = H
2b: R1 = H
R4
R2
N
HN
HN
N
4f: R1 = H,
R2 = 4-COOCH3-Ph
R1
R1
N
f
d
R3
R3
N
N
N
H
H
6a-6h
5
5a: R1 = NO2, R2 = 4-COOCH3-Ph, R3 = 4-PhOPh
5b: R1 = NH2, R2 = 4-COOCH3-Ph, R3 = 4-PhOPh
5c: R1 = NO2, R2 = CH2COOCH3, R3 = 4-PhOPh,
6a, 6b, 6h: R4 = 4-COOH-Ph
e
6c: R4 = CH2COOH
6d, 6e: R4 = (CH2)2COOH
6f: R4 = (CH2)3COOH
5d: R1 = NO2, R2 = (CH2)2COOCH3, R3 = 4-PhOPh
5e: R1 = NO2, R2 = (CH2)2COOCH3, R3 = 3,5-CF3-PhCH2
5f: R1 = NO2, R2 = (CH2)3COOCH3, R3 = 3,5-CF3-PhCH2
5g: R1 = NO2, R2 = 4-PhOPh, R3 = 4-COOCH3-Ph
5h: R1 = H, R2 = 4-COOCH3-Ph, R3 = 4-PhOPh
6g: R3 = 4-COOH-Ph, R4 = 4-PhOPh
Scheme 1. Reagents and conditions: (a) urea, microwave; (b) R1 = H, POCl3, N,N-dimethylaniline, reflux; R1 = NO2, POCl3/PCl5, reflux; (c) R2NH2, DIEA, i-PrOH, rt; (d)
R3 = aromatic amine, concd HCl(aq), acetone, reflux; R3 = 3,5-CF3-PhCH2, i-PrOH, reflux/microwave; (e) Pd/C, HFIP, rt; (f) LiOH or KOH, THF/CH3OH/H2O, reflux.

L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
2799
OH
R1
OH
HN
HN
N
Cl
N
O2N
O2N
O2N
N
N
N
a
b
N
N
Cl
Cl
H
8
7
3a
8a: R1 = 4-PhOPh
8b: R1 = PhCH2
8c: R1 = 3-F-Ph
O
O
O
O
HN
N
P
HN
P
BnO OBn
HO OH
O2N
O2N
N
N
c
d
R1
R1
N
N
N
H
H
9a-9c
10a-10c
Scheme 2. Reagents and conditions: (a) 2-aminoethanol, DIEA, i-PrOH, rt; (b) R1 = 4-phenoxylphenylamine or 3-fluorophenylamine, concd HCl, acetone/water, reflux;
R1 = benzyl amine, i-PrOH, reflux; (c) dibenzyl diisopropylphosphoramidite, 1H-tetrazole, H2O2, DMF/DCM, rt; (d) TFA, H2O, rt.
R1
N
R1
N
R1
N
HN
N
HN
N
HN
O2N
O2N
O2N
b
O
a
R2
NH2
N
H
N
H
N
4
Cl
11
12
11a: R1 = 4-COOCH3-Ph
4a: R1 = 4-COOCH3-Ph
12a: R1 = 4-COOCH3-Ph, R2 = 3-F-Ph
12b: R1 = 4-COOCH3-Ph, R2 = 4-Cl-Ph
12c: R1 = 4-COOCH3-Ph, R2 = 3,4-Cl-Ph
12d: R1 = 4-COOCH3-Ph, R2 = 3-CF3-Ph
12e: R1 = 4-COOCH3-Ph, R2 = 4-CF3-Ph
12f: R1 = 4-COOCH3-Ph, R2 = PhCH2CH2
12g: R1 = 4-COOCH3-Ph, R2 = cyclohexyl
12h: R1 = (CH2)2COOCH3, R2 = 3,4-Cl-Ph
11b: R1 = (CH2)2COOCH3
4c: R1 = (CH2)2COOCH3
R3
HN
N
O2N
c
N
O
R2
N
H
N
H
13
13a-13g: R3 = 4-COOH-Ph
13h: R3 = (CH2)2COOH
Scheme 3. Reagents and conditions: (a) NH3/ethanol, microwave; (b) R2NCO, dioxane, microwave; (c) KOH, methanol/water, reflux.
Table 1
The chemical structures and inhibitory activities on hPin1 of compounds 6a–6h
Table 2
The chemical structures and inhibitory activities on hPin1 of compounds 10a–10c
R4
O
O
HN
N
P
HN
N
HO OH
R1
O2N
N
N
R3
R1
N
H
N
H
R1
IC50 (lM)
a
Compound
R1
R3
R4
IC50 (lM)
a
Compound
10a
10b
10c
4-PhOPh
PhCH2
3-F-Ph
9.75
46.5
NA
6a
6b
6c
6d
6e
6f
NO2
NH2
NO2
NO2
NO2
NO2
NO2
H
4-PhOPh
4-PhOPh
4-PhOPh
4-PhOPh
3,5-CF3-PhCH2
3,5-CF3-PhCH2
4-COOH-Ph
4-PhOPh
4-COOH-Ph
4-COOH-Ph
CH2COOH
(CH2)2COOH
(CH2)2COOH
(CH2)3COOH
4-PhOPh
4.87
23.05
16.4
31.4
16.4
33.2
9.46
9.81
a
Compound dose (lM) required to inhibit the Pin1 activity by 50%.
6g
6h
4-COOH-Ph
quinazoline analogues and Pin1. By comparison with compound
6a, the inhibitory activity of compound 6g, which has 4-carboxyl-
phenyl as R3 and 4-phenoxylphenyl as R4 group, has shown an IC50
a
Compound dose (lM) required to inhibit the Pin1 activity by 50%.
of 9.46 lM.
addition, the effect of the substituents at C-6 position of quinazo-
lines on the inhibition was investigated preliminarily. The inhibi-
tory activity of compound 6b with an amino substituent
It has been disclosed that the charge–charge interactions be-
tween phosphate inhibitors and the active site is essential for
Pin1 inhibition.12,13 Since the initial modification of quinazoline
scaffold did not bring about a strong Pin1 inhibitor, we attempted
to integrate a phosphate into the quinazoline skeleton, thus pro-
viding compounds 10a–10c. Indeed, compound 10a (IC50
(IC50 = 23.05
that of compound 6a. Compound 6h is devoid of a nitro group,
and exhibited an IC50 of 9.81 M. Therefore, we rationalized that
lM) was decreased to about 1/5 in comparison with
l
the installment of hydrogen acceptor rather than hydrogen donor
is advantageous to the interaction between the synthesized
9.75
l
M) was a more potent Pin1 inhibitor than the corresponding
M). However, the incorpo-
carboxylate counterpart 6d (IC50 31.4
l

2800
L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
Table 3
series. Based on the analysis of the predicted binding mode and
SAR studies of the titled compounds, we envisioned that discon-
nection of quinazoline scaffold might facilitate the phenyl frag-
ment to extend into the prolyl pocket and therefore enhancing
the hydrophobic contacts. In addition, incorporation of a hydrogen
bond donor fragment on the appropriate location of the benzene
ring might assist in the binding orientation and binding affinity
via the hydrogen bond interactions with the side chain of
GLN131 and Ser154.
The chemical structures and inhibitory activities on hPin1 of compounds 13a–13h
R3
HN
O2N
N
O
R2
N
N
H
N
H
R2
R3
IC50 (lM)
a
Compound
13a
13b
13c
13d
13e
13f
3-F-Ph
4-Cl-Ph
4-COOH-Ph
4-COOH-Ph
4-COOH-Ph
4-COOH-Ph
4-COOH-Ph
4-COOH-Ph
4-COOH-Ph
(CH2)2COOH
13.4
9.0
4. Conclusion
3,4-Cl-Ph
3-CF3-Ph
4-CF3-Ph
PhCH2CH2
Cyclohexyl
3,4-Cl-Ph
2.90
8.50
5.60
52.5
54.2
>100
In summary, a series of novel quinazoline-based chemical enti-
ties were synthesized as potent Pin1 inhibitors with IC50 value at
micromolar level. The SAR and binding mode of the titled com-
pounds were explored preliminarily, and that will shed light for
the discovery of more potent novel small molecule Pin1 inhibitors.
13g
13h
a
Compound dose (lM) required to inhibit the Pin1 activity by 50%.
5. Experimetal
5.1. Chemistry
ration of phosphate into the quinazoline skeleton did not give rise
to a significant enhance in potency as anticipated, presumably due
to the longer linker between the phosphate and the quinazoline
5.1.1. General
motif. Compound 10b (IC50 46.5 lM) was about 1/5 as active as
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. 1H NMR (300 MHz) on a Varian
Mercury 300 spectrometer was recorded in DMSO-d6 or CDCl3.
Chemical shifts are reported in d (ppm) units relative to the inter-
nal standard tetramethylsilane (TMS). High resolution mass spec-
tra (HRMS) were obtained on an Agilent Technologies LC/MSD
TOF spectrometer. All chemicals and solvents used were of reagent
grade without purified or dried before use. All the reactions were
monitored by thin-layer chromatography (TLC) on pre-coated sil-
ica gel G plates at 254 nm under a UV lamp. Column chromatogra-
phy separations were performed with silica gel (200–300 mesh).
compound 10a, suggesting that the bulky hydrophobic group at
2-position of quinazolines would positively contribute to the
binding.
After some modification on 4- and 6-substituents, 4-carboxyl-
phenylamino and 6-nitro group were kept intact and a variety of
hydrophobic moieties were introduced into 2-position of quinazo-
line via a urea linker, as shown in Table 3. All compounds 13a–13g
have shown the binding affinity to Pin1 with an IC50 value ranging
from 2.9 to 54.2
lM. Compound 13c exhibited the most potent
inhibitory activity with an IC50 value of 2.9
lM. It has been demon-
strated that aromatic hydrophobic substituents are more favorable
for the binding than the alkyl hydrophobic groups since com-
5.1.2. 4-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)benzoic acid (6a)
pounds 13a–13e (IC50 2.9–13.4
eties always presented higher inhibitory activity than that of 2-
alkyl substituted quinazolines (compound 13f: IC50 52.5 M and
13g: IC50 54.2 M). The inhibitory activity was increased in the or-
lM) with substituted phenyl moi-
5.1.2.1. 6-Nitroquinazoline-2,4-(1H, 3H)-dione (2a). The mix-
ture of 2-amino-5-nitrobenzoic acid (10.0 g, 0.055 mol) and urea
(32.2 g, 0.54 mol) was stirred at 150 °C for 10 h. The reaction mix-
ture was cooled to 100 °C and then water (50 mL) was added to
quench the reaction. The crude product was obtained by filtration,
and then washed with water (50 mL Â 3). After dried under vac-
uum condition, compound 2a was obtained as yellow solid (9.1 g,
80%); mp >300 °C; 1H NMR (DMSO-d6) d 7.24 (d, J = 9.0 Hz, 1H,
ArH), 8.36 (dd, J1 = 9.0 Hz, J2 = 2.7 Hz, 1H, ArH), 8.55 (d, J = 2.7 Hz,
1H, ArH).
l
l
der of 13c > 13e > 13b > 13a further proved that the bulky hydro-
phobic fragments on 2-substituents are favorable for the
interactions. Surprisingly, replacement of 4-carboxylphenylamino
group in compound 13c with carboxylethylene (compound 13h,
IC50 >100 lM) resulted in a loss of activity completely.
In order to get some insight for further structure-based modifi-
cation of quinazolines Pin1 inhibitor, the binding mode was inves-
tigated using FlexX algorithm implemented in SYBYL 7.2.19 The
coordinates of X-ray co-crystal structure of a carboxylate inhibitor
(reference molecule) with Pin1 (pdb code: 3JYJ) reported by Pfizer
in 201013 was employed for docking the synthesized compounds.
The binding modes of most of the evaluated target compounds
are somewhat similar to that of the reference molecule and exem-
plified by the representative compound 6a as shown in Figure 1.
The carboxylate on 4-substituents anchored with the positive
charged amino side chain of Arg69 and Lys63 via the crucial
charge–charge interaction, the other two hydrophobic moieties
are adjustable to fit into the hydrophobic pockets. The quinazoline
scaffold was positioned in the hydrophobic prolyl pocket and the
6-nitro group formed hydrogen bonds with OH on the side chain
of Ser154 and NH on both side chain and amide bond of GLN131.
Nevertheless, in comparison with the disposition of the benzene
ring of reference molecule, the quinazoline scaffold orientated
slightly outward. The 2-substituents on quinazoline extended to
the shallow hydrophobic region. However, the orientation of this
hydrophobic contact is somewhat promiscuous within the whole
5.1.2.2. 2,4-Dichloro-6-nitroquinazoline (3a). The reaction mix-
ture of compound 2a (0.78 g, 3.78 mmol), PCl5 (4.11 g, 19.7 mmol)
and POCl3 (16 mL) was stirred at reflux for 6.5 h. The excess POCl3
was removed by evaporation. The residue was dissolved in ice
water, and then the solution pH was adjusted to pH 5–6 with sat-
urated NaHCO3. The water phase was extracted with EtOAc
(60 mL Â 5) and the organic layer was dried over anhydrous
Na2SO4, concentrated to give the crude product which was purified
by column chromatography on silica gel (petroleum ether/
EtOAc = 40:1) to afford compound 3a as white solid (0.58 g, 63%);
mp 122–124 °C; 1H NMR (CDCl3) d: 8.18 (d, J = 9.0 Hz, 1H), 8.76
(dd, J1 = 9.3 Hz, J2 = 2.1 Hz, 1H), 9.18 (d, J = 1.8 Hz, 1H).
5.1.2.3. Methyl 4-(2-chloro-6-nitroquinazolin-4-ylamino)ben-
zoate (4a). To
a stirred solution of compound 3a (0.50 g,
2.05 mmol) in i-PrOH (8 mL) and CH2Cl2 (1.5 mL) was added
methyl 4-aminobenzoate (0.34 g, 2.25 mmol) and DIEA (0.42 g,

L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
2801
Figure 1. FlexX-modeled binding mode of compound 6a (carbon atoms colored orange) in comparison with the crystal structure (3JYJ in PDB)13 of a carboxylate Pin1
inhibitor (carbon atoms colored cyan). H-Bonding interactions are presented with yellow line. The chemical structure of reference molecule is presented. Molecular image
was generated with UCSF Chimera.20
3.28 mmol). The reaction mixture was stirred at room temperature
for 5 h. After filtration, compound 4a was obtained as yellow solid
(0.68 g, 93%); mp 279–282 °C; 1H NMR (DMSO-d6) d (ppm): 10.98
(br s, 1H, NH), 9.71(d, J = 2.4 Hz, 1H, ArH), 8.60 (dd, J1 = 9.3 Hz,
J2 = 2.4 Hz, 1H, ArH), 8.06 (d, J = 9.0 Hz, 2H, ArH), 7.99 (d,
J = 9.0 Hz, 2H, ArH), 7.91 (d, J = 9.0 Hz, 1H, ArH), 3.86 (s, 3H, OCH3).
(DMSO-d6) d (ppm): 10.73 (br s, 1H, NH), 10.11 (br s, 1H, NH),
7.85–7.93 (m, 4H, ArH), 7.57 (s, 1H, ArH), 7.37–7.47 (m, 5H, ArH),
7.27 (d, J = 8.7 Hz, 1H, ArH), 7.16 (t, J = 7.2 Hz, 1H, ArH), 7.01 (d,
J = 8.4 Hz, 4H, ArH), 3.81 (s, 3H, OCH3); HRMS (ESI): m/z, calcd for
C
28H24N5O3 [M+H+]: 478.1874; found 478.1874.
5.1.3.2. 4-(6-Amino-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)benzoic acid (6b). The reaction mixture of compound
5b (0.072 g, 0.15 mmol) and LiOHÁH2O (0.063 g, 1.5 mmol) in water
(6.0 mL) and THF (3.0 mL) was refluxed for 4.5 h. The solvent was
removed by evaporation and the residue was dissolved in water
(5.0 mL), which was cooled in ice-water bath and adjusted to pH
2.0 with diluted HCl solution. The resulting mixture stood for over-
night. After filtration, the title compound 6b was obtained as brick
red solid (0.06 g, 85%); mp 208–210 °C; 1H NMR (DMSO-d6) d
(ppm): 10.15 (br s, 1H, NH), 9.56 (br s, 1H, NH), 7.97 (d,
J = 8.7 Hz, 2H, ArH), 7.91 (d, J = 8.7 Hz, 2H, ArH), 7.65 (d,
J = 7.8 Hz, 2H, ArH), 7.46 (s, 1H, ArH), 7.38 (t, J = 6.3 Hz, 3H, ArH),
7.21 (d, J = 8.7 Hz, 1H, ArH), 7.11 (t, J = 7.5 Hz, 1H, ArH), 6.97–
5.1.2.4. Methyl 4-(6-nitro-2-(4-phenoxyphenylamino)quinazo-
lin-4-ylamino)benzoate (5a). To a stirred suspension of com-
pound 4a (0.46 g, 1.27 mmol) in acetone (1.5 mL) and water
(2.5 mL) was added 4-phenoxyphenylamine (0.13 g, 2.06 mmol)
and co-NMR (DMSO-d6) d: 10.98 (s, 1H, NH), concd HCl (24 drops).
The reaction mixture was refluxed for 10 h and then cooled to
room temperature. The crude product was obtained by filtration
and recrystalized with ethanol. Compound 5a was obtained as yel-
low solid (0.56 g, 87%); mp 234–236 °C; 1H NMR (DMSO-d6) d
(ppm): 11.32 (br s, 1H, NH), 10.68 (br s, 1H, NH), 9.63 (s, 1H,
ArH), 8.55 (d, J = 7.8 Hz, 1H, ArH), 7.55–8.01 (m, 7H, ArH), 7.40 (t,
J = 8.4 Hz, 2H, ArH), 7.16 (t, J = 7.5 Hz, 1H, ArH), 7.00–7.04 (m, 4H,
ArH), 3.82 (s, 3H, OCH3); HRMS (ESI): m/z, calcd for C28H22N5O5
[M+H+]: 508.1615, found 508.1621.
7.01 (m, 4H, ArH); HRMS (ESI): m/z, calcd for
C27H22N5O3
[M+H+]: 464.1717, found 464.1712.
5.1.2.5. 4-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-yla-
mino)benzoic acid (6a). The reaction mixture of compound 5a
(0.15 g, 1.0 mmol) and LiOHÁH2O (0.11 g, 9.0 mmol) in water
(12 mL) and THF (4.0 mL) was refluxed for 6.5 h. The solvent was
removed by evaporation and the residue was dissolved in water
(10 mL), which was cooled in ice-water bath and adjusted to pH
1.5 with diluted HCl. The resulting mixture was stirred for 5 h.
After filtration, the title compound 6a was obtained as brick red so-
lid (0.13 g, 92%); mp >300 °C; 1H NMR (DMSO-d6) d (ppm): 10.36
(br s, 1H, NH), 9.75 (s, 1H, NH), 9.54 (d, J = 2.1 Hz, 1H, ArH), 8.37
(dd, J1 = 9.0 Hz, J2 = 1.8 Hz, 1H, ArH), 7.94 (m, 6H, ArH), 7.52 (d,
J = 9.3 Hz, 1H, ArH), 7.37 (t, J = 7.8 Hz, 2H, ArH), 7.08 (t, J = 7.2 Hz,
1H, ArH), 7.10 (d, J = 7.8 Hz, 4H, ArH); HRMS (ESI): m/z, calcd for
5.1.4. 2-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino) acetic acid (6c)
5.1.4.1. Methyl 2-(2-chloro-6-nitroquinazolin-4-ylamino) ace-
tate (4b). To a suspension of 2,4-dichloro-6-nitroquinazoline 3a
(0.1 g, 0.41 mmol) in i-PrOH (3 mL) was added glycine methyl ester
hydrochloride (0.061 g, 0.49 mmol) and DIEA (0.11 g, 0.82 mol).
The resulting mixture was stirred at room temperature for 0.5 h
and filtered to provide product 4b as light yellow solid (0.077 g,
63%); mp 161–162 °C; 1H NMR: (CDCl3) d (ppm): 8.81 (s, 1H,
ArH), 8.50 (dd, J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, ArH), 7.82 (d, J = 9.0 Hz,
1H, ArH), 4.49 (d, J = 4.8 Hz, 2H, CH2), 3.91 (s, 3H, OCH3); HRMS
(ESI): m/z, calcd for
C
11H10ClN4O4 [M+H+]: 297.0385, found
297.0392.
C
27H20N5O5 [M+H+]: 494.1459, found 494.1457.
5.1.4.2. Methyl 2-(6-nitro-2-(4-phenoxyphenylamino)quinazo-
lin-4-ylamino)acetate (5c). Following the preparation protocol
of Section 5.1.2.4, the reaction mixture of compound 4b (0.1 g,
0.34 mmol), 4-phenoxyphenylamine (0.10 g, 0.55 mmol) and con-
cd HCl (24 drops) in acetone (4 mL) and water (5 mL) was refluxed
for 3 h to give compound 5c as brow solid (0.09 g, 61%); mp 203–
205 °C; 1H NMR (DMSO-d6) d (ppm): 9.36 (s, 1H, ArH), 8.55 (d,
J = 9.3 Hz, 1H, ArH), 7.71 (d, J = 9.3 Hz, 1H, ArH), 7.56 (d,
J = 6.6 Hz, 2H, ArH), 7.41 (t, J = 7.5 Hz, 2H, ArH), 7.15 (t, J = 7.5 Hz,
5.1.3. 4-(6-Amino-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)benzoic acid (6b)
5.1.3.1. Methyl 4-(6-amino-2-(4-phenoxyphenylamino)quinaz-
olin-4-ylamino)benzoate (5b). The mixture of compound 5a
(0.35 g, 0.69 mmol) and 10% Pd/C (0.14 g) in HFIP (15 mL) was
hydrogenated at room temperature and 1 atm for 3.5 h. Pd/C was
filtered off and the filtrate was concentrated to give compound
5b as yellow solid (0.33 g, 100%); mp 232–234 °C; 1H NMR

2802
L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
1H, ArH), 7.02–7.05 (m, 4H, ArH), 4.31 (d, J = 4.8 Hz, 2H, CH2), 3.61
(s, 3H, OCH3); HRMS (ESI): m/z, calcd for C23H20N5O5 [M+H+]:
446.1459, found 446.1451.
10 min. The mixture was cooled to room temperature and the
crude product was precipitated. After filtration and recrystalliza-
tion with ethanol, compound 5e was obtained as yellow crystals
(0.25 g, 75%); mp 186–187 °C; 1H NMR (DMSO-d6) d (ppm): 9.06
(br s, 1H, NH), 8.82 (br s, 0.5H, NH), 8.61 (br s, 0.5H, NH), 7.95–
8.24 (m, 5H, ArH), 7.25 (d, J = 9.3 Hz, 1H, ArH), 4.71 (d, J = 5.7 Hz,
2H, ArCH2), 3.53–3.69 (m, 5H, OCH3 and CH2), 2.73 (t, J = 6.6 Hz,
2H, CH2); HRMS (ESI): m/z, calcd for C21H18F6N5O4 [M+H+]:
518.1257, found 518.1255.
5.1.4.3. 2-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-yla-
mino)acetic acid (6c). The reaction mixture of compound 5c
(0.05 g, 0.11 mmol) and NaOH (0.045 g, 1.12 mmol) in water
(4 mL) and THF (2 mL) was stirred at room temperature for 2 h,
compound 6c was obtained as brow solid (0.043 g, 88%); mp
>300 °C; 1H NMR (DMSO-d6) d (ppm): 12.08 (br s, 1H, COOH),
9.69 (br s, 1H, NH), 9.19 (d, J = 2.4 Hz, 1H, ArH), 8.33 (dd,
J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, ArH), 7.80 (m, 2H, ArH), 7.45 (d,
J = 9.0 Hz, 1H, ArH), 7.36 (t, J = 8.1 Hz, 2H, ArH), 7.09 (t, J = 7.5 Hz,
1H, ArH), 6.96 (d, J = 8.4 Hz, 4H, ArH), 4.21 (m, 2H, NCH2); HRMS
5.1.6.2. 3-(2-(3,5-Bis(trifluoromethyl)benzylamino)-6-nitroqui-
nazolin-4-ylamino)propanoic acid (6e). The reaction mixture of
compound 5e (0.05 g, 0.10 mmol) and NaOH (0.04 g, 0.96 mmol) in
water (4 mL) and THF (2 mL) was stirred at room temperature for
1.5 h, compound 6e was obtained as off-white solid (0.04 g, 84%);
mp >300 °C; 1H NMR (DMSO-d6) d (ppm): 12.24 (br s, 1H, OH),
9.54 (br s, 1H, NH), 9.24 (s, 0.6H, ArH), 9.12 (s, 0.4H, ArH), 8.76
(br s, 0.4H, NH), 8.62 (br s, 0.6H, NH), 8.39 (d, J = 9.0 Hz, 0.6H,
ArH), 8.27 (d, J = 9.3 Hz, 0.4H, ArH), 8.09 (s, 2H, ArH), 7.98 (s, 1H,
ArH), 7.48 (d, J = 8.7 Hz, 0.6H, ArH), 7.38 (d, J = 8.4 Hz, 0.4H, ArH),
4.78 (br s, 2H, ArCH2), 3.64 (m, 2H, CH2), 2.67 (m, 1H, CH2), 2.49
(m, 1H, CH2); HRMS (ESI): m/z, calcd for C20H16F6N5O4 [M+H+]:
504.1101, found 504.1110.
(ESI): m/z, calcd for
C
22H18N5O5 [M+H+]: 432.1302, found
432.1292.
5.1.5. 3-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)propanoic acid (6d)
5.1.5.1. Methyl 2-(2-chloro-6-nitroquinazolin-4-ylamino) pro-
panoate (4c). To a suspension of 2,4-dichloro-6-nitroquinazoline
3a (0.4 g, 1.64 mmol) in i-PrOH (10 mL) was added methyl 3-amino
propanoate hydrochloride (0.27 g, 1.97 mmol) and DIEA (0.42 g,
3.28 mmol). The resulting mixture was stirred at room tempera-
ture for 0.5 h and filtered to provide compound 4c as light yellow
solid (0.48 g, 93%); mp 178–179 °C; 1H NMR (CDCl3) d (ppm): 8.72
(d, J = 2.1 Hz, 1H, ArH), 8.50 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H, ArH),
7.86 (d, J = 9.6 Hz, 1H, ArH), 4.02 (q, J = 5.4 Hz, 2H, CH2), 3.76 (s,
3H, CH3), 2.80 (t, J = 5.7 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for
5.1.7. 4-(2-(3,5-Bis(trifluoromethyl)benzylamino)-6-
nitroquinazolin-4-ylamino)butanoic acid (6f)
5.1.7.1. Ethyl 2-(2-chloro-6-nitroquinazolin-4-ylamino)butano-
ate (4d). To a suspension of 2,4-dichloro-6-nitroquinazoline 3a
(0.1 g, 0.41 mmol) in i-PrOH (3 mL) was added ethyl 4-amino but-
anoate hydrochloride (0.054 g, 0.49 mmol) and DIEA (0.11 g,
0.82 mmol). The resulting mixture was stirred at room tempera-
ture for 0.5 h and filtered to provide product 4d as light yellow so-
lid (0.11 g, 84%); mp 149–150 °C; 1H NMR (CDCl3) d (ppm): 8.87 (s,
1H, ArH), 8.66 (d, J = 9.3 Hz, 1H, ArH), 8.01 (br s, 1H, NH), 7.83 (d,
J = 9.0 Hz, 1H, ArH), 4.25 (q, J = 7.2 Hz, 2H, CH2), 3.73 (m, 2H,
CH2), 2.62 (m, 2H, CH2), 2.12 (m, 2H, CH2), 1.30 (t, J = 7.5 Hz, 3H,
CH3); HRMS (ESI): m/z, calcd for C14H16ClN4O4 [M+H+]: 339.0854,
found 339.0855.
C
12H12ClN4O4 [M+H+]: 311.0542, found 311.0549.
5.1.5.2. Methyl 2-(6-nitro-2-(4-phenoxyphenylamino)quinazo-
lin-4-ylamino)propanoate (5d). Following the preparation pro-
tocol of Section 5.1.2.4, the reaction mixture of compound 4c
(0.05 g, 0.17 mmol), 4-phenoxyphenylamine (0.51 g, 0.27 mmol)
and concd HCl (4 drops) in acetone (2 mL) and water (3 mL) was
refluxed for 2 h to give compound 5d as yellow solid (0.07 g,
92%); mp 185–187 °C; 1H NMR (DMSO-d6) d (ppm): 10.64 (br s,
1H, NH), 10.08 (br s, 1H, NH), 9.32 (d, J = 1.8 Hz, 1H, ArH), 8.53
(d, J = 8.7 Hz, 1H, ArH), 7.70 (d, J = 9.3 Hz, 1H, ArH), 7.63 (d,
J = 9.0 Hz, 2H, ArH), 7.40 (t, J = 8.1 Hz, 2H, ArH), 7.17 (t, J = 7.5 Hz,
1H, ArH), 7.03–7.06 (m, 4H, ArH), 3.77 (m, 2H, CH2), 3.58 (s, 3H,
OCH3), 2.78 (t, J = 6.9 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for
5.1.7.2. Ethyl 4-(2-(3,5-bis(trifluoromethyl)benzylamino)-6-
nitroquinazolin-4-ylamino) butanoate (5f). The reaction mix-
ture of compound 4d (0.3 g, 0.89 mmol) and 3,5-bis(trifluoro-
methyl)benzylamine (1.08 g, 4.43 mmol) in isopropanol (5 mL)
was heated by microwave (power 200 W, temperature 110 °C)
for 45 min. The mixture was cooled to room temperature and the
crude product was precipitated. After filtration and recrystalliza-
tion with ethanol, compound 5f was obtained as yellow crystals
(0.32 g, 67%); mp 172–173 °C; 1H NMR (DMSO-d6) d (ppm): 9.11
(br s, 0.5H, NH), 9.07 (br s, 0.5H, NH), 8.73 (br s, 0.5H, NH), 8.55
(br s, 0.5H, NH), 8.22 (d, J = 9.6 Hz, 1H, ArH), 7.95–8.12 (m, 4H,
ArH), 7.26 (d, J = 9.3 Hz, 1H, ArH), 4.71 (d, J = 5.4 Hz, 2H, ArCH2),
3.95–4.02 (m, 2H, CH2), 3.51 (m, 1H, CH), 3.41 (m, 1H, CH), 2.41
(m, 1H, CH), 2.19 (m, 1H, CH), 1.92 (m, 1H, CH), 1.69 (m, 1H, CH),
1.11–1.13 (m, 3H, CH3); HRMS (ESI): m/z, calcd for C23H22F6N5O4
[M+H+]: 546.1570, found 546.1561.
C
24H22N5O5 [M+H+]: 460.1615, found 460.1614.
5.1.5.3. 3-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-yla-
mino)propanoic acid (6d). The reaction mixture of compound
5d (0.2 g, 0.44 mmol) and NaOH (0.17 g, 4.34 mmol) in water
(10 mL) and THF (8 mL) was stirred at room temperature for
30 min, compound 6d was obtained as red solid (0.18 g, 96%);
mp 266–268 °C; 1H NMR (DMSO-d6) d (ppm): 12.27 (br s, 1H,
COOH), 9.76 (br s, 1H, NH), 9.18 (d, J = 1.8 Hz, 1H, ArH), 8.94 (br
s, 1H, NH), 8.32 (dd, J1 = 9.6 Hz, J2 = 2.1 Hz, 1H, ArH), 7.87 (d,
J = 8.1 Hz, 2H, ArH), 7.44 (d, J = 9.3 Hz, 1H, ArH), 7.40 (t,
J = 8.1 Hz, 2H, ArH), 7.36 (t, J = 8.4 Hz, 2H, ArH), 7.08 (t, J = 7.2 Hz,
1H, ArH), 6.95–7.00 (m, 4H, ArH), 3.74 (m, 2H, CH2), 2.70 (t,
J = 6.9 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for C23H20N5O5
[M+H+]: 446.1459, found 446.1465.
5.1.7.3. 4-(2-(3,5-Bis(trifluoromethyl)benzylamino)-6-nitroqui-
nazolin-4-ylamino)butanoic acid (6f). The reaction mixture of
compound 5f (0.05 g, 0.09 mmol) and NaOH (0.367 g, 0.92 mmol)
in water (3 mL) and THF (2 mL) was refluxed for 1.5 h, compound
6f was obtained as light yellow solid (0.033 g, 69%); mp 266–
268 °C; 1H NMR (DMSO-d6) d (ppm): 10.04 (s, 1H, NH), 9.29 (s,
1H, ArH), 8.99 (t, J = 4.8 Hz, 1H, NH), 8.52 (d, J = 9.0 Hz, 1H, ArH),
8.14 (s, 2H, ArH), 7.99 (s, 1H, ArH), 7.68 (d, J = 9.3 Hz, 1H, ArH),
4.96 (d, J = 4.8 Hz, 2H, CH2), 3.54 (m, 2H, CH2), 2.20 (t, J = 6.6 Hz,
5.1.6. 3-(2-(3,5-Bis(trifluoromethyl)benzylamino)-6-
nitroquinazolin-4-ylamino)propanoic acid (6e)
5.1.6.1. Methyl 3-(2-(3,5-bis(trifluoromethyl)benzylamino)-6-
nitroquinazolin-4-ylamino) propanoate (5e). The reaction mix-
ture of compound 4c (0.2 g, 0.64 mmol) and 3,5-bis(trifluoro-
methyl)benzylamine (0.78 g, 3.22 mmol) in isopropanol (4 mL)
was heated by microwave (power 50 W, temperature 110 °C) for

L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
2803
2H, CH2), 1.73 (q, J = 6.6 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for
21H18F6N5O4 [M+H+]: 518.1257, found 518.1267.
extracted with EtOAc and the organic layer was dried over anhy-
drous MgSO4, concentrated to give the crude product which was
purified by column chromatography on silica gel (petroleum
ether/EtOAc = 50:1) to afford compound 3b, which was recrystal-
ized with methanol as yellow solid (0.49 g, 80%); mp 116–117 °C;
HRMS (ESI): m/z, calcd for C8H5Cl2N2 [M+H+]: 198.9829 found
198.9832.
C
5.1.8. 4-(6-Nitro-4-(4-phenoxyphenylamino)quinazolin-2-
ylamino)benzoic acid (6g)
5.1.8.1.
2-Chloro-6-nitro-N-(4-phenoxyphenyl)quinazolin-4-
amine (4e). To a suspension of 2,4-dichloro-6-nitroquinazoline
3a (0.50 g, 2.03 mmol) in i-PrOH (30 mL) was added 4-phenoxy-
phenylamine (0.45 g, 2.43 mmol) and DIEA (0.32 g, 2.48 mmol).
The resulting mixture was stirred at room temperature for 8 h
and concentrated under vacuum. The residue was dissolved in
EtOAc, washed with saturated NaHCO3 aqueous solution and brine
and dried over anhydrous MgSO4. The crude product obtained after
concentration was purified with column chromatography to afford
product 4e as red solid (0.64 g, 80%); mp 222–223 °C; 1H NMR
(acetone-d6) d (ppm): 9.13 (s, 1H, NH), 8.79 (d, J = 2.4 Hz, 1H,
NH), 8.44 (dd, J1 = 9.3 Hz, J 2=2.7 Hz, 1H, ArH), 8.03 (d, J = 8.4 Hz,
2H, ArH), 7.67 (d, J = 9.3 Hz, 1H, ArH), 7.37 (t, J = 8.1 Hz, 2H, ArH),
7.10 (t, J = 7.2 Hz, 1H, ArH), 7.03 (t, J = 8.7 Hz, 4H, ArH).
5.1.9.3.
Methyl
4-(2-chloroquinazolin-4-ylamino)benzoate
(4f). To a stirred solution of compound 3b (0.10 g, 0.50 mmol)
in i-PrOH (2.5 mL) and CH2Cl2 (0.5 mL) was added methyl 4-ami-
nobenzoate (0.08 g, 0.55 mmol) and DIEA (0.14 mL). The reaction
mixture was stirred at room temperature for 45 h. After filtration,
compound 4f was obtained as white solid (0.105 g, 67%); mp 217–
219 °C; 1H NMR (acetone-d6, d ppm) d 9.65 (s, 1H, NH), 8.49 (d,
J = 8.1 Hz, 1H), 8.10 (d, J = 9.0 Hz, 2H, ArH), 8.04 (d, J = 9.0 Hz, 2H,
ArH), 7.91 (t, J = 7.2 Hz, 1H, ArH), 7.77 (d, J = 8.1 HZ, 1H, ArH),
7.65 (t, J = 7.2 Hz, 1H, ArH), 3.88 (s, 3H, OCH3).
5.1.9.4. Methyl 4-(2-(4-phenoxyphenylamino)quinazolin-4-yla-
mino)benzoate (5h). To a stirred suspension of compound 4f
(0.05 g, 0.16 mmol) in acetone (1.5 mL) and water (2.5 mL) was
added 4-phenoxyphenylamine (0.047 g, 0.25 mmol) and concd
HCl (3 drops). The reaction mixture was refluxed for 7.5 h and then
cooled to room temperature. The crude product was obtained by
filtration and washed with acetone. Compound 5h was obtained
as yellow solid (0.069 g, 94%); mp 216–218 °C; 1H NMR (DMSO-
d6) d (ppm): 11.18 (s, 1H, NH), 10.55 (s, 1H, NH), 8.73 (d,
J = 8.4 Hz, 1H, ArH), 7.90–7.93 (m, 5H, ArH), 7.67 (d, J = 8.4 Hz,
1H, ArH), 7.55 (t, J = 7.5 Hz, 1H, ArH), 7.39–7.47 (m, 4H, ArH),
7.18 (t, J = 7.2 Hz, 1H, ArH), 7.02–7.05 (m, 4H, ArH), 3.82 (s, 3H,
OCH3); HRMS (ESI): m/z, calcd for C28H23N4O3 [M+H+]: 463.1765,
found 463.1764.
5.1.8.2. Methyl 4-(6-nitro-4-(4-phenoxyphenylamino)quinazo-
lin-2-ylamino)benzoate (5g). Following the preparation protocol
of Section 5.1.2.4, the reaction mixture of compound 4e (0.2 g,
0.51 mmol), methyl 4-aminobenzoate (0.12 g, 0.76 mmol) and con-
cd HCl (8 drops) in acetone and water was refluxed for 6.5 h to give
the crude product, which was recrystallized from ethanol to afford
compound 5g as yellow solid (0.19 g, 74%); mp 260–261 °C; 1H
NMR (DMSO-d6) d (ppm): 11.29 (br s, 1H, NH), 10.78 (br s, 1H,
NH), 9.58 (d, J = 2.1 Hz, 1H, ArH), 8.53 (dd, J1 = 9.0 Hz, J2 = 2.1 Hz,
1H, ArH), 7.67–7.82 (m, 7H, ArH), 7.44 (t, J = 8.1 Hz, 2H, ArH),
7.19 (t, J = 7.5 Hz, 1H, ArH), 7.05–7.12 (m, 4H, ArH), 3.81 (s, 3H,
OCH3); HRMS (ESI): m/z, calcd for C28H22N5O5 [M+H+]: 508.1615,
found 508.1619.
5.1.8.3. 4-(6-Nitro-4-(4-phenoxyphenylamino)quinazolin-2-yla-
mino)benzoic acid (6g). The reaction mixture of compound 5g
(0.05 g, 0.1 mmol) and KOH (0.112 g, 2.0 mmol) in water (12 mL)
and CH3OH (25 mL) was refluxed for 11 h, compound 6g was ob-
tained as yellow solid (0.044 g, 90%); mp >300 °C; 1H NMR
(DMSO-d6) d (ppm): 10.39 (s, 1H, NH), 10.01 (s, 1H, NH), 9.50 (d,
J = 1.8 Hz, 1H, ArH), 8.41 (dd, J1 = 9.3 Hz, J2 = 2.1 Hz, 1H, ArH),
7.78–7.93 (m, 6H, ArH), 7.58 (d, J = 9.0 Hz, 1H, ArH), 7.44 (t,
J = 7.8 Hz, 2H, ArH), 7.07–7.18 (m, 5H, ArH); HRMS (ESI): m/z, calcd
for C27H20N5O5 [M+H+]: 494.1459, found 494.1462.
5.1.9.5.
4-(2-(4-Phenoxyphenylamino)quinazolin-4-ylami-
no)benzoic acid (6h). The reaction mixture of compound 5h
(0.10 g, 0.22 mmol) and LiOHÁH2O (0.082 g, 1.95 mmol) in water
(4 mL), methanol (18 mL) and THF (2.0 mL) was refluxed for 2 h.
The solvent was removed by evaporation and the residue was dis-
solved in water, which was cooled in ice-water bath and adjusted
to pH 2.0 with diluted HCl. The resulting mixture was stirred for
2 h. After filtration, the title compound 6h was obtained as yellow
solid (0.095 g, 98%); mp 224–226 °C; 1H NMR (DMSO-d6) d (ppm):
13.10 (br s, 1H, COOH), 11.14 (s, 1H, NH), 10.56 (s, 1H, ArH), 8.73
(d, J = 8.1 Hz, 1H, ArH), 7.88–7.99 (m, 5H, ArH), 7.67 (d, J = 8.4 Hz,
1H, ArH), 7.54 (t, J = 7.8 Hz, 1H, ArH), 7.43 (q, J = 9.3 Hz, 4H, ArH),
7.16 (t, J = 7.2 Hz, 1H, ArH), 7.00–7.05 (m, 4H, ArH); HRMS (ESI):
m/z, calcd for C27H21N4O3 [M+H+]: 449.1608, found: 449.1610.
5.1.9. 4-(2-(4-Phenoxyphenylamino)quinazolin-4-
ylamino)benzoic acid (6h)
5.1.9.1. Quinazoline-2,4-(1H,3H)-dione (2b). The mixture of 2-
aminobenzoic acid (0.5 g, 3.65 m mol) and urea (2.19 g, 0.36 mol)
was stirred at 150 °C for 6 h. The reaction mixture was cooled to
100 °C and then water (5 mL) was added to quench the reaction.
The crude product was obtained by filtration, and then washed
with water (3 mL Â 3) and methanol (3 mL Â 3) to give the crude
product 0.41 g, which was dissolved in heated NaOH aqueous solu-
tion, then cooled to 0 °C and adjusted pH 6 with diluted HCl aque-
ous solution. After stirred 20 min, the mixture was filtered and
compound 2b was obtained as white solid (0.37 g, 65%), which
was used directly without further purification.
5.1.10. 2-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)ethyl dihydrogen phosphate (10a)
5.1.10.1.
2-(2-Chloro-6-nitroquinazolin-4-ylamino)ethanol
(7). To a stirred solution of compound 3a (1.12 g, 5.0 mmol) in
i-PrOH (11 mL) was added 2-aminoethanol (0.37 g, 6.0 mmol)
and DIEA (0.97 g, 7.5 mmol). The reaction mixture was stirred at
room temperature for 7 h. After filtration, compound 7 was ob-
tained as yellow solid (1.30 g, 97%); mp 155–156 °C; 1H NMR
(DMSO-d6) d (ppm): 12.22 (s, 1H, ArNH), 9.95 (s, 1H, ArNH), 9.28
(t, 1H, NH), 9.19 (d, J = 1.8 Hz, 1H, ArH), 8.30 (dd, J1 = 9.0 Hz,
J2 = 1.8 Hz, 1H, , ArH), 8.04 (s, 1H, ArH), 7.68 (d, J = 9.3 Hz, 1H,
ArH), 7.54 (s, 2H, ArH), 3.68 (m, 2H, NCH2), 2.65 (t, J = 6.6 Hz, 2H,
CH2CO).
5.1.9.2. 2,4-Dichloroquinazoline (3b). The reaction mixture of
compound 2b (0.50 g, 3.09 mmol), POCl3 (4.3 mL) and N,N-dimeth-
ylaniline (1.6 mL) was stirred at reflux for 7 h. The excess POCl3
was removed by evaporation. The residue was dissolved in EtOAc
which was washed with cold diluted HCl aqueous solution in order
to remove the N,N-dimethylaniline. The organic phase was
adjusted to pH 5–6 with saturated NaHCO3. The water phase was
5.1.10.2.
2-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)ethanol (8a). To a stirred suspension of compound 7
(0.29 g, 1.07 mmol) in acetone (10 mL) and water (4.0 mL) was

2804
L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
added 4-phenoxyphenylamine (0.32 g, 1.72 mmol) and concd HCl
(0.64 g, 6.42 mmol). The reaction mixture was refluxed for 1 h
and then cooled to room temperature. Compound 8a was obtained
as yellow solid (0.44 g, 99%); mp 226–228 °C; 1H NMR (DMSO-d6) d
(ppm): 10.67 (br s, 1H, NH), 10.12 (br s, 1H, NH), 9.41 (d, J = 2.1 Hz,
1H, Ar), 8.53 (dd, J1 = 9.0 Hz, J2 = 1.8 Hz, 1H, ArH), 7.70 (d, J = 9.3 Hz,
1H, ArH), 7.62 (d, J = 8.4 Hz, 2H, ArH), 7.40 (t, J = 7.5 Hz, 2H, ArH),
7.15 (t, J = 7.2 Hz, 1H, Ar), 7.05 (m, 4H, ArH), 3.65 (m, 4H, CH2CH2);
HRMS (ESI): m/z, calcd for C22H20N5O4 [M+H+]: 418.1510, found
418.1514.
dibenzyl diisopropylphosphoramidite (0.31 g, 0.9 mmol) and 1H-
tetrazole (0.067 g, 0.96 mmol) was stirred for 40 min. Then 30%
H2O2 (0.24 g, 2.1 mmol) was added and stirred for 50 min to pro-
vide compound 9b (0.27 g, 74%); mp 113–115 °C; 1H NMR (CDCl3)
d (ppm): 8.75 (s, 1H, ArH), 8.29 (d, J = 7.8 Hz, 1H, ArH), 7.29 (m,
16H, ArH), 5.05 (d, J = 6.9 Hz, 4H, 2CH2), 4.70 (s, 2H, CH2), 4.18 (s,
2H, CH2), 3.73 (s, 2H, CH2). HRMS (ESI): m/z, calcd for C31H31N5O6P
[M+H+]: 600.2006, found: 600.2012.
5.1.11.3.
2-(2-(Benzylamino)-6-nitroquinazolin-4-ylamino)
ethyl dihydrogen phosphate (10b). Following the preparation
protocol of Section 5.1.10.4, the mixture of compound 9b (0.12 g,
0.2 mmol), TFA (2.17 g, 19 mmol) and water (0.018 g, 1 mmol)
was stirred at room temperature for 5 h to provide compound
10b as yellow solid (0.08 g, 95%); mp 196–198 °C; 1H NMR
(DMSO-d6, 500 MHz) d (ppm): 9.10 (br s, 1H), 8.21 (d, J = 7.5 Hz,
1H, ArH), 7.34 (d, J = 7.5 Hz, 2H, ArH), 7.29 (t, J = 7.5 Hz, 3H, ArH),
7.20 (d, J = 7.5 Hz, 2H, ArH), 4.56 (m, 2H, CH2), 4.13 (m, 2H, CH2),
3.63 (m, 2H, CH2); HRMS (ESI): m/z, calcd for C17H19N5O6P
[M+H+]: 420.1067, found: 420.1065.
5.1.10.3. Dibenzyl 2-(6-nitro-2-(4-phenoxyphenylamino)qui-
nazolin-4-ylamino)ethyl phosphate (9a). To a suspension of
compound 8a (0.104 g, 0.25 mmol) in dried DCM (3 mL) and DMF
(0.5 mL) was added dibenzyl diisopropylphosphoramidite (0.13 g,
0.38 mmol) and 1H-tetrazole (0.028 g, 0.4 mmol), respectively.
The resulting reaction mixture was stirred at room temperature
for 1 h, and then 30% H2O2 (0.099 g, 0.88 mmol) was added. The
reaction solution was stirred for another 35 min and then diluted
with EtOAc (60 mL). The organic mixture was washed water
(60 mL Â 5) and saturated NaHCO3 aqueous solution. The water
phase was extracted with EtOAc (60 mL Â 4), and the combined or-
ganic phase was dried over anhydrous Na2SO4, concentrated to
give the crude product which was purified by column chromatog-
raphy on silica gel (DCM/methanol = 100:1) to afford compound
9a, which was recrystalized with petroleum ether and DCM to give
the orange solid (0.15 g, 90%); mp 128–129 °C; 1H NMR (CDCl3) d
(ppm): 8.94 (s, 1H, ArH), 8.40 (d, J = 9.3 Hz, 1H, ArH), 8.17 (t, 1H,
NH), 8.06 (d, J = 7.2 Hz, 1H, ArH), 7.64–7.68 (m, 3H, ArH), 7.50 (d,
J = 8.1 Hz, 1H, ArH), 7.27–7.41 (m, 10H, ArH), 7.11 (t, J = 7.2 Hz,
1H, ArH), 7.02 (d, J = 8.7 Hz, 4H, ArH), 5.08 (d, J = 9.0 Hz, 2H, ArCH2),
5.06 (d, J = 9.6 Hz, 2H, ArCH2), 4.26 (m, 2H, CH2), 3.80 (m, 2H, CH2);
HRMS (ESI): m/z, calcd for C36H33N5O7P [M+H+]: 678.2112, found
678.2118.
5.1.12. 2-(2-(3-Fluorophenylamino)-6-nitroquinazolin-4-
ylamino)ethyl dihydrogen phosphate (10c)
5.1.12.1. 2-(2-(3-Fluorophenylamino)-6-nitroquinazolin-4-yla-
mino)ethanol (8c). The reaction mixture of compound
7
(0.54 g, 2 mmol), 3-fluorophenylamine (0.36 g, 3.2 mmol) and con-
cd HCl (1.2 g, 12 mmol) was refluxed for 30 min. After filtration,
compound 8c (0.55 g, 80%) was obtained as white solid; mp 241–
243 °C; 1H NMR (DMSO-d6) d (ppm): 10.85 (br s, 1H), 10.18 (br s,
1H), 9.43 (d, J = 2.1 Hz, 1H, ArH), 8.53 (dd, J1 = 9.0 Hz, J2 = 2.1 Hz,
1H, ArH), 7.62–7.71 (m, 2H, ArH), 7.38–7.48 (m, 2H, ArH), 6.99–
7.04 (m, 1H, ArH), 3.66–3.71 (m, 4H, CH2CH2); HRMS (ESI): m/z,
calcd for C16H15FN5O3 [M+H+]: 344.1153, found 344.1157.
5.1.12.2. Dibenzyl 2-(2-(3-fluorophenylamino)-6-nitroquinazo-
lin-4-ylamino)ethyl phosphate (9c). Following the preparation
protocol of Section 5.1.10.3, the mixture of compound 8c (0.12 g,
5.1.10.4.
2-(6-Nitro-2-(4-phenoxyphenylamino)quinazolin-4-
ylamino)ethyl dihydrogen phosphate (10a). The reaction mix-
ture of compound 9a (0.24 g, 0.35 mmol), TFA (3.79 g, 33.
25 mmol) and water (0.032 g, 1.75 mmol) was stirred at room tem-
perature for 4.5 h. The excess TFA was removed by evaporation, the
residue was treated with EtOAc and the yellow solid precipitated.
After filtration, the yellow solid was recrystalized with DMF and
water to afford compound 10a (0.16 g, 92%); mp 230–231 °C; 1H
NMR (DMSO-d6) d (ppm): 9.25 (br s, 1H), 9.19 (s, 1H, ArH), 8.25
(d, J = 8.4 Hz, 1H, ArH), 7.80 (br s, 3H), 6.91–7.40 (m, 10H, ArH),
4.16 (m, 2H, CH2), 3.78 (m, 2H, CH2); HRMS (ESI): m/z, calcd for
0.35 mmol),
dibenzyl
diisopropylphosphoramidite
(0.18 g,
0.52 mmol) and 1H-tetrazole (0.039 g, 0.56 mmol) was stirred for
1 h. Then 30% H2O2 (0.14 g, 1.22 mmol) was added and stirred for
15 min to provide compound 9c (0.18 g, 87%); mp 152–154 °C; 1H
NMR (CDCl3) d (ppm): 8.44 (d, J = 2.1 Hz, 1H, ArH), 8.35 (dd,
J1 = 9.0 Hz, J2 = 2.1 Hz, 1H, ArH), 7.86 (d, J = 11.4 Hz, 1H, ArH), 7.66
(br s, 1H), 7.55 (d, J = 9.3 Hz, 1H, ArH), 7.19–7.36 (m, 12H, ArH), 6.75
(t, J = 7.5 Hz, 1H, ArH), 5.08 (d, J = 9.0 Hz, 2H, ArCH2), 5.06 (d,
J = 9.3 Hz, 2H, ArCH2), 4.30 (m, 2H, CH2), 3.81 (m, 2H, CH2); HRMS
(ESI): m/z, calcd forC30H28FN5O6P [M+H+]:604.1756, found604.1759.
C
22H21N5O7P [M+H+]: 498.1173, found: 498.1175.
5.1.11. 2-(2-(Benzylamino)-6-nitroquinazolin-4-ylamino)ethyl
dihydrogen phosphate (10b)
5.1.11.1. 2-(2-(Benzylamino)-6-nitroquinazolin-4-ylamino)eth-
5.1.12.3. 2-(2-(3-Fluorophenylamino)-6-nitroquinazolin-4-yla-
mino)ethyl dihydrogen phosphate (10c). Following the prepara-
tion protocol of Section 5.1.10.4, the mixture of compound 9c
(0.12 g, 0.2 mmol), TFA (2.17 g, 19 mmol) and water (0.018 g,
1 mmol) was stirred at room temperature for 5 h to provide the ti-
tle compound 10c as yellow solid (0.08 g, 100%); mp 237–239 °C;
1H NMR (DMSO-d6) d (ppm): 10.09 (br s, 1H), 9.23 (s, 1H, ArH),
8.32 (d, J = 9.0 Hz, 1H, ArH), 7.85 (m, 1H, ArH), 7.57 (d, J = 7.5 Hz,
1H, ArH), 7.48 (d, J = 7.5 Hz, 1H, ArH), 7.27–7.34 (m, 1H, ArH),
6.79 (t, J = 7.8 Hz, 1H, ArH), 4.15 (m, 2H, CH2), 3.79 (m, 2H, CH2);
HRMS (ESI): m/z, calcd for C16H16FN5O6P [M+H+]: 424.0817, found:
424.0815.
anol (8b). To
a stirred suspension of compound 7 (0.46 g,
1.7 mmol) in i-PrOH (10 mL) was added benzyl amine (0.64 g,
5.95 mmol). The reaction mixture was refluxed for 3 h. The crude
product was obtained after filtration and recrystalized with etha-
nol to afford the desired product 8b as light yellow solid (0.49 g,
85%); mp 194–196 °C; 1H NMR (DMSO-d6) d (ppm): 9.11 (d,
J = 2.4 Hz, 1H, ArH), 8.74 (br s, 0.5H), 8.49 (br s, 0.5H), 8.20 (d,
J = 9.0 Hz, 1H, ArH), 8.01 (br s, 0.5H), 7.84 (br s, 0.5H), 7.42 (m,
1H, ArH), 7.17–7.35 (m, 5H, ArH), 4.74 (m, 1H, OH), 4.56 (s, 2H,
ArCH2), 3.62–3.64 (m, 4H, CH2CH2); HRMS (ESI): m/z, calcd for
C
17H18N5O3 [M+H+]: 340.1404, found: 340.1406.
5.1.13. 4-(2-(3-(3-Fluorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13a)
5.1.11.2. Dibenzyl 2-(2-(benzylamino)-6-nitroquinazolin-4-yla-
mino)ethyl phosphate (9b). Following the preparation protocol
of Section 5.1.10.3, the mixture of compound 8b (0.20 g, 0.6 mmol),
5.1.13.1. Methyl 4-(2-amino-6-nitroquinazolin-4-ylamino)ben-
zoate (11a). The reaction mixture of compound 4a (0.54 g,
1.5 mmol) and the newly prepared saturated NH3 ethanol solution

L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
2805
was irradiated by microwave (power 50 W, temperature 90 °C) for
135 min. The desired compound 11a (0.5 g, 98%) was obtained as
yellow solid after filtration and washed with ethanol. Mp 295–
296 °C; 1H NMR (DMSO-d6) d (ppm): 10.19 (s, 1H, NH), 9.45 (d,
J = 2.1 Hz, 1H, ArH), 8.30 (dd, J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, ArH),
8.18 (d, J = 8.7 Hz, 2H, ArH), 7.96 (d, J = 8.7 Hz, 2H, ArH), 7.34 (d,
J = 9.6 Hz, 1H, ArH), 7.18 (s, 2H, NH2), 3.84 (s, 3H, OCH3); HRMS
J = 6.6 Hz, 2H, ArH); HRMS (ESI): m/z, calcd for C22H16N6O4Cl
[M+H+]: 479.0865, found 479.0865.
5.1.15. 4-(2-(3-(3,4-Dichlorophenyl)ureido)-6-nitroquinazolin-
4-ylamino)benzoic acid (13c)
5.1.15.1. Methyl 4-(2-(3-(3,4-dichlorophenyl)ureido)-6-nitro-
quinazolin-4-ylamino)benzoate (12c). The solution of com-
pound 11a (0.034 g, 0.1 mmol) and 3,4-dichlorophenyl isocyanate
(0.056 g, 0.3 mmol) in dioxane (1 mL) was heated by microwave
(power 150 W, temperature 120 °C) for 15 min. After filtration,
compound 12c (0.028 g, 53%) was obtained as yellow solid; mp
273–275 °C; 1H NMR (DMSO-d6) d (ppm): 12.07 (s, 1H, NH),
10.57 (s, 1H, NH), 10.48 (s, 1H, NH), 9.60 (d, J = 2.1 Hz, 1H, ArH),
8.46 (dd, J1 = 9 Hz, J2 = 2.1 Hz, 1H, ArH), 8.25 (d, J = 8.7 Hz, 2H,
ArH), 7.88–7.96 (m, 4H, ArH), 7.51 (s, 2H, ArH), 3.84 (s, 3H,
OCH3); HRMS (ESI): m/z, calcd for C23H17Cl2N6O5 [M+H+]:
527.0632, found 527.0625.
(ESI): m/z, calcd for
340.1044.
C
16H13N5O4 [M+H+]: 340.1040, found
5.1.13.2. Methyl 4-(2-(3-(3-fluorophenyl)ureido)-6-nitroquinaz-
olin-4-ylamino)benzoate (12a). The solution of compound 11a
(0.034 g, 0.1 mmol) and 3-fluorophenyl isocyanate (0.04 g,
0.3 mmol) in dioxane (1 mL) was heated by microwave (power
150 W, temperature 120 °C) for 15 min. After filtration, compound
12a (0.026 g, 54%) was obtained as yellow solid; mp 242–243 °C;
1H NMR (DMSO-d6) d (ppm): 12.05 (s, 1H, NH), 10.53 (s, 1H, NH),
10.39 (s, 1H, NH), 9.59 (d, J = 2.1 Hz, 1H, Ar), 8.44 (dd, J1 = 9 Hz,
J2 = 1.8 Hz, 1H, ArH), 8.27 (d, J = 8.7 Hz, 2H, ArH), 7.89–7.94 (m,
3H, ArH), 7.53 (d, J = 11.4 Hz, 1H, ArH), 7.26–7.33 (m, 2H, ArH),
6.86 (t, J = 8.4 Hz, 1H, ArH), 3.84 (s, 3H, OCH3); HRMS (ESI): m/z,
calcd for C23H18FN6O5 [M+H+]: 477.1317, found 477.1317.
5.1.15.2. 4-(2-(3-(3,4-Dichlorophenyl)ureido)-6-nitroquinazo-
lin-4-ylamino)benzoic acid (13c). The reaction mixture of com-
pound 12c (0.105 g, 0.2 mmol) and KOH (0.152 g, 2.72 mmol) in
methanol (2 mL) and water (1.5 mL) was refluxed for 50 min, and
then diluted with water (5 mL) and cooled with ice bath. The solu-
tion was adjusted to pH 2.0 with diluted HCl and stood for 1 h. The
crude product was obtained after filtration and recrystalized with
DMF/H2O to give compound 13c (0.095 g, 73%) as yellow solid;
mp >300 °C; 1H NMR (DMSO-d6) d (ppm): 12.12 (s, 0.5H, NH),
11.07 (s, 0.5H, NH), 10.66 (s, 0.5H, NH), 10.54 (s, 0.5H, NH), 9.67
(s, 0.5H, ArH), 9.61 (s, 0.5H, ArH), 8.51–8.58 (m, 1H, ArH), 8.26
(d, J = 8.1 Hz, 1H, ArH), 7.95–8.05 (m, 4H, ArH), 7.64 (d, J = 9.0 Hz,
1H, ArH), 7.33 (s, 2H, ArH); HRMS (ESI): m/z, calcd for
5.1.13.3. 4-(2-(3-(3-Fluorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13a). The reaction mixture of compound
12a (0.105 g, 0.22 mmol) and KOH (0.168 g, 2.99 mmol) in metha-
nol (2 mL) and water (1.5 mL) was refluxed for 30 min, and then di-
luted with water (5 mL) and cooled with ice bath. The solution was
adjusted to pH 2.0 with diluted HCl and stood for 2 h. The crude
product was obtained after filtration and recrystalized with DMF/
H2O to give compound 13a (0.075 g, 83%) as yellow solid; mp
>300 °C; 1H NMR (DMSO-d6) d (ppm): 12.12 (s, 1H, NH), 10.64 (s,
1H, NH), 10.47 (s, 1H, NH), 9.68 (s, 1H, ArH), 8.52 (d, J = 9.0 Hz,
1H, ArH), 8.27 (d, J = 8.4 Hz, 2H, ArH), 8.02 (s, 1H, ArH), 7.97 (d,
J = 8.7 Hz, 2H, ArH), 7.58 (d, J = 11.1 Hz, 1H, ArH), 7.32 (m, 2H,
ArH), 6.89 (m, 1H, ArH); HRMS (ESI): m/z, calcd for C22H16N6O5F
[M+H+]: 463.1160, found 463.1172.
C
22H15Cl2N6O5 [M+H+]: 513.0475, found 513.0466.
5.1.16. 4-(2-(3-(3-Trifluorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13d)
5.1.16.1. Methyl 4-(2-(3-(3-trifluorophenyl)ureido)-6-nitroqui-
nazolin-4-ylamino)benzoate (12d). The solution of compound
11a (0.034 g, 0.1 mmol) and 3-trifluorophenyl isocyanate
(0.056 g, 0.3 mmol) in dioxane (1 mL) was heated by microwave
(power 150 W, temperature 120 °C) for 10 min. After filtration,
the title compound 12d (0.037 g, 70%) was obtained as yellow so-
lid; mp 275–276 °C; 1H NMR (DMSO-d6) d ppm: 12.14 (s, 1H, NH),
10.55 (s, 1H, NH), 10.45 (s, 1H, NH), 9.60 (d, J = 2.1 Hz, 1H, ArH),
8.46 (dd, J1 = 9 Hz, J2 = 2.1 Hz, 1H, ArH), 8.26 (d, J = 8.7 Hz, 2H,
ArH), 8.10 (s, 1H, ArH), 7.92 (d, J = 8.7 Hz, 3H, ArH), 7.67 (d,
J = 8.7 Hz, 1H, ArH), 7.53 (t, J = 7.5 Hz, 1H, ArH), 7.38 (d,
J = 7.8 Hz, 1H, ArH), 3.84 (s, 3H, OCH3); HRMS (ESI): m/z, calcd for
5.1.14. 4-(2-(3-(4-Chlorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13b)
5.1.14.1. Methyl 4-(2-(3-(4-chlorophenyl)ureido)-6-nitroqui-
nazolin-4-ylamino)benzoate (12b). The solution of compound
11a (0.034 g, 0.1 mmol) and 4-chlorophenyl isocyanate (0.046 g,
0.3 mmol) in dioxane (1 mL) was heated by microwave (power
150 W, temperature 120 °C) for 15 min. After filtration, compound
12b (0.036 g, 73%) was obtained as yellow solid; mp 251–253 °C;
1H NMR (DMSO-d6) d (ppm): 12.04 (s, 1H, NH), 10.64 (s, 1H, NH),
10.45 (s, 1H, NH), 9.67 (s, 1H, ArH), 8.51 (d, J = 8.1 Hz, 1H, ArH),
8.32 (d, J = 8.1 Hz, 2H, ArH), 7.96 (d, J = 8.1 Hz, 3H, ArH), 7.63 (d,
J = 8.7 Hz, 2H, ArH), 7.36 (d, J = 7.8 Hz, 2H, ArH), 3.86 (s, 3H,
OCH3); HRMS (ESI): m/z, calcd for C23H18ClN6O5 [M+H+]:
493.1027, found 493.1022.
C
24H18F3N6O5 [M+H+]: 527.1285, found 527.1290.
5.1.16.2. 4-(2-(3-(3-Trifluorophenyl)ureido)-6-nitroquinazolin-
4-ylamino)benzoic acid (13d). The reaction mixture of com-
pound 12d (0.132 g, 0.25 mmol) and KOH (0.19 g, 3.4 mmol) in
methanol (2 mL) and water (1.5 mL) was refluxed for 30 min, and
then diluted with water (15 mL) and cooled with ice bath. The
solution was adjusted to pH 2.0 with diluted HCl and stood for
2.5 h. The crude product was obtained after filtration and recrystal-
ized with DMF/H2O to give compound 13d (0.064 g, 50%) as yellow
solid; mp >300 °C; 1H NMR (pyridine-d5) d (ppm): 12.65 (br s, 1H,
NH), 12.43 (s, 1H, NH), 11.65 (br s, 1H, NH), 9.53 (s, 1H, ArH), 8.57
(d, J = 8.7 Hz, 1H, ArH), 8.42 (d, J = 8.4 Hz, 2H, ArH), 8.05 (d,
J = 7.5 Hz, 2H, ArH), 7.93 (d, J = 9.3 Hz, 1H, ArH), 7.77 (m, 1H,
ArH), 7.48 (t, J = 7.5 Hz, 1H, ArH), 7.40 (d, J = 7.2 Hz, 1H, ArH);
HRMS (ESI): m/z, calcd for C23H16N6O5F3 [M+H+]: 513.1128, found
513.1130.
5.1.14.2. 4-(2-(3-(4-Chlorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13b). The reaction mixture of compound
12b (0.108 g, 0.22 mmol) and KOH (0.168 g, 2.99 mmol) in metha-
nol (2 mL) and water (1.5 mL) was refluxed for 4.5 h, and then di-
luted with water (5 mL) and cooled with ice bath. The solution was
adjusted to pH 2.0 with diluted HCl and stood for 1 h. The crude
product was obtained after filtration and recrystalized with DMF/
H2O to give compound 13b (0.095 g, 93%) as yellow solid; mp
>300 °C; 1H NMR (CD3OD-d4) d (ppm): 9.48 (d, J = 1.8 Hz, 1H,
ArH), 8.60 (dd, J1 = 6.6 Hz, J2 = 1.8 Hz, 1H, ArH), 8.07 (d, J = 6.6 Hz,
2H, ArH), 7.94 (d, J = 6.6 Hz, 2H, ArH), 7.92 (s, 1H, ArH), 7.58 (d,
J = 6.9 Hz, 1H, ArH), 7.36 (d, J = 6.6 Hz, 2H, ArH), 7.21 (d,

2806
L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
5.1.17. 4-(2-(3-(4-Trifluorophenyl)ureido)-6-nitroquinazolin-4-
ylamino)benzoic acid (13e)
0.1 mmol) and cyclohexyl isocyanate (0.038 g, 0.3 mmol) in diox-
ane (1 mL) was heated by microwave (power 150 W, temperature
120 °C) for 105 min. After filtration, compound 12g (0.035 g, 76%)
was obtained as yellow solid; mp 272–275 °C; 1H NMR (DMSO-
d6) d (ppm): 10.55 (s, 1H, NH), 9.94 (s, 1H, NH), 9.63 (d,
J = 2.4 Hz, 1H, ArH), 9.45 (d, J = 9.3 Hz,1H, NH), 8.50 (dd,
J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, ArH), 8.27 (d, J = 9.0 Hz, 2H, ArH), 7.99
(d, J = 8.7 Hz, 2H, ArH), 7.67 (d, J = 9.6 Hz, 1H, ArH), 3.85 (s, 3H,
OCH3), 3.65 (m, 1H, NCH), 1.82 (m, 2H, CH2), 1.65 (m, 2H, CH2),
1.22–1.57 (m, 6H, (CH2)3); HRMS (ESI): m/z, calcd for C23H25N6O5
[M+H+]: 465.1881, found 465.1877.
5.1.17.1. Methyl 4-(2-(3-(4-trifluorophenyl)ureido)-6-nitroqui-
nazolin-4-ylamino)benzoate (12e). The solution of compound
11a (0.034 g, 0.1 mmol) and 4-trifluorophenyl isocyanate
(0.056 g, 0.3 mmol) in dioxane (1 mL) was heated by microwave
(power 150 W, temperature 120 °C) for 15 min. After filtration,
compound 12e (0.03 g, 57%) was obtained as yellow solid; mp
242–243 °C; 1H NMR (DMSO-d6) d (ppm): 12.22 (s, 1H, NH),
10.60 (s, 1H, NH), 10.50 (s, 1H, NH), 9.62 (d, J = 2.1 Hz, 1H, ArH),
8.49 (dd, J1 = 9 Hz, J2 = 2.1 Hz, 1H, ArH), 8.28 (d, J = 9.0 Hz, 2H,
ArH), 7.91–7.96 (m, 3H, ArH), 7.75 (d, J = 8.7 Hz, 2H, ArH), 7.30
(d, J = 8.7 Hz, 2H, ArH), 3.84 (s, 3H, OCH3); HRMS (ESI): m/z, calcd
for C24H18F3N6O5 [M+H+]: 527.1285, found 527.1290.
5.1.19.2. 4-(2-(3-Cyclohexylureido)-6-nitroquinazolin-4-ylami-
no)benzoic acid (13g). The reaction mixture of compound 12g
(0.093 g, 0.2 mmol) and KOH (0.152 g, 2.72 mmol) in methanol
(2 mL) and water (1.5 mL) was refluxed for 60 min, and then di-
luted with water (10 mL) and cooled with ice bath. The solution
was adjusted to pH 2.5 with diluted HCl and extracted with EtOAc
(50 mL Â 3). The organic layer was concentrated to produce the
crude product, which was recrystalized with DMF/H2O to give
compound 13g (0.074 g, 82%) as yellow solid; mp >290 °C; 1H
NMR (DMSO-d6) d (ppm): 10.55 (s, 1H, NH), 9.93 (s, 1H, NH),
9.43 (s, 1H, ArH), 8.47 (d, J = 9.0 Hz, 1H, ArH), 8.20 (m, 2H, ArH),
7.94–7.98 (m, 3H, ArH), 7.67 (d, J = 9.0 Hz, 1H, ArH), 5.55 (m, 1H,
CONH), 3.60 (m, 1H, NCH), 1.01–1.80 (m, 10H, CH2); HRMS (ESI):
m/z, calcd for C22H23N6O5 [M+H+]: 451.1724, found 451.1711.
5.1.17.2. 4-(2-(3-(4-Trifluorophenyl)ureido)-6-nitroquinazolin-
4-ylamino)benzoic acid (13e). The reaction mixture of compound
12e (0.132 g, 0.25 mmol) and KOH (0.19 g, 3.4 mmol) in methanol
(2.5 mL) and water (2.0 mL) was refluxed for 2 h, and then diluted
with water (5 mL) and cooled with ice bath. The solution was ad-
justed to pH 2.0 with diluted HCl and stood for 2 h. The crude prod-
uct was obtained after filtration and recrystalized with DMF/H2O to
give compound 13e (0.107 g, 88%) as yellow solid; mp >250 °C; 1H
NMR (pyridine-d5) d (ppm): 12.63 (br s, 1H, NH), 12.41 (s, 1H,
NH), 11.70 (br s, 1H, NH), 9.55 (s, 1H, ArH), 8.57 (dd, J = 8.7 Hz,
1H, ArH), 8.42 (d, J = 8.4 Hz, 2H, ArH), 8.05 (d, J = 7.5 Hz, 2H, ArH),
7.93 (d, J1 = 6.6 Hz, J2 = 1.5 Hz, 1H, ArH), 8.43 (d, J = 6.3 Hz, 2H,
ArH), 8.04 (d, J = 6.3 Hz, 2H, ArH), 8.00 (m, 2H, ArH), 7.92 (d,
J = 6.9 Hz, 1H, ArH), 7.73 (d, J = 6.3 Hz, 2H, ArH); HRMS (ESI): m/z,
calcd for C23H16N6O5F3 [M+H+]: 513.1128, found 513.1122.
5.1.20. 3-(2-(3-(3,4-Dichlordrophenyl)ureido-6-
nitroquinazolin-4-ylamino)propanoic acid (13h)
5.1.20.1. Methyl 3-(2-amino-6-nitroquinazolin-4ylamino)pro-
panoate (11b). The reaction mixture of compound 4c (0.093 g,
0.3 mmol) and the newly prepared saturated NH3 ethanol solution
(4 mL) was irradiated by microwave (power 50 W, temperature
90 °C) for 3 h. Compound 11b (0.6 g, 69%) was obtained as yellow
solid after filtration and washed with ethanol. Mp 265–266 °C;
1H NMR (DMSO-d6) d (ppm): 9.05 (d, J = 2.1 Hz, 1H, ArNH), 8.54
(t, J = 4.8 Hz, 1H, NH), 8.19 (dd, J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, ArH),
7.20 (d, J = 9.6 Hz, 1H, ArH), 6.86 (br s, 2H, NH2), 3.68 (q,
J = 6.6 Hz, 2H, CH2), 3.61 (s, 3H, OCH3), 2.74 (t, J = 7.2 Hz, 2H, CH2).
5.1.18. 4-(6-Nitro-2-(3-phenethylureido)quinazolin-4-ylamino)
benzoic acid (13f)
5.1.18.1. Methyl 4-(6-nitro-2-(3-phenethylureido)quinazolin-4-
ylamino)benzoate (12f). The solution of compound 11a (0.034 g,
0.1 mmol) and phenethyl isocyanate (0.074 g, 0.5 mmol) in diox-
ane (1 mL) was heated by microwave (power 150 W, temperature
120 °C) for 15 min. After filtration, compound 12f (0.039 g, 80%)
was obtained as yellow solid; mp 275–277 °C; 1H NMR (DMSO-
d6) d (ppm): 10.51 (s, 1H, NH), 9.97 (s, 1H, NH), 9.59 (d,
J = 2.1 Hz, 1H, ArH), 9.44 (t, J = 6.0 Hz, 1H, NH) 8.46 (dd, J1 = 9 Hz,
J2 = 2.1 Hz, 1H, ArH), 8.27 (d, J = 8.4 Hz, 2H, ArH), 7.96 (d,
J = 8.7 Hz, 2H, ArH), 7.45 (d, J = 9.3 Hz, 1H, ArH), 7.23–7.34 (m,
5H, ArH), 3.84 (s, 3H, OCH3), 3.49–3.55 (m, 2H, CH2), 2.79–2.84
(t, J = 6.9 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for C25H23N6O5
[M+H+]: 487.1724, found 487.1729.
5.1.20.2. Methyl 3-(2-(3-(3,4-dichlordrophenyl)ureido-6-nitro-
quinazolin-4-ylamino)propanate (12h). The solution of com-
pound 11b (0.035 g, 0.12 mmol) and 3,4-dichlorophenyl
isocyanate (0.068 g, 0.36 mmol) in dioxane (1 mL) was heated by
microwave (power 150 W, temperature 120 °C) for 5 min. After fil-
tration, compound 12h (0.056 g, 97%) was obtained as yellow so-
lid; mp 259–261 °C; 1H NMR (DMSO-d6) d (ppm): 12.29 (s, 1H,
NH), 10.06 (s, 1H, NH), 9.22 (d, J = 2.4 Hz, 1H, ArH), 9.15 (t, 1H,
NH), 8.39 (dd, J1 = 9.3 Hz, J2 = 2.1 Hz, 1H, ArH), 8.06 (d, J = 1.8 Hz,
1H, ArH), 7.82 (d, J = 9.0 Hz, 1H, ArH), 7.54 (s, 2H, ArH), 3.73 (q,
J = 6.6 Hz, 2H, CH2), 3.62 (s, 3H, OCH3), 2.82 (t, J = 6.9 Hz, 2H,
CH2); HRMS (ESI): m/z, calcd for C19H17Cl2N6O5 [M+H+]:
479.0632, found 479.0634.
5.1.18.2. 4-(6-Nitro-2-(3-phenethylureido)quinazolin-4-ylami-
no)benzoic acid (13f). The reaction mixture of compound 12f
(0.107 g, 0.22 mmol) and KOH (0.168 g, 2.99 mmol) in methanol
(2 mL) and water (1.5 mL) was refluxed for 30 min, and then diluted
with water (10 mL) and cooled with ice bath. The solution was ad-
justed to pH 2.5 with diluted HCl and stood for 1 h. The crude prod-
uct was obtained after filtration and recrystalized with DMF/H2O to
give compound 13f (0.088 g, 85%) as yellow solid; mp >300 °C; 1H
NMR (DMSO-d6) d (ppm): 12.80 (br s, 1H, COOH), 10.50 (s, 1H,
NH), 9.97 (s, 1H, NH), 9.44 (br s, 1H, CONH), 8.46 (d, J = 9.3 Hz, 1H,
ArH), 8.22 (d, J = 9.0 Hz, 2H, ArH), 7.96 (d, J = 8.7 Hz, 2H, ArH), 7.46
(d, J = 9.0 Hz, 1H, ArH), 7.23–7.31 (m, 5H, ArH), 3.51–3.53 (m, 2H,
CH2), 2.84 (t, J = 7.2 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for
5.1.20.3. 3-(2-(3-(3,4-Dichlordrophenyl)ureido-6-nitroquinazo-
lin-4-ylamino)propanoic acid (13h). The reaction mixture of
compound 12h (0.048 g, 0.1 mmol) and KOH (0.076 g, 1.36 mmol)
in methanol (1 mL) and water (0.7 mL) was refluxed for 3.5 h, and
then diluted with water (5 mL) and cooled with ice bath. The solu-
tion was adjusted to pH 2.0 with diluted HCl and stood for 1 h. The
crude product was obtained after filtration and washed with water
to give compound 13h (0.043 g, 95%) as yellow solid; mp 240–
242 °C; 1H NMR (DMSO-d6) d (ppm): 12.22 (s, 1H, NH), 9.95 (s,
1H, NH), 9.28 (s, 1H, NH), 9.19 (d, J = 1.8 Hz, 1H, ArH), 8.30 (dd,
J1 = 9.0 Hz, J2 = 1.8 Hz, 1H, , ArH), 8.04 (s, 1H, ArH), 7.68 (d,
J = 9.3 Hz, 1H, ArH), 7.54 (s, 2H, ArH), 3.68 (m, 2H, CH2), 2.65 (t,
C
24H21N6O5 [M+H+]: 473.1567, found 473.1565.
5.1.19. 4-(2-(3-Cyclohexylureido)-6-nitroquinazolin-4-ylamino)
benzoic acid (13g)
5.1.19.1. Methyl 4-(2-(3-cyclohexylureido)-6-nitroquinazolin-4-
ylamino)benzoate (12g). The solution of compound 11a (0.034 g,

L. Zhu et al. / Bioorg. Med. Chem. 19 (2011) 2797–2807
2807
J = 6.6 Hz, 2H, CH2); HRMS (ESI): m/z, calcd for C18H15Cl2N6O5
synthesized title compounds were built using the Sybyl Sketcher
model and fully minimized with the Powell method (Tripos force
field and Gasteiger–Huckel charges) to an energy gradient of
0.05 kcal/(mol Å). The 30 final docked conformations were ranked
according to their binding free energy. The docking mode was cho-
sen on the basis of binding affinity rank.
[M+H+]: 465.0475, found 465.0478.
5.2. Biological evaluation
5.2.1. Protein expression and purification
The Pet28a-Pin1 plasmid was a gift from Professor Joseph P.
Noel (The Salk Institute for Biological Studies, La Jolla, California).
The N-terminally His6-tagged Pin1 was expressed at 22 °C in Esc-
heirchia coli strain BL21 following induction at an optical density
of 0.6 (600 nm) with 0.5 mM IPTG for 20 h in terrific broth. Cells
were resuspended in 25 mM Tris–Cl, 500 mM NaCl, 10 mM imidaz-
Acknowledgments
This work is supported by the National Natural Science Founda-
tion of China (No. 30500634), NSF of Beijing (No. 7102112) and Na-
tional Program of New Drug Innovation and Production (No.
2009ZX09301-003-1-1).
ole, 100 lg/mL cocktail, 0.5 mg/mL lysozyme. Following sonication
at 4 °C, the soluble supernatant was loaded onto an Ni-NTA (Qia-
gen) column and washed with 10 bed volumes of washing buffer
(50 mM imidazole, 500 mM NaCl, 20 mM Tris–Cl). His6-Pin1 was
eluted with 400 mM imidazole, 500 mM NaCl, 20 mM Tris–Cl and
condensed by ultrafiltration (Millipore 5 kDa) with 20 mM Tris–
Cl, 100 mM NaCl, 5 mM DTT.
References and notes
1. Lu, K. P.; Hanes, S. D.; Hunter, T. Nature 1996, 380, 544.
2. Yaffe, M. B.; Schutkowski, M.; Shen, M.; Zhou, X. Z.; Stukenberg, P. T.; Rahfeld, J.
U.; Xu, J.; Kuang, J.; Kirschner, M. W.; Fischer, G.; Cantley, L. C.; Lu, K. P. Science
1997, 278, 1957.
3. Shen, M.; Stukenberg, P. T.; Kirschner, M. W.; Lu, K. P. Genes Dev. 1998, 12, 706.
4. Lu, K. P.; Zhou, X. Z. Nat. Rev. Mol. Cell Biol. 2007, 8, 904.
5. Bao, L.; Kimzey, A.; Sauter, G.; Sowadski, J. M.; Lu, K. P.; Wang, D. G. Am. J.
Pathol. 2004, 164, 1727.
6. Ayala, G.; Wang, D. G.; Wulf, G.; Frolov, A.; Li, R.; Sowadski, J.; Wheeler, T. M.;
Lu, K. P.; Bao, L. Cancer Res. 2003, 63, 6244.
7. Rippmann, J. F.; Hobbie, S.; Daiber, C.; Guilliard, B.; Bauer, M.; Birk, J.; Nar, H.;
Garin-Chesa, P.; Pettig, W. J.; Schnapp, A. Cell Growth Differ. 2000, 11, 409.
8. Ryo, A.; Uemura, H.; Ishiguro, H.; Saitoh, T.; Yamaguchi, A.; Perrem, K.; Kubota,
Y.; Lu, K. P.; Aoki, I. Clin. Cancer Res. 2005, 11, 7523.
9. Zhang, C. J.; Zhang, Z. H.; Xu, B. L.; Wang, Y. L. Acta Pharm. Sinica 2008, 43, 9.
10. Xu, G. G.; Etzkorn, F. A. Drug News Perspect. 2009, 22, 399.
11. Mori, T.; Uchida, T. Curr. Enzyme Inhib. 2010, 6, 46.
12. Guo, C.; Hou, X.; Dong, L.; Dagostino, E.; Greasley, S.; Ferre, R.; Marakovits, J.;
Johnson, M. C.; Matthews, D.; Mroczkowski, B.; Parge, H.; VanArsdale, T.;
Popoff, I.; Piraino, J.; Margosiak, S.; Thomson, J.; Los, G.; Murry, B. W. Bioorg.
Med. Chem. Lett. 2009, 19, 5613.
13. Dong, L.; Marakovits, J.; Hou, X.; Guo, C.; Greasley, S.; Dagostino, E.; Ferre, R.;
Johnson, M. C.; Kraynov, E.; Thomson, J.; Pathak, V.; Murry, B. W. Bioorg. Med.
Chem. Lett. 2010, 20, 2210.
5.2.2. Pin1 PPIase assay and IC50 measurements of Pin1 inhibitors
PPIase activities were measured at 10 °C JASCO V-650 spectro-
photometer using protease-coupled assay according to Wang
et al.17,18 Suc-Ala-Glu-Pro-Phe-4-nitroanilide in 0.47 M LiCl/trifluo-
roethanol was used as the substrate. In brief, the assay buffer
(840
solution), and inhibitors (10
were preequilibrated in the cuvette at 10 °C for 30 min. Then,
100 L of ice-cooled chymotrypsin (60 mg/mL in 0.001 M HCl)
was added and mixed immediately. Additional 40 L of substrate
lL of 35 mM HEPES at PH 7.8), Pin1 (10
lL of 850 lg/mL stock
lL of varying concentrations in DMSO)
l
l
was added to start the assay and the reaction was monitored by
absorbance at 390 nM for 90 s. The data was analyzed by Graphpad
Prism 5.01. DMSO was used as solvent for compounds 6a–6h and
10a–10c, and pyridine for compounds 13a–13h.
14. Potter, A. J.; Ray, S.; Gueritz, L.; Nunns, C. L.; Bryant, C. J.; Scrace, S. F.;
Matassova, N.; Baker, L.; Dokurno, P.; Robinson, D. A.; Surgenor, A. E.; Davis, B.;
Murry, J. B.; Richardson, C. M.; Moore, J. D. Bioorg. Med. Chem. Lett. 2010, 20,
586.
15. Potter, A. J.; Oldfield, V.; Nunns, C.; Fromont, C.; Ray, S.; Northfield, C. J.; Bryant,
C. J.; Scrace, S. F.; Robinson, D.; Matossova, N.; Baker, L.; Dokurno, P.; Surgenor,
A. E.; Davis, B.; Richardson, C. M.; Murray, J. B.; Moore, J. D. Bioorg. Med. Chem.
Lett. 2010, 20, 6483.
5.3. Computational studies
All calculations and manipulations were performed using FlexX
software package integrated in SYBYL 7.219, running on SGI Fuel
workstation. The X-ray crystal structure of Pin1 complexed with
a
carboxylate inhibitor was retrieved from PDB (PDB code:
16. Ranganathan, R.; Lu, K. P.; Hunter, T.; Noel, J. P. Cell 1997, 89, 875.
17. Wang, X. J.; Xu, B.; Mullins, A. B.; Neiler, F. K.; Etzkorn, F. A. J. Am. Chem. Soc.
2004, 126, 15533.
3JYJ).13 In FlexX docking, the Receptor Description File (RDF) de-
scribed the active site environment. It contains the information
about the protein, its amino acids, the active site, non-amino acid
residues, and specific torsion angles. The active site was defined
as all residues within 6.5 Å radius of the cocrystallized reference
molecule. The default Sybyl/FlexX parameters were used. The
ˇ
18. Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colón-Bonilla, E.; Rich, D. H. Biochemistry
1991, 30, 6127.
19. SYBYL 7.2, Tripos Inc., 1699 South Hanley Road, St. Louis, MO 631444, USA.
20. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.;
Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.