Design and Synthesis of New Templates Derived from Pyrrolopyrimidine as Selective Multidrug-Resistance-Associated Protein Inhibitors in Multidrug Resistance

Article abstract of DOI:10.1021/jm0310129

In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further syn

Full text of DOI:10.1021/jm0310129

J . Med. Chem. 2004, 47, 1339-1350
1339
Design a n d Syn th esis of New Tem p la tes Der ived fr om P yr r olop yr im id in e a s
Selective Mu ltid r u g-Resista n ce-Associa ted P r otein In h ibitor s in
Mu ltid r u g Resista n ce
Shouming Wang,*,† Nan Chi Wan,† J ohn Harrison,† Warren Miller,† Irina Chuckowree,† Sukhjit Sohal,†
Timothy C. Hancox,† Stewart Baker,† Adrian Folkes,† Francis Wilson,† Deanne Thompson,‡ Simon Cocks,‡
Hayley Farmer,§ Anthony Boyce,§ Caroline Freathy,§ J an Broadbridge,§ J ohn Scott,§ Paul Depledge,§
Richard Faint,§ Prakash Mistry,§ and Peter Charlton§
Department of Medicinal Chemistry, Department of Pharmacology, Analytical Department, Xenova Ltd.,
957 Buckingham Avenue, Slough, Berkshire SL1 4NL,U.K.
Received August 21, 2003
In our continued effort to identify selective MRP1 modulators, we have developed two novel
templates, 3 and 4, through rational drug design by identifying the key pharmacophore
interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR
work on these novel templates gave a number of potent MRP1 modulators with great selectivity
against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several
compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies
were demonstrated.
Multidrug resistance (MDR) mediated by P-glycopro-
tein (Pgp) or multidrug resistance associated protein
(MRP) remains a major obstacle for successful treat-
ment of cancer. Circumvention of Pgp and MRP trans-
port is important for high efficacy of anticancer drugs.
While several Pgp inhibitors have entered clinical trials,
the development of specific MRP1 inhibitors is still in
its infancy.1
Previously, we have reported the selective inhibitory
activities of pyrrolopyrimidine analogues (1) against
MRP1.2 Our SAR studies in this series revealed an
important pharmacophoric interaction of the functional
groups at the 7-position as probable H-bond acceptors.
However, SAR exploration of the lipophilic cyclohexyl
group attached at the 5- and 6-position was not straight-
forward. Our new template design strategy is based on
the fusion of the side chain at the 6-position, by ring-
opening the cyclohexyl group, with the functional groups,
such as nitrile 1 or thiazole group 2 at the 7-position,
into an aromatic pyridyl analogue 3 or its isostere, the
nitro analogue 4.
This manipulation would allow the key pharmacophore
interaction at the 7-position of template 1 or 2 to be
retained and the conformation of the template to be
more restricted. Moreover, readily amenable chemistry
can be devised for templates 3 and 4 to allow us to
explore the regions previously unexplored on template
represented by 1 or 2. Here, we disclose our synthesis
and SAR work on templates 3 and 4.
from indole aminoesters 5 because this would allow us
to adopt the protocols developed by us previously2
(Scheme 1). Direct nitration or sulfonylation of chloro-
pyrimidine intermediate 6 gave the functionalized
intermediates 7 at the 6-position of template 6, which
were converted into sulfonamides or nitro analogues 8
as follows. Thus, the sulfonyl chloride (7, Y ) SO2Cl, R′
) H) was reacted with a variety of primary and
secondary amines at 0 °C in the presence of triethyl-
amine, yielding the corresponding chloropyrimidine
sulfonamides, which were subsequently coupled with
3,4-difluorophenethylpiperazine (Q) to give a library of
sulfonamides (8, Y ) SO2NR1R2). The 6-nitro analogue
(8, Y ) NO2) was similarly prepared through the
coupling of the 6-nitrochloropyrimidine intermediate (7,
Y ) NO2) with the side chain Q. Alkylations on the
indole nitrogen of analogues 9 can also be achieved
successfully with NaH as base to give a number of
analogues 10. Following a literature method,3 a number
of 3-substituted indole pyrimidines 12 were prepared
from the indole amino esters 5 and acetonitrile deriva-
tives, such as chloroacetonitrile, with HCl gas and
subsequent alkylations, chlorinations, and aminations.
Ch em istr y
At first, we embarked on the synthesis and SAR
exploration of template 4 and its analogues, starting
* To whom correspondence should be addressed. Present address:
Trigen Ltd., Emmanuel Kaye Building, Manresa Road, London SW3
6LR, U.K. Phone: +44 (0) 2073518319. Fax: +44(0)2073518392.
E-mail: swang@trigen.co.uk.
† Department of Medicinal Chemistry.
‡ Analytical Department.
§ Department of Pharmacology.
10.1021/jm0310129 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/07/2004

1340 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
Sch em e 1a
Sch em e 3a
a
Reagents and conditions: (a) Y ) NO2, NaNO3, H2SO4, 0°C
to room temp, 93%; Y ) SONR1R2, (i) ClSO3H, 0 °C to reflux, 70-
95%, (ii) R1R2NH, DCM, TEA, 0°C, 28-77%; (b) XCH2CN, HCl
(g), dioxane, room temp, 38-87%; (c) NaH, R′′X, DMF, 90%; (d)
13, TEA, DMF, 100 °C, 27-99%; (e) X ) Cl, R1R2NH, Na2CO3,
EtOH, reflux, 44-77%; (f) POCl3, TEA‚HCl (0.4 equiv), reflux, 60-
93%.
Sch em e 2a
a
Reagents and conditions: (a) 18, NaH, DMF, 40 °C, 61%; (b)
H2, 10% Pd-C, EtOH, room temp, 96%; (c) (i) Me2NCH(MeO)2,
DMF, 100 °C; (ii) NH3, EtOH, 100 °C, 91% (two steps); (d)
m-CPBA, CHCl3, room temp, 24 h, 100%; (e) Na/MeOH, reflux,
80%; (f) POCl3, Et3N‚HCl, reflux, 54-95%; (g) 13, DMF, 100 °C,
71-95%; (h) ClCH2CN, HCl(g), dioxane, 0 °C to room temp, 81%;
(j) XR ) OMe, Na/MeOH, reflux, 60%; XR ) morpholine, K2CO3,
EtOH, 54%; (k) 13, DMF, 50 °C, o/n, 90-95%; (m) R1R2NH, DMF,
100 °C, 55-98%; (n) R ) Me, MeZnCl, Pd(Ph3P)4, THF, 67 °C,
85%.
a
Reagents and conditions: (a) glycine ethyl ester HCl salt,
K2CO3, CH3CN, reflux, 16 h, 19%; (b) (BOC)2O, Et3N, DMAP,
DCM, room temp, 16 h, 100%; (c) NaH, THF, 0 °C to room temp,
80%.
The syntheses of template 3 and its analogues are
outlined in Scheme 3.
In each case, we retained the previously optimized side
chain 13 in the analogue compounds 8-10 and 12.
Most of the indole amino esters 5, such as those when
R ) MeO at the 5- and 6-position, can be prepared from
readily available starting materials with reported pro-
cedures,4 while those with electron-withdrawing groups
at the phenyl ring were prepared by novel methods as
shown in Scheme 2.
Thus, 4-nitrile indole amino ester 16 was prepared
from the reaction of 2,3-dicyanofluorobenzene 14 with
glycine ester and base under refluxing conditions and
subsequent cyclization of 15 by triethylamine. Alterna-
tively, acetamide 175 can be coupled with bromoethyl
acetate 18 in the presence of NaH to give the cyclized
amino ester 19 directly. Notably, both N-Boc and acetyl
groups were cleaved during the subsequent pyrimidone
formation step with ammonia under bomb conditions.2
Both nitrile and nitro groups in 16 and 19 served as
versatile handles for further functional group manipula-
tions using standard methodologies at the final ana-
logue stage after the piperazine side chain was intro-
duced into the template (see Experimental Section).
Thus, methylaminochloropyridine nitrile 206 was
alkylated with bromoethyl acetate 18 and simultaneous
cyclization gave the amino ester 21, which was dechlo-
rinated by hydrogenolysis to give 22. Methoxypyridine
analogue 25 was also obtained by the displacement of
the chloro group of 21 with sodium methoxide.
Following the protocols described earlier, amino esters
22 and 25 were converted into the corresponding pyrim-
idones 23 and 28, respectively. m-CPBA oxidation of
pyrimidone 23 gave the N-oxide intermediate 24, which
was converted into the regioisomeric dichloro intermedi-
ates 27a (minor) and 27b (major) upon treatment with
POCl3. The chloro group on the pyrimidine ring of 27b
was selectively replaced by the piperazine amine side
chain 13 at 50 °C to give the chloropyridine analogue
29. This was then used as a key intermediate for
reaction with a diverse set of amines to give a series of
analogues 30. Direct alkylation of compound 29 was also
achieved with palladium-catalyzed couplings,7 yielding
analogues 32. Intermediates with improved aqueous
solubility (31) were achieved by the displacement of the
chloro group of 28 with amines, such as morpholine, or

Templates from Pyrrolopyrimidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1341
Sch em e 4a
(human non-small-cell lung carcinoma) and in the Pgp
expressing cell line EMT6/AR1.0 (murine mammary
carcinoma). Most of the active compounds were then
subjected to the secondary single-dose potentiation
assay.
To screen out compounds that inhibit P450 enzymes
to avoid any potential drug-drug interactions, selective
potent compounds were also subjected to CYP 3A4
inhibition studies. The CYP 3A4 isozyme was chosen
initially for this study because it is one of the major
oxidative enzymes among the family of P450 enzymes.12
Table 1 highlights the results of the MRP1 and Pgp
accumulation and MRP1 and Pgp single-dose potentia-
tion (sdpa) and CYP 3A4 assays.
a
Reagents and conditions: (a) Me2NCOCl, TMSCN, DCM, room
temp, 92%; (b) NaH, 18, THF, 0 °C to room temp, 88%.
Compound 43, bearing no H-bond acceptor functional
group, showed poor or moderate activity against MRP1
and Pgp, whereas 6-nitro analogue 44 showed potent
inhibitory activity against MRP1 in both accumulation
and single-dose potentiation assays with good selectivity
against Pgp. These results validated our hypothesis on
template 4. Further library syntheses on alkoxy, aceta-
midoyl, amide, or sulfonamide linkages did not show
improved activity as exemplified by compounds 45, 46,
48, and 49, although 6-carboxy ester analogue 47 was
equipotent with 44. Further SAR studies on the 5-posi-
tion revealed a number of compounds with good activity
(50-54); in particular, the 5-nitro analogue (54) and
carboxyamide analogue (52) are as potent as compound
44 with good selectivity against Pgp. Further derivati-
zation of amide 52 to secondary amides such as 53 or
analogues with electron-donating groups such as 55 did
not give improved potency. Analogues with 7- or 8-sub-
stitutions showed reduced activity (56, 57). The additive
substitution effect at the 5- and 6-position is not very
significant (58).
Sch em e 5a
a
Reagents and conditions: (a) sarcosine ethyl ester‚HCl, K2CO3,
DMF, 85 °C, 32-52%; (b) Cs2CO3 or KO-t-Bu, CH3CN, room temp
to 50 °C, 48-70%.
Many of the above active compounds were evaluated
in CYP 3A4 assays. Unfortunately, nearly all the
compounds so far showed significant inhibitory activity
against CYP 3A4 as shown in Table 1. Previously, we
have demonstrated potent MRP1 activity in the pyrrolo-
pyrimidine series,2 and here, CYP 3A4 activity was not
a serious problem in general. Close comparison of the
two templates 1 and 4 revealed that the latter has a
much flatter conformation, while the template 1 has a
puckered cyclohexyl ring (Figure 1). It was tentatively
assumed that this particular steric group might be
responsible for the general inactivity of the template 1
against CYP 3A4. Having observed that introducing
substituents at the 3-position of the pyrimidine ring was
not detrimental to MRP1 activity,2 we sought to intro-
duce a steric group at the 3-position of the pyrimidine
ring as shown in Scheme 1. Indeed, compounds 59 and
60 did not show any significant CYP3A4 activity, but
much of the MRP1 activity was retained. This finding
was also applied successfully to overcome CYP 3A4
activity for most of the active compounds later. To
summarize the above results, it is clear that H-bond
acceptors at the 5- or 6-position are important for
pharmacophore interactions.
alkoxide groups. Analogues 26 and 33 were obtained
by the standard protocols described earlier from pyrimi-
dones 23 and 31, respectively.
To extend the SAR studies on the 6-pyridyl template
3, the corresponding 5-pyridyl and 8-pyridyl analogues
were also synthesized according to Schemes 4 and 5.
Thus, the key 5-pyridyl amino ester 36 was synthe-
sized from the acetamidopyridine N-oxide8 34 with its
cyanation to compound 35, following a literature pro-
cedure.9 The subsequent alkylation and cyclization of
35 to 36 and eventual synthesis of the analogue 37 was
carried out by the protocols described earlier. Similarly,
8-pyridyl analogues 41 and 42 were prepared from the
readily accessible starting materials 38 (X ) CN,10 X )
CO2Et11). The key intermediate esters 36 and 40 were
reacted with N,N-dimethylformamide dimethyl acetal
and subsequent heating with ammonia in ethanol to
give the corresponding pyrimidones, which were con-
verted into chloropyrimidines with POCl3. These were
then reacted with the side chain Q to give 37 and 42,
using the same conditions as described in Schemes 1-3.
Analogues 41 were prepared using the same synthetic
route as for compound 33 in Scheme 3.
Resu lts a n d Discu ssion
Next we turned our attention to the SAR studies on
the indole N-substitutions at the 9-position with either
5- or 6-nitro analogues 54 or 44 or 5-amide analogue
52 as the parent molecules. A library of compounds was
prepared via a parallel synthesis approach. Table 2
Our initial SAR study was focused on the substitution
of the phenyl ring of the indolopyrimidine template 4.
The compounds were evaluated in drug accumulation
assays both in the MRP1 expressing cell line COR.L23/R

1342 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
Ta ble 1. Inhibitory Activitiesa of Indolopyrimidine Analogues in MRP1, Pgp, and CYP 3A4 Assays
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
CYP3A4
compd
R
R′
MRP1 (accb)
Pgp (accb)
MRP1 (sdpac)
Pgp (sdpac)
43
44
45
46
47
48
49
5-CH3
6-NO2
6-MeO
6-NHCOCH3
6-CO2CH3
6-CONH2
H
H
H
H
H
H
H
1.235
0.133
0.82
2.71
0.113
3.44
21.6
9.23
8.3
>40
>40
>40
3.31
0.632
0.051
0.608
ndd
ndd
9.25
12.58
ndd
1.738
ndd
ndd
ndd
0.059
0.731
2.53
ndd
ndd
ndd
0.235
0.542
ndd
ndd
50
51
52
53
54
55
56
57
58
59
5-Cl
5-CN
5-CONH2
5-CONHPy-4
5-NO2
5-NH2
7-MeO
7-MeO,8-NO2
5-NO2,6-MeO
6-NO2
H
H
H
H
H
H
H
H
H
0.367
0.277
0.183
0.65
0.183
1.9
3.15
3.45
0.078
0.672
17.2
11.6
28.3
0.167
0.169
0.065
0.708
0.059
0.708
ndd
>1.0
>1.0
3.32
ndd
ndd
ndd
1.36
24.55
18.04
27.86
>40
ndd
0.78
6.62
ndd
1.53
1.79
ndd
ndd
ndd
>40
ndd
ndd
>40
0.156
0.107
>1.0
>1.0
14.3
>100
60
6,8-di-NO2
0.118
8.22
0.019
1.48
42.26
a
All the IC50 values in this text represent the mean of a minimum of three experiments. Variations among the results were less than
10%. acc: accumulation assay. c sdpa: single-dose potentiation assay. nd: not determined.
b
d
prepared according to the chemistry shown in Scheme
3. As shown in Table 3, compound 70 is the most simple
analogue of template 3, which showed very good MRP1
inhibitory activity with remarkable selectivity against
Pgp in both accumulation and single-dose potentiation
assays. More lipophilic benzyl substitution at the 9-N
position gave compounds with slightly reduced activity
(71). Therefore, we focused the rest of our SAR studies
on the 9-N-methyl analogues. The key intermediate
5-chloro compound 29 showed potent activity against
MRP1 with a significant level of CYP3A4 activity, while
a similar magnitude of potency was observed in the
corresponding alkoxy analogues 72 and 73; these ana-
logues did not show any level of CYP3A4 activity at 100
µM. Replacement of the 5-chloro group with diverse
amines gave a number of very potent analogues (74-
77) with acceptable levels of CYP 3A4 activity; even the
5-methyl analogue 78 showed a similar in vitro profile.
However, the 7-chloro-substituted analogue 79 resulted
in reduction of MRP1 activity. For those compounds
with a certain degree of CYP 3A4 activity like 29,
introduction of a steric group at the 3-position of the
pyrimidine ring gave compounds with a much reduced
level of CYP 3A4 activities while the MRP1 activities
were retained (80, 81).
F igu r e 1. Superimposition of templates 1 (green) and 4
(yellow) carried out in Sybyl, version 6.9, using the default
setting of the GASP module.
shows the assay results on some of the typical examples.
N-alkylations of the 5-nitro analogue 54 did not result
in loss of potency (61-63). The 4-pyridylmethylene
alkylated analogue 64 showed much improved MRP1
activity in both accumulation and single-dose potentia-
tion assays; its MRP1 selectivity over Pgp is maintained
in these assays as well. N-alkylations of the 5-amide
analogue 52 gave consistently more active compounds
65-67, whereas N-alkylations of the 6-nitro analogue
44 did not yield compounds with much improved activity
(68). Unfortunately, most of the above compounds
showed significant inhibitory activity against CYP 3A4
except compound 61, which showed no activity in this
assay. The reason for this is not yet understood.
However, by use of the same strategy as previously with
the introduction of a morpholine group at the 3-position,
the CYP 3A4 activity was significantly diminished to
an acceptable level (69).
We also investigated the SAR of the corresponding
5- or 8-pyridyl templates. The results on selected
examples are illustrated in Table 4. The 5-pyridyl
analogue 82 showed slightly reduced activity compared
with the 6-pyridyl analogue 70 in both MRP1 ac-
cumulation and potentiation assays. Active functional
groups, such as nitrile and amides identified in template
4, were introduced into the 5-position of the 8-pyridyl
template, resulting in a number of potent MRP1 inhibi-
tors (83, 85, 86). Further introduction of substitutents
A library of 6-pyridylindolopyrimidine compounds was

Templates from Pyrrolopyrimidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1343
Ta ble 2. SAR Studies on Analogues with 9-N Substitutions
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
compd
R
R1
CH3
Ph(CH2)2
CH2Py-3
CH2Py-4
CH3
CH2CO2Et
CH2Py-4
CH2CO2Et
CH3
R2
MRP1 (accb)
Pgp (accb)
MRP1 (sdpac)
Pgp (sdpac)
CYP3A4
61
62
63
64
65
66
67
68
69
5-NO2
5-NO2
5-NO2
5-NO2
5-CONH2
5-CONH2
5-CONH2
6-NO2
H
H
H
H
H
H
H
H
0.147
0.222
0.621
0.08
0.062
0.122
0.27
10.0
6.94
6.94
6.14
>40
9.2
>40
10.5
>40
0.231
0.0409
0.0265
0.0137
0.024
0.045
0.02
>1.0
0.443
0.294
0.236
1.2
0.651
0.85
1.02
>100
11.79
8.10
5.78
0.26
nd
0.20
15.0
31.31
0.138
0.23
0.078
0.029
5-CONH2
0.608
a
All the IC50 values in this text represent the mean of a minimum of three experiments. Variations among the results were less than
10%. acc: accumulation assay. c sdpa: single-dose potentiation assay.
b
Ta ble 3. SAR Studies on 6-Pyridineindolopyrimidine Series
IC50 (µM)a
IC50 (µM)a
IC50 (µM)
IC50 (µM)a
IC50 (µM)a
CYP3A4
compd
R
R1
R2
MRP1 (accb)
Pgp (accb)
MRP1 (sdpac)
Pgp (sdpac)
70
71
29
72
73
74
75
76
77
78
79
80
81
H
H
5-Cl
CH3
CH2Ph
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
0.044
0.545
0.13
22.5
8.4
0.024
0.048
0.041
0.063
0.027
0.012
0.028
0.015
0.010
0.021
0.087
0.013
0.017
4.7
1.11
1.22
1.71
0.95
1.34
1.90
0.78
2.1
17.85
ndd
9.44
ndd
ndd
ndd
ndd
ndd
ndd
ndd
ndd
ndd
ndd
ndd
5-MeO
0.172
0.265
0.166
0.115
0.106
0.071
0.033
1.20
>100
>100
26.41
53.81
18.48
20.80
>100
ndd
5-iPrO
5-NMe2
5-NHMe
5-NHCH2Py-2
morpholine
5-CH3
7-Cl
5-Cl
5-Cl
1.2
>10
CH2OMe
0.244
0.09
2.0
2.47
36.82
20.03
a
All the IC50 values in this text represent the mean of a minimum of three experiments. Variations among the results were less than
10%. acc: accumulation assay. c sdpa: single-dose potentiation assay. nd: not determined.
b
d
into the pyridine ring had some effects; however, they
were not dramatic (84, 87). The morpholine methylene
side chain was introduced at the 3-position of the
pyrimidine ring of analogue 86 to overcome its potent
activity against CYP3A4 with retained MRP1 activity
(88).
On the basis of the SAR obtained from the two
template series 3 and 4, a number of compounds were
subjected to pharmacokinetics studies. This study was
carried out with Balb/C mice dosed at 20 mg/kg intra-
venously (iv) or 50 mg/kg intraperitoneally (ip) or orally
(po). Table 5 illustrates the results of some early
examples (more detailed studies will be published
elsewhere).
As shown in Table 5, 52 showed a good level of
absorption following ip or po administration. Com-
pounds 52, 60, 69, and 77 showed a range of half-lives
from 2 to 7 h depending on the routes of administration.
A wide range of Cmax and AUC values was observed with
the above compounds.
demonstrate the in vivo efficacy for these new classes
of compounds in our xenograft models in the early stage
of the project, we selected analogues 52 (XR13097) and
60 (XR13287) for in vivo xenograft studies. The signifi-
cant level of CYP 3A4 activity of compound 52 was not
a major concern for us at this stage, although CYP
activity remains a major criterion for selecting clinical
candidates to avoid potential drug-drug interactions.
The study was carried out with CD1 athymic mice
bearing subcutaneous COR L23/R tumors (n ) 6).
Compounds 52 (XR13097) and 60 (XR13287) were
administered ip (50 mg/kg) 30 min before and 4 h after
vincristine (0.6 mg/kg iv) on days 0 and 5 (Figure 2).
As shown in Figure 2, 52 significantly enhanced the
antitumor efficacy of vincristine when compared with
vincristine alone [from day 5 onward for 52 and day 10
onward for 60 (graft not shown)]. This was reflected in
an optimal T/C % ratio of 19.3 for compound 52 plus
vincristine (compared with 83.4 and 84.2 for vincristine
alone and compound 52 alone, respectively) and 45.7
for compound 60 plus vincristine (compared with 73.6
To understand the compounds’ characteristics and to

1344 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
Ta ble 4. SAR Studies on 5- and 8-Pyridylindolopyrimidine Analogues
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
CYP3A4
compd
R1
R2
R3
X
MRP1 (accb)
MRP1 (sdpac)
Pgp (sdpac)
82
83
84
85
86
87
88
0.27
0.094
0.045
0.19
0.067
0.012
0.011
0.010
7.56
>10
19.3
ndd
>100
0.21
1.23
ndd
H
H
H
H
H
NO2
H
H
H
H
H
H
CN
CN
CONH2
CONH2
CONH2
CONH2
0.355
1.255
0.223
0.11
0.088
0.097
CH3
CH3
H
CH3
H
0.356
>10
>10
6.04
1.91
33.69
a
All the IC50 values in this text represent the mean of a minimum of three experiments. Variations among the results were less than
10%. acc: accumulation assay. c sdpa: single-dose potentiation assay. nd: not determined.
b
d
Ta ble 5. Pharmacokinetic Studies in Micea
activity with good selectivity against Pgp. The amenable
chemistry developed allowed the use of parallel synthe-
sis techniques to generate a library of analogues to
explore the templates on positions previously difficult
to access in the pyrrolopyrimidine series. We have
identified a solution to overcome the CYP 3A4 inhibition
problem on some of the active compounds by introducing
steric groups at the 3-position of the pyrimidine ring of
templates 3 and 4. Many compounds from these series
demonstrated good pharmacokinetic profiles and in vivo
efficacies in our xenograft models, exemplified by our
early compounds 52 and 60. More detailed efficacy
studies on some more analogues will be published
elsewhere.
dose (mg/kg) route Cmax (µM) AUC (µg‚h/mL) t1/2 (h) F (%)
Compound 52
20
50
50
iv
ip
po
37
84
13
94
76
123
4.6
2.6
6.0
81
52
Compound 60
20
50
iv
ip
9
5.1
30.57
2.4
6.86
26.43
Compound 69
20
iv
15
14
3.03
Compound 77
20
iv
11.88
6.32
1.97
a
Cmax, maximum plasma concentration; AUC, area under the
concentration-time curve; F, % bioavailability.
Exp er im en ta l Section
Meth od s a n d Ma ter ia ls. Reagents, starting materials, and
solvents were purchased from common commercial suppliers
and used as received or distilled from the appropriate drying
agent. Reactions requiring anhydrous conditions were per-
formed under an atmosphere of nitrogen or argon. Precoated
aluminum-backed silica gel 60 F254 plates with a layer thick-
ness of 0.25 mm were used for thin-layer chromatography, and
the stationary phase for preparative column chromatography
using medium pressure was silica gel 60, mesh size 40-60 µm
from E. Merck, Darmstadt, Germany.
NMR spectra were obtained using a Bruker ACF 400
operating at 400 MHz and the 1H shifts in ppm were calibrated
to that of residual CHCl3 in CDCl3 at 7.26 ppm. Mass spectra
were obtained in the indicated mode using a Finnigan SSQ
710L machine. Melting points were determined using an
electrothermal 9100 series apparatus.
The compound purity of all compounds tested in biological
systems was assessed as being >95% using HPLC using both
a water/acetonitrile gradient containing 0.02% TFA at 30 °C
on a Waters symmetry C18, 5 µM, 150 mm × 3.9 mm column
and a water/actonitrile gradient containing 0.05% phosphoric
acid at 30 °C on a LiChrospher RP-8, 5 µM, 250 mm × 4.6
mm column. UV photodiode array detection was applied.
Microanalyses were performed on a representation of com-
pounds by MEDAC Ltd. U.K. Where analyses are indicated
only by the symbols of the elements, results obtained were
within 0.4% of the theoretical values.
F igu r e 2. Xenograft curves depicting tumor growth inhibition
of subcutaneous COR L23/R cell in CD1 NUDE mice under
coadminstration of compound 52 or XR13097 with vincristine.
and 94.0 for vincristine alone and compound 60 alone,
respectively). In contrast, vincristine, 52, or 60 alone
had no significant effect on tumor growth. In addition,
the treatments with combination schedules were well
tolerated (loss of body weight less than 13%).
Con clu sion s
Gen er a l P r oced u r e for P r ep a r a t ion of An a logu es
8-10.2,13 A mixture of 1-chloro-9H-2,4,9-triazafluorene 6 (1.0
equiv), 1-[2-(3,4-difluorophenyl)ethyl]piperazine (1.2 equiv),
and triethylamine (1.1 equiv) was heated in dimethylforma-
mide at 80 °C overnight. The reaction mixture was cooled and
poured onto ice/water and the precipitated solid was collected
Through rational drug design by identifying the key
pharmacophore interactions in the pyrrolopyrimidine
template series exemplified by 1 and 2, we have
synthesized and investigated two novel template series
3 and 4, which showed very potent MRP1 modulating

Templates from Pyrrolopyrimidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1345
by filtration and triturated from hot ethyl acetate to yield the
desired title compounds 8 and 9.
3-Am in o-1-m eth yl-4-m eth oxy-1H-pyr r olo[3,2-c]pyr idin e-
2-ca r boxylic Acid Eth yl Ester (25). Sodium (0.417 g, 18.14
mmol, 3 equiv) was dissolved in methanol (20 mL) at room
temperature, to which was added 3-amino-4-chloro-1-methyl-
1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid ethyl ester (21)
(1.533 g, 6.05 mmol, 1.0 equiv). The mixture was heated at
reflux overnight. The solvent was removed, and water was
added to the residue. The resulting precipitate was filtered,
washed with water, and dried in vacuo to give the title
Gen er a l P r oced u r e for N-9 Alk yla tion of 1-{4-[2-(3,4-
Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9H -2,4,9-t r ia za -
flu or en e (9). To a suspension of NaH (1.1 equiv) in DMF
cooled to 0 °C was added a solution of the corresponding
triazafluorene derivatives 9 (1.0 equiv) in DMF dropwise over
10 min. The resultant mixture was stirred at 0 °C for a further
40 min before addition of an alkyl halide (1.1 equiv), and the
mixture was stirred at room temperature for 2 h. Purification
by flash chromatography with EtOAc/petrol/EtOH gave 10.
1
compound 25 (1.128 g, 79%). H NMR (CDCl3) δ 3.75 (s, 3H,
CH3O), 4.83 (s, 3H, CH3O), 4.0 (s, 3H, CH3N), 5.40 (br s, 2H,
NH2), 6.60 (d, 1H, PyH), 7.75 (d, 1H, PyH).
(2,3-Dicya n op h en yl)ca r ba m ic Acid Eth yl Ester (15).
3-Flouro-1,2-dicyanobenzene (2.48 g, 17.03 mmol) and glycine
ethyl ester HCl salt (1.05 equiv, 2.49 g, 17.88 mmol) were
dissolved in acetonitrile (25 mL). To this was added potassium
carbonate (2.2 equiv, 5.18 g, 37.47 mmol), and the reaction
mixture was refluxed overnight. The volatiles were removed
in vacuo, and the residue was taken up in DCM and washed
with water. The organic layer was dried (MgSO4), filtered, and
evaporated in vacuo. Subsequent purification by flash chro-
matography afforded the title compound as a beige solid (0.84
g, 19%). 1H NMR (CDCl3) δ 1.22 (t, 3H, J ) 7.1 Hz, CH3), 3.90
(d, 2H, J ) 5.4 Hz, CH2), 4.23 (q, 2H, J ) 7.1 Hz, CH2), 5.35
(br, 1H, NH), 6.70 (d, 1H, J ) 8.7 Hz, ArH), 6.98 (d, 1H, J )
8.7 Hz, ArH), 7.39 (t, 1H, J ) 8.8 Hz, ArH).
1,5-Dich lor o-9-m eth yl-9H-2,4,6,9-tetr aazaflu or en e (27b)
a n d 1,7-Dich lor o-9-m et h yl-9H-2,4,6,9-t et r a a za flu or en e-
(27a ). To a suspension of 9-methyl-2,9-dihydro-2,4,6,9-tet-
raazafluoren-1-one (23) (6.40 g, 32.0 mmol), prepared from
3-amino-1-methyl-1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid
ethyl ester (22) by standard protocol,2 in chloroform (500 mL)
at 0 °C was added m-chlorobenzoic acid (16.6 g, purity 77%
max), and the mixture was stirred at room temperature for
16 h. The material was then collected by filtration and washed
thoroughly with ether to give the N-oxide as pale-cream solid
24 (6.90 g, 99%). To this material was added triethylamine
hydrochloride (2.94 g, 21.3 mmol) and phosphorus oxychloride
(70 mL), and the mixture was heated under reflux for 2 h.
The majority of the phosphorus oxychoride was then removed
by distillation at reduced pressure. The residue was diluted
with toluene, and the solid was collected by filtration. The
residue was suspended in water, and the pH was adjusted to
7 by the addition of saturated aqueous sodium hydrogen
carbonate. The white solid was collected by filtration, washed
with water, and dried to give compound 27b (3.62 g, 54%). 1H
NMR (CDCl3) δ 4.20 (s, 3H, NCH3), 7.38 (d, 1H, J ) 6.0 Hz,
PyH), 8.50 (d, 1H, J ) 6.0 Hz, PyH), 9.03 (s, 1H, PymH).
The toluene phosphorus oxychloride residue was poured into
a vigorously stirred ice/water mixture, the pH was adjusted
to 7, and the mixture was then extracted with ethyl acetate
to give traces of the 5- and 7-chloropyridines. These were
purified by chromatography (50% ethyl acetate/petrol) to afford
compound 27a (145 mg, 1.8%). 1H NMR (CDCl3) δ 4.14 (s, 3H,
CH3), 7.42 (s, 1H, PyH), 8.88 (s, 1H, PymH), 9.44 (s, 1H, PyH).
5-Ch lor o-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-9-m eth yl-9H-2,4,6,9-tetr a a za flu or en e (29). To a solu-
tion of 1,5-dichloro-9-methyl-9H-2,4,6,9-tetraazafluorene 27b
(1.52 g. 6.01 mmol, 1.0 equiv) in DMF (30 mL) was added
triethylamine (0.92 mL, 6.61 mmol. 1.1 equiv) and the pip-
erazine side chain (1.68 g, 6.61 mmol, 1.1 equiv). The solution
was heated at 50 °C for 20 h, and the solvent was removed in
vacuo. The residue was partitioned between saturated aqueous
sodium hydrogen carbonate (60 mL) and chloroform (3 × 80
mL). The combined organic layers were washed with brine (50
mL), separated, and dried (MgSO4). The residue was triturated
in ethyl acetate to give a pale-cream solid (2.10 g, 79%). 1H
NMR (CDCl3) δ 2.59 (t, 2H, J ) 7.6 Hz, CH2), 2.66 (br s, 4H,
2 × CH2), 2.73 (t, 2H, J ) 7.6 Hz, CH2), 3.45 (br s, 4H, 2 ×
CH2), 3.97 (s, 3H, CH3), 6.85 (m, 1H, ArH), 7.03-6.91 (m, 2H,
ArH), 7.25 (d, 1H, J ) 5.8 Hz, PyH), 8.36 (d, 1H, J ) 5.8 Hz,
PyH), 8.83 (s, 1H, PymH); MS (DCI/NH3) m/z 443 (M + H)+.
3-Am in o-4-cya n oin d ole-1,2-d ica r boxylic Acid 1-ter t-
Bu tyl Ester 2-Eth yl Ester (16). (2,3-Dicyanophenyl)carbamic
acid ethyl ester (453 mg, 1.71 mmol) was dissolved in anhy-
drous DCM (5 mL), and to this was added triethylamine (1
equiv, 240 µL), BOC2O (1.2 equiv, 447 mg, 2.05 mmol), and
DMAP (0.1 equiv, 20 mg, 0.17 mmol). The reaction mixture
was stirred at room temperature overnight, diluted with DCM,
and washed with water. The organic layer was dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified
by flash chromatography to give the title compound as a light-
1
yellow oil (606 mg, 100%), which crystallized on standing. H
NMR (CDCl3) δ 1.18 (t, 3H, J ) 7.2 Hz, CH3), 1.20 (s, 9H,
t-BOC), 4.05 (q, 2H, J ) 7.1 Hz, CH2), 4.12-4.20 (br, 2H, NH2),
7.60-7.64 (m, 2H, ArH), 7.72-7.82 (m, 1H, ArH).
1-Acetyl-3-a m in o-4-n itr o-1H-in d ole-2-ca r boxylic Acid
(19). To a suspension of NaH (21 mg, 1.1 equiv) in THF (10
mL) at 0 °C was added a solution of N-(2-cyano-3-nitrophenyl)-
acetamide (100 mg, 0.49 mmol) in THF (10 mL) dropwise over
15 min. The resultant mixture was stirred at 0 °C for a further
40 min before addition of ethyl bromoacetate (0.06 mL, 1.1
equiv), and the mixture was allowed to warm to room tem-
perature and stirred for 2 h. Brine was added to the mixture,
the organic phase was collected, the aqueous layer was
extracted with EtOAc, and the combined organic fractions were
dried (MgSO4) and concentrated to give a red solid (126 mg,
1
88%). H NMR (CDCl3) δ 1.34 (t, 3H, J ) 7.1 Hz, CH3), 2.41
(s, 3H, CH3), 4.34 (q, 2H, J ) 7.1 Hz, CH2), 5.60 (br s, 2H,
NH2) 7.49 (dd, 1H, J ) 7.9 Hz, J ) 8.5 Hz, ArH), 7.90 (d, 1H,
J ) 7.9 Hz, ArH), 7.76 (d, 1H, J ) 8.5 Hz, ArH).
3-Am in o-4-ch lor o-1-m eth yl-1H-p yr r olo[3,2-c]p yr id in e-
2-ca r boxylic Acid Eth yl Ester (21). 21 was synthesized from
2-chloro-4-methylaminopyridine-3-nitrile (20) according to a
reported method.6
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-5-
m eth yl-9H-2,4,9-tr ia za flu or en e (43). 43 was synthesized by
the standard protocols2,13 from 3-amino-1-benzoyl-4-methyl-
1H-indole-2-carboxylic acid ethyl ester, which was prepared
from 2-amino-6-methylbenzonitrile using a reported proce-
dure.14 Mp 180-180.5 °C; 1H NMR (CDCl3) δ 2.6-2.74 (m, 6H,
3 × CH2), 2.81-2.85 (m, 2H, CH2), 3.05 (s, 3H, CH3), 3.87-
3.89 (m, 4H, 2 × CH2), 6.93-6.96 (m, 1H, ArH), 7.03-7.12
(m, 3H, 3 × ArH), 7.33 (d, 1H, J ) 8.2 Hz, ArH), 7.44 (t, 1H,
J ) 7.7 Hz, ArH), 7.96 (s, 1H, NH), 8.75 (s, 1H, PymH); MS
m/z 408.5 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-6-n i-
tr o-9H-2,4,9-tr ia za flu or en e (44). Sodium nitrate (0.26 g,
3.08 mmol, 1.1 equiv) was dissolved in concentrated sulfuric
acid (3 mL) with a few drops of water. This solution was added
dropwise to a stirring solution of commercially available
3-Am in o-1-m eth yl-1H-p yr r olo[3,2-c]p yr id in e-2-ca r box-
ylic Acid Eth yl Ester (22). To a solution of 3-amino-4-chloro-
1-methyl-1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid ethyl es-
ter (9.95 g, 39.2 mmol) in ethanol (400 mL) was added 10%
palladium on carbon (800 mg) as a slurry in ethanol under
argon. The mixture was purged with hydrogen and stirred for
24 h at atmospheric pressure. The mixture was purged with
argon and filtered through Celite, and the ethanol was
removed in vacuo. The residue was partitioned between
saturated aqueous sodium hydrogen carbonate (200 mL) and
ethyl acetate (3 × 250 mL). The combined organic layers were
dried over MgSO4 and the solvent was removed in vacuo to
give the title compound as a yellow solid (8.28 g, 96%). 1H NMR
(CDCl3) δ 1.28 (t, 3H, J ) 7.1 Hz, CH3), 4.35 (q, 2H, J ) 7.1
Hz, CH2), 3.95 (s, 3H, CH3), 5.19 (br s, 2H, NH2), 7.30 (d, 1H,
PyH), 7.25 (s, 1H, PyH), 8.38 (d, 1H, PyH), 8.95 (s, 1H, PymH).

1346 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
1-chloro-9H-2,4,9-triazafluorene (0.57 g, 2.8 mmol, 1.0equiv)
in concentrated sulfuric acid (20 mL) at 0 °C. The reaction
mixture was allowed to warm slowly to room temperature and
stirred overnight. The reaction mixture was poured onto ice,
and the precipitated solid was collected by filtration to yield
1-chloro-6-nitro-9H-2,4,9-triazafluorene (0.68 g, 98%). This
compound (0.265 g, 1.07 mmol) was treated according to the
general procedure to give 44 (0.351 g, 75%). 1H NMR (DMSO-
d6) δ 2.60 (m, 6H, 3 × CH2), 2.80 (m, 2H, CH2), 3.85 (m, 4H,
2 × CH2), 7.10 (m, 1H, ArH), 7.35 (m, 2H, ArH), 7.75 (d, 1H,
J ) 9.1 Hz, ArH), 8.40 (dd, 1H, J ) 9.0, 2.3 Hz, ArH), 8.55 (s,
1H, PymH), 8.90 (d, 1H, J ) 2.3 Hz, ArH), 12.05 (s, br, 1H,
NH); MS m/z: 439.3 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-6-
m eth oxy-9H-2,4,9-tr ia za flu or en e (45). Compound 45 was
prepared by treatment of 6 (when R ) 6-MeO) the side chain
13 according to a general procedure.2 1H NMR (CDCl3) δ 2.65
(m, 6H, 3 × CH2), 2.80 (m, 2H, CH2), 3.90 (s, 3H, OCH3), 3.95
(m, 4H, 2 × CH2), 6.95 (m, 1H, ArH), 7.05 (m, 2H, ArH), 7.20
(m, 1H, ArH), 7.40 (d, 1H, J ) 8.9 Hz, ArH), 7.70 (s, 1H, ArH),
8.20 (s, 1H, NH), 8.70 (s, 1H, PymH); MS m/z 424.1 (M + H)+.
collected and dried to give 1-{4-[2-(3,4-difluorophenyl)ethyl]-
piperazin-1-yl}-9H-2,4,9-triazafluorene-6-carboxylic acid as a
white solid (89 mg, 31%).
To a suspension of 1-{4-[2-(3,4-difluorophenyl)ethyl]piper-
azin-1-yl}-9H-2,4,9-triazafluorene-6-carboxylic acid (85 mg,
0.19 mmol) in DMF (3 mL) was added 1′,1-carbonyldiimidazole
(63 mg, 0.36 mmol, 2 equiv). The mixture was stirred for 1 h
at room temperature before addition of concentrated aqueous
ammonia (18.2 µL, 0.46 mmol, 2.4 equiv) and stirring over-
night. Water was added and extracted with ethyl acetate. The
organic layer was washed with brine and dried (Na2SO4), and
the solvent was removed in vacuo to give a crude product.
Purification by flash chromatography (2-20% MeOH/CH2Cl2)
gave 48 as a white solid (25 mg, 30%). Mp 303-304 °C; 1H
NMR (DMSO-d6) δ 2.42-2.52(m, 6H, 3 × CH2), 2.64 (t, 2H, J
) 7.5 Hz, CH2), 3.64 (t, 4H, J ) 4.5 Hz, 2 × CH2), 6.95 (m,
1H, CH), 7.08 (s, br, 1H, NH2), 7.28 (m, 2H, 2 × CH), 7.5 (d,-
1H, CH, J ) 8.7 Hz), 7.90 (br dd, 2H, CH + NH2), 8.34 (s, 1H,
CH), 8.57 (s, 1H, CH), 11.38 (br, s, 1H, NH); MS m/z 437 (M
+ H)+.
1-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-6-(pyr -
r olid in e-1-su lfon yl)-9H-2,4,9-tr ia za flu or en e (49). 1-Chloro-
9H-2,4,9-triazafluorene 6 (when R ) R′ ) H) (183 mg, 0.76
mmol) was added portionwise to chlorosulfonic acid (2 mL) at
0 °C. After 2 h, the mixture was heated to 100 °C and
maintained at this temperature for 1 h. The reaction mixture
was then cooled and poured onto ice/water carefully and the
resulting precipitate was collected by filtration to give 1-chloro-
9H-2,4,9-triazafluorene-6-sulfonyl chloride (221 mg, 96%). 1H
NMR (DMSO-d6) δ 7.55 (d, 1H, J ) 8.5 Hz, ArH), 7.88 (d, 1H,
J ) 8.5 Hz, ArH), 8.37 (s, 1H, PymH), 8.79 (s, 1H, ArH), 12.37
(s br, 1H, NH).
1-Chloro-9H-2,4,9-triazafluorene-6-sulfonyl chloride (83.6
mg, 0.28 mmol), pyrrolidine (44 µL, 0.54 mmol, 2 equiv), and
triethylamine (39 µL, 0.28 mmol, 1 equiv) were stirred together
in dichloromethane (3 mL) at room temperature. After the
mixture was stirred overnight, the reaction was quenched with
water. The mixture was then extracted into dichloromethane
and dried (Na2SO4), and the solvent was removed in vacuo to
yield a residue that was triturated with diethyl ether to give
1-chloro-6-(pyrrolidine-1-sulfonyl)-9H-2,4,9-triazafluorene (37.6
mg, 38%). 1H NMR (CDCl3/MeOD-d4) δ 1.67-1.72 (m, 4H, 2
× CH2), 3.22-3.27 (m, 4H, 2 × CH2), 7.68 (d, 1H, J ) 8.7 Hz,
ArH), 8.04 (d, 1H J ) 8.7 Hz, ArH), 8.82 (s, 1H, NH), 8.87 (s,
1H, PymH).
Subsequent treatment of 1-chloro-6-(pyrrolidine-1-sulfonyl)-
9H-2,4,9-triazafluorene according to standard protocol2 gave
product 49 (37 mg, 42%). Mp 238-239 °C; 1H NMR (CDCl3) δ
1.68-1.75 (m, 4H, 2 × CH2), 2.58-2.76 (m, 8H, 4 × CH2),
3.00-3.06 (m, 4H, 2 × CH2), 3.69-3.73 ((m, 4H, 2 × CH2),
6.82 (m, 1H, ArH), 6.90-7.00 (m, 2H, 2 × ArH), 7.44 (d, 1H,
J ) 8.6 Hz, ArH), 7.80 (d, 1H, J ) 8.6 Hz, ArH), 8.56 (s, 1H,
PymH), 8.63 (s, 1H, ArH), 8.88 (s, 1H, NH); MS m/z 527.4 (M
+ H)+.
N-4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en -6-yl)a ceta m id e (46). To a stirring sus-
pension of 1-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-1-yl}-9H-
2,4,9-triazafluoren-6-ylamine (0.05 g, 0.12 mmol) [prepared
from 1-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-1-yl}-6-nitro-
9H-2,4,9-triazafluorene 44 using hydrogenolysis (see method
for 55)] in anhydrous dichloromethane (3 mL) was added acetic
anhydride (11.5 µL, 0.12 mmol) followed by triethylamine (17
µL, 0.12 mmol). The mixture was stirred overnight at room
temperature under
a nitrogen atmosphere. The reaction
mixture was filtered to collect a white solid, which was further
purified by flash chromatography (silica gel, 5% MeOH/CHCl3)
to give product 46 (40 mg, 67%). Mp 234.5 °C; 1H NMR
(DMSO-d6) δ 2.18 (s, 3H, CH3), 2.56-2.72 (m, 6H, 3 × CH2),
2.80 (t, 2H, J ) 7.4 Hz, CH2), 3.70-3.88 (m, 4H, 2 × CH2),
7.08-7.18 (m, 1H, CH), 7.28-7.42 (m, 2H, 2 × CH), 7.53 (d,
1H, CH, J ) 8.8 Hz), 7.64 (dd, 1H, CH, J ) 1.8, 7.0 Hz), 8.45
(s, 2H, 2 × CH), 9.98 (s, 1H, NH), 11.18 (s, 1H, NH); MS m/z
451 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en e-6-ca r boxylic Acid Meth yl Ester (47).
2-Fluoro-5-formylbenzonitrile (9.22 g, 61.83 mmol) and pyri-
dinium dichromate (2 equiv, 123.65 mmol, 46.52 g) were
stirred in DMF (90 mL) at room temperature overnight. The
reaction mixture was diluted with ethyl acetate and washed
with water (×3), the organic layer was dried (MgSO4) and
filtered, and the volatiles were removed in vacuo to give
3-cyano-4-fluorobenzoic acid as a grayish solid (8.21 g, 80%).
3-Cyano-4-fluorobenzoic acid (8.21 g, 49.70 mmol) was sus-
pended in methanol (80 mL). To this was added acetyl chloride
(4 equiv, 14 mL), and the reaction mixture was stirred at room
temperature for 2 days. Then the reaction volume was reduced
in vacuo, and the precipitate was collected by filtration, washed
with water, and air-dried to give 3-cyano-4-fluorobenzoic acid
methyl ester as a white solid (7.25 g, 82%). Subsequent
treatment of 3-cyano-4-fluorobenzoic acid methyl ester accord-
ing to Scheme 2 and standard protocols2,13 afforded 47 (375
mg, 98%). Mp. 260-261 °C; 1H NMR (CDCl3/MeOD-d4) δ 2.56-
2.68 (m, 6H, 3 × CH2), 2.72-2.80 (m, 2H, CH2), 3.84-3.90 (m,
4H, 2 × CH2), 3.90 (s, 3H, MeO), 6.82-6.90 (m, 1H, ArH),
6.94-7.05 (m, 2H, ArH), 7.47 (d, 1H, J ) 8.6 Hz, ArH), 8.14
(d, 1H, J ) 8.6 Hz, ArH), 8.52 (s, 1H, ArH), 8.93 (s, 1H, PymH);
MS m/z 452 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en e-6-ca r boxylic Acid Am id e (48). To a
stirring solution of 1-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-
1-yl}-9H-2,4,9-triazafluorene-6-carboxylic acid methyl ester
(47) (0.3 g, 0.66 mmol) in ethanol (3 mL) was added a solution
of sodium hydroxide (53.2 mg, 1.32 mmol, 2 equiv). The
mixture was heated to reflux for 5 days before concentration
in vacuo. The resulting solid was dissolved in water, and the
solution was acidified by dropwise addition of concentrated
hydrochloric acid until a precipitate formed. The solid was
5-Ch lor o-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-9H-2,4,9-tr ia za flu or en e (50). 50 was synthesized from
2-chloro-6-fluorophenylnitrile according to Scheme 2 and a
general protocol.2 1H NMR (DMSO-d6) δ 2.65 (m, 6H, 3 × CH2),
2.75 (t, 2H, CH2), 3.75 (t, 4H, 2 × CH2), 7.05 (m, 1H, ArH),
7.21 (d, 1H, J ) 6.5 Hz, ArH), 7.30 (m, 2H, ArH), 7.50 (dd,
1H, J ) 6.5 Hz, 6.5 Hz, ArH), 7.55 (d, 1H, J ) 8.0 Hz, ArH),
8.50 (s, 1H, PymH), 11.60 (s, 1H, NH); MS m/z 428.1 (M +
H)+.
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en e-5-ca r bon itr ile (51). 51 was synthesized
from 3-amino-4-cyanoindole-1,2-dicarboxylic acid 1-tert-butyl
ester 2-ethyl ester (16) according to the general protocol.2 Mp
1
233-234 °C; H NMR (CDCl3/MeOD-d4) δ 2.75-2.88 (m, 6H,
3 × CH2), 2.90-3.00 (m, 2H, CH2), 4.00-4.08 (m, 4H, 2 × CH2),
7.00-7.04 (m, 1H, ArH), 7.12-7.20 (m, 2H, 2 × ArH), 7.63 (t,
1H, J ) 7.7 Hz, ArH), 7.2 (d, 1H, J ) 8.3 Hz, ArH), 7.89 (d,
1H, J ) 8.3 Hz, ArH), 8.80 (s, 1H,); MS m/z 419 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en e-5-ca r boxylic Acid Am id e (52). A mix-

Templates from Pyrrolopyrimidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1347
ture of compound 51 (0.5 g, 1.19 mmol), sodium hydroxide
solution (1 M, 1.2 mL), and 30% hydrogen peroxide solution
(0.7 mL) was stirred in methanol (30 mL) for 3 days. Saturated
sodium carbonate solution was added, resulting in a precipi-
tate, which was collected by filtration to yield a crude product
that was purified using flash chromatography to yield the title
protocol2 to give 58 (83.7 mg, 66%). Mp 276-277 °C; 1H NMR
(DMSO-d6) δ 2.63-2.70 (m, 6H, 3 × CH2), 2.77-2.81 (m, 2H,
CH2), 3.78 (m, 4H, 2 × CH2), 3.92 (s, 3H, CH3), 7.10-7.15 (m,
1H, ArH), 7.23-7.32 (m, 2H, 2 × ArH), 7.52 (d, 1H, J ) 8.9
Hz, ArH), 7.72 (d, 1H, J ) 9.3 Hz, ArH), 8.32 (s, 1H, PymH),
11.52 (s, 1H, NH); MS m/z 469.3 (M + H)+.
1
compound (0.475 g, 90%). Mp 274-276 °C; H NMR (DMSO-
1-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-3-m or -
p h olin -4-ylm eth yl-6-n itr o-9H-2,4,9-tr ia za flu or en e (59). To
a solution of 3-chloromethyl-2,9-dihydro-2,4,9-triazafluoren-
1-one 11 (when X ) Cl, R ) R′ ) H) (502 mg, 2.15 mmol, 1.0
equiv) in concentrated sulfuric acid (2 mL) at 0 °C was added
a solution of sodium nitrate (206.4 mg, 2.36 mmol, 1.1 equiv)
in concentrated sulfuric acid (2 mL) and water (1 drop)
dropwise. The resultant was stirred at 0 °C for 2 h before ice
was added. The precipitate obtained was collected by filtration,
washed with water, and dried to give 3-chloromethyl-6-nitro-
2,9-dihydro-2,4,9-triazafluoren-1-one as a yellow solid (560 mg,
d6) δ 2.54-2.68 (m, 6H, 3 × CH2), 2.72-2.80 (m, 2H, CH2),
3.28-3.35 (m, 4H, 2 × CH2), 7.04-7.10 (m, 1H, ArH), 7.25-
7.34 (m, 2H, 2 × ArH), 7.60 (t, 1H, J ) 7.8 Hz, ArH), 7.72 (s,
br, 1H, NH), 7.80 (d, 1H, J ) 7.8 Hz, ArH), 8.10 (d, 1H, J )
7.8 Hz, ArH), 8.50 (s, 1H, PymH), 11.70 (s br, 1H, NH), 11.72
(s br, 1H, NH); MS m/z 437 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en e-5-ca r boxylic Acid P yr id in -4-yla m id e
(53). 1-{4-[2-(3,4-Difluorophenyl)ethyl]piperazin-1-yl}-9H-
2,4,9-triazafluorene-5-carboxylic acid amide 52 (100 mg, 0.28
mmol) and 4-bromopyridine hydrochloride (1.1 equiv, 49 mg)
were suspended in 1,4-dioxane (1.5 mL). To this was added
cesium carbonate (2.5 equiv, 185 mg), Pd2(dba)3 (2 mol % Pd,
0.0023 mmol, 2 mg), and xanthphos (1.5 equiv to Pd, 0.0035
mmol, 2 mg). After 3 days at 100 °C, the reaction mixture was
diluted with DCM, washed with water (×2), and dried (Na2SO4),
and the solvent was removed in vacuo to yield a residue, which
was purified by column chromatography to give the title
compound as a light-yellow solid (16 mg, 11%). Mp 262-263
°C; 1H NMR (CDCl3/MeOD-d4) δ 2.45-2.52 (m, 6H, 3 × CH2),
2.60-2.63 (m, 2H, CH2), 3.75-3.80 (m, 4H, 2 × CH2), 6.68-
6.71 (m, 1H, ArH), 6.80-6.95 (m, 2H, 2 × ArH), 7.45 (t, 1H, J
) 8.0 Hz, ArH), 7.60 (d, 1H, J ) 8.2 Hz, ArH), 7.78 (d, 2H, J
) 5.0 Hz, ArH), 8.18 (d, 1H, J ) 7.5 Hz, ArH), 8.25 (d, 2H, J
) 6.5 Hz, ArH), 8.45 (s, 1H,PymH), 14.95 (s br, 1H, CONH);
MS m/z 514 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-9H-5-
n itr o-9H-2,4,9-tr ia za flu or en e (54). 54 was synthesized from
compound 19 according to the standard protocols.2,13 1H NMR
(DMSO) δ 2.75-2.85 (m, 6H, 3 × CH2), 2.95 (m, 2H, CH2),
4.00 (m, 4H, 2 × CH2), 7.26 (m, 1H, ArH), 7.50 (m, 2H, 2 ×
ArH), 7.84 (dd, 1H, J ) 8.1 Hz, J ) 7.5 Hz, ArH), 7.94 (d, 1H,
J ) 7.5 Hz, ArH), 8.14 (d, 1H, J ) 8.1 Hz, ArH), 8.64(s, 1H,
ArH), 12.14 (br s, 1H, NH); MS m/z 439.3 (M + H)+.
1
93%). H NMR (DMSO-d6) δ 4.62 (s, 2H, CH2), 7.62 (d, 1H, J
) 9.1 Hz, ArH), 8.24 (d, 1H, J ) 9.2 Hz, ArH), 8.75 (s, 1H,
ArH), 8.95 (s, 1H, NH), 9.07 (s, 1H, NH).
3-Chloromethyl-6-nitro-2,9-dihydro-2,4,9-triazafluoren-1-
one (296 mg,1.06 mmol), morpholine (0.14 mL, 1.6 mmol, 1.5
equiv), and anhydrous sodium carbonate (99 mg, 0.94 mmol,
1.4 equiv) were refluxed in absolute ethanol (5 mL) for 1.5 h.
The reaction mixture was cooled to room temperature, water
was added, and the resultant was extracted into ethyl acetate.
The organic layer was dried (Na2SO4), and the solvent was
removed in vacuo. 3-Morpholin-4-ylmethyl-6-nitro-2,9-dihydro-
2,4,9-triazafluoren-1-one (175 mg, 50%) was isolated and used
without further purification. 1H NMR (CDCl3/MeOD-d4) δ
2.59-2.62 (m, 4H, 2 × CH2), 3.64 (s, 2H, CH2), 3.74-3.77 (m,
4H, 2 × CH2), 7.55 (d, 1H, J ) 9.2 Hz, ArH), 8.32 (d, 1H, J )
9.2 Hz, ArH), 9.07 (s, 1H, ArH), 9.27 (s, 1H, NH), 9.38 (s, 1H,
NH).
Following the general protocols,2,3 the title compound 59 was
obtained (34%). Mp 110-111 °C; 1H NMR (CDCl3) δ 2.48-
2.68 (m, 12H, 6 × CH2), 3.60-3.63 (m, 4H, 2 × CH2), 3.77 (s,
2H, CH2), 3.89-3.91 (m, 4H, 2 × CH2), 6.78-6.80 (m, 1H,
ArH), 6.86-6.98 (m, 2H, 2 × ArH), 7.29 (d, 1H, J ) 9.1 Hz,
ArH), 8.06 (d, 1H, J ) 9.1 Hz, ArH), 8.92 (d, 1H, J ) 2.0 Hz,
ArH), 10.45 (s br, 1H, NH); MS m/z 538.4 (M + H)+.
1-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-3-m or -
ph olin -4-ylm eth yl-6,8-din itr o-9H-2,4,9-tr iazaflu or en e (60).
Compound 60 was prepared in an analogous manner to 59
except 3-chloromethyl-6,8-dinitro-2,9-dihydro-2,4,9-triazafluo-
ren-1-one was prepared in 92% yield from 3-chloromethyl-2,9-
dihydro-2,4,9-triazafluoren-1-one (2.32 g, 9.94 mmol) using 2.1
equiv of sodium nitrate (2.0 equiv, 20.23 mmol, 1.72 g) and
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9H -
2,4,9-tr ia za flu or en -5-yla m in e (55). Compound 54 (70 mg,
0.16 mmol) was stirred in MeOH (4 mL) in the presence of
10% Pd/C (cat.) under H2 balloon for 2 h. The mixture was
filtered through a pad of Celite and evaporated to dryness to
1
give the title compound 55 (30 mg, 46%). H NMR (CDCl3) δ
2.72-2.81 (m, 6H, 3 × CH2), 2.89 (m, 2H, CH2), 3.93 (br s,
4H, 2 × CH2), 5.46 (br s, 2H, NH2), 6.54 (d, 1H, J ) 7.7 Hz,
ArH), 6.86 (d,1H, J ) 8.0 Hz, ArH), 7.01 (m, 1H, ArH), 7.17
(m, 2H, 2 × ArH), 7.38 (dd, 1H, J ) 7.7 Hz, J ) 8.0 Hz, ArH),
7.83 (br s, 1H, NH), 8.70 (s, 1H, ArH); MS m/z 409.3 (M +
H)+.
1
stirring overnight at room temperature. H NMR (DMSO-d6)
δ 4.84 (s, 2H, CH2), 9.18 (s, 1H, ArH), 9.30 (s, 1H, ArH), 13.32
(s, br, 1H, NH), 13.45 (s, br, 1H, NH).
Subsequent displacement of chloride with morpholino group
(as per 59) and further reactions using general protocols2,3 as
1
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-7-
m eth oxy-9H-2,4,9-tr ia za flu or en e (56). 56 was synthesized
from compound 6 (when R ) 7-MeO, R′ ) H) according to
general protocol.2 1H NMR (CDCl3) δ 2.70 (m, 2H, CH2), 2.75
(m, 4H, 2 × CH2), 2.85 (m, 2H, CH2), 3.90 (m, 4H, 2 × CH2),
3.98 (s, 3H, CH3), 6.95 (m, 3H, 3 × ArH), 7.10 (m, 2H, ArH),
8.0 (s, 1H, NH), 8.18 (d, 1H, J ) 8.5 Hz, ArH), 8.70 (s, 1H,
PymH); MS m/z 424.4 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-7-
m eth oxy-8-n itr o-9H-2,4,9-tr ia za flu or en e (57). 57 was syn-
thesized from 1-chloro-7-methoxy-9H-2,4,9-triazafluorene us-
ing the nitration method described for compound 44. 1H NMR
(DMSO-d6) δ 2.45 (m, 6H, 3 × CH2), 2.70 (m, 2H, CH2), 3.70
(m, 4H, 2 × CH2), 3.90 (s, 3H, CH3O), 6.90 (m, 1H, ArH), 7.12
(d, 1H, J ) 8.9 Hz, ArH), 7.20 (m, 2H, ArH), 8.20 (d, 1H, J )
8.9 Hz, ArH), 8.38 (s, 1H, PymH); MS m/z 424.4 (M + H)+.
above gave product 60 (80% overall yield). Mp 69-70 °C; H
NMR (CDCl3) δ 2.60-2.68 (m, 10H, 5 × CH2), 2.74-2.78 (m,
2H, CH2), 3.73-3.75 (m, 4H, 2 × CH2), 3.80 (s, 2H, CH2), 3.91-
3.93 (m, 4H, 2 × CH2), 6.85-6.92 (m, 1H, ArH), 6.96-7.04
(m, 2H, 2 × ArH), 9.25 (d, 1H, J ) 2.0 Hz, ArH), 9.51 (d, 1H,
J ) 2.0 Hz, ArH), 10.03 (s br, 1H, NH); MS m/z 583.4 (M +
H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-5-n itr o-9H-2,4,9-tr ia za flu or en e (61). Compound 61
was prepared by treatment of compound 54 with iodomethane
under the general alkylation procedure. 1H NMR (CDCl3) δ
2.84 (m, 2H, CH2), 2.92 (m, 4H, 2 × CH2), 2.99 (m, 2H, CH2),
3.75 (m, 4H, 2 × CH2), 4.26 (s, 3H, CH3), 7.11 (m, 1H, ArH),
7.24 (m, 2H, ArH), 7.85 (dd, 1H, J ) 7.8 Hz, J ) 8.1 Hz, ArH),
7.94 (d, 1H, J ) 8.1 Hz, ArH), 8.07 (d, 1H, J ) 7.8 Hz, ArH),
9.01 (s, 1H, ArH); MS m/z 453.1 (M + H)+
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-6-
m eth oxy-5-n itr o-9H-2,4,9-tr ia za flu or en e (58). 1-Chloro-6-
methoxy-9H-2,4,9-triazafluorene (300 mg, 1.288 mmol) was
nitrated (99%) (see 59). The resulting 1-chloro-6-methoxy-5-
nitro-9H-2,4,9-triazafluorene was then treated per the general
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-5-n i-
tr o-9-p h en eth yl-9H-2,4,9-tr ia za flu or en e (62). Compound
62 was prepared by treatment of compound 54 with (2-
bromoethyl)benzene following the general alkylation proce-
1
dure. H NMR (CDCl3) δ 2.55 (m, 2H, CH2), 2.66 (m, 4H, 2 ×

1348 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
CH2), 2.73 (m, 2H, CH2), 3.34 (m, 6H, 3 × CH2), 4.7 (t, 2H, J
) 6.9 Hz, CH2), 6.63 (m, 2H, 2 × ArH), 6.85 (m, 1H, ArH),
6.98 (m, 5H, 5 × ArH), 7.61 (dd, 1H, J ) 8.2 Hz, J ) 7.9 Hz,
ArH), 7.74 (d, 1H, J ) 8.2 Hz, ArH), 7.81 (d, 1H, J ) 7.65,
ArH), 8.69 (s, 1H, ArH); MS m/z 543.5 (M + H)+.
compound 12 (R ) 5-NO2, R′ ) CH3), followed by NaOH/MeOH
hydrolysis. 1H NMR (acetone-d6) δ 2.62-2.89 (m, 12H, 6 ×
CH2, including HOD), 3.60 (m, 4H, 2 × CH2), 3.66-3.68 (m,
4H, 2 × CH2), 3.84 (s, 2H, CH2), 4.14 (s, 3H, CH3), 7.13 (m, H,
ArH), 7.19-7.31 (m, 2H, 2 × ArH), 7.75 (t, H, ArH), 7.88 (d,
H, J ) 8.3 Hz, ArH), 8.33 (d, H, ArH, J ) 7.5 Hz); MS m/z
550.5 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-9H-2,4,6,9-tetr a a za flu or en e (70). 70 was synthe-
sized from compound 22 using the general protocols.2,13 1H
NMR (CDCl3) δ 2.65 (m, 8H, 4 × CH2), 3.41 (br s, 4H, 2 ×
CH2), 3.91 (s, 3H, CH3), 6.94 (m, 3H, ArH), 7.27 (d, 1H, J )
5.9 Hz, PyH), 8.58 (d, 1H, J ) 5.9 Hz, PyH), 8.9 (s, 1H, PymH),
9.47 (s, 1H, PyH); MS m/z 409.4 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-5-n i-
tr o-9-pyr idin -3-ylm eth yl-9H-2,4,9-tr iazaflu or en e (63). Com-
pound 63 was prepared by treatment of compound 54 with
3-(iodomethyl)pyridine following the general alkylation pro-
cedure. 1H NMR (CDCl3) δ 2.56 (m, 6H, 3 × CH2), 2.70 (m,
2H, CH2), 5.71 (s, 2H, CH2), 6.85 (m, 1H, ArH), 6.9-7.2 (m,
4H, 4 × ArH), 7.5-7.6 (m, 2H, ArH), 7.75 (m, 1H, ArH), 8.47
(m, 1H, ArH), 8.83 (s, 1H, ArH); MS m/z 530.5 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-5-n i-
tr o-9-pyr idin -4-ylm eth yl-9H-2,4,9-tr iazaflu or en e (64). Com-
pound 64 was prepared by treatment of compound 54 with
4-(chloromethyl)pyridine in the presence of KI following the
general alkylation procedure. 1H NMR (CDCl3) δ 2.53 (m, 6H,
3 × CH2), 2.69 (m, 2H, CH2), 3.37 (m, 4H, 2 × CH2), 5.71 (s,
2H, CH2), 6.68 (m, 3H, ArH and d, J ) 4.5 Hz, 2 × PyH), 6.97
(m, 2H, ArH), 7.40 (d, 1H, J ) 8.3 Hz, ArH), 7.53 (dd, 1H, J )
8.3 Hz, J ) 7.3 Hz, ArH), 7.80 (d, 1H, J ) 7.3 Hz, ArH), 8.48
(d, 2H, J ) 4.5 Hz, PyH), 8.83 (s, 1H, ArH); MS m/z 530.5 (M
+ H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-9H-2,4,9-tr ia za flu or en e-5-ca r boxylic Acid Am id e
(65). Compound 65 was prepared by treatment of compound
51 with iodomethane following the general alkylation proce-
dure and then hydrolysis with NaOH/MeOH following the
procedure similar to that for 52. Mp 234-235 °C; 1H NMR
(CDCl3) δ 2.57 (m, 2H, CH2), 2.66 (m, 4H, 2 × CH2), 2.75 (m,
2H, CH2), 3.43 (m, 4H, 2 × CH2), 4.00 (s, 3H, CH3), 7.06 (m,
1H, ArH), 7.22-7.37 (m, 2H, 2 × ArH), 7.74 (dd, 1H, J ) 7.7
Hz, J ) 8.1 Hz), 7.83 (br s, 1H, NH), 7.91 (d, 1H, J ) 8.1 Hz,
ArH), 8.14 (d, 1H, J ) 7.8 Hz, ArH), 8.64 (s, 1H, ArH), 11.48
(br s, 1H, NH); MS m/z 451.1 (M + H)+.
9-Ben zyl-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-9H-2,4,6,9-tetr a a za flu or en e (71). 71 was synthesized
1
as 70. H NMR (CDCl3) δ 2.50 (m, 6H, 3 × CH2), 2.65 (m, 2H,
CH2), 3.35 (m, 4H, 2 × CH2), 5.58 (s, 2H, CH2), 6.75 (m, 1H,
ArH), 6.90 (m, 4H, ArH), 7.10 (d, 1H, J ) 5.9 Hz, PyH), 7.15
(m, 3H, ArH), 8.45 (d, 1H, J ) 5.9 Hz, PyH,), 8.70 (s, 1H,
PymH), 9.45 (s, 1H, PyH); MS m/z 485.4 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-5-
m eth oxy-9-m eth yl-9H-2,4,6,9-tetr a a za flu or en e (72). So-
dium (0.021 g, 0.91 mmol) was dissolved in MeOH (5 mL), to
which was added compound 29 (0.06 g, 0.135 mmol). The
mixture was then heated at reflux for 2 h. The solvent was
removed under vacuum, water was added to the residue, and
the resulting solid was filtered and dried to give the title
compound 72 (0.0409 g, 69% yield). 1H NMR (CDCl3) δ 2.60
(m, 2H, CH2), 2.67 (m, 4H, 2 × CH2), 2.72 (m, 2H, CH2), 3.40
(m, 4H, 2 × CH2), 3.95 (s, 3H, NCH3), 4.20 (s, 3H, CH3O), 6.88
(m, 1H, ArH), 6.92 (d, 1H, J ) 6.0 Hz, PyH), 7.0 (m, 2H, ArH),
8.15 (d, 1H, J ) 6.0 Hz, PyH), 8.78 (1H, s, PymH); MS (DCI/
NH3) m/z 439.1 (M + H)+.
1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-5-iso-
p r op oxy-9-m et h yl-9H -2,4,6,9-t et r a a za flu or en e (73). 73
was synthesized as 72. 1H NMR (CDCl3) δ 1.50 (d, 6H, J )
6.2 Hz, 2 × CH3), 2.60 (m, 2H, CH2), 2.65 (m, 4H, 2 × CH2),
2.72 (m, 2H, CH2), 3.40 (m, 4H, 2 × CH2), 3.92 (s, 3H, CH3),
5.60 (m, 1H, CH), 6.82 (m, 1H, ArH), 6.85 (d, 1H, J ) 6.0 Hz,
PyH), 7.0 (m, 2H, ArH), 8.12 (d, 1H, J ) 6.0 Hz, PyH), 8.80 (s,
1H, PymH); MS (DCI/NH3) m/z 467.5 (M + H)+.
(5-Ca r ba m oyl-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er -
a zin -1-yl}-2,4,9-tr ia za flu or en -9-yl)a cetic Acid Eth yl Ester
(66). Compound 66 was prepared by treatment of compound
52 with ethyl bromoacetate following the general alkylation
1
procedure. H NMR (CDCl3) δ 1.12 (t, 3H, J ) 6.9 Hz, CH3),
2.52-2.68 (m, 6H, 3 × CH2), 2.70-2.73 (m, 2H, CH2), 3.36-
3.43 (m, 4H, 2 × CH2), 4.12 (q, 2H, J ) 6.9 Hz, CH2), 5.18 (s,
2H, CH2), 6.05 (br s, 1H, NH), 6.82-6.87 (m, 1H, ArH), 6.93-
7.01 (m, 2H, 2 × ArH), 7.41 (d, 1H, J ) 7.5 Hz, ArH), 7.64 (t,
1H, J ) 7.5 Hz, ArH), 8.40 (d, 1H, J ) 7.5 Hz, ArH), 8.69 (s,
1H, ArH), 12.15 (br s, 1H, NH); MS m/z 523.0 (M + H)+.
1-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-9-pyr -
idin -4-ylm eth yl-9H-2,4,9-tr iazaflu or en e-5-car boxylic Acid
Am id e (67). Compound 67 was prepared by treatment of
compound 52 with 4-(chloromethyl)pyridine in the presence
of KI following the general alkylation procedure. 1H NMR
(CDCl3) δ 2.50-2.58 (m, 6H, 3 × CH2), 2.68-2.71 (m, 2H, CH2),
3.37-3.41 (m, 4H, 2 × CH2), 5.70 (s, 2H, CH2), 6.10 (br s, 1H,
NH), 6.82-6.86 (m, 3H, 3 × ArH), 6.95-7.05 (m, 2H, 2 × ArH),
7.38 (d, 1H, J ) 8.2 Hz, ArH), 7.60 (t, 1H, J ) 8.2 Hz), 8.42 (d,
1H, J ) 8.2 Hz, ArH), 8.46 (d, 2H, J ) 4.5 Hz, 2 × ArH), 8.72
(s, 1H, ArH), 12.24 (br s, 1H, NH); MS m/z 528.5 (M + H)+.
(1-{4-[2-(3,4-Diflu or op h en yl)eth yl]p ip er a zin -1-yl}-6-n i-
tr o-2,4,9-tr ia za flu or en -9-yl)a cetic Acid Eth yl Ester (68).
Compound 68 was prepared by treatment of the 6-nitro
derivative of compound 6 (R ) 6-NO2, R′ ) H) with ethyl
bromoacetate following the general alkylation procedure and
subsequent coupling with 13 using the general protocol.2 1H
NMR (CDCl3) δ 1.25 (t, 3H, J ) 6.9 Hz, CH3), 2.70-2.85 (m,
8H, 4 × CH2), 3.50 (m, 4H, 2 × CH2), 4.25 (q, 2H, J ) 6.9 Hz,
CH2), 5.25 (s, 2H, CH2), 6.95 (m, 1H, ArH), 7.10 (m, 2H, ArH),
7.40 (d, 1H, J ) 9.1 Hz, ArH), 8.50 (dd, 1H, J ) 9.1 Hz, 2.2
Hz, ArH), 8.90 (s, 1H, PrmH), 9.30 (d, 1H, J ) 2.2 Hz, ArH);
MS m/z 525.5 (M + H)+.
Gen er a l P r oced u r e for Rea ction s of Am in es w ith 29
(Sch em e 3, Step m ). P r ep a r a tion of 74-77. To the amine
(10 equiv) in n-butanol (2 mL) (gaseous amines were presatu-
rated in n-butanol) was added 5-chloro-1-{4-[2-(3,4-difluo-
rophenyl)ethyl]piperazin-1-yl}-9-methyl-9H-2,4,6,9-tetraaza-
fluorene (29) (50 mg, 0.11 mmol) and triethylamine (0.023 mL,
0.165 mmol, 1.5 equiv). The reaction mixture was heated at
100 °C for 48 h. After the mixture was cooled to room
temperature, volatiles were removed in vacuo. The residue was
partitioned between water (60 mL) and chloroform (3 × 80
mL). The combined organic layers were washed with brine (50
mL), separated, and dried (MgSO4). The residue was either
triturated or columned to give target compounds in 34-98%
yields.
(1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9-
m et h yl-9H -2,4,6,9-t et r a a za flu or en -5-yl)d im et h yla m in e
(74). This compound was prepared using the general method
above. The crude material was purified by chromatography
(5% f 10% methanol in chloroform) to give the title compound
as a white solid (38 mg, 74%). Mp 166-168 °C; 1H NMR
(CDCl3) δ 2.68 (t, 2H, J ) 6.3 Hz, CH2), 2.75 (br s, 4H, 2 ×
CH2), 2.82 (t, 2H, J ) 7.7 Hz, CH2), 3.3 (s, 6H, 2 × NCH3),
3.50 (br s, 4H, 2 × CH2), 3.96 (s, 3H, CH3), 6.73 (d, 1H, J )
6.1 Hz, CH), 6.89 (m, 1H, ArH), 7.11-7.00 (m, 2H, ArH), 8.2
(d, 1H, J ) 6.3 Hz, PyH), 8.7 (s, 1H, PymH); MS (DCI/NH3)
m/z 452.1 (M + H)+.
(1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-9-
m eth yl-9H-2,4,6,9-tetr a a za flu or en -5-yl)m eth yla m in e (75).
This compound was prepared using the general method above.
The crude material was purified by trituration with diethyl
ether to give the title compound as a white solid (42 mg, 85%).
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-3-m or p h olin -4-ylm eth yl-9H-2,4,9-tr ia za flu or en e-
5-ca r boxylic Acid Am id e (69). Compound 69 was prepared
by treatment of the 5-cyano-3-methylmorpholine derivative of
1
Mp 166-168 °C; H NMR (CDCl3) δ 2.68 (t, 2H, J ) 6.3 Hz,

Templates from Pyrrolopyrimidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1349
CH2), 2.75 (br s, 4H, 2 × CH2), 2.82 (t, 2H, J ) 7.7 Hz, CH2),
3.15 (d, 3H, J ) 5.1 Hz, NCH3), 3.40 (br s, 4H, 2 × CH2), 3.96
(s, 3H, NCH3), 6.6 (d, 1H, J ) 6.2 Hz, PyH), 6.7 (m, 1H, NH),
6.9 (m, 1H, ArH), 7.0-7.11 (m, 2H, ArH), 8.2 (d, 1H, J ) 6.0
Hz, PyH), 8.65 (s, 1H, PymH); MS (DCI/NH3) m/z 438.3 (M +
H)+.
(1-{4-[2-(3,4-Difluorophenyl)ethyl]piperazin-1-yl}-9-methyl-
9H2,4,6,9-tetr a a za flu or en -5-yl)p yr id in -2-ylm eth yla m in e
(76). This compound was prepared using the general method
above. The crude material was purified by chromatography
(4% methanol in chloroform) to give the title compound as a
white solid (20 mg, 34%). Mp 174-176 °C; 1H NMR (CDCl3) δ
2.68 (t, 2H, J ) 6.3 Hz, CH2), 2.75 (br s, 4H, 2 × CH2), 2.82 (t,
2H, J ) 7.7 Hz, CH2), 3.40 (br s, 4H, 2 × CH2), 3.96 (s, 3H,
NCH3), 5.08 (d, 2H, J ) 5.9 Hz, CH2), 6.68 (d, 1H, J ) 5.9 Hz,
PyH), 6.9 (m, 1H, ArH), 6.98-7.11 (m, 2H, ArH), 7.12-7.21
(m, 1H, PyH), 7.4 (d, 1H, J ) 7.9 Hz, PyH), 7.6 (t, 2H, J ) 5.9
Hz, PyH), 8.17 (d, 1H, J ) 6.4 Hz, PyH), 8.63 (d, 1H, J ) 5.9
Hz, PyH), 8.7 (s, 1H, PymH); MS (DCI/NH3) m/z 515.4 (M +
H)+.
3.40 (m, 4H, 2 × CH2), 3.70 (m, 4H, 2 × CH2), 3.80 (s, 2H,
CH2), 3.85 (s, 3H, CH3), 6.70 (m, 1H, ArH), 6.90 (m, 2H, ArH),
7.18 (d, 1H, J ) 5.88 Hz, PyH), 8.30 (d, 1H, J ) 5.88 Hz, PyH);
MS m/z 543.2 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-9H-2,4,5,9-tetr a a za flu or en e (82). 82 was prepared
from compound 36 by standard protocols described in Schemes
2 and 4 and in the literature.2,13 Mp 195-197 °C; 1H NMR
(MeOD-d4) δ 2.86 (m, 4H, 2 × CH2), 2.96 (m, 4H, 2 × CH2),
3.63 (m, 4H, 2 × CH2), 3.97 (s, 3H, CH3), 7.05 (m, 1H, ArH),
7.10 (m, 2H, 2 × ArH), 7.54 (dd, 1H, J ) 4.5 Hz, J ) 8.4 Hz,
ArH), 8.04 (d, 1H, J ) 8.4 Hz, ArH), 8.35 (d, 1H, J ) 4.5 Hz,
ArH), 8.58 (s, 1H, ArH); MS m/z 409.4 (M + H)+.
8-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m et h yl-9H -1,5,7,9-t et r a a za flu or en e-4-ca r b on it r ile (83).
[(3,4-Dicyanopyridin-2-yl)methylamino]acetic acid ethyl ester
(39)11 was prepared from 2-chloro-3,4-dicyanopyridine (38)10
(2.8 g, 17.09 mmol). A mixture of the above ester 39 and cesium
carbonate (3.07 g, 2.0 equiv) was heated in dry acetonitrile
(20 mL) at 50 °C. After 2 h, ice was added and the orange
precipitate that formed was collected, washed with water, and
dried to give 3-amino-4-cyano-1-methyl-1H-pyrrole[2,3-b]pyr-
idine-2-carboxylic acid ethyl ester 40 (1.07 g, 4.36 mmol, 92%).
Treatment of 40 according to general protocols2,13 gave the title
compound 83 (347 mg, 67%). Mp 180-181 °C; 1H NMR (CDCl3)
δ 2.49-2.53 (m, 2H, CH2), 2.59 (m, 4H, 2 × CH2), 2.63-2.67
(m, 2H, CH2), 3.41 (m, 4H, 2 × CH2), 3.97 (s, 3H, CH3), 6.74-
6.77 (m, H, ArH), 6.85-6.93 (m, 2H, 2 × ArH), 7.37 (d, H, J )
4.9 Hz, ArH,), 8.61 (d, H, J ) 4.9 Hz, ArH,), 8.70 (s, H, ArH);
MS m/z 435 (M + H)+.
1-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-5-m or ph olin -4-yl-9H-2,4,6,9-tetr aazaflu or en e (77).
This compound was prepared using the general method above.
The crude material was purified by chromatography (4%
methanol in chloroform) to give the title compound as a white
1
solid (35 mg, 62%). Mp 138-140 °C; H NMR (CDCl3) δ 2.68
(t, 2H, J ) 6.3 Hz, CH2), 2.75 (br s, 4H, 2 × CH2), 2.82 (t, 2H,
J ) 7.7 Hz, CH2), 3.40 (br s, 4H, 2 × CH2), 3.85 (br s, 4H, 2 ×
CH2), 3.96 (s, 3H, NCH3), 4.01 (m, 4H, 2 × CH2), 6.85 (d, 1H,
J ) 5.9 Hz, PyH), 6.9 (m, 1H, ArH), 6.98-7.11 (m, 2H, ArH),
8.2 (d, 1H, J ) 5.9 Hz, PyH), 8.65 (s, 1H, PymH); MS (DCI/
NH3) m/z 494.5 (M + H)+.
8-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-2,9-
d im eth yl-9H-1,5,7,9-tetr a a za flu or en e-4-ca r bon itr ile (84).
1-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-5,9-
d im eth yl-9H-2,4,6,9-tetr a a za flu or en e (78). To a solution of
5-chloro-1-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-1-yl}-9-
methyl-9H-2,4,6,9-tetraazafluorene 29 (50 mg, 0.11 mmol) in
THF (1.5 mL) was added palladium tetrakistriphenylphos-
phine (5 mol %, 6.5 mg) followed by methylzinc chloride (0.34
mL, 0.68 mmol, 6 equiv, 2.0 M solution in THF). The solution
was heated at reflux for 24 h and then poured into an aqueous
solution of EDTA (300 mg) in water (10 mL), and the pH was
adjusted to 7 by addition of potassium carbonate powder. The
solution was extracted with ethyl acetate (3 × 20 mL), the
combined organic layers were separated and dried (MgSO4),
and the solvent was removed in vacuo. The crude material was
purified by chromatography (5% f 10% methanol in chloro-
form) to give the title compound as a white solid (41 mg, 85%).
1
84 was synthesized as 83. Mp 178-179 °C; H NMR (CDCl3/
D2O) δ 2.66-2.83 (m, 11H, CH3 + 4 × CH2), 3.55 (m, 4H, 2 ×
CH2), 4.10 (s, 3H, CH3), 4.72 (s br, 2H, NH2), 6.93 (m, H, ArH),
7.02-7.10 (m, 2H, 2 × ArH), 7.40 (s, H, ArH), 8.85 (s, H, ArH);
MS m/z 448 (M + H)+.
8-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-2,9-
d im eth yl-9H-1,5,7,9-tetr a a za flu or en e-4-ca r boxylic Acid
Am id e (85). 85 was synthesized from 84 using the procedure
1
described for 52. Mp 259-260 °C; H NMR (CDCl3) δ 2.57-
2.63 (m, 2H, CH2), 2.70-2.89 (m, 9H, 3 × CH2 + CH3), 3.50
(m, 4H, 2 × CH2), 4.05 (s, 3H, CH3), 6.17 (s, br, H, NH), 6.84-
6.86 (m, H, ArH), 6.94-7.03 (m, 2H, 2 × ArH), 8.00 (s, H, ArH),
8.61 (s, H, ArH), 11.97 (s, H, NH); MS m/z 466 (M + H)+.
8-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m et h yl-9H -1,5,7,9-t et r a a za flu or en e-4-ca r b oxylic Acid
Am id e (86). 86 was synthesized from 83 using the procedure
1
Mp 168-170 °C; H NMR (CDCl3) δ 2.59 (m, 2H, CH2), 2.68
(br, s, 4H, 2 × CH2), 2.75 (t, 2H, J ) 7.7 Hz, CH2), 3.14 (s, 3H,
CH3), 3.45 (br, s, 4H, 2 × CH2), 3.94 (s, 3H, NCH3), 6.86 (m,
1H, ArH), 6.99 (m, 2H, ArH), 7.15 (d, 1H, J ) 5.9 Hz, PyH),
8.48 (d, 1H, J ) 5.9 Hz, PyH), 8.76 (s, 1H, PymH); MS (DCI/
NH3) m/z 423 (M + H)+.
1
described for 52. Mp 253-254 °C; H NMR (CDCl3) δ 2.60-
2.63 (m, 2H, CH2), 2.70 (m, 4H, 2 × CH2), 2.73-2.77 (m, 2H,
CH2), 3.53 (m, 4H, 2 × CH2), 4.09 (s, 3H, CH3), 66.12 (s, br, H,
NH), 6.84-6.87 (m, H, ArH), 6.97-7.03 (m, 2H, 2 × ArH), 8.15
(d, H, J ) 5.0 Hz, ArH), 8.64 (s, H, NH), 8.77 (d, H, J ) 5.0
Hz, ArH), 12.01 (s, H, NH); MS m/z 452 (M + H)+.
7-Ch lor o-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-9-m eth yl-9H-2,4,6,9-tetr a a za flu or en e (79). This com-
pound was prepared from 27a using the same method as for
29. 1H NMR (CDCl3) δ 2.59 (m, 2H, CH2), 2.67 (br s, 2H, CH2),
2.75 (br s, 4H, 2 × CH2), 3.47 (br s, 4H, 2 × CH2), 3.92 (s, 3H,
CH3), 6.87 (m, 1H, ArH), 7.04-6.93 (m, 2H, ArH), 7.34 (s, 1H,
PyH), 8.70 (s, 1H, PrmH), 9.23 (s, 1H, PyH); MS (DCI/NH3)
m/z 442 (M + H)+.
8-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-2,9-
d im et h yl-3-n it r o-9H -1,5,7,9-t et r a a za flu or en e-4-ca r b ox-
ylic Acid Am id e (87). 87 was prepared from 2-chloro-6-
methyl-5-nitropyridine-3,4-dicarbonitrile in a manner analo-
gous to that of 83, followed by the procedure for 52 (46 mg,
1
70%). Mp 217-219 °C; H NMR (CDCl3) δ 2.60-2.64 (m, 2H,
CH2), 2.68 (s, 3H, CH3), 2.68-2.72 (m, 4H, 2 × CH2), 2.74-
2.78 (m, 2H, CH2), 3.50-3.55 (m, 4H, 2 × CH2), 4.10 (s, 3H,
CH3), 6.10 (s, br, 1H, NH), 6.83-6.88 (m, 1H, ArH), 6.98-7.04
(m, 2H, 2 × ArH), 8.67 (s, 1H, ArH), 11.67 (br s, 1H, NH); MS
m/z 511 (M + H)+.
8-{4-[2-(3,4-Diflu or op h e n yl)e t h yl]p ip e r a zin -1-yl}-9-
m eth yl-6-m or p h olin -4-ylm eth yl-9H-1,5,7,9-tetr a a za flu o-
r en e-4-ca r boxylic Acid Am id e (88). 88 was prepared from
compound 40 (Scheme 5) according to standard protocol.3 Mp
195-196 °C; 1H NMR (CDCl3) δ 2.56-2.63 (m, 6H, 3 × CH2),
2.69 (m, 4H, 2 × CH2), 2.73-2.73 (m, 2H, CH2), 3.52 (m, 4H,
2 × CH2), 3.68-3.70 (m, 4H, 2 × CH2), 3.81 (s, 2H, CH2), 4.04
(s, 3H, CH3), 6.12 (s, br, H, NH), 6.84-6.86 (m, H, ArH), 6.95-
5-Ch lor o-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-3-m eth oxym eth yl-9-m eth yl-9H-2,4,6,9-tetr a a za flu o-
r en e (80). 80 was synthesized from compound 25 using the
literature protocol.3 1H NMR (CDCl3) δ 2.60 (m, 2H, CH2), 2.70
(m, 4H, 2 × CH2), 2.80 (m, 2H, CH2), 3.50 (m, 4H, 2 × CH2),
3.58 (s, 3H, CH3O), 4.0 (s, 3H, CH3N), 4.75 (s, 2H, CH2), 6.90
(m, 1H, ArH), 7.05 (m, 2H, ArH), 7.30 (d, 1H, J ) 5.8 Hz, PyH),
8.40 (d, 1H, J ) 5.8 Hz, PyH); MS m/z 488.1 (M + H)+
5-Ch lor o-1-{4-[2-(3,4-d iflu or op h en yl)eth yl]p ip er a zin -
1-yl}-9-m e t h yl-3-m or p h olin -4-ylm e t h yl-9H -2,4,6,9-t e t -
r a a za flu or en e (81). 81 was synthesized from 25 using the
literature protocol.3 1H NMR (CDCl3) δ 2.51 (m, 2H, CH2), 2.60
(m, 4H, 2 × CH2), 2.65 (m, 2H, CH2), 2.70 (m, 4H, 2 × CH2),

1350 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
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7.03 (m, 2H, 2 × ArH), 8.13 (d, H, J ) 5.0 Hz, ArH), 8.75 (d,
H, J ) 5.0 Hz, ArH), 12.27 (br s, H, NH); MS m/z 551 (M +
H)+.
Biology Assa ys.2 In Vitr o Assa ys. Drug accumulation
assays (MRP1 and Pgp) have been described and referenced
in detail in our previous communication.2
CYP 3A4 In h ibition Assa y. This was carried out according
to a literature protocol.2,15
Sin gle-Dose P oten tia tion Assa y (MRP 1). The ability of
modulators to potentiate drug cytotoxicity in COR.L23 cells
was assessed at a single dose of the cytotoxic drug doxorubicin,
selected to provide a differential window of activity between
the parental cell line COR.L23/P (80-100% growth inhibitory
activity) and the drug-selected resistant cell line COR.L23/R
(0-10% growth inhibitory activity). Addition of the MRP-1
modulator to the resistant cell line effected a dose-dependent
potentiation of drug cytotoxicity to levels observed with the
parental cell line.
Briefly, cells were seeded into 96-well plates at (2-4) × 102
cells per 100 µL/well and incubated for 2 h at 37 °C. Varying
concentrations of modulator (or solvent control) were subse-
quently added at 50 µL/well, in quadruplicate for each
concentration, and incubated for an additional hour before
addition of 50 µL/well doxorubicin to a final concentration of
100 nM. After incubation for 5 days, cell proliferation was
assessed by addition of 10 µL/well alamarBlue. Viable cell
number, proportional to fluorescence (excitation λ 560/15 nm;
emission λ 590/35 nm), was determined after further incuba-
tion for 5 h. IC50 values were calculated for the modulator in
the presence of a single dose of cytotoxic, using media-only
wells as background and untreated, resistant cells as being
representative of 100% viability.
Similar protocols were applied for Pgp single-dose potentia-
tion assays with the use of Pgp expressing murine mammary
carcinoma EMT6/AR1.0 subline.
P h a r m a cok in etic Stu d ies a n d in Vivo Effica cy Stu d -
ies. Protocols for these studies have been described in detail
in our early communication.2
Ack n ow led gm en t. The authors thank Mrs. Xiaol-
ing Cockcroft from Chemovation Ltd. (U.K.) for model-
ing the work in this project.
Refer en ces
(1) (a) Persidis, A. Cancer multidrug resistance. Nat. Biotechnol.
1999, 17, 94-95. (b) Norman, B. H. Inhibitors of MRP1-mediated
multidrug resistance. Drugs Future 1998, 23 (9), 1001-1013.
J M0310129