Discovery and development of 2,7-Disubstituted-Pyrrolo[2,1-f][1,2,4]triazines. A New Class of Anaplastic Lymphoma Kinase(ALK)*Inhibitors with in-vivo Anti-tumor Efficacy

1. 2,7-Disubstituted-Pyrrolo[2,1-f][1,2,4]triazines
A New Class Anaplastic Lymphoma Kinase (ALK)
Inhibitors with in-vivo Anti-tumor Efficacy
Gregory J. Wells, Ph.D.
N
R
N
N N
H
R'
Gregory J. Wells, Ph.D.

2. General
 Kinome - the set of protein kinases in an organism’s genome.
 Kinases - enzymes that catalyze phosphorylation (from ATP) of
amino acids, which fall into several groups and families:
• ones that phosphorylate serine and threonine;
• ones that phosphorylate tyrosine;
• some that phosphorylate both (eg. MAP2K and GSK families).
 Essential for control and regulation of most biochemical pathways
• 518 known human protein kinases
• 155 crystal structures solved
• 160+ associated with human diseases
• 14 small molecule inhibitors approved since 2001
 Among the most active areas of targeted drug discovery in the past decade
Gregory J. Wells, Ph.D.

3. ALK is a member of the TKL branch of the Protein Kinase Phylogenetic Tree
(Reprinted with permission from Cell Signaling Technology, Inc.)
Gregory J. Wells, Ph.D.

4. ALK – Background
 Anaplastic Lymphoma Kinase (ALK) is a cell membrane-spanning
receptor tyrosine kinase with MW = 180 kDa
 Most abundantly expressed in neonatal and adult brains, suggesting
a possible role in brain and CNS development (Oncogene, 2001,
20, 5623)
 Member of the insulin receptor (IR) family with IGF-1R, IRR, LTK
and c-Ros
 Binds to insulin receptor substrate 1 (IRS1); activates MAPK
signaling pathway by activating the Ras-extracellular signal
regulated kinase (ERK) pathway
Gregory J. Wells, Ph.D.

5. Relation to Anaplastic Large-Cell Lymphoma (ALCL)
 ALK is implicated in the progression of certain tumors; approximately 60-70%
of anaplastic large cell lymphomas (ALCL) are associated with a chromosome
mutation containing a classical t (2;5) or other translocation that generates a mutant
oncogenic fusion protein (NPM-ALK) consisting of nucleophosmin (NPM) and the
intracellular domain of ALK
 NPM-ALK possesses a constitutively active tyrosine kinase domain responsible for
its oncogenic property through activation of downstream effectors
 ALCL is a subset of high-grade non-Hodgkin’s lymphomas (NHLs) that typically
presents as an aggressive systemic disease with ~50% 5-year survival rate after
chemotherapy; predominant in children and young adults with about 2,500 new
cases per year in US
 Aberrant expression of constitutively active ALK is directly implicated in the
pathogenesis of ALCL and ALK inhibition can markedly impair the growth of
ALK+ lymphoma cells
 Additionally, the EML4–ALK fusion gene has been identified in non-small-cell lung
cancer (NSCLC) patients (Nature, 2007, 561) and represents another in a list of
ALK fusion proteins that are promising targets for ALK inhibitor therapy.
Gregory J. Wells, Ph.D.

6. Competitor ALK Inhibitors
N
N HN
F
O
N Cl N
N
N N N N
N N NH O N H H
H
O S O O NH
O
Novartis (NVP-TAE684) GlaxoSmithKline
ALK IC50 = 3 nM ALK IC50 = 0.5 nM
H
N
N
O
Cl
N
N O O
F N
H
N N CN N NH N
H Cl O
H2N N
O
O
Chugai Pharma. Chembridge O
Pfizer
(Crizotinib)
ALK IC50 = 2 nM ALK IC50 = 0.2 µM
ALK IC50 = 2 nM
Gregory J. Wells, Ph.D.

7. Previously reported Cephalon ALK inhibitors
H N F
N O O
N O
HN N N F
N N
N
N N
H
Wan, Cheng, Blood, 1617 (2006) Milkiewicz, Bioorg. Med. Chem., 4351 (2010)
ALK IC50 = 2nM ALK IC50 = 10nM
Karpas Cell IC50 = 20nM Karpas Cell IC50 = 150nM
O O
N
N Cl
N Cl O O N
N
N
N N N
N N N H H
H H
O CONH2
O
Mesaros, Bioorg. Med. Chem., 463 (2011) Ott, Med. Chem. Lett., 493 (2010)
ALK IC50 = 4nM ALK IC50 = 14nM
Karpas Cell IC50 = 50nM Karpas Cell IC50 = 45nM
Gregory J. Wells, Ph.D.

8. ALK Activity Is Essential for the
Proliferation of ALK+ ALCL Cells in Culture
H
N O
H
N O N ALK IC50
(Me)2N O N
N
Enzyme 3.7 nM
Cellular 10-30 nM
CEP-14513
― 30 100 300 (nM) ― 30 100 300 (nM)
p-NPM-ALK p-NPM-ALK
NPM-ALK NPM-ALK
120 K562 cells
120 120
Karpas-299 Sup-M2
R e la tiv e liv in g c e lls
R e la t iv e liv in g c e lls
90
R e la t iv e liv in g c e lls
90 90
60
60 60
30
30 30
0
0 0
1 10 100 1000 1 10 100 1000
1 10 100 1000
CEP-14513 (nM) CEP-14513 (nM) CEP-14513 (nM)
Gregory J. Wells, Ph.D.

9. Conceptualization of Pyrrolotriazines as Potential ALK Inhibitors
Cl
N
N
HN N NH N
HN N
Gregory J. Wells, Ph.D.

10. Precedents in the Medicinal Chemistry Literature
Purine/Adenosine mimics as C-linked nucleosides Quinazoline mimics as kinase inhibitors
NH2
H
N
N N
HO N HO N HO NH
O O O
N N
H S
N F
N
N OH
HO OH HO OH N N
N
Heterocycles, 569 (1992) Tet. Lett., 5339 (1994) JMC, 4059 (2004) JMC, 2143 (2006)
Meijo University Albert Einstein College of Medicine VEGFR-2 (BMS)
Substituted Aminopyrazole hinge-binding motif H
N O
H O HN
N F
N
NH2
O N
HN
F
N F N
N O
N
N N
N N
H H
BMCL, 1945 (2007)
JMC, 7360 (2009) c-MET (BMS)
IGF-1R (BMS)
Gregory J. Wells, Ph.D.

11. Crystal Structure of NVP-TAE684 bound to ALK
Cl
N
HN N NH O
O S
O
N
NVP-TAE684
N
N
Gregory J. Wells, Ph.D.

12. Docking of a 2,7-pyrrolo[2,1-f][1,2,4]triazine analog with ALK
N
N
HN N O
O
N
N
Gregory J. Wells, Ph.D.

13. Synthesis of a Key Intermediate
2-Methylthio-7-bromo-pyrrolo[2,1-f][1,2,4]triazine
OEt
O
Benzoyl N
Chloroamine OEt thioisocyanate H 2N NaOH
OEt O HN
O N NH
N N THF
KO-t-Bu, THF N
H O
O NH2 S S N
H
1 2 3
4
Cl Cl Cl Cl Br
Br
1. MeI, THF NBS N N
N
+ N
+
2. POCl3 N N N N
THF- MeOH (2/1) S N S N
S N S N
Br Br
5 6 7 8
Br Br
N N N
1. NaBH4, IPA, 55oC
N + N
+ N
S N S N S N
2. DDQ, DCM
Br Br
10 11
9
Gregory J. Wells, Ph.D.

14. Targets from 2,7-Orthogonally Functionalized Intermediate
Method A
Pd(OAc)2, Ph3P
N 1. m-CPBA, DCM N
N N N
S N R2 HN N
N R2
S N B(OH)2 NH2
Br 2.
R2 R1
9
12 µW, DIPEA, NMP 13
R1
(R2 = OMe, -NHSO2Me)
1. m-CPBA, DCM
2. K2CO3, MeI
N
N
S N N SO2Me
O
14
Gregory J. Wells, Ph.D.

15. Targets from 2,7-Orthogonally Functionalized Intermediate
Method B
1. m-CPBA, DCM N
Pd(OAc)2, PH3P N
N
N
N N HN N
S N 2. R1-NH2,mW HN N R2
B(OH)2
Br Br R1
DIEPA, NMP R1
9 15 R2 16
R1 =
O
MeO OMe
OMe N N N
N
O N N O
Gregory J. Wells, Ph.D.

16. SAR of the 2-Position with 7-Phenyl-pyrrolo[2,1-f][1,2,4]triazine
N
2
R1 N
N N
H
Cpd. R1 ALK IC50 (nM)a IR IC50 (nM)a
OMe
MeO
17 212 ± 70 295 ± 95
MeO
O
18 N >2000 194 ± 45
N
19 N 48 ± 8 35 ± 3
O
20 N
38 ± 11 19 ± 6
a
IC50 values ± SD reported as the average of ≥ 3 determinations
Gregory J. Wells, Ph.D.

17. SAR of the 2-Aryl-phenyl Group at C7
N
N
N
N
N N 7 R2
H
Cpd. R2 ALK IC50 (nM)a IR IC50 (nM)a
21 -OMe 20 ± 2 123 ± 37
22 -C(O)NH2 726 ± 113 326 ± 102
23 -SO2CH3 883 ± 233 > 2000
24 -NHSO2CH3 260 ± 57 1874 ± 485
25 -N(Me)SO2CH3 7±2 366 ± 143
a
IC50 values ± SD reported as the average of ≥ 3 determinations
Gregory J. Wells, Ph.D.

18. In Vitro Profiles of Early Best Analogs
Liver microsome, t1/2(min) CYP inhibition IC50 (µM)a KINOMEscan® F (%)
(Mouse, Rat, Human) 3A4 S(90) @ 1 mM (Rat)
N
N
N
N N
N 8, 14, 40 9.4 0.524 4
H OMe
21
N
N
N
N
N N N
H SO2CH3
30, 21, 40 14.6 0.614 --
25
a
CYPs (1A2, 2C9, 2C19, 2D6): IC50 all > 30 µM
 Desired: Improved kinase selectivity and oral bioavailability
Gregory J. Wells, Ph.D.

19. Optimized 2,7-Disubstituted Analogs
R1
N
N
N 7
H 2 N R2
O
ALK Cell IR KINOMEScan® F(%)
Eg. R1 R2 IC50 (nM)a IC50 (nM)b IC50 (nM)a S(90) @ 1 µM (Rat)
26 N -OCH 9±3 100 391 ± 80 0.085 --
N
HO
27 N -OCH 5 ± 21 100 206 ± 49 0.067 8
N
HO
28 N
-OCH 4±1 80 160 ± 36 0.067 6
N
O
29 -OCH 9±3 85 357 ± 95 0.050 34
N
N
N
30 -OCH 9±1 70 266 ± 65 0.164 38
N
N
a
IC50 values ± SD reported as the average of ≥ 3 determinations
b
IC50 values reported as a mean of at least two determinations
Gregory J. Wells, Ph.D.

20. Optimized 2,7-Disubstituted Analogs
R1
N
N
N 7
H 2 N R2
O
ALK Cell IR KINOMEScan® F(%)
Eg. R1 R2 IC50 (nM)a IC50 (nM)b IC50 (nM)a S(90) @ 1 µM (Rat)
31 N
-N(Me)SO2Me 9±2 200 934 ± 323 0.187 --
N
HO
32 N -N(Me)SO2Me 10 ± 2 60 1137 ± 398 0.187 38
N
HO
33 N -N(Me)SO2Me 15 ± 5 80 1171 ± 169 0.194 41
N
O
34 N -N(Me)SO2Me 7±2 50 809 ± 299 0.187 47
N
a
IC50 values ± SD reported as the average of ≥ 3 determinations; b IC50 values reported as a mean of at least two determinations;
c
Kinase selectivity was determined using the Ambit Bioscience KINOMEscan® technology, and is expressed as S(90), the fraction
of kinases inhibited >90% when screened at 1 μM across a panel of 256 kinases.
Gregory J. Wells, Ph.D.

21. Anti-tumor Efficacy Comparison of Best Analogs
O
10mg/kg, po, bid 30 mg/kg, po, bid 55 mg/kg, po, bid
N (%TGI) (%TGI)
N
N
N
N
H
N OMe No significant effect 60 98
O
29
HO
N
N
N
N
H
N
N
N SO2CH3
35% TGI 81 98
O
32
O
N
N
N
N N
N
N
No significant effect 50 96
H SO2CH3
O
34 a
TGI = tumor growth inhibition; calculated from tumor volume on final day
relative to vehicle control group
Gregory J. Wells, Ph.D.

22. MTS Assay of 32 in ALK-positive and ALK-negative cell lines
HO
N
N
N
N
N N N
H SO2CH3
O
ALK positive lines
ALK negative lines
Compound Conc. (nM)
Gregory J. Wells, Ph.D.

23. In-vivo Efficacy of 32 on Tumor Volume
in ALK positive SUP-M2 Xenografts in Scid Mice
HO
N
N
N
N
N N N
H SO2CH3
O
Gregory J. Wells, Ph.D.

24. Plasma and Tumor Levels of 32 in ALK
Positive SUP-M2 Xenografts in Scid Mice
HO
N
N
N
N
N N N
H SO2CH3
O
Gregory J. Wells, Ph.D.

25. Pharmacokinetics of Lead Compound 32
HO
N
N
N
N
N N N
H SO2CH3
O
Gregory J. Wells, Ph.D.

26. Single Oral Dose PK/PD for Lead Compound 32
HO
N
N
N
N
N N N
H SO2CH3
O
Gregory J. Wells, Ph.D.

27. In vitro ADME and Safety Pharmacology Profile
HO
N
N
N
N
N N N
H SO2CH3
O
 t1/2 > 40 min (Mouse, Rat, Human liver microsomes)
 Caco-2 permeability assay: Papp (AB) = 20.3 x 10-6 cm/s; PDR < 2
 Plasma protein binding: Mouse (91%); Rat (68%); Humans (83%)
 hERG (patch clamp): IC50 = 9.1 μM
 CYP (1A2, 2C9, 2C19, 2D6, 3A): IC50 >12 μM
 Compound nominated for pre-development
and late stage pre-clinical safety studies
Ott, Wells; JMC 6328 (2011)
Gregory J. Wells, Ph.D.

28. Unusual Mass Spectral Characteristics of Pyrrolo[2,1-f][1,2,4]triazines
O
N
N
N
N
N N OMe
H
O
29
(MW = 514)
- Infused alone - Infused w/ascorbic acid
 Apparent formation of semiquinonediimine radical and quinonediimine ions
(m/z = 514, 513, resp.) supported by their suppression with ascorbic acid
 Evidence to suspect (potentially toxic) reactive metabolite formation
 Similar observations with related pyrrolotriazine during metabolic studies
Gregory J. Wells, Ph.D.

29. Proposed Mechanism of Ion Formation during ES-MS of Compound 29
Gregory J. Wells, Ph.D.

30. UV Chromatograms from Microsomal Incubation of 32 containing GSH
HO
N
N
N
N
N N N
H SO2CH3
O
(0 min)
(60 min)
Gregory J. Wells, Ph.D.

31. LC/MS Spectra of GSH Adduct of 32 Formed During Microsomal Incubation
Proposed fragmentation pattern
Wells-Knecht, Wells; Chem. Res. Toxicol., 1994 (2011)
Gregory J. Wells, Ph.D.

32. Conflicting ADME vs. Analytical Trapping Experiments
 At this stage…
Lead compound 32 showed good F, TGI, LMS, and acceptable safety
pharmacology profile in several animal models.
 However…
Bioactivation/glutathione (GSH) trapping experiments in liver microsomes with
a subset of analogs identified apparent NADPH-mediated oxidation products of
reactive and potentially toxic quinone diimines.
 Prompting…
Further structural modifications on the suspected sites of oxidation to attenuate
potential formation of toxic metabolites to produce a “cleaner” drug candidate.
Gregory J. Wells, Ph.D.

33. Strategies to Mitigate Bioactivation/Glutathione Formation
Shift piperazine ring from para to meta
to avoid formation of p-iminoquinone
Incorporate EWGs on phenyl ring
to lower oxidation potential
HO
N Incorporate small groups at C5
N
N
to block potentially reactive site
N
N N N
H SO2CH3
O
Replace heterocycle with piperidine to remove
the p-nitrogen and lower the oxidation potential
Gregory J. Wells, Ph.D.

34. Syntheses of Fluorophenyl- and m-Piperazine-Containing Analogs
R2 HO HO
HN R2 N R2 N R2
X a b c
N N
N
R4 NO2
R4 NO2 R4 NO2 R4 NH2
R5
R5 R5 R5
35 38
36 37
+
N R2
N R3 d or e N
N e N
N
+ HO N R1
R4 N N
N R6 N
N
R1
R1
H
R5
NH2
O
41 42 39
40
Reagents and conditions: (a) (i) for X = F (inhibitors 11 and 13), N-Boc-piperazine, K2CO3, DMF, 60 °C, 60−90%, for
a
X = Br (inhibitor 12), N-Boc-piperazine, Pd2(dba)3, XantPhos, Cs2CO3, 1,4-dioxane, 100 °C, 66%; (ii) TFA, DCM, rt,
80−97%; (b) (S)-2-methyl-oxirane, MeOH, 42−82%; (c) H2, Pd/C, MeOH, 87−97%; (d) 9a (R1 = N(Me)SO2Me), DIPEA,
1-methoxy-2-propanol, microwave, 200 °C, 8%; (e) (i) 9b, (F3CSO2)2NPh, DIPEA, DMF, 0 °C → rt; (ii) 8 or 10, 85 °C,
30−47%.
Gregory J. Wells, Ph.D.

35. F- and m-Substitution on Aminophenyl Group Reduced GSH Adduct Formation
R2
R3
N
HO N
N A: N
R4 N N B: N
R1 N
H
R5
IC50 (nM)a
Cpd R1 R2 R3 R4 R5 ALK ALK %GSH
enzyme cell adductsb
32 -N(Me)SO2Me H A H -OMe 10 ± 2 60 61
43 -N(Me)SO2Me H A F -OMe 56 ± 16 -- 12
44 -N(Me)SO2Me F A H -OMe 14 ± 3 200 13
45 -N(Me)SO2Me F A H F 91 ± 19 -- 0.8
46 -N(Me)SO2Me B H H -OMe 28 ± 8 -- 3.9c
47 -OMe B H H -OMe 187 ± 64 -- 2.3c
IC50 values reported as the average of at least two separate determinations; standard deviations are indicated where at least
a
three determinations were made. bRelative to the tested compound. cDue to O-demethylation and iminoquinone formation/trapping
Gregory J. Wells, Ph.D.

36. Synthesis of Piperidine Analogs
t-Boc R7
N N
t-Boc Cl
N a b, c, d
B
O +
NO2
NO2 NH2
O O
O O
+
t-Boc R7
N N R
e, b R e
N N
or
N N
NH2 f, e N N H2N N
O H Ar Ar
O
g, b when R, R7 = H and Ar = o-methoxyphenyl
O
R8 N
N
N
N N O
H
O
Reagents and conditions: (a) Pd(PPh3)4, KHCO3, water/1,4-dioxane, 80 °C, 97%; (b) TFA, DCM, rt, 95%; (c) for inhibitors 22, 23,
a
25, 27, 28, epoxide, MeOH, 42−80%; for inhibitors 31−40, BrCH2CONHMe or ICH2CONH2, Cs2CO3, MeCN, 75 °C, 71−80%; (d) H2,
Pd/C, MeOH, > 90%; (e) (i) 20a, (F3CSO2)2NPh, DIPEA, DMF, 0 °C →rt; (ii) aniline (e.g., 19 or 21), 85 °C, 13−58%; (f) LiAlH4, THF,
reflux, 92%; (g) N-Boc-aminoacid, EDCI, HOBt, DIPEA, DMF, 50−60%.
Gregory J. Wells, Ph.D.

37. Piperidine Analogs – Potency and Liver Microsome Stability
R7
N
N
N
N N R1
H
O
ALK IC50 (nM)a Liver microsome t1/2 (min)b
Cmpd R1 R7 Enzyme Cellular M R Mo H
HO
48 -N(Me)SO2Me 6±1 60 > 40 21 <5 <5
HO
49 -N(Me)SO2Me 6±2 70 > 40 11 <5 <5
50 -N(Me)SO2Me H 10 ± 4 40 > 40 22 <5 7
HO
51 -OMe 3.4 ± 0.9 100 > 40 > 40 21 >4
a
IC50 values reported as the average of at least two separate determinations; standard deviations
are indicated where at least three determinations were made. b Relative to the tested compound.
Gregory J. Wells, Ph.D.

38. Piperidine Analogs – Potencies, Selectivity, and Rat PK
R7
N
N
N
N N
H Ar
O
IC50 (nM)a Rat PK iv data
Cmpd R7 Ar ALK ALK IR S(90)b T1/2 (h) CL Rat F%
Enzyme Cellular Enzyme (mL/min/kg)
O
52 Me- 6±2 70 222 ± 84 0.10 1.4 29 3
HO
53 O 3±1 80 149 ± 55 0.09 1.4 62 16
F
HO O 3±1 30 164 ± 42 0.06 2 39 15
54
HO
O O 7±2 150 579 ± 194 0.09 2.6 25 13
55 H2N
O O 11 ± 5 200 335 ± 87 -- 2.5 32 15
H
56 N
O
H 10 ± 4 100 161 ± 47 0.04 -- -- --
N
57
O
H2N
O
6±2 100 222 ± 81 0.03 3.4 12 43
58
O
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least
three determinations were made. b Kinase selectivity was determined using the Ambit Bioscience KINOMEscan technology, and is
expressed as S(90), the fraction of kinases inhibited >90% when screened at 1 μM across a panel of 256 kinases.
Gregory J. Wells, Ph.D.

39. Piperidine Analogs – Potencies, Selectivity, and Rat PK (cont.)
R7
N
N
N
N N
H Ar
O
IC50 (nM)a Rat PK iv data
Cmpd R7 Ar ALK ALK IR S(90)b T1/2 (h) CL Rat F%
Enzyme Cellular Enzyme (mL/min/kg)
H2N O
58 6±2 100 222 ± 81 0.03 3.4 12 43
O
O
59 H2N 12 ± 4 500 2967 ± 184 -- -- -- --
O O
60 H2N CN 19 ± 6 250 747 ± 274 -- -- -- --
O
O
61 H2N 1060 ± 460 -- > 10,000 -- -- -- --
N
O
62 H2N O 9±4 150 410 ± 141 0.06 -- -- --
O
N
O
H2N
63 N S 15 ± 4 80 552 ± 62 0.13 -- -- --
O
O
a
IC50 values are reported as the average of at least two separate determinations; standard deviations are indicated where at least
three
determinations were made. bKinase selectivity was determined using the Ambit Bioscience KINOMEscan technology, and is
expressed as S(90), the fraction of kinases inhibited >90% when screened at 1 μM across a panel of 256 kinases.
Gregory J. Wells, Ph.D.

40. Piperidine Analogs – Potency and Rat PK for 5-Substituted Analogs
H2N
N R
O 5
N
N O
N N N S
H
O
O
IC50 (nM)a Liver microsome t1/2 (min)b
Cmpd R ALK enzyme ALK Cell M R Mo H Rat F%
64 H 15 ± 4 80 29 <5 <5 <5 --
65 OH 11 ± 1 -- <5 <5 <5 <5 --
66 Me 6±2 70 > 40 31 6 18 24
67 Cl 5±1 70 > 40 > 40 15 37 41
IC50 values are reported as the average of at least two separate determinations; standard deviations are
indicated where at least three determinations were made. bM = mouse; R = rat; Mo = monkey; H = human.
Mesaros, Thieu, Wells; JMC, 2012, 115
Gregory J. Wells, Ph.D.

41. Pharmacokinetic Parameters of 58 in S-D Rats and Scid Mice
H2N
N
O
N
N
N N O
H
O
Gregory J. Wells, Ph.D.

42. PK/PD for 58 in Sup-M2 Xenografts in Scid Mice
H2N
N
O
N
N
N N O
H
O
Inhibition of NPM-ALK, single dose Compound levels in plasma and tumor
30mg/kg sol's in PEG400
Gregory J. Wells, Ph.D.

43. Antitumor Efficacy of 58 in Scid Mice
H2N
N
O
N
N
N N O
H
O
Gregory J. Wells, Ph.D.

44. Pharmacological Milestones in the Development
of Pyrrolotriazines as ALK Inhibitors
Efficacy Metabolic
Potency Selectivity
(TGI) Stability
N
N
N
? ?
N
N
H
N
 
HO
N
N
N
O
N
H
N
N
N SO CH
2 3
   
H2N
N
O
N
N
N
N
O
   
H
O
Gregory J. Wells, Ph.D.

45. Acknowledgements
Chemistry Biology Oncology
Henry Breslin Jay Theroff Lisa Aimone Hong Chang
James Diebold Gregory Ott Mark Albom Mangeng Cheng
Arup Ghose Jonathan Parrish Thelma Angeles Lihui Lu
Diane Gingrich Rabi Tripathy Laura Gwinn Matt Quail
Robert Hudkins Ted Underiner Beverly Holskin Wendy Wan
Joseph Lisko Jason Wagner Zeqi Huang Ashley Wohler
Robert McHugh Linda Weinberg Kristen Murray Bruce Ruggeri
Eugen Mesaros Gregory Wells Damaris Steele
Karen Milkiewicz Craig Zificsak Kelli Zeigler DDS/Analytical
Tho Thieu Bruce Dorsey Sherri Meyer
Mark Ator Joe Herman
Deborah Luciani
Kevin Wells-Knecht
Mehran Yazdanian
Gregory J. Wells, Ph.D.

46. Supplementary slides
Gregory J. Wells, Ph.D.

47. ERK Pathway
Orton, Biochem J. 2005, v.392(Pt. 2), 249.
Gregory J. Wells, Ph.D.

48. MAPK/ERK Pathway (alternate view)
Gregory J. Wells, Ph.D.

49. Comparison of Crystal Structure of NVP-TAE684 and
Docking of a 2,7-pyrrolo[2,1-f][1,2,4]triazine analog
Cl
N
N
HN N NH O
N
O S HN N O
O
O
N
N
NVP-TAE684
N
N
N
Gregory J. Wells, Ph.D.

50. Structure-Activity of Select Fused Pyrrolocarbazole Derivatives
H
N
O
R2 N
N
N
R1
ALK IC50 (nM)
CEP R1 R2 Enzyme Cell
11719 iBu HOCH2CC- 1.1 10 to 30
11834 iBu HON=C- 3.1 10 to 30
14083 nPr Ph(Me)NC(O)NH- 1.6 10 to 30
14513 iBu 3.7 10 to 30
11673 CH2cPr 428 >3000
11988 Et iBuON=C(Me)- 21838 >30000
Gregory J. Wells, Ph.D.

51. ALK Activity Is Essential for the
Proliferation of ALK+ ALCL Cells in Culture
H
N O
H
N O N
O N
(Me)2N
N
― 30 100 300 ― 30 100 300
(nM) (nM)
120 p-NPM-ALK 120
p-NPM-ALK
Sudhl-1
Sup-M2
NPM-ALK
R e la tiv e liv in g c e lls
R e la t iv e liv in g c e lls
90 90 NPM-ALK
60 60
30 30
0 0
1 10 100 1000 1 10 100 1000
CEP-14513 (nM) CEP-14513 (nM)
― 30 100 300
(nM)
p-NPM-ALK
120
120 K562 cells
Karpas-299
NPM-ALK
R e la tiv e liv in g c e lls
R e la t iv e liv in g c e lls
90 90
60 60
30 30
0 0
1 10 100 1000 1 10 100 1000
CEP-14513 (nM) CEP-14513 (nM)
Gregory J. Wells, Ph.D.