Studies on Pyrrolopyrimidines as Selective Inhibitors of Multidrug-Resistance-Associated Protein in Multidrug Resistance

Article abstract of DOI:10.1021/jm031011g

Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. Whi

Full text of DOI:10.1021/jm031011g

J . Med. Chem. 2004, 47, 1329-1338
1329
Articles
Stu d ies on P yr r olop yr im id in es a s Selective In h ibitor s of Mu ltid r u g-Resista n ce-
Associa ted P r otein in Mu ltid r u g Resista n ce
Shouming Wang,*,† Adrian Folkes,† Irina Chuckowree,† Xiaoling Cockcroft,† Sukhjit Sohal,† Warren Miller,†
J ohn Milton,† Stephen P. Wren,† Nigel Vicker,† Paul Depledge,‡ J ohn Scott,‡ Lyndsay Smith,‡ Hazel J ones,‡
Prakash Mistry,‡ Richard Faint,‡ Deanne Thompson,§ and Simon Cocks§
Department of Medicinal Chemistry, Department of Pharmacology, and Analytical Department, Xenova Ltd.,
957 Buckingham Avenue, Slough, Berkshire SL1 4NL, U.K.
Received August 21, 2003
Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated
protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp
and MRP transport 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. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel
and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed
the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the
lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on
activity. Modifications to the nitrile group at the 7-position resulted in the identification of
analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy
has been demonstrated by xenograft studies on selected compounds.
The appearance of tumor cells resistant to a range of
cytotoxic drugs is a serious problem in cancer chemo-
therapy. This phenomenon is called multidrug resis-
tance (MDR). One form of MDR can be caused by
members of the ATP-binding cassette (ABC) family of
transport proteins.1 These are large polytopic membrane
proteins that actively transport drugs out of cells,
resulting in a decreased intracellular drug concentra-
tion. In humans, two ABC transporters have been
identified that cause resistance in tumor cells: P-
glycoprotein (Pgp) (MDR1)2 and the multidrug resis-
tance associated protein (MRP1).3 Pgp transports drugs
in an unmodified form, whereas MRP1 transports drugs
conjugated to the anionic ligands glutathione (GSH),
glucuronide, or sulfate4 or transports them in an
unmodified form, probably together with GSH.5 Among
those cytotoxics transported by MRP1 are various
natural product oncolytics, such as vinca alkaloids,
epipodophyllotoxins, anthracyclines, and camptoth-
ecins,6 most of which are also substrates for Pgp
transport,7 although taxanes are apparently not subject
to MRP1 mediated resistance.8 Furthermore, MRP1
transports leukotriene C4 (LTC4) as substrate in an
ATP-dependent fashion with high efficiency.9
overexpressed at the same time in drug-resistant cells.12
The correlation between drug resistance and expression
of the drug efflux pumps, Pgp and MRP1, has spurred
considerable efforts in the development of inhibitors of
Pgp and MRP1.13 Recently, Robert has reviewed a
number of MDR reversing agents in clinical trials,14
including MS-209, a dual modulator against both Pgp
and MRP.15 Among the specific potent inhibitors of Pgp
entered into clinical evaluation are GF120918,16
LY335979,17 OC144-093,18 and XR9576.19 However, the
development of specific MRP1 modulators is still in its
infancy,13b although Eli Lilly has reported the raloxifene
analogues 120 and isooxazoloquinoline analogues 221 as
selective MRP1 inhibitors.
Previously, we have reported the dual inhibitory
activities of quinazolinone analogues 3 against Pgp and
MRP1.22 In our continued effort to develop selective
MRP1 inhibitors, we screened our in-house chemical
library against the target. This led to the identification
of the pyrrolopyrimidine analogue 4 as the lead mol-
ecule. Compound 4 is a highly selective MRP1 inhibitor
with an IC50 of 3.9 µM (concentration resulting in 50%
inhibition of tritiated daunomycin efflux by cells) in drug
accumulation assays using the MRP1 expressing cell
line COR.L23/R and with an IC50 greater than 50 µM
The differential expression and tissue/tumor specifici-
ties of Pgp and MRP1 have been reviewed recently,10,11
although it is also known that Pgp and MRP can be
* 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.
‡ Department of Pharmacology.
§ Analytical Department.
10.1021/jm031011g CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/07/2004

1330 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
Sch em e 2a
for Pgp activity (EMT6/AR1.0 subline). Here, we report
our SAR studies of pyrrolopyrimidine analogues as novel
and selective MRP1 inhibitors.
Our lead optimization strategy for this series was
initially aimed at the following: (i) replacement of
benzylthio group at the 4-position by amines in order
to enhance diversity and remove the metabolic liability
of the sulfur atom; (ii) modification of the nitrile group
at the 7-position into other functional groups to define
their importance of binding in this region; (iii) introduc-
tion of substituents at the 2-position to identify potential
new binding interactions in this area. The above posi-
tions were chosen for structural modification because
they explore different areas of space. Other consider-
ations were the ease of synthetic access and the avail-
ability of reagents and functional groups suitable for
manipulation. The common intermediate 11 (Scheme 1)
allows rapid exploration of the 4-position, and the
2-position is easily modified as outlined in Scheme 2. A
variety of structurally diverse commercially available
amines were identified to react with the common
intermediate 11 to probe what functionality is tolerated
in this region. The nitrile group at the 7-position is a
versatile moiety for functional group manipulation as
indicated in Scheme 3.
a
Reagents: (a) (i) TMP, n-BuLi, -78 °C, THF, (ii) n-Bu3SnCl,
70% (two steps); (b) R1R2NH, i-PrOH, Et3N, reflux, 72%-95%; (c)
I2, THF, room temp (15), 81%; ArI, Pd(Ph3P)4, CuI, TEA, THF,
argon, reflux, 73-85%.
Sch em e 3a
Ch em istr y
At first, we investigated the possibility of replacing
the thiobenzyl side chain with amines because this
would allow the rapid introduction of diversity through
parallel synthesis. A library of such compounds (12)
were prepared via the key intermediate, chloropyrimi-
dine 11, following a literature protocol23 (Scheme 1).
Sch em e 1a
a
Reagents: (a) RMgBr, THF, 60 °C, 75-85%; (b) (i) NaHS,
DMF, 60 °C, 5 days; (ii) R′CH(X)COR′′, EtOH, 100 °C, 82-93%;
(c) NH2OH‚HCl, K2CO3, EtOH, reflux, 82%; (d) RCOCl, 60 °C, 18%;
(e) H2, Pd/C, room temp, 72%; (f) (MeO)2CHCH2CH(MeO)2,
Dowtherm, 175 °C, 83%; (g) NaOH (1.0 equiv, 1 M), H2O2 (27%, 5
equiv), MeOH, room temp, 85%; (h) RCHO, Et3SiH, TFA, CH3CN,
reflux, 25-90%.
The synthesis of 2-substituted pyrrolopyrimidine
analogue is depicted in Scheme 2.
Chloropyrimidine 11 was lithiated with n-BuLi in the
presence of 2,2,6,6-tetramethylpiperidine (TMP) in THF
at -78 °C, followed by quenching with tri-n-butyltin
chloride to give compound 13 in 70% yield. The subse-
quent replacement of the chloro group with amines gave
the intermediate products 14. These were converted into
either the 2-iodo analogue 15 or 2-aryl analogues 16
under Stille coupling conditions.24
To ensure a convergent route to rapid analogue
generation, we focused on the transformation of the
nitrile group of 12, as shown in Scheme 3. Thus, a
number of functional groups were introduced, including
ketones (17), thiazoles (18), oxadiazoles (20), pyrim-
idines (22), and amides (23, 24). The preparation of the
a
Reagents: (a) EtOH, room temp, 97%; (b) BrCH2CO2Et,
K2CO3, DMF, 100 °C, 55%; (c) (MeO)2CHN(CH3)2, DMF, 100 °C,
90%; (d) NH3, EtOH, bomb, 110°C, 80%; (e) POCl3, TEA‚HCl (0.4
equiv), reflux, 85%; (f) R1R2NH, DMF, 100 °C, 82-95%.

Pyrrolopyrimidines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1331
Ta ble 1. Inhibitory Activitya of 4-Substituted Compounds in
Accumulation Assays for MRP1(L23/R) and Pgp (EMT6/AR1.0)
Ta ble 2. Inhibitory Activity of Substituted Phenethylpipera-
zine Compounds in Accumulation Assays for Pgp(EMT6/AR1.0)
and MRP1(L23/R)
compd
X
IC50 (µM)/MRP1a
IC50 (µM)/Pgpa
30
34
35
36
37
38
39
40
41
42
H
2-Cl
3-Cl
4-Cl
2-NO2
3-NO2
4-NO2
3-MeO
4-MeO
3,4-F,F
0.69
0.955
0.92
0.399
1.415
0.41
0.523
2.30
9.63
10.6
17.9
9.2
>50
9.665
12.94
16.2
ndb
>50
0.229
10.52
a
All the IC50 values in this text represent the mean of a
minimum of three experiments. Variations among the results were
b
less than 10%. nd: not determined.
MRP1 activity and good selectivity (29, 30). Further
modifications of the piperazine ring, such as different
size ring structures with or without additional func-
tional groups (data not shown) and its N-alkyl side
chain, did not give any improvement in potency and
selectivity (31-33). Given that both indole and phenyl
analogues 29 and 30 showed similar activity and
selectivity, it was decided to examine the SAR of the
phenyl ring of analogue 30 in more detail. Some of the
data in this area is illustrated in Table 2.
As shown in Table 2, ortho-substituted analogues 34
and 37 appeared to be marginally less active than the
parent analogue 30 in MRP1 modulation. Compounds
with electron-withdrawing groups at the 3- or 4-position
(36, 38, 39) gave better MRP1 activity than those with
electron-donating groups (40, 41). In particular, the 3,4-
difluorophenethyl analogue 42 appeared to be 3-fold
more potent than the parent compound 30. Conse-
quently, this side chain and the readily available
phenethylpiperazine side chain present in compound 30
were retained for further optimization work in other
areas of the lead molecule 4.
Next, we turned our attention to the SAR studies on
2-substituted pyrrolopyrimidine analogues, which were
conveniently prepared according to the chemistry de-
scribed in Scheme 2. Table 3 exemplifies some of the
active analogues in this area.
Compared with the activity of compound 30, the
introduction of iodo and aryl groups at the 2-position of
the pyrimidine ring was not detrimental to activity, and
the MRP1 selectivity over Pgp was retained (43-45).
The corresponding pyridine analogues (46, 47) showed
good MRP1 activity comparable to that of compound 42,
with 47 showing >2-fold improvement in potency. The
more sterically hindered 2-methylphenyl-substituted
compound 48 showed 3-fold less potency than compound
42. Therefore, substitution at this position was generally
well tolerated and this position was identified as a good
point for attaching solubilizing groups without reducing
the inherent MRP1activity (results to be published).
Although we had made significant progress in finding
selective and potent MRP1 inhibitors, our early evalu-
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%.
secondary amides (24) were achieved from the primary
amides (23) using modified literature procedures.25
Resu lts a n d Discu ssion
By use of the synthetic method described in Scheme
1, a library of compounds (12) were prepared and
evaluated in drug accumulation assays using both
MRP1 expressing cell line COR.L23/R and a Pgp
expressing cell line EMT6/AR1.0. The initial screening
of this library of compounds uncovered a number of
active compounds that showed significant MRP1 inhibi-
tory activity and selectivity against Pgp. Some repre-
sentative examples are illustrated in Table 1.
Replacement of the benzylthio group within lead
molecule 4 with primary and secondary amines ap-
peared to retain the MRP1 inhibitory activity and some
selectivity against Pgp as represented by compounds 25,
26, and 28. In general, amide or sulfonamide linkages
resulted in a decrease of MRP1 activity and poor
selectivity over Pgp (27). However, compounds bearing
a piperazine linkage exhibited significantly improved

1332 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
Ta ble 3. Inhibitory Activity for 2-Substituted Pyrrolopyrimi-
dine Compounds in Accumulation Assays for Pgp(EMT6/AR1.0)
and MRP1(L23/R)
Ta ble 4. Inhibitory Activity for 7-Substituted Pyrrolopyrimi-
dine Compounds in Accumulation Assays for Pgp(EMT6/AR1.0)
and MRP1(L23/R)
IC50 (µM)/ IC50 (µM)/
compd
R
Ar
MRP1a
Pgpa
30
43
44
45
46
47
48
H
I
Ph
Ph
Ph
Ph
0.69
9.63
13.05
5.0
0.887
0.715
0.57
0.218
0.105
0.62
4-CH3CONHPh Ph
3-Pyr
4-Pyr
2-CH3-Ph
23.7
3,4-(F,F)-Ph
3,4-(F,F)-Ph
3,4-(F,F)-Ph
ndb
1.31
17.99
a
All the IC50 values in this text represent the mean of a
minimum of three experiments. Variations among the results were
b
less than 10%. nd: not determined.
ation of some of the active compounds suggested only a
limited degree of metabolic stability with poor pharma-
cokinetic profiles. Pharmacokinetic studies of two of our
early lead compounds, 30 and 42, showed very poor half-
lives of less than 30 min after intravenous administra-
tion. We speculated that the nitrile group at the
7-position on template 4 could be a metabolic weak point
and sought to identify a replacement for the nitrile
group according to the chemistry described in Scheme
3. Some of the examples are shown in Table 4.
Conversion of the nitrile into ketones and the corre-
sponding methyl ester gave compounds 49-51 with
reduced MRP1 activity when compared with analogue
30. Transformation of the nitrile into various hetero-
cycles, such as thiazole, oxadiazole, and pyrimidine (52-
54), gave compounds with similar activity. With the
more active side chain, 3,4-difluorophenethylpiperazine,
at the 4-position, the simple amide analogues 55 and
56 appeared to be 2-fold more potent than the corre-
sponding nitrile analogue 42. Further parallel synthesis
revealed more secondary amides with significantly
enhanced potency against MRP1 and good selectivity
over Pgp (57-59). To further demonstrate the MRP1
inhibitory activity, many compounds were subjected to
potentiation assays.26 Table 5 illustrates the data on
several of these compounds (55-59). Most of the com-
pounds showed approximately 100 nM potency (55-58),
while 59 was 2-fold less potent in this particular assay.
To summarize the above results, it is clear that the
functional groups at the 7-position are critical for
activity, probably as hydrogen bond acceptors in phar-
macophoric interactions.
It is widely recognized that the cytochrome P450s
(CYP) are a major class of oxidative enzymes involved
in drug metablolism.27 Because any potential MRP1
modulators will be coadministered with cytotoxics, it is
important to gain some understanding on potential
drug-drug interaction of a molecule before it reaches
the later stages of drug development. Therefore, we
subjected a number of active compounds to CYP assays,
particularly CYP 3A4 (Table 5) because it is considered
the major oxidative enzyme for cytotoxic drugs and is
expressed at a high level relative to the other hepatic
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%.
Ta ble 5. Inhibitory Activity of Compounds (55-59) in MRP
Potentiation Assay (EC50) and Drug Metabolism Studies with
CYP 3A4 Assay (IC50
)
compd
EC50 (µM, PA)a
IC50 (µM, CYP3A4)a
55
56
57
58
59
0.117
0.10
0.15
0.097
0.235
45.8
>100
2.44
30.15
9.73
a
All the EC50 and IC50 values in this text represent the mean
of a minimum of three experiments. Variations among the results
were less than 10%.
CYPs.28 As the data in Table 5 show, most of these
compounds showed little inhibition of CYP 3A4 and
these compounds were further evaluated in pharmaco-
29

Pyrrolopyrimidines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1333
Ta ble 6. Pharmacokinetic Studies in Balb/C Micea
dose (mg/kg) route Cmax (µM) AUC (µg‚h/mL) t1/2 (h) F (%)
Compound 55
ments with the combination schedules were well toler-
ated (loss of body weight less than 15%).
Con clu sion
20
20
50
iv
ip
po
28
4.43
8.37
25
13.06
44.92
1.47
6.35
6.86
52
71.6
In the present report we have described some syn-
thesis procedures and SAR studies of our lead MRP1
inhibitor 4 through structural modifications at the 2-,
4-, and 7-position of the pyrrolopyrimidine template.
Through this study, we established that substitution at
the 2-position was not detrimental to activity and 3,4-
difluorophenylethylpiperazine was a well tolerated side
chain at the 4-position, whereas various H-bond accep-
tor groups were identified at the 7-position to confer
activity through crucial pharmacophoric interactions. As
a result, a number of novel, potent, and selective MRP1
modulators with good pharmacokinetic profiles were
identified. Their in vivo efficacies were also demon-
strated in xenograft models, exemplified by compound
55, XR12890. Further in vivo studies with different
cytotoxic agents and this compound will be published
elsewhere.
Compound 56
20
20
50
iv
ip
po
18.31
7.34
5.73
16.94
14.55
21.12
2.3
1.84
7.9
85
50
Compound 57
20
20
iv
iv
4
12
0.8
2.5
Compound 58
11.39
7.38
Compound 59
20
50
50
iv
ip
po
24.49
32.49
14.26
40.65
46.31
56.37
6.72
4.48
3.75
45.6
55.5
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
1
operating at 400 MHz, and the H shift values, in ppm, were
F igu r e 1. Xenograft curves depicting tumor growth inhibition
of subcutaneous COR L23/R cell in CD1 NUDE mice under
coadminstration of compound 55, XR12890, with vincristine.
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 on 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 the
compounds by MEDAC Ltd., U.K. Where analyses are indi-
cated only by the symbols of the elements, results obtained
were within 0.4% of the theoretical values.
kinetics studies. This was carried out with Balb/C mice
dosed at either 20 or 50 mg/kg intravenously (iv),
intraperitoneally (ip), or orally (po) (Table 6).
Compounds 55, 56, and 59 showed oral bioavailability
of >50% when dosed at 50 mg/kg (Table 6). Moreover,
the plasma concentrations of these compounds were 10-
fold greater than their corresponding IC50 values for
several hours after dosing. When given iv at 20 mg/kg,
compound 57 had the lowest half-life and Cmax of the
compounds in this series and had a less attractive
profile than other members of this series.
Ch em ist r y. Gen er a l P r oced u r e for t h e Syn t h esis of
P yr r olop yr im id in es 12 a n d Com p ou n d s 25, 26, 28-42,
Exem p lified by P r ep a r a tion of Com p ou n d 30. 4-(4-
P h en eth ylp ip er a zin e-1-yl)-5,6,7,8-tetr a h yd r o-1,3,4b-tr i-
a za flu or en e-9-ca r bon itr ile (30). A mixture of 4-chloro-
5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carbonitrile (11) (80
mg, 0.343 mmol), 1-(2-phenylethyl)piperazine (1.1 equiv, 72
mg), and triethylamine (1.1 equiv, 53 µL, 0.377 mmol) was
heated in DMF (0.5 mL) at 100 °C for 2 h. The reaction mixture
was cooled and poured onto ice/water and the precipitated solid
was collected by filtration to yield the desired title compound
To assess the in vivo efficacy of this class of com-
pounds, several compounds were chosen on the basis of
their pharmacokinetic data for in vivo xenograft studies
with CD1 athymic mice bearing subcutaneous COR
L23/R tumors. Figure 1 illustrates the xenograft curves
for tumor growth inhibition of one of the compounds
(55), XR12890. The antitumor activity of vincristine (0.6
mg/kg iv, q5d × 2) was significantly (p < 0.01) potenti-
ated by the coadminstration of 50 mg/kg of XR12890
intraperitoneally or 100 mg/kg orally (graph not shown).
The maximum T/C percent ratios for tumor volumes in
animals treated with vincristine plus XR12890 ip or po
were 37% or 35%, respectively. In contrast, vincristine
or XR12890 alone had no significant effect on tumor
growth in this MRP1-resistant tumor model. The treat-
1
(94 mg, 70%). Mp 176-177 °C; H NMR (CDCl3) δ 1.97-2.13
(m, 4H, 2 × CH2), 2.64-2.80 (m, 6H, 3 × CH2), 2.81-2.92 (m,
2H, CH2), 3.22 (2H, t, J ) 6.4 Hz), 3.38-3.50 (m, 4H, 2 × CH2),
4.45 (2H, t, J ) 5.5 Hz), 7.16-7.36(m, 5H, 5 × ArH); MS m/z
387 (M + H)+.
4-P h en et h yla m in o-4a ,5,6,7,8,9a -h exa h yd r o-1,3,4b -t r i-
a za flu or en e-9-ca r bon itr ile (25). Mp 182-183 °C; 1H NMR
(CDCl3) δ 1.84-1.94 (m, 2H, CH2), 2.0-2.10 (m, 2H, CH2), 3.02
(t, 2H, J ) 6.6 Hz, CH2), 3.08 (t, 2H, J ) 6.3 Hz, CH2), 3.99 (q,

1334 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
2H, J ) 5.2 Hz), 4.02 (t, 2H, J ) 6.2 Hz), 4.84 (brs, 1H, NH),
7.18-7.40 (5H, m, 5 × ArH), 8.45 (s, 1H, ArH); MS m/z 318
(M + H)+.
2H, J ) 5.9 Hz, CH2), 7.12-7.25 (m, 2H 2 × ArH), 7.21-7.34
(1H (under solvent peak), ArH), 7.33-7.40 (d, 1H, J ) 7.6 Hz,
ArH), 8.61 (s, 1H, ArH); MS m/z 421.3 (M + H)+.
4-(Met h ylp h en et h yla m in o)-5,6,7,8-t et r a h yd r o-1,3,4b -
tr ia za flu or en e-9-ca r bon itr ile (26). Mp 128-129 °C; 1H
NMR (CDCl3) δ 1.90 (m, 4H, 2 × CH2), 2.95 (t, 2H, CH2), 2.98
(s, 3H, CH3), 3.15 (t, 2H, CH2), 3.65 (t, 2H, CH2), 4.00 (t, 2H,
CH2), 7.10-7.25 (m, 5H, ArH), 8.60 (s, 1H, PyrmH); MS m/z
332 (M + H)+.
{4-[2-(3-Ch lor op h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (35). Mp 205-
206 °C; 1H NMR (CDCl3) δ 1.88-2.18 (m, 4H, 2 × CH2), 2.48-
2.98 (m, 8H, 4 × CH2), 3.10-3.52 (m, 6H, 3 × CH2), 4.22-
4.42 (m, 2H, CH2), 7.00-7.35 (m, 4H, 4 × ArH), 8.60 (s, 1H,
ArH); MS m/z 421.3 (M + H)+.
N-(9-Cya n o-5,6,7,8-t et r a h yd r o-1,3,4b -t r ia za flu or en -4-
yl)-C-p h en ylm eth a n esu lfon a m id e (27). To a solution of
4-chloro-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carboni-
trile (11) (0.073 g, 0.343 mmol) in dioxane (6 mL) was added
ethanol (12 mL) saturated with ammonia at 0 °C. The mixture
was heated with stirring in a sealed tube at 140 °C for 20 h.
The reaction mixture was evaporated in vacuo, the residue was
triturated with hot methanol, and the creamy solid was filtered
as 4-amino-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carbo-
nitrile (415 mg, 65%). A mixture of the above product (73 mg,
0.343 mmol) and benzylsulfonyl chloride (1.5 equiv, 98 mg)
and DMAP (cat.) was heated in pyridine (1.5 mL) at 50-70
°C for 4 h. Pyridine was removed in vacuo, and the residue
was taken up in ethyl acetate, washed with saturated NaH-
CO3, dried over MgSO4, and concentrated in vacuo. After
purification on column chromatography with dichloromethane,
brownish crystals were obtained (20 mg, 16%). Mp 128-129
°C; 1H NMR (CDCl3) δ 1.95-2.00 (m, 2H, CH2), 2.03-2.10 (m,
2H, CH2), 3.10 (t, 2H, J ) 6.4 Hz, CH2), 4.40 (s, 2H, CH2),
4.45 (t, 2H, J ) 6.1 Hz, CH2), 7.20-7.30 (m, 3H, 3 × ArH),
7.45 (d, 2H, J ) 8.0 Hz), 7.60 (s, 1H, ArH), 11.55 (br s, 1H,
NH); MS m/z 368 (M + H)+.
4-{4-[2-(4-Ch lor op h en yl)et h yl]p ip er a zin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (36). H
1
NMR (CDCl3) δ 1.99-2.12 (m, 4H, 2 × CH2), 2.62-2.75 (m,
6H, 3 × CH2), 2.77-2.86 (m, 2H, CH2), 3.22 (t, 2H, J ) 6.3
Hz, CH2), 3.42 (t, 4H, J ) 4.8 Hz, 2 × CH2), 4.35 (2H, t, J )
5.9 Hz, CH2), 7.12-7.20 (d, 2H, J ) 8.4 Hz, CH2), 7.22-7.31
(d, 2H, 2 × ArH), 8.61 (s, 1H, ArH); MS m/z 421.2 (M + H)+.
4-{4-[2-(2-Nitr op h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (37). 1H NMR
(CDCl3) δ 1.99-2.12 (m, 4H, CH2), 2.67-2.78 (m, 6H, 3 × CH2),
3.14 (t, 2H, J ) 6.6 Hz, CH2), 3.20 (t, 2H, J ) 6.6 Hz, CH2),
3.42 (t, 4H, J ) 4.8 Hz, 2 × CH2), 4.35 (2H, t, J ) 5.9 Hz,
CH2), 7.35-7.44 (m, 2H, 2 × ArH), 7.52 (t, 1H, J ) 8.7 Hz,
ArH), 7.9-7.98 (d, 1H, J ) 8.2 Hz), 8.61 (s, 1H, ArH); MS m/z
432.2 (M + H)+.
4-{4-[2-(3-Nitr op h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (38). Mp 194-
195 °C; 1H NMR (CDCl3) δ 1.99-2.15 (m, 4H, CH2), 2.69-
2.83 (m, 6H, 3 × CH2), 2.96 (t, 2H, J ) 7.6 Hz, CH2), 3.20 (t,
2H, J ) 6.4 Hz, CH2), 3.38-3.58 (m, 4H, 2 × CH2), 4.36 (t,
2H, J ) 5.6 Hz, CH2), 7.47 (t, 1H, J ) 7.9 Hz, ArH), 7.59 (1H,
d, J ) 7.6 Hz, ArH), 8.10 (t, 2H, J ) 8.0 Hz, 2 × ArH), 8.62 (s,
1H, ArH); MS m/z 432.3 (M + H)+.
4-{4-[2-(4-Nitr op h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (39). 1H NMR
(DMSO-d6) δ 1.87-2.0 (m, 4H, CH2), 2.62-2.7 (m, 4H, 2 ×
CH2), 2.90 (t, 2H, J ) 7.2 Hz, CH2), 3.12 (2H, t, J ) 6.5 Hz,
CH2), 3.25-3.34 (m, 6H, 3 × CH2), 4.34 (t, 2H, J ) 5.8 Hz,
CH2), 7.54 (d, 2H, J ) 8.7 Hz, 2 × ArH), 8.13 (2H, t, J ) 8.7
Hz, 2 × ArH), 8.50 (s, 1H, ArH); MS m/z 432.3 (M + H)+.
4-[2-(3,4-Dih yd r o-1H -isoq u in olin -2-yl)e t h yla m in o]-
5,6,7,8-tetr ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (28).
1H NMR (CDCl3) δ 1.70-1.80 (m, 4H, 2 × CH2), 2.85-2.93
(m, 6H, 3 × CH2), 3.02 (t, 2H, CH2), 3.70-3.75 (m, 4H, 2 ×
CH2), 4.20 (t, 2H, CH2), 6.25 (br s, 1H, NH), 7.02 (d, 1H, J )
7.5 Hz, ArH), 7.12-7.20 (m, 3H, ArH), 8.45 (s, 1H, ArH); MS
m/z 373 (M + H)+.
4-{4-[2-(1H-In d ol-3-yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (29). Mp 223-
1
232 °C; H NMR (DMSO-d6) δ 1.85-2.05 (m, 4H, CH2), 2.6-
4-{4-[2-(3-Meth oxyp h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (40). Mp
181-182 °C; 1H NMR (CDCl3) δ 1.97-2.11(m, 4H, CH2), 2.62-
2.76 (m, 6H, 3 × CH2), 2.77-2.87 (m, 2H, CH2), 3.21 (t, 2H, J
) 6.3 Hz, CH2), 3.36-3.50 (m, 4H, CH2), 3.80 (s, 3H, CH3),
4.36 (2H, t, J ) 5.4 Hz, CH2), 6.70-6.86 (m, 3H, 3 × ArH),
7.21 (1H, t, J ) 7.7 Hz, ArH), 8.63 (s, 1H, ArH); MS m/z 417.3
(M + H)+.
2.75 (m, 6H, 3 × CH2), 2.87 (t, 2H, J ) 8.4 Hz), 3.15 (t, 2H, J
) 7.3 Hz), 3.32-3.4 (m, 4H, 2 × CH2), 4.32-4.40 (m, 2H, CH2),
7.00 (t, 1H, J ) 7.1 Hz), 7.05 (t, 1H, J ) 7.1 Hz, ArH), 7.19 (s,
1H, ArH), 7.32 (d, 1H, J ) 8.1 Hz, ArH), 7.53 (d, 1H, J ) 7.8
Hz), 8.48 (s, 1H, ArH), 10.78 (s, 1H, ArH); MS m/z 426.2 (M +
H)+.
4-[4-(3-P h en ylpr opyl)piper azin -1-yl]-5,6,7,8-tetr ah ydr o-
1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (31). Mp 156-158 °C;
1H NMR (CDCl3) δ 1.8-1.92 (m, 2H), 2.99-2.15 (m, 4H, CH2),
2.39-2.50 (t, 2H, J ) 7.6 Hz, CH3), 2.59-2.72 (m, 6H, 3 ×
CH2), 3.18-3.25 (t, 2H, J ) 6.3 Hz), 3.38-3.45 (m, 4H, 2 ×
CH2), 4.31-4.42 (t, 2H, J ) 6.0 Hz), 7.13-7.32 (m, 5H, 5 ×
ArH), 8.62 (s, 1H, ArH); MS m/z 401.3 (M + H)+.
4-{4-[2-(4-Meth oxyp h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-
1
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (41). H
NMR (DMSO-d6) δ 1.88-2.01 (m, 4H, 2 × CH2), 2.52-2.59
(m, 2H, CH2), 2.59-2.68 (m, 2H, CH2), 2.68-2.74 (2H, m, CH2),
3.12 (t, 2H, J ) 6.2 Hz, CH2), 3.22-3.41 (m, 6H, 3 × CH2),
3.69 (s, 3H, CH3), 4.35 (t, 2H, J ) 5.0 Hz), 6.82 (d, 2H, J ) 8.7
Hz, ArH), 7.16 (t, 2H, J ) 8.7 Hz, ArH), 8.45 (s, 1H, ArH); MS
m/z 417.3 (M + H)+.
4-(4-P h en ylm eth a n esu lfon ylp ip er a zin -1-yl)-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (32). Mp 239-
241 °C; 1H NMR (DMSO-d6) δ 1.85-1.97 (m, 4H, CH2), 3.10-
3.18 (t, 2H, J ) 6.2 Hz), 3.25-3.35 (m, 8H, 4 × CH2), 3.21-
3.3(m, 2H, CH2), 4.3-4.39 (2H, t, J ) 5.8 Hz), 4.50 (s, 2H,
CH2), 7.32-7.45 (m, 5H, 5 × ArH), 8.51 (s, 1H, ArH); MS m/z
437.3 (M + H)+. Anal. (C22H24N6O2S‚0.5H2O) C, H, N.
4-[4-(2-Oxo-2-p h en ylet h yl)p ip er a zin -1-yl]-5,6,7,8-t et -
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (33). Mp 171-
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-5,6,7,8-
1
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (42). H
NMR (CDCl3) δ 1.92-2.12 (4H, m, 2 × CH2), 2.55-2.7 (m, 6H,
3 × CH2), 2.72-2.82 (m, 2H, CH2), 3.21 (t, 2H, J ) 6.4 Hz),
3.42 (t, 4H, J ) 4.8 Hz, 2 × CH2), 4.35 (2H, t, J ) 5.9 Hz),
6.83-6.93 (m, 1H, ArH), 6.98-7.1 (2H, m, 2 × ArH), 8.61 (s,
1H, ArH); MS m/z 423.3 (M + H)+.
4-Ch lor o-2-tr ibu tylsta n n a n yl-5,6,7,8-tetr a h yd r o-1,3,4b-
tr ia za flu or en e-9-ca r bon itr ile (13). n-BuLi (28 mL, 69.83
mmol, 2.5 M solution in hexane) was added to a solution of
2,2,6,6-tetramethylpiperidine (11.78 mL, 69.83 mmol) in THF
(100 mL) at -78 °C. To this was added 4-chloro-5,6,7,8-
tetrahydro-1,3,4b-triazafluorene-9-carbonitrile (3.24 g, 13.97
mmol) in THF (100 mL). After the mixture was stirred for 30
min, tributyltin chloride (18.95 mL, 69.83 mmol) was added.
After 1 h, the reaction mixture was quenched by addition of
ammonium chloride solution. The organics were extracted into
1
173 °C; H NMR (DMSO-d6) δ 1.85-1.97 (m, 4H, 2 × CH2),
2.70-2.76 (m, 4H, 2 × CH2), 3.09-3.15 (2H, t, J ) 6.2 Hz,
CH2), 3.41-3.49 (m, 4H, 2 × CH2), 3.95 (s, 2H, CH2), 4.32-
4.4 (t, 2H, J ) 5.7 Hz, CH2), 7.52 (2H, t, J ) 7.5 Hz, ArH),
7.62 (1H, t, J ) 7.4 Hz, ArH), 8.02 (d, 2H, J ) 7.2 Hz, ArH),
8.5 (s, 1H, ArH); MS m/z 401.3 (M + H)+.
4-{4-[2-(2-Ch lor op h en yl)et h yl]p ip er a zin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (34). Mp
156-158 °C; 1H NMR (CDCl3) δ 1.99-2.12 (m, 4H, 2 × CH2),
2.67-2.78 (m, 6H, 2 × CH2), 2.98-3.07 (m, 2H, CH2), 3.20 (t,
2H, J ) 6.4 Hz, CH2), 3.42 (t, 4H, J ) 4.8 Hz, CH2), 4.35 (t,

Pyrrolopyrimidines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1335
ethyl acetate and dried (MgSO4) and the solvent was removed
in vacuo to yield an orange solid (5.1 g, 70%, used crude in
next step).
CH2), 3.48 (m, 4H, 2 × CH2), 4.42 (t, 2H, J ) 5.4 Hz, CH2),
6.90-7.40 (m, 6H, 6 × ArH), 8.03 (m, 1H,ArH); MS m/z 513
(M + H)+.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
49 a n d 50 Is Exem p lified by Th a t for 49. 1-[4-(4-P h en -
eth ylp ip er a zin -1-yl)-5,6,7,8-tetr a h yd r o-1,3,4b-tr ia za flu o-
r en -9-yl]p r op a n -1-on e (49). To a solution of ethylmagnesium
bromide (1.0 M solution in tert-butyl methyl ether, 3.0 equiv,
1.16 mmol, 1.16 mL) and dry tetrahydrofuran (0.5 mL) was
added a solution of 4-(4-phenethylpiperazine-1-yl)-5,6,7,8-
tetrahydro-1,3,4b-triazafluorene-9-carbonitrile (150 mg, 0.39
mmol) in THF (2 mL) dropwise. The reaction mixture was
heated to 60 °C for 5 h. The reaction mixture was then cooled,
quenched with water, extracted into ethyl acetate, and dried
(MgSO4), and the solvent was removed in vacuo. The residue
was purified using flash chromatography to yield the title
compound as a white solid (49) (43.5 mg, 27%). Mp 129-130
Gen er a l P r oced u r e for th e Syn th esis of 14. 4-{4-
P h en eth ylp ip er a zin -1-yl}-2-tr ibu tylsta n n a n yl-5,6,7,8-tet-
r ah ydr o-1,3,4b-tr iazaflu or en e-9-car bon itr ile (14a). 4-Chlo-
ro-2-tributylstannanyl-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-
9-carbonitrile (13) and 1-[2-phenethyl]piperazine were reacted
together with the standard protocol described above to yield
4-{4-phenethylpiperazin-1-yl}-2-tributylstannanyl-5,6,7,8-tet-
rahydro-1,3,4b-triazafluorene-9-carbonitrile (70% yield). 1H
NMR (CDCl3) δ 0.90-1.70 (m, 29H, 3 × n-Bu), 2.00 (m, 4H, 2
× CH2), 2.70 (m, 6H, 3 × CH2), 2.87 (m, 2H, CH2), 3.18 (t, 2H,
J ) 6.6 Hz, CH2), 3.42 (m, 4H, 2 × CH2), 4.35 (t, 2H, J ) 5.3
Hz, CH2), 7.30 (m, 5H, ArH); MS m/z 676.5 (M + H)+.
2-Iodo-4-(4-ph en eth ylpiper azin -1-yl)-5,6,7,8-tetr ah ydr o-
1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (43). A mixture of 4-{4-
phenethylpiperazin-1-yl}-2-tributylstannanyl-5,6,7,8-tetrahydro-
1,3,4b-triazafluorene-9-carbonitrile (14a ) (200 mg, 0.384 mmol)
and I2 (1.5 equiv) in THF (2 mL) was stirred at room
temperature for 2 h. The reaction was then quenched with
saturated Na2S2O3, the mixture was extracted with dichlo-
romethane, washed with sodium bicarbonate solution, and
dried (MgSO4), and the solvent was removed in vacuo to yield
the crude product, which was purified using flash chromatog-
raphy (EtOAc/DCM, 1:1) to yield the title compound (43) (0.106
g, 70%). 1H NMR (CDCl3) δ 1.90-2.00 (m, 4H, 2 × CH2), 2.60-
2.65 (m, 6H, 3 × CH2), 2.75 (m, 2H, CH2), 3.10 (t, 2H, CH2),
3.35-3.40 (m, 4H, 2 × CH2), 4.20 (t, 2H, CH2), 7.10-7.30 (m,
5H, 5 × ArH); MS m/z 513.2 (M + H)+.
1
°C; H NMR (CDCl3) δ 1.22 (t, 3H, J ) 7.3 Hz, CH3), 1.92-
2.05 (m, 4H, 2 × CH2), 2.65-2.75 (m, 6H, 3 × CH2), 2.82-
2.88 (m, 2H, CH2), 3.33-3.40 (m, 6H, 3 × CH2), 3.43 (t, 2H, J
) 6.6 Hz, CH2), 4.45 (t, 2H, J ) 5.0 H, CH2), 7.16-7.32 (m,
5H, 5 × ArH), 8.65 (s, 1H, ArH); MS m/z 418 (M + H). Anal.
(C25H31N5O) C, H, N.
[4-(4-P h en eth ylpiper azin -1-yl)-5,6,7,8-tetr ah ydr o-1,3,4b-
tr ia za flu or en -9-yl]-p h en ylm eth a n on e (50). Mp 140-142
°C; 1H NMR (CDCl3) δ 1.95-2.02 (m, 2H, CH2), 2.03-2.12 (m,
2H, CH2), 2.68-2.78 (m, 6H, 3 × CH2), 2.85-2.90 (m, 2H, CH2),
3.37 (t, 2H, J ) 6.7 Hz, CH2), 3.40-3.46 (m, 4H, 2 × CH2),
4.46 (t, 2H, J ) 5.6 Hz, CH2), 7.20-7.28 (m, 3H, 3 × ArH),
7.30 (t, 2H, J ) 7.4 Hz, 2 × ArH), 7.44 (t, 2H, J ) 7.4 Hz, 2 ×
ArH), 7.54 (t, 1H, J ) 7.4 Hz, ArH), 7.89 (d, 2H, J ) 7.1 Hz,
2 × ArH), 8.51 (s, 1H, ArH); MS m/z 466 (M + H)+.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
44-48 Is Exem p lified by th e Meth od for 46. 4-{4-[2-(3,4-
Diflu or oph en yl)eth yl]piper azin -1-yl}-2-pyr idin -3-yl-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (46). A
mixture of 4-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-1-yl}-2-
tributylstannanyl-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-
carbonitrile (14b) (360 mg, 0.506 mmol), 3-iodopyridine (106
mg, 1.0 equiv), toluene (3.7 mL), Pd(PPh3)4 (7.7 mg, 10 mol
%), and copper iodide (19.4 mg, 20 mol %) was heated to reflux
for 1 day. The reaction mixture was then cooled, diluted with
ethyl acetate, washed with sodium bicarbonate solution, and
dried (MgSO4) and the solvent was removed in vacuo to yield
the crude product, which was purified using flash chromatog-
raphy to yield the title compound (46) (8 mg, 5%). Mp 206-
4-(4-P h en eth ylpiper azin -1-yl)-5,6,7,8-tetr ah ydr o-1,3,4b-
tr ia za flu or en e-9-ca r boxylic Acid Meth yl Ester (51). A
mixture of 4-(4-phenethylpiperazin-1-yl)-5,6,7,8-tetrahydro-
1,3,4b-triazafluorene-9-carboxylic acid amide (300 mg, 0.74
mmol) [prepared as for compound 55], acetyl chloride (0.75
mL), and methanol (4 mL) was heated to 50 °C for 4 days.
The solvent was then removed in vacuo, the residue was
dissolved in dichloromethane, washed with sodium bicarbonate
solution, and dried (MgSO4), and the solvent was removed in
vacuo to yield a solid, which was purified using flash chroma-
tography to yield the title compound as an off-white solid (36
mg, 12%). Mp 154-155 °C; 1H NMR (CDCl3) δ 1.90-2.00 (m,
4H, 2 × CH2), 2.60-2.70 (m, 6H, 3 × CH2), 2.78-2.82 (m, 2H,
CH2), 3.29-3.38 (m, 6H, 3 × CH2), 3.91 (s, 3H, CH3), 4.35 (t,
2H, J ) 5.8 Hz, CH2), 7.12-7.25 (m, 5H, 5 × ArH), 8.68 (s,
1H, ArH); MS m/z 420 (M + H)+; HPLC 97.64%.
1
207 °C; H NMR (CDCl3) δ 1.99 (m, 4H, 2 × CH2), 2.63 (m,
6H, 3 × CH2), 2.74 (m, 2H, CH2), 3.13 (m, 2H, CH2), 3.45 (m,
4H, CH2), 4.29 (m, 2H, CH2), 6.87 (m, 1H,), 7.00 (m, 2H, ArH),
7.32 (m, 1H, ArH), 8.59 (dd, 1H, J ) 4.8 and 1.7 Hz, ArH),
8.71 (m, 1H, ArH), 9.61 (s, 1H, ArH); MS m/z 500.4 (M + H)+.
9-(4,5-Dim eth ylth ia zol-2-yl)-4-(4-p h en eth ylp ip er a zin -
1-yl)-5,6,7,8-tetr a h yd r o-1,3,4b-tr ia za flu or en e (52). To a
solution of 4-(4-phenethylpiperazine-1-yl)-5,6,7,8-tetrahydro-
1,3,4b-triazafluorene-9-carbonitrile (30) (3.3 g, 8.55 mmol) in
dimethylformamide (30 mL) was added sodium hydrosulfide
hydrate (4.7 g, 42.73 mmol). The reaction mixture was heated
at 60 °C for 5 days. After cooling, the reaction mixture was
extracted into ethyl acetate, washed with water, and dried
(MgSO4) and the solvent was removed in vacuo to yield the
crude product, which was purified using flash chromatography
to yield the intermediate 4-(4-phenethylpiperazin-1-yl)-5,6,7,8-
tetrahydro-1,3,4b-triazafluorene-9-carbothioic acid amide (2.20
g, 61%). A mixture of 4-(4-phenethylpiperazin-1-yl)-5,6,7,8-
tetrahydro-1,3,4b-triazafluorene-9-carbothioic acid amide (70
mg, 0.167 mmol), triethylamine (1.2 equiv, 0.2 mmol, 28µL),
and 3-bromo-2-butanone (1.2 equiv, 0.2 mmol, 30.2 mg) was
heated to 100 °C for 2.5 h. The volatiles were then removed in
vacuo, and the residue was triturated with water and filtered
to yield a solid, which was purified using flash chromatography
(2% MeOH in DCM) to yield the title compound as a beige
solid (39.2 mg, 49%). Mp 150-152 °C; 1H NMR (CDCl3) δ 1.98-
2.10 (m, 4H, 2 × CH2), 2.38 (s, 3H,CH3), 2.41 (s, 3H, CH3),
2.66-2.75 (m, 6H, 3 × CH2), 2.82-2.90 (m, 2H, CH2), 3.42-
3.48 (m, 4H, 2 × CH2), 3.54 (t, 2H, J ) 6.3 Hz, CH2), 4.35 (t,
2H, J ) 5.1 Hz, CH2), 7.20-7.32 (m, 5H, 5 × ArH), 8.71 (s,
1H, ArH); MS m/z 473 (M + H)+. Anal. (C27H32N6S) C, H, N.
4-(4-P h en eth ylp ip er a zin -1-yl)-2-p h en yl-5,6,7,8-tetr a h y-
d r o-1,3,4b-tr ia za flu or en e-9-ca r bon itr ile (44). Mp 271-272
1
°C; H NMR (CDCl3) δ 2.15-1.95 (m, 4H, 2 × CH2), 2.75 (m,
6H, 3 × CH2), 2.88 (m, 2H, CH2), 3.21 (m, 2H, CH2), 3.51 (m,
4H, 2 × CH2), 4.34 (m, 2H, CH2), 7.47-7.22 (m, 8H, 8 × ArH),
8.55 (m, 2H, 2 × ArH); MS m/z 463 (M + H)+.
N-{4-[9-Cya n o-4-(4-p h en et h ylp ip er a zin -1-yl)-5,6,7,8-
t et r a h yd r o-1,3,4b -t r ia za flu or en -2-yl]p h en yl}a cet a m id e
(45). Mp 281-283 °C; 1H NMR (DMSO-d6) δ 1.90 (m, 4H, 2 ×
CH2), 2.09 (s, 3H, CH3), 2.60 (m, 2H, CH2), 2.64 (m, 4H, 2 ×
CH2), 2.75 (m, 2H, CH2), 3.08 (m, 2H, CH2), 3.39 (m, 4H, 2 ×
CH2), 4.33 (m, 2H, CH2), 7.30-7.14 (m, 5H, 5 × ArH), 7.69
(m, 2H, 2 × ArH), 8.32 (m, 2H, 2 × ArH), 10.12 (br s, 1H,
NH); MS m/z 520 (M + H)+.
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-2-pyr -
idin-4-yl-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carbonitrile
(47). Mp 205-207 °C; 1H NMR (DMSO-d6) δ 3.48-2.92 (m,
14H, 7 × CH2), 3.88 (m, 4H, 2 × CH2), 4.26 (2H, m, CH2), 6.99
(m, 1H, ArH), 7.24 (m, 3H, 3 × ArH), 8.42 (m, 2H, 2 × ArH),
8.73 (m, 2H, ArH), 10.94 (m, 1H, ArH); MS m/z 500 (M + H)+.
4-{4-[2-(3,4-Diflu or op h en yl)et h yl]p ip er a zin -1-yl}-2-o-
tolyl-5,6,7,8-tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r bon i-
tr ile (48). 1H NMR (CDCl3) δ 2.00-2.18 (m, 4H, 2 × CH2),
2.62-2.90 (m, 11H, 4 × CH2, CH3), 3.26 (t, 2H, J ) 6.2 Hz,

1336 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Wang et al.
9-(5-Meth yl[1,2,4]oxa d ia zol-3-yl)-4-(4-p h en eth ylp ip er -
a zin -1-yl)-5,6,7,8-tetr a h yd r o-1,3,4b-tr ia za flu or en e (53). A
mixture of 4-(4-phenethylpiperazine-1-yl)-5,6,7,8-tetrahydro-
1,3,4b-triazafluorene-9-carbonitrile (725 mg, 1.7 mmol), potas-
sium carbonate (1.04 g, 7.5 mmol), and hydroxylamine hydro-
chloride (457 mg, 6.6 mmol) in ethanol (16 mL) was heated to
reflux overnight. The reaction mixture was reduced in vacuo
and triturated with water, and a precipitate was collected. This
was triturated with hot dichloromethane to yield the desired
amidoxime (19) (430 mg, 60%). A mixture of the above
amidoxime (188 mg, 0.44 mmol) and acetyl chloride (1.5 equiv,
48 µL, 0.683 mmol) in pyridine (1.5 mL) was heated to 60 °C
for 6 h. The reaction mixture was cooled, extracted into
dichloromethane, washed with sodium bicarbonate solution,
and dried (MgSO4) and the solvent was removed in vacuo to
yield the crude product, which was purified by flash chroma-
tography to yield the title compound as a beige solid (37 mg,
5%). Mp 79 °C; 1H NMR (CDCl3) δ 2.00-2.14 (m, 4H, 2 × CH2),
2.70 (s, 3H, CH3), 2.70-2.80 (m, 6H, 2 × CH2), 2.83-2.92 (m,
2H, CH2), 3.40-3.52 (m, 6H, 3 × CH2), 4.40 (t, 2H, J ) 6.0
Hz, CH2), 7.20-7.33 (m, 5H, 5 × ArH), 8.77 (s, 1H, ArH); MS
m/z 444 (M + H)+.
4-(4-P h en eth ylp ip er a zin -1-yl)-9-p yr im id in -2-yl-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e (54). To N-hydroxy-4-(4-
phenethylpiperazin-1-yl)-5,6,7,8-tetrahydro-1,3,4b-triazafluo-
rene-9-carboxamidine (19) (430 mg, 1.02 mmol) in acetic acid
(5 mL) was added acetic anhydride (1.6 equiv, 154 µL, 1.63
mmol), and the mixture stirred for 10 min. Palladium (10%)
on carbon was then added (110 mg), and the mixture was
stirred under a positive pressure of hydrogen for 5 h. The
reaction mixture was then filtered through Celite, and the
volatiles were removed in vacuo. The residue was dissolved
in dichloromethane, washed with sodium bicarbonate solution,
and dried (MgSO4) and the solvent was removed in vacuo to
yield the desired amidine (21) (382 mg, 95%). This was
converted into the dihydrochloride salt by treatment with HCl
in methanol.
amide (55) (62 mg, 0.14 mmol) in acetonitrile (1.5 mL) was
added 2,3-dimethoxybenzaldehyde (70 mg, 0.42 mmol), trieth-
ylsilane (75 µL, 0.42 mmol), and trifluoroacetic acid (51 µL,
0.546 mmol). The reaction mixture was heated to reflux for
18 h. The solvent was then removed in vacuo, the residue was
dissolved in ethyl acetate, washed with sodium bicarbonate
solution, and dried (MgSO4), and the solvent was removed in
vacuo to yield the crude product. This was recrystallized from
dichloromethane/hexane to yield the desired title compound
1
(76 mg, 92%). Mp 177-178 °C; H NMR (CDCl3) δ 1.95-2.01
(m, 4H, 2 × CH2), 2.60-2.72 (m, 6H, 3 × CH2), 2.78-2.82 (m,
2H, CH2), 3.36 (br, 4H, 2 × CH2), 3.55 (t, 2H, J ) 6.3 Hz, CH2),
3.83 (s, 3H, CH3), 3.90 (s, 3H, CH3), 4.30 (t, 2H, J ) 5.8 Hz,
CH2), 4.72 (d, 2H, J ) 6.0 Hz, CH2), 6.80 (d, 1H, J ) 6.0 Hz,
ArH), 6.84-6.86 (m, 1H, ArH), 6.98-7.10 (m, 4H, ArH), 8.50
(s, 1H, ArH), 9.35 (t, 1H, ArH); MS m/z 591 (M + H)+; HPLC
96.25%.
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r boxylic Acid Eth -
yla m id e (56). Mp 162-163 °C; 1H NMR (CDCl3) δ 1.44 (t,
3H, J ) 7.2 Hz, CH3), 2.08-2.18 (m, 4H, 2 × CH2), 2.77-2.90
(m, 6H, 3 × CH2), 2.92-2.98 (m, 2H, CH2), 3.52-3.60 (m, 4H,
2 × CH2), 3.65-3.73 (m, 4H, 2 × CH2), 4.50 (t, 2H, J ) 6.0
Hz, CH2), 7.02-7.08 (m, 1H, ArH), 7.14-7.28 (m, 2H, 2 ×
ArH), 8.70 (s, 1H, ArH), 9.05 (br s, 1H, NH); MS m/z 469 (M
+ H)+.
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r boxylic Acid (P yr -
id in -2-ylm eth yl)a m id e (58). Mp 147-149 °C; 1H NMR
(CDCl3) δ 1.88-2.00 (m, 4H, 2 × CH2), 2.08-2.17 (m, 6H, 3 ×
CH2), 2.20-2.26 (m, 2H, CH2), 3.32-3.38 (m, 4H, 2 × CH2),
3.48 (t, 2H, J ) 6.4 Hz, CH2), 4.28 (t, 2H, J ) 6.0 Hz, CH2),
4.78 (2H, d, J ) 5.9 Hz, CH2), 6.82-6.86 (m, 1H, ArH), 6.93-
7.04 (m, 2H, ArH), 7.10 (t, 1H, J ) 7.6 Hz, ArH), 7.30 (d, 1H,
J ) 7.6 Hz, ArH), 7.56 (t, 1H, J ) 7.6 Hz, ArH), 8.50 (s, 1H,
ArH), 8.50-8.55 (d, 1H, J ) 7.6 Hz, ArH), 9.56 (m, 1H, CONH);
MS m/z 532 (M + H)+. Anal. (C29H31F2N7O) C, H, N.
To 4-(4-phenethylpiperazin-1-yl)-5,6,7,8-tetrahydro-1,3,4b-
triazafluorene-9-carboxamidine dihydrochloride salt (21) (105
mg, 0.22 mmol) in Dowtherm (2 mL) was added potassium
carbonate (30.4 mg, 0.44 mmol) and malonaldehyde bis-
(dimethylacetal) (120 µL, 0.265 mmol). The reaction mixture
was heated to 175 °C for 5 h. The reaction mixture was cooled,
diluted with ethyl acetate, extracted into 2 M HCl, neutralized
(NaHCO3), extracted into dichloromethane, and dried (MgSO4)
and the solvent was removed in vacuo to yield crude product,
which was purified using flash chromatography to yield the
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-5,6,7,8-
tetr a h yd r o-1,3,4b-tr ia za flu or en e-9-ca r boxylic Acid P yr -
id in -3-yla m id e (59).25b A mixture of 4-{4-[2-(3,4-difluorophen-
yl)ethyl]piperazin-1-yl}-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carboxylic
acid amide (55) (100 mg, 0.227 mmol) and 3-bromopyridine
(1.1 equiv, 0.25 mmol) was stirred in 1,4-dioxane (1.5 mL). To
this was added cesium carbonate (1.5 equiv, 111 mg), tris-
(dibenzylideneacetone)dipalladium (2 mg), and xanthphos (2
mg). The reaction mixture was heated to 100 °C overnight.
After cooling, the reaction mixture was extracted into dichlo-
romethane, washed with water, and dried (MgSO4) and the
solvent was removed in vacuo to yield the crude product, which
was purified using flash chromatography to yield the title
compound (5 mg, 4%). Mp 227-228 °C; 1H NMR (CDCl3) δ
1.90-2.03 (m, 4H, 2 × CH2), 2.60-2.71 (m, 6H, 3 × CH2),
2.72-2.78 (m, 2H, CH2), 3.38-3.43 (m, 4H, 2 × CH2), 3.55 (t,
2H, J ) 6.0 Hz, CH2), 4.32 (t, 2H, J ) 6.0 Hz, CH2), 6.82-
6.88 (m, 1H, ArH), 6.95-7.04 (m, 2H, ArH), 7.22-7.25 (m, 1H,
ArH), 8.23-8.28 (m, 2H, ArH), 8.55 (s, 1H, ArH), 8.80 (s, 1H,
ArH); MS m/z 518 (M + H)+.
Biology Assa ys. Dr u g Accu m u la tion Assa y (MRP ).
Compounds were assayed for inhibition of MRP1-dependent
transport of the radiolabeled cytotoxic agent and MRP sub-
strate, daunomycin. 1 × 104 COR.L23/R cells (MRP expressing
human non-small-cell lung carcinoma MDR subline) were
seeded 48 h prior to assay into 96-well opaque culture plates.
Compounds were serially diluted over a range of concentra-
tions from 100 to 0.015 µM in assay medium containing
tritiated daunomycin at 0.3 µCi/mL and incubated with
COR.L23/R cells at 37 °C for 2 h before washing and deter-
mination of cell-associated radioactivity. Results are expressed
as an IC50 for daunomycin accumulation, where 100% ac-
cumulation is that observed in the presence of the known
MRP1 modulator verapamil30 at 100 µM.
1
title compound (80 mg, 83%). Mp 164 °C; H NMR (CDCl3) δ
2.05-2.20 (m, 4H, 2 × CH2), 2.78-2.90 (m, 6H, 3 × CH2),
2.93-2.98 (m, 2H, CH2), 3.50 (m br, 4H, 2 × CH2), 3.60 (t, 2H,
J ) 6.7 Hz, CH2), 4.50 (t, 2H, J ) 5.8 Hz, CH2), 7.15 (t, 1H, J
) 4.8 Hz, CH2), 7.28-7.40 (m, 5H, 5 × ArH), 8.88 (s, 1H, ArH),
8.95 (d, 2H, J ) 4.8 Hz, 2 × ArH); MS m/z 440 (M + H)+.
4-{4-[2-(3,4-Diflu or oph en yl)eth yl]piper azin -1-yl}-5,6,7,8-
tetr ah ydr o-1,3,4b-tr iazaflu or en e-9-car boxylic Acid Am ide
(55). A solution of KOH (465 mg, 8.3 mmol) in water (2 mL)
was added to a suspension of 4-{4-[2-(3,4-difluorophenyl)ethyl]-
piperazin-1-yl}-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-car-
bonitrile (500 mg, 1.1 mmol) in ethanol (6 mL). The reaction
mixture was stirred for 6 h at reflux and then cooled. The
precipitate was collected by filtration and washed with ethanol
to yield the title compound as a white solid (179 mg, 37%).
Mp 257-259 °C; 1H NMR (CDCl3) δ 2.00-2.15 (m, 4H, 2 ×
CH2), 2.66-2.82 (m, 6H, 3 × CH2), 2.84-2.88 (m, 2H, CH2),
3.48 (m br, 4H, 2 × CH2), 3.59 (t, 2H, J ) 6.2 Hz, CH2), 4.43
(t, 2H, J ) 5.9 Hz, CH2), 5.52 (s br, 1H, NH), 6.99-7.02 (m,
1H, ArH), 7.08-7.17 (m, 2H, ArH), 8.63 (s, 1H, ArH), 8.84 (s
br, 1H, NH); MS m/z 441 (M + H)+; HPLC 95.23%.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
56-58 Is Exem p lified by Th a t for 57. 4-{4-[2-(3,4-Diflu o-
r op h en yl)eth yl]p ip er a zin -1-yl}-5,6,7,8-tetr a h yd r o-1,3,4b-
tr ia za flu or en e-9-ca r boxylic Acid 2,3-Dim eth oxyben zyl-
a m id e (57).25a To 4-{4-[2-(3,4-difluorophenyl)ethyl]piperazin-
1-yl}-5,6,7,8-tetrahydro-1,3,4b-triazafluorene-9-carboxylic acid
P oten tia tion Assa y. The ability of modulators to potenti-
ate the cytotoxicity of doxorubicin was evaluated in the COR.
L23/R cell line as outlined previously.26 IC50 values for doxo-

Pyrrolopyrimidines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1337
rubicin (concentration resulting in 50% inhibition of cell
growth) were calculated from plotted results using untreated
cells as 100%. EC50 values for modulators (concentration
required to give 50% of full reversal) were obtained from
graphs of potentiation index (ratio of IC50 of cytotoxic drug
alone to IC50 of cytotoxic drug in the presence of modulator)
plotted against concentration of modulator.
Similar protocols26 were applied for Pgp assays with the use
of Pgp expressing murine mammary carcinoma EMT6/AR1.0
subline.
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Inhibition of CYP 3A4 activity was measured by determin-
ing the conversion of testosterone to 6â-hydroxytestosterone
in the presence and absence of modulators according to a
literature protocol.31 Briefly, modulators (0.1-100 µM) were
incubated at 37 °C with human liver microsomes (0.1 mg/mL)
in the presence of testosterone (110 µM). The reaction was
initiated by the addition of a NADPH generating system and
stopped by addition of methanol after 10 min. Supernatants
were analyzed by HPLC using an acetonitrile/water gradient
containing 0.05% orthophosphoric acid on an Intersil ODS-2
column (4.6 mm × 250 mm, 5 µm) with UV detection at 300
nm. IC50 (concentration resulting in 50% inhibition) values
were calculated from graphs of percent control activity versus
drug concentration.
P h a r m a cok in et ic St u d ies. All animal experimentation
was performed to U.K. Home Office regulations, and the
UKKCCCR guidelines were adhered to throughout the studies.
Compounds were administered intravenously, intraperito-
neally , or orally at 20-50 mg/kg to female Balb/c mice (three
to four animals per time point). Blood samples were collected
from anesthetized animals by cardiac puncture at various
times between 0.03 and 24 h and centrifuged to prepare
plasma, which was stored at -40 °C until analysis. Samples
(100 µL) were extracted with methanol (300 µL) at -40 °C
(15 min incubation), and the supernatants were analyzed by
HPLC as outlined above. The area under the concentration-
time curves (AUC) and elimination half-life (t1/2) of the
compounds were calculated using noncompartmental analysis
using PCModfit (Gamms Ltd., Hertfordshire, U.K.). AUC
values were calculated using the linear trapezoidal rule up to
the peak concentration and the logarithmic trapezoidal rule
for postpeak concentration using PCModfit.
In Vivo Effica cy Stu d ies. The efficacy of compounds
(modulators) was evaluated using the resistant COR L23/R
cell xenografts. Female CD1 athymic mice were inoculated
subcutaneously with 1 × 106 COR/L23R cells in 100 µL of
phosphate buffered saline. When the tumors reached a mean
diameter of 0.4-0.6 cm, the animals were randomized into
groups of six and treated with vincristine (iv) or modulator
(ip or po) alone or in combination on days 0 (start of treatment)
and 5. The modulators were administered once before (typi-
cally at -30 min) and once after (typically at 4 h) the cytotoxic
drug administration. Control animals were treated with
vehicle alone. Tumor volumes and body weights were mea-
sured at least three times per week as outlined previously.32
The mean relative tumor volume was calculated using the
tumor volume on the first day of treatment (day 0). The ratio
(mean relative tumor volume of the treated group)/(the mean
relative tumor volume of control group) × 100 (T/C, %) was
calculated each time the tumors were measured. Statistical
analysis was performed using two-way ANOVA with Bonfer-
roni posttests.
Ack n ow led gm en t. We thank Drs. Francis Wilson
and Peter Charlton for proofreadings and valuable
comments.
Note Ad d ed a fter ASAP P ostin g. This manuscript
was released ASAP on 2/7/2004 with an incorrect
reference citation three lines below structures 1 and 2.
The correct version was posted on 2/10/2004.
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