First SAR study for overriding NRAS mutant driven ... - ACS Publications

Subscriber access provided by University of South Dakota

Article

First SAR study for overriding NRAS mutant driven acute myeloid leukemia Hanna Cho, Injae Shin, Eunhye Joo, seunghye choi, Wooyoung Hur, HAELEE KIM, EUNMI HONG, Nam Doo Kim, Hwan Geun Choi, Nathanael S Gray, and Taebo Sim J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00882 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 78 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

Journal of Medicinal Chemistry

First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid Leukemia

Hanna Cho,†,1 Injae Shin,†,1 Eunhye Joo,† Seunghye Choi,† Wooyoung Hur,‡ Haelee Kim,§ Eunmi Hong,§ Nam Doo Kim,§,∥ Hwan Geun Choi,§ Nathanael S. Gray,⊥,# and Taebo Sim†, ‡,*

†

KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro,

Seongbuk-gu, Seoul 02841, Republic of Korea. ‡

Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5

Hwarangro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea. §

Daegu-Gyeongbuk Medical Innovation Foundation, 2387 dalgubeol-daero, Suseong-gu, Daegu

42019, Republic of Korea. ∥

NDBio Therapeutics Inc., 32 Songdogwahak-ro, Yeonsu-gu, Incheon 21984, Republic of Korea.

⊥

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

#

Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston,

MA 02115, USA. 1

These authors are equally contributed to this work.

ABSTRACT GNF-7, a multi-targeted kinase inhibitor, served as a dual kinase inhibitor of ACK1 and GCK, which provided a novel therapeutic strategy for overriding AML expressing NRAS mutation. This SAR study with GNF-7 derivatives, designed to target NRAS mutant-driven AML, led to identification of the extremely potent inhibitors, 10d, 10g and 11i, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK. These substances strongly suppress proliferation of mutant NRAS expressing AML cells via apoptosis and AKT/mTOR signaling

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 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

blockade. Compound 11i is superior to GNF-7 in terms of kinase-inhibitory activity, cellular activity and differential cytotoxicity. Moreover, 10k possessing a favorable mouse pharmacokinetic profile prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors for overriding NRAS mutant-driven AML.

Introduction The small GTPase, RAS, plays the role of a molecular switch that regulates signal transduction in response to extracellular stimuli. In this role, it participates in diverse cellular events such as cell cycle, differentiation and survival. Malfunctioning RAS is strongly related to tumorigenesis. Mutations in the RAS gene, which encode over-active RAS proteins, are found in 30% of all human tumors. Among the three mutants, including HRAS, KRAS and NRAS, KRAS is most frequently present in various types of cancers including lung (35%), colon (45%) and pancreatic (90%).1 NRAS mutations are also common in acute myeloid leukemia (AML)2, appearing in 10% of AML patients.3 AML has remained a devastating and life-threatening disease with poor prognosis,4 especially for patients 65 years or older whose median overall survival is less than 1 year.5 Therefore, there exists an unmet medical need to create further innovative therapeutic strategies for overcoming AML even though enormous efforts6 have been devoted to developing targeted therapies for AML treatment including FLT3 inhibitors, PLK inhibitors, IDH inhibitors, DNMT inhibitor, LSD1 inhibitor, DOT1L inhibitors, HDAC inhibitors, BET inhibitors, BCL-2 inhibitors. The high relevance and crucial role of its mutations in cancers has made RAS an attractive therapeutic target for cancer treatment.7 However, despite being subjected to intensive efforts over several decades, no efficacious pharmacological RAS inhibitors have been identified. This is caused by the extraordinarily high affinity of RAS for its substrates, GTP and GDP, as well as the presence of a surface in this protein that does not participate in high-affinity interactions with small molecules.8


Page 2 of 78

Page 3 of 78 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


Thus, other strategies have been explored to inhibit the function of hyper-activated RAS.9 One involves targeting effectors such as RAF10, MEK11 and ERK12 in canonical RAS13 and PI3K/AKT14 pathways. Although many substances, which function in this manner are under clinical evaluation, rapid drug resistance and complex feedback mechanisms are major hurdles for this approach to drug development.15 Blocking RAS localization by preventing farnesylation of RAS has also been investigated. In spite of displaying in vitro anti-cancer effects, farnesyltransferase inhibitors (FTIs)16 generally have poor clinical outcomes. Furthermore, genetic approaches have been employed to identify new target genes that interact with oncogenic RAS in a lethal manner. Although this strategy led to identification of STK33 as an effective target, more significant effort is required to discover active candidates.17 As part of a program to explore a promising anticancer strategy that focuses on blocking signalling by the oncogenic NRAS mutation, we recently identified dual inhibition of ACK1 and GCK as a novel therapeutic target to overcome NRAS mutant-driven acute myeloid leukemia (AML).3 Both ACK1 and GCK are non-canonical effectors of the RAS pathway. Activated cdc42-associated kinase 1 (ACK1 or TNK2) is a non-receptor tyrosine kinase originally identified as an inhibitory regulator of cdc42. Recent studies have highlighted the oncogenic properties of ACK118 by showing that it directly phosphorylates Tyr176 of AKT, thus, activates AKT in a PI3K-independent manner.19 Germinal center kinase (GCK/MAP4K2) belongs to the STE20 family of serine/threonine kinases associated with the JNK pathway and inflammatory process.20 In a previous study, we showed that the multi-kinase inhibitor GNF-721, 22 effectively and selectively suppresses proliferation of primary AML patient-derived cells harboring mutant NRAS as well as AML cell lines expressing mutant NRAS. Through mechanistic analysis using small molecule kinase inhibitors and RNA interference studies and in situ kinase profiling (KiNativ method), we ascertained that the effect of GNF-7 on NRAS mutant AML cells is associated with a synthetic lethal interaction between ACK1 and GCK. This synthetically lethal effect of GNF-7 significantly reduces the disease burden and prolongs overall survival in an AML xenotransplantation mice model.3



The current study was designed to discover new inhibitors of ACK1 and GCK, which are more potent and/or less toxic than GNF-7, in order to overcome AML caused by the NRAS mutant. In the effort described below, a structure activity relationship (SAR) study was carried out exploring the effects on NRAS mutant AML cells as well as ACK1 and GCK enzymes of GNF-7 derivatives, which possess the dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold. We observed that among the 29 synthetic GNF-7 derivatives prepared in this study, 10d and 11i exhibit much higher enzymatic inhibition and cellular activities compared with GNF-7. Moreover, 11i clearly exceeds GNF-7 in terms of kinase-inhibitory activity, cellular potency and differential cytotoxicity. We observed that another derivative, 10k having a favorable mouse PK profile significantly prolonged life-span of Ba/F3-NRAS-G12D injected mice without prominent toxicity found in GNF-7, which makes 10k superior to GNF-7.

RESULT AND DISCUSSION

Figure 1. Structure of GNF-7 (left) and representative GNF-7 analogues (right).

General procedure for synthesis of GNF-7 derivatives By employing a more efficient synthetic route for the preparation GNF-7 than the one previously reported,21 we were able to generate derivatives that contain a variety of different moieties at the R1, R2, and R3 position identified in Figure 1, Schemes 1-3 and Tables 1-3. The synthetic route


Page 4 of 78

Page 5 of 78 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


developed to prepare GNF-7 along with 10a-k, and 11a-i, given in Scheme 1, begins with chlorination of the commercially available pyrimidine derivative 1 using POCl3 and DIPEA to yield the corresponding chloride 2.23 Reaction of 2 with 2-methyl-5-nitroaniline produced aniline derivative 3, which upon reaction with methylamine provided 4. Cyclic urea formation promoted by reaction of 4 with triphosgene formed the key intermediate 5, which was utilized in routes to generate GNF-7 and derivatives containing various R1 and R2 replacements. Reduction of the nitro group in 5 to give 6 followed by acylation with 3-(trifluoromethyl)benzoyl chloride provided amide 7, which through Buckwald amination with various aniline derivatives is transformed to GNF-7 and R1-substituted analogues 10a-k. Similarly, a sequence involving reaction of 5 with 6-methylpyridin-3-amine (giving 8), nitro group reduction (giving 9) and amide coupling with various carboxylic acids produced R2substituted analogues 11a-i. A modification of the routes shown in Scheme 1 was used to prepare R3-substituted analogues. As shown in Scheme 2, pyrimidine trichloride 2 was first converted to 12, which contains N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benzamide group present in GNF-7 and a handle to introduce various R3 substituents to create analogues 13a-e. In addition, cyclic urea forming reactions of 13a-e produced 14a-e, which were then transformed to 15a-e by using Buchwald amination with 6methylpyridin-3-amine.

Scheme 1. Synthesis of GNF-7, 10a-k and 11a-ia



a

Reagent and condition: (A) POCl3, DIPEA, toluene, 110 oC, 77%; (B) 2-methyl-5-

nitroaniline, NaI, K2CO3, acetone, 50 oC, 74%; (C) MeNH2 (2 M in MeOH solution), DIPEA, 1,4dioxane, 60

o

C, 77%; (D) triphosgene, TEA, THF, 70

o

C, 63%; (E) Fe powder, NH4Cl,

THF/MeOH/H2O, 80 oC, 87%; (F) 3-(trifluoromethyl)benzoyl chloride, K2CO3, DCM, rt, 73%; (G) various amines, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 oC, 13-84% or ammonia solution, DMSO, 150 oC, 28%; (H) 6-methylpyridin-3-amine, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 oC, 65%; (I) Fe powder, NH4Cl, THF/MeOH/H2O, 80 oC, 81%; (J) various carboxylic acids, HATU, DIPEA, DMF, rt, 30-61%.

Scheme 2. Synthesis of 15a-ea

a

Reagent and condition: (A) N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benzamide,

NaI, K2CO3, acetone, 50 oC, 69%; (B) various amines, THF, 60 oC or aniline, TEA, n-butanol, 120 oC;


Page 6 of 78

Page 7 of 78 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


(C) triphosgene, TEA, THF, 70 oC; (D) 6-methylpyridin-3-amine, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 oC, 12-27% over 3 steps.

Scheme 3. Synthesis of 17a-ea

a

Reagent

and

condition:

(A)

4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-

(trifluoromethyl)benzoic acid, HATU, DIPEA,DMF, rt, 65%; (B) various amines, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 oC, 34-62%.

Structure-Activity Relationships IC50 values for all synthetic GNF-7 analogues against both ACK1 and GCK kinases were determined using an in vitro biochemical kinase assay. The growth inhibitory activities (GI50’s) of these substances in the NRAS-G12D transformed Ba/F3 cell line were also estimated. The first phase of this effort, focusing on optimization of the R1 substituent, (Table1) showed that 10a, containing an aniline head group, displays a 6-fold lower activity (IC50 = 47 nM) against GCK compared with that of GNF-7 (IC50 = 8 nM) while its potency against ACK1 (IC50 = 37 nM) is nearly equal to that of GNF-7. This finding is consistent with information gained from a previous docking study,3 which suggested that the pyridine nitrogen in GNF-7 contributes to binding with GCK through hydrogen bonding with Tyr27. ACK1 lacks a residue analogous to Tyr27 in GCK. Both 10b containing the 4-(4-ethylpiperazin-1-yl)-phenylamine head group that is present in the pan-FGFR inhibitor BGJ398,24 and 10c containing a 6-(4-ethylpiperazin-1-yl)pyridin-3-amine group were found to have a kinase-inhibitory activity against GCK that is in the range of that of the



parent GNF-7, indicating that the nitrogen on the ethylpiperazine ring could form a hydrogen bond with Tyr27 of GCK. Similarly, replacement of ethylpiperazine group in 10c with piperazinyl ethanone (10d) and ethylpiperazinyl methanone groups (10e) led to a significant increase in kinase-inhibitory activity (10d, GCK IC50 = 1 nM, ACK1 IC50 = 8 nM; 10e, GCK IC50 = 2 nM, ACK1 IC50 = 13 nM). Taken together, these results demonstrate that extended head groups in GNF-7 analogues may enable additional interaction with both ACK1 and GCK, and thus increase binding affinity. Compared with that of GNF-7, the anti-proliferative activities of 10d and 10e on Ba/F3NRAS-G12D cells were moderately greater or the same as GNF-7 (GI50’s of 0.059 µM and 0.107 µM respectively) in spite of their higher enzymatic inhibition activities. Compared to GNF-7, 10a-e exhibited lower differential cytotoxicities against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells which serves as a control. It is of note that compared with GNF-7, 10d possesses increased kinase-inhibitory activity against both ACK1 (8 nM of IC50) and GCK (1 nM of IC50) and a higher cellular activity on Ba/F3-NRAS-G12D cells. We also explored analogues that contain an aminopyrazole R1 substituent that is present in NG25, a dual TAK1 and GCK inhibitor.25 The kinase-inhibitory and cellular activities of 10f, bearing a 1-methyl-pyrazole group, are comparable to those of GNF-7 and to other substances (10b-e) containing a piperazinyl group. Notably, 10g exhibited a slightly higher enzymatic inhibitory activity (GCK IC50 = 5 nM, ACK1 IC50 = 9 nM) relative to GNF-7, which indicates that 1,3-dimethyl pyrazole group may fit in the hydrophobic pocket adjacent to the hinge region in the kinases. Also, the cellular activity and differential cytotoxicity of 10g on Ba/F3 cells are comparable to those of GNF-7. In contrast to analogues with aromatic groups at the R1 position, derivatives containing aliphatic amine groups (10h, 10i and 10j) generally displayed lower kinase-inhibitory activities and anti-proliferative activities on Ba/F3-NRAS-G12D cells, whereas 10h bearing a cyclopropylamine group has similar IC50 and GI50 values to those of aniline bearing 10a. This observation suggests that the cyclopropyl moiety, which has pseudo double bond character,26 can participate in pi-pi stacking interactions in the hydrophobic pocket near the hinge region. In contrast, 10i having a bulky and


Page 8 of 78

Page 9 of 78 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


flexible cyclohexyl group has substantially diminished potency compared with those of 10h and 10j. Compared with GNF-7, 10k exhibited a comparable inhibitory activity against ACK1 and a 3-fold less potency against GCK (IC50 = 28 nM). Overall, the data gained from the SAR study with analogues containing different R1 groups suggest that aromatic amine groups containing an additional hydrogen bonding acceptor have enhanced kinase-inhibitory activities against both ACK1 and GCK.

Table 1. Enzymatic inhibitory activities against ACK1 and GCK and anti-proliferative activities on Ba/F3- NRAS-G12D cells

IC50 (nM)a Entry

Ba/F3 cells GI50 (µM)b

R1 ACK1

GCK

NRAS-G12D

Parental

GNF-7

25

8

0.081 ± 0.038

1.033 ± 0.331

10a

37

47

0.380 ± 0.168

1.786 ± 0.217

10b

20

8

0.083 ± 0.005

0.559 ± 0.039

10c

28

7

0.071 ± 0.025

0.264 ± 0.000

10d

8

1

0.059 ± 0.023

0.402 ± 0.149



a

Page 10 of 78

10e

13

2

0.107 ± 0.021

0.728 ± 0.458

10f

20

6

0.100 ± 0.049

0.352 ± 0.034

10g

9

5

0.129 ± 0.002

2.064 ± 0.534

10h

35

42

0.547 ± 0.070

5.063 ± 0.173

10i

60

925

2.452 ± 0.803

5.221 ± 1.147

10j

21

33

0.290 ± 0.055

3.286 ± 1.243

10k

18

28

0.223 ± 0.075

2.114 ± 0.282

Radiometric biochemical kinase assay results. bGI50 represents the concentration at which a

compound causes half-maximal growth inhibition. NRAS-G12D-transformed Ba/F3 cells and parental Ba/F3 cells were treated with inhibitors for 72 h in a dose escalation. Average GI50 values with SD (n = 3, duplicate) are shown.

Our attention next focused on optimization of the R2 group (Table 2). The inhibitory effect of the aryl-CF3 group in GNF-7 was assessed using the unsubstituted analogue 11a. Interestingly, the inhibitory potency of 11a against GCK (IC50 = 1080 nM) is greatly less than that of GNF-7 while its activity against ACK1 (IC50 = 21 nM) is the same as that of GNF-7, indicating that CF3 group is essential for kinase-inhibitory activity on GCK but not ACK1. The 4-methyl aryl group containing analogue, 11b, has a 5-fold reduced kinase-inhibitory activity against GCK (IC50 = 39 nM) and antiproliferative activity (GI50 = 0.387 µM) on Ba/F3-NRAS-G12D cells. In contrast, the in vitro potencies of 11b against ACK1 (IC50 = 20 nM) and parental Ba/F3 cells (GI50 = 0.870 µM) were retained.


Page 11 of 78 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


The effects of additional substituents at the meta or para positions of the tail aryl ring in GNF-7 were explored using 11c-i. The results showed that addition of meta-morpholine (11c) and 4piperidinol moiety (11d) resulted in slight decreases in inhibitory activity against GCK (11c, IC50 = 45 nM; 11d, IC50 = 72 nM) and anti-proliferation of Ba/F3-NRAS-G12D cells (11c, GI50 = 0.284 µM; 11d, GI50 = 0.118 µM). Moreover, introduction of a meta-4-methylimidazole group (11e) also brought about a slight reduction in kinase-inhibitory activities (GCK IC50 = 27 nM, ACK1, IC50 = 53 nM) and cellular Ba/F3-NRAS-G12D cellular activity (GI50 = 0.238 µM) compared with that of GNF-7. In exploring the effect para-aryl substituents, we found that 11f possessing para-pyrrole moiety has a marginally increased activity against ACK1 (IC50 = 10 nM) but not against GCK (IC50 = 24 nM), while 11g containing a para-piperazine moiety has retained kinase-inhibitory activity against both ACK1 and GCK (ACK1 IC50 = 28 nM, GCK IC50 = 10 nM). Based on these results, we conclude that para- rather than meta-substitution is more suitable for altering inhibitory activities against both ACK1 and GCK. Indeed, 11h, having a piperazinylmethylphenyl substituent, has a 2-fold increased potency (IC50 = 5 nM) against GCK kinase compared to that of 11g. Furthermore, 11i, containing a 3(dimethylamino)pyrrolidinemethylphenyl group, has enhanced kinase-inhibitory activities against both ACK1 (IC50 = 6 nM) and GCK (IC50 = 2 nM) compared with GNF-7. Also, 11i has a significantly increased anti-cellular proliferation potency (GI50 = 0.023 µM) on Ba/F3-NRAS-G12D relative to GNF-7. It should be emphasized that 11i compared to GNF-7 possesses a higher differential cytotoxicity against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells.

Table 2. Enzymatic inhibitory activities against ACK1 and GCK and anti-proliferative activities on Ba/F3-NRAS-G12D cells



Entry


ACK1

GCK

NRAS-G12D

Parental

25

8

0.081 ± 0.038

1.033 ± 0.331

21

1080

1.466 ± 0.170

2.697 ± 0.057

11b

20

39

0.387 ± 0.069

0.870 ± 0.163

11c

26

45

0.284 ± 0.152

1.649 ± 0.146

11d

40

72

0.118 ± 0.035

0.671 ± 0.207

11e

53

27

0.238 ± 0.188

>5

11f

10

24

0.069 ± 0.024

0.317 ± 0.072

11g

28

10

0.341 ± 0.014

0.685 ± 0.032

11h

26

5

0.316 ± 0.094

0.818 ± 0.021

11i

6

2

0.023 ± 0.152

0.636 ± 0.115

GNF-7

11a

a

R2

IC50 (nM)a

Page 12 of 78




Page 13 of 78 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


The effects of R3 groups on inhibitory activities were explored by replacing the methyl group in GNF-7 with more sterically bulky aliphatic groups, phenyl group and benzyl group. The results show that as the size of the R3 substituent increases (15a-c, 15e), both enzymatic activities and cellular activities decrease. Although the phenyl group in 15d causes a slightly increased kinaseinhibitory activity against ACK1 (IC50 = 15 nM), it causes a decreased activity against GCK and cellular activity compared with GNF-7 in which R3 substituent is methyl group. The

observations

made

in

the

SAR

study

suggest

that

3-

(dimethylamino)pyrrolidinemethylphenyl and methyl are optimal R2 and R3 groups, respectively. Moreover, the results indicate that that the R1 substituents in 10d-g are superior to that in GNF-7 in terms of enzymatic inhibitory activities. As a result, the activities of analogues 17a-d, containing a combination of the optimal R2/R3 substituent and the R1 substituents present 10d-g, were assessed. Contrary to expectations, compared with those of 11i, none of these compounds exhibited enhanced enzymatic inhibitory activities against ACK1 or GCK (Table 3). Compared with GNF-7, 17a-d have lower differential cytotoxicities against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells even though they displayed comparable cellular activities against BaF3-NRAS-G12D cells.

Table 3. Enzymatic inhibitory activities against ACK1 and GCK and anti-proliferative activities on Ba/F3-NRAS-G12D cells

IC50 (nM)a Entry

GNF-7

R1

R2


R3

methyl

ACK1

GCK

NRAS-G12D

Parental

25

8

0.081 ± 0.038

1.033 ± 0.331



a

Page 14 of 78

15a

ethyl

23

34

0.125 ± 0.067

0.708 ± 0.001

15b

cyclopropyl

36

63

0.095 ± 0.019

0.649 ± 0.141

15c

cyclohexyl

94

497

2.385 ± 0.408

>5

15d

phenyl

15

55

0.475 ± 0.098

3.304 ± 0.675

15e

benzyl

40

165

0.356 ± 0.011

>5

17a

methyl

25

16

0.041 ± 0.002

0.187 ± 0.076

17b

methyl

21

14

0.094 ± 0.054

0.704 ± 0.098

17c

methyl

25

8

0.068 ± 0.027

0.414 ± 0.075

17d

methyl

11

17

0.123 ± 0.052

0.509 ± 0.021




Page 15 of 78 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


Based on the results of the SAR study presented above, the anti-proliferative activities of selected analogues in the human AML cell lines, OCI-AML3 (NRAS-Q61L) and U937 (NRAS wt), were assessed (Table 4). In this assessment, a higher differential cytotoxic effect on OCI-AML3 (NRAS mt) cell lines relative to U937 (NRAS wt) is a positive finding. GI50’s on OCI-AML3 cells were found to be in the range of 0.030 µM to 0.466 µM. The four GNF-7 derivatives, 10b, 10c, 10f and 11i, have remarkable potencies against OCI-AML3 cells (GI50’s range from 0.030 to 0.042 µM) and reasonable anti-proliferative activities against U937 cells (GI50 = ca. 1 µM). Based on their cellular selectivities against OCI-AML3 cells (NRAS mt) over U937 cells (NRAS wt), five derivatives, 10c, 10e, 11b, 11f and 11i, were found to be superior to GNF-7. This is especially true for 11f, which displayed an exceptional cellular selectivity (ca. 100-fold) against OCI-AML3 cells over U937 cells. It is worth recalling that 11i exhibits the highest differential cytotoxicity against Ba/F3NRAS-G12D cells relative to parental Ba/F3 cells. Thus, this effort has led to the identification of promising new GNF-7 analogues that have strong anti-proliferative activities and high cellular selectivities against NRAS mutant expressing AML cell lines. It should be noted that 11i clearly surpasses GNF-7 in terms of cellular potency and selectivity on NRAS mutation cells (Ba/F3-NRASG12D and OCI-AML3) as well as enzymatic inhibitory activities against ACK1 and GCK.

Table 4. Anti-proliferative activities of the selected GNF-7 analogues against OCI-AML3 and U937 cells

Entry

OCI-AML3 (NRAS Q61L) GI50 (µM)a

U937 (NRAS wt) GI50 (µM)a

GNF-7

0.219

±

0.058

4.377

±

0.081

10b

0.040

±

0.002

0.839

±

0.317

10c

0.030

±

0.001

1.412

±

0.276

10d

0.179

±

0.017

2.483

±

0.499

10e

0.122

±

0.040

6.479

±

0.383



a

Page 16 of 78

10f

0.042

±

0.031

1.125

±

0.129

10g

0.208

±

0.034

2.463

±

1.679

10j

0.367

±

0.065

8.531

±

0.039

10k

0.466

±

0.120

4.967

±

1.052

11b

0.394

±

0.025

19.375

±

1.775

11c

0.260

±

0.064

2.805

±

0.134

11d

0.143

±

0.002

2.180

±

0.785

11f

0.267

±

0.092

11i

0.037

±

0.005

>25 1.973

±

0.182

GI50 represents the concentration at which a compound causes half-maximal growth inhibition. OCI-

AML3 and U937 cells were treated with inhibitors for 72 h. Average GI50 values with SD (n = 3, duplicate) are shown.

Microsomal stability and mouse PK profiling With selected compounds (10b, 10c, 10d, 10e, 10f, 10g, 10k and 11i) displaying distinctive in vitro potency, we evaluated their microsomal stability to estimate metabolic clearance in liver. The remaining amount of each compound after 30 minutes incubation in liver microsomes of human, dog, rat, and mouse was measured (Table 5). Briefly, the microsomal stabilities of compounds with 5membered aromatic pyrazole ring (10f, 10g) or the primary amine (10k) as head group are comparable to those of GNF-7. The presence of piperazine moiety in head group (10b, 10c, 10d and 10e) caused poor microsomal stability. 11i possessing additional substituents at meta position of GNF7 tail showed the little reduced stability compared with GNF-7. We next assessed inhibitory activities of the compounds against four cytochromes P450 (CYPs) and found that 10k had little or no inhibition effects on CYP1A2, CYP2C9, CYP2D6, and CYP3A4 at 10 µM. Especially, 10k is far superior to GNF-7 in terms of CYP2C9 inhibition profile. We also investigated the mouse pharmacokinetic property (PO, 10 mg/kg) of 10k (Table 6). Compound 10k exhibited excellent oral


Page 17 of 78 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


systemic exposure (AUCinf = 52464.8 ng hr/mL, AUClast = 36363.7 ng hr/mL, Cmax = 2406.7 ng/mL), thus we chose this compound for in vivo efficacy study using mouse leukemia model.

Table 5. Microsomal stabilitya and CYP inhibitionb profile

Microsomal stability (%)

CYP remaining activity (%)

Entry

a

Human

Dog

Rat

Mouse

1A2

2C9

2D6

3A4

GNF-7

78.6

55.3

84.8

80.5

93.2

27.0

68.4

>100

10b

46.7

21.3

19.7

20.9

92.1

34.3

79.8

>100

10c

29.8

21.4

19.2

21.2

89.7

23.0

68.0

>100

10d

47.3

38.0

75.4

49.1

87.2

15.6

83.1

88.5

10e

24.6

23.3

38.3

24.2

81.8

29.0

32.8

94.2

10f

78.8

71.3

98.4

73.3

88.0

42.9

78.8

>100

10g

69.2

69.9

82.3

74.7

88.2

38.3

72.0

>100

10k

84.4

67.9

81.3

80.0

89.2

68.1

82.2

> 100

11i

57.5

43.1

76.5

58.7

67.5

55.1

4.9

21.6

Liver microsomal stability (% remaining during 30 minutes at 1 µM). b% of control activity at 10 µM.

Table 6. Mouse oral PK property of 10ka

Parameter

10k

Dose (mg/kg)

10

AUCinf (ng hr/ml)

52464.8 ± 20913.0

AUClast ( ng hr/ml)

36363.7 ± 5340.0



a

Cmax (ng /ml)

2406.7 ± 434.7

t1/2 (hr)

12.4 ± 7.9

tmax (hr)

2.7 ± 1.2

MRTinf (hr)

18.1 ± 11.3

10k was formulated as a solution in 10% DMAC, 10 % Tween80, 80% HP-β-CD (20% in water).

Docking study of 10d, 10k and 11a To gain an insight into the binding mode of the GNF-7 analogues, molecular docking studies were performed using GNF-7, 10d, 10k, and 11a and the kinases ACK1 and GCK (Figure 2). The results suggest that these four inhibitors make a hinge contact through a pair of hydrogen bonds with Ala208 of ACK1 and Cys93 of GCK, respectively. The amide linker in these compounds also participates in interactions with ACK1 and GCK through a pair of H-bonds, which is a typical binding mode of type II kinase inhibitors. While the pyridine nitrogen of GNF-7 forms a hydrogen bond with the hydroxyl group of Tyr27 in GCK, no such interaction exists with Phe137 of ACK1, which might responsible for the higher potency of GNF-7 against GCK than against ACK1 (Figure 2a). The nitrogen on piperazinylethanone moiety of 10d contributes additional hydrogen bonding interactions with Asp215 of ACK1 and Glu100 of GCK respectively (Figure 2b). This interaction might serve as a driving force for improved binding affinity to each of the kinases. Especially, the four hydrogen bonds existing between 10d and Cys93/Tyr27/Glu100 in GCK could be the source of its exceptional inhibitory activity against GCK (IC50 = 1 nM). It is of note that 10k possesses high inhibitory activities against both ACK1 and GCK even though it does not participate in hydrophobic interactions in the hinge region besides a pair of hinge contact (Figure 2c). The trifluoromethylphenyl group in the tail parts of GNF-7, 10d, and 10k is likely ideal for binding in the hydrophobic pocket of GCK. The results of a previous docking study of NG25 on GCK also demonstrated that the hydrophobic pocket composed of Ile64 and Gln60 provides a suitable binding site for the trifluoromethylphenyl group.25 In this regard, the unsubstituted phenyl tail of 11a, lacking the CF3 moiety, might be too small to


Page 18 of 78

Page 19 of 78 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


tightly interact with GCK (Figure 2d). Accordingly, 11a displays a greatly decreased activity against GCK (IC50 = 1080 nM). Unlike GCK, no such binding site exists in ACK1 for interaction with the CF3. Therefore, 11a, which lacks the CF3 group, has a similar level of inhibitory activity against ACK1 compared with GNF-7.

Figure 2. Binding models of inhibitors on ACK1 (left) and GCK (right). (a) GNF-7, (b) 10d, (c) 10k and (d) 11a

X-ray co-crystal structure of ACK1-10d complex We determined X-ray co-crystal structure of 10d bound to the kinase domain of ACK1 (Table 7 and Figure 3). Unfortunately, X-ray co-crystal structure of GCK-10d complex could not be obtained. The X-ray co-crystal structure reveals that 10d is bound on ACK1 as a typical Type II kinase inhibitor. The amide linker of 10d forms hydrogen bonds with the K158-E177 salt bridge as well as with the backbone amino group of D270 at a distance of 3.1 and 2.9 Å. The trifluoromethylphenyl group of 10d binds to the hydrophobic pocket, thereby forcing the DFG F271 out of the ATP binding pocket. The nitrogen on the pyrimidine ring and the NH group between the pyrimidine and the pyridine make a pair of hydrogen bonds with the A208 in the hinge region at a distance of 2.6 and 2.8 Å. The pyrimidine ring orients for π- π interaction with F271. The side chains



Page 20 of 78

of the L184, L189, L243, F248, I190 and L259 improve binding affinity of 10d through hydrophobic interaction. In contrast to molecular docking analysis (Figure 2b) suggesting a hydrogen bond interaction between the piperazine moiety of 10d and D215-ACK1, the X-ray co-crystal structure reveals that a water molecule forms a hydrogen bonding with the nitrogen on piperazine of 10d at a distance of 3.3 Å as well as with the backbone amino group of S212 at a distance of 2.5 Å. This water-mediated hydrogen bond network contributes to the affinity of 10d on ACK1 and leads to the higher potency of 10d against ACK1 compared with GNF-7.

Table 7. Data collection and refinement statistics for ACK1 in complex with 10da

ACK1-10d Wavelength (Å)

0.97950

Resolution range (Å)

50.00 - 2.20 (2.24 - 2.20)

Space group

P 1 21 1

Unit cell a, b, c

71.18 43.12 93.89

α, β, γ

90 98.34 90

Multiplicity

3.8 (2.9)

Completeness (%)

98.2 (87.0)

Mean I/sigma (I)

18.64 (1.36)

Wilson B-factor

40.26

R-merge

0.147 (0.906)

R-work

0.2060 (0.2996)

R-free

0.2566 (0.3242)

Number of atoms

4074

Macromolecules

3885

Ligands

96


Page 21 of 78 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


Water

93

Protein residues

481

RMS (bonds)

0.009

RMS (angles)

1.44

Ramachandran favoured (%)

96.76

Ramachandran outlier (%)

0.22

Clash score

4.57

Average B-factor

50.69

Macromolecules

50.78

Ligands

50.62

Solvent

46.99

a

Statistics for the highest-resolution shell is shown in parentheses.



Figure 3. X-ray crystal structure of 10d bound to ACK1 (PDB code 5ZXB). (a) Overall fold of the ACK1-10d complex structure. Some residues (135-137, 161-169, and 272-292) are missing in the structure due to a lack of electron density. (b) Protein residues are represented by white, and 10d is designated by gold. Sulfur is displayed in yellow, nitrogen in blue, oxygen in red, and fluoride in cyan. Lys158 is eliminated for clarity.

Kinase selectivity profile of 10k In order to assess the kinase selectivity of 10k, kinome-wide inhibition profiling was performed at 1 µM (Figure 4 and Table S1). Among a total of 351 kinases, 33 kinases were inhibited more than 90%, including a number of tyrosine kinases (c-Src, YES, LCK, BMX, LYN, FYN, FMS, FGR, CSK, HCK, RET, ABL2, PDGFRβ, DDR2, DDR1, EphB1, KDR, BLK, ABL1, PDGFRα, JAK1, JAK2, FGFR2, FGFR1, EphA1), SIK1, RAF family (ARAF, BRAF, RAF1), and p38 family (p38α, 38β) as well as ACK1 and GCK. This indicates that 10k is a multi-targeted kinase inhibitor, like GNF-7. 10k is capable of inhibiting several kinases besides ACK1 and GCK, suggesting that offtarget effects are associated with the anti-proliferative activity of 10k.


Page 22 of 78

Page 23 of 78 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


Figure 4. (a) Kinome-wide selectivity profiling of 10k at 1 µM. Kinases showing > 90% inhibition are indicated with red circles. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). (b) The list of 33 kinases out of 351 kinases showing > 90% inhibition.

To investigate whether off-targets also contribute to the anti-proliferative activities of nonselective GNF7 analogues, we knocked down both ACK1 and GCK in OCI-AML3 cells using electroporation method and assessed the anti-proliferative activities of several analogues (Figure S1). The double siRNA-mediated ACK1/GCK knockdown in OCI-AML3 cells resulted in about 40% inhibition of cell growth. Treatment of ACK1/GCK dual inhibitors to the double knockdown OCIAML3 cells further suppressed the cell growth. The knockdown experiment results suggest that other targets besides ACK1 and GCK are involved in the anti-proliferative activities of these compounds.

Effects on AKT/mTOR and GCK signalings



In order to validate the results of the SAR study in a cellular context, western blot analysis in Ba/F3-NRAS-G12D and OCI-AML3 cell lines was performed. Considering both in vitro potency and metabolic stability, we chose derivatives 10d, 10e, 10g, 10k and 11i. After 2 h treatment with each compound, the cells were subjected to western blot analysis. As a consequence of our previous observation that GNF-7 suppresses AKT/mTOR signalling and GCK downstream in Ba/F3-NRASG12D cells,3 we examined the phosphorylation level of p70S6K1, AKT, JNK and p38 in both Ba/F3NRAS-G12D and OCI-AML3 cell lines (Figure 5a and 5b). All five compounds at 1 µM were found to attenuate phosphorylation of p70S6K1, JNK and p38, and moderately suppressed phosphorylation of AKT (S473). Analogue 10d which possesses an excellent in vitro potency, dramatically abolished phosphorylation of downstream molecules which is consistent to our SAR study. Similar results were observed using OCI-AML3 cells. All compounds (10d, 10e, 10g, 10k, 11i and GNF-7) caused a decreased level of phosphorylation of p70S6K1, AKT (S473), JNK and p38 (Figure 5b). Compound 10k displayed a moderate suppression of AKT/mTOR signalling while it completely abolished GCK signalling at 1 µM. We further examined dose dependent inhibition of compound 10d and 10k in OCI-AML3 (Figure 5c). 10d and 10k showed dose-dependent diminish of p70S6K1, AKT and p38 phosphorylation. This finding indicates that the kinase inhibition data gained in the SAR study correlates with the activities in Ba/F3 cells transformed with the NRAS mutation and human OCIAML3 cell line expressing the NRAS mutation.


Page 24 of 78

Page 25 of 78 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


Figure 5. Effect on downstream signalling inhibition. The phospho-p70S6K1, phospho-AKT (S473), phospho-JNK phospho-p38 levels in Ba/F3-NRAS-G12D (a) and OCI-AML3 (b) treated with indicated compounds for 2 h. Compounds at 1 µM attenuated phosphorylation of each molecule. Actin was used for loading control. (c) 10d and 10k block both AKT/mTOR signalling and GCK signalling in a dose-dependent manner in OCI-AML3.

Induction of apoptosis and cell cycle arrest in NRAS mutant cell lines To determine if the anti-proliferative effect of the new GNF-7 analogues is mainly responsible for apoptosis and cell cycle arrest, we examined the level of pro-apoptotic markers (cleaved PARP and cleaved caspase 3) and anti-apoptotic markers (bcl-2 and MCL1) by using western blot analysis. Analogues 10d and 10k were explored to assess their ability to induce apoptosis in both Ba/F3-NRAS-G12D and OCI-AML3 cells. For this purpose, cells were treated for 24 h with indicated concentrations of compounds (Figure 6a). In both Ba/F3-NRAS-G12D and OCI-AML3 cells, 10d at 1 µM was found to dramatically increase the levels of both cleaved PARP and cleaved caspase 3 and diminished bcl-2 and MCL1 in a similar manner as does GNF-7. Compound 10k increased apoptotic markers in OCI-AML3 at 1 µM and significantly increases the level of apoptotic markers in Ba/F3NRAS-G12D cells at 5 µM (Figure S2b). Flow cytometry analysis was carried out next using 1 µM of each analogue and by staining apoptotic cells with annexin V and propidium iodide (Figure 6b and Figure S3). Treatment with 10d led to a noticeable increase of apoptotic cells while treatment with 10k slightly increases the number of apoptotic cells in Ba/F3-NRAS-G12D cells, a finding that is consistent with the western blot results. In OCI-AML3, apoptotic cells were increased up to 15-30% by treatment with 10k and 10d. It is noteworthy that 10d displays better or similar apoptotic induction effect in Ba/F3-NRAS-G12D and OCI-AML3 cells compared to GNF-7. We also observed dosedependent apoptosis occurs by treatment with 10k in both cell lines. (Figure 6c and Figure S2a). Next, we analysed cell cycle phase after treatment of each compounds (Figure 7). G0-G1 arrest was mainly induced by 10d at 1 µM. It was compatible with that of GNF-7. 10k at 1 µM also



increased G0-G1 population. Taken together, these compounds suppressed cell proliferation via apoptosis and cell cycle arrest.

Figure 6. Effect on the cell apoptosis. (a) Western blot for apoptotic molecules in Ba/F3-NRAS-G12D (upper panel) and OCI-AML3 (low panel) treated with indicated compounds for 24 h. Cleaved PARP form was induced by each compound at 1 µM in OCI-AML3. Cleaved caspase 3 was increased by 1 µM of 10d and GNF-7 in Ba/F3-NRAS-G12D cells (upper panel). Actin was used for loading control. (b) % of apoptotic cells detected by FACS analysis in Ba/F3-NRAS-G12D (upper panel) and OCIAML3 (low panel). Apoptotic cells were determined via dual-staining of FITC-conjugated annexin V and propidium iodide after 24 h treatment of indicated compounds at 1 µM. All experiments were performed in three times, and bar graph represents the average (n=3) and SD. One-way ANOVA; ** P < 0.05. (c)

Dose dependent increase of apoptotic cells by compound 10k in Ba/F3-NRAS-G12D

(upper panel) and OCI-AML3 (low panel). Each cell line was treated with indicated concentrations of 10k for 24 h and subjected to flow cytometry analysis. All experiments were performed in three times, and bar graph represents the average (n=3) and SD. One-way ANOVA; ** P < 0.05.


Page 26 of 78

Page 27 of 78 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


Figure 7. Induction of G0-G1 arrest after compound treatment. (a-b) OCI-AML3 were treated with indicated concentration of each compound for 24 h and the percentage of each cell cycle phase were analysed by Flow cytometry after propidium iodide staining. 1 µM of each compound significantly increase population of G0-G1 phase.

Suppression of anchorage independent growth To investigate if 10d, 10k and 11i block tumorigenesis of NRAS mutated AML cell line, we carried out soft agar assay in OCI-AML3 (Figure 8). Incubation with each compound at two different concentrations (0.1 and 1 µM) for 14 days suppressed anchorage-independent growth in terms of both colony number and colony size of OCI-AML3 (Figure 8b and 8c). Especially, it is noteworthy that 1 µM of 10d completely reduced both colony number and colony size compared to the DMSO treated cells. Furthermore, the effect caused by 1 µM of 10d is superior to that promoted by GNF-7, indicating that 10d is more effective than GNF-7 in inhibiting anchorage-independent growth of OCIAML3. 10k and 11i also diminished colony number and size at 1 µM while they moderately decreased colony formation at 0.1 µM concentration, indicating that blockade of ACK1 and GCK activity prevents tumorigenesis of NRAS mutant driven AML cell line.



Figure 8. Anchorage independent growth inhibition of 10d, 10k and 11i in OCI-AML3. (a) Cells embedded in 0.7% low melting agar were incubated with the indicated concentrations of compounds for 2 weeks and colonies were observed (n = 3 experiment per condition). (b) Average number of colonies per well was automatically counted using Image J software and shown in bar-graph. (+/- SD, One way ANOVA; **** p < 0.0001, *** p < 0.0005, ** p < 0.05). (c) Average size of colonies per well was automatically measured using Image J software and shown in bar-graph. (+/- SD, One way ANOVA; **** p < 0.0001, *** p < 0.0005, ** p < 0.05).

In vivo efficacy study The anti-leukemic efficacies of 10k (15 mg/kg/day) and GNF-7 (8 mg/kg/day) compared with a vehicle control were evaluated using the Ba/F3-NRAS-G12D/Luciferase-bearing blood circulating mouse model. Analogue 10k was found to display a desirable mouse pharmacokinetic profile (data not shown) for oral administration. Differences in tumor engraftment and progression among the different treatment groups (po, qd) were monitored by using bio-luminescence imaging (Figure 9a) and quantitative bio-photonic imaging analysis (Figure 9b). Derivative 10k (15 mg/kg/day)


Page 28 of 78

Page 29 of 78 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


diminished more than 50% of leukemic cells at day 20, which is comparable to the efficacy of GNF-7 (8 mg/kg/day). The incidence of death due to leukemia progression was also evaluated following administration of 10k (po, qd) (Figure 9c). Similar to the results arising from the blood circulating model, 10k (15 mg/kg/day) has a level of life-span extension that is similar to that of GNF-7 (8 mg/kg/day). Moreover, the 10k (25 mg/kg/day)-treated group shows a median survival of 35 d, which is almost 50% longer compared with the vehicle control group. In GNF-7 (8 mg/kg/day)-treated group, noticeable body weight loss (> 15%) was observed which is a common symptom of in vivo toxicity. However, 10k (15 mg/kg/day)-treated group exhibited a very small weight change (Figure 9d). It should be noted that 10k is superior to GNF-7 as prominent toxicity was found in mice treated with GNF-7. The anti-leukemic efficacy of 10k was also assessed in AML xenograft model of OCI-AML3 cells. The tumor volumes of the group treated with vehicle, GNF-7 (8 mg/kg/day), 10k (15 mg/kg/day) and 10k (25 mg/kg/day) were 100%, 53.8%, 41.9%, and 14.9% respectively. Overall, the dosedependent anti-leukemia effect of 10k has been demonstrated using life-span extension and OCIAML3 xenograft AML models.



Figure 9. Anti-leukemic efficacy of vehicle alone, GNF-7, and 10k in Ba/F3-NRAS-G12D blood circulating model and OCI-AML3 xenograft model. (a) Bioluminescence images of mice administered with vehicle only, GNF-7, or 10k once daily (n=4 per cohort). Immunodeficient mice were tail intravenously injected with Ba/F3-NRAS-G12D/Luc cells and treated with vehicle, GNF-7 (8 mg/kg/day, po, qd), or 10k (15 mg/kg/day, po, qd) for 3 w. Bioluminescence images were obtained once every two days using IVIS spectrum (PerkinElmer) after administration of luciferin (150 mg/kg). (b) Ba/F3-NRAS-G12D/Luc cell progression was monitored by quantitative biophotonic imaging analysis of control and compound-treatment groups. Quantitation from the in vivo imaging using Opti-view software (Version 2.02, ART Inc.). Results represent Mean ± SEM, n=4 per group, **p

First SAR study for overriding NRAS mutant driven ... - ACS Publications

Recommend Documents