Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d] pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Article abstract of DOI:10.1016/j.bmc.2012.08.050

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d] pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.

Full text of DOI:10.1016/j.bmc.2012.08.050

Bioorganic & Medicinal Chemistry 20 (2012) 6770–6789
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeting conserved water molecules: Design of
4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using
fragment-based screening and structure-based optimization
Nicholas G. M. Davies a, Helen Browne a, Ben Davis a, Martin J. Drysdale a, Nicolas Foloppe a,
Stephanie Geoffrey a, Ben Gibbons a , Terance Hart a, Roderick Hubbard a, Michael Rugaard Jensen b,
Howard Mansell a, Andrew Massey a, Natalia Matassova a, Jonathan D. Moore a, James Murray a,
Robert Pratt a, Stuart Ray a, Alan Robertson a, Stephen D. Roughley a, Joseph Schoepfer b, Kirsten Scriven a,
Heather Simmonite a, Stephen Stokes a, Allan Surgenor a, Paul Webb a, Mike Wood a, Lisa Wright a,
Paul Brough a,
⇑
a Vernalis Ltd, Granta Park, Great Abington, Cambridge CB21 6GB, UK
b Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 4 September 2012
Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we
describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were
identified following structure-driven optimization of purine hits revealed by NMR based screening of a
proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to
the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as mea-
sured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance
assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in
binding affinity with minimal increase in molecular weight. Some compounds in this chemical series
inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client
proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demon-
strated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based
design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved
water molecules in drug design.
Keywords:
Hsp90
Fragment-based screening
Cancer
Structure-based design
Conserved water molecules
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
to disrupt multiple oncogenic pathways12 by an indirect attack on
critical proteins and has become an attractive target for cancer ther-
Heat shock protein 90 (Hsp90) is a member of a class of proteins
known as molecular chaperones that, via a complex ATP-driven
mechanism, play a critical role in the maturation, stability and func-
tion of so called ‘client’ proteins within the cell.1–5 Heat shock pro-
teins are over-expressed under conditions of cellular stress such as
an increase in temperature or oxidation potential, but also have a
critical role within non-stressed cells in maintaining homeostasis.
Chaperone proteins are abundant in cells, comprising between 5%
and 10% of cellular proteins and Hsp90 may represent 1% of the to-
tal even under non stressed conditions. Well over 100 clients of
Hsp90 have been identified6 including many proteins that are in-
volved in pathways associated with cancer pathology7–10 and the
hallmark traits of cancer.11 Thus inhibition of Hsp90 has potential
apy. The abundance and role of chaperones in homeostasis contrib-
uted to early concerns as to the suitability of Hsp90 as a drug target.
However, the inherent selectivity of Hsp90 inhibitors for cancer
cells versus normal cells appears to provide a therapeutic window
for these agents. Rationalization for this selectivity includes the
prevalence of a high affinity form of Hsp90 in tumor cells,13 and
the ‘addiction’ or greater reliance of cancer cells on oncogenic client
proteins (especially the activated mutated forms).14 More recently
Hsp90 inhibition has attracted research interest in non-oncology
therapeutic areas such as inflammation and neurodegeneration.
Pioneering studies of Hsp90 inhibition characterized the natural
products geldanamycin15–17 and radicicol18,19 (Fig. 1) as competi-
tive binders to an ATP site on the N-terminal domain of Hsp90.20
These studies paved the way for the identification of semi-synthetic
inhibitors such as 17-AAG21 and 17-DMAG22 (5a and 5b; Fig. 2)
which progressed into clinical evaluation. Identification of
⇑
Corresponding author.
E-mail address: p.brough@vernalis.com (P. Brough).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.050

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6771
O
O
Cl
N
O
O
S
O
N
NH2
N
N
N
H
O
Me
O
O
N
N
N
H
Cl
N
H2N
N
O
H2N
N
S
H
H
O
N
O
HO
Me
MeO
Me
N
O
N
8
7
MeO
Me
OH
O
H2N
Cl
O
OH
O
OH
9: NVP-BEP800
N
OCONH2
geldanamycin
S
N
N
radicicol
PU3
N
N
N
N
N
N
N
Figure 1. Structures of the natural product Hsp90 inhibitors geldanamycin and
radicicol and the synthetic inhibitor PU3.
N
N
N
H
H
N
H
N
11
12
10
Figure 3. Fragment hits (7, 8 and 10–12) identified by 1H NMR screening, and the
pre-clinical Hsp90 inhibitor NVP-BEP800 (9). Fragments 10–12 were starting points
for Hsp90 inhibitors reported here.
H2N
O
Cl
N
O
H
N
N
N
N
N
H2N
OH
HO
N
O
N
H
N
N
amino-pyrimidine such as 1, PU3, 8 and 9 and the natural co-factor
analogue ADP-NP.40 Figure 4A shows the different orientations of
the purine moieties in fragment 11 and PU3 when they bind the
O
N
O
OH
F3C
O
ATP site of Human Hsp90a. Such differences probably arise because
1: BIIB021
2: SNX2112
3: NVP-AUY922
compounds 10–12 do not have an exocyclic NꢀH amino group
available to hydrogen-bond to Asp93, contrary to compounds 1, 8
and 9. Importantly, the new binding mode presented by the purine
in fragments 10–12, and the associated vectors for derivatisation,
suggested that they could be exploited in the design of novel
Hsp90 inhibitors. This has been achieved (see below), illustrating
the great versatility of fragments as starting points for medicinal
chemistry.
These structural data combined with in-house experience with
earlier series led to the design and synthesis of simple 4-aryl deriv-
atives (13a–c) that displayed moderate affinity to Hsp90 (Table 1).
The imidazole moiety in compounds 10–12 and 13a–c donates
a key hydrogen-bond to the carboxylate of Asp93 (Fig. 4A). This
requires that the purine adopts a favourable tautomer on the imid-
azole nitrogens, while the alternative tautomer removes this
interaction. To force location of the proton on the nitrogen hydro-
gen-bonding to Asp93, we synthesized an analogous series of
4-aryl-[2,3-d]pyrrolopyrimidines (14a–c, Table 1), since these
compounds necessarily contain the required N7–H hydrogen bond
donor. Yet, the replacement of N with C–H in the purine to pyrrol-
opyrimidine modification was clearly detrimental to binding affin-
ity (Table 1). This was attributed to a change in the hydrogen-bond
interactions between the ligand cores (purine or pyrrolopyrimi-
dine) and the structurally conserved water molecules (Fig. 4A) that
have been characterized in many ligand-Hsp90 structures.
O
R
N
N
O
Me
N
N
H
O
HO
Me
MeO
Me
HO
N
OH
O
MeO
Me
N
N
N
OH
H
OCONH2
OH
O
6: Ganetespib
5a: 17-AAG R = NHCH 2CH=CH2
5b: 17-DMAG R = NHCH 2CH2NMe2
4: AT13387
Figure 2. Disclosed structures of Hsp90 inhibitors in clinical trials.
synthetic inhibitors of Hsp90 function, such as the purine derivative
PU323 (Fig. 1) has been followed by extensive research in the
field24–28 and led to a number of synthetic small molecule inhibitors
entering the clinic29,30 (Fig. 2).
We have previously disclosed a novel series of Hsp90 inhibitors
based on a 4-aryl-5-cyanopyrrolopyrimidine scaffold.31 The rele-
vance of this core for Hsp90 inhibition has since been indepen-
dently confirmed.32 Here, we provide
a full account of the
structure-based design and detailed biological characterization of
compounds from this chemotype.
2. Hit identification, fragment evolution and compound design
The purine to pyrrolopyrimidine modification replaces an
OꢀHꢁ ꢁ ꢁN hydrogen bond between water 2 and purine with an
OꢀHꢁ ꢁ ꢁHꢀC5 steric clash between water and pyrrolopyrimidine,
probably explaining the loss of affinity for compounds 14a–c.
However, this analysis raised the possibility that water 2 may be
replaced to positive effect with respect to affinity by substituting
the pyrrolopyrimidine core at position 5. This reasoning was based
on the realization that (a) waters 2 and 3 are in a semi-hydropho-
bic environment (Leu48, Ile91 and Val186), a priori not the most
favorable environment for water molecules; (b) displacing
‘trapped’ water molecules may sometimes have an entropic advan-
tage;41 (c) replacing a water may enable additional favorable direct
interactions between ligand and protein. Computational chemistry
identified a number of small groups that had potential for the
investigation of this SAR with the cyano group being prioritized
from the outset. The cyano group was preferred because it was pre-
dicted that it would maintain a key hydrogen-bond with the Asn51
side chain (Figs. 4B and 5). Indeed, analysis of the hydrogen-bond
network formed by the conserved waters strongly suggested that
Hit identification remains a significant challenge in drug discov-
ery and fragment-based screening has emerged as an efficient way
to sample chemical space that can complement or replace other Hit
ID techniques such as high throughput and in silico screening.33–36
Combining fragment screening with structure-based design can
provide a powerful strategy for drug discovery37 and indeed frag-
ment-based approaches have been extensively used in Hsp90 dis-
covery programmes.38
We recently described a series of 2-aminothieno[2,3-d]pyrimi-
dine inhibitors (exemplified by 9; Figs. 3 and 6) that were designed
by combining structural information from hits identified from frag-
ment-based screening campaigns (e.g., 7 and 8; Fig. 3) with that
derived from in silico screening hits.39
Other hits identified from the fragment screening included
purine compounds 10–12 (Fig. 3). Interestingly, X-ray structures
revealed that these purine fragments bound to Hsp90 in an orienta-
tion different from that previously seen with amino-purines and

6772
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
A
B
Leu48
Leu48
Val186
Asn51
Val186
Asn51
2
3
3
Ile91
Ile91
4
4
Asp93
Asp93
1
1
Figure 4. Design of the cyanopyrrolopyrimidine core from the initial amino-purine fragment hit, illustrated with X-ray crystal structures of the N-terminal domain of human
Hsp90 in complex with representative compounds. The hydrogens were modeled on the X-ray coordinates so as to maximize the number of hydrogen-bonds between
a
compound, conserved waters and protein. Hydrogen bonds are depicted as green dotted lines. For consistency, the water molecules are numbered as previously assigned.32
Panel A: The initial fragment hit 11 [PDB code: 4FCP] (orange carbons, ball and stick) bound to the ATPase site of Hsp90, overlaid with the Hsp90 inhibitor PU3 [PDB code:
1UYM] (light blue carbons, sticks). The adenine moiety of 11 adopts an orientation different from the orientation of the adenine in PU3. The four conserved water molecules
are shown and numbered with red labels. Residues Leu48, Ile91 and Val186 line the cavity which contains conserved water molecules 2–4. Water 2 accepts a hydrogen-bond
from the side-chain amide of Asn51 and donates a hydrogen-bond to a nitrogen of 11. Panel B: The cyanopyrrolopyrimidine 15a [PDB code: 4FCQ] (magenta carbons, ball and
stick), overlaid with the initial purine fragment 11 [PDB code: 4FCP] (orange carbons, sticks). Conserved water 2 was displaced by the nitrile group of 15a, which accepts a
hydrogen-bond from the side-chain amide of Asn51. Note also the plausible unconventional hydrogen-bond donated by the C6ꢀH group of 15a to water 3.
Table 1
2.9 Å
Binding affinities of selected purines, pyrrolopyrimidines and pyrazolopyrimidines by
fluorescence polarization (FP), surface plasmon resonance (SPR) and isothermal
A
titration calorimetry (ITC) assaysa
Y
Y
Y
Y
X
S
X
S
X
X
S
CN
CN
N
5
N
3
N
N
N
2.5 Å
N
N
H
N
H
N
N
N
B
C
N
S
N
N
H
H
1
13
14
15
16
ID
X
Y
FP Kib
(lM)
SPR Kd
(l
M)
ITC Kd
(
lM)
13a
13b
13c
14a
14b
14c
15a
15b
15c
16
Me
Me
H
Me
Me
H
Me
Me
H
Cl
0.661
12.8
24.5
>200
>200
>200
>20
—
2.73
—
—
—
F
2.30
1.4 Å
CN
Me
F
CN
Me
F
>20
>100
>100
>100
—
0.195
0.101
0.430
1.22
0.156
0.251
—
0.385
0.204
5.23
CN
Me
Me
38
—
Figure 5. Simple geometric considerations regarding the displacement of water
molecules by substituents of aryl groups. Three model systems are shown, after
geometry optimization with the Merck molecular force-field44: (A) hydrogen-bond
(green dotted line) between pyridine and a water molecule; the Oꢁ ꢁ ꢁN distance is
2.9 Å; (B) benzonitrile, with a N„CꢀC covalently bonded length of 2.5 Å; (C)
phenol, with a OꢀC bond length of 1.4 Å. The aromatic rings are aligned vertically
across the three systems, to facilitate the visual comparison of the noted lengths.
a
All results mean of at least two independent determinations.
Ki calculated from IC50 values.
b
the oxygen of water 2 accepted a hydrogen-bond from the side-
chain amide group of Asn51. Modeling indicated that the nitrile
would project its nitrogen to a position similar to that of the
oxygen of water 2. Other, shorter groups such as methyl, chlorine
or hydroxyl would displace the water molecule, but without
replacing its interaction with Asn51 (Fig. 5). In addition, a cyano
substituent had the potential to further polarize the N7–H bond,
enhancing the N7–Hꢁ ꢁ ꢁAsp93 hydrogen-bond.
distance is about 2.9 Å. Importantly, this distance is similar to
the corresponding distance of 2.5 Å obtained with a cyano group
(Fig. 5B). Thus, a cyano substituent puts its nitrogen in a position
similar to that of the displaced water oxygen. Hence, the cyano
substituent has the right length to provide an equivalent of the
Oꢁ ꢁ ꢁN interaction, especially if the water oxygen is also accepting
a hydrogen-bond from a third group (e.g., Asn51 in Fig. 4A). In con-
trast, a hydroxyl substituent (Fig. 5C) is too short to bring its oxy-
gen in a position equivalent to that of the water oxygen. In other
words, a hydroxyl substituent is not expected to be a very good
substitute for the initial hydrogen-bonded water, either sterically
or electronically. This is worth noting since, based on the obvious
analogy between a water and a hydroxyl, chemical intuition might
suggest a hydroxyl group to displace the hydrogen-bonded water
molecule. However, simple geometric considerations indicate that
The present compound evolution suggests some generally
interesting geometrical insights regarding the practical displace-
ment of a water molecule which donates a hydrogen bond to a het-
eroatom acceptor in an aryl ring. This refers to water 2 donating a
hydrogen bond to an imidazole nitrogen in compound 11 (Fig. 4A),
and the replacement of this interaction with a nitrile group as in
compound 15a (Fig. 4B). This situation can be represented more
generally with the simplified model compounds shown in Fig. 5.
Figure 5A shows a water molecule donating a hydrogen-bond to
a pyridine nitrogen; as expected, the Oꢁ ꢁ ꢁN hydrogen-bonding

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6773
a nitrile group may frequently be a more suitable choice to dis-
place/replace a neighbouring water molecule. Indeed, there are
other examples where a nitrile group added to a ligand was found
to be very efficient way to displace water molecules.42 In addition,
the nitrile is a generally biocompatible functional group, incorpo-
rated in many drugs.43
optimization on this series. Encouragingly, the high affinity of the
new series led to moderate anti-proliferative activity by in vitro
cancer cell proliferation assays (15a GI50 = 11.6 lM, BT474 cell
line). Subsequent activity focused on enhancing cellular activity
and achieving a compound profile suitable for oral dosing.
We selected compounds 15a–c (Table 1) as initial target com-
pounds, based on their structural analogy to 13a–c and 14a–c.
The conservation of substituents X and Y across the sub-series
13–16 enabled interpretation of SAR pertaining to the variation
in the fused bicyclic core. Data presented in Table 1 showed that,
compared to analogous purines, 5-cyanopyrrolo[2,3-d]pyrimidines
displayed a significant and consistent enhancement of affinity, by
three independent assay techniques.
We also considered a number of other 5-position substituents
on the pyrrolo[2,3-d]pyrimidine core. As expected, data presented
in Tables 1 and 2 show that no analogue matched the exquisite
affinity of the cyano compounds 15a–c, for reasons that can be
understood by simple molecular recognition considerations. It is
important to note that the nitrile provides a significant affinity gain
with minimal addition of molecular weight to the compound and
thus contributes to the high ligand efficiency observed (LE for
15b = 0.41 kcal/mol/heavy atom).
A small set of analogous 5-cyanopyrazolopyrimidines were also
made (e.g., 16, Table 1) but these compounds had considerably
lower affinity for Hsp90 than the analogous pyrrolopyrimidines.
This reduction in binding affinity may be explained by an electro-
static clash between the oxygen of conserved water molecule 3 and
the introduced N-6 atom in the pyrazolopyrimidines. Instead, in
the pyrrolopyrimidines there is a favourable interaction with
hydrogen-bond character between the pyrrole C6-H and the water
oxygen (Fig. 4). Modeling of small aliphatic substituents on C6 was
attempted, with the intention to displace conserved water 3. How-
ever, this exercise suggested that these substituents may clash
with the protein and therefore these compounds were not made.
A recent publication reporting an independent discovery of pyrrolo
[2,3-d]pyrimidines has confirmed the reduced interest of such
substituents.32
3. Compound optimisation and in vitro characterisation
Inspection of the crystal structures of Hsp90-bound cyanopyr-
rolopyrimidines and overlays with ligands from other series such
as thienopyrimidine 9 (Fig. 6) indicated there were two main vec-
tors for the elaboration of the cyanopyrrolopyrimidine series; (a)
the 50 position of the 4-aryl group (as used to effect in the pro-
gramme leading to identification of 9) and (b) the 2-position of
the pyrrolopyrimidine core, via sulphur or other linking atom. Sub-
stitution at the para position of the 4-aryl group was limited to
small moieties (Table 2) due to steric constraints imposed by the
protein. Figure 6 shows the overlay of 15a with 9, illustrating these
points. Subsequent elaboration via either or both the preferred vec-
tors afforded a number of compounds with high affinity for Hsp90
and greatly improved anti-proliferative properties (Table 3).
These initial modifications however, generated many com-
pounds with poor metabolic stability and an undesirable hERG
binding profile (Table 4) which was possibly due to the incorpora-
tion of an alkyl amino functionality. However the S-acetamide
compounds such as 33 (Table 3) had a somewhat more promising
metabolic profile and further optimization identified the S-acetam-
ide series 36a–36r (Table 5). The data in Table 5 demonstrates that
excellent anti-proliferative activity could be achieved in com-
pounds that did not contain an amino solubilising group. This sig-
nificantly reduced the propensity for hERG binding as seen for
compounds 36q and 36r in Table 4. Compound 36r was further
characterized by isothermal titration calorimetry (ITC) and surface
plasmon resonance (SPR). The ITC-derived Kd for 36r was <3 nM
Asp93
Following the successful strategy of targeting water 2 with
cyanopyrrolopyrimidines 15a–c, we initiated a programme of lead
4
Table 2
1
Binding affinities of selected 5-substituted pyrrolo[2,3-d]pyrimidines by fluorescence
polarization (FP), surface plasmon resonance (SPR) and isothermal titration calorim-
etry (ITC) assaysa
Y
Me
R
N
N
H
W
N
ID
W
Y
R
FP Kib lM) SPR Kd
(
lM) ITC Kd (lM)
14a SMe
14b SMe
Me
F
Me Br
Me Me
Me CF3
Me Cyclo-
propyl
H
H
>200
>200
>200
>200
2.64
>50
>100
>100
>20
19.3
55
—
—
—
—
—
—
Figure 6. Vectors available for elaboration of the cyanopyrrolopyrimidine series
17
18
19
20
SMe
SMe
SMe
SMe
when bound to Hsp90a (grey surface). The cyanopyrrolopyrimidine series is
represented by 15a [PDB code: 4FCQ] (magenta carbons, ball and stick). It is
overlaid on compound 9 [PDB code: 2WI7] (green carbons, sticks), a representative
>100
of
a previously reported 2-aminothieno[2,3-d]pyrimidine series of extensively
elaborated Hsp90 inhibitors.39 The overlay is based on X-ray structures, on which
hydrogens were modeled and shown selectively (white). The protein is represented
as a grey molecular surface around the biding site, to emphasize the tight structural
fit between Hsp90 and the cyanopyrrolopyrimidine chemotype. In this context, the
two positions (indicated with black vectors) particularly amenable for derivatisa-
tion of the cyanopyrrolopyrimidine were the 2-position of the pyrrolopyrimidine
core and the 50-position of the 4-aryl group. Asp93 is shown for orientation, with
selected hydrogen bonds (green dotted lines). Conserved water molecules 1 and 4
are labeled.
21
22
SMe
H
F
CONH2
>200
2.83
0.358
0.628
4.07
>100
1.88
0.430
2.81
4.26
2.22
—
Me CN
1.62
0.251
0.725
3.69
1.83
15b SMe
F
F
F
F
CN
CN
CN
CN
23
24
25
OMe
NHMe
Et
1.72
a
All results mean of at least two independent determinations.
Ki calculated from IC50 values.
b

6774
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
Table 3
Affinity (FP) and anti-proliferative data (BT474 cells) for elaborated cyanopyrrolo[2,3-d]pyrimidinesa
Y
O
R1
Cl
CN
N
N
H
W
N
ID
W
Y
R1
FP Kib (nM)
GI50 (BT474) (nM)
26
27
SMe
SMe
Cl
Cl
4
2
48
31
N
CH2CH2NEt2
28
29
SMe
SMe
OMe
OMe
3
31
47
F
N
F
12
N
F
30
31
32
Cl
Me
<1
7
72
26
N
N
N
S
S
OMe
Cl
Me
Me
62
2010
O
S
H
N
33
Cl
CH2CH2NEt2
8
78
O
34
35
CN
Me
<1
80
N
S
S
F
OMe
172
388
N
F
a
All results mean of at least two independent determinations.
Ki calculated from IC50 values.
b
which was readily converted to the SEM protected halide 42. At
this point Suzuki cross coupling reactions could be used to intro-
duce the 4-aryl moieties, followed by 3-bromination and conver-
sion to 3-cyano compounds via Pd-mediated cyanation (42 to 15
via 47; Scheme 1). However this reaction proved capricious and
we routinely used the longer, but more robust route shown, via
the carboxylic acid 44.
Table 4
hERG bindinga and patch clampb data for selected compounds
ID
hERG binding IC50
(lM)
hERG patch clamp IC50
(
lM)
26
27
30
33
34
36q
36r
3.64
1.71
1.40
3.14
1.07
>30
—
—
—
17.8
—
>30
>30
The compounds shown in Table 5 and compounds 30–35 in Ta-
ble 3 were synthesized by a similar general route (Scheme 2)
which utilized the SMe moiety as a versatile chemical handle by
converting it to the electrophilic sulphone. Suzuki cross-coupling
of 46 (Scheme 1) with MOM protected boronic acid 48a (formed
in situ from the corresponding bromide), afforded the 4-aryl deriv-
ative 49. Oxidation of the sulphide to corresponding sulphone 50
enabled displacement by ethyl thioglycolate followed by elabora-
tion to the desired amides 53. Intermediate 50 could also be re-
acted with oxygen, carbon and nitrogen based nucleophiles,
though these compounds were not as potent as the analogous
sulphides. Removal of the MOM protecting group followed by
either alkylation with alkyl iodides then TBAF mediated SEM re-
moval or sole SEM removal (as appropriate) provided compounds
36(a–r). Several compounds from this series used the MEM pro-
tected compound 48b as a starting point, with the subsequent
transformation as described in scheme 2 affording similar chemical
yields.
>30
a
hERG binding IC50 determined in a 3H-dofetilide assay.
Effect on ionic currents in voltage-clamped HEK293 cells that stably express the
b
hERG channel.
and the enthalpy of binding (
D
H) was ꢀ10.3 kcal/mol versus
ꢀ5.5 kcal/mol for the less elaborated compound 15b. The SPR-de-
rived Kd for 36r was 0.4 nM with a measured dissociation rate (Koff
of 0.0006 sꢀ1 (vs 0.20 sꢀ1 for 15b).
)
One compound that showed potent anti-proliferative effects
across a range of cell lines was the N,N-dimethyl amide derivative
36r. Figure 7 shows the X-ray crystal structure for compound 36r
bound to Hsp90a. This demonstrates that the same binding pose
of the core is maintained through series elaboration and highlights
further interactions such as a water-mediated interaction from
methoxy substituents to Phe138. Interestingly there is a similar
interaction made from a phenolic oxygen in thienopyrimidine 9.39
Pyrrolopyrimidine compounds substituted with other groups at
the 5-position made use of key intermediates 47a and 44
(Scheme 3). Thus the 5-bromo compound 47a was a versatile inter-
mediate for coupling and lithiation chemistry to furnish com-
pounds 18–20, whilst 55 was employed via Suzuki coupling to
afford 21. Intermediate 57 enabled the preparation of the 2-OMe,
4. Synthetic chemistry
The pyrrolo[2,3-d]pyrimidine scaffold was constructed via a
modified literature route45 (Scheme 1) generating compound 41,

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6775
Table 5
Binding affinity (FP) and anti-proliferative data (three cancer cell lines) for S-acetimido-4-aryl-5-cyanopyrrolo[2,3-d]pyrimidinesa
OMe
O
R
Cl
CN
N
R1R2N
N
H
S
N
O
36
ID
R
NR1R2
FP Kib (nM)
BT474 GI50 (nM)
HCT116 GI50 (nM)
MDA-MB-468 GI50 (nM)
36a
36b
36c
Me
H
<1
<1
<1
69
102
25
60
1160
20
62
236
23
NH
NH
Et
NH
36d
36e
36f
36g
36h
36i
36j
36k
36l
36m
36n
36o
36p
36q
36r
H
Me
Et
H
Me
Et
Me
Me
H
Me
H
Me
Me
H
i-PrNH
EtNH
EtNH
3
2
161
141
44
128
121
84
81
136
85
45
21
31
56
238
42
40
117
50
65
81
98
728
153
172
28
178
59
64
152
84
77
102
178
183
124
28
<1
<1
<1
<1
<1
5
t-BuNH
t-BuNH
i-PrNH
i-PrNH
F(CH2)2NH
F(CH2)2NH
CF3CH2NH
Azetidine
Azetidine
MeNH
2
<1
<1
<1
<1
<1
<1
35
33
23
30
42
131
32
Me2N
Me2N
20
34
Me
a
All results mean of at least two independent determinations.
Ki calculated from IC50 values.
b
similar to those already described but with some variety in the se-
quence of the compound construction. Mitsunobu reactions or
phenolic alkylations with amino-alkyl halides were used to pre-
pare the 50 aryl phenolic substituents, either prior to or post cou-
Asp93
3
4
pling to the cyano-pyrrolopyrimidine scaffold. Scheme
5
illustrates how the analogous purines, 5-H pyrrolopyrimidines
and pyrazolo-pyrimidines from Table 1 were prepared. Com-
pounds 14a–c used the same coupling and de-protection strategy
as already described for 15a–c, but using the intermediate 42.
Cross coupling reactions were also used to construct purines
13a–c and pyrazolo derivative 16.
1
Asn51
Phe138
5. Pharmacodynamic and pharmacokinetic studies
To establish that the cyanopyrrolo[2,3-d]pyrimidine class of
compounds retained the molecular signature of known Hsp90
inhibitors, we studied the effect of selected compounds 36q, 36r
and 28 on cellular pharmacodynamic biomarkers compared to
the thienopyrimidine compound 9. Figure 8 shows that the repre-
sentative compounds gave the expected depletion of the client pro-
teins Her2, pHer2, pAKT and AKT and induction of the Hsp70 co-
chaperone.
The pharmacokinetic (PK) profile following oral dosing was
evaluated for cell active compounds 36q and 36r in the mouse (Ta-
ble 6). In summary 36r was bioavailable via the oral route of
administration. The exposure of 36r in plasma was moderate and
the half life (>1 h) can be considered reasonable for the mouse.
In contrast however 36q was very poorly bioavailable via the oral
route of administration (Table 7). The exposure of 36q in plasma
and kidney was very low and the half life could not be determined;
36q was detected also in muscle, but at levels below the limit of
quantification. The compounds differ by only one methyl group
(methoxy in 36r to hydroxyl in 36q) and glucuronidation of the
free phenolic OH in 36q during first pass metabolism may explain
Figure 7. X-ray structure of cyanopyrrolopyrimidine 36r [PDB code: 4FCR]
(magenta carbons, ball and stick) bound to Hsp90 (grey surface). Hydrogen atoms
a
were modeled on the X-ray structure and are shown selectively. The surface
rendering of the protein emphasizes the excellent structural fit between Hsp90 and
compound 36r. Selected hydrogen-bonds (green dotted lines) are shown, in
particular between 36r, Asp93, Asn51, and water molecules of particular interest.
Structurally conserved water molecules 1, 3 and 4 are labeled. A water molecule is
also shown, which hydrogen-bonds the two methoxy groups of 36r, the backbone
of Phe138 and the side-chain of Asn51. The Asn51 side-chain also hydrogen-bonds
the nitrile of 36r.
NHMe and ethyl analogues of the 2-S-methyl derivative 15b (23–
25, Table 2), by again making use of the sulphone moiety for nucle-
ophilic displacement reactions (Scheme 3).
Other elaborated compounds from Table 3 were prepared as
shown in Scheme 4. The procedures and transformations are

6776
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
OH
N
O
O
O
b
Br
a
O
O
N
+
O
O
O
O
O
CN
CN
HS
NH2
40
39
37
38
OH
N
Cl
c
N
d
N
e
N
N
S
SiMe3
S
N
H
O
42
41
Cl
Br
Cl
Cl
CO2H
CONH2
O
N
g
f
N
N
N
SiMe3
S
N
N
SiMe3
N
S
N
SiMe3
O
S
N
O
43
44
45
h
Cl
N
CN
Ar
Ar
N
Br
CN
i,e
j,k
i,k
N
42
N
N
N
SiMe3
S
N
O
N
H
SiMe3
S
N
S
O
46
15
15a: Ar = 2,4-dimethylphenyl
15b: Ar = 4-fluoro-2-methylphenyl
15c: Ar = 4-cyanophenyl
47
47a: Ar = 2,4-dimethylphenyl
Scheme 1. Synthesis of 4-aryl-5-cyanopyrrolopyrimidines. Reagents and conditions: (a) Bu4NBr, K2CO3, DMF, 90 °C, 40–55%; (b) thiourea, NaOEt, EtOH, reflux, 50%; (c) (i)
NaOH (aq), dimethylsulphate, rt, (ii) HCl (aq), rt, 92%; (d) (i) POCl3, 100 °C, 81%, (ii) SEM-chloride, NaH, DMF, 0 °C to rt, 86%; (e) NBS, DMF, rt, 70%; (f) n-BuLi, THF, CO2, ꢀ78 °C
to rt, 91%; (g) (i) CO2Cl2, CH2Cl2, DMF (cat), (ii) NH3 (aq), rt, 77%; (h) TFAA, Et3N, CH2Cl2, 0 °C to rt, 86%; (i) ArB(OH)2, NaHCO3, Pd2Cl2(PPh3)2, DMF, 80 °C; (j) CuCN, dppf,
Pd2(dba)3, 1,4-dioxane, 100 °C; (k) TBAF, H2N(CH2)2NH2, THF, 50 °C.
O
N
O
N
O
N
O
O
O
O
O
O
O
O
O
c
a
b
Cl
R3
Cl
CN
Cl
CN
46
CN
+
Cl
N
N
O
N
B(OH)2
48a: R3 = Me
(48b: R3 = CH2CH2OMe)
N
RO
SiMe3
N
S
SiMe3
S
N
SiMe3
S
O
O
O
49
O
50
O
O
N
51: R = Et
52: R = H
O
d
O
N
OH
CN
O
O
g
e
OR
f
Cl
Cl
CN
Cl
CN
N
N
N
R1R2N
R1R2N
N
N
SiMe3
S
SiMe3
S
N
R1R2N
O
N
H
O
S
O
O
O
53
53a: R1=R2=Me
54
54a: R1=R2=Me
36
Scheme 2. Synthesis of 4-aryl-2-acetamido-5-cyanopyrrolo[2,3-d]pyrimidines. Reagents and conditions: (a) PdCl2(PPh3)2, NaHCO3, DMF, 80 °C, 1.5 h, 98%; (b) mCPBA,
CH2Cl2, 0 °C to rt, 1 h, 80%; (c) ethyl thioglycolate, NaH, THF, 0 °C to rt, 1 h, 96%; (d) NaOH (aq), rt, 2 h; (e) HNR1R2, HBTU, MeCN, rt; (f) PPTS, i-PrOH, 85 °C, 16 h; (g) (i) RI,
Cs2CO3, DMF, rt, 2 h and/or (ii) TBAF, H2N(CH2)2NH2, THF, 40 °C.
the significant difference in PK profiles. This hypothesis is sup-
ported by UGT intrinsic clearance (UGT CLint) data from a micro-
somal incubation assay formatted to measure clearance via
glucuronidation by UGT.46 In this assay the UGT intrinsic clearance
Ligand-Hsp90 X-ray crystallography in conjunction with molecular
modeling studies led to rational displacement of a selected con-
served water molecule by the inhibitors, which increased their
binding affinity efficiently with the addition of a single nitrile
group. Importantly, this affinity gain was obtained with minimal
addition of molecular weight, and this decisively influenced the
choice of core for subsequent elaboration by medicinal chemistry.
Further structure-based optimization led to a series of high affinity
compounds, examples of which displayed sub 100 nM GI50 values
in anti-proliferative assays in several human cancer cell lines
in vitro. One compound was additionally established to be bio-
available in the mouse following oral dosing. The study highlights
the importance of precise consideration of water molecules in
for 36r was 4
lL/min/mg. This contrasts with a much higher value
(UGT CLint = 73
l
L/min/mg) for 36q (corresponding to a high
extraction ratio of 0.8).
6. Conclusion
In summary we have identified a series of 4-aryl-5-cyanopyrrol-
o[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, using start
points from a 1H NMR screen of a proprietary fragment library.

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6777
CF3
N
N
N
N
H
S
N
S
N
H
19
18
c,a
b,a
a
d,a
Br
Br
N
N
N
N
N
H
SiMe3
S
N
S
N
N
S
N
O
H
17
47a
20
F
F
O
N
O
e
a
NH2
O
NH2
45
N
N
S
SiMe3
S
N
N
H
N
55
21
f
F
F
F
g
1. h or i or j
2. a
CN
CN
CN
N
N
N
N
SiMe3
N
H
S
N
X
N
N
SiMe3
S
N
O
O
O
O
23: X = O
24: X = NH
25: X = CH2
56
57
Scheme 3. Synthesis of 5- and 2-substituted pyrrolo[2,3-d]pyrimidines. Reagents and conditions: (a) TBAF, H2N(CH2)2NH2, THF, 40 °C; (b) (i) n-BuLi, THF, ꢀ78 °C, (ii) MeI,
ꢀ78 °C to rt; (c) CF3CO2Na, CuI, DMF, toluene, 170 °C, 16 h; (d) cyclopropyl boronic acid, Pd(OAc)2, P(Cy)3, K3PO4, toluene, H2O, 100 °C, 2 h; (e) 2-methyl-4-fluoro-phenyl
boronic acid, NaHCO3, Pd2Cl2(PPh3)2, DMF, 80 °C; (f) TFAA, Et3N, CH2Cl2, 0 °C to rt; (g) mCPBA, CH2Cl2, 0 °C to rt, 1 h; (h) KOtBu, MeOH, THF, 0 °C; (i) MeNH2, THF, DMF, 100 °C,
5 h; (j) EtMgBr, Et2O, 0 °C.
ligand–protein binding and the potentially special role of the ni-
trile group in displacing and replacing such molecules.
were detected using chemiluminescence and imaged on an LAS-
4000 (Fuji Instruments). GAPDH levels were determined to show
equal protein loading.
7. Experimental
7.3. Pharmacokinetics
7.1. Fluorescence polarization assay
Male Balb-C mice received a single 5 mg/Kg dose of compound
via oral gavage in 1% methyl cellulose (w/v). Plasma was prepared
from terminal blood samples for analysis. LC–MS/MS was used for
quantitative bioanalysis and pharmacokinetic analysis was per-
formed using WinNonLin (Pharsight).
The ability of compounds to compete with a fluorescently la-
beled probe for binding to full length human Hsp90b was deter-
mined essentially as described previously47 but with the following
modifications: firstly, a modified probe (VER-51001) was used that
binds to Hsp90 with a greater affinity that that described previously,
allowing the accurate determination of IC50 for higher affinity com-
pounds; secondly, a N-terminally truncated form of human Hsp90-
alpha was used due to its more robust performance in FP assays.
Synthetic procedures for VER-51001 and details relating o the FP as-
say can be found in the Supplementary data section.
7.4. Glucuronidation assay
Rate of glucuronidation was measured by incubating test com-
pounds in human liver microsomes supplemented with MgCl2, ala-
methicin and UDPGA.
A time course was run over 30 min.
Individual wells for each time point were stopped by the addition
of acetonitrile, samples were centrifuged and the supernatant ana-
lyzed by LC–MS/MS. The rate of compound disappearance was
scaled to intrinsic clearance (UGT CLint) and a UGT extraction ratio
was generated for rank-ordering.
7.2. Cell growth and client protein degradation
Growth inhibition assays using the SRB method were under-
taken as previously described.48 Her2-postive BT474 breast cancer
cells were treated with the indicated concentrations of Hsp90
inhibitors for 24 h and client protein degradation determined by
western blotting. Her2 (Abcam), pHer2 (Y1248, Abcam), AKT (Cell
Signalling Technologies), pAKT (S473, Cell Signalling Technologies),
HSP70 (Stressgen) and GAPDH (Cell Signalling Technologies) levels
7.5. Isothermal titration calorimetry (ITC)
The ITC measurements were performed using an iTC200 instru-
ment (Microcal, GE Healthcare). The feedback mode was ‘low’ with

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N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
O
O
N
OH
O
N
O
a
Cl
CN
Cl
CN
N
N
N
SiMe3
S
N
O
SiMe3
S
O
60
b
49
X
N
X
N
N
O
26: X=Cl, R =
27: X=Cl, R =
X
R
O
R
O
R
Cl
g
N
c
CN
+
Cl
46
CN
Cl
N
F
N
28: X=OMe, R =
29: X=OMe, R =
N
B(OH)2
N
SiMe3
S
F
O
N
H
S
N
F
61
61a: X=OMe, R =
F
N
N
61b: X=Cl, R = Me
61c: X=OMe, R =
61d: X=CN, R = Me
F
F
d
Cl
N
30: X=Cl, R=Me, R1 =
X
O
X
O
R
O
31: X=OMe, R=Me, R1 =
N
R
Cl
CN
H
N
f,g
e,g
Cl
CN
Cl
N
N
, R1=
CN
33: X=Cl, R =
N
O
N
O
N
H
N
O
N
N
SiMe3
S
N
N
F
F
34: X=CN, R=Me, R1 =
35: X=OMe, R =
R1
N
H
S
N
O
O
32
62
N
, R1=
62b: X=Cl, R = Me
62f: X=OMe, R = Me
62e: X=CN, R = Me
Scheme 4. Synthesis of elaborated 5-cyano-pyrrolo[2,3-d]pyrimidines. Reagents and conditions: (a) PPTS, i-PrOH, 85 °C, 3 h, 82%; (b) DIAD, PPh3, THF, ROH, 0 °C to rt; (c)
NaHCO3, Pd2Cl2(PPh3)2, DMF, 80 °C; (d) mCPBA, CH2Cl2, 0 °C to rt; (e) R1SH, Et3N, 100 °C, 30 min.; (f) Et2N(CH2)2OH, Et3N, DMF, 100 °C, 30 min.; (g) TBAF, H2N(CH2)2NH2, THF,
40 °C.
R2
Cl
N
Cl
N
13a: R1 = Me, R2 = Cl
N
a
N
N
b,c
R2
N
N
R1
S
13b: R1 = Me, R2 = F
13c: R1 = H, R2 = CN
N
H
Cl
Cl
N
N
O
N
H
N
63
Cl
N
14a: R1 = Me, R2 = Me
14b: R1 = Me, R2 = F
14c: R1 = H, R2 = CN
R1
S
d, e
N
N
SiMe3
N
S
N
H
O
N
42
O
Cl
O
CN
N
NH2
CN
N
HN
f
g
N
N
16
N
N
S
N
N
H
S
N
H
N
H
S
N
64
65
Scheme 5. Synthesis of scaffold analogues of 5-cyano-pyrrolo[2,3-d]pyrimidines. Reagents and conditions: (a) (i) PTSA, EtOAc, (ii) 3,4-dihydro-2H-pyran, 50 °C, NH3 (aq),
73%; (b) ArB(OH)2, K2CO3, DMF, (Ph3P)4Pd, 110 °C, microwave; (c) NaSMe, DMF, 120°C, microwave, 15 min; (d) ArB(OH)2, NaHCO3, Pd2Cl2(PPh3)2, DMF, 80 °C; (e) TBAF,
H2N(CH2)2NH2, THF, 40 °C; (f) POCl3, N,N-dimethylaniline,
D, 4 h; (g) 2,4-dimethylphenylboronic acid, K3PO4 (aq), Pd(dppf)2Cl2, 1,4-dioxane, 90 °C, 6 h.
reference power setting of 4
1000 rpm and thermostated at 25 °C. All experiments were per-
l
Calsꢀ1. The cell was stirred at
respectively, and 300 s spacing. The first ‘waste’ injection of
1.2 L was discarded in all cases. All data were fitted to a one site
l
formed using the dialysis buffer (see below) with 1% DMSO (v/v).
model using the provided software.
All experiments were conducted with 10
and 100–200 M protein in the syringe. The experiments were
conducted with 8–16 injections, with volumes of 4.75–2.37 L,
l
M ligand in the cell
Protein (6 mg/ml) was dialysed with stirring overnight at 4 °C,
in 12.5 mM phosphate pH 7.4, 37.5 mM NaCl, 0.5 mM EDTA,
0.05% Triton-100. Upon recovery the protein was filtered
l
l

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6779
Ni2+ were included on the chip to serve as controls for non-specific
binding and refractive index changes. The sensor surface was
regenerated between experiments as advised by manufacture with
additional injections of 0.1 mg/ml Trypsin, 0.5 M Imidazole and 45%
DMSO (all at a flow rate of 15 l
L minꢀ1 60 s) to eliminate any
carry-over of protein and/or analyte. For each concentration in
the titration series the surface was prepared as described above.
The concentration series was formed using doubling dilutions from
the top concentration of at least 5–10 fold above the Kd. All sample
measurements were performed at a flow rate of 35 l .
L minꢀ1
Injection times ranged from 60 to 480 s, dissociations times ranged
from 60 to 480 s. Data processing was performed using BIAevaluation
1.1 software (BIAcore GE Healthcare Bio-Sciences Corp.). Sensor-
grams were double referenced prior to global fitting of the concen-
tration series to a single step kinetic model.
Figure 8. Western blot showing effects on PD biomarkers in BT474 cells of Hsp90
inhibitors 9, 36q, 36r and 28 at 0.1, 0.3 and 1.0 M compound concentration after
24 h incubation. GAPDH was used as a loading control.
l
7.7. Chemistry
Table 6
7.7.1. 4-Chloro-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxy
methyl)-7H-pyrrolo[2,3-d]pyrimidine (42)
PK profile for 36r following dosing at 5 mg/Kg po in Balb-C mouse
Parameter
Plasma
Muscle
Kidney
To a mixture of sodium hydride (60% dispersion in mineral oil;
276 mg; 6.89 mmol) in DMF (10 mL) at 0 °C was added drop-wise a
solution of 4-chloro-2-methylsulfanyl-7H-pyrrolo[2,3-d]pyrimi-
dine [prepared as detailed in Ref. 45] (1.145 g; 5.74 mmol) in
anhydrous DMF (20 mL). When the addition was complete, 2-
(trimethylsilyl)ethoxymethyl chloride (1.32 ml; 7.46 mmol) was
added drop-wise and the reaction mixture was stirred at 0 °C for
1.5 h then allowed to warm to ambient temperature. The reaction
mixture was partitioned between water (100 mL) and ethyl acetate
(100 mL). The organic phase was separated, dried over Na2SO4 and
then filtered and the filtrate solvents evaporated in vacuo. The
crude pro0duct was purified by flash chromatography on silica
gel (70 g) eluting with a solvent gradient of 0–5% ethyl acetate in
hexane to afford the title compound 42 (1.73 g, 91%) as a colourless
oil: LC–MS (method A) tR = 2.88 min; m/z = 332, 330 [M+H]+; 1H
AUC (0–1, ng/mL h)
Half life (h)
Cmax (ng/mL)
297
1.4
239
0.553
0.25
—
231
2.4
68
0.158
0.25
0.8
6915
1.2
5389
12.48
0.25
23.3
Cmax
(l
M)
Tmax (h)
Tissue/plasma ratio
Table 7
PK profile for 36q following dosing at 5 mg/Kg po in Balb-C mouse
Parameter
Plasma
Kidney
AUC (0–2 h, ng/mL h)
Half life (h)
Cmax (ng/mL)
16
ND
11
0.025
0.25
—
41
ND
31
0.072
0.50
2.6
Cmax
(l
M)
NMR (400 MHz, DMSO-d6):
d
ꢀ0.10 (s, 9H), 0.84 (t, 2H,
Tmax (h)
Tissue/plasma ratio
J = 8.1 Hz), 2.53 (s, 3H), 3.52 (t, 2H, J = 8.1 Hz), 5.58 (s, 2H), 6.62
(d, 1H, J = 3.6 Hz), 7.69 (d, 1H, J = 3.6 Hz).
ND = not determined.
7.7.2. 5-Bromo-4-chloro-2-methylsulfanyl-7-(2-trimethylsila
nyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (43)
To a solution of 4-chloro-2-methylsulfanyl-7-(2-trimethylsila-
(0.22 lM spin filter). The protein concentration was determined by
UV absorbance spectroscopy at 280 nM using an extinction coeffi-
cient of 15900. The dialysis buffer was degassed; this was then
used for subsequent preparation of protein and ligand solutions
for the titration experiments. For each experiment the ligands were
freshly prepared from a 20 mM DMSO stock solution.
nyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine
(42,
0.5 g,
1.516 mmol) in DMF (14 mL) at ꢀ78 °C was added drop-wise a
solution of N-bromosuccinimide (270 mg, 1.52 mmol) in DMF
(6 mL). After 5 min the reaction was allowed to warm to ambient
temperature. The solution was partitioned between EtOAc
(2 ꢂ 40 mL) and water (40 mL). The combined organics were
passed through a hydrophobic frit and evaporated in vacuo. The
crude product was purified by flash chromatography on SiO2
(50 g) eluting with hexane-5% EtOAc/hexane (gradient) to afford
the title compound 43 (433 mg, 70%) as a colourless solid: LC–
MS (method A) tR = 3.11 min; m/z = 410, 408 [M+H]+.
7.6. Surface plasmon resonance (SPR)
SPR measurements were performed on a BIAcore T200 instru-
ment (BIAcore GE Healthcare). All experiments were performed
on Series S NTA chips (certified) according to provider’s protocols
with 10 mM HEPES pH 7.4, 150 mM NaCl, 25 M EDTA, 0.05%
Tween-20 and 1% DMSO as a running buffer. The surface was gen-
erated using HSP90 aa9-236 construct containing on its N-terminus
three consecutive hexa-Histidine tags (mhhhhhhhhgatgstagsg-
tagstgasgastggtgathhhhhhhhddddkspmghhhhhhhhhhssghiddddk).
Introduction of the multiple oligo-Histidine tag led to robust gener-
ation of a stable Hsp90 surface with no detectable drift of a protein
from a chip for at least 30 min. The use of double his-tagged pro-
teins to generate stable surfaces has been previously described in
Ref. 49. First, 0.5 mM Ni2+ was been injected into the experimental
channel till 60RU. Subsequently 100 nM HSP90 was injected over
7.7.3. 4-Chloro-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxy
methyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid (44)
To a solution of n-butyl lithium (2.5 M in hexanes, 0.24 ml,
0.59 mmol) in THF (0.5 ml) at ꢀ78 °C was added slowly drop-wise
a solution of 5-bromo-4-chloro-2-methylsulfanyl-7-(2-trimethyls-
ilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (43, 200 mg,
0.489 mmol) in THF (2 mL). After 2 min, crushed solid CO2 (excess)
was added and the mixture was left to warm to ambient tempera-
ture. Acetic acid (2 mL) was added, then water (20 mL) and the
mixture extracted with EtOAc (2 ꢂ 20 mL). The combined organics
were passed through a hydrophobic frit and evaporated in vacuo to
the sensor at 10
bound on the surface. Reference surfaces without immobilized
l
L minꢀ1 till saturation level of 1500 RU stably

6780
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
afford the title compound 44 (167 mg, 91%) as a colourless solid:
LC–MS (method A) tR = 2.66 min; m/z = 374 [M+H]+.
was allowed to cool to ambient temperature, and partitioned be-
tween EtOAc (2 ꢂ 20 mL) and sat. NaHCO3 (aq) solution (20 mL).
The combined organics were passed through a hydrophobic frit
and evaporated in vacuo to give a crude solid (100 mg). The crude
product was purified by flash chromatography on SiO2 (20 g) elut-
ing with hexane to 10% EtOAc/hexane (gradient) to afford the SEM
protected intermediate, 4-(2,4-dimethyl-phenyl)-2-methylsulfa-
nyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrim-
idine-5-carbonitrile (10 mg, 0.024 mmol). This product was
dissolved in THF (0.4 mL), then ethylenediamine (0.005 ml,
0.071 mmol) and tetrabutylammonium fluoride (TBAF, 1.0 M solu-
tion in THF, 0.15 mL, 0.142 mmol) were added sequentially. The
reaction mixture was heated at 50 °C overnight, allowed to cool
to ambient temperature and then was partitioned between EtOAc
(2 ꢂ 10 mL) and water (10 mL). The combined organics were
passed through a hydrophobic frit and evaporated in vacuo. The
resultant crude product was purified by flash chromatography on
SiO2 (5 g) eluting with hexane to 40% EtOAc/hexane (gradient) to
afford the title compound 15a (5 mg, 72%) as an off-white solid:
LC–MS (method A) tR = 2.44 min; m/z = 295 [M+H]+; 1H NMR
(400 MHz, CD3OD): d 2.26 (s, 3H), 2.43 (s, 3H), 2.65 (s, 3H), 7.19
(d, 1H, J = 7.7 Hz), 7.23 (s, 1H), 7.30 (d, 1H, J = 7.7 Hz), 8.11 (s,
1H) NH not obsd; HRMS, Calcd for C16H13N4S [MꢀH]ꢀ. Found
293.0875 requires 293.0861; HPLC (from Method A) 100%
(tR = 2.41 min).
7.7.4. 4-Chloro-2-methylsulfanyl-7-(2-trimethylsilanyl-
ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid
amide (45)
To a solution of 4-chloro-2-methylsulfanyl-7-(2-trimethylsila-
nyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid
(44, 100 mg, 0.268 mmol) in CH2Cl2 (1.5 mL) was added oxalyl
chloride (2 M in CH2Cl2, 0.17 mL, 0.349 mmol) followed by a few
drops of DMF. After 10 min the reaction mixture was evaporated
in vacuo, then re-dissolved in CH2Cl2 (3 mL). Aqueous ammonia
solution (2 mL) was added and the mixture was stirred vigorously
for 15 min. Water (10 mL), and CH2Cl2 (10 mL) were added and the
resultant phases separated. The aqueous phase was extracted with
further CH2Cl2 (15 mL). The combined organics were passed
through a hydrophobic frit and evaporated in vacuo. The crude
product was purified by flash chromatography on SiO2 (20 g) elut-
ing with CH2Cl2-5% MeOH/CH2Cl2 (gradient) to afford the title
compound 45 (77 mg, 77%) as a yellow solid: LC–MS (method A)
tR = 2.47 min; m/z = 375, 373 [M+H]+.
7.7.5. 4-Chloro-2-methylsulfanyl-7-(2-trimethylsilanyl-
ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (46)
To a solution of 4-chloro-2-methylsulfanyl-7-(2-trimethylsila-
nyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid
amide (45, 73 mg, 0.196 mmol) in CH2Cl2 at 0 °C was added Et3N
followed by TFAA (0.03 mL, 0.21 mmol) slowly drop-wise. The stir-
red reaction mixture was then allowed to warm to ambient tem-
perature. Further CH2Cl2 (5 mL) was then added and the organic
phase washed with sat. NaHCO3 (aq) solution (15 mL). The organic
layer was passed through a hydrophobic frit and evaporated in va-
cuo. The crude product was purified by flash chromatography on
SiO2 (20 g) eluting with hexane-20% EtOAc/hexane (gradient) to af-
ford the title compound 46 (60 mg, 86%) as a colourless solid: LC–
MS (method A) tR = 2.84 min; m/z = 357, 355 [M+H]+.
7.7.8. 4-(2-Methyl-4-fluoro-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (15b)
A mixture of 4-chloro-2-methylsulfanyl-7-(2-trimethylsilanyl-
ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
54 mg, 0.152 mmol), 2-methyl-4-fluorophenylboronic
(46,
acid
(30 mg, 0.198 mmol), Pd2Cl2(PPh3)2 (5 mg, 0.0076 mmol), NaHCO3
aqueous solution (1 M, 0.46 ml, 0.456 mmol) and DMF (3 mL) was
degassed by bubbling N2 through the mixture for 5 min. The reac-
tion was then heated under a nitrogen atmosphere at 80 °C for 3 h.
The mixture was allowed to cool and was then partitioned be-
tween EtOAc (2 ꢂ 15 mL) and brine (15 mL). The combined organ-
ics were passed through a hydrophobic frit and evaporated in
vacuo. The crude product was purified by flash chromatography
on SiO2 (20 g) eluting with hexane-20% EtOAc/hexane (gradient)
to afford the title compound 15b (54 mg, 83%) as a colourless solid.
(LC–MS (method A) tR = 2.91 min; m/z = 429 [M+H]+. To the cross
coupling product (50 mg, 0.126 mmol) in THF (1 mL) was added
ethylenediamine (0.025 ml, 0.378 mmol) followed by TBAF (1.0 M
solution in THF, 0.76 mL, 0.756 mmol). The reaction mixture was
heated at 50 °C overnight. The reaction was allowed to cool to
ambient temperature and was then partitioned between EtOAc
(2 ꢂ 15 mL) and water (15 mL). The combined organics were
passed through a hydrophobic frit and evaporated in vacuo. The
resultant crude product was purified by flash chromatography on
SiO2 (20 g) eluting with 10% EtOAc/hexane to 40% EtOAc/hexane
(gradient) to afford the title compound 15b (19 mg, 51%) as a col-
ourless solid: LC–MS (method A) tR = 2.40 min; m/z = 299 [M+H]+;
1H NMR (400 MHz, DMSO-d6): d 2.22 (s, 3H); 2.57 (s, 3H), 7.15–
7.20 (m, 1H), 7.26 (dd, 1H, J = 10.1, 2.2 Hz), 7.46 (dd, 1H, J = 8.6,
6.1 Hz); 8.44 (s, 1H), 13.19 (br s, 1H); HRMS, Calcd for C15H12N4SF
[M+H]+. Found 299.0760 requires 299.0767; HPLC 97.5%
(tR = 1.21 min).
7.7.6. 5-Bromo-4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-
trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine
(47a)
To a solution of 4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-
trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (42)
(100 mg, 0.25 mmol) in CH2Cl2 (3 mL) at 0 °C was added drop-wise
a solution of N-Bromosuccinimide in CH2Cl2 (45 mg, 0.25 mmol) in
DCM (3 mL). After 5 min the reaction was allowed to warm to
ambient temperature. The solution was evaporated in vacuo and
the residue was partitioned between EtOAc (2 ꢂ 20 mL) and sat.
sodium thiosulfate solution (20 mL). The combined organics were
passed through a hydrophobic frit and evaporated in vacuo. The
crude was applied to a column of SiO2 (20 g) eluting with hex-
ane-5% EtOAc/hexane (gradient) to afford title compound
(100 mg, 84%) as a colourless oil: LC–MS (method B) tR = 5.92 min;
m/z = 480, 478 [M+H]+.
7.7.7. 4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (15a)
5-Bromo-4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-tri-
methylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (47a,
90 mg, 0.188 mmol), CuCN (67 mg, 0.753 mmol), 1,1’-bis(diphenyl-
phosphino)ferrocene (dppf) (17 mg, 0.03 mmol), tris(dibenzyli-
deneacetone)dipalladium(0) (Pd2(dba)3) (7 mg, 0.04 mmol) and
1,4-dioxane (1.5 mL) were combined and heated at 100 °C over-
night. The reaction had not gone to completion so further amounts
of CuCN (67 mg), dppf (17 mg) and Pd2(dba)3 (7 mg) were added
and the reaction heated for a further 2 h. The reaction mixture
7.7.9. 4-(4-Cyano-phenyl)-2-methylsulfanyl-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (15c)
The title compound was prepared by the method outlined for
15b, using 4-cyanophenyl boronic acid and 46 to afford 15c
(9 mg, 35%) as
a pale yellow solid: LC–MS (method A)
tR = 3.56 min; m/z = 290 [MꢀH]ꢀ; 1H NMR (400 MHz, DMSO-d6):
d 2.61 (s, 3H); 8.05 (d, 2H, J = 8.2 Hz), 8.07 (d, 2H, J = 8.2 Hz);

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6781
8.56 (s, 1H), 13.32 (br s, 1H); HRMS, Calcd for C15H10N5S [M+H]+.
Found 292.0654 requires 292.0657; HPLC 95.4% (tR = 1.15 min).
(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
ylsulfanyl]-acetic acid ethyl ester (51, 1.32 g, 2.23 mmol) in MeOH
(20 mL) and stirred at ambient temperature for 2 h. The mixture
was then evaporated in vacuo, water (100 mL) was added and
the resultant solution acidified to pH 5 by cautious addition of
1.2 N aq HCl solution. This mixture was then extracted with EtOAc
(2 ꢂ 100 mL). The combined organics were dried (Na2SO4) and
evaporated in vacuo to afford the title compound 52 (1.27 g,
100%) as a yellow foam: LC–MS (method A) tR = 2.63 Min; m/
z = 567, 565 [M+H]+.
7.7.10. 4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-2-
methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (49)
To a solution of 1-bromo-2-chloro-4-methoxy-5-methoxyme-
thoxy-benzene 48a (prepared as outlined in SI section; 2.27 g,
8.06 mmol) in THF (50 mL) at ꢀ78 °C under N2, was added tri-iso-
propylborate (3.72 mL, 16.12 mmol) followed by nBuLi (2.5 M solu-
tion in hexanes, 4.2 mL, 10.48 mmol,) drop-wise. The reaction was
then allowed to warm to room temperature and then evaporated in
vacuo. DMF (20 mL), 4-chloro-2-methylsulfanyl-7-(2-trimethylsi-
lanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(46, 2.0 g, 5.64 mmol), NaHCO3 (1 N aq solution, 24.2 mL,
24.2 mmol), and PdCl2(PPh3)2 (280 mg, 0.4 mmol) were added
sequentially and the resultant mixture was degassed by bubbling
N2 through the mixture for 5 min. The reaction was then heated
at 80 °C for 1.5 h and the resultant mixture partitioned between
EtOAc (250 mL) and brine (250 mL). The organic phase was dried
(Na2SO4) and evaporated in vacuo to afford a crude oil. This was
purified by flash chromatography on SiO2 (100 g) eluting with hex-
ane to 30% EtOAc/hexane (gradient) to afford the title compound
49 (3.04 g, 98%) as a yellow oil: LC–MS (method A) tR = 2.84 min;
m/z = 523, 521 [M+H]+.
7.7.14. 2-[4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-
5-cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-ylsulfanyl]-N,N-dimethyl-acetamide (53a)
To a solution of [4-(2-chloro-4-methoxy-5-methoxymethoxy-
phenyl)-5-cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo
[2,3-d]pyrimidin-2-ylsulfanyl]-acetic acid (52, 500 mg, 0.885
mmol) in anhydrous MeCN (10 mL), were added sequentially tri-
ethylamine (0.19 mL, 1.33 mmol), dimethylamine (2.0 M solution
in THF, 0.67 mL, 1.33 mmol,) and HBTU (O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium
hexafluorophosphate,
504 mg,
1.33 mmol). After 1 h the reaction mixture was partitioned be-
tween EtOAc (2 ꢂ 50 mL) and aqueous NH4Cl solution (50 mL).
The combined organics were dried (Na2SO4) and evaporated in va-
cuo. The resultant crude oil purified by flash chromatography on
SiO2 (50 g) eluting with hexane to 80% EtOAc/hexane (gradient)
to afford the title compound 53 (470 mg, 89%) as a white foam:
LC–MS (method A) tR = 2.67 min; m/z = 594, 592 [M+H]+.
7.7.11. 4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-2-
methanesulfonyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (50)
To an ice cooled solution of 4-(2-chloro-4-methoxy-5-methoxy-
methoxy-phenyl)-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxy-
methyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (49, 3.04 g,
5.5 mmol) in CH2Cl2 (100 mL) was added mCPBA (4.93 g, 22 mmol)
dissolved in CH2Cl2 (70 mL) drop-wise. After the addition was com-
plete the reaction was allowed to warm to ambient temperature
and was then washed sequentially with aq sodium thiosulfate
solution (150 mL) and saturated aq NaHCO3 sol. (170 mL). The or-
ganic layer was dried (Na2SO4) filtered and the filtrates evaporated
in vacuo to afford a crude oil, which was purified by flash chroma-
tography on SiO2 (50 g) eluting with hexane to 40% EtOAc/hexane
(gradient) to afford the title compound 50 (2.5 g, 80%) as a yellow
foam: LC–MS (method A) tR = 2.62 min; m/z = 555, 553 [M+H]+.
7.7.15. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-ylsulfanyl]-N,N-dimethyl-acetamide (54a)
To a solution of 2-[4-(2-chloro-4-methoxy-5-methoxymethoxy-
phenyl)-5-cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo
[2,3-d]pyrimidin-2-ylsulfanyl]-N,N-dimethyl-acetamide (53, 628
mg, 1.06 mmol) in iPrOH (10 mL) PPTS (pyridinium p-toluenesulfo-
nate) (53 mg, 0.212 mmol) was added and the mixture heated at
85 °C under N2 overnight. The reaction was allowed to cool and
partitioned between EtOAc (2 ꢂ 20 mL) and NH4Cl solution
(20 mL). The dried (Na2SO4) organics were evaporated in vacuo
to give a crude oil. This was applied to a cartridge of SiO2 (50 g)
eluting with CH2Cl2-5% MeOH/CH2Cl2 (gradient) to afford the title
compound 54 (411 mg, 71%) as a white foam: LC–MS (method A)
tR = 2.53 min; m/z = 548, 550 [M+H]+.
7.7.12. [4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-5-
cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-ylsulfanyl]-acetic acid ethyl ester (51)
7.7.16. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N,N-dimethyl-
acetamide (36q)
Ethyl thioglycolate (0.31 mL, 2.78 mmol) was added to a mix-
ture of NaH (46 mg, 1.15 mmol, 60% dispersion in mineral oil) in
THF (10 mL) at 0 °C under an N2 atmosphere. A solution of 4-(2-
chloro-4-methoxy-5-methoxymethoxy-phenyl)-2-methanesulfonyl-
7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile (50, 1.28 g, 2.32 mmol) in THF (10 mL) was then
added drop-wise. The reaction was then allowed to warm to ambi-
ent temperature before being partitioned between EtOAc
(2 ꢂ 100 mL) and aq NH4Cl sol. (100 mL). The organic layer was
dried (Na2SO4) and evaporated in vacuo to afford a crude oil, which
was purified by flash chromatography on SiO2 (70 g) eluting with
hexane-40% EtOAc/hexane (gradient) to afford the title compound
(1.32 g, 96%) as a yellow oil: LC–MS (method A) tR = 2.84 min; m/
z = 595, 593 [M+H]+.
To 2-[4-(2-chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-7-
(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
ylsulfanyl]-N,N-dimethyl-acetamide (60 mg, 0.109 mmol) under
N2, anhydrous THF (2 mL), ethylenediamine (0.022 mL,
0.327 mmol) and 1.0 M TBAF in THF solution (0.65 mL, 0.65 mmol)
were added sequentially. The mixture was heated at 40 °C over-
night. The reaction mixture was allowed to cool and was then par-
titioned between EtOAc (30 mL) and water (30 mL). The organic
layer was dried (Na2SO4), filtered and the filtrates evaporated in
vacuo to give a crude oil (ca 60 mg). This was applied to a cartridge
of SiO2 (10 g) eluting with hexane-100% EtOAc (gradient) to afford
the title compound 36q (16 mg, 35%) as a colourless solid: LC–MS
(method A) tR = 1.86 min; m/z = 418, 420 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 2.97 (s, 3H), 3.18 (s, 3H), 3.93 (s, 3H), 4.19
(s, 2H), 6.89 (s, 1H), 7.09 (s, 1H), 8.08 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 34.4 (CH2), 36.5 (CH3), 38.2 (CH3), 56.7
(CH3), 86.8 (C), 113.7 (CH), 114.3 (C), 115.1 (C), 118.1 (CH), 124.0
(C), 128.4 (C), 136.9 (CH), 146.9 (C), 151.2 (C), 153.9 (C), 159.4
7.7.13. [4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-5-
cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-ylsulfanyl]-acetic acid (52)
1 N aq NaOH (4.46 mL, 4.46 mmol) was added to a solution of
[4-(2-chloro-4-methoxy-5-methoxymethoxy-phenyl)-5-cyano-7-

6782
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
(C), 166.4 (C), 171.0 (C); HRMS, Calcd for C18H17ClN5O3S [M+H]+.
Found 418.0723 requires 418.0741; HPLC 97.2% (tR = 0.98 min).
pale-yellow solid: LC–MS (method A) tR = 2.01 min; m/z = 458,
460 [M+H]+; 1H NMR (400 MHz, CD3OD) d 0.36–0.40 (m, 2H),
0.63–0.68 (m, 2H), 1.40 (t, 3H, J = 7.0 Hz), 2.59–2.65 (m, 1H), 3.88
(s, 2H), 3.91 (s, 3H), 4.09 (q, 2H, J = 7.0 Hz), 7.08 (s, 1H), 7.13 (s,
1H), 8.11 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 6.5 (CH2), 15.0
(CH3), 23.7 (CH), 35.6 (CH2), 56.8 (CH3), 66.1 (CH2), 86.8 (C),
114.1 (CH), 114.4 (C), 115.2 (C), 116.3 (CH), 125.8 (C), 127.9 (C),
137.2 (CH), 148.7 (C), 152.8 (C), 154.0 (C), 159.4 (C), 166.1 (C),
7.7.17. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N,N-dimethyl-acetamide
(36r)
To a mixture of 2-[4-(2-chloro-5-hydroxy-4-methoxy-phenyl)-
5-cyano-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]
pyrimidin-2-yl-sulfanyl]-N,N-dimethyl-acetamide (54: NR1R2 =
NMe2, 80 mg, 0.15 mmol), Cs2CO3 (71 mg, 0.22 mmol) and anhy-
drous DMF (2 mL) under N2 was added methyl iodide (0.01 mL,
0.15 mmol). The reaction mixture was stirred at ambient tempera-
ture for 2 h, then partitioned between EtOAc (20 mL) and aq NH4Cl
solution (20 mL). The organic layer was washed with brine (20 mL)
then dried (Na2SO4) filtered and filtrate solvents evaporated in va-
cuo to afford a yellow oil, (96 mg, 100%) which was de-protected
with TBAF and ethylene diamine by the method utilized for 36q
above. Purification by flash chromatography (hexanes/EtOAc)
afforded title compound 36r as a colourless solid: LC–MS (method
A) tR = 1.99 min; m/z = 434, 432 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 2.96 (s, 3H), 3.20 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 4.22
(s, 2H), 7.09 (s, 1H), 7.13 (s, 1H), 8.10 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 34.4 (CH2), 36.5 (CH3), 38.2 (CH3), 56.8
(CH3), 56.8 (CH3), 86.5 (C), 114.0 (CH), 114.4 (C), 115.1 (CH),
115.4 (C), 125.8 (C), 128.0 (C), 137.6 (CH), 149.4 (C), 152.5 (C),
154.4 (C), 159.2 (C), 166.1 (C), 170.9 (C); HRMS, Calcd for
172.9 (C); HRMS, Calcd for
458.1042 requires 458.1054; HPLC 96.9% (tR = 1.11 min).
C
21H21ClN5O3S [M+H]+. Found
7.7.21. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-isopropyl-
acetamide (36d)
The title compound was prepared by the methods outlined for
36q. Purification by flash chromatography, eluting with DCM/
MeOH (9:1) afforded 36d (52 mg, 50%) as a pale-brown solid:
LC–MS (method A) tR = 1.66 min, m/z = 432 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 1.03 (d, 6H, J = 6.6 Hz), 3.88 (s, 2H), 3.93 (sept,
1H, J = 6.6 Hz), 3.93 (s, 3H), 6.94 (s, 1H), 7.10 (s, 1H), 8.10 (s, 1H);
13C NMR (100.6 MHz, CD3OD) d 22.4 (CH3), 35.8 (CH2), 43.0 (CH),
56.8 (CH3), 86.9 (C), 113.8 (CH), 114.5 (C), 115.0 (C), 118.1 (CH),
124.1 (C), 128.3 (C), 137.2 (CH), 146.9 (C), 151.2 (C), 153.9 (C),
159.5 (C), 166.2 (C), 170.5 (C); HRMS, Calcd for C19H19ClN5O3S
[M+H]+. Found 432.0878 requires 432.0897; HPLC 91.0%
(tR = 1.03 min).
C
19H19ClN5O3S [M+H]+. Found 432.0887 requires 432.0879; HPLC
100% (tR = 1.05 min).
7.7.22. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-ethyl-acetamide (36e)
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with EtOAc
afforded 36e (15 mg, 14%) as a pale yellow solid: LC–MS (method
A) tR = 1.70 min, m/z = 432 [M+H]+; 1H NMR (400 MHz, DMSO-d6)
d 0.97 (t, 3H, J = 7.3 Hz), 3.08 (m, 2H), 3.80 (s, 3H), 3.87 (s, 3H),
3.91 (s, 2H), 7.13 (s, 1H), 7.19 (s, 1H), 8.08 (t, 1H, J = 5.0 Hz), 8.44
(s, 1H), 13.08 (br s, 1H); HRMS, Calcd for C19H19ClN5O3S [M+H]+.
Found 432.0879 requires 432.0897; HPLC 97.1% (tR = 1.05 min).
7.7.18. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-cyclopropyl-acetamide
(36a)
The title compound was prepared by the methods outlined for
36r, via the MEM protected intermediate 48b. Purification by flash
chromatography, eluting with DCM to 5% MeOH in DCM (gradient)
afforded 36a (30 mg; 51%) as a yellow solid: LC–MS (method A)
tR = 2.02 min; m/z = 444, 446 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) d 0.36 (m, 2H), 0.58 (m, 2H), 2.60 (m, 1H), 3.81 (s, 3H), 3.87
(s, 3H), 3.88 (s, 2H); 7.13 (s, 1H); 7.19 (s, 1H); 8.17 (d, 1H,
J = 4.0 Hz); 8.45 (s, 1H); 13.14 (br s, 1H); HRMS, Calcd for
7.7.23. 2-[4-(2-Chloro-5-ethoxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-ethyl-acetamide
(36f)
C
20H19ClN5O3S [M+H]+. Found 444.0879 requires 444.0897; HPLC
100% (tR = 1.06 min).
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with EtOAc fol-
lowed by trituration with diethyl ether afforded 36f (30 mg, 26%)
as a pale-yellow solid: LC–MS (method A) tR = 1.80 min, m/
z = 448, 446 [M+H]+; 1H NMR (400 MHz, DMSO-d6) d 0.97 (t, 3H,
J = 7.3 Hz), 1.33 (t, 3H, J = 6.7 Hz), 3.07 (m, 2H), 3.87 (s, 3H), 3.91
(s, 2H), 4.05 (q, 2H, J = 6.7 Hz), 7.10 (s, 1H), 7.18 (s, 1H), 8.07 (t,
1H, J = 5.0 Hz), 8.43 (s, 1H), 13.10 (br s, 1H); HRMS, Calcd for
7.7.19. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-cyclopropyl-
acetamide (36b)
The title compound was prepared by the methods outlined for
36q, via the MEM protected intermediate 48b. Purification by flash
chromatography, eluting with DCM to 4% MeOH in DCM (gradient)
afforded 36b (30 mg, 42%) as a colourless solid: LC–MS (method A)
tR = 1.90 min; m/z = 430, 432 [M+H]+; 1H NMR (400 MHz, CD3OD) d
0.37–0.40 (m, 2H), 0.63–0.68 (m, 2H), 2.60–2.66 (m, 1H), 3.87 (s,
2H), 3.94 (s, 3H), 6.93 (s, 1H), 7.10 (s, 1H), 8.10 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 6.5 (CH2), 23.7 (CH), 35.6 (CH2), 56.8
(CH3), 86.8 (C), 113.8 (CH), 114.5 (C), 115.1 (C), 118.1 (CH), 124.1
(C), 128.3 (C), 137.2 (CH), 146.9 (C), 151.2 (C), 153.9 (C), 159.5
(C), 166.1 (C), 173.0 (C); HRMS, Calcd for C19H17ClN5O3S [M+H]+.
Found 430.0730 requires 430.0741; HPLC 100% (tR = 0.99 min).
C
20H21ClN5O3S [M+H]+. Found 446.1040 requires 446.1054; HPLC
100% (tR = 1.03 min).
7.7.24. N-tert-Butyl-2-[4-(2-chloro-5-hydroxy-4-methoxy-
phenyl)-5-cyano-7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-
acetamide (36g)
The title compound was prepared by the methods outlined for
36q. Purification by flash chromatography, eluting with 5% MeOH
in DCM afforded 36g (36 mg, 62%) as an off-white solid: LC–MS
(method A) tR = 3.00 min, m/z = 446 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 1.22 (s, 9H), 3.83 (s, 2H), 3.93 (s, 3H), 6.96 (s, 1H), 7.10
(s, 1H), 8.11 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 28.7 (CH3),
36.6 (CH2), 52.3 (C), 56.8 (CH3), 86.9 (C), 113.8 (CH), 114.4 (C),
115.0 (C), 118.2 (CH), 124.2 (C), 128.3 (C), 137.1 (CH), 147.0 (C),
151.3 (C), 153.8 (C), 159.5 (C), 166.3 (C), 170.7 (C); HRMS, Calcd
for C20H21ClN5O3S [M+H]+. Found 446.1035 requires 446.1054;
HPLC 100% (tR = 1.10 min).
7.7.20. 2-[4-(2-Chloro-5-ethoxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-cyclopropyl-
acetamide (36c)
The title compound was prepared by the methods outlined for
36r, via the MEM protected intermediate 48b. Purification by flash
chromatography, eluting with heaxane to 80% EtOAc in hexane
(gradient) afforded the title compound 36c (27 mg, 41%) as a

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6783
7.7.25. N-tert-Butyl-2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-
cyano-7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-acetamide
(36h)
8.35 (m, 1H), 8.42 (s, 1H), 9.56 (s, 1H), 13.0–13.20 (br s, 1H); HRMS,
Calcd for
18H16ClFN5O3S [M+H]+. Found 436.0632 requires
436.0646; HPLC 100% (tR = 0.97 min).
C
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with 5% MeOH
in DCM afforded 36h (9 mg, 21%) as an off-white solid: LC–MS
(method A) tR = 3.13 min, m/z = 460 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 1.22 (s, 9H), 3.85 (s, 2H), 3.87 (s, 3H), 3.92 (s, 3H), 7.14
(s, 1H), 7.67 (s, 1H), 8.13 (s, 1H); HRMS, Calcd for C21H23ClN5O3S
[M+H]+. Found 460.1195 requires 460.1210; HPLC 95.3%
(tR = 1.17 min).
7.7.30. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-(2,2,2-trifluoro-
ethyl)-acetamide (36m)
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with 1% MeOH
in DCM afforded 36m (51 mg, 81%)) as a colourless solid: LC–MS
(method B) tR = 3.23 min, m/z = 472 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 3.89 (q, 2H, JH–F = 9.4 Hz), 3.93 (s, 3H), 4.00 (s, 2H),
6.90 (s, 1H), 7.09 (s, 1H), 8.10 (s, 1H); 13C NMR (100.6 MHz, CD3OD)
d 35.5 (CH2), 41.7 (CH2, q, JC–F = 34.6 Hz), 56.8 (CH3), 86.9 (C),
113.8 (CH), 114.5 (C), 115.0 (C), 118.1 (CH), 124.1 (C), 125.7 (C, q,
JC–F = 278.2 Hz), 128.3 (C), 137.1 (CH), 146.9 (C), 151.2 (C), 153.8
(C), 159.6 (C), 165.8 (C), 172.1 (C); HRMS, Calcd for
7.7.26. 2-[4-(2-Chloro-5-ethoxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-isopropyl-
acetamide (36i)
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with EtOAc
afforded 36i (51 mg, 39%) as a pale-brown solid: LC–MS (method
A) tR = 2.09 min, m/z = 462, 460 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 1.03 (d, 6H, J = 6.6 Hz), 1.40 (t, 3H, J = 7.0 Hz), 3.90 (s,
2H), 3.91 (s, 3H), 3.93 (sept, 1H, J = 6.6 Hz), 4.09 (q, 2H,
J = 7.0 Hz), 7.09 (s, 1H), 7.13 (s, 1H), 8.11 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 15.0 (CH3), 22.4 (CH3), 35.8 (CH2), 43.0
(CH), 56.8 (CH3), 66.1 (CH2), 86.8 (C), 114.2 (CH), 114.4 (C),
115.2 (C), 116.3 (CH), 125.8 (C), 127.9 (C), 137.3 (CH), 148.7 (C),
152.8 (C), 154.0 (C), 159.4 (C), 166.1 (C), 170.4 (C); HRMS, Calcd
for C21H23ClN5O3S [M+H]+. Found 460.1215 requires 460.1210;
HPLC 100% (tR = 1.15 min).
C
18H14ClF3N5O3S [M+H]+. Found 472.0431 requires 472.0458;
HPLC 97.3% (tR = 1.05 min).
7.7.31. 2-(2-Azetidin-1-yl-2-oxo-ethylsulfanyl)-4-(2-Chloro-5-
hydroxy-4-methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-
carbonitrile (36n)
The title compound was prepared by the methods outlined for
36q. Purification by flash chromatography, eluting with Et3N/
EtOH/EtOAc (0:0:1–1:5:44 gradient) afforded 36n (48 mg, 39%))
as a yellow solid: LC–MS (method B) tR = 2.97 min, m/z = 430, 432
[M+H]+; 1H NMR (400 MHz, CD3OD) d 2.08–2.16 (m, 2H), 3.77 (s,
2H), 3.92 (s, 3H), 3.95–3.99 (m, 2H), 4.29–4.33 (m, 2H), 6.94 (s,
1H), 7.10 (s, 1H), 8.06 (s, 1H); HRMS, Calcd for C19H17ClN5O3S
[M+H]+. Found 430.0730 requires 430.0741; HPLC 95.6%
(tR = 0.98 min).
7.7.27. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-isopropyl-acetamide
(36j)
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with EtOAc fol-
lowed by trituration with diethyl ether afforded 36j (53 mg; 38%)
7.7.32. 2-(2-Azetidin-1-yl-2-oxo-ethylsulfanyl)-4-(2-Chloro-4,5-
dimethoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(36o)
as
a pale-brown solid: LC–MS (method A) tR = 2.00 min, m/
z = 448, 446 [M+H]+; 1H NMR (400 MHz, CD3OD) d 1.03 (d, 6H,
J = 6.6 Hz), 3.87 (s, 3H), 3.90 (s, 2H), 3.91 (s, 3H), 3.93 (sept, 1H,
J = 6.6 Hz), 7.11 (s, 1H), 7.13 (s, 1H), 8.12 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 22.4 (CH3), 35.8 (CH2), 43.0 (CH), 56.8
(CH3), 86.7 (C), 114.0 (CH), 114.5 (C), 115.1 (CH), 115.3 (C), 125.8
(C), 127.9 (C), 137.5 (CH), 149.5 (C), 152.6 (C), 154.1 (C), 159.3
(C), 166.1 (C), 170.4 (C); HRMS, Calcd for C20H21ClN5O3S [M+H]+.
Found 446.1039 requires 446.1054; HPLC 98.5% (tR = 1.10 min).
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with Et3N/
EtOH/EtOAc (0:0:1–1:5:44 gradient) afforded 36n (60 mg, 49%)
as a yellow solid LC–MS (method B) tR = 3.10 min, m/z = 444, 446
[M+H]+; 1H NMR (400 MHz, CD3OD) d 2.09–2.17 (m, 2H), 3.81 (s,
2H), 3.84 (s, 3H), 3.89 (s, 3H), 3.93–3.97 (m, 2H), 4.32–4.36 (m,
2H), 7.10 (s, 1H), 7.12 (s, 1H), 8.07 (s, 1H); HRMS, Calcd for
C
20H19ClN5O3S [M+H]+. Found 444.0878 requires 444.0897; HPLC
97.3% (tR = 1.04 min).
7.7.28. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-(2-fluoro-ethyl)-
acetamide (36k)
7.7.33. 2-[4-(2-Chloro-4,5-dimethoxy-phenyl)-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-methyl-acetamide
(36p)
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with EtOAc/hex-
ane (2:1) afforded 36k (25 mg, 30%) as a brown solid: LC–MS
(method A) tR = 2.94 min, m/z = 450 [M+H]+; 1H NMR (400 MHz,
CDCl3) d 3.51-3.60 (m, 2H,), 3.90-3.98 (m, 8H), 4.32–4.47 (m, 2H),
7.00 (s, 1H,), 7.05 (s, 1H,), 7.38 (m, 1H), 7.81 (s, 1H), 10.60-11.00
The title compound was prepared by the methods outlined for
36r. Purification by flash chromatography, eluting with DCM to
4% MeOH in DCM (gradient) afforded 36p (10 mg, 18%) as a brown
solid: LC–MS (method A) tR = 1.59 min, m/z = 418, 420 [M+H]+; 1H
NMR (400 MHz, CD3OD) d 2.72 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H),
3.93 (s, 2H), 7.08 (s, 1H), 7.13 (s, 1H), 8.12 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 26.8 (CH3), 35.5 (CH2), 56.8 (CH3), 86.8
(C), 114.0 (CH), 114.4 (C), 115.0 (CH), 115.2 (C), 125.8 (C), 127.9
(C), 137.2 (CH), 149.4 (C), 152.6 (C), 154.0 (C), 159.5 (C), 165.9
(C), 171.9 (C); HRMS, Calcd for C18H17ClN5O3S [M+H]+. Found
418.0727 requires 418.0741; HPLC 100% (tR = 1.00 min).
(br s, 1H); HRMS, Calcd for
C
19H18ClFN5O3S [M+H]+. Found
450.0775 requires 450.0803; HPLC 95.5% (tR = 1.04 min).
7.7.29. 2-[4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-5-cyano-
7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-N-(2-fluoro-ethyl)-
acetamide (36l)
The title compound was prepared by the methods outlined for
36q. Purification by flash chromatography, eluting with EtOAc/
hexane (2:1) to EtOAc (gradient) afforded 36l (25 mg, 53%) as a col-
ourless solid: LC–MS (method A) tR = 2.85 min, m/z = 436 [M+H]+;
1H NMR (400 MHz DMSO-d6) d 3.35 (m, 1H), 3.42 (m, 1H), 3.86
(s, 3H), 3.95 (s, 2H), 4.32–4.37(m, 2H), 6.90 (s, 1H), 7.15 (s, 1H),
7.7.34. 5-Bromo-4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine (17)
To
a solution of 5-bromo-4-(2,4-dimethyl-phenyl)-2-meth-
ylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidine (47a, 500 mg, 1.05 mmol) in THF (4 mL) was added a

6784
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
solution of tetrabutylammonium fluoride (1.0 M in THF, 6.3 mL,
6.3 mmol), followed by ethylene diamine (0.21 mL, 3.14 mmol).
The mixture was heated under a nitrogen atmosphere for 24 h, al-
lowed to cool and partitioned between EtOAc (50 mL) and water
(50 mL). The organic phase was separated and passed through a
hydrophobic frit and filtrates solvent removed in vacuo. The crude
product was purified by flash chromatography on silica gel (50 g),
eluting with 30–40% EtOAc in hexane gradient to afford the title
compound 17 (288 mg, 79%) as a colourless solid: LC–MS (method
B) tR = 4.44 min, m/z = 350, 348 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 2.08 (s, 3H), 2.38 (s, 3H), 2.59 (s, 3H), 7.09–7.14 (m,
3H), 7.33 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 14.2 (CH3),
19.9 (CH3), 21.4 (CH3), 89.5 (C), 113.1 (C), 126.4 (CH), 126.9
(CH), 130.5 (CH), 131.6 (CH), 134.3 (C), 137.4 (C), 140.2 (C), 153.3
(C), 162.0 (C), 166.2 (C); HRMS, Calcd for C15H15BrN3S [M+H]+.
Found 348.0160 requires 348.0170; HPLC 97.4% (tR = 1.38 min).
7.04–7.15 (m, 3H); 8.08 (s, 1H). 10.80 (br s, 1H); HRMS, Calcd for
16H15N3F3S [M+H]+. Found 338.0938 requires 338.0939; HPLC
96.7% (tR = 1.39 min).
C
7.7.37. 5-Cyclopropyl-4-(2,4-dimethyl-phenyl)-2-methylsulfa
nyl-7H-pyrrolo[2,3-d]pyrimidine (20)
5-Bromo-4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-tri-
methylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (47a,
100 mg, 0.209 mmol), Pd(OAc)2 (3 mg, 0.01 mmol), P(Cy)3 (57 mg,
0.418 mmol), K3PO4 (170 mg, 0.80 mmol), cyclopropylboronic acid
(25 mg, 0.30 mmol), toluene (1.0 ml) and water (0.05 ml) were
combined under N2 atmosphere and the mixture was degassed
by bubbling N2 through the mixture for 5 min. The reaction mix-
ture was then heated at 100 °C for 2 h, allowed to cool to RT and
was then partitioned between EtOAc (2 ꢂ 15 mL) and water
(15 mL). The organics were dried (Na2SO4) and passed through a
hydrophobic frit and evaporated in vacuo. The resultant crude
product was purified by flash chromatography on SiO2 (10 g) elut-
ing with hexane-5% EtOAc/hexane (gradient) to afford the desired
product and some dehalogenated product (17 mg). This compound
mixture was de-protected using the method outlined for 17 and
the crude product was purified by preparative HPLC (performed
at pH 4) to afford the title compound 20 (4 mg, 6%) as an off-white
solid: LC–MS (method A) tR = 2.70 min, m/z = 310 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 0.37–0.39 (m, 4H), 1.03–1.10 (m, 1H), 2.10
(s, 3H), 2.34 (s, 3H), 2.52 (s, 3H), 7.00–7.01 (m, 1H), 7.10 (d, 1H,
J = 8.0 Hz), 7.14 (s, 1H), 7.21 (d, 1H, J = 8.0 Hz), 11.73 (s, 1H); HRMS,
Calcd for C18H20N3S [M+H]+. Found 310.1368 requires 310.1378;
HPLC 99.4% (tR = 1.41 min).
7.7.35. 4-(2,4-dimethyl-phenyl)-5-methyl-2-methylsulfanyl-
7H-pyrrolo[2,3-d]pyrimidine (18)
To a solution of n-butyl lithium (2.5 M, 0.10 mL, 0.253 mmol) in
anhydrous THF (2 mL) cooled in a CO2-acetone bath under a nitrogen
atmosphere was added a solution of 5-bromo-4-(2,4-dimethyl-
phenyl)-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-
7H-pyrrolo[2,3-d]pyrimidine (47a,110 mg, 0.23 mmol) in anhydrous
THF (1.4 mL) drop-wise. When addition was complete, methyl
iodide (72 lL, 1.15 mmol) was added and the reaction mixture stir-
red for 5 min, the cooling bath was removed and reaction mixture
allowed to warm to ambient temperature. The reaction mixture
was partitioned between sat. (aq) NH4Cl solution and EtOAc and
the organic phase was passed through a hydrophobic frit and sol-
vents removed in vacuo to give an oil which was purified by flash
chromatography on SiO2 eluting with a 0 to 10% gradient of EtOAc
in hexane to afford intermediate product as a colourless oil (80 mg;
84%). The SEM group was removed by the procedure described for
17 above and resulting the crude product was purified by flash
chromatography on silica gel eluting with a gradient of o to to
25% EtOAc in hexane followed by trituration with diethyl ether
to afford the title compound 18 (36 mg, 56%) as a colourless solid:
LC–MS (method A) tR = 2.84 min, m/z = 284 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 1.70 (d, 3H, J = 1.1 Hz), 2.07 (s, 3H), 2.38 (s,
3H), 2.59 (s, 3H), 6.98 (q, 1H, J = 1.1 Hz), 7.10–7.15 (m, 3H); 13C
NMR (100.6 MHz, CD3OD) d 11.2 (CH3), 14.1 (CH3), 19.6 (CH3),
21.3 (CH3), 111.9 (C), 114.8 (C), 123.8 (CH), 127.1 (CH), 129.9
(CH), 131.7 (CH), 136.0 (C), 136.9 (C), 140.1 (C), 154.4 (C), 161.7
7.7.38. 4-(4-Fluoro-2-methy-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine-5-carboxylic acid amide (21)
The title compound was prepared by the methods outlined for
17. Thus 55 (65 mg, 0.15 mmol) was reacted with TBAF and ethy-
lenediamine in THF and the product purified by flash chromatogra-
phy on silica gel eluting with DCM to 4% MeOH/DCM (gradient) to
afford the title compound 21 (17 mg, 36%) as a colourless solid: LC–
MS (method A) tR = 1.92 min, m/z = 317 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 2.19 (s, 3H), 2.61 (s, 3H), 6.98–7.03 (m, 1H),
7.05 (dd, 1H, JH-H = 2.5 Hz, JH–F = 10.0 Hz), 7.32 (dd, 1H, JH-
H = 8.4 Hz, JH–F = 5.9 Hz), 7.82 (s, 1H); 13C NMR (100.6 MHz, CD3OD)
d 14.2 (CH3), 20.0 (CH3, d, JC–F = 1.2 Hz), 112.2 (C), 112.9 (C), 113.3
(CH, d, JC–F = 21.8 Hz), 117.7 (CH, d, JC–F = 21.7 Hz), 130.4 (CH),
132.2 (CH, d, JC–F = 8.8 Hz), 135.8 (C, d, JC–F = 3.0 Hz), 140.6 (C, d,
JC–F = 8.2 Hz), 155.1 (C), 161.1 (C), 164.5 (C, d, JC–F = 246.0 Hz),
166.4 (C), 168.4 (C); HRMS, Calcd for C15H14N4OFS [M+H]+. Found
317.0860 requires 317.0872; HPLC 97.9% (tR = 1.00 min).
(C), 164.6 (C); HRMS, Calcd for
C
16H18N3S [M+H]+. Found
284.1221 requires 284.1221; HPLC 94.4% (tR = 1.36 min).
7.7.36. 4-(2,4-Dimethyl-phenyl)-2-methylsulfanyl-5-trifluoro
methyl-7H-pyrrolo[2,3-d]pyrimidine (19)
7.7.39. 4-(2,4-Dimethyl-phenyl)-7H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile (22)
5-Bromo-4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-tri-
methylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (47a,
100 mg, 0.209 mmol), CuI (80 mg, 0.418 mmol), sodium trifluoro-
acetate (57 mg, 0.418 mmol), toluene (0.5 ml) and DMF (1 mL)
were combined under N2 atmosphere and heated to 170 °C over-
night. The reaction mixture was allowed to cool to RT and was then
partitioned between EtOAc (2 ꢂ 15 mL) and water (15 mL). The
organics were passed through a hydrophobic frit and evaporated
in vacuo. The resultant crude product was purified by flash chro-
matography on silica gel (20 g) eluting with hexane-6% EtOAc/hex-
ane (gradient) to afford the desired protected product together
with small quantity of dehalogenated product. The SEM group
was removed by the procedure described for 17 above and the
resulting crude product was purified by preparative HPLC (pH 4)
to afford title compound 19 (7 mg, 10%) as an off-white solid:
LC–MS (method A) tR = 2.68 min, m/z = 338 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 1.98 (s, 3H); 2.34 (s, 3H); 2.55 (s, 3H);
The title compound was prepared as outlined in the SI section,
to afford the title compound 22 (75 mg, 59%) as a colourless solid:
LC–MS (method A) tR = 2.03 min, m/z = 249 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 2.19 (s, 3H), 2.40 (s, 3H), 7.17 (d, 1H,
J = 7.8 Hz), 7.21 (s, 1H), 7.27 (d, 1H, J = 7.8 Hz), 8.27 (s, 1H), 8.91
(s, 1H); 13C NMR (100.6 MHz, CD3OD) d 19.7 (CH3), 21.4 (CH3),
86.4 (C), 114.9 (C), 117.3 (C), 127.4 (CH), 130.6 (CH), 132.2 (CH),
133.9 (C), 137.3 (C), 138.7 (CH), 141.1 (C), 153.2 (C), 153.5 (CH),
162.1 (C); HRMS, Calcd for C15H13N4 [M+H]+. Found 249.1146 re-
quires 249.1140; HPLC 100% (tR = 1.05 min).
7.7.40. 4-(4-Fluoro-2-methyl-phenyl)-2-methanesulfonyl-7-(2-
trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile (57)
To a solution of 4-[(4-fluoro-2-methyl-phenyl]-2-methylsulfa-
nyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrim-
idine-5-carbonitrile 56 (189 mg, 0.44 mmol) prepared as outlined

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6785
in 15b (part 1) in CH2Cl2 (8.5 mL) at 0 °C was added drop-wise a
solution of mCPBA (396 mg, 1.76 mmol) in CH2Cl2 (8.5 mL). After
addition was complete the reaction was allowed to warm to ambi-
ent temperature. After 1 h the reaction mixture was washed with
5% sodium thiosulphate (aq) solution (20 mL) and the aqueous
layer was extracted with further CH2Cl2 (20 mL). The combined
organics were then washed with sat. NaHCO3 (aq) solution.
(40 mL) then passed through a hydrophobic frit and the filtrate
evaporated in vacuo. The resultant crude product was purified by
flash chromatography on SiO2 (25 g) eluting with 20–40% EtOAc
in hexane (gradient) to afford the title compound 57 (187 mg,
92%) as a colourless oil: LC–MS (method A) tR = 2.65 min, m/
z = 461 [M+H]+.
(C), 164.6 (C, d, JC–F = 246.5 Hz); HRMS, Calcd for C15H13N5F
[M+H]+. Found 282.1148 requires 282.1155; HPLC 100%
(tR = 1.08 min).
7.7.43. 2-Ethyl-4-(4-fluoro-2-methyl-phenyl)-7H-pyrrolo[2,3-d]
pyrimidine-5-carbonitrile (25)
EtMgBr (3.0 M solution in Et2O, 0.04 ml, 0.11 mmol,) was added
to a stirred ice-bath cooled solution of 4-[(4-fluoro-2-methyl-phe-
nyl]-2-methanelsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile 57 (50 mg, 0.11 mmol).
After 20 min. the reaction was partitioned between EtOAc
(2 ꢂ 15 ml) and water (15 ml). The combined organic phase was
then passed through a hydrophobic frit and evaporated in vacuo to
give the crude protected product which was de-protected with tet-
rabutylammonium fluoride using the method outlined for 17. Puri-
fication was by flash chromatography on silica gel (10 g) eluting
with CH2Cl2 to 6% MeOH/CH2Cl2 (gradient) to afford the title com-
7.7.41. 4-(4-Fluoro-2-methyl-phenyl)-2-methoxy-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (23)
To a mixture of KOtBu (20 mg, 0.17 mmol) in THF (1 mL) at 0 °C
under N2, was added MeOH (0.007 mL, 0.17 mmol) followed by
drop-wise addition of a solution of 4-(4-fluoro-2-methyl-phenyl)-
2-methanesulfonyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo
[2,3-d]pyrimidine-5-carbonitrile (57, 40 mg, 0.087 mmol) in THF
(0.5 mL). After 30 min. the reaction mixture was partitioned be-
tween EtOAc (2 ꢂ 10 ml) and sat. NaHCO3 solution (15 mL). The
organics were then passed through a hydrophobic frit and evapo-
rated in vacuo to give the crude protected product, which was
de-protected with tetrabutylammonium fluoride using the method
outlined for compound 17. Purification was by flash chromatogra-
phy on silica gel (10 g) eluting with 10 to 50% EtOAc in hexane
(gradient) to afford the title compound 23 (12 mg, 48%) as a white
solid: LC–MS (method A) tR = 2.16 min, m/z = 283 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 2.26 (s, 3H), 4.06 (s, 3H), 7.04–7.10 (m, 1H),
7.13 (dd, 1H, JH-H = 2.5 Hz, JH–F = 9.9 Hz), 7.40 (dd, 1H, JH-
H = 8.4 Hz, JH–F = 5.8 Hz), 8.04 (s, 1H); 13C NMR (100.6 MHz, CD3OD)
d 19.9 (CH3, d, JC–F = 1.1 Hz), 55.5 (CH3), 86.3 (C), 112.4 (C), 113.6
(CH, d, JC–F = 21.9 Hz), 115.2 (C), 118.1 (CH, d, JC–F = 21.9 Hz),
132.6 (CH, d, JC–F = 9.0 Hz), 133.0 (C, d, JC–F = 3.1 Hz), 136.7 (CH),
140.6 (C, d, JC–F = 8.3 Hz), 155.5 (C), 163.0 (C), 164.2 (C), 164.9 (C,
d, JC–F = 247.0 Hz); HRMS, Calcd for C15H12N4OF [M+H]+. Found
283.0994 requires 283.0995; HPLC 100% (tR = 1.11 min).
pound (24 mg, 79%) as
a beige solid: LC–MS (method A)
tR = 2.20 min, m/z = 281 [M+H]+; 1H NMR (400 MHz, CD3OD) d 1.39
(t, 3H, J = 7.6 Hz), 2.23 (s, 3H), 3.04 (q, 2H, J = 7.6 Hz), 7.06–7.11
(m, 1H), 7.14 (dd, 1H, JH-H = 2.6 Hz, JH–F = 9.9 Hz), 7.41 (dd, 1H,
JH-H = 8.4 Hz, JH–F = 5.8 Hz), 8.18 (s, 1H); 13C NMR (100.6 MHz,
CD3OD) d 13.5 (CH3), 19.9 (CH3, d, JC–F = 1.2 Hz), 33.0 (CH2), 86.0
(C), 113.6 (CH, d, JC–F = 21.9 Hz), 114.8 (C), 115.0 (C), 118.1 (CH, d,
JC–F = 21.8 Hz), 132.6 (CH, d, JC–F = 8.9 Hz), 133.3 (C, d,
JC–F = 3.1 Hz), 137.8 (CH), 140.7 (C, d, JC–F = 8.6 Hz), 154.0 (C), 160.9
(C), 164.8 (C, d, JC–F = 247.2 Hz), 168.2 (C); HRMS, Calcd for
C
16H14N4F [M+H]+. Found 281.1199 requires 281.1202; HPLC
98.7% (tR = 1.14 min).
7.7.44. 4-[2,4-Dichloro-5-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-2-
methylsulfanyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(26)
To a solution of 1-[2-(2,4-Dichloro-5-iodo-phenoxy)-ethyl]-
pyrrolidine (58) [preparation method in SI] (200 mg, 0.518 mmol)
in THF at ꢀ78 °C under an N2 atmosphere was added triisopropyl
borate (0.24 mL, 1.04 mmol) followed by nBuLi (2.5 M in hexanes,
0.27 ml, 0.67 mmol) drop-wise. The reaction was allowed to warm
to RT and was then evaporated in vacuo to give the crude boronic
acid. This was combined with 4-chloro-2-methylsulfanyl-7-(2-tri-
methylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-car-
bonitrile (46) (92 mg, 0.26 mmol), 1.0 M NaHCO3 sol. (0.8 mL,
0.78 mmol), PdCl2(PPh3)2 (9 mg, 0.01 mmol) and DMF (6 mL). The
mixture was degassed by bubbling N2 through the mixture for
5 min. and was subsequently heated at 80 °C for 2 h under an N2
atmosphere. The reaction was allowed to cool before being parti-
tioned between EtOAc (2 ꢂ 60 ml) and aqueous NH3 solution
(60 mL). The combined organics were dried (Na2SO4) and evapo-
rated in vacuo to give a crude oil. This was purified by flash chro-
matography on silica gel (50 g) eluting with CH2Cl2 to 5% MeOH/
CH2Cl2 (gradient) to afford the protected product as yellow oil,
160 mg (still impure). This was de-protected using the method
outlined for 17 and purified by flash chromatography on silica
gel (20 g) to afford the title compound 26 (49 mg, 42%) as a yellow
solid: LC–MS (method A) tR = 1.83 min, m/z = 450, 448 [M+H]+; 1H
NMR (DMSO-d6) d 1.68 (m, 4H), 2.60 (m, 7H), 2.89 (t, 2H,
J = 5.5 Hz), 4.22 (t, 2H, J = 5.5 Hz), 7.18 (s, 1H), 7.42 (s, 1H), 8.46
(s, 1H); HRMS, Calcd for C20H20N5OSCl2 [M+H]+. Found 448.0764
requires 448.0766; HPLC 98.6% (tR = 1.7 min).
7.7.42. 4-(4-Fluoro-2-methyl-phenyl)-2-methylamino-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (24)
To a solution of 4-(4-fluoro-2-methyl-phenyl)-2-methanesulfo-
nyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrim-
idine-5-carbonitrile (57) (59 mg, 0.109 mmol) in DMF (2 mL) was
added
a solution of methylamine (2.0 M in THF, 0.11 mL,
0.218 mmol). The reaction mixture was heated at 100 °C for 2 h,
a further 1.0 mL of the methylamine solution was added and heat-
ing continued for 3 h, the reaction mixture was then partitioned
between EtOAc (20 mL) and sat. NaHCO3 (aq) solution (15 mL).
The phases were separated and organics were then passed through
a hydrophobic frit and evaporated in vacuo to give the crude pro-
tected product which was purified by flash chromatography on sil-
ica gel (20 g) eluting with 0–35% EtOAc in hexane (gradient) to
afford 40 mg of SEM protected product. De-protection of the SEM
group was as outlined for 17 above, followed by purification by
flash chromatography on silica gel (10 g) eluting with 0–60% EtOAc
in hexane (gradient) to afford title compound 24 (18 mg, 60%) as a
colourless solid: LC–MS (method A) tR = 2.08 min, m/z = 282
[M+H]+; 1H NMR (400 MHz, CD3OD) d 2.25 (s, 3H), 2.96 (s, 3H),
7.01–7.06 (m, 1H), 7.09 (dd, 1H, JH-H = 2.6 Hz, JH–F = 9.9 Hz), 7.35
(dd, 1H, JH-H = 8.4 Hz, JH–F = 5.9 Hz), 7.75 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 19.9 (CH3), 28.6 (CH3), 86.0 (C), 108.9 (C),
7.7.45. 4-[2,4-Dichloro-5-(2-diethylamino-ethoxy)-phenyl]-2-
methylsulfanyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(27)
The title compound was prepared by the methods outlined for
26 using 2-(2,4-dichloro-5-iodo-phenoxy)-ethyl]-diethyl-amine.
Purification by flash chromatography, eluting with 0–15% MeOH
in DCM (gradient) afforded 27 (31 mg, 63%) as a yellow solid:
113.4 (CH, d, JC–F = 21.9 Hz), 115.7 (C), 117.9 (CH, d, JC–F
=
21.8 Hz), 132.2 (CH, d, JC–F = 9.0 Hz), 133.8 (C, d, JC–F = 3.0 Hz),
134.1 (CH), 140.4 (C, d, JC–F = 8.2 Hz), 155.7 (C), 162.0 (C), 162.3

6786
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
LC–MS (method A) tR = 1.86 min, m/z = 452, 450 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) d 0.99 (t, 6H, J = 7.0 Hz), 2.58 (s, 3H), 2.62
(q, 4H, J = 7.0 Hz), 2.90 (br t, 2H, J = 5.4 Hz), 4.17 (br t, 2H,
J = 5.4 Hz), 7.42 (s, 1H), 8.47 (s, 1H), 8.47 (s, 1H), 12.83 (br s, 1H);
HRMS, Calcd for C20H22N5OSCl2 [M+H]+. Found 450.0920 requires
450.0922; HPLC 95.6% (tR = 1.04 min).
(s, 3H), 4.17 (t, 2H, J = 5.3 Hz), 7.12 (s, 1H), 7.15 (s, 1H), 8.08 (s,
1H); 13C NMR (100.6 MHz, CD3OD) d 14.3 (CH3), 36.6 (CH2, t,
JC–F = 24.9 Hz), 54.0 (CH2, t, JC–F = 3.7 Hz), 55.3 (CH2), 56.8 (CH3),
63.4 (CH2, t, JC–F = 29.4 Hz), 69.3 (CH2), 86.6 (C), 113.8 (C), 114.2
(CH), 115.4 (C), 116.9 (CH), 126.4 (C), 127.9 (C), 131.3 (C, t,
JC–F = 247.2 Hz), 136.8 (CH), 148.4 (C), 153.0 (C), 154.2 (C), 159.1
(C), 168.2 (C); HRMS, Calcd for C21H21N5O2SClF2 [M+H]+. Found
480.1056 requires 480.1073; HPLC 100% (tR = 1.12 min).
7.7.46. 4-(2-Chloro-5-hydroxy-4-methoxy-phenyl)-2-
methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (60)
7.7.50. 4-(2,4-dichloro-5-methoxy-phenyl)-2-methylsulfanyl-7-
(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (61b)
A
solution of 49 (510 mg, 0.98 mmol) and PPTS (50 mg
0.196 mmol) in isopropanol (7 mL) was heated to 85 °C for 3 h then
allowed to cool. The reaction mixture was then diluted with
dichloromethane (30 mL) and washed with sat NaCl (aq) solution
(2 ꢂ 20 mL) then the organic phase was dried over magnesium sul-
fate and filtered. The filtrate solvents were evaporated and the
residual product was purified by flash chromatography on silica
gel (50 g) eluting with 0 to 30% EtOAc in hexane (gradient) to af-
ford title compound 60 (382 mg, 82%) as a colourless solid: LC–
MS (method A) tR = 2.76 min, m/z = 479, 477 [M+H]+;
The title compound was prepared by the methods outlined for
15b. Thus 46 (390 mg, 1.1 mmol) was reacted with 2-chloro-4,5-
dimethoxyphenyl boronic acid [compound 59, see Supplementary
data for preparation] (1.37 mmol) and the intermediate product
purified by flash chromatography on silica gel (70 g) eluting with
hexane-20% EtOAc/hexane (gradient) to afford the title compound
61e (260 mg, 48%) as a yellow oil: LC–MS (method A) tR = 2.96 min,
m/z = 497, 495 [M+H]+.
7.7.47. 4-{2-Chloro-5-[2-((S)-3-fluoro-pyrrolidin-1-yl)-ethoxy]-
4-methoxy-phenyl}-2-methylsulfanyl-7-(2-trimethylsilanyl-
ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
(61a)
A mixture of 60 (75 mg, 0.157 mmol), 2-((S)-3-fluoro-pyrroli-
din-1-yl)-ethanol (31 mg, 0.236 mmol) and triphenyl phosphine
(62 mg, 0.236 mmol) in THF (2 mL) was stirred at ambient temper-
ature for 10 min. The mixture was cooled with an ice-water bath
7.7.51. 4-(2,4-Dichloro-5-methoxy-phenyl)-2-methanesulfonyl-
7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (62b)
To a solution of 61b (230 mg, 0.468 mmol) in DCM (8 mL)
cooled with an ice-water bath was added drop-wise a solution of
m-chloroperoxybenzoic acid (70%, 420 mg, 1.87 mmol) in DCM
(8 mL). The reaction mixture was then allowed to warm to room
temperature and after 1 h diluted with DCM (20 mL) and then
washed with 5% (w/v) aq sodium thiosulphate solution. The phases
were separated and the aqueous phase was re-extracted with DCM
(20 mL). The combined organics were washed with sat Na2CO3(aq)
solution, dried over sodium sulphate and filtered. The filtrate was
evaporated in vacuo to afford desired product as a yellow solid
(230 mg, 90%): LC–MS (method A) tR = 2.73 min, m/z = 529, 527
[M+H]+.
and a solution of DIAD (46 lL, 0.236 mmol) in anhydrous THF
(2 mL) was added drop-wise. The mixture was then stirred at
ambient temperature until the reaction was complete, and then di-
luted with EtOAc (20 mL) and water (20 mL). The organic phase
was washed with sat Na2CO3 (aq) solution, sat. NaCl (aq) then dried
over MgSO4. The crude product was purified by flash chromatogra-
phy on silica gel (25 g) eluting with 20% EtOAc in DCM. Further
purification was via an SCX-II ion exchange column (Biotage, elut-
ing with DCM, then 7 N NH3 in methanol). This afforded the title
compound 61a (36 mg, 39%) as a solid: LC–MS (method B)
tR = 3.75 min, m/z = 592, 594 [M+H]+.
7.7.52. 4-(2,4-Dichloro-5-methoxy-phenyl)-2-(2-diethylamino-
ethylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (30)
A mixture of 62b (50 mg, 0.095 mmol), 2-diethylaminoethane-
thiol hydrochloride (64 mg, 0.38 mol) and triethylamine in DMF
(2 mL) was heated at 100 °C for 30 min. then allowed to cool. The
mixture was partitioned between EtOAc (40 mL) and aqueous
ammonia solution (40 mL), and phases separated. The organic phase
dried over Na2SO4 and solvent was removed in vacuo to afford a
brown oil (120 mg). This product was de-protected with TBAF
according to methods utilized for compound 17, and purified by
flash chromatography on silica gel (20 g) eluting with 0 to 20%
MeOH in DCM (gradient) to afford the title compound 30 (26 mg,
61%) as a yellow solid: LC–MS (method A) tR = 1.84 min, m/z = 450,
452 [M+H]+; 1H NMR (400 MHz, CD3OD) d 1.12 (t, 6H, J = 7.2 Hz),
2.83 (q, 4H, J = 7.2 Hz), 3.05–3.09 (m, 2H), 3.36–3.39 (m, 2H), 3.93
(s, 3H), 7.26 (s, 1H), 7.61 (s, 1H), 8.14 (s, 1H); 13C NMR
(100.6 MHz, CD3OD) d 11.1 (CH3), 27.4 (CH2), 48.3 (CH2), 53.1
(CH2), 57.2 (CH3), 86.1 (C), 114.2 (C), 115.3 (C), 115.7 (CH), 125.2
(C), 125.9 (C), 131.8 (CH), 135.4 (C), 138.2 (CH), 154.7 (C), 155.5
(C), 158.3 (C), 166.5 (C); HRMS, Calcd for C20H22N5SOCl2 [M+H]+.
Found 450.0920 requires 450.0922; HPLC 97.5% (tR = 1.05 min).
7.7.48. 4-{2-Chloro-5-[2-((S)-3-fluoro-pyrrolidin-1-yl)-ethoxy]-
4-methoxy-phenyl}-2-methylsulfanyl-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (28)
Compound 61a (36 mg, 0.06 mmol) was de-protected by the
method used for 17. Purification was by prep HPLC (pH 4) affording
the title compound 28 (4.4 mg, 16%) as a colourless solid: LC–MS
(method A) tR = 1.72 min, m/z = 464, 462 [M+H]+; 1H NMR (CD3OD)
d 1.97-2.12 (m, 1H), 2.14-2.31 (m, 1H), 2.62 (s, 3H), 2.70-2.77 (m,
1H), 2.94 (ddd, 1H, J = 31.6, 12.4, 4.8 Hz), 3.03-3.27 (m, 4H), 3.92
(s, 3H), 4.22 (t, 2H, J = 5.6 Hz), 5.21 (dm, 1H, J = 55 Hz), 7.15 (s,
1H), 7.16 (s, 1H), 8.09 (s, 1H); HRMS, Calcd for C21H22N5O2SClF
[M+H]+. Found 462.1176 requires 462.1167; HPLC 95.3%
(tR = 0.96 min).
7.7.49. 4-{2-Chloro-5-[2-(3,3-difluoro-pyrrolidin-1-yl)-ethoxy]-
4-methoxy-phenyl}-2-methylsulfanyl-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (29)
The title compound was prepared by the methods outlined for
61a using 2-(3,3-difluoro-pyrrolidin-1-yl)-ethanol to afford 61c,
followed by de-protection with TBAF as per compound 17 and
purification by flash chromatography, eluting with 0–4% MeOH
in DCM (gradient) to afford 29 (36 mg, 74%) as a colourless solid:
LC–MS (method A) tR = 2.07 min, m/z = 482, 480 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 2.19–2.30 (m, 2H), 2.62 (s, 3H), 2.88 (t, 2H,
J = 7.1 Hz), 2.92 (t, 2H, J = 5.3 Hz), 3.07 (t, 2H, JH–F = 13.4 Hz), 3.92
7.7.53. 4-(2-Chloro-4,5-dimethoxy-phenyl)-2-(2-diethylamino-
ethylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (31)
The title compound was prepared by the methods outlined for
preparation of compound 30 (Scheme 4). Final purification by flash
chromatography on silica gel (10 g) eluting with 0 to 8% MeOH in
DCM (gradient) afforded the title compound 31 (22 mg, 60%) as a
yellow solid: LC–MS (method A) tR = 1.68 min, m/z = 446 [M+H]+;

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6787
1H NMR (400 MHz, DMSO-d6) d 0.97 (t, 6H, J = 7.0 Hz), 2.63 (m, 4H),
2.86 (m, 2H), 3.28 (m, 2H), 3.80 (s, 3H), 3.86 (s, 3H), 7.16 (s, 1H),
7.19 (s, 1H), 8.44 (s, 1H), 12.60 (br s, 1H); HRMS, Calcd for
(25 g) eluting with 0–60% EtOAC in hexane followed by purifica-
tion by prep HPLC, afforded the title compound 35 as a colourless
solid: LC–MS (method A) tR = 2.33 min, m/z = 510, 508 [M+H]+; 1H
NMR (CD3OD) d 0.65 (d, 6H, J = 7.1 Hz), 1.39-1.51 (m, 2H), 2.06-
2.14 (m, 4H), 2.27 (t, 2H, J = 13.4 Hz), 3.12 (s, 3H), 3.20-3.28 (m,
1H), 3.36 (t, 2H, J = 5.3 Hz), 6.31 (s, 1H), 6.35 (s, 1H), 7.29 (s, 1H);
HRMS, Calcd for C23H25N5O2SClF2 [M+H]+. Found 508.1390 re-
quires 508.1386; HPLC 100% (tR = 1.26 min).
C
21H23N5O2SCl [MꢀH]ꢀ. Found 444.1271 requires 444.1261; HPLC
99.1% (tR = 0.93 min).
7.7.54. 4-(2,4-Dichloro-5-methoxy-phenyl)-2-(2-diethylamino-
ethoxy)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (32)
The title compound was prepared by the methods outlined for
preparation of compound 30 (Scheme 4) utilizing 2-diethylamino-
ethanol at the appropriate step. Final purification by flash chroma-
tography on silica gel (10 g) eluting with 1:10 Et3N/DCM to
1:10:89 Et3N/MeOH/DCM (gradient) afforded the title compound
32 (16 mg, 28%) as a cream-coloured solid: LC–MS (method A)
tR = 1.75 min, m/z = 436, 434 [M+H]+; 1H NMR (400 MHz, CD3OD)
d 1.11 (t, 6H, J = 7.2 Hz), 2.75 (q, 4H, J = 7.2 Hz), 3.03 (t, 2H,
J = 6.1 Hz), 3.93 (s, 3H), 4.60 (t, 2H, J = 6.1 Hz), 7.25 (s, 1H), 7.61
(s, 1H), 8.08 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 11.3 (CH3),
48.8 (CH2), 52.1 (CH2), 57.2 (CH3), 66.3 (CH2), 86.4 (C), 112.7
(C), 115.3 (C), 115.5 (CH), 125.2 (C), 125.9 (C), 131.8 (CH), 135.3
(C), 137.5 (CH), 155.5 (C), 155.7 (C), 159.8 (C), 163.4 (C); HRMS,
7.7.58. 4-[(4-Fluoro-2-methyl-phenyl]-2-methylsulfanyl-7-(2-
trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-
5-carboxylic acid amide (55)
The title compound was prepared by the methods outlined for
15b (step 1). Thus 45 (60 mg, 0.161 mmol) was reacted with 2-
methyl-4-fluorophenyl boronic acid (32 mg, 0.209 mmol) and the
product purified by flash chromatography on silica gel eluting with
20–65% EtOAc in hexane (gradient) to afford the title compound 55
(65 mg, 90%) as a colourless oil: LC–MS (method A) tR = 2.67 min,
m/z = 447 [M+H]+.
Calcd for
C
20H20N5O2Cl2 [MꢀH]ꢀ. Found 432.0987 requires
7.7.59. 2,6-Dichloro-9-(tetrahydro-pyran-2-yl)-9H-purine (63)
To anhydrous ethyl acetate (25 mL) under nitrogen atmosphere
was added 2,6-dichloropurine (3 g, 16 mmol) and p-toluenesul-
phonic acid (48 mg, 0.22 mmol), this mixture was warmed to
432.0994; HPLC 100% (tR = 1.00 min).
7.7.55. 2-{5-Cyano-4-[2,4-dichloro-5-(2-diethylamino-ethoxy)-
phenyl]-7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl}-N-methyl-
acetamide (33)
50 °C and stirred as
a
suspension. 3,4-Dihydro-2H-pyran
(1.63 mL, 17.9 mmol) was added drop-wise over 10 min and the
resulting mixture stirred at 50 °C for 1 h, then cooled to ambient
temperature. Aqueous ammonia (0.880, 2 mL) was added and the
solution stirred for 5 min. The reaction mixture was diluted with
ethyl acetate and washed twice with water. The phases were sep-
arated and the organic phase dried over MgSO4 and filtered. The fil-
trate solvents were removed in vacuo to give a pale-yellow solid,
which was suspended in refluxing hexane for 15 min, allowed to
cool for 1 h and filtered and dried to afford title compound (63)
(3.19 g, 73%) as a colourless solid: Rf 0.76 CH2Cl2/MeOH (10:1)
SiO2; 1H NMR (400 MHz, DMSO-d6) d 1.55 (m, 2H), 1.75 (m, 1H),
2 (m, 2H), 2.25 (m, 1H), 3.75 (m, 1H), 4.00 (m, 1H), 5.80 (dd, 1H),
8.95 (s, 1H).
The title compound was prepared by the methods outlined for
preparation of compound 30 (Scheme 4) utilizing 2-diethylamino-
ethanol and 2-mercapto-N-methylacetamide for the appropriate
steps. Final purification by prep HPLC (pH 9) afforded the title com-
pound 33 (7 mg, 30%) as a colourless solid (formate salt): LC–MS
(method A) tR = 1.70 min, m/z = 509, 507 [M+H]+; 1H NMR
(400 MHz, CD3OD) d 1.31 (t, 6H, J = 7.3 Hz); 2.73 (s, 3H); 3.20 (q,
4H, J = 7.3 Hz); 3.49 (t, 2H, J = 5.0 Hz), 3.95 (s, 2H), 4.43 (t, 2H,
J = 5.0 Hz); 7.34 (s, 1H); 7.69 (s, 1H); 8.18 (s, 1H); 8.53 (s, 1H);
HRMS, Calcd for C22H23N6O2SCl2 [MꢀH]ꢀ. Found 505.0967 requires
505.0980; HPLC 94.8% (tR = 0.95 min).
7.7.56. 4-(2-Chloro-4-cyano-5-methoxy-phenyl)-2-(2-
diethylamino-ethylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-5-
carbonitrile (34)
7.7.60. General procedure A: cross-coupling reaction with 63
To a solution of 2,6-dichloro-9-(tetrahydro-pyran-2-yl)-9H-
purine (63, 350 mg, 1.28 mmol) in DMF (10 mL) was added the
appropriate boronic acid (1.41 mmol) and potassium carbonate
(532 mg, 3.85 mmol) and the resulting solution then sparged with
nitrogen gas. tetrakis(triphenylphosphine)palladium (74 mg,
0.06 mmol) was added and the mixture heated to 110 °C for 1 h
in a microwave synthesizer. The reaction mixture was cooled to
ambient temperature, diluted with ethyl acetate and washed with
saturated brine solution. Organics were dried over MgSO4 then
purified via silica gel chromatography, eluting with a gradient of
0–50% EtOAc in hexane.
The title compound was prepared by the methods outlined for
preparation of compound 30 (Scheme 4) utilizing 2-chloro-4-cya-
no-5-methoxyphenylboronic acid 66 [see Supplementary data sec-
tion for synthesis] and diethylaminoethanethiol for the
appropriate steps. Final purification by flash chromatography on
silica gel (20 g) eluting with 0–10% MeOH in DCM (gradient) affor-
ded the title compound 34 (25 mg, 66%) as yellow solid: LC–MS
(method A) tR = 1.75 min, m/z = 441 [M+H]+; 1H NMR (400 MHz,
CD3OD) d 1.16 (t, 6H, J = 7.2 Hz), 2.93 (q, 4H, J = 7.2 Hz), 3.16–
3.20 (m, 2H), 3.40–3.43 (m, 2H), 3.99 (s, 3H), 7.42 (s, 1H), 7.90 (s,
1H), 8.18 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 10.8 (CH3),
27.1 (CH2), 48.5 (CH2), 52.9 (CH2), 57.6 (CH3), 85.9 (C), 105.2
(C), 114.1 (C), 115.1 (C), 115.5 (C), 115.8 (CH), 125.5 (C), 135.5
(CH), 138.5 (CH), 141.6 (C), 154.8 (C), 157.5 (C), 161.3 (C), 166.3
(C); HRMS, Calcd for C21H22N6OSCl [M+H]+. Found 441.1251 re-
quires 441.1264; HPLC 94.9% (tR = 0.97 min).
7.7.61. General procedure B: preparation of 13a–c
The 4-aryl purine products from 63 (general procedure A)
(0.20 mmol) in dimethylformamide (2 mL) were treated with so-
dium thiomethoxide (18 mg, 0.26 mmol) and then heated to
120 °C for 15 min in a microwave synthesizer. The reaction mix-
ture was diluted with ethyl acetate and washed with saturated so-
dium bicarbonate solution followed by saturated brine solution.
Organics were dried over MgSO4 and concentrated in vacuo. The
residue was applied to a Biotage SCX-2 (silica bound sulphonic
acid) ion exchange column, and washed with methanol and prod-
uct eluted off with 7 N NH3 in methanol solution. Products were
purified by preparative HPLC at pH 4.
7.7.57. 4-{2-Chloro-5-[2-(3,3-difluoro-pyrrolodin-1-yl)-ethoxy]-
4-methoxy-phenyl}-2-isopropylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (35)
The title compound was prepared by the methods outlined for
preparation of compound 30 (Scheme 4) utilizing 2-(3,3-difluoro-
pyrrolidin-1-yl)-ethanol and isopropylthiol at the appropriate
steps. Final purification by flash chromatography on silica gel

6788
N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
7.7.62. 6-(4-Chloro-2-methyl-phenyl)-2-methylsulfanyl-9H-
purine (13a)
[M+H]+; 1H NMR (400 MHz, CD3OD + DMSO-d6) d 2.33 (s, 3H),
2.62 (s, 3H), 6.30 (d, 1H, J = 3.5 Hz), 7.06–7.11 (m, 1H), 7.15 (dd,
1H, JH-H = 2.5 Hz, JH–F = 10.0 Hz), 7.31 (d, 1H, J = 3.5 Hz), 7.50 (dd,
1H, JH-H = 8.4 Hz, JH–F = 6.0 Hz); 13C NMR (100.6 MHz, CD3OD + DM-
SO-d6) d 14.3 (CH3), 20.4 (CH3, d, JC–F = 1.5 Hz), 101.4 (CH), 113.6
(CH, d, JC–F = 21.7 Hz), 115.3 (C), 118.5 (CH, d, JC–F = 21.4 Hz),
126.7 (CH), 132.7 (CH, d, JC–F = 8.9 Hz), 134.6 (C, d, JC–F = 2.9 Hz),
Prepared by general procedures A then B to afford title com-
pound 13a (12 mg, 20%) as a colourless solid: LC–MS (method A)
tR = 2.33 min, m/z = 291 [M+H]+; 1H NMR (400 MHz, CD3OD + DM-
SO-d6) d 2.36 (s, 3H), 2.63 (s, 3H), 7.38 (dd, 1H, J = 8.3, 1.9 Hz), 7.44
(d, 1H, J = 1.9 Hz), 7.59 (d, 1H, J = 8.3 Hz), 8.35 (s, 1H); 13C NMR
(100.6 MHz, CD3OD + DMSO-d6) d 14.6 (CH3), 20.4 (CH3), 127.0
(CH), 127.4 (C, br), 131.8 (CH), 133.0 (CH), 134.9 (C), 136.5 (C),
140.6 (C), 145.8 (CH), 156.9 (C, br), 156.9 (C, br), 166.9 (C); HRMS,
Calcd for C13H12N4SCl [M+H]+. Found 291.0467 requires 291.0471;
HPLC 96.2% (tR = 1.19 min).
140.8 (C, d, JC–F = 8.3 Hz), 154.5 (C), 160.0 (C), 164.4 (C, d, JC–F
=
246.7 Hz), 164.9 (C); HRMS, Calcd for C14H13N3SF [M+H]+. Found
274.0819 requires 274.0814; HPLC 98.6% (tR = 1.28 min).
7.7.67. 4-(2-Methylsulfanyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl-
benzonitrile (14c)
7.7.63. 6-(4-Fluoro-2-methyl-phenyl)-2-methylsulfanyl-9H-
purine (13b)
Prepared by general procedures A then B to afford title com-
pound 13b (11 mg, 20%) as a colourless solid: LC–MS (method A)
tR = 2.19 min, m/z = 275 [M+H]+; 1H NMR (400 MHz, CD3OD) d
The title compound was prepared by the methods outlined for
15b. Thus 42 (100 mg, 0.303 mmol) was reacted with 4-cyano-
phenyl boronic acid (58 mg, 0.394 mmol), followed by de-protec-
tion (TBAF) and final product purification by flash chromatography
on silica gel (20 g) eluting with 0–50% EtOAc in hexane (gradient)
to afford the title compound 14c (20 mg, 25%) as a yellow solid:
LC–MS (method A) tR = 2.37 min, m/z = 267 [M+H]+; 1H NMR
2.36 (s, 3H), 2.62 (s, 3H), 7.06–7.11 (m, 1H), 7.14 (dd, 1H, JH-H
=
2.5 Hz, JH–F = 9.9 Hz), 7.59 (dd, 1H, JH-H = 8.4 Hz, JH–F = 5.9 Hz),
8.33 (s, 1H); 13C NMR (100.6 MHz, CD3OD) d 14.5 (CH3), 20.5
(CH3, d, JC–F = 1.5 Hz), 113.7 (CH, d, JC–F = 21.7 Hz), 118.5 (CH, d,
JC–F = 21.6 Hz), 127.4 (C, br), 132.4 (C, d, JC–F = 2.9 Hz), 133.4 (CH,
d, JC–F = 8.9 Hz), 141.4 (C, d, JC–F = 8.4 Hz), 145.7 (CH), 157.0 (C,
br), 157.2 (C), 164.9 (C, d, JC–F = 247.8 Hz), 167.1 (C); HRMS, Calcd
for C13H12N4SF [M+H]+. Found 275.0763 requires 275.0767; HPLC
100% (tR = 1.11 min).
(400 MHz, CD3OD + DMSO-d6)
d 2.68 (s, 3H), 6.86 (d, 1H,
J = 3.7 Hz), 7.48 (d, 1H, J = 3.7 Hz), 7.98 (d, 2H, J = 8.4 Hz), 8.37 (d,
2H, J = 8.4 Hz); 13C NMR (100.6 MHz, CD3OD + DMSO-d6) d 14.5
(CH3), 101.2 (CH), 113.6 (C), 114.4 (C), 119.7 (C), 127.9 (CH),
130.7 (CH), 133.8 (CH), 143.5 (C), 155.6 (C), 155.7 (C), 165.2 (C);
HRMS, Calcd for C14H11N4S [M+H]+. Found 267.0699 requires
267.0704; HPLC 100% (tR = 1.21 min).
7.7.64. 4-(2-Methylsulfanyl-9H-purin-6-yl)-benzonitrile (13c)
Prepared by general procedures A then B to afford title com-
pound 13c (8 mg, 15%) as an off-white solid: LC–MS (method A)
tR = 2.22 min, m/z = 268 [M+H]+; 1H NMR (400 MHz, CD3OD + DM-
SO-d6) d 2.71 (s, 3H), 7.99 (d, 2H, J = 8.3 Hz), 8.44 (s, 1H), 8.98 (d,
2H, J = 8.3 Hz); 13C NMR (100.6 MHz, CD3OD + DMSO-d6) d 14.8
(CH3), 115.0 (C), 119.7 (C), 128.4 (C, br), 131.3 (CH), 133.6 (CH),
141.2 (C), 145.7 (CH), 152.0 (C), 157.0 (C, br), 166.5 (C); HRMS,
Calcd for C13H10N5S [M+H]+. Found 268.0663 requires 268.0657;
HPLC 96.9% (tR = 1.12 min).
7.7.68. 4-Chloro-6-methylsulfanyl-1H-pyrazolo[3,4-
d]pyrimidine-3-carbonitrile (65)
6-Methylsulfanyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrim-
idine-3-carboxylic acid amide (prepared according to the literature
procedure described in Ref. 50; 380 mg, 1.69 mmol) was added to a
stirring mixture of phosphorus oxychloride (15 mL) and N,N-
dimethylaniline (1.5 mL). The reaction mixture was heated to re-
flux where it was held for 4 h and then allowed to cool to RT.
The excess POCl3 was removed in vacuo and the residue was added
in portions to rapidly stirring ice/H2O, and the pH then adjusted to
two by the careful addition of ammonia solution. The acidic aque-
ous mixture was extracted with ethyl acetate (3 ꢂ 25 mL) and the
combined extracts were washed with water (30 mL), brine
(2 ꢂ 30 mL), dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel (25 g) and the
desired product eluted using 30% ethyl acetate/iso-hexane. The
pure fractions containing product were combined and concen-
trated in vacuo to yield the title compound 65 (185 mg, 49%) as a
pale-yellow solid.
7.7.65. 4-(2,4-Dimethyl-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine (14a)
The title compound was prepared by the methods outlined for
15b. Thus 42 (100 mg, 0.303 mmol) was reacted with 2,4-di-
methyl-phenyl boronic acid (55 mg, 0.364 mmol), followed by
de-protection (TBAF) and final product purification by flash chro-
matography on silica gel (20 g) eluting with 0–20% EtOAc in hex-
ane (gradient) to afford the title compound 14a (25 mg, 32%) as a
colourless solid: LC–MS (method B) tR = 4.18 min, m/z = 270
[M+H]+; 1H NMR (400 MHz, CD3OD) d 2.26 (s, 3H), 2.38 (s, 3H),
2.60 (s, 3H), 6.26 (d, 1H, J = 3.6 Hz), 7.13 (d, 1H, J = 7.7 Hz), 7.18
(s, 1H), 7.26 (d, 1H, J = 3.6 Hz), 7.32 (d, 1H, J = 7.7 Hz); 13C NMR
(100.6 MHz, CD3OD) d 14.2 (CH3), 20.1 (CH3), 21.3 (CH3), 101.7
(CH), 115.3 (C), 126.3 (CH), 127.4 (CH), 130.5 (CH), 132.6 (CH),
135.4 (C), 137.4 (C), 140.4 (C), 154.4 (C), 161.4 (C), 164.9 (C);
HRMS, Calcd for C15H16N3S [M+H]+. Found 270.1065 requires
270.1065; HPLC 96.9% (tR = 1.33 min).
7.7.69. 4-(2,4-Dimethyl-phenyl)-6-methylsulfanyl-1H-
pyrazolo[3,4-d]pyrimidine-3-carbonitrile (16)
4-Chloro-6-methylsulfanyl-1H-pyrazolo[3,4-d]pyrimidine-3-
carbonitrile (65, 75 mg, 0.33 mmol), 2,4-dimethylphenylboronic
acid (65 mg, 0.43 mmol), 2 M K3PO4 solution (1 mmol, 0.5 mL)
and 1,4-dioxane (3 mL) were charged into a flask and thoroughly
degassed. [1,10-bis(diphenylphosphino)ferrocene]dichloro-palla-
dium(II) (13.5 mg, 0.0167 mmol,) was added and the reaction mix-
ture was heated at 90 °C for 6 h. After cooling, the reaction mixture
was partitioned between ethyl acetate and brine. The organic layer
was separated and the aqueous layer extracted with a further por-
tion of ethyl acetate. The combined ethyl acetate extracts were
washed with saturated sodium hydrogen carbonate solution
(2 ꢂ 30 mL) then brine (2 ꢂ 30 mL), dried (MgSO4) and concen-
trated in vacuo. The residue was purified by flash chromatography
on silica gel (10 g) and the product eluted using 20% ethyl acetate
in iso-hexane. The fractions containing product were combined
and concentrated in vacuo and the residue further purified by
7.7.66. 4-(4-Fluoro-2-methyl-phenyl)-2-methylsulfanyl-7H-
pyrrolo[2,3-d]pyrimidine (14b)
The title compound was prepared by the methods outlined for
15b. Thus 42 (200 mg, 0.61 mmol) was reacted with 2-fluoro-4-
methyl-phenyl boronic acid (112 mg, 0.72 mmol), followed by
de-protection (TBAF) and final product purification by flash chro-
matography on silica gel (20 g) eluting with 0 to 20% EtOAc in hex-
ane (gradient) to afford the title compound 14b (17 mg, 50%) as a
colourless solid: LC–MS (method A) tR = 2.49 min, m/z = 274

N. G. M. Davies et al. / Bioorg. Med. Chem. 20 (2012) 6770–6789
6789
22. Kaur, G.; Belotti, D.; Burger, A. M.; Fisher-Nielson, K.; Borsotti, P.; Riccardi, E.;
Thillainathan, J.; Hollingshead, M.; Sausville, E. A.; Giavazzi, R. Clin. Cancer Res.
2004, 10, 4813.
23. Chiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.; Zheng, F. F.; Sepp-
Lorenzino, L.; Rosen, N. Chem. Biol. 2001, 8, 289.
24. Patel, H. J.; Modi, S.; Chiosis, G.; Taldone, T. Expert Opin. Drug Discovery 2011, 6, 559.
25. Taldone, T.; Sun, W.; Chiosis, G. Bioorg. Med. Chem. 2009, 17, 2225.
26. Drysdale, M. J.; Brough, P. A. Curr. Top. Med. Chem. (Sharjah, United Arab
Emirates) 2008, 8, 859.
preparative HPLC at pH 4 to afford the title compound 16 (4.3 mg,
4%) as a colourless solid: LC–MS (method A) tR = 2.55 min, m/
z = 296 [M+H]+; 1H NMR (400 MHz, CD3OD) d 2.31 (s, 3H), 2.42
(s, 3H), 2.65 (s, 3H), 7.20 (d, 1H), 7.24 (s, 1H), 7.37 (d, 1H); HRMS,
Calcd for C15H14N5S [M+H]+. Found 296.0965 requires 296.0970;
HPLC (from method A) 97.3% (tR = 2.51 min).
27. Messaoudi, S.; Peyrat, J. F.; Brion, J. D.; Alami, M. Anticancer Agents Med. Chem.
2008, 8, 761.
Acknowledgment
28. Janin, Y. L. Drug Discovery Today 2010, 15, 342.
29. Biamonte, M. A.; Van de Water, R.; Arndt, J. W.; Scannevin, R. H.; Perret, D.; Lee,
W. C. J. Med. Chem. 2010, 53, 3.
We thank Loic Le Strat for compound purification and analysis.
30. Gao, Z.; Garcia-Echeverria, C.; Jensen, M. R. Curr. Opin. Drug Discov. Dev. 2010,
13, 193.
Supplementary data
31. Brough, P. A.; Drysdale, M. J.; Davies, N. G. M.; Foloppe, N. N.; Stokes, S. PCT Int.
Appl. WO2007104944, 2007.
32. Kung, P. P.; Sinnema, P. J.; Richardson, P.; Hickey, M. J.; Gajiwala, K. S.; Wang,
F.; Huang, B.; McClellan, G.; Wang, J.; Maegley, K.; Bergqvist, S.; Mehta, P. P.;
Kania, R. Bioorg. Med. Chem. Lett. 2011, 21, 3557.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.050.
33. Hajduk, P. J.; Greer, J. Nat. Rev. Drug Discovery 2007, 6, 211.
34. Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. J. Med. Chem. 2008, 51,
3661.
35. Jhoti, H.; Leach, A. R. Structure-Based Drug Discovery; Springer: Dordretcht, The
Netherlands, 2007.
36. Hubbard, R. E.; Davis, B.; Chen, I.; Drysdale, M. J. Curr. Top. Med. Chem. 2007, 7,
1568.
37. Foloppe, N. Future Med. Chem. 2011, 3, 1111.
38. Roughley, S. D.; Hubbard, R. E. J. Med. Chem. 2011, 54, 3989.
39. Brough, P. A.; Barril, X.; Borgognoni, J.; Chene, P.; Davies, N. G. M.; Davis, B.;
Drysdale, M. J.; Dymock, B.; Eccles, S. A.; Garcia-Echeverria, C.; Fromont, C.;
Hayes, A.; Hubbard, R. E.; Jordan, A. M.; Jensen, M. R.; Massey, A.; Merrett, A.;
Padfield, A.; Parsons, R.; Radimerski, T.; Raynaud, F. I.; Robertson, A.; Roughley,
S. D.; Schoepfer, J.; Simmonite, H.; Sharp, S. Y.; Surgenor, A.; Valenti, M.; Walls,
S.; Webb, P.; Wood, M.; Workman, P.; Wright, L. J. Med. Chem. 2009, 52, 4794.
40. PDB codes 1: 3060; PU3: 1UY6; 8: 2WI1; 9: 2WI7.
41. Dunitz, J. D. Science (Washington, DC, 1883) 1994, 264, 670.
42. Chen, J. M.; Xu, S. L.; Wawrzak, Z.; Basarab, G. S.; Jordan, D. B. Biochemistry
1998, 37, 17735.
43. Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. J. Med. Chem. 2010,
53, 7902.
44. Halgren, T. A. J. Comput. Chem. 1996, 17, 490.
45. Davoll, J. J. Chem. Soc. 1960, 131.
46. Fisher, M. B.; Campanale, K.; Ackermann, B. L.; VandenBranden, M.; Wrighton,
S. A. Drug Metab. Dispos. 2000, 28, 560.
47. Howes, R.; Barril, X.; Dymock, B. W.; Grant, K.; Northfield, C. J.; Robertson, A. G.;
Surgenor, A.; Wayne, J.; Wright, L.; James, K.; Matthews, T.; Cheung, K. M.;
McDonald, E.; Workman, P.; Drysdale, M. J. Anal. Biochem. 2006, 350, 202.
48. Kelland, L. R.; Sharp, S. Y.; Rogers, P. M.; Myers, T. G.; Workman, P. J. Natl.
Cancer Inst. 1999, 91, 1940.
49. Potter et al Bioorg. Med. Chem. Lett. 2010, 20, 586.
50. Petrie, C. R.; Cottam, H. B.; Mckernan, P. A.; Robins, R. K.; Revankar, G. R. J. Med.
Chem. 1985, 28, 1010.
References and notes
1. Mosser, D. D.; Morimoto, R. I. Oncogene 2004, 23, 2907.
2. Pearl, L. H.; Prodromou, C. Annu. Rev. Biochem. 2006, 75, 271.
3. Obermann, W. M.; Sondermann, H.; Russo, A. A.; Pavletich, N. P.; Hartl, F. U. J.
Cell Biol. 1998, 143, 901.
4. Panaretou, B.; Prodromou, C.; Roe, S. M.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.;
Pearl, L. H. EMBO J. 1998, 17, 4829.
5. Pearl, L. H.; Prodromou, C.; Workman, P. Biochem. J. 2008, 410, 439.
6. For a list, see the web page maintained by D. Picard: http://www.picard.ch/
downloads/Hsp90interactors.pdf (accessed June 2012).
7. Whitesell, L.; Lindquist, S. L. Nat. Rev. Cancer 2005, 5, 761.
8. Maloney, A.; Workman, P. Expert Opin. Biol. Ther. 2002, 2, 3.
9. Kamal, A.; Boehm, M. F.; Burrows, F. J. Trends Mol. Med. 2004, 10, 283.
10. Chiosis, G.; Vilenchik, M.; Kim, J.; Solit, D. Drug Discovery Today 2004, 9, 881.
11. Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57.
12. Workman, P. Cancer Lett. 2004, 206, 149.
13. Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.; Fritz, L. C.; Burrows,
F. J. Nature 2003, 425, 407.
14. da Rocha, D. S.; Friedlos, F.; Light, Y.; Springer, C.; Workman, P.; Marais, R.
Cancer Res. 2005, 65, 10686.
15. DeBoer, C.; Meulman, P. A.; Wnuk, R. J.; Peterson, D. H. J. Antibiot. (Tokyo) 1970,
23, 442.
16. Supko, J. G.; Hickman, R. L.; Grever, M. R.; Malspeis, L. Cancer Chemother.
Pharmacol. 1995, 36, 305.
17. Neckers, L.; Schulte, T. W.; Mimnaugh, E. Invest. New Drugs 1999, 17, 361.
18. Delmotte, P.; Delmotte-Plaque, J. Nature 1953, 171, 344.
19. Soga, S.; Shiotsu, Y.; Akinaga, S.; Sharma, S. V. Curr. Cancer Drug Targets 2003, 3,
359.
20. Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.; Pearl, L. H. J.
Med. Chem. 1999, 42, 260.
21. Schulte, T. W.; Neckers, L. M. Cancer Chemother. Pharmacol. 1998, 42, 273.