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Challenges and Perspectives on the Development of Small-molecule EGFR Inhibitors against T790M-mediated Resistance in Non-small-cell Lung Cancer Zhendong Song, Yang Ge, Changyuan Wang, Shanshan Huang, Xiaohong Shu, Kexin Liu, Youwen Zhou, and xiaodong ma J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00840 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 16, 2016

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Journal of Medicinal Chemistry

Challenges and Perspectives on the Development of Small-molecule EGFR

Inhibitors

against

T790M-mediated

Resistance

in

Non-small-cell Lung Cancer Zhendong Song,a Yang Ge,a Changyuan Wang,a Shanshan Huang,a Xiaohong Shu,a Kexin Liu,a Youwen Zhoub and Xiaodong Maa,∗

a

College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.

b

Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC,

V5Z 4E8, Canada.

ABSTRACT: Due to the development of drug-resistance mutations, particularly the “gatekeeper” threonine790-to-methionine790 (T790M) mutation in the ATP-binding pocket of the epidermal growth factor receptor (EGFR), the current generation of EGFR tyrosine kinase inhibitors lost their clinical efficacy. Recently, a large number of small-molecule inhibitors with striking inhibitory potency against EGFR mutants with the T790M change have been identified. In particular, the inhibitors rociletinib and osimertinib, which can selectively target both sensitizing mutations and the T790M resistance, while sparing the wild-type (WT) form of the receptor, have been designated as breakthrough therapies in the treatment of mutant non-small-cell lung cancer (NSCLC) by the US FDA in 2014. We hope that this perspective on the small-molecule EGFR T790M inhibitors, along with their discovery strategies, will assist in the design of future T790M-containing EGFR inhibitors with high levels of selectivity over WT EGFR, broad kinase selectivity, and desirable physicochemical properties. Key words: NSCLC; EGFR; T790M; Cancer; Inhibitor 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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INTRODUCTION Lung cancer remains the most common cancer worldwide, with approximately 1.8 million new cases annually. It is also the most common cause of cancer-related deaths worldwide,

with

a

5-year

survival

rate

of

less

than

20%

(http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx). In particular, non-small-cell lung cancer (NSCLC), the most common lung cancer (accounting for almost 85%), has a 5-year survival rate of less than 15%.1–3 As the most valuable therapeutic targets for NSCLC, epidermal growth factor receptors (EGFRs) play an indispensable role in cancer cell proliferation, survival, adhesion, migration, and differentiation.4–8 Gefitinib9 and erlotinib10,11 were the first generation of EGFR inhibitors approved for the treatment of NSCLC by the US FDA in 2002 and 2004, respectively. By inhibiting the kinase domain of EGFR and disrupting the oncogenic cell signaling, both drugs achieved remarkable benefits in those patients carrying the so-called “sensitizing mutations” such as L858R and the exon-19 deletion.12,13 However, in about 50% of patients with EGFR alteration, a single amino acid threonine790-to-methionine790 mutation (T790M) often occurs, greatly limiting the efficacy of these drugs in clinical use.14–17 In the mutated methionine residue (Figure 1), its bulkier side chain is thought to sterically impede the binding of these reversible inhibitors and disrupt the formation of a crucial water-mediated hydrogen bond between the inhibitor and the T790M of EGFR.18–20 In another proposed mechanism, the T790M-containing mutants have an increased affinity for ATP, resulting in reduced cellular potency for the ATP-competitive inhibitors. Second-generation EGFR inhibitors containing an electrophilic Michael addition receptor moiety that can covalently alkylate the conserved cysteine residue (Cys797) close to the ATP binding site of the EGFR were developed to overcome the resistance caused by the T790M.21 Although the formation 2


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of the covalent bond greatly facilitates the occupation of the ATP site by these irreversible inhibitors, thus achieving higher selectivity for the EGFR-family tyrosine kinase relative to the reversible inhibitors, their clinical efficacy still has been limited by associated skin rash and gastrointestinal toxicity, possibly due to their activity against the wild-type (WT) EGFR.22–24

a

b M790

T790

M793

M793

C797

C797

Figure 1. (a) the crystallographically determined binding of erlotinib to wild-type (WT) EGFR. (b) the T790M mutation leads to steric hindrance of erlotinib binding owing to the presence of the bulkier methionine side chain (magenta) in the ATP-kinase-binding pocket.20

O N O

N O

N

HN

N

HN O HN F3C

N

O N

NH

N

N

NH N

2 (osimertinib)

1 (rociletinib)

Figure 2. Novel inhibitors rociletinib and osimertinib for the treatment of EGFR T790M mutation-related NSCLC. 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Recently, a family of small-molecule inhibitors, including the novel clinical candidates 1 (rociletinib, also known as CO-1686, AVL-301, CNX-419) 25–30, and 2 (osimertinib, also known as AZD9291)31–35 have been developed to overcome the EGFR T790M mutation-related resistance (Figure 2). These inhibitors not only effectively target the sensitizing mutations and the T790M resistance mutation, but also have better selectivity for T790M-containing EGFR mutants over WT EGFR, whose inhibition can lead to dose-limiting toxicities including skin rash and diarrhea.36,37 For their excellent biological properties, compounds 1 and 2 were designated breakthrough therapies in the treatment of mutant NSCLC by the US FDA in 2014, and the phase III clinical trials are underway now to assess their safety and efficacy.38,39

Figure 3. (a) X-ray structure of inhibitor 340 complexed with EGFR T790M (PDB code：3IKA). (b) “U-shaped” binding conformation of inhibitor 3.41

The co-crystal structure (PDB code: 3IKA) of the EGFR T790M kinase domain in complex with 3 (WZ4002)40 indicated that a “U-shaped” configuration of a 4


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pyrimidine core, together with an aniline ring bearing a hydrophilic group and an acrylamide moiety, is propitious to bind with EGFR target (Figure 3).41 A great number of small molecules were designed and synthesized as selective EGFR inhibitors against the EGFR T790M mutation based on this binding mechanism.42,43 Focusing on these molecules and reviewing their design strategies along with their typical anti-cancer activity will provide valuable clues for the further development of more active EGFR T790M inhibitors.

DIPHENYLPYRIMIDINES WZ4002 and Its Analogues Cl

N

Met790

N

HN

O

Val726

O

Leu718 Leu792

NH Met793

N

O

N

WZ4002 Pro794

3 (WZ4002) IC50 (H1975 cellDM) = 47 nM (Ba/F3 cellDM) = 8 nM (Ba/F3 cellAM) = 3 nM (Ba/F3 cellWT) = 113 nM (PC9 GRdel E746_A750/T790M) = 14 nM Selectivity (DM/WT) = 14

Gly796

Cys797

Figure 4. The structure of inhibitor 3 and its binding sites with EGFR T790M (PDB code： 3IKA).40 DM = L858R/T790M double mutations, WT = wild-type, AM = exon 19 del E746_A750 activating mutation.

5



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Cl

N HN

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N

Cl

N HN

O

N

S

NH

NH N

N

O

O

N

N

5 (WZ8040)

4 (WZ3146)

IC50 (H1975 cellDM) = 9 nM (Ba/F3 cellDM) = 9 nM (Ba/F3 cellAM) = 2 nM (Ba/F3 cellWT) = 32 nM Selectivity (DM/WT) = 4

IC50 (H1975 cellDM) = 29 nM (Ba/F3 cellDM) = 5 nM (Ba/F3 cellAM) = 2 nM (Ba/F3 cellWT) = 24 nM Selectivity (DM/WT) = 5

Figure 5. Structures of inhibitors 4,5 and their activities against NSCLC cell lines.

By screening an irreversible kinase inhibitor library specifically against the T790M-mutated EGFR, in 2009, Jänne et al. at the Dana-Farber Cancer Institute, together with the scientists at Gray’s lab, successfully identified a set of effective pyrimidine-based EGFR T790M inhibitors (Figure 5), including compounds 3, 4 (WZ3146)40, and 5 (WZ8040)40. In vitro, all these compounds were able to inhibit the

growth

of

both

gefitinib-resistant

(H1975

cellL858R/T790M(DM)

cellL858R/T790M(DM)) and gefitinib-sensitive (Ba/F3 celldel

E746_A750(AM)

and

Ba/F3

) cell lines at

nanomolar concentrations. Compared with gefitinib, they were 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against WT EGFR. Among them, compound 3 was least potent against WT EGFR (IC50 (Ba/F3 cell) = 113 nM), but was effective against EGFR T790M (IC50

(Ba/F3 cell)

= 8 nM). In vivo,

compound 3 is also very effective in mouse lung cancer models harboring either double mutant EGFR L858R/T790M or EGFR del E746_A750/T790M. These results 6


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indicated that these mutant-selective irreversible EGFR kinase inhibitors are remarkably more effective and better tolerated than quinazoline-based first-generation and second-generation EGFR inhibitors. In addition, Gray et al. first determined the crystal structure of compound 3 bound to EGFR T790M (PDB code: 3IKA), revealing a structural basis for the increased potency and mutant selectivity of these agents (Figure 4). Inhibitor 3 represented a major breakthrough in the search for effective inhibitors against T790M-mediated resistance in NSCLC. Unfortunately, it remained an experimental inhibitor and did not enter clinical studies. Rociletinib and Its Analogues

CF3

N HN

N

NH

O NH N

EGFR WT EGFR

a 10,000 GI50 nmol/L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O

1,000 EGFR mutated 100

N 10

O 1 (rociletinib) Selectivity (DM/WT) = 22

5 9 8 R 31 7 99 97 82 PC -EP A4 I-12 I-H35 H1 C 2 C C N N HC C8 HC

7



NCI-H1975 b 2,000

Vehicle Rociletinib (10 mg/kg) Rociletinib (30 mg/kg)

Afatinib (20 mg/kg) Erlotinib (100 mg/kg)

Rociletinib (100 mg/kg)

1,500 Tumor volume Mean mm3 ± SEM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1,000

500

0

5

10

15

Figure 6. Structures of rociletinib and its anticancer activities in cells (a) and in a xenograft nude mouse model (b).

Inhibitor 1, derived from compound 3, was first discovered by Avila Therapeutics, Inc. (“Avila”), a biotechnology company developing targeted covalent drugs that treat diseases through protein silencing. In May 2010, the Clovis Oncology, Inc. (“Clovis”) entered into a worldwide license agreement with Avila to discover, develop and commercialize a covalent inhibitor of mutant forms of the EGFR gene product. Nevertheless, in March 2012, Avila was acquired by Celgene Corporation (“Celgene”), so Clovis entered into an exclusive license agreement with Gatekeeper Inc. to acquire exclusive rights under patent applications associated with mutant EGFR inhibitors and methods of treatment. It is currently being evaluated in phase Ⅲ clinical trials in EGFR-mutant NSCLC (ClinicalTrials.gov, NCT02322281). It is the first agent in clinical development for the treatment of T790M-positive NSCLC, offering potentially potent inhibition of mutant EGFR, while avoiding the on-target toxicity produced by the inhibition of the WT EGFR (Figure 6).

8


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N HN

Cl

N

Cl HN N

N N

O

N N

O

N

NH NH

O

O N 7 IC50(H1975 CellDM) = 96 nM (A431 CellWT) = 2300 nM Selectivity (DM/WT) = 240 LogD7.4 = 3.6

6 IC50(H1975 CellDM) = 33 nM (A431 CellWT) = 1280 nM Selectivity (DM/WT) = 390 LogD7.4 > 4.3 Cl

N HN

N

HN

N N

O

N

NH

O

NH N

Cl

N

NH O

N

N

O

N 9 IC50(H1975 CellDM) = 6 nM (A431 CellWT) = 770 nM Selectivity (DM/WT) = 140 LogD7.4 = 3.7

8 IC50(H1975 CellDM) = 19 nM (A431 CellWT) = 1200 nM Selectivity (DM/WT) = 620 LogD7.4 = 4.1

Figure 7. Structures and biological properties of inhibitors 6−9.

Osimertinib and Its Analogues As previously reported,44 the inhibition of WT EGFR is not thought to contribute to the clinical efficacy of EGFR inhibitors, but is believed to be responsible for the side-effects of skin rash and diarrhea. With the aim of identifying a double-mutant-selective template, Ward et al. synthesized a large number of molecules targeting the double mutant form of EGFR tyrosine kinase. In 2013, their efforts in this area culminated in the identification of a pyrollopyridine derivative 645, which exhibits not only reasonable potency in double- and activating-mutant cell 9



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assays (IC50 of 33 nM and 150 nM, respectively), but also an improved margin to WT EGFR (390-fold higher). However, the high lipophilicity (Log D7.4 > 4.3), which often translated into poor physicochemical properties and pharmacokinetics (PK), hinders its further development. In contrast, although compound 745 (IC50 = 96 nM), which bears a basic acrylamide-based side chain, showed a small decrease in activity of 3-fold compared with the natural equivalent (analogue 6), it did show a marked reduction in LogD7.4 (value of 3.6). Based on this scaffold, a set of small molecules featuring a piperazine substituent on the para position of the aniline ring were prepared. The representative inhibitors within this series were the analogues 845 and 945, which were more potent than 7 in double mutant cell assays (19 nM and 6 nM, respectively). The particular compound 8 still exhibited a good selectivity for the double mutant over the WT of 620-fold. Moreover, in contrast with 945, not only did it offer modest exposure when dosed orally at 50 mg/kg in SCID mice, but also has good aqueous solubility (LogD7.4 = 4.1) (Figure 7). The overall potency, selectivity, and physical property profiles of analogue 8 make it a good starting point.

Table 1. Antiproliferative activities of 10a−f in WT and mutant NSCLC cell lines Cl

N HN

N

N N

O NH R

O 10a－f

Compd. R IC50(DM cell, nM)a

10a

10b

10c

N N

N

N O

16

10d

NH2

N

N

NH N

O

10e

N

N

N

10f N

N

O

19

31

2

10


0.6

4

N

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IC50(WT cell, nM)b

10000

1600

6060

357

145

938

IC50(AM cell, nM)c

73

28

211

16

2

21

LogD7.4

2.6

--

2.2

3.1

2.8

3.3

Selectivity(DM/WT)

625

84

195

179

242

235

a

double mutations (DM) cell = H1975 cell harboring L858R/T790M double mutations. bwild-type

(WT) cell = LoVo cell. cactivating mutation (AM) cell = PC9 cell harboring activating mutant exon 19 del E746_A750.

Table 2. Antiproliferative activities of analogues 11a−e in WT and mutant NSCLC cell lines R2

N

N

N

HN

HN

1 N R

O

N

NH

NH N

N

O

N

O N

N 11a－e

O

2 (osimertinib)

2

11a

11b

11c

11d

11e

R1/R2

Me/H

Me/CN

Me/Cl

H/Cl

Me/Me

H/H

IC50(DM cell, nM)

15

0.9

2

0.2

1

2

IC50(WT cell, nM)

480

46

58

11

71

33

IC50(AM cell, nM)

17

1

2

0.6

2

2

LogD7.4

3.4

2.7

3.3

3.3

2.8

2.9

Selectivity(DM/WT)

32

51

29

55

71

15.5

11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tumor growth inhibition (%)


134%

140 120

H1975L858R/T790M A431WT

Osimertinib

160 148%

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Gefitinib 119%

120% 102% 79%

80

65%

60

62%

40 8%

20 10

100 2.5 5 Dose in mg/kg/day for 7 days

6.25

Figure 8. In vivo efficacy profiles (% tumor growth inhibition) of osimertinib and gefitinib.

Table 3. In vitro pharmacokinetic parameters for typical inhibitors 2, 10d−f and 11a−e 2

10d

10e

10f

11a

11b

11c

11d

11e

45

22

60

59

62

27

12

--

--

45

13

10

24

6

7

1

--

--

0.28/0

1.3/0.

3.3/1.

1.4/0.38

--

--

--

rat HW iv Cl (mL min-1 kg-1) rat HW po F (%) Mouse AUC/Cmax (µM·h/µM),10mg/

0.203/0 0/0

.11

31

2

.047

kg po

In 2014, Finlay et al. further developed the hit compound 835, while performing a body of work (analogues 10a−f35, Table 1) focused on preparing amides of the distal piperazine nitrogen of 8. Apparently, compared with analogues 10d−f, inhibitors 10a−c showed more reasonable potency profiles, particularly against the activating mutant cell lines. In additional compounds 11a−e35 (Table 2), where the basic side chain was replaced with amines such as those in 10d and 10f, it can be seen that incorporation of the newly identified basic side chains conferred high cellular potency to them. With these highly encouraging in vitro data in hand, the PK properties of 12


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typical compounds were investigated (Table 3). Notably, compound 2 demonstrated improved rat PK, particularly exhibiting the highest value (F) of 45%. In addition, in in vivo experiments using mouse xenograft models, inhibitor 2 produced more durable and more profound responses in EGFR NSCLC tumors than the first-generation EGFR tyrosine kinase inhibitors (TKIs) (Figure 8). Eventually, this contribution resulted in the identification of a novel clinical candidate, namely osimertinib, which is highly potent and selective towards the mutated forms of EGFR (DM EGFR IC50 = 15 nM, AM EGFR IC50 = 17 nM, WT EGFR IC50 = 480 nM). As a third-generation EGFR inhibitor, compound 2 dramatically reduces the side-effects encountered with currently available medicines, and shows promise for patients with advanced lung cancers. Due to these excellent biological properties, inhibitor 2 has advanced into phase III trails (ClinicalTrials.gov, NCT02474355), and it is widely believed that it will be approved for the market in the near future. Phenylsulfonylfuroxan-based Anilinopyrimidines

13



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EGFR inhibitor backbone Cl N NO-donor O OO N+ N SO2Ph

HN

N

OO N+ O N SO2Ph

O

HN

NH N

O

O

O

O

Ο

Ο 12

13 DM

DM

IC50(EGFR Enzyme ) = 47 nM (EGFR EnzymeWT) = 292 nM (H1975 cellDM) = 29 nM Selectivity (DM/WT) = 6

IC50(EGFR Enzyme ) = 16 nM (EGFR EnzymeWT) = 52 nM (H1975 cellDM) = 43 nM Selectivity (DM/WT) = 3

Cl

N HN

N

O

N

N linker

O

N

O

NH N

Cl

N

ON+ O N

PhO2S

Ο O

O

SO2Ph N+ ON O

N

Cl

N

O HN

N

N

O

O

Ο

N R

N H

N

14a R = Me IC50(EGFR EnzymeDM) = 19 nM (EGFR EnzymeWT) = 130 nM (H1975 cellDM) = 52 nM Selectivity (DM/WT) = 7

N

N 15 IC50(EGFR EnzymeDM) = 263 nM (EGFR EnzymeWT) = 201 nM (H1975 cellDM) = 108 nM

14b R = CH2=CHCO IC50(EGFR EnzymeDM) = 45 nM (EGFR EnzymeWT) = 350 nM (H1975 cellDM) = 55 nM Selectivity (DM/WT) = 8

Figure 9. (Phenylsulfonyl)furoxan-based NO-releasing compounds 12−15 as potent and selective EGFR L858R/T790M inhibitors.

14


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3,000

Vehicle WZ4002 (8 mg/kg)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Gefitinib (16 mg/kg)

2,000

13 (14 mg/kg)

1,500 1,000 500

0

8 10 2 4 6 12 Days post initial treatment (d)

14

Figure 10. In vivo antitumor effect of compound 13 in a human NSCLC (H1975) xenograft nude mouse model.

Taking into consideration that the high levels of nitric oxide (NO) generated from NO donors can not only induce apoptosis and inhibit metastasis of tumor cells but also sensitize tumor cells to chemotherapy, radiation, and immunotherapy in vitro and in vivo,46,47 Zhang et al. used a hybrid strategy and designed a variety of (phenylsulfonyl)furoxan-based NO-releasing compounds48 that exhibit selective antitumor activity (1249 and 1349, Figure 9). The enzymatic assays showed that compound 13 had a strong and selective EGFR L858R/T790M kinase inhibitory activity (IC50 = 47 nM). Although its potency against the mutant enzyme is 60-fold lower than that of 3 (IC50 = 8 nM), compound 13 still exhibited slightly enhanced antiproliferative eﬀects on the NSCLC cell lines HCC827 (IC50 = 7 nM) and H1975 (IC50 = 29 nM), which harbors an EGFR mutation. Further in vivo evaluation showed that compound 13 substantially inhibited tumor growth (60%) in an H1975 xenograft mouse model. Nevertheless, compared with 3, it is less powerful in vivo (Figure 10). This investigation also revealed that inhibitor 13 released high levels of NO in H1975 cells but not in normal human cells, and its activity was diminished by pretreatment 15



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with a NO scavenger. Moreover, compound 13 induced apoptosis of H1975 and HCC827 cells more strongly than compound 3. Together, these results suggest that analogue 13 is a good candidate to serve as an effective treatment of EGFR-mutated NSCLC patients. To search for more potent NO-donating EGFR inhibitors, Zhang et al. synthesized a series of phenylsulfonylfuroxan-based anilinopyrimidine derivatives in 2014 (Figure 9). Fortunately, two of the active analogues which were identified, namely 14a50 (IC50 = 52 nM) and 14b50 (IC50 = 55 nM), were comparable with 3 in inhibiting H1975 cell harboring the EGFR L858R/T790M mutation. Both 14a and 14b greatly inhibited EGFR activation and downstream signaling in H1975 cells. Analogue 14b, in particular, also produced high levels of NO in H1975 cells but not in normal human cells, and its antiproliferative activity was diminished by the NO scavenger hemoglobin. The strong antiproliferative activity of 14b may be attributed to the synergic effects of the high levels of NO production and the inhibition of EGFR and downstream signaling in the cancer cells. In 2014, Zhang et al. also reported another series of EGFR inhibitors containing 2-anilinopyrimidine as a scaffold and 3-aminopropamide as a “protected” warhead. Within this class of inhibitors, compound 1551 (Figure 9) displayed the strongest antiproliferative effects on both EGFR L858R/T790M kinase and H1975 cells, with IC50 values of 263 nM and 108 nM, respectively. Additionally, inhibitor 15 dose-dependently produced inhibitory effects on EGFR downstream signaling in H1975 cells. In fact, in the presence of arginine, the 3-aminopropamide group in this chemotype was converted to the active acrylamide present in inhibitor 3, and hence covalently binds to the Cys797 of the EGFR T790M mutant to exert its inhibitory activity. Overall, these findings, based on a hybrid strategy, provide a proof of 16


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principle of the feasibility of using NO-releasing designed compounds as potent and selective inhibitors of EGFR for treatment of NSCLC.

HETEROCYCLOPYRIMIDINES Five-membered Heterocyclopyrimidines

N

N HN

N

N H

HN

N

N

N

N

16 IC50(EGFR EnzymeDM) = 0.978 µM IC50(H1975 cellDM) = 18.06 µM

N

N

NH

NH N

N R

17a R = cyclopentyl IC50(H1975 cellDM) = 0.36 µM 17b R = cyclohexyl IC50(H1975 cellDM) = 0.39 µM

Figure 11.Structures of inhibitors 17a,b and their activities against the EGFR L858R/T790M.

Using a virtual screening technology, in 2012, Yang et al. found two very active compounds, namely 17a52 and 17b52 (Figure 11), which exhibited significant in vitro antitumor potency against the HCC827 and H1975 NSCLC cell lines. Further evaluation for potency and selectivity in enzymatic assays and in vivo anti-NSCLC studies indicated that compound 17a not only possesses high potency against both EGFR-activating and -resistant mutations, but also has a good spectrum of kinase selectivity across the kinome. In vivo antitumor activity assays showed that an once-daily oral dose of compound 17a at 5 mg/kg for 15 days led to complete tumor regression in the HCC827 xenograft model and at 50 mg/kg exhibited substantial tumor inhibition in the H1975 xenograft model. Furthermore, the good PK properties of inhibitor 17a made it worthy of further development. 17



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Page 18 of 55

HN N N HN

N N

O

H N

N O NH

N

N

O

O

NH

N 19 18 AM IC50(PC9 CellAM) = 300 nM IC50(PC9 Cell ) = 500 nM (PC9 GR CellE746_A750/T790M) = 600 nM (PC9 GR CellE746_A750/T790M) = 500 nM

Figure 12. Structures of inhibitors 18,19 and their cellular antiproliferative activities.

In 2011, Gray et al. extensively modified the diphenylpyrimidine scaffold, and prepared several classes of covalent EGFR kinase inhibitors (Figure 12) through a systematic structure-activity relationship (SAR) study based on a mutant EGFR-dependent cellular proliferation assays. For instance, the analogue 1853 with considerable potency (IC50 = 600 nM) against the PC9 growth-resistant GR cells (EGFR E746_A750/T790M) was obtained by replacing the original pyrimidine core with a purine template. Another representative analogue, namely 1953, characterized by a pyrrolopyrimidine core, was also effective, with an IC50 value of 500 nM against PC9 GR cells. Overall, this contribution reveals two novel scaffolds which could be used to discover effective EGFR T790M inhibitors.

18


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N N

N N N

N

F N

N N

N F

N

NH

HN

N N

O

NH O

N N

O

N

N

O F

NH HN

HN

N

N

N N

O N

20 21 22 IC50(H1975 Cell) = 6 nM IC50(H1975 CellDM) = 7 nM IC50(H1975 Cell) = 6 nM (H3255 Cell) = 2 nM (H3255 Cell) = 5 nM (H3255 CellAM) = 2 nM (A549 Cell) = 194 nM (A549 Cell) = 142 nM (A549 CellWT and k-Ras) = 178 nM Selectivity (DM/WT) = 32 Selectivity (DM/WT) = 24 Selectivity (DM/WT) = 25

Figure 13. Structures of purine derivatives 20−22 and their activities against NSCLC cell lines.

By designing and synthesizing a large number of 2,6-substituted purine analogues, Pfizer′s researchers successfully discovered three molecules (20−2254,55, Figure 13) with single digit nanomolar activity against both the WT and mutant NSCLC cell lines. Importantly, all of them also displayed 24 to 32-fold selectivity for the EGFR L858R/T790M mutant over WT EGFR. However, their detailed biological properties and SAR information were not described. Six-membered Heterocyclopyrimidines Table 4. 2-Oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives 23a−h and their biological activities

Cl

N

N

Cyclization HN

N

O

HN

O

2′

N

N

R2 O

1′

R1

NH N

N

3′

O

N

NH

4′

N

O

N 3

23a－h 19



Compd

R1

R2

Kinase inhibition

Antiproliferation

(IC50, nM)

(IC50, nM)

T790 WT

H1975 DM

A431 cell

M

cell

23a

2′-MeO

Me

1.16

6.56

3.14

750

47

23b

H

Me

0.43

1.66

1.19

550

45

23c

3′-MeO

Me

0.41

1.67

2.04

360

24

23d

2′-MeO

Ph

0.30

0.51

0.98

110

16

23e

2′-MeO

2-naphthyl

0.80

1.34

2.44

570

30

23f

2′-MeO

4-phenoxyphenyl

1.52

2.03

3.57

1200

48

23g

2′-MeO

Bn

0.29

0.67

0.93

170

14

23h

2′-MeO

4-benzoxyphenyl

1.67

2.43

6.31

1150

33

6.18

3.77

1.88

1390

48

3

2400

Vehicle Gefitinib (50 mg/kg) 23g (20 mg/kg) 23g (10 mg/kg)

2100 1800 Tumor volume Mean mm3 ± SEM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 55

1500 1200 900 600 300 0

2

4 6 10 8 12 Days post initial treatment (d)

14

Figure 14. In vivo antitumor effect of compound 23g in a human NSCLC (H1975) xenograft nude mouse model.

Applying the conformational constraint strategy, Ding et al. designed and synthesized a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives 20


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(23a−h41, Table 4) as potential EGFR inhibitors in 2012. Among them, analogues 23d and 23g were the most effective inhibitors, with IC50 values in the subnanomolar range against all types of EGFR kinase, including the clinical resistance-related EGFR T790M mutant. Western blot analysis of the activation of EGFR and downstream signaling in cancer cells harboring different mutants of EGFR indicated that compounds 23d and 23g inhibited the phosphorylation of EGFR in a dose-dependent manner in both the H1975 NSCLC cells bearing EGFR L858R-T790M and HCC827 cells harboring EGFR del E746_A750. In addition, cell-based assays indicated that both of them were also able to inhibit gefitinib-sensitive HCC827 cells and gefitinib-resistant H1975 cells with a better potency than that of inhibitor 3. Importantly, 23d and 23g only showed moderate or minimal cytotoxicity to normal HL-7702 and HLF-1 cells, indicating that they might possess a high safety index. An additional in vivo antitumor efficacy study by orally dosing at 30 mg/kg/day demonstrated that compound 23g effectively inhibited the tumor growth in an EGFR L858R/T790M-driven human NSCLC xenograft mouse model of H1975 (Figure 14). Taken together, these studies provide a validated hit with a different chemical scaffold for further development of EGFR inhibitors to overcome the EGFR T790M mutation-related clinical resistance to gefitinib or erlotinib.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N

Page 22 of 55

N

HN

N

N

O

O NH N

O

N

23g Knowledge-based hybrid & Conformational constrain O

O N HN R

N N

N N

N

1

HN

N

N N

NH R

O

N

N

N

NH

NH N

N

O

O

2

HN

N

N

O

N

O

O N

26a R1 = MeO; N R2 = 4-Methylpiperazin-1-yl 24 26b R1 = MeO; R2 = Morpholino 26c R1 = MeO; R2 = 4-(Methylamino)piperazin-1-yl 26d R1 = EtO; R2 = 4-Methylpiperazin-1-yl

25

Figure 15. Compounds 24−26 bearing the pyrimido[4,5-d]pyrimidin-4(1H)-one scaffold as potential EGFR L858R/T790M inhibitors.

Table 5. Biological activities of pyrimido[4,5-d]pyrimidin-4(1H)-one derivatives 24−26

EGFR binding affinity (Kd, nM)

Antiproliferation (IC50, µM)

Compd. WT

Selectivity

A431

H1975

Selectivity

(WT:DM)

cell

cell

(DM:WT)

DM

24

110

1.1

100

3.567

0.307

12

25

17

0.88

20

1.536

0.648

3

26a

140

1.8

78

2.983

0.143

21

26b

950

6.1

156

>30

0.476

63

26c

310

2.6

119

14.530

0.086

169

22


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26d

420

3.1

136

5.254

0.396

13

3

1.2

0.3

4

1.042

0.055

19

With the aim of searching for inhibitors that selectively target EGFR T790M double mutants, in 2013, Ding et al. designed compounds 2456 and 2556 (Figure 15) featuring a pyrimido[4,5-d]pyrimidin-4(1H)-one core to increase the selectivity. As seen in Table 5, both inhibitors 24 and 25 bound effectively to EGFR L858R/T790M with Kd values of 1.1 nM and 0.88 nM, respectively. Additionally, they moderately inhibited the T790M-mutated H1975 cells with IC50 values of 0.307 nM and 0.648 nM, respectively. Nevertheless, the two inhibitors only exhibited 3- to 12-fold selectivity towards EGFR L858R/T790M mutants over WT EGFR. Using conformational constraints, thereby to improve ligand selectivity for a molecular target, a chemotype of pyrimido[4,5-d]pyrimidin-4(1H)-one based series of EGFR L858R/T790M inhibitors (26a–d)56, with higher selectivity than the WT kinase, were eventually discovered. The four rigid compounds 26a–d not only bind effectively to EGFR L858R/T790M, but also strongly repress the growth of the mutant H1975 cells at low-nanomolar concentrations. As the most active inhibitor, 26c was able to inhibit the growth of H1975 cells within 0.086 µM concentrations, along with 169-fold less potency against NSCLC cells with WT EGFR. Compared with pyrimido[4,5-d]pyrimidin-4(1H)-one-based

inhibitor

26c

displayed

3, the superior

selectivity over WT EGFR, and may serve as an attractive hit for future development.

23



N HN

N N

N

N O

HN

N N

N

O

O NH N

N

R

O N

O

N

N

23g 27a R = Cl IC50(EGFR EnzymeDM) = 36 nM Aqueous thermodynamic (H1975 cellDM) = 89 nM solubility (pH6.8) = 4 µg/mL Solubility (pH6.8) = 215 µg/mL 27b R = Me IC50(EGFR EnzymeDM) = 9 nM (H1975 cellDM) = 47 nM Solubility (pH6.8) = 1265 µg/mL

Figure 16. Compounds 27a,b bearing an 2-Oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl scaffold as potential EGFR L858R/T790M inhibitors.

2,000 Vehicle 27b (10 mg/kg)


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Page 24 of 55

27b (30 mg/kg)

1000 500

0

2

10 12 4 6 8 Days post initial treatment (d)

14

Figure 17. Antitumor efficacy of compound 27b in a human NSCLC (H1975) xenograft nude mouse model.

With the availability of these effective inhibitors, Ding et al. further modified compound 23g to improve its aqueous solubility and oral absorption properties. Fortunately,

structural

optimizations

of

the

2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl template led to the identification of one 24


Page 25 of 55

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of the most promising compounds 27b57 (Figure 16), which had an aqueous solubility of 1265 µg/mL, approximately 300-fold greater than that of the original lead compound 3 (4 µg/mL). Compound 27b potently inhibits the enzymatic function of EGFR L858R/T790M, with an IC50 value of 9 nM, and suppresses the proliferation of gefitinib-resistant H1975 human NSCLC cells, with an IC50 value of 47 nM. Additionally, this compound also exhibits good pharmacokinetic properties, with an oral bioavailability of 30%. Although 27b is about 9-fold less potent than 23g, its superior aqueous solubility contributes to its

promising antitumor efficacy without

obvious signs of toxicity following an EGFR L858R/T790M-driven human H1975 xenograft implantation in an in vivo mouse model (Figure 17). Altogether, these results

suggest

that

structure

optimization

of

the

2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl scaffold is an efficient strategy in the development of anti-gefitinib-resistant agents.

N

N HN

N

N

O

R1 NH R2

O

28a R1 = H, R2 = MeO IC50 (EGFR EnzymeDM) = 5.6 nM (EGFR EnzymeWT) = 13.8 nM (H1975 CellDM) = 63.8 nM 1 28b R = H, R2 = N-Methylpiperazine IC50 (EGFR EnzymeDM) = 0.7 nM (EGFR EnzymeWT) = 1.2 nM (H1975 CellDM) = 62.2 nM 1 28c R = MeO, R2 = N-Methylpiperazine IC50 (EGFR EnzymeDM) = 1.1 nM (EGFR EnzymeWT) = 3.8 nM (H1975 CellDM) = 59.0 nM

Figure 18. Structures of pteridin-7(8H)-one-based EGFR L858R/T790M inhibitors 28a–c.

In 2013, Li et al. discovered a novel pteridin-7(8H)-one EGFR-inhibitor scaffold, and successfully transformed it into irreversible inhibitors of the EGFR T790M mutant using a computational scaffold-hopping strategy. Within this class of inhibitors, various compounds (28b−c)58 exhibited competitive inhibitory enzymatic activity against both the WT and T790M/L858R EGFR mutants at subnanomolar 25



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Page 26 of 55

concentrations. Specifically, analogues 28b and 28c (Figure 18), have excellent enzyme inhibitory activity, with subnanomolar IC50 values for the WT (IC50 = 1.2 nM and 3.8 nM, respectively) and the T790M/L858R double mutant EGFRs (IC50 = 0.7 nM and 1.1 nM, respectively). In addition, the 28b and 28c analogues exhibited potent cellular antiproliferative activity against both the gefitinib-sensitive (IC50 = 8.6 nM and 4.0 nM, respectively) and -resistant cancer cell lines (IC50 = 63.8 nM and 59.0 nM, respectively). Furthermore, an in vivo antitumor efficacy study demonstrated that compound 28c significantly inhibited tumor growth and induced tumor stasis in an EGFR T790M/L858R-driven human NSCLC xenograft mouse model of H1975 at 20 mg/kg/day. This work indicated that the molecular 3D similarity can be remarkably useful in scaffold hopping with a carefully conceived design strategy.

Table 6. Typical oxopyrido[2,3-d]pyrimidine derivatives 29a−d and their biological properties R1 N N

HN

N

O

O NH 2

R

O 29a－d

Compd.

29a

R1/R2

H

IC50(H1975 cellDM, nM)

3

4

6

4

15

IC50(HCC827 cellAM, nM)

3

12

12

9

6

IC50(A431cellWT, nM)

50

386

521

1054

348

Selectivity(DM/WT)

17

97

87

264

23

29d N

N

Me

29c N

N

Me

29d N N

26


Me

2 N O

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More recently, Wurz`s group59 and Ding`s60 team almost at the same time independently identified the novel 7-oxopyrido[2,3-d]pyrimidinyl scaffold

as

covalent T790M mutant-selective EGFR inhibitors. The representative compounds 29a−d59 were described as presented in Table 6. As a hit, compound 29a exhibited single digit nanomolar activity in the EGFR mutant cell line, as well as moderate selectivity (DM/WT = 17) against the A431 cell line (IC50 = 50 nM). On the basis of this backbone, a methyl group was introduced in the 5-position of the pyridine ring, which resulted in a superior analogue 29b that was roughly equipotent to compound 1 in the H1975 (IC50 = 4 nM) and HCC827 cell lines (IC50 = 12 nM), and 97-fold more selective against the WT A431 cell line (IC50 = 386 nM). On the other hand, the analogues 29c and 29d, which were identified by opening the N-methylpiperazine ring, were equipotent to agent 29b in inhibiting mutant cell lines, and had higher selective (DM/WT = 264) against the A431 cell line (IC50 = 1054 nM). In an H1975 xenograft model, oral dosing of compound 29d at 30 mg/kg daily for 2 weeks resulted in significant tumor growth inhibition with no observed loss in body weight. Furthermore, in an exploratory 4-day rat toxicology study, no hepatobiliary toxicity was observed. Apparently, inhibitor 29d demonstrated more potential potency in suppressing the functions of EGFR T790M mutant than the inhibitor 2. Seven-membered Heterocyclopyrimidines

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 55

O Cl

N HN

N

N

N

Cyclization O

N

HN

O

N O

O NH N

R

NH N

O

N

O

N WZ4002

30a R = H IC50(EnzymeDM) = 9 nM (EnzymeWT) = 5 nM (H1975 cellDM) = 89 nM 60 30b R = F IC50(EnzymeDM) = 14 nM (EnzymeWT) = 6 nM (H1975 cellDM) = 69 nM

Figure 19. Analogues 30a,b featuring a 5,8-Dioxo-pyrimido[4,5-e][1,4]diazepine template as potential EGFR L858R/T790M inhibitors.

al.

identified

5,8-dioxo-pyrimido[4,5-e][1,4]diazepine

derivatives

In

2013,

Ding

et

a as

new potent

class EGFR

of

T790M

inhibitors (Figure 19). Within this series of EGFR inhibitors, most have a strong ability to inhibit a panel of EGFR mutants, including the clinical resistance related T790M mutants, at nanomolar concentrations. Two typical compounds in this class, namely 30a,b61, are able to repress the EGFR L858R/T790M kinase with IC50 values of 9 and 14 nM, respectively. Furthermore, both of them also displayed comparable potency (89 and 69 nM, respectively) to that of 3 in suppressing the proliferation of H1975 cells. Yet, 30a,b inhibitors scarcely show any particular selectivity for the EGFR L858R/T790M mutant over WT EGFR.

4-METHOXYPIPERIDINE-SUBSTITUTED PYRIMIDINES

28


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N N N

N N

N

NH

NH O

N

N

O

NH

N

Cl

O

31

32 DM

DM

EGFR Kiapp = 16 nM EGFRWT Kiapp = 367 nM Selectivity (DM/WT) = 22

EGFR Kiapp = 14 nM EGFRWT Kiapp = 743 nM Selectivity (DM/WT) = 53 LogD7.4 = 5.2

LogD7.4 = 4.1

N

O

N

N

N N

N

NH

N

O

N

N

N

NH O

N

H2N

NH N NH N

N N

N 33

N

N H

Cl

35

34

EGFRDM Kiapp = 33 nM EGFRWT Kiapp = 1100 nM Selectivity (DM/WT) = 35 LogD7.4 = 2.7

EGFRDM Kiapp = 12 nM EGFRDM Kiapp = 18 nM WT EGFR Kiapp = 317 nM EGFRWT Kiapp = 611 nM Selectivity (DM/WT) = 26 Selectivity (DM/WT) = 33 LogD7.4 = 2.4 LogD7.4 = 3.6 Figure 20. Diaminopyrimidine derivatives 31−3561 as potential EGFR L858R/T790M inhibitors.

Using a high-throughput biochemical screen, compound 3162, derived from a scaffold of the Jak2/Tyk2 inhibitor, was identified as a moderately potent inhibitor of both T790M mutant form of EGFR by Hanan et al. in 2014. Interestingly, compound 31 exhibited high level of selectivity for mutant EGFR T790M/L858R over WT EGFR (53 times). To advance the initial hit into a viable lead series, a number of parameters

were

optimized,

generating

a

new

series

of

noncovalent

diaminopyrimidine-based T790M EGFR inhibitors 32–3562 (Figure 20). Although the selectivity of these inhibitors was not superior to that of hit 32, the bicyclic scaffolds 32–35 enable a substantial improvement in decreasing lipophilicity with LogD values 29



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Page 30 of 55

ranging from 2.4 to 4.1, making them promising leads in the search for selective and noncovalent inhibitors of T790M-containing EGFR mutants. In addition, the X-ray crystal structures revealed two distinct binding models which enabled the design of potent T790M EGFR inhibitors with high levels of selectivity over the WT EGFR.

TETRAHYDROPYRIDOTHIENO-[2,3-d]PYRIMIDINES N

S

N NH

N

S

N 36 HO N

Cys797 addition O

O

N HO

N NH

38 NH

IC50(Enzyme assayDM) = 100 nM

37

Figure 21. Discovery of tetrahydropyridothieno-[2,3-d]pyrimidine derivative 38 as a potential EGFR L858R/T790M inhibitor.

Applying a knowledge-based design approach, in 2010 Hsieh et al. discovered a novel tetrahydropyridothieno-[2,3-d] pyrimidine scaffold 3863 as an EGFR kinase inhibitor. The development of inhibitor 38 was achieved by designing in an unusual S-chiral 2-phenyl-2-aminoethanol side chain (analogue 3763) identified from a counter-screening approach and then introducing a Michael acceptor group into the tricyclic core through a diversity point “NH” in hit 3663 (Figure 21). Inhibitor 38 showed more than 300-fold enhancement of the EGFR WT kinase inhibition along with more than 3000-fold enhancement of the HCC827 antiproliferative activity compared to hit 36. In addition, compound 38 was also able to inhibit the 30


Page 31 of 55

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gefitinib-resistant double mutant EGFR T790M/L858R kinase at nanomolar concentrations (IC50 = 100 nM). Even though the potency of inhibitor 38 in inhibiting mutant NSCLC cells was not reported, this compound provides a new scaffold warranting further modification.

4-ANILINOQUINAZOLINES O

O

N N

NH NH

O

F Cl

N

39 (Afatinib)

IC50(EnzymeWT) = 0.50 nM (EnzymeT790M) = 0.97 nM (H1975 CellDM) = 0.90 µM

N

Ο

N

O

O

N

N

NH NH

O

NH NH

F

O

F

F

F Cl

Cl

N

40 IC50(EnzymeWT) = 0.20 nM (EnzymeT790M) = 6.70 nM (H1975 CellDM) = 0.41 µM

N

41 IC50(EnzymeWT) = 2.60 nM (EnzymeT790M) = 16.40 nM (H1975 CellDM) = 0.29 µM

Figure 22. 4-Anilinoquinazoline derivatives 39−41 as potential EGFR L858R/T790M inhibitors.

Compound 39 (afatinib)64–68, a second-generation EGFR inhibitor, improved the inhibitory potency against the gefitinib-resistant EGFR L858R/T790M (IC50 = 0.97 nM). On the basis of its novel aniline-quinazoline backbone, in 2014, Yu et al. 31



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Page 32 of 55

replaced the acrylamide function with an alternative and less reactive electrophile, by preparing a family of quinazoline analogues bearing fluoro-substituted olefins, to enhance the activity against the EGFR T790M. Among the newly designed quinazoline derivatives, compounds 4069 and 4169 were the best inhibitors against the drug-resistant H1975 cells, with IC50 values of 0.41 µM and 0.29 µM, respectively (Figure 22). Compared with afatinib, both of them showed a weaker ability to inhibit the T790M-mutated EGFR, but approximately 2- to 3-fold higher activity against the mutant cells harboring the T790M mutation. Despite these disappointing results, the aniline-quinazoline scaffold may be value for discovery of more potent inhibitors with enhanced activity and selectivity.

PYRIDONE Table 7. Novel pyridone derivatives and their physicochemical properties and cellular potency O

H N

H N

O

O

O

O NH HN

N

O NH HN

HO

N

N

O N

Cl

Compd

NH HN

O

N 42

43

44

42

43

44

0.065

0.019

0.015

23

LogD

--

3.5

2.7

TMLR Ki (μM)a wtEGFR Selectb pEGFR H1975 EC50 (μM)a

H N

a

Ki and EC50 data are an average of at least two independent experiments. bRatio of wtEGFR Kiapp over EGFR(TMLR) Kiapp. cRatio of pEGFR EC50 in H292 cells over pEGFR EC50 in H1975 cells. 32


Page 33 of 55

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Beginning with a nonselective high-throughput screening hit 4270, which lacked selectivity over WT EGFR, Bryan et al. recently identified a set of noncovalent pyridone-based T790M EGFR inhibitors recently. Within this series, compounds 4370 and 4470 showed the best biochemical potency, ligand efficiency, and WT EGFR selectivity relative to initial hit 42. Nevertheless, both of them showed low micromolar to submicromolar activity in cellular activity. Compound 44 also demonstrated 23-fold cellular selectivity over WT EGFR when comparing pEGFR inhibition in the TMLR cell line H1975 vs the WT EGFR-driven cell line H292, meeting the goal of a selective tool compound with improved kinase selectivity for evaluation in cellular assays. In addition, 44 showed desirable physicochemical properties, improved solubility, and moderate in vitro stability highlighting the promise of this series for further development (Table 7).

OTHER SCAFFOLDS TAS-291371 and TAS-12172-74 (structures not disclosed) are two potent and mutant-selective EGFR inhibitors that were discovered by Taiho Pharmaceutical Co., Ltd. TAS-2913 was able to inhibit EGFR mutants in subnanomolar range with more than 10-fold selectivity over WT EGFR. Moreover, TAS-2913 caused significant tumor growth inhibition in the H1975 xenograft model. Partial tumor regression was observed at over 100 mg/kg, whereas it did not cause body weight loss. While TAS-121 also inhibits kinase activity of EGFR mutants harboring T790M in a sub-nano molar range. In a cell proliferation assay, TAS-121 suppresses growth of NCI-H1975 and HCC827 cells with higher potency than that of a normal cell which 33



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Page 34 of 55

grows in an EGF-dependent manner. By contrast, TAS-121 demonstrated higher selectivity than TAS-2913, and may represent a new therapeutic strategy in the treatment of NSCLC resistant to currently available EGFR/TKIs. Its further pre-clinical evaluations and development are ongoing.

O N

Cl

N N

O HN

N

N 45 (EGF816)

Figure 23. Structure of novel NSCLC clinical candidate 45.

In addition, there are several extraordinary compounds that have been advanced to clinical studies (Table 8). For example, HM6171375,76 (structure not disclosed, Hanmi Pharmaceutical Co., Ltd. Seoul, South Korea), an orally available small-molecule with good efficacy in vivo models, especially those with concurrent T790M mutations, is currently being tested in phase II clinical trials (ClinicalTrials.gov, NCT02444819, NCT02485652). Preparations have begun for a broader Phase III trial program to be initiated in 2016. With the aim of achieving first market authorisation for HM61713 for patients with EGFR mutation-positive NSCLC by 2017 in the U.S, Boehringer Ingelheim

partnered

with Hanmi

Pharmaceutical to

develop

commercialise HM61713 in July 2015. Table 8. Novel mutant-selective EGFR inhibitors that have been advanced to clinical trials Drugs

Status

Trial identifiers

Sponsor

HM61713

phase II

NCT02444819, NCT02485652

Hanmi Pharma

EGF816

phase I/II

NCT02108964

Novartis 34


Co., Ltd.

and

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PF-06747775

phase I/II

NCT02349633

Pfizer

Avitinib

phase I

NCT02330367

Acea Bio (Hangzhou) Co., Ltd.

ASP8273

phase I

NCT02113813

Astellas Pharma Inc

Compound 45 (EGF816)77-79, discovered by Novartis, is another novel mutant-selective EGFR inhibitor, being tested in phase I/II clinical trials (ClinicalTrials.gov,

NCT02108964)

(Figure

23).

Meanwhile,

PF-06747775 (structure not disclosed, Pfizer) which already started phase I/II clinical evaluations (ClinicalTrials.gov, NCT02349633), is another EGFR-mutant specific kinase inhibitor in development for NSCLC patients with acquired resistance to EGFR inhibition related to the occurrence of the T790M mutation. Avitinib80 (structure not disclosed) maleate (ClinicalTrials.gov, NCT02330367) and ASP827381 (structure not disclosed,

ClinicalTrials.gov,

NCT02113813)

are

two

novel

third-generation, mutant-selective EGFR inhibitors, which are undergoing clinical trials (phase I) and have yielded promising preliminary results. As these agents are selective towards the mutant forms of EGFR, their toxicity profiles may be more favorable compared to non-selective EGFR inhibitors, which also inhibit the EGFR WT form. However, their structures and in vitro properties have not yet been disclosed.

CONCLUSIONS Since the first-generation EGFR inhibitors gefitinib and erlotinib were approved for the treatment of NSCLC disease in 2002 and 2004, respectively, a number of more effective anti-EGFR drugs have been discovered and evaluated in clinical studies. However, in approximately 50% of patients with clinically acquired resistance, a

35



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Page 36 of 55

single T790M point mutation at the “gatekeeper” of EGFR appeared after repeated treatments with the first-generation EGFR inhibitors, making these agents lose major efficacy in treating NSCLC disease. Therefore, searching for agents particularly effective against mutant EGFR T790M remains urgent. Recently, a large number of small molecules, including the novel clinical candidates, rociletinib, osimertinib and HM81713 have been discovered as strong inhibitors of the EGFR T790M mutant kinase. In particular, rociletinib and osimertinib which displayed promising efficiency in clinical studies, represent the leading third-generation irreversible EGFR inhibitors in clinical development. It is hoped that their approval will provide big breakthrough in the treatment of patients with EGFRT790M-mutated NSCLC. More excitingly, continuous efforts focused on this field have also generated a number of even more selective inhibitors, such as compounds 29a-d. Most of these highly active inhibitors are structurally characterized by a pyrimidine core, modifications of which using a conformational constraint strategy are highly effective to overcome the acquired resistant mutations. Despite the clinical efficacy of the pyrimidine-based EGFR inhibitors rociletinib and osimertinib, it is fully anticipated that patients will ultimately develop acquired resistance to these agents. EGFR L718Q, L844V, and C797S have been identified as three major drug resistance mutations for these third-generation inhibitors. Unfortunately, the C797S mutation, in the presence of Del 19 or L858R and T790M, causes resistance to all current EGFR inhibitors.82-84 Therefore, cysteine point mutations may constitute a recurring liability for a broad range of covalent drugs in the future. In addition, a more detailed understanding of the mechanisms of acquired resistance and whether cross resistance will occur to all irreversible pyrimidine based and to existing EGFR inhibitors is still urgent. Overall, to

develop

more

active

selective

EGFR

T790M

36


inhibitors,

these

low

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molecular-weight inhibitors with different chemical skeletons are of great value.

AUTHOR INFORMATION Corresponding Author ∗

Tel:

+86-411-86110419.

Fax:

+86-411-86110419.

E-mail

address:

[email protected] Notes The authors declare no competing financial interest. Biographies Zhendong Song obtained his B.Sc. (Hons.) in Pharmacy (2015) at Dalian Medical University, China. During his undergraduate study, he did considerable work on designing and synthesizing effective NSCLC inhibitors. Now he is studying for a Master's degree at Dalian Medical University. His research interests lie in the general areas of medicinal chemistry, drug discovery and development, lead optimization, and structure-based design of anticancer agents, with primary focus on the structure-based design of mutant EGFR T790M inhibitors. Yang Ge received his B.Sc. (Hons.) from Binzhou Medical University, China, in 2014. Then he moved to Dalian Medical University, concentrating on drug research and development. He is currently studying for a Master's degree at Dalian Medical University. His research interests also lie in the general areas of medicinal chemistry, drug discovery and development, and lead optimization, with primary focus on the structure-based design of anticancer drugs Changyuan Wang received his B.Sc. (Hons.) from the College of Pharmacy, Dalian Medical University, China. After he graduated in 2004, he worked at this school, focusing on screening potential inhibitors against HIV, HCV, and various cancers. His research interests include the development of anticancer screening platforms, animal 37



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Page 38 of 55

models, and viral pathogenicity of human and animal viruses. Shanshan Huang received her B.Sc. (Hons.) from the College of Pharmacy, Dalian Medical University, China, in 1996. Since then, she has worked at this school, and applied himself to screen potential inhibitors against HIV, HCV, and various cancers. Using cell- and enzyme-based assays and animal models, she has discovered several drug candidates for clinical exploration. Xiaohong Shu received her B.S. degree in Natural Pharmaceutical Chemistry from Shengyang Pharmaceutical University, and received M.S. and Ph.D. degree

in

Biochemistry and Molecular Biology from Dalian Medical University in China.

The

focus of her research is on the molecular pharmacology of cancer. She is a co-author of more than 40 publications in international

journals

and

a

first

or corresponding author in the journals such as Biochemical Pharmacology, PLoS ONE, Neurotherapeutics and Current Drug Metabolism, and 15 book chapters. She has

been

got professor’s qualification of Pharmacology

in

Dalian

Medical

since December, 2012. Kexin Liu received his B.S. degree in Clinical Medicine and M.S. degree in Pharmacology from Dalian medical University in China and Ph. D. degree in Pharmacy in the University of Tokyo (Laboratory of Yuichi Sugiyama, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences) in Japan. The focus of his research is on the molecular pharmacokinetics of transport in liver, intestine, kidney as well as transporter-mediated DDI and MDR. Prof. Liu is a co-author of more than 170 publications in international journals and a first or corresponding author in the famous journals such as Hepatology, Drug Metab Dispos, J. Pharmacol. Exp. Ther.， and 50 book chapters as well as 30 review articles. 38


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Youwen Zhou earned his PhD in Molecular Genetics at the State University of New York, followed by his medical degree at the University of Toronto in 1995. He completed his residency training specializing in dermatology at the University of British Columbia (UBC). Now, he is a Professor in the Department of Dermatology and Skin Science, UBC. He is also the Director of the Chieng Genomics Centre and Laboratory of Predictive Medicine and Therapeutics in the Vancouver Coastal Health Research Institute. His main scientific research is to investigate the pharmacology of anti-cancer agents. He has been the principal investigator in over 20 research projects, and is a supervisor for both the Experimental Medicine Graduate Program and the Clinical Dermatology Fellowship at the UBC. Xiaodong Ma received his Ph. D. in Organic Chemistry from Fudan University in Shanghai, China, in 2012. After working as a chemical R& D engineer in PetroChina Co. Ltd. for two years, he became an Assistate Professor at Dalian Medical University, China, in 2014.

His current research mainly concentrates on the general areas of

medicinal chemistry, drug discovery and development, and mechanistic enzymology, with primary efforts on the structure-based design of NSCLC inhibitors. Until now, he has designed and synthesized a great number of small-molecule EGFR T790M inhibitors worthy of further development as potential anti-NSCLC agents.

ACKNOWLEDGEMENTS This work was supported in part by grants from the National Natural Science Foundation of China (No. 81402788), and the PhD Start-up Fund of Natural Science Foundation of Liaoning Province, China (No. 20141115).

ABBREVIATIONS USED EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer; TKIs, tyrosine kinase inhibitos; PK, pharmacokinetics; T790M, threonine-to-methionine; 39



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NO, nitric oxide; SAR, structure-activity relationship; WT, wild-type; DM, double mutations; AM, activating mutant

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Small molecules

EGFRT790M

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Challenges and Perspectives on the Development ... - ACS Publications

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