7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: Novel and highly selective protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.035

High throughput screening of the Roche compound collection led to the identification of diaminopyrroloquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloquinazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good mouse PK properties.

Full text of DOI:10.1016/j.bmcl.2012.10.035

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7518–7522
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: Novel and highly selective
protein tyrosine phosphatase 1B inhibitors
⇑
Adrian Wai-Hing Cheung , Bruce Banner, Jolly Bose, Kyungjin Kim, Shiming Li, Nicholas Marcopulos,
Lucja Orzechowski, Joseph A. Sergi, Kshitij C. Thakkar, Bing-Bing Wang, Weiya Yun,
Catherine Zwingelstein, Steven Berthel, Andrée R. Olivier
Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High throughput screening of the Roche compound collection led to the identification of diaminopyrro-
loquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloqui-
nazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to
give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good
mouse PK properties.
Received 22 August 2012
Revised 3 October 2012
Accepted 8 October 2012
Available online 16 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PTP1B inhibitor
Protein tyrosine phosphatase
Phosphatase selectivity
Pharmacokinetic
PTP1B, the first protein tyrosine phosphatase (PTP) cloned and
fully characterized, became an attractive drug target for diabetes
and obesity when two independent research teams1,2 disclosed
in 1999/2000 that PTP1B knockout mice showed enhanced insulin
sensitivity, lower plasma glucose and insulin levels, as well as
resistance to weight gain compared to control mice when fed high
fat diets. Recent reports also suggest a role for PTP1B in cancer.3,4
Despite significant medicinal chemistry efforts during the last
decade in the pharmaceutical industry, there has been a dearth
of small molecule PTP1B inhibitors entering clinical trials.5 This
is likely due to the highly polar phosphotyrosine-binding pocket
as well as limited opportunity for hydrophobic interactions in
the surrounding protein surface.5,6 An analysis by van Huijsduijnen
of 63 small, structurally different and potent PTP1B inhibitors
showed that most violate the Lipinski rules, are close to or exceed
the molecule weight cutoff of 500 Da and/or have PSA values great-
er than 160 A2.6 Since van Huijsduijnen’s analysis in 2004, progress
has been made in the design of PTP1B inhibitors in terms of cellular
and/or in vivo activities.5
other phosphatases (except T-cell phosphatase) and lowered blood
glucose in diet-induced obese (DIO) mice acutely as well as sub-
chronically (5 days, oral q.d. dosing).7,8 Since compound 1 is struc-
turally quite distinct from other reported PTP1B active site binders,
we carried out NMR study using 15N-labeled PTP1B and concluded
that compound 1 binds near the active site but does not penetrate
into the phosphate sub-pocket, and does not induce the closed
conformation of the WPD loop. The mixed competitive binding
kinetics of compound 1 is consistent with the proposed binding
model.9
One approach taken in modifying the diaminopyrroloquinazo-
line series during lead optimization involves the removal of the
pyrrole ring and replacement of C-8 with a nitrogen which led to
pyrido[2,3-d]pyrimidine-2,4-diamine series (Fig. 1). This paper
describes the synthesis and SAR of the pyrido[2,3-d]pyrimidine-
2,4-diamine series of PTP1B inhibitors leading to the discovery of
compounds 5 and 24 which are potent PTP1B inhibitors, selective
for PTP1B over other phosphatases (except T-cell phosphatase)
and possess good mouse PK properties.10,11
High throughput screening of the Roche compound collection
identified a series of diaminopyrroloquinazolines (exemplified by
compound 1, Fig. 1) as novel PTP1B inhibitors.7,8 Compound 1
showed good cell based potency, good selectivity for PTP1B over
Various 4-N-substituted analogs (compounds 5–8) were pre-
pared according to Scheme 1. Coupling of commercially available
2,4-diamino-6-hydroxypyrimidine with 3-dimethylamino-1-(2-
trifluoromethyl-phenyl)-propenone 2 (prepared from 1-(2-trifluo-
romethyl-phenyl)-ethanone and N,N-dimethylformamide dimethyl
acetal)12,13 gave compound 3.14,15 Chlorination of compound 3
(giving compound 4),16 followed by microwave (MW) assisted
⇑
Corresponding author.
E-mail address: adriancheungdr@gmail.com (A.Wai-Hing Cheung).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.035

A.W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7518–7522
7519
high-throughput strategy was devised in which o-fluoro group of
intermediate C (e.g., compound 21, prepared in gram-quantity fol-
lowing a modified version of Scheme 1) was displaced by a number
of nucleophiles under harsh conditions to give analogs 22–29
(Scheme 4). Examples of nucleophiles used in the fluoride displace-
ment22 reaction include, but not limited to, primary or secondary
amines, alcohols, phenols, methanethiolate, benzenethiol and
1H-imidazole.
R1
R2
N
N
N
N
5
4
6
7
N
3 N
2
S
H2N
N
1
N
8
H2N
pyrido[2,3-d]pyrimidine-2,4-diamine
1
Analogs 31–34, in which the 6-position was modified, were pre-
pared by Friedlander condensation (Scheme 5) of compound 20
and modified acetophenones 30.
Figure 1. Structures of compound 1 and pyrido[2,3-d]pyrimidine-2,4-diamine.
All the new analogs prepared were tested in PTP1B assay as pre-
viously described23 and the data are summarized in Tables 1–4.
The values reported are the average of at least two separate exper-
iments and it is typical for duplicate values to be within twofold of
each other.
To investigate the SAR at the N-4 position, we kept the 7-substi-
tuent fixed as 2-trifluoromethyl-phenyl group because of its excel-
lent PTP1B activity (to be discussed below) and chemical
compatibility with the synthetic route. A library of over 35 primary
and secondary amines (not shown) was screened at N-4 and the
most PTP1B potent analogs (compounds 5–8) are shown in Table 1.
Of all the N-4 amines studied, monomethylated analog 5 showed
the best PTP1B potency.
Keeping the N-methyl group identified above fixed at the 4-po-
sition, we next investigated the SAR of the 7-phenyl group.
Employing the synthetic routes shown in Schemes 2 and 3, a num-
ber of mono-substituted 7-phenyl analogs were prepared in which
substituents such as chloro-, methyl-, trifluoromethyl- and meth-
oxy- were systematically scanned at the o-, m- and p- positions
displacement of the 4-chloro group with various amines gave com-
pounds 5–8 with different 4-amino groups.
Various 7-substituted phenyl analogs (compounds 9–18) were
prepared according to Schemes 2 and 3. Starting with a variety of
substituted acetophenones,17 the corresponding 3-dimethyl-
amino-1-propenones A (Scheme 2) were prepared which then
went through a similar sequence as shown in Scheme 1 to give
compounds 9–18. This synthetic scheme is low throughput for
7-phenyl exploration but is useful for compound scale-up.
An alternative and higher throughput method of preparing
compounds 9–18 is depicted in Scheme 3. Methylamine displace-
ment of commercially available 6-chloro-2,4-diaminopyrimidine
gave compound 1918 which was then formylated to give com-
pound 20.19 Friedlander condensation20,21 reactions of compound
20 with substituted acetophenones B17 were carried out in a paral-
lel synthesis fashion to give compounds 9–18.
To prepare 7-o,o0-disubstituted-phenyl analogs in which one of
the o-substituents is highly electron-deficient (e.g., CF3, Cl, F), a
OH
N
F
OH
F
F
F
N
F
F
N
acetic acid
+
H2N
N
N
reflux, 2 days
57% yield
O
H2N
N
NH2
2
3
R1
R2
Cl
N
N
F
F
R1RNH, Et2iPrN
n-butanol
POCl3, iPr2EtN
35oC, 18 h
N
F
F
N
F
F
150oC, MW, 20 m
25%-60% yield
H2N
N
N
H2N
N
4
5-8
Scheme 1. Synthesis of various 4-N-substituted 7-(2-trifluoromethyl-phenyl)-pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 5–8).
N
O
NH
N
OH
N
R3
N
R3
R6
R4
R5
N
R4
R5
+
H2N
N
H2N
NH2
R7
R7
R6
A
9-18
Scheme 2. Synthesis of various 7-substituted phenyl N-4-methyl-pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 9–18).

7520
A.W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7518–7522
Cl
N
NH
25% aq.
CH3NH2
1. DMF, POCl3
0 oC to 40 o
C
N
N
150 o
C
2. H2O, NaOH
90 oC
sealed tube
H2N
NH2
H2N
N
NH2
47% yield
33% yield
19
NH
NH
O
R3
solid KOH
ethanol
R4
R5
R3
R6
N
N
H
O
+
100 oC
sealed tube
R4
R5
N
N
H2N
NH2
H2N
N
R7
R6
25-55% yield
R7
9-18
B
20
Scheme 3. Alternative synthesis of various 7-substituted phenyl N-4-methyl-pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 9–18).
NH
N
NH
N
N
R3
Nu-H
K2CO3
N
R3
H2N
N
H2N
N
1-methyl-2-pyrrolidinone
190oC
Nu
sealed tube
F
R3 = Cl, CF3, F
R3 = Cl, CF3, F
25-50% yield
22-29
C (e.g. 21)
Scheme 4. Synthesis of various 7-o,o0-disubstituted phenyl N-4-methyl-pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 22–29).
R8
NH
O
NH
R8
solid KOH
ethanol
N
H
N
O
F
F
+
100 o
C
NH2
H2N
N
N
N
F
F
H2N
sealed tube
F
20-60% yield
30
20
31-34
F
Scheme 5. Synthesis of various 6-substituted 7-(2-trifluoromethyl-phenyl)-pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 31–34).
of 7-phenyl. The SAR (data not shown) showed that mono-o-
substituted analogs consistently gave better PTP1B potencies than
the corresponding mono-m- and mono-p-substituted analogs. A
subset of mono-o-substituted analogs (9–14) are shown in Table 2
to illustrate the SAR. Two of the most PTP1B potent o-substitutents
are o-CF3 (5) and o-Br (13) while the other o-substituents gave
compounds which are two- to fivefold less potent in PTP1B assay
(Table 2), compared to compounds 5 and 13.
Table 1
PTP1B activities of various 4-N-substituted 7-(2-trifluoromethyl-phenyl)-pyrido[2,3-
d]pyrimidine-2,4-diamine (compounds 5–8)
R1
R2
N
N
H2N
N
N
F
F
We next investigated the preference for disubstituted 7-phenyl
group and using chloro-substitution as a test case, we prepared all
four possible combinations containing at least one o-chloro group
(2,3-diCl 15, 2,4-diCl 16, 2,5-diCl 17 and 2,6-diCl 18). Of the four
isomers, the two more potent disubstituted isomers (16 and 18)
showed about twofold improvement in PTP1B potency compared
to mono-substituted 14.
F
Compound
R1
R2
PTP1B, IC50 (lM)
5
6
7
8
CH3
CH2CH3
CH2CH2CH3
CH2CH2OH
H
H
H
H
0.48
1.34
2.43
1.85
Having demonstrated that o,o0-disubstitution gave the most po-
tent analogs and -CF3 is one of the best o-substitutents, we next try

A.W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7518–7522
7521
Table 2
Table 4
PTP1B activities of various N-4-methyl 7-substituted phenyl pyrido[2,3-d]pyrimidine-
2,4-diamine (compounds 9–18)
PTP1B activities of various 6-substituted N-4-methyl 7-(2-trifluoromethyl-phenyl)-
pyrido[2,3-d]pyrimidine-2,4-diamine (compounds 31–34)
H3C
NH
H3C
NH
N
R3
R6
R8
N
R4
R5
H2N
N
N
H2N
N
N
F
F
R7
F
Compound
R3
R4
R5
R6
R7
PTP1B, IC50 (lM)
5
9
CF3
CH3
OCH3
NO2
CO2H
Br
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
0.48
1.66
2.79
1.37
2.39
0.53
1.16
1.18
0.61
0.96
0.51
Compound
R8
PTP1B, IC50 (lM)
10
11
12
13
14
15
16
17
18
5
H
CH3
CH2CH2CH3
CO2H
SO2CH3
0.48
0.50
0.27
0.25
1.00
31
32
33
34
Cl
to prepare a series of 7-o,o0-disubstituted-phenyl analogs in which
one of the o-substituents is -CF3. Gram-quantity of intermediate 21
was prepared according to a modified version of Scheme 1 and its
o-fluoro group was displaced by a number of nucleophiles. Some of
the representative examples (compounds 22–27) are shown in Ta-
ble 3. Interestingly, none of the newly prepared o,o0-disubstituted
analogs showed significantly improved PTP1B potency compared
to compound 21. However, the range of o0-substituents introduced
via fluoride displacement allowed the tuning of lipophilicity and
basicity which in turn impacted the cellular potency, physico-
chemical (e.g., permeability, solubility) and PK properties of the
resulting analogs.
Table 3
PTP1B activities of various N-4-methyl 7-o,o0-disubstituted phenyl pyrido[2,3-
d]pyrimidine-2,4-diamine (compounds 21–29)
H3C
NH
N
R3
The superior PTP1B potency of -CF3 as an o-substituent is illus-
trated by three o,o0-disubstituted analogs (27–29) which share pyr-
rolidine as the common o0-substituent. Analog 27 with o’-CF3 is
slightly but reproducibly more potent than compound 28 (with
o0-Cl) and 29 (with o0-F).
To investigate the SAR at the 6-position, we fixed the 4-substi-
tuent as N-methyl and the 7-substituent as 2-trifluoromethyl-phe-
nyl group. Of the limited 6-substituents explored, a relatively
narrow range (about fourfold) of PTP1B potency was observed (Ta-
ble 4). For example, 6-substituents such as n-propyl (32) and car-
boxylic acid (33) led to about twofold improvement in PTP1B
potency while 6-substituent such as methylsulfone led to a two-
fold drop in PTP1B potency, compared to compound 5.
Based on PTP1B potency and ADME properties (e.g., aqueous
solubility, metabolic stability, and Caco-2 permeability, data not
shown), two analogs described above (compounds 5 and 24) were
chosen and tested in a mouse PK study (Table 5). Compound 5 was
found to have moderate volume of distribution, moderately high
clearance and good oral bioavailability in mouse. Compared to
compound 5, compound 24 has lower volume of distribution, im-
proved clearance and slightly lower oral bioavailability.
Compounds 5 and 24 were assayed against a panel of ten phos-
phatases and both compounds showed >100fold selectivity against
nine phosphatases but only two- to threefold selectivity against T-
cell phosphatase.24
H2N
N
N
R7
Compound
R3
R7
PTP1B, IC50 (lM)
5
CF3
CF3
CF3
CF3
H
F
0.48
0.26
0.56
0.35
21
22
23
OCH3
SCH3
24
25
CF3
CF3
0.20
1.15
O
O
N
26
27
28
29
CF3
CF3
Cl
1.35
0.43
0.82
0.62
N
N
N
N
In summary, high throughput screening of the Roche compound
collection led to the identification of diaminopyrroloquinazoline
series as a novel class of PTP1B inhibitors. Structural modification
of diaminopyrroloquinazoline series resulted in pyrido[2,3-
d]pyrimidine-2,4-diamine series which was further optimized to
give compounds 5 and 24 as potent, selective (except T-cell
F

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A.W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7518–7522
Table 5
PK parameters of compounds 5 and 24 after IV and PO administration to C57 mouse
IV arma
PO arma
Compound IV, Dose (mg/kg) CL (mL/min/kg) Vd (L/kg) AUC extrap. (ng h/mL) PO, Dose (mg/kg) Cmax (ng/mL) AUC extrap. (ng h/mL) t1/2 (h) F (%)
5
24
5
10
61
24
1.8
0.9
1370
6970
50
50
3530
2200
8990
10980
7.2
1.3
66
32
a
A dose of drug was either intravenously (5 mg/kg, DMA/Cremophor/PEG 400/acetate buffer for compound 5; 5 mg/kg, DMA/PEG 400/HPBCD in water for compound 24)
injected into the tail vein of male C57 mouse (n = 4) or orally (50 mg/kg, Klucel/Tween 80 suspension for both compounds) administered using an intubation tube (n = 4).
Plasma samples were collected up to 24 h after intravenous or oral administration. The plasma concentrations of compound 5 and 24 were determined by LCMS.
April 1, 2004; American Chemical Society: Washington, DC, 2004; MEDI 053;
(b) Lineweaver–Burke plot of compound 5 is deposited as Supplementary data.
10. Thakkar, K. C.; Berthel, S.; Banner, B.; Bose, J.; Cheung, A.; Kim, K; Li, S.; Mackie,
G.; Marcopulos, N.; Orzechowski, L.; Olivier, A. R.; Sergi, J. A.; Spence, C.; Wang,
B-B.; Yun, W.; Zwingelstein, C. Abstracts of Papers, 239th National Meeting of
the American Chemical Society, San Francisco, CA, March 21–25, 2010;
American Chemical Society: Washington, DC, 2010; MEDI 181
11. Berthel, S. J.; Cheung, A. W-H.; Kim, K.; Li, S.; Thakkar, K. C.; Yun, W. WO
2007009911; CAN 146:184481.
phosphatase) PTP1B inhibitors with good mouse PK properties.
Further reports detailing the studiesof diaminopyrroloquinazoline
and related series are in preparation.
Acknowledgments
The authors would like to thank Jia Kui Li for formulation, Drs.
Navita Mallalieu and Aruna Railkar for carrying out the rodent PK
studies. We would also like to thank Roche Physical Chemistry
Department for spectroscopic measurements and interpretations
and Dr. Francisco Talamas for critical reading of the manuscript.
12. Tseng, S. S.; Epstein, J. W.; Brabander, H. J.; Francisco, G. J. Heterocycl. Chem.
1987, 24, 837.
13. Moyroud, J.; Chene, A.; Guesnet, J.-L.; Mortier, J. Heterocycles 1996, 43, 221.
14. Robins, R. K.; Hitchings, G. H. J. Am. Chem. Soc. 1958, 80, 3449.
15. Troschuetz, R.; Dennstedt, T. Arch. Pharm. 1994, 327, 221.
16. Ife, R. J.; Brown, T. H.; Blurton, P.; Keeling, D. J.; Leach, C. A.; Meeson, M. L.;
Parsons, M. E.; Theobald, C. J. J. Med. Chem. 1995, 38, 2763.
17. Various substituted acetophenones could be prepared: (a) from substituted
benzoic acids, see e.g. (a) Jorgenson, M. J. Org. React. 1970, 18, 1; from
substituted benzaldehydes, see e.g. (b) Tanouchi, T. et al J. Med. Chem. 1981, 24,
1149; from substituted phenol triflates, see e.g. (c) Garrido, F.; Raeppel, S.;
Mann, A.; Lautens, M. Tetrahedron Lett. 2001, 42, 265; from substituted aryl
iodides, see e.g. (d) Cacchi, S.; Fabrizi, G.; Gavazza, F.; Goggiamani, A. Org. Lett.
2003, 5, 289.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
035.
18. Elion, G. B.; Hitchings, G. H. J. Am. Chem. Soc. 1953, 75, 4311.
19. Delia, T. J.; Otteman, R. Heterocycles 1983, 20, 1805.
20. Perandones, F.; Soto, J. L. J. Heterocycl. Chem. 1998, 35, 413.
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References and notes
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22. Vlasov, V. M. J. Fluorine Chem. 1993, 61, 193.
23. Human PTP1B (1-321) was cloned from
a human cDNA library using
conventional molecular biology techniques. The cDNA sequence was
identical to the published human PTP1B sequence (Accession number
M33689). The protein was expressed and purified from Escherichia coli as
described by Barford D. et al. J. Mol. Biol. 1994, 239, 726. To measure PTP1B
inhibitory activity, a tyrosine phosphorylated peptide based on the amino acid
sequence of insulin receptor tyrosine autophosphorylation site 1146
(TRDI(pY)E) was used as substrate. The reaction conditions were as follows:
PTP1B (0.5–2 nM) was incubated with compound for 15 min in buffer
containing 37.5 mM Bis-Tris buffer pH 6.2, 140 mM NaCl, 0.05% BSA and
5. Combs, A. P. J. Med. Chem. 2010, 53, 2333.
6. van Huijsduijnen, R. H.; Sauer, W. H. B.; Bombrun, A.; Swinnen, D. J. Med. Chem.
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7. Thakkar, K. C.; Bose, J.; Berthel, S.; Dong, H.; Emerson, D.; Fry, D.; Gillespie, P.;
Li, S.; Olivier, A. R.; Orzechowski, L.; Pflieger, P.; Sergi, J. A.; Yun, W. Abstracts of
Papers, 227th National Meeting of the American Chemical Society, Anaheim,
CA, March 28–April 1, 2004; American Chemical Society: Washington, DC,
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8. Thakkar, K. C.; Berthel, S.; Bose, J.; Banner, B.; Dvorozniak, M.; Grimsby, J.;
Mackie, G.; Olivier, A. R.; Orzechowski, L.; Spence, C.; Sergi, J. A. Abstracts of
Papers, 227th National Meeting of the American Chemical Society, Anaheim,
CA, March 28–April 1, 2004; American Chemical Society: Washington, DC,
2004; MEDI 049.
2 mM DTT. The reaction was started by the addition of 50 lM substrate. After
20 min at room temperature (22–25 °C), the reaction was stopped with KOH
and the amount of free phosphate measured using Malachite Green as
previously described (Harder et al. Biochem. J. 1994, 298, 395).
24. A panel of phosphatases were screened for selectivity: PTP1B, LAR, PTP
CD45, PTP , PTP , HCP, SHP2, TC, YOP. To measure general PTPase inhibitory
activity across panel of PTPases, the substrate 6,8-difluoro-4-
a, PTPb,
e
c
a
methylumbelliferyl phosphate (DiFMUP; from Molecular Probes) was used at
the Km for each enzyme. The buffer conditions were identical as in the
Malachite Green assay (Ref. 23). The reaction was stopped with KOH.
Fluorescense of the de-phosphorylated product was monitored (Excitation:
360 nm/Emission: 460 nm).
9. (a) Thakkar, K. C.; Bose, J.; Banner, B.; Dvorozniak, M.; Fry, D.; Grimsby, J.;
Mackie, G.; Olivier, A. R.; Spence, C.; Sergi, J. A. Abstracts of Papers, 227th
National Meeting of the American Chemical Society, Anaheim, CA, March 28–