Pyrrolopyrimidine-inhibitors with hydantoin moiety as spacer can explore P4/S4 interaction on plasmin

Article abstract of DOI:10.1016/j.bmc.2014.02.002

In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique feature of the new line of the plasmin inhibitors is

Full text of DOI:10.1016/j.bmc.2014.02.002

Bioorganic & Medicinal Chemistry 22 (2014) 2339–2352
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Pyrrolopyrimidine-inhibitors with hydantoin moiety as spacer can
explore P4/S4 interaction on plasmin
b
c
d
a
a
Naoki Teno a, , Keigo Gohda , Keiko Wanaka , Yuko Tsuda , Takuya Sueda , Yukiko Yamashita ,
⇑
Tadamune Otsubo a
a Hiroshima International University, Faculty of Pharmaceutical Sciences, 5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112, Japan
b Computer-Aided Molecular Modeling Research Center, Kansai (CAMM-Kansai), 5-1-7, Ohmichidori, Nagata-ku, Kobe 653-0833, Japan
c Kobe Research Projects on Thrombosis and Haemostasis, 3-15-18, Asahigaoka, Tarumi-ku, Kobe 655-0033, Japan
d Kobe Gakuin University, Faculty of Pharmaceutical Sciences, 1-1-3, Minatojima, Chuo-ku, Kobe 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two
motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique
feature of the new line of the plasmin inhibitors is that the interaction between the plasmin inhibitors
and key subsites in plasmin can be controlled by a spacer like hydantoin. The application of the novel
chemotype is demonstrated by 1n and provides further evidence on the importance of hydantoin as
the spacer.
Received 28 November 2013
Revised 30 January 2014
Accepted 3 February 2014
Available online 24 February 2014
Keywords:
Plasmin inhibitors
P4 moiety
Ó 2014 Elsevier Ltd. All rights reserved.
Scaffold
Pyrrolopyrimidine
Hydantoin
Structure–activity relationship
1. Introduction
in therapeutic strategies aimed at inhibiting tumor growth.4 Addi-
tionally, plasmin inhibitors, which have a different mode-of-action
Plasminogen is the main component of the fibrinolytic system
and can be converted to the active enzyme, plasmin, by distinct
plasminogen activators, which leads to fibrinolysis. Plasmin
directly degrades extracellular matrix components including the
glycoproteins fibronectin, laminin, and elastin, as well as proteo-
glycans.1 Failure of the extracellular matrix is crucial for cancer
invasion and metastasis. It is accomplished by the concerted action
of several proteases, including the serine protease plasmin and a
number of matrix metalloproteases (MMPs). The MMPs and plas-
minogen activator-plasmin systems have also been implicated in
experimental vascular tumor formation.2 The activity of each of
these proteases is regulated by an array of activators, inhibitors
and cellular receptors,3 resulting in serious diseases that arise from
an imbalance in physiological substances. Thus, antiproteolysis,
which is performed by plasmin inhibitors, has become a key target
from prior anti-cancer drugs, have become regarded as a potential
therapy for the treatment of cancer.4 However, the necessary
proof-of-concept validation of the therapeutic usefulness of plas-
min inhibitors has not been provided. This would be due to the lack
of bioavailable and low-molecular weight plasmin inhibitors.
The number of reports on plasmin inhibitors has recently in-
creased. The peptidic aldehyde has been recently utilized as an irre-
versible warhead of plasmin inhibitor consisting of a tetrapeptide.5
The S3 pocket of plasmin is electropositive due to the guanidino
group, Arg719, which donates a hydrogen bond to a P3 methionine
residue of the peptidic aldehyde inhibitors. These observations for
the P3/S3 interaction were in agreement with a previous report
published by Backes et al.6 As distinct types of plasmin inhibitors
were developed, it has been noted that a macrolide structure of
peptidomimetic plasmin inhibitors interacts with the S2 and S3
binding pockets of plasmin,7 however, they were not sufficiently
selective to avoid activity toward plasma kallikrein (PK) which
shares a high degree of sequence homology with plasmin. Our
efforts in this area have recently focused on the optimization of
peptidic and non-peptidic plasmin inhibitors having the nitrile
warhead in Lys (P1 moiety) since P1/S1 interaction primarily
Abbreviations: MMP, matrix metalloproteinase; PK, plasma kallikreine; UK, urokinase;
DABCO, 1,4-diazabicyclo[2.2.2]octane; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DKP,
diketopiperazine; <Glu, pyroglutamic acid.
⇑
Corresponding author. Tel.: +81 823 73 8942; fax: +81 823 73 8981.
E-mail address: n-teno@ps.hirokoku-u.ac.jp (N. Teno).
http://dx.doi.org/10.1016/j.bmc.2014.02.002
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
contributes to its affinity for plasmin.8,9 New non-peptidic P2 and
P3 moieties enhanced the inhibitory potency toward plasmin with
the selectivity over PK and urokinase (UK).8 Through these studies,
we are able to acquire more information on P2/S2 and P3/S3 inter-
actions from the investigation on inhibitors using a substrate li-
brary, while the plasmin inhibitors containing P4 moiety have not
been studied in this context other than recent aldehyde-containing
inhibitors.5 Since a few information on P4/S4 interaction between
plasmin and molecule and on how the interaction affects the po-
tency of plasmin inhibition and the selectivity against PK and UK
are available,5,6 we sought to exploit structure–activity relation-
ships (SAR) developed from studies using P4-containing inhibitors.
With regard to the novel type of cysteine proteases (cathepsins
K and S) inhibitors, heterocyclic core structures like purine and
pyrimidine were utilized as the scaffold in which the cathepsin K
and S inhibitors possessed a nitrile warhead for the P2 and P3
moieties.10–13 The structural features of the core structure gave
us a clue to the discovery of the novel chemotype for plasmin
inhibitors unlike previously published plasmin inhibitors. Since
the heterocyclic structure of pyrrolopyrimidine10–13 has three po-
tential attachment sites, we considered it as the prime scaffold
for plasmin inhibitors in order to exploit the SAR toward P4/S4
interaction (Fig. 1). Therefore, our strategy toward the SAR studies
on P4/S4 interaction for plasmin inhibitors included the assess-
ment of pyrrolopyrimidine as the scaffold that would allow the
attachment of P1 and P4 substituents.
Fmoc group of 6a–6t with 20% piperidine in DMF provided the de-
sired products 1a–t, respectively.
The derivatives having imidazolidinone (1u and 1v) or diketopi-
perazine (DKP) (1w) as a spacer were prepared in the same way as
for 1a as shown in Scheme 2.
Commercially available or synthetic aniline derivatives were
alkylated by ethyl bromoacetae in the presence of K2CO3 or diiso-
propyl ethylamine and subsequently the N-alkylated aniline deriv-
atives were treated with sodium cyanate in acetic acid15 to yield
hydantoin derivatives 2d–2f, 2h, 2i, 2k, 2l and 2n–2t. Commer-
cially available aniline derivatives were alkylated by 2-chloroethyl
isocyanate16 and formation of the imidazolidinone ring was carried
out by using sodium hydride to give 2u and 2v. The building block
with DKP (2w) was prepared based on the reference.17 Experimen-
tal procedures for the synthesis and characterization (1H NMR) of
2d–2f, 2h, 2i, 2k, 2l and 2n–2w are available in Supplementary
data.
We improved the method10,12,15 for preparing the building
block, methyl 6-(bromomethyl)-7-methyl-pyrrolo[2,3-d]pyrimi-
dine-2-carboxylate (3), that suits our goals depicted as in Scheme 3.
Regioselective substitution of the chloride at the 4-position of
5-bromo-2,4-dichloropyrimidine gave the intermediate 3a. The
second chloride was exchanged with cyanide to give the pyrimi-
dine 3b. The nitrile group of 3b was converted to methyl ester
(3c) using 5–10% HCl/MeOH. Bromide 3c was coupled with
2-prop-2-ynyloxy-tetrahydro-pyran18 by using Pd(PPh3)2Cl2 and
CuI as catalysts to yield 3d. Formation of the pyrrolopyrimidine
ring (3e) was carried out by using DBU, followed by removal of
tetrahydropyran using 6 M HCl and MeOH to give 3f. Alcohol 3f
was converted to the bromide 3.
2. Chemistry
The synthesis of plasmin inhibitors 1a–1t containing pyrrolo-
pyrimidine as the scaffold is illustrated in Scheme 1. The synthetic
approach allowed us to access compounds with structural diversity
by using an almost unlimited choice of commercially available, 2a
or synthetic hydantoin derivatives, 2b,14 2c,14 2d–2f, 2h, 2i, 2k, 2l
and 2n–2t. Condensation of 1-methylhydantoin (2a) with 3 was
performed by using sodium hydride to yield 4a. Other hydantoin
analogues, 2b–2f, 2h, 2i, 2k, 2l and 2n–2t, were coupled with the
common intermediate 3 in DMF in the presence of K2CO3 to afford
4b–4f, 4h, 4i, 4k, 4l and 4n–4t. Regardless of the structure of R (in
the case of 4f, 4h, 4i, 4k and 4q–4t) in Scheme 1, the hydrolysis of
methylester and hydantoin were detected simultaneously by
hydrolysis with LiOHꢀH2O. However, the product immediately
rearranged to form the hydantoin moiety by using 1,10-carbodiim-
idazole (CDI). In contrast, the methylesters of 4a–4e, 4l and 4n–4p
were readily hydrolyzed and their hydantoins were intact in the
same way for 4f, 4h, 4i, 4k and 4q–4t shown above. The hydro-
lyzed 4a–4f, 4h, 4i, 4k, 4l, 4n–4t were coupled with 5 to give the
corresponding 6a–6f, 6h, 6i, 6k, 6l and 6n–6t, respectively. Treat-
ment of 6f, 6i and 6l with trifluoroacetic anhydride and triethyl-
amine9 yielded the corresponding 6g, 6j and 6m. Removal of
Another building block with nitrile moiety as the warhead were
prepared from the starting material, H-Lys(Fmoc)-NH2 as shown in
Scheme 4. The N-terminal group was protected by trityl group (5a)
and subsequent treatment with trifluoroacetic anhydride and tri-
ethylamine9 yielded 5b. The acid labile group of 5b was removed
to give the building block 5 as the P1 moiety.
3. Results and discussion
The strategy that we pursued, schematically outlined in Figure 1,
was to find a heterocyclic scaffold which can combine the Lys-ni-
trile as the P1-warhead and additionally extend the P2, P3 or P4
group. We first focused on analyzing synthetically feasible hetero-
aromatic compounds combined with Lys-nitrile by using com-
puter-assisted molecular modeling to see how they are tolerated
in an active site of plasmin. Molecular modeling of pyrropyrimi-
dine-Lys-nitrile (7) into the active site of plasmin (Fig. 2) suggested
that pyrropyrimidine at the
a amino group of Lys would be direc-
ted toward non-prime sites other than the S1 pocket. Additionally,
the 6-position of pyrrolopyrimidine could be used to introduce the
Figure 1. Schematic outline for developing new chemotype of plasmin inhibitors.

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2341
Scheme 1. Reagents and conditions: (A) 60% NaH, DMF, rt, 15 h; (B) K2 CO3, DMF, rt, 5 h; (C) LiOH, H2O, THF/MeOH/H2O, rt, 3–15 h; (D) CDI, DMF, rt, 15 h; (E) H-Lys(Fmoc)-
CN(5), HOAt, WSCIꢀHCI, DMF, rt, 15 h; (F) (CF3CO)2O, Et3N, THF/DMF, 0 °C, 5–10 min; (G) 20% Piperidine/DMF, rt, 40 min; (H) Preparative HPLC.
substituents, which should extend into the non-prime subsites,
suggesting an application of pyrrolopyrimidine as the scaffold.
As a simple entry onto the pyrrolopyrimidine scaffold, we de-
signed 1a with hydantoin, which would enable to the introduction
of a wide range of substituents at 1-position of hydantoin. Pyrrol-
opyrimidine-based inhibitor 1a exhibited no inhibitory activity
against plasmin (Table 1) and molecular modeling of 1a (pink) sug-
gested that 1-methylhydantoin moiety on pyrrolopyrimidine di-
rected toward the S2 and S3 pockets (Fig. 3). Replacement of
methyl with 4-fluorobenzene (1b) showed an increased potency
against plasmin. The binding mode of 1b (green) complexed with
plasmin was shown to be different from 1a (pink) as depicted in
Figure 3. 4-Fluorobenzene linearly extended from hydantoin
pointed toward the S4 pocket unlike the 1-methylhydantoin moi-
ety in 1a but did not reach so deep into the S4 subsite. Indeed,
replacement of 4-fluorobenzene with 4-fluorobenzyl that en-
hanced free rotation fully lost the inhibitory activity against plas-
min (data not shown). Compound 1c inhibited plasmin at a
similar level to that of 1b while having the selectivity against PK
(1b vs 1c). A similar effect of the introduction of the aromatic moi-
ety on the potency and selectivity was also found in another deriv-
ative, 1d. As indicated in Figure 3, two Arg residues (Arg719 and
Arg767) are located close to the S4 subsite and their positive
charge could make a favorable electrostatic interaction with the
functional group in the P4 interacting region of 1e–1j (Table 2).
Compound 1i with the amide substituents at the meta-position
of the aromatic ring marginally increased their potency for plasmin
compared to 1b–1d yet retained the selectivity against PK and UK,
but other compounds 1e–1h and 1j caused no substantial changes
in the potency against a targeted enzyme. Extension by one meth-
ylene and oxygen groups onto the P4 moiety as in 1k–1l lost their
inhibitory activity against plasmin (Table 2). Conversion of amide
(1l) to nitrile (1m) resulted in the retention of potency to a similar
level to that of 1b.
In order to pursue the size of the S4 subsite, we focused on sub-
stituents at the 4-position of benzene extended from hydantoin. In
this context, 1n–1q were designed to elucidate whether the appro-
priate residue at the 4-position of the benzene was well tolerated
in the S4 subsite of plasmin as listed in Table 3. Inhibitors 1n
and 1o displayed potent plasmin inhibition while having a signifi-
cant selectivity for plasmin over PK and UK. Molecular modeling of
1n (yellow) suggested that an aromatic ring occupied the S4 pocket
in plasmin in Figure 4a. Replacement of the aromatic ring with a
picolyl group (1p) marginally decreased the potency for plasmin

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N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
Scheme 2. Reagents and conditions: (A) 60% NaH, DMF, rt, 15 h; (B) LiOHꢀH2O, THF/MeOH/H2O, rt, 3–15 h; (C) H-Lys(Fmoc)-CN(5), HOAt, WSCIꢀHCI, DMF, rt, 15 h; (D) 20%
Piperidine/DMF, rt, 40 min; (E) Preparative HPLC.
Scheme 3. Reagents and conditions: (A) Methylamine, MeOH, 0 °C, 20 min ? rt, 3 h; (B) NaCN, DABCO, DMSO, 60 °C, 2 h; (C) 5–10% HCI/MeOH; (D) 2-Prop-2-ynyloxy-
tetrahydro-pyran, Et3N, Cul, (Ph3P)2PdCl2, DMF, 80 °C, 4 h; (E) DBU, DMF, 100 °C, 2 h; (F) 6 M HCI, MeOH, rt, 3 h; (G) CBr4, Ph3P, CH2Cl2, 0 °C, 0.5 h ? rt, 15 h.
Scheme 4. Reagents and conditions: (A) Tr-Cl, Et3N, CH2Cl2, rt, 4 h; (B) (CF3CO)2O, Et3N, THF, 0 °C, 5 min; (C) CH3COOH, Trifluoroethanol, CH2Cl2, rt, 4 h.
compared to 1n but caused no substantial change in the selectivity
against untargeted enzymes. The introduction of quinoline (1q) re-
sulted in the loss of the inhibitory activity against plasmin. The
amide moiety was introduced as H-acceptor on the P4 moiety of
1n and 1p (1r–1t in Table 3). Inhibitors 1r having the amide group
on the external aromatic ring inhibited plasmin nearly equipotent

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2343
Figure 3. Compounds 1a (pink) and 1b (green) docked into the plasmin active site.
Table 2
Inhibition of plasmin, plasma kallikrein and urokinase by 1b and 1e–1m
Figure 2. Pyrrolopyrimidine core structure 7 (pink) docked into the plasmin active
site.
Table 1
Inhibition of plasmin, plasma kallikrein and urokinase by 1a–1d
Compounds
R
IC50 (lM)
Plasmin
Plasma
Urokinase
kallikrein
1b
1e
130
260
740
>1000
>400
>1000
Compounds
R
IC50 (lM)
Plasmin
>1000
Plasma kallikrein
>1000
Urokinase
>1000
1f
160
>1000
>1000
1a
1b
CH3
130
740
>1000
1g
1h
210
220
>1000
>1000
>1000
>1000
1c
180
330
>1000
>1000
>1000
>1000
1d
1i
1j
100
220
250
>1000
>1000
>1000
>1000
>1000
>1000
to 1p, but 1s and 1t showed a decreased trend for potency against
plasmin. Molecular modeling of 1n and 1s implied that the P4 moi-
eties of both compounds were directed toward the S4 pocket of
plasmin (see Supplementary data). However, a detailed breakdown
showed that the external aromatic rings of both compounds were
pointed in the different direction and the internal aromatic ring of
1s was slightly displaced from the surface of the S4 subsite com-
pared to that of 1n. The results on the P4 moieties suggest that
the S4 pocket in plasmin possesses a deep binding site but a fused
aromatic ring is not acceptable to the pocket (1n, 1o, 1p vs 1q).
1k
1l
400
130
>1000
>1000
>1000
>1000
1m

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N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
Table 3
Table 4
Inhibition of plasmin, plasma kallikrein and urokinase by 1c and 1n–1t
Inhibition of plasmin, plasma kallikrein and urokinase by 1b, 1n and 1u–1w
Compounds
R
IC50 (lM)
Compounds
R
IC50 (lM)
Plasmin Plasma Urokinase
kallikrein
Plasmin
Plasma
kallikrein
Urokinase
1b
1u
1n
1v
130
510
51
740
>1000
>1000
>400
1c
1n
1o
180
51
>1000
>1000
>1000
>1000
>400
>400
>1000
>1000
>1000
64
1p
1q
87
>1000
>1000
>1000
>400
>250
>200
>250
1w
960
>1000
>1000
1r
1s
1t
99
>1000
>1000
>1000
>1000
>1000
>1000
510
170
Derivatives with imidazolidinone (1u and 1v) or DKP (1w) as
the spacer were prepared to determine how the binding mode
and the inhibitory activity against plasmin change by the type of
spacer employed. Compounds 1u and 1v, which differ from 1b
and 1n only by the replacement of carbonyl with methylene, had
decreased potency against plasmin, respectively, as listed in Ta-
ble 4. Molecular modeling supported that 4-fluorobenzene in 1u
(pink) occupied the S2 and S3 pockets (Fig. 4b) unlike that in 1b
(sky blue). Likewise, as depicted in Figure 4a, phenoxybenzene
Given the results described above, it seems that the preferences in
the S4 subsite have no distinctive feature. However, Swedberg
et al. has disclosed that the S4 pocket tends to prefer a Lys residue
in the peptidic aldehyde inhibitors.5 On the basis of their report,
alternative P4 moieties combined with pyrrolopyrimidine scaffold
will be covered in future publications.
combined with hydantoin in 1n (yellow, IC50 = 51
pointed toward the S4 subsite whereas those in 1v (sky blue, IC50
>250 M for plasmin) and 1w (blue, IC50 = 960 M for plasmin) di-
rected toward other pockets, S2 and S3, suggesting that 1v and 1w
lM for plasmin)
l
l
Figure 4. (a) Compounds 1n (yellow), 1v (sky blue) and 1w (blue) docked into the plasmin active site. (b) Compounds 1b (sky blue) and 1u (pink) docked into the plasmin
active site.

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2345
did not much interact with them. The results reveal that phenoxy-
benzene in 1n is accepted by the S4 subsite in plasmin because
hydantoin directs phenoxybenzene to interact with the S4 pocket;
however, imidazolidinone and DKP in 1v and 1w do not allow for
the interaction between phenoxybenzene and the S4 subsite.
Appropriate moieties extending from the pyrrolopyrimidine
scaffold engaged with S4 and S1 pockets in the active site of plas-
min when having hydantoin as the spacer. The application of pyr-
rolopyrimidine scaffold as the novel plasmin inhibitors has been
demonstrated by the derivatives represented by 1n.
yield. Rf = 0.15 (n-hexane/EtOAc = 5:1). 1H NMR (600 MHz, CDCl3):
d 8.12 (s, 1H), 5.58 (br s, 1H), 3.10 (d, J = 4.8 Hz, 3H).
5.1.2. 5-Bromo-4-methylamino-pyrimidine-2-carbonitrile (3b)
At room temperature, to a solution of NaCN (3.3 g, 67.3 mmol)
in H2O (10 ml) was added successively DMSO (30 ml), DABCO
(1.2 g, 11.2 mmol), and a solution of 3a (10 g, 44.9 mmol) in DMSO
(50 ml). The mixture was stirred for 2 h at 60 °C, poured into an ice
water, and extracted with EtOAc. The combined extracts were
washed with brine, dried over MgSO4 and concentrated under re-
duced pressure. The residue was purified by silica gel column chro-
matography to give 7.0 g of 3b in 74% yield. Rf = 0.18 (n-hexane/
EtOAc = 3:1). 1H NMR (600 MHz, CDCl3): d 8.31 (s, 1H), 5.69 (br s,
1H), 3.11 (d, J = 4.8 Hz, 3H).
4. Conclusion
In the course of our exploration of the SAR studies on the P4/S4
interaction, we designed, synthesized and evaluated a new series
of plasmin inhibitors that have two motifs, the P1 and P4 moieties,
arranged in the prrrolopyrimidine scaffold. The representative
inhibitor 1n of this series revealed the application of pyrrolopyrim-
idine scaffold as the novel plasmin inhibitors. With respect to the
selectivity, it turned out that the structure of this series was not
totally tolerated for PK and UK. In addition, the interaction be-
tween plasmin inhibitors and the key pockets of plasmin could
be controlled by the type of modified heterocyclic moiety extend-
ing away from the pyrrolopyrimidine (Fig. 4a and b). In this way, a
novel structural class of plasmin inhibitors may provide a starting
point for the design of the potent inhibitors having S4/P4 interac-
tion. We anticipate that the SAR developed from our studies of the
new chemotypes described in our report will lead to further dis-
coveries of novel inhibitors for plasmin as well as other serine
proteases.
5.1.3. Methyl 5-bromo-4-methylamino-pyrimidine-2-
carboxylate (3c)
To 3b (7 g, 32.8 mmol) was added hydrochloric acid (5–10%
methanol solution, 70 ml) and the mixture was stirred for 15 h at
room temperature. The solvent was evaporated down and the res-
idue was extracted with EtOAc. The combined extracts were
washed with brine, dried over MgSO4 and concentrated under re-
duced pressure. The residue was purified by silica gel column chro-
matography to give 6.4 g of 3c in 80% yield. Rf = 0.15 (n-hexane/
EtOAc = 1:2). 1H NMR (600 MHz, CDCl3): d 8.40 (s, 1H), 5.61 (br s,
1H), 4.00 (s, 3H), 3.18 (d, J = 4.8 Hz, 3H).
5.1.4. Methyl 4-methylamino-5-(3-tetrahydropyran-2-
yloxyprop-1-ynyl)pyrimidine -2-carboxylate (3d)
At room temperature, a solution of 3c (6.4 g, 26.2 mmol) and
2-prop-2-ynyloxy-tetrahydro-pyran17 (5.5 g, 39.3 mmol) in DMF
(65 ml) was treated with triethylamine (11 ml, 78.5 mmol), CuI
(0.5 g, 2.6 mmol) and (Ph3P)2PdCl2 (0.9 g, 1.3 mmol). The mixture
was stirred for 4 h at 80 °C, poured into an ice water, and extracted
with EtOAc. The combined extracts were washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to give 6.7 g of
3d in 84% yield. Rf = 0.48 (n-hexane/EtOAc = 1:3). 1H NMR
(600 MHz, CDCl3): d 8.34 (s, 1H), 5.94 (br s, 1H), 4.87–4.86 (m,
1H), 4.55 (dd, J = 16.2 Hz, 7.2 Hz, 2H), 4.00 (s, 3H), 3.92–3.88 (m,
1H), 3.59–3.57 (m, 1H), 3.16 (d, J = 4.8 Hz, 3H), 1.86–1.77 (m,
3H), 1.69–1.58 (m, 3H).
5. Experimental
5.1. Materials and methods
All chemicals were purchased from Tokyo Chemical Industry
Co., Ltd or Wako Pure Chemical Industries, Ltd and used without
further purification. All protected amino acids and the coupling re-
agents were purchased from Watanabe Chemical Industries, Ltd.
1H NMR experiments were recorded on a JMTC-600 (JEOL Ltd)
600 NMR spectrometer with CDCl3, DMSO-d6 or D2O as solvents.
Chemical shifts are expressed in parts per million (ppm, d) and re-
ferred to the solvent signal. HRMS spectra were recorded on The
AccuTOF (JMS-T100LC) equipped with an electrospray ion source
(JEOL Ltd). The preparative HPLC system consisted of a SHINADZU
CBM-20A System Controller and a SHINADZU Pump Unit LC-20AT,
a SHIMADZU In-Line Degasser DGU-20A3R, a SHIMADZU SPD-20A
Absorbance Detector, a SHIMADZU FCV-11AL Valve Unit and a SHI-
MADZU FRC-10A Fraction Collector (SHIMADZU Corporation). The
absorbance detector was operated at 254 nm. The mobile phase
for preparation and analysis was a combinations of water (A) and
acetonitrile (B), both containing 0.1% TFA, and the flow rate was
5.1.5. Methyl 7-methyl-6-(tetrahydropyran-2-
yloxymethyl)pyrrolo[2,3-d]pyrimidine-2-carboxylate (3e)
At room temperature, a solution of 3d (6.74 g, 22 mmol) in DMF
(45 ml) was treated with DBU (1.1 ml, 7.5 mmol). The mixture was
stirred for 2 h at 100 °C, poured into an ice water, and extracted
with EtOAc. The combined extracts were washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to give 5.1 g of
3e in 76% yield. Rf = 0.12 (n-hexane/EtOAc = 3:1). 1H NMR
(600 MHz, CDCl3): d 9.05 (s, 1H), 6.65 (s, 1H), 4.96 (d, J = 13.2 Hz,
1H), 4.74–4.72 (m, 2H), 4.09 (s, 3H), 3.99 (s, 3H), 3.91–3.87 (m,
1H), 3.60–3.57 (m, 1H), 1.84–1.83 (m, 1H), 1.76–1.72 (m, 1H),
1.64–1.57 (m, 4H).
8
and 1 ml/min, respectively. TSK gel ODS columns
(21.5 ꢁ 300 mm for preparation and 4.6 ꢁ 150 mm for analysis,
TOSOH Corporation) were used.
5.1.1. 5-Bromo-2-chloro-N-methyl-pyrimidin-4-amine (3a)
Methylamine (9.8 M MeOH solution) (8.9 ml, 87.7 mmol) was
added dropwise at 0 °C over 20 min to a solution of 5-bromo-
2,4-dichloropyrimidine (10 g, 43.9 mmol) in MeOH (150 ml). After
stirring for 20 min at 0 °C, the mixture was warmed to room
temperature, stirred for 3 h, and evaporated down. The residue
was suspended in EtOAc, washed with satd NaHCO3 and brine,
dried with MgSO4, and evaporated down. The residue was chro-
matographed on silica gel column to give 10 g of 3a in quantitative
5.1.6. Methyl 6-(hydroxymethyl)-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (3f)
At room temperature, a solution of 3e (4.0 g, 13.1 mmol) in
MeOH (120 ml) was treated with 6 M HCl (4.4 ml, 26.2 mmol), stir-
red for 3 h at room temperature, treated with NaHCO3 (2.2 g,
26.2 mmol). The solvents were concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy to give 1.6 g of desired 3f in 55% yield. Rf = 0.60 (CH2Cl2/
MeOH = 9:1). 1H NMR (600 MHz, CDCl3): d 9.03 (s, 1H), 6.60 (s,

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N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
1H), 4.96 (d, J = 5.4 Hz, 2H), 4.09 (s, 3H), 4.01 (s, 3H), 1.97–1.96 (m,
NMR (600 MHz, DMSO-d6): d 9.09 (s, 1H), 7.85 (s, 4H), 7.35 (s,
1H).
2H), 6.82 (s, 1H), 5.03 (s, 2H), 4.65 (s, 2H), 3.94 (s, 3H), 3.92 (s, 3H).
5.1.7. Methyl 6-(bromomethyl)-7-methyl-pyrrolo[2,3-d]pyrim
idine-2-carboxylate (3)
5.1.13. Methyl 6-[[3-(4-carbamoylphenyl)-2,5-dioxo-
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4f)
Compound 4f was prepared in a manner similar to that de-
scribed for 4b in 81% yield. Rf = 0.74 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.08 (s, 1H), 7.97 (br s, 1H), 7.94 (d,
J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.35 (br s, 1H), 6.82 (s,
1H), 5.03 (s, 2H), 4.64 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H).
A solution of CBr4 (4.8 g, 14.5 mmol) in CH2Cl2 (10 ml) was
added dropwise over 15 min to a solution of 3f (1.6 g, 7.23 mmol)
and Ph3P (1.9 g, 7.23 mmol) in CH2Cl2 (50 ml) at 0 °C. After stirring
for 30 min at 0 °C, the mixture was warmed to room temperature,
stirred for 3 h. The mixture was diluted with CH2Cl2, washed with
satd NaHCO3 and brine, dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography to give 1.7 g of 3 in 90% yield. Rf = 0.55 (EtOAc).
1H NMR (600 MHz, CDCl3): d 9.07 (s, 1H), 6.71 (s, 1H), 4.68 (s,
2H), 4.09 (s, 3H), 4.02 (s, 3H).
5.1.14. Methyl 6-[[2,5-dioxo-3-(3-
sulfamoylphenyl)imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxylate (4h)
Compound 4h was prepared in a manner similar to that de-
scribed for 4b in 56% yield. Rf = 0.62 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.09 (s, 1H), 8.32 (s, 1H), 7.73–7.71
(m, 1H), 7.64–7.59 (m, 2H), 7.47 (s, 2H), 6.82 (s, 1H), 5.03 (s, 2H),
4.64 (s, 2H), 3.94 (s, 3H), 3.92 (s, 3H).
5.1.8. Methyl 7-methyl-6-[(3-methyl-2,5-dioxo-imidazolidin-1-
yl)methyl]pyrrolo[2,3-d]pyrimidine-2-carboxylate (4a)
To a solution of 1-methylhydantoin (80 mg, 0.70 mmol) in DMF
(3 ml), 60% NaH (30 mg, 0.84 mmol) was added at 0 °C and the
mixture was stirred at room temperature for 30 min. Compound
3 (100 mg, 0.35 mmol) in DMF (2 ml) was added at 0 °C and the
reaction mixture was stirred for 15 h at ambient temperature.
The reaction mixture was quenched with satd NH4Cl and extracted
with EtOAc. The combined extracts were washed with brine, dried
over MgSO4 and evaporated down. The residue was purified by sil-
ica gel column chromatography to give 67 mg of 4a in 30% yield.
Rf = 0.25 (EtOAc/MeOH = 95:5). 1H NMR (600 MHz, CDCl3): d 9.03
(s, 1H), 6.74 (s, 1H), 4.91 (s, 2H), 4.08 (s, 3H), 4.05 (s, 3H), 3.94
(s, 2H), 3.03 (s, 3H).
5.1.15. Methyl 6-[[3-(3-carbamoylphenyl)-2,5-dioxo-
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4i)
Compound 4i was prepared in a manner similar to that de-
scribed for 4b in 77% yield. Rf = 0.35 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.08 (s, 1H), 8.05 (br s, 2H), 7.91 (d,
J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.49–7.51 (m, 2H), 6.18 (s,
1H), 5.03 (s, 2H), 4.65 (s, 2H), 3.95 (s, 3H), 3.92(s, 3H).
5.1.16. Methyl 6-[[3-(4-acetonyloxyphenyl)-2,5-dioxo-
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4k)
Compound 4k was prepared in a manner similar to that de-
scribed for 4b in 69% yield. Rf = 0.75 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.08 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H),
6.96 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 5.00 (s, 2H), 4.83 (s, 2H), 4.58
(s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 2.16 (s, 3H).
5.1.9. Methyl 6-[[3-(4-fluorophenyl)-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxylate
(4b)
To a solution of 2b14 (68 mg, 0.35 mmol) in DMF (10 ml), K2CO3
(150 mg, 1.1 mmol) and 3 (100 mg, 0.35 mmol) were added at
ambient temperature. The reaction mixture was stirred for 15 h
at ambient temperature. The reaction solvent was evaporated
and the residue was extracted with EtOAc. The combined extracts
were washed with water and brine, dried over MgSO4 and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography to give 90 mg of 4b in 65% yield.
Rf = 0.48 (EtOAc), 1H NMR (600 MHz, CDCl3): d 9.08 (s, 1H), 7.70–
7.68 (m, 2H), 7.29 (t, J = 9.0 Hz, 2H), 6.79 (s, 1H), 5.01 (s, 2H),
4.61 (s, 2H), 3.94 (s, 3H), 3.92 (s, 3H).
5.1.17. Methyl 6-[[3-[4-(2-amino-2-oxo-ethoxy)phenyl]-2,5-
dioxo-imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (4l)
Compound 4l was prepared in a manner similar to that de-
scribed for 4b in 77% yield. Rf = 0.14 (EtOAc). 1H NMR (600 MHz,
DMSO-d6): d 9.08 (s, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.55 (br s, 1H),
7.43 (br s, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 5.00 (s, 2H),
4.58 (s, 2H), 4.43 (s, 2H), 3.94 (s, 3H), 3.92 (s, 3H).
5.1.10. Methyl 6-[[3-(4-methoxyphenyl)-2,5-dioxo-
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4c)
5.1.18. Methyl 6-[[2,5-dioxo-3-(4-phenoxyphenyl)imidazolidin-
1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxylate
(4n)
Compound 4n was prepared in a manner similar to that de-
scribed for 4b in 60% yield. Rf = 0.48 (EtOAc). 1H NMR (600 MHz,
CDCl3): d 9.05 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.8 Hz,
2H), 7.12 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 8.4 Hz, 2H), 6.99 (t,
J = 7.8 Hz, 2H), 6.81 (s, 1H), 5.02 (s, 2H), 4.37 (s, 2H), 4.09 (s, 3H),
4.08 (s, 3H).
Compound 4c was prepared from 2c14 and 3 in a manner similar
to that described for 4b in 74% yield. Rf = 0.15 (EtOAc). 1H NMR
(600 MHz, DMSO-d6): d 9.08 (s, 1H), 7.65–7.62 (m, 2H), 6.99 (d,
J = 9.0 Hz, 2H), 6.78 (s, 1H), 5.00 (s, 2H), 4.58 (s, 2H), 3.94 (s, 3H),
3.92 (s, 3H), 3.76 (s, 3H).
5.1.11. Methyl 6-[[2,5-dioxo-3-(4-propylphenyl)imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxylate
(4d)
Compound 4d was prepared in a manner similar to that de-
scribed for 4b in 81% yield (containing impurity). Rf = 0.72 (EtOAc).
5.1.19. Methyl 6-[[3-[4-(4-methoxyphenoxy)phenyl]-2,5-dioxo-
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4o)
5.1.12. Methyl 6-[[2,5-dioxo-3-(4-sulfamoylphenyl)imidazoli
din-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carboxylate (4e)
Compound 4o was prepared in a manner similar to that de-
scribed for 4b in 65% yield. Rf = 0.48 (EtOAc). 1H NMR (600 MHz,
DMSO-d6):
d 9.05 (s, 1H), 7.60 (d, J = 9.0 Hz, 2H), 6.97 (d,
Compound 4e was prepared in a manner similar to that de-
J = 8.4 Hz, 2H), 6.95–6.93 (m, 4H), 6.76 (s, 1H), 4.98 (s, 2H), 4.57
(s, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.73 (s, 3H).
scribed for 4b in 29% yield. Rf = 0.55 (EtOAc/MeOH = 9:1). 1H

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2347
5.1.20. Methyl 6-[[2,5-dioxo-3-[4-(4-
5.1.27. Methyl 6-[[2,5-dioxo-4-(4-phenoxyphenyl)piperazin-1-
pyridylmethoxy)phenyl]imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxylate (4p)
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxylate
(4w)
Compound 4p was prepared in a manner similar to that de-
scribed for 4b in 42% yield. Rf = 0.35 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.08 (s, 1H), 8.58 (d, J = 5.4 Hz, 2H),
7.58 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 5.4 Hz, 2H), 7.08 (d, J = 9.0 Hz,
2H), 6.78 (s, 1H), 5.21 (s, 2H), 5.00 (s, 2H), 4.58 (s, 2H), 3.93 (s,
3H), 3.92 (s, 3H).
Compound 4w was prepared in a manner similar to that de-
scribed for 4a in 67% yield. Rf = 0.52 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.13 (s, 1H), 7.43 (t, J = 7.8 Hz, 2H),
7.39 (d, J = 9.0 Hz, 2H), 7.20–7.19 (m, 1H), 7.07–7.05 (m, 4H),
6.87 (s, 1H), 4.97 (s, 2H), 4.44 (s, 2H), 4.14 (s, 2H), 3.93 (s, 3H),
3.85 (s, 3H).
5.1.28. 9H-Fluoren-9-ylmethyl N-[(5S)-6-amino-6-oxo-5-
(tritylamino)hexyl]carbamate (5a)
5.1.21. Methyl 6-[[2,5-dioxo-3-[4-(6-quinolyloxy)phenyl]
imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-
2-carboxylate (4q)
Compound 4q was prepared in a manner similar to that de-
scribed for 4b in 82% yield. Rf=0.58 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.09 (s, 1H), 8.83 (d, J = 3.0 Hz, 1H), 8.27
(d, J = 8.4 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H),
7.55 (dd, J = 3.0, 6.0 Hz, 1H), 7.51 (dd, J = 4.2 Hz, 1H), 7.35 (d,
J = 3.0 Hz, 1H), 7.25 (d, J = 9.6 Hz, 2H), 6.80 (s, 1H), 5.03 (s, 2H),
4.65 (s, 2H), 3.95 (s, 3H), 3.93 (s, 3H).
H-Lys(Fmoc)-NH2ꢀHCl (2.41 g, 5.97 mmol) was suspended in
CH2Cl2 (60 ml), and triethylamine (1.75 ml, 12.5 mmol) and trityl
chloride (1.83 g, 6.57 mmol) were added under ice cold conditions.
The mixture was stirred at room temperature for 6 h. The reaction
mixture was quenched with saturated ammonium chloride and ex-
tracted with ethyl acetate. The combined extracts were washed
with water and brine, dried over magnesium sulfate and concen-
trated under reduced pressure to give 3.9 g of crude 5a (containing
impurity) in 98% yield. Rf = 0.21 (n-hexane/EtOAc = 1:2).
5.1.22. Methyl 6-[[3-[4-(2-carbamoylphenoxy)phenyl]-2,5-
dioxo-imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (4r)
Compound 4r was prepared in a manner similar to that de-
scribed for 4b in 48% yield. Rf=0.65 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.08 (s, 1H), 7.72 (d, J = 6.0 Hz, 1H), 7.68
(d, J = 9.0 Hz, 2H), 7.67 (br s, 1H), 7.57 (br s, 1H), 7.45 (t,
J = 6.6 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 9.0 Hz, 2H), 6.84
(d, J = 8.4 Hz, 1H), 6.79 (s, 1H), 5.02 (s, 2H), 4.62 (s, 2H), 3.94 (s,
3H), 3.92 (s, 3H).
5.1.29. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-
(tritylamino)pentyl]carbamate (5b)
Compound 5a (3.9 g, 6.41 mmol) was dissolved in THF (80 ml),
and triethylamine (3.6 ml, 25.6 mmol) and trifluoroacetic anhy-
dride (1.3 ml, 9.6 mmol) were added under ice cold conditions.
The mixture was stirred under the same conditions for 5 min.
The reaction mixture was quenched with saturated ammonium
chloride and extracted with ethyl acetate. The combined extracts
were washed with water and brine, dried over magnesium sulfate
and concentrated under reduced pressure to give 3.7 g of desired
5b in 98% yield. Rf = 0.85 (n-hexane/EtOAc = 1:1). 1H NMR
(600 MHz, DMSO-d6): d 7.91–7.89 (m, 2H), 7.69–7.68 (m, 2H),
7.47–7.41 (m, 8H), 7.38–7.21 (m, 13H), 4.31 (d, J = 4.2 Hz, 2H),
4.22 (t, J = 6.6 Hz, 1H), 3.29–3.25 (m, 1H), 2.93 (q, J = 6.0 Hz, 2H),
1.69–1.27 (m, 6H).
5.1.23. Methyl 6-[[3-(3-carbamoyl-4-phenoxy-phenyl)-2,5-
dioxo-imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (4s)
Compound 4s was prepared in a manner similar to that de-
scribed for 4b in 74% yield. Rf=0.80 (EtOAc/MeOH = 8:2). 1H NMR
(600 MHz, DMSO-d6): d 9.09 (s, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.71
(dd, J = 9.0, 3.0 Hz, 1H), 7.69 (br s, 1H), 7.66 (br s, 1H), 7.40 (t,
J = 7.8 Hz, 2H), 7.15 (t, J = 7.8 Hz, 1H), 7.01 (t, J = 7.8 Hz, 3H), 6.80
(s, 1H), 5.02 (s, 2H), 4.64 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H).
5.1.30. 9H-Fluoren-9-ylmethyl N-[(5S)-5-amino-5-cyano-
pentyl]carbamate (5)
Compound 5b (3.7 g, 6.29 mmol) was dissolved in CH2Cl2
(60 ml), and trifluoroehanol (15 ml) and acetic acid (15 ml) were
added at room temperature. The mixture was stirred under the
same conditions for 4 h. The mixture was evaporated and the res-
idue was purified by silica gel column chromatography to give
1.9 g of desired 5 in 87% yield. Rf = 0.61 (CH2Cl2/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 7.91 (d, J = 7.2 Hz, 2H), 7.70 (d,
J = 7.2 Hz, 2H), 7.43 (t, J = 6.6 Hz, 2H), 7.36–7.28 (m, 4H), 7.23–
7.20 (m, 1H), 4.31 (d, J = 6.6 Hz, 2H), 4.23 (t, J = 7.2 Hz, 1H), 3.66
(t, J = 7.2 Hz, 1H), 3.00–2.98 (m, 2H), 1.62–1.58 (m, 2H), 1.42–
1.39 (m, 4H).
5.1.24. Methyl 6-[[3-[3-carbamoyl-4-(4-pyridylmethoxy)phenyl]
-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (4t)
Compound 4t was prepared in a manner similar to that de-
scribed for 4b in 70% yield. Rf=0.52 (CH2Cl2/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.08 (s, 1H), 8.60 (d, J = 6.0 Hz, 2H), 8.04
(d, J = 3.0 Hz, 1H), 7.76 (br s, 1H), 7.69 (br s, 1H), 7.64–7.62 (m,
1H), 7.48 (d, J = 5.4 Hz, 2H), 7.19 (d, J = 9.0 Hz, 1H), 6.78 (s, 1H),
5.35 (s, 2H), 5.00 (s, 2H), 4.60 (s, 2H), 3.94 (s, 3H), 3.92 (s, 3H).
5.1.31. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[7-methyl-6-
[(3-methyl-2,5-dioxo-imidazolidin-1-yl)methyl]pyrrolo[2,3-
d]pyrimidine-2-carbonyl]amino]pentyl]carbamate (6a)
5.1.25. Methyl 6-[[3-(4-fluorophenyl)-2-oxo-imidazolidin-1-yl]
methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxylate (4u)
Compound 4u was prepared in a manner similar to that de-
scribed for 4a in 25% yield. Rf = 0.30 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.07 (s, 1H), 7.61 (q, J = 4.8 Hz, 2H),
7.18 (d, J = 9.0 Hz, 2H), 6.75 (s, 1H), 4.72 (s, 2H), 3.89 (s, 3H),
3.84–3.82 (m, 5H), 3.42 (t, J = 8.4 Hz, 2H).
To a solution of 4a (67 mg, 0.21 mmol) in MeOH/THF/H2O
(2 ml/2 ml/2 ml), LiOHꢀH2O (13 mg, 0.32 mmol) was added at room
temperature and the mixture was stirred at room temperature for
3 h. The reaction mixture was neutralized with 1 M HCl and evap-
orated down. The residue in DMF (2 ml) was added to a solution of
3 (100 mg, 0.25 mmol) and triethylamine (35 ml, 0.25 mmol) in
DMF (3 ml) at 0 °C. Subsequently, HOAt (55 mg, 0.42 mmol) and
WSCIꢀHCl (80 mg, 0.42 mmol) were added to the former solution
at 0 °C. The reaction mixture was evaporated down and H2O was
added to the residue to afford powder. The residue was purified
by silica gel column chromatography to give 61 mg of desired 6a
5.1.26. Methyl 7-methyl-6-[[2-oxo-3-(4-
phenoxyphenyl)imidazolidin-1-yl]methyl]pyrrolo[2,3-
d]pyrimidine-2-carboxylate (4v)
Compound 4v was prepared in a manner similar to that de-
scribed for 4a in 31% yield (containing impurity). Rf = 0.15 (EtOAc).

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N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
in 45% yield. Rf = 0.18 (EtOAc/MeOH = 95:5). 1H NMR (600 MHz,
DMSO-d6): 9.58 (d, J = 7.8 Hz, 1H), 9.04 (s, 1H), 7.88 (d,
J = 7.2 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H),
7.33–7.31 (m, 3H), 6.64 (s, 1H), 4.97 (q, J = 7.8 Hz, 1H), 4.90 (s,
2H), 4.30–4.28 (m, 2H), 4.21–4.18 (m, 1H), 4.09 (s, 2H), 3.93 (s,
3H), 3.00–2.99 (m, 2H), 2.91 (s, 3H), 2.01–1.97 (m, 2H), 1.46–1.39
(m, 4H).
perature and the mixture was stirred at room temperature for 3 h.
The reaction mixture was neutralized with 1 M HCl and evaporated
down. The reaction mixture was evaporated down and the residue
d
was dissolved in DMF (20 ml). To
a solution, CDI (55 mg,
0.34 mmol) was added at room temperature and stirred at ambient
temperature for 15 h. The reaction mixture was evaporated down.
The residue in DMF (2 ml) was added to a solution of 3 (100 mg,
0.24 mmol) and triethylamine (34 ll, 0.24 mmol) in DMF (3 ml)
5.1.32. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-(4-
fluorophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6b)
at 0 °C. Subsequently, HOAt (65 mg, 0.48 mmol) and WSCIꢀHCl
(92 mg, 0.48 mmol) were added to the former solution at 0 °C.
The mixture was stirred at room temperature for 15 h. The reaction
mixture was evaporated down and H2O was added to the residue
to afford powder. The residue was purified by silica gel column
chromatography to give 53 mg of desired 6f in 41% yield.
Rf = 0.18 (EtOAc/MeOH = 9:1). 1H NMR (600 MHz, DMSO-d6): d
9.60 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.97–7.94 (m, 3H), 7.88 (d,
J = 7.8 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H),
7.41 (t, J = 7.8 Hz, 2H), 7.35–7.32 (m, 4H), 6.79 (s, 1H), 5.02 (s,
2H), 4.99–4.97 (m, 1H), 4.65 (s, 2H), 4.30–4.27 (m, 2H), 4.21–
4.19 (m, 1H), 3.96 (s, 3H), 3.06–2.97 (m, 2H), 2.01–1.98 (m, 2H),
1.46–1.39 (m, 4H).
Compound 6b was prepared in a manner similar to that de-
scribed for 6a in 87% yield. Rf = 0.48 (EtOAc). 1H NMR (600 MHz,
DMSO-d6):
d 9.58 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.88 (d,
J = 7.8 Hz, 2H), 7.71–7.68 (m, 4H), 7.41 (t, J = 7.2 Hz, 2H), 7.33–
7.27 (m, 5H), 6.77 (s, 1H), 5.01 (s, 2H), 4.97 (q, J = 7.2 Hz, 1H),
4.62 (s, 2H), 4.30–4.28 (m, 2H), 4.20–4.19 (m, 1H), 3.96 (s, 3H),
3.00–2.99 (m, 2H), 2.00–1.98 (m, 2H), 1.46–1.39 (m, 4H).
5.1.33. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-(4-
methoxyphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-
5.1.37. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-(4-
cyanophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6g)
carbonyl]amino]pentyl]carbamate (6c)
Compound 6c was prepared in a manner similar to that de-
scribed for 6a in 73% yield. Rf = 0.81 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.58 (d, J = 7.8 Hz, 1H), 9.05 (s, 1H), 7.97
(s, 1H), 7.87 (d, J = 7.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.56 (d,
J = 8.4 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.34–7.31 (m, 2H), 7.01 (d,
J = 9.0 Hz, 2H), 6.76 (s, 1H), 5.00 (s, 2H), 4.99–4.97 (m, 1H), 4.59
(s, 2H), 4.31–4.27 (m, 2H), 4.23–4.18 (m, 1H), 3.96 (s, 3H), 3.76 (s,
3H), 3.02–2.97 (m, 2H), 2.02–2.00 (m, 2H), 1.49–1.40 (m, 4H).
Compound 6f (18 mg, 0.024 mmol) was dissolved in THF/DMF
(4 ml/0.5 ml), and triethylamine (14
ll, 0.097 mmol) and trifluoro-
acetic anhydride (5 l, 0.037 mmol) were added under ice cold
l
conditions. The mixture was stirred under the same conditions
for 5 min. The reaction mixture was quenched with satd NH4Cl
and extracted with EtOAc. The combined extracts were washed
with water and brine, dried over magnesium sulfate and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography to give 14 mg of desired 6g in 82%
yield. Rf = 0.23 (EtOAc), 1H NMR (600 MHz, DMSO-d6): d 9.58 (d,
J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.92–7.86 (m, 7H), 7.66 (d, J = 7.8 Hz,
2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33–7.30 (m, 2H), 6.80 (s, 1H), 5.03
(s, 2H), 4.99–4.97 (m, 1H), 4.66 (s, 2H), 4.30–4.27 (m, 2H), 4.21–
4.19 (m, 1H), 3.96 (s, 3H), 3.06–2.97 (m, 2H), 2.01–1.98 (m, 2H),
1.46–1.39 (m, 4H).
5.1.34. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-(4-propylphenyl)imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6d)
Compound 6d was prepared in a manner similar to that de-
scribed for 6a in 73% yield. Rf = 0.65 (EtOAc). 1H NMR (600 MHz,
DMSO-d6):
d 9.58 (d, J = 8.4 Hz, 1H), 9.04 (s, 1H), 7.88 (d,
J = 7.2 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H),
7.41 (t, J = 6.6 Hz, 2H), 7.33–7.32 (m, 3H), 7.23 (d, J = 7.8 Hz, 2H),
6.76 (s, 1H), 5.00 (s, 2H), 4.99–4.97 (m, 1H), 4.60 (s, 2H), 4.31–
4.27 (m, 2H), 4.23–4.19 (m, 1H), 3.96 (s, 3H), 3.02–2.97 (m, 2H),
2.62–2.59 (m, 2H), 2.00–1.98 (m, 2H), 1.58 (q, J = 7.2 Hz, 2H),
1.46–1.39 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H).
5.1.38. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-(3-sulfamoylphenyl)imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6h)
Compound 6h was prepared in a manner similar to that de-
scribed for 6f in 59% yield. Rf = 0.67 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.58 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 8.32
(s, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.72–7.61 (m, 5H), 7.47 (s, 2H),
7.42 (m, 6H), 6.80 (s, 1H), 5.03 (s, 2H), 4.98–4.95 (m, 1H), 4.65 (s,
2H), 4.30–4.27 (m, 2H), 4.21–4.19 (m, 1H), 3.97 (s, 3H), 3.03–2.99
(m, 2H), 2.01–1.98 (m, 2H), 1.46–1.41 (m, 4H).
5.1.35. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-(4-sulfamoylphenyl)imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6e)
Compound 6e was prepared in a manner similar to that de-
scribed for 6a in 31% yield. Rf = 0.75 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.58 (d, J = 9.0 Hz, 1H), 9.05 (s, 1H),
7.88 (d, J = 7.2 Hz, 2H), 7.85 (s, 4H), 7.67 (d, J = 7.2 Hz, 2H), 7.41
(t, J = 7.8 Hz, 2H), 7.35–7.32 (m, 4H), 6.80 (s, 1H), 5.03 (s, 2H),
4.99–4.97 (m, 1H), 4.66 (s, 2H), 4.30–4.27 (m, 2H), 4.21–4.19 (m,
1H), 3.96 (s, 3H), 3.06–2.97 (m, 2H), 2.01–1.98 (m, 2H), 1.46–1.38
(m, 4H).
5.1.39. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-(3-
carbamoylphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]-5-cyano-
pentyl]carbamate (6i)
Compound 6i was prepared in a manner similar to that de-
scribed for 6f in 60% yield. Rf = 0.40 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.59 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 8.05
(br s, 2H), 7.88–7.92 (m, 3H), 7.66–7.68 (m, 3H), 7.49–7.52 (m,
2H), 7.39–7.42 (m, 2H), 7.32 (br s, 3H), 6.79 (s, 1H), 5.02 (s, 2H),
4.98 (br s, 1H), 4.66 (s, 2H), 4.29 (br s, 2H), 4.19 (br s, 1H), 3.97
(s, 3H), 3.00 (d, J = 6.0 Hz, 2H), 1.98 (m, 2H), 1.40–1.46 (m, 4H).
5.1.36. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-(4-
carbamoylphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]-5-cyano-
pentyl]carbamate (6f)
To a solution of 4f (72 mg, 0.17 mmol) in MeOH/THF/H2O (4 ml/
5 ml/4 ml), LiOHꢀH2O (8.6 mg, 0.26 mmol) was added at room tem-

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2349
5.1.40. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-(3-
5.1.46. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-[4-(4-pyridylmethoxy)phenyl]imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6p)
cyanophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6j)
Conversion of 6i–6j was performed in a manner similar to that
described for 6g in 79% yield. Rf = 0.30 (EtOAc). 1H NMR (600 MHz,
DMSO-d6): d 9.59 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 8.08 (br s, 2H),
7.88 (d, J = 7.8 Hz, 2H), 7.64–7.68 (m, 4H), 7.41 (t, J = 7.8 Hz, 2H),
7.32 (m, 3H), 6.80 (s, 1H), 5.02 (s, 2H), 4.98 (br s, 1H), 4.65 (s,
2H), 4.29 (br s, 2H), 4.19 (br s, 1H), 3.96 (s, 3H), 3.00 (m, 2H),
2.00 (m, 2H), 1.40–1.46 (m, 4H).
Compound 6p was prepared in a manner similar to that de-
scribed for 6a in 41% yield. Rf = 0.60 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.59 (d, J = 8.4 Hz, 1H), 9.04 (s, 1H),
8.58 (d, J = 4.8 Hz, 2H), 7.88 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 7.8 Hz,
2H), 7.58 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 4.8 Hz, 2H), 7.41 (t,
J = 7.8 Hz, 2H), 7.34–7.30 (m, 3H), 7.09 (d, J = 4.8 Hz, 2H), 6.75 (s,
1H), 5.21 (s, 2H), 5.00 (s, 2H), 4.98–4.96 (m, 1H), 4.59 (s, 2H),
4.29–4.27 (m, 2H), 4.20–4.18 (m, 1H), 3.96 (s, 3H), 3.00–2.99 (m,
2H), 2.01–1.98 (m, 2H), 1.46–1.40 (m, 4H).
5.1.41. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-(4-
acetonyloxyphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]-5-cyano-
pentyl]carbamate (6k)
Compound 6h was prepared in a manner similar to that de-
scribed for 6f in 17% yield (containing impurity). Rf = 0.70
(EtOAc/MeOH = 9:1).
5.1.47. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-[4-(6-quinolyloxy)phenyl]imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6q)
Compound 6q was prepared in a manner similar to that de-
scribed for 6f in 41% yield. Rf = 0.75 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.58 (d, J = 7.8 Hz, 1H), 9.06 (s, 1H), 8.84–
8.83 (m, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.88
(d, J = 9.0 Hz, 2H), 7.75 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H),
7.55 (dd, J = 3.0 Hz, 6.0 Hz, 1H), 7.52–7.50 (m, 1H), 7.41 (t,
J = 6.6 Hz, 2H), 7.36–7.31 (m, 4H), 7.25 (d, J = 9.0 Hz, 2H), 6.73 (s,
1H), 5.03 (s, 2H), 4.98–4.96 (m, 1H), 4.66 (s, 2H), 4.30–4.28 (m,
2H), 4.21–4.19 (m, 1H), 3.97 (s, 3H), 3.00–2.99 (m, 2H), 2.01–1.98
(m, 2H), 1.46–1.40 (m, 4H).
5.1.42. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-[4-(2-amino-2-
oxo-ethoxy)phenyl]-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]-5-cyano-
pentyl]carbamate (6l)
Compound 6l was prepared in a manner similar to that de-
scribed for 6a in 52% yield. Rf = 0.48 (CH2Cl2/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.58 (d, J = 7.8 Hz, 1H), 9.04 (s, 1H),
7.88 (d, J = 7.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.58–7.55 (m, 3H),
7.42–7.39 (m, 3H), 7.33–7.31 (m, 3H), 7.01 (d, J = 9.6 Hz, 2H),
6.76 (s, 1H), 5.00 (s, 2H), 4.98–4.95 (m, 1H), 4.59 (s, 2H), 4.43 (s,
2H), 4.30–4.28 (m, 2H), 4.20–4.19 (m, 1H), 3.96 (s, 3H), 3.00–2.99
(m, 2H), 2.01–1.96 (m, 2H), 1.46–1.40 (m, 4H).
5.1.48. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-[4-(2-
carbamoylphenoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]-5-cyano-pentyl]carbamate (6r)
Compound 6r was prepared in a manner similar to that de-
scribed for 6f in 88% yield. Rf = 0.75 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.58 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.88
(d, J = 7.8 Hz, 2H), 7.73–7.66 (m, 6H), 7.46–7.39 (m, 3H), 7.33–
7.30 (m, 4H), 7.21 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H), 6.84
(d, J = 7.8 Hz, 1H), 6.77 (s, 1H), 5.01 (s, 2H), 4.98–4.96 (m, 1H),
4.63 (s, 2H), 4.30–4.28 (m, 2H), 4.21–4.19 (m, 1H), 3.96 (s, 3H),
3.00–2.99 (m, 2H), 2.01–1.98 (m, 2H), 1.46–1.40 (m, 4H).
5.1.43. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-[4-
(cyanomethoxy)phenyl]-2,5-dioxo-imidazolidin-1-yl]methyl]-
7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6m)
Conversion of 6l–6m was performed in a manner similar to that
described for 6g in 83% yield. Rf = 0.50 (EtOAc). 1H NMR (600 MHz,
DMSO-d6): d 9.58 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.97 (s, 1H), 7.88
(d, J = 7.8 Hz, 2H), 7.68–7.65 (m, 3H), 7.41 (t, J = 7.2 Hz, 2H), 7.33–
7.31 (m, 3H), 7.14 (d, J = 9.6 Hz, 2H), 6.76 (s, 1H), 5.19 (s, 2H), 5.00
(s, 2H), 4.98–4.95 (m, 1H), 4.61 (s, 2H), 4.30–4.28 (m, 2H), 4.20–
4.18 (m, 1H), 3.96 (s, 3H), 3.00–2.99 (m, 2H), 2.01–1.96 (m, 2H),
1.46–1.41 (m, 4H).
5.1.49. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-(3-carbamoyl-
4-phenoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]-5-cyano-
pentyl]carbamate (6s)
Compound 6s was prepared in a manner similar to that de-
scribed for 6f in 53% yield. Rf = 0.80 (EtOAc/MeOH = 9:1). 1H NMR
(600 MHz, DMSO-d6): d 9.59 (d, J = 7.8 Hz, 1H), 9.50 (s, 1H), 8.04
(br s, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.66–7.72 (m, 5H), 7.39–7.41
(m, 4H), 7.31–7.33 (m, 3H), 7.15 (t, J = 7.8 Hz, 1H), 7.00–7.03 (m,
3H), 6.78 (s, 1H), 5.02 (s, 2H), 4.98 (br s, 1H), 4.65 (s, 2H), 4.29
(br s, 2H), 4.19 (br s, 1H), 3.96 (s, 3H), 3.00 (m, 2H), 1.98 (m, 2H),
1.41–1.46 (m, 4H).
5.1.44. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-3-(4-phenoxyphenyl)imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6n)
Compound 6n was prepared in a manner similar to that de-
scribed for 6a in 85% yield. Rf = 0.83 (EtOAc). 1H NMR (600 MHz,
DMSO-d6):
d 9.59 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H), 7.88 (d,
J = 7.2 Hz, 2H), 7.70–7.66 (m, 4H), 7.40–7.38 (m, 4H), 7.33–7.30
(m, 3H), 7.15–7.12 (m, 3H), 7.00 (d, J = 7.8 Hz, 2H), 6.77 (s, 1H),
5.01 (s, 2H), 4.98–4.96 (m, 1H), 4.63 (s, 2H), 4.29–4.27 (m, 2H),
4.20–4.18 (m, 1H), 3.96 (s, 3H), 3.00–2.99 (m, 2H), 2.01–1.96 (m,
2H), 1.46–1.40 (m, 4H).
5.1.50. 9H-Fluoren-9-ylmethyl N-[(5S)-5-[[6-[[3-[3-carbamoyl-
4-(4-pyridylmethoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]-5-cyano-pentyl]carbamate (6t)
Compound 6t was prepared in a manner similar to that de-
scribed for 6f in 89% yield. Rf = 0.48 (CH2Cl2/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.59 (d, J = 9.0 Hz, 1H), 9.05 (s, 1H),
8.59 (d, J = 5.4 Hz, 2H), 8.05 (d, J = 2.4 Hz, 1H), 7.88 (d, J = 7.8 Hz,
2H), 7.77 (br s, 1H), 7.70 (br s, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.64–
7.63 (m, 1H), 7.48 (d, J = 6.6 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33–
7.31 (m, 3H), 7.19 (d, J = 9.6 Hz, 1H), 6.76 (s, 1H), 5.35 (s, 2H),
5.1.45. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-[4-(4-
methoxyphenoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-
carbonyl]amino]pentyl]carbamate (6o)
Compound 6o was prepared in a manner similar to that de-
scribed for 6a in 83% yield (containing impurity). Rf = 0.52 (EtOAc).

2350
N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
5.00 (s, 2H), 4.98–4.96 (m, 1H), 4.60 (s, 2H), 4.30–4.28 (m, 2H),
4.21–4.19 (m, 1H), 3.96 (s, 3H), 3.00–2.99 (m, 2H), 2.01–1.98 (m,
2H), 1.46–1.40 (m, 4H).
(s, 3H), 2.86 (t, J = 7.8 Hz, 2H), 2.03–1.90 (m, 2H), 1.64–1.56 (m,
2H), 1.49–1.41 (m, 2H), HR-ESI-MS [M+Na]+ 527.21589 for C25H28-
N8NaO4 (calcd 527.21312).
5.1.51. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[3-(4-
fluorophenyl)-2-oxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6u)
Compound 6u was prepared in a manner similar to that de-
scribed for 6a in 96% yield (including impurity). Rf = 0.17 (EtOAc).
5.1.57. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-(4-
propylphenyl)imidazolidin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxamide (1d)
Compound 1d was prepared in a manner similar to that de-
scribed for 1a in 46% yield. 1H NMR (600 MHz, DMSO-d6): d 9.62
(d, J = 7.8 Hz, 1H), 9.08 (s, 1H), 7.66 (br s, 2H), 7.57 (d, J = 8.4 Hz,
2H), 7.24 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 5.03–5.02 (m, 1H), 5.01
(s, 2H), 4.61 (s, 2H), 3.98 (s, 3H), 2.82–2.80 (m, 2H), 2.55 (t,
J = 7.8 Hz, 2H), 2.02–1.99 (m, 2H), 1.61–1.56 (m, 4H), 1.47–1.45
(m, 2H), 0.89 (t, J = 7.8 Hz, 3H), HR-ESI-MS [M+H]+ 517.26512 for
5.1.52. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[7-methyl-6-
[[2-oxo-3-(4-phenoxyphenyl)imidazolidin-1-yl]methyl]
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6v)
C27H33N8O3 (calcd 517.26756).
Compound 6v was prepared in a manner similar to that de-
scribed for 6a in 75% yield (including impurity). Rf = 0.43 (EtOAc/
MeOH = 9:1).
5.1.58. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-(4-
sulfamoylphenyl)imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1e)
5.1.53. 9H-Fluoren-9-ylmethyl N-[(5S)-5-cyano-5-[[6-[[2,5-
dioxo-4-(4-phenoxyphenyl)piperazin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carbonyl]amino]pentyl]carbamate
(6w)
Compound 6a was prepared in a manner similar to that de-
scribed for 6w in 96% yield. Rf = 0.51 (EtOAc/MeOH = 9:1). 1H
NMR (600 MHz, DMSO-d6): d 9.60 (d, J = 8.4 Hz, 1H), 9.10 (s, 1H),
7.89 (d, J = 7.2 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.45–7.40 (m, 6H),
7.33–7.31 (m, 3H), 7.19 (t, J = 7.2 Hz, 1H), 7.07–7.05 (m, 4H), 6.86
(s, 1H), 4.99–4.96 (m, 3H), 4.44 (s, 2H), 4.30–4.28 (m, 2H), 4.21–
4.19 (m, 1H), 4.14 (s, 2H), 3.88 (s, 3H), 3.00–2.99 (m, 2H), 2.01–
1.98 (m, 2H), 1.46–1.40 (m, 4H).
Compound 1e was prepared in a manner similar to that de-
scribed for 1a in 55% yield. 1H NMR (600 MHz, D2O): d 8.79 (s,
1H), 7.72 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 6.61 (s, 1H),
4.93–4.91 (m, 3H), 4.49 (s, 2H), 3.77 (s, 3H), 2.84 (t, J = 7.8 Hz,
2H), 1.96–1.91 (m, 2H), 1.58–1.49 (m, 2H), 1.45–1.42 (m, 2H),
HR-ESI-MS [M+H]+ 554.19632 for C24H28N9O5S (calcd 554.19341).
5.1.59. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(4-
carbamoylphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1f)
Compound 1e was prepared in a manner similar to that de-
scribed for 1a in 51% yield. 1H NMR (600 MHz, D2O): d 8.85 (s,
1H), 7.67–7.65 (m, 2H), 7.48–7.47 (m, 2H), 6.70 (s, 1H), 4.94–
4.91 (m, 3H), 4.48 (s, 2H), 3.80 (s, 3H), 2.84 (t, J = 7.2 Hz, 2H),
1.97–1.92 (m, 2H), 1.59–1.47 (m, 2H), 1.44–1.42 (m, 2H), HR-ESI-
MS [M+H]+ 518.22796 for C25H28N9O4 (calcd 518.22642).
5.1.54. N-[(1S)-5-Amino-1-cyano-pentyl]-7-methyl-6-[(3-
methyl-2,5-dioxo-imidazolidin-1-yl)methyl]pyrrolo[2,3-
d]pyrimidine-2-carboxamide (1a)
Compound 6a (60 mg, 0.095 mmol) was dissolved in 20% piper-
idine in DMF (940
ll) at room temperature. The reaction mixture
5.1.60. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(4-
was stirred under the same conditions for 40 min. The mixture
was evaporated and pale yellow products were precipitated by
ether. The products were purified with HPLC system (column;
cyanophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1g)
Compound 1g was prepared in a manner similar to that de-
scribed for 1a in 61% yield. 1H NMR (600 MHz, D2O): d 8.86 (s,
1H), 7.62–7.60 (m, 2H), 7.52–7.49 (m, 2H), 6.70 (s, 1H), 4.93–4.90
(m, 3H), 4.47 (s, 2H), 3.81 (s, 3H), 2.84 (t, J = 7.8 Hz, 2H), 1.98–
1.91 (m, 2H), 1.60–1.57 (m, 2H), 1.46–1.42 (m, 2H), HR-ESI-MS
[M+H]+ 500.21735 for C25H26N9O3 (calcd 500.21586).
TSK gel ODS-120T,
U
21.5 ꢁ 300 mm, eluent CH3CN containing
0.1% TFA/H2O containing 0.1% TFA) to give 1a in 32% yield. 1H
NMR (600 MHz, D2O): d 8.91 (s, 1H), 6.69 (s, 1H), 4.97 (t,
J = 7.2 Hz, 1H), 4.85 (s, 2H), 4.02 (s, 2H), 3.81 (s, 3H), 2.86–2.65
(m, 2H), 2.84 (s, 3H), 2.00–1.94 (m, 2H), 1.61–1.58 (m, 2H), 1.47–
1.44 (m, 2H), HR-ESI-MS [M+H]+ 413.20471 for C19H25N8O3 (calcd
413.20496).
5.1.61. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-(3-
sulfamoylphenyl)imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1h)
Compound 1h was prepared in a manner similar to that de-
scribed for 1a in 47% yield. 1H NMR (600 MHz, D2O): d 8.86 (s,
1H), 7.97 (s, 1H), 7.56–7.53 (m, 2H), 7.47–7.44 (m, 1H), 6.73 (s,
1H), 4.94–4.92 (m, 3H), 4.50 (s, 2H), 3.82 (s, 3H), 2.84 (t,
J = 7.8 Hz, 2H), 1.98–1.91 (m, 2H), 1.60–1.56 (m, 2H), 1.46–1.42
(m, 2H), HR-ESI-MS [M+H]+ 554.19408 for C24H28N9O5S (calcd
554.19341).
5.1.55. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(4-
fluorophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1b)
Compound 1b was prepared in a manner similar to that de-
scribed for 1a in 41% yield. 1H NMR (600 MHz, D2O): d 8.90 (s,
1H), 7.35–7.33 (m, 2H), 7.02 (t, J = 9.0 Hz, 2H), 6.74 (s, 1H), 4.96
(t, J = 7.2 Hz, 1H), 4.93 (s, 2H), 4.45 (s, 2H), 3.83 (s, 3H), 2.86 (t,
J = 7.2 Hz, 2H), 2.01–1.92 (m, 2H), 1.61–1.58 (m, 2H), 1.47–1.44
(m, 2H), HR-ESI-MS [M+H]+ 493.21387 for C24H26FN8O3 (calcd
493.21119).
5.1.62. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(3-
carbamoylphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1i)
5.1.56. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(4-
methoxyphenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1c)
Compound 1c was prepared in a manner similar to that de-
scribed for 1a in 52% yield. 1H NMR (600 MHz, D2O): d 8.90 (s,
1H), 7.24 (d, J = 9.0 Hz, 2H), 6.84 (d, J = 9.6 Hz, 2H), 6.76 (s, 1H),
4.95 (t, J = 7.8 Hz, 1H), 4.91 (s, 2H), 4.40 (s, 2H), 3.83 (s, 3H), 3.66
Compound 1i was prepared in a manner similar to that de-
scribed for 1a in 43% yield. 1H NMR (600 MHz, D2O): d 8.81 (br s,
1H), 7.79 (s, 1H), 7.58 (br s, 1H), 7.47 (br s, 1H), 7.40 (br s, 1H),
6.63 (s, 1H), 4.93 (s, 3H), 4.50 (s, 2H), 3.79 (s, 3H), 2.84 (br s, 2H),
1.93 (br s, 2H), 1.59 (br s, 2H), 1.43 (br s, 2H). HR-ESI-MS
[M+Na]+ 540.20996 for C25H27N9NaO4 (calcd 540.20837).

N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
2351
5.1.63. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(3-
cyanophenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1j)
5.1.69. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-[4-(4-
pyridylmethoxy)phenyl]imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1p)
Compound 1j was prepared in a manner similar to that de-
scribed for 1a in 38% yield. 1H NMR (600 MHz, D2O): d 8.83 (s,
1H), 7.83 (s, 1H), 7.66 (br s, 1H), 7.43 (br s, 2H), 6.64 (s, 1H), 4.94
(s, 3H), 4.48 (s, 2H), 3.80 (s, 3H), 2.84 (br s, 2H), 1.96 (br s, 2H),
1.59 (br s, 2H), 1.44 (br s, 2H). HR-ESI-MS [M+Na]+ 522.20008 for
Compound 1p was prepared in a manner similar to that de-
scribed for 1a in 42% yield. 1H NMR (600 MHz, D2O): d 8.83 (s,
1H), 8.57 (d, J = 7.2 Hz, 2H), 7.95 (d, J = 6.0 Hz, 2H), 7.28 (d,
J = 8.4 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 6.64 (s, 1H), 5.32 (s, 2H),
4.93 (t, J = 7.8 Hz, 1H), 4.90 (s, 2H), 4.41 (s, 2H), 3.79 (s, 3H), 2.84
(t, J = 7.8 Hz, 2H), 1.98–1.92 (m, 2H), 1.60–1.56 (m, 2H), 1.45–
1.42 (m, 2H), HR-ESI-MS [M+H]+ 587.25798 for C30H32N9O4 (calcd
582.25772).
C25H25N9NaO3 (calcd 522.19780).
5.1.64. 6-[[3-(4-Acetonyloxyphenyl)-2,5-dioxo-imidazolidin-1-
yl]methyl]-N-[(1S)-5-amino-1-cyano-pentyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1k)
Compound 1k was prepared in a manner similar to that de-
scribed for 1a in 44% yield. 1H NMR (600 MHz, D2O): d 8.85 (s,
1H), 7.24 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 6.69 (s, 1H),
4.93 (t, J = 7.2 Hz, 1H), 4.89 (s, 2H), 4.75 (s, 2H), 4.39 (s, 2H), 3.80
(s, 3H), 2.84 (t, J = 7.8 Hz, 2H), 2.10 (s, 3H), 1.98–1.91 (m, 2H),
1.60–1.56 (m, 2H), 1.46–1.42 (m, 2H), HR-ESI-MS [M+H]+
547.24336 for C27H31N8O5 (calcd 547.24174).
5.1.70. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-[4-(7-
quinolyloxy)phenyl]imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1q)
Compound 1q was prepared in a manner similar to that de-
scribed for 1a in 40% yield. 1H NMR (600 MHz, D2O): d 8.87 (s,
1H), 8.79 (d, J = 5.4 Hz, 1H), 8.66–8.65 (m, 1H), 8.00 (d, J = 9.6 Hz,
1H), 7.80 (t, J = 6.6 Hz, 1H), 7.67 (br s, 1H), 7.43–7.42 (m, 2H),
7.31 (s, 1H), 7.05–7.04 (m, 2H), 6.80 (s, 1H), 4.96–4.93 (m, 3H),
4.48 (s, 2H), 3.85 (s, 3H), 2.84 (t, J = 7.8 Hz, 2H), 2.00–1.912 (m,
2H), 1.60–1.56 (m, 2H), 1.46–1.42 (m, 2H), HR-ESI-MS [M+H]+
618.25698 for C33H32N9O4 (calcd 618.25772).
5.1.65. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-[4-(2-amino-2-
oxo-ethoxy)phenyl]-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1l)
Compound 1l was prepared in a manner similar to that de-
scribed for 1a in 44% yield. 1H NMR (600 MHz, D2O): d 8.86 (s,
1H), 7.29 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.68 (s, 1H),
4.94 (t, J = 7.8 Hz, 1H), 4.91 (s, 2H), 4.46 (s, 2H), 4.42 (s, 2H), 3.80
(s, 3H), 2.84 (t, J = 7.2 Hz, 2H), 1.97–1.91 (m, 2H), 1.60–1.56 (m,
2H), 1.45–1.42 (m, 2H), HR-ESI-MS [M+H]+ 548.23666 for
5.1.71. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-[4-(2-
carbamoylphenoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide
(1r)
Compound 1r was prepared in a manner similar to that de-
scribed for 1a in 62% yield. 1H NMR (600 MHz, DMSO-d6): d 9.62
(d, J = 8.4 Hz, 1H), 9.09 (s, 1H), 7.73–7.65 (m, 7H), 7.58 (br s, 1H),
7.45 (t, J = 6.6 Hz, 1H), 7.22 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 8.4 Hz,
2H), 6.85 (d, J = 8.4 Hz, 1H), 6.79 (s, 1H), 5.02–5.00 (m, 3H), 4.63
(s, 2H), 3.97 (s, 3H), 2.82–2.80 (m, 2H), 2.02–2.00 (m, 2H), 1.60–
1.58 (m, 2H), 1.47–1.44 (m, 2H), HR-ESI-MS [M+Na]+ 632.23626
for C31H31N9NaO5 (calcd 632.23458).
C26H30N9O5 (calcd 548.23699).
5.1.66. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-[4-
(cyanomethoxy)phenyl]-2,5-dioxo-imidazolidin-1-yl]methyl]-
7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1m)
Compound 1m was prepared in a manner similar to that de-
scribed for 1a in 60% yield. 1H NMR (600 MHz, D2O): d 8.87 (s,
1H), 7.32 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.71 (s, 1H),
4.94 (t, J = 7.8 Hz, 1H), 4.91 (s, 2H), 4.85 (s, 2H), 4.42 (s, 2H), 3.81
(s, 3H), 2.85 (t, J = 7.2 Hz, 2H), 1.98–1.92 (m, 2H), 1.60–1.56 (m,
2H), 1.45–1.42 (m, 2H), HR-ESI-MS [M+Na]+ 552.20948 for C26H27-
N9NaO4 (calcd 552.20837).
5.1.72. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(3-carbamoyl-4-
phenoxy-phenyl)-2,5-dioxo-imidazolidin-1-yl]methyl]-7-
methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide (1s)
Compound 1s was prepared in a manner similar to that de-
scribed for 1a in 47% yield. 1H NMR (600 MHz, DMSO-d6): d 9.63
(d, J = 7.8 Hz, 1H), 9.09 (s, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.66–7.72
(m, 5H), 7.40 (t, J = 7.8 Hz, 2H), 7.16 (t, J = 7.8 Hz, 1H), 7.02 (t,
J = 9.0 Hz, 3H), 6.80 (s, 1H), 5.03 (s, 2H), 5.02 (br s, 1H), 4.65 (s,
2H), 3.99 (s, 3H), 2.82 (m, 2H), 2.01 (m, 2H), 1.59 (m, 2H), 1.46
(m, 2H). HR-ESI-MS [M+Na]+ 632.23530 for C31H31N9NaO5 (calcd
632.23458).
5.1.67. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-3-(4-
phenoxyphenyl)imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1n)
Compound 1n was prepared in a manner similar to that de-
scribed for 1a in 54% yield. 1H NMR (600 MHz, DMSO-d6): d 9.63
(d, J = 8.4 Hz, 1H), 9.09 (s, 1H), 7.70–7.68 (m, 4H), 7.40 (t,
J = 7.2 Hz, 2H), 7.16–7.11 (m, 3H), 7.00 (d, J = 7.2 Hz, 2H), 6.79 (s,
1H), 5.04–5.00 (m, 3H), 4.63 (s, 2H), 3.99 (s, 3H), 2.82–2.80 (m,
2H), 2.02–2.00 (m, 2H), 1.61–1.58 (m, 2H), 1.47–1.45 (m, 2H),
HR-ESI-MS [M+Na]+ 589.22935 for C30H30N8NaO4 (calcd
589.22877).
5.1.73. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-[3-carbamoyl-4-
(4-pyridylmethoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide
(1t)
Compound 1t was prepared in a manner similar to that de-
scribed for 1a in 52% yield. 1H NMR (600 MHz, D2O): d 8.85 (s,
1H), 8.60 (d, J = 4.8 Hz, 2H), 7.94 (d, J = 4.8 Hz, 2H), 7.69 (s, 1H),
7.46–7.44 (m, 1H), 6.98–6.97 (m, 1H), 6.68 (s, 1H), 5.43 (s, 2H),
4.94–4.90 (m, 3H), 4.43 (s, 2H), 3.81 (s, 3H), 2.84 (t, J = 7.2 Hz,
2H), 1.96–1.92 (m, 2H), 1.59–1.57 (m, 2H), 1.44–1.42 (m, 2H),
HR-ESI-MS [M+H]+ 625.26459 for C31H33N10O5 (calcd 625.26354).
5.1.68. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-[4-(4-
methoxyphenoxy)phenyl]-2,5-dioxo-imidazolidin-1-
yl]methyl]-7-methyl-pyrrolo[2,3-d]pyrimidine-2-carboxamide
(1o)
Compound 1o was prepared in a manner similar to that de-
scribed for 1a in 62% yield. 1H NMR (600 MHz, DMSO-d6): d 9.63
(d, J = 8.4 Hz, 1H), 9.09 (s, 1H), 7.66–7.63 (m, 4H), 7.02–6.96 (m,
6H), 6.78 (s, 1H), 5.03–5.00 (m, 3H), 4.61 (s, 2H), 3.98 (s, 3H),
3.76 (s, 3H), 2.83–2.80 (m, 2H), 2.02–2.00 (m, 2H), 1.61–1.58 (m,
2H), 1.47–1.44 (m, 2H), HR-ESI-MS [M+H]+ 597.25750 for
5.1.74. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[3-(4-
fluorophenyl)-2-oxo-imidazolidin-1-yl]methyl]-7-methyl-
pyrrolo[2,3-d]pyrimidine-2-carboxamide (1u)
Compound 1u was prepared in a manner similar to that de-
C31H33N8O5 (calcd 597.25739).
scribed for 1a in 45% yield. 1H NMR (600 MHz, D2O): d 8.88 (s,

2352
N. Teno et al. / Bioorg. Med. Chem. 22 (2014) 2339–2352
1H), 7.25 (q, J = 4.8 Hz, 2H), 6.98 (t, J = 8.2 Hz, 2H), 6.73 (s, 1H), 4.95
(t, J = 7.2 Hz, 1H), 4.59 (s, 2H), 3.76–3.73 (m, 5H), 3.38 (t, J = 8.4 Hz,
2H), 2.84 (t, J = 7.8 Hz, 2H), 1.98–1.92 (m, 2H), 1.58–1.56 (m, 2H),
1.46–1.42 (m, 2H), HR-ESI-MS [M+H]+ 479.23274 for C24H28FN8O2
(calcd 479.23192).
inhibitor that reduced the absorbance at 405 nm by 50% compared
with the absorbance measured under the same conditions without
inhibitor.
Acknowledgements
5.1.75. N-[(1S)-5-Amino-1-cyano-pentyl]-7-methyl-6-[[2-oxo-3-
(4-phenoxyphenyl)imidazolidin-1-yl]methyl]pyrrolo[2,3-
d]pyrimidine-2-carboxamide (1v)
The work was supported by Grant-in-Aid for Scientific Research
(C, 24590154) from the Ministry of Education, Science, Sports and
Culture of Japan. We are grateful to Dr. Lawrence H. Lazarus for
critically reading the manuscript.
Compound 1v was prepared in a manner similar to that de-
scribed for 1a in 38% yield. 1H NMR (600 MHz, DMSO-d6): d 9.63
(d, J = 9.0 Hz, 1H), 9.11 (s, 1H), 7.66–7.64 (m, 4H), 7.39 (t,
J = 7.8 Hz, 2H), 7.12 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.97
(d, J = 8.4 Hz, 2H), 6.79 (s, 1H), 5.02–5.00 (m, 1H), 4.77 (s, 2H),
3.90–3.86 (m, 5H), 2.82–2.80 (m, 2H), 2.02–1.99 (m, 2H), 1.61–
1.58 (m, 2H), 1.47–1.44 (m, 2H), HR-ESI-MS [M+H]+ 553.26677
for C30H33N8O3 (calcd 553.26756).
Supplementary data
Supplementary data (experimental procedures for the synthesis
and characterization (1H NMR) of 2d–2f, 2h, 2i, 2k, 2l, 2n–2w)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2014.02.002.
5.1.76. N-[(1S)-5-Amino-1-cyano-pentyl]-6-[[2,5-dioxo-4-(4-
phenoxyphenyl)piperazin-1-yl]methyl]-7-methyl-pyrrolo[2,3-
d]pyrimidine-2-carboxamide (1w)
Compound 1w was prepared in a manner similar to that de-
scribed for 1a in 32% yield. 1H NMR (600 MHz, D2O): d 8.99 (s,
1H), 7.24 (t, J = 7.8 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 7.04 (t,
J = 6.6 Hz, 1H), 6.94–6.89 (m, 5H), 4.97 (t, J = 7.8 Hz, 1H), 4.88 (s,
2H), 4.38 (s, 2H), 4.15 (s, 2H), 3.80 (s, 3H), 2.84 (t, J = 7.2 Hz, 2H),
2.00–1.92 (m, 2H), 1.61–1.57 (m, 2H), 1.47–1.43 (m, 2H), HR-ESI-
MS [M+H]+ 581.26157 for C31H33N8O4 (calcd 581.26193).
References and notes
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Y.; Yamamoto, R.; Yagita, H.; Okumura, K.; Nishikori, M.; Wanaka, K.; Tsuda, Y.;
Okada, Y.; Nakauchi, H.; Heissig, B.; Hattori, K. Leukemia 2012, 26, 332.
3. Danø, K.; Behrendt, N.; Høyer-Hansen, G.; Johnsen, M.; Lund, L. R.; Ploug, M.;
Rømer, J. Thromb. Haemost. 2005, 93, 676.
4. Szende, B.; Okada, Y.; Tsuda, Y.; Horvath, A.; Bökönyi, G.; Okamoto, S.; Wanaka,
K.; Kéri, G. In Vivo 2002, 16, 281.
5. Swedberg, J. E.; Harris, J. M. Biochemistry 2011, 50, 8454.
6. Backes, B. J.; Harris, J. L.; Leonetti, F.; Craik, C. S.; Ellman, J. A. Nat. Biotechnol.
2000, 18, 187.
7. Saupe, S. M.; Steinmetzer, T. J. Med. Chem. 2012, 55, 1171.
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5.2. Docking studies
9. Teno, N.; Otsubo, T.; Gohda, K.; Wanaka, K.; Sueda, T.; Ikeda, K.; Hijikata-
Okunomiya, A.; Tsuda, Y. J. Peptide Sci. 2012, 18, 620.
Plasmin coordinates used in this study were prepared from mi-
cro-plasmin complex with streptokinase (PDB ID: 1BML) structure.
The preparation procedure was described in detail in Ref. 19. The
docked conformation of compound 3 was deduced by Monte Carlo
multiple minimum method20 with the BatchMin molecular
mechanics engine21 with standard parameters. The MMFF94s
parameter was used as the force field.22 The MMFF and AMBER9423
atomic charges were loaded for inhibitor and enzyme atoms,
respectively. Manipulation of molecular structures was done using
the MAESTRO 9.0 molecular modeling package (Schrodinger Inc.,
Portland, USA) on a single Pentium IVcentral processing unit
(3.0 GHz) computer with Linux/CentOS4.0.
10. Teno, N.; Miyake, T.; Ehara, T.; Irie, O.; Sakaki, J.; Ohmori, O.; Gunji, H.;
Matsuura, N.; Masuya, K.; Hitomi, Y.; Nonomura, K.; Horiuchi, M.; Gohda, K.;
Iwasaki, A.; Umemura, I.; Tada, S.; Kometani, M.; Iwasaki, G.; Cowan-Jacob, S.
W.; Missbach, M.; Lattmann, R.; Betschart, C. Bioorg. Med. Chem. Lett. 2007, 17,
6096.
11. Teno, N.; Irie, O.; Miyake, T.; Gohda, K.; Horiuchi, M.; Tada, S.; Nonomura, K.;
Kometani, M.; Iwasaki, G.; Betschart, C. Bioorg. Med. Chem. Lett. 2008, 18, 2599.
12. Teno, N.; Masuya, K.; Ehara, T.; Kosaka, T.; Miyake, T.; Irie, O.; Hitomi, Y.;
Matsuura, N.; Umemura, I.; Iwasaki, G.; Fukaya, H.; Toriyama, K.; Uchiyama, N.;
Nonomura, K.; Sugiyama, I.; Kometani, M. J. Med. Chem. 2008, 51, 5459.
13. Teno, N.; Masuya, K. Curr. Top. Med. Chem. 2010, 10, 752.
14. Kumar, V.; Rana, H.; Sankolli, M.; Kaushik, M. P. Tetrahedron Lett. 2011, 52,
6148.
15. Betschart, C.; Hayakawa, K.; Irie, O.; Sakaki, J.; Iwasaki, G.; Lattmann, R.;
Missbach, M.; Teno, N. WO 2003020721, 2003.
16. Chang, C.-S.; Lin, Y.-T.; Shih, S.-R.; Lee, C.-C.; Lee, Y.-C.; Tai, C.-L.; Tseng, S.-N.;
Chern, J.-H. J. Med. Chem. 2005, 48, 3522.
5.3. Inhibition of plasmin, plasma kallikrein, and urokinase
17. Cain, J. P.; Mayorov, A. V.; Cai, M.; Wang, H.; Tan, B.; Chandler, K.; Lee, Y.;
Petrov, R. R.; Trivedi, D.; Hruby, V. J. Bioorg. Med. Chem. Lett. 2006, 16, 5462.
18. Trost, B. M.; Jonasson, C.; Wuchrer, M. J. Am. Chem. Soc. 2001, 123, 12736.
19. Gohda, K.; Teno, N.; Wanaka, K.; Tsuda, Y. J. Enzyme Inhib. 2012, 27, 571.
20. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc. 1989, 111, 4379.
21. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
22. Halgren, T. A. J. Comput. Chem. 1996, 17, 490.
23. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.;
Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995,
117, 5179.
24. Tada, M.; Tsuda, Y.; Wanaka, K.; Hijikata-Okunomiya, A.; Horie, N.; Okamoto,
U.; Okamoto, S.; Okada, Y. Biol. Pharm. Bull. 2001, 24, 520.
Human plasmin was purchased from Chromogenix (Sweden)
and human urokinase from Green Cross (Japan). Human plasma
kallikrein was purified from human plasma.24 H-D-Val-Leu-Lys-
pNA (S-2251), H-D-Phe-Pro-Arg-pNA (S-2302), and <Glu-Gly-Arg-
pNA (S-2444) were purchased from Chromogenix (Sweden).
Inhibitory activities were determined by measuring the effect of
the inhibitor on the hydrolytic activities of enzyme on synthetic
peptide substrate. Plasmin activity was measured with 0.3 mM
S-2251, plasma kallikrein with 0.2 mM S-2302, and urokinase with
0.1 mM S-2444. The IC50 value was taken as the concentration of