Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor

Abstract

We have designed and synthesized 9H-pyrimido[4,5-b]indole-containing compounds to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small molecules showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compound, 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, 31 achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-negative breast cancer xenograft models in mice. Determination of the co-crystal structure of 31 with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-containing proteins shows that 31 is a highly selective inhibitor of BET proteins. Our data show that 31 is a potent, selective and orally active BET inhibitor.

Graphical Abstract

INTRODUCTION

Acetylation of lysine residues at the histone tail plays a central role in the dynamic regulation of chromatin-based gene transcription. Bromodomain-containing proteins (BRDs) function as one of epigenetic “readers” by binding to acetyl lysine residues in the histone tail and regulate gene expression.^1,2 Bromodomain and extra-terminal (BET) proteins are one sub-family of BRDs which has four members, BRD2, BRD3, BRD4, and testis-specific BRDT. BET proteins have been implicated in a variety of human diseases, including NUT midline carcinoma,³ MLL-fusion leukemia,⁴ inflammatory diseases,⁵ and acute heart failure.⁶

Structurally, BET proteins consist of two tandem bromodomains (BD1 and BD2) both of which bind to acetyl-lysine (Ac-K) residues in the histone tails. Recently, small molecule BET inhibitors, such as JQ-1(1, Figure 1),³ have been discovered that bind to the acetyl-lysine binding pocket in the BET proteins.^{3,5,7–17,18,19} Potent and selective BET inhibitors are powerful tools with which to investigate the role of BET proteins in different biological and pathological processes.^{3–6,18,20–27} In preclinical studies, BET inhibitors have demonstrated their therapeutic potential in the treatment of human cancers, inflammation, HIV infection, acute heart failure, and other disorders. Several BET inhibitors, including OTX-015 (2),¹⁸ CPI-0610 (3)²⁸ I-BET762 (4) (Figure 1) are currently being evaluated in human clinical trials for the treatment of different cancers. Recently obtained clinical data have provided an important proof of the concept that BET small-molecule inhibitors may have a therapeutic potential for the treatment of several forms of human cancer.^29–32

Figure 1:

Representative, Previously Reported Potent BET Bromodomain Inhibitors

Our laboratory previously reported the design of a new class of BET bromodomain inhibitors containing a tricyclic 5H-pyrido[4,3-b]indole structure, exemplified by RX-37 (6).³³ This compound binds to BET proteins with nanomolar affinities and demonstrates high selectivity over other bromodomain-containing proteins.³³ It potently inhibits cell growth in the MV4;11 and MOLM-13 acute leukemia cell lines harboring mixed lineage leukemia 1 (MLL1) fusion protein and has a high specificity for the K562 leukemia cell line harboring BCR-ABL fusion protein.³³ Hence, 6 represents a promising lead compound for further optimization toward our goal of development of a new class of potent, selective, efficacious BET inhibitors for the treatment of human cancers and other medical conditions.

Our previously reported synthetic routes to 6 are not suited to large scale synthesis, and this has limited the exploration of its structure-activity relationships (SAR) and potential in vivo evaluation of this class of compounds. We have therefore decided to develop alternative tricyclic core structures, which allowed us to explore the SAR more extensively and comprehensively. In the present study, we report our efforts to obtain potent, efficacious, and orally active BET inhibitors. This has resulted in the discovery of compound 31, which binds to BET proteins with high affinities and potently inhibits cell growth in acute leukemia cell lines harboring the MLL1 fusion protein and in a panel of human breast cancer cell lines. Significantly, compound 31 has an excellent oral pharmacokinetic profile and orally administered, effectively inhibits tumor growth in mice.

RESULTS AND DISCUSSION

Analysis of the co-crystal structure of 6 in a complex with BRD4 BD2 protein shows that one additional nitrogen atom can be inserted into the 5H-pyrido[4,3-b]indole structure (Table 1) without causing any unfavorable interactions with the protein. This led to the design of compounds containing the 9H-pyrimido[4,5-b]indole core structure, which can be more efficiently synthesized than the core structure of 6 as shown in Schemes 1 and 2.

Table 1.

Design of BET inhibitors containing a 9H-pyrimido[4,5-b]indole tricyclic core structure

Compound	BRD4 BD1		BRD4 BD2
	IC50 (nM)	Ki (nM)	IC50 (nM)	Ki (nM)
1	46.7±4.4	14.9±2.6	42.2±9.1	12.0±3.5
4	145±22	46.4±6.4	77.5±15.0	23.0±4.2
5	31.7±7.7	9.0±2.9	226±44	74.8±8.6
6	75.5±6.2	24.7±1.0	36.5±8.9	12.2±1.6
7	1125±92	387±46	1760±396	476±86
8	39.2±11.6	12.0±3.5	26.4±5.3	10.4±1.1

Scheme 1:

Synthesis of key intermediate 7

Scheme 2:

Synthesis of common intermediate, S10

We first synthesized compound 7 to test this new core for the design of potent BET inhibitors. This compound binds to recombinant human BRD4 BD1 and BD2 proteins with K_i = 387 nM and 476 nM, respectively (Table 1), in our competitive fluorescence-polarization assays³³. Since this compound still lacks a moiety to interact with the well-defined “WPF” hydrophobic pocket in the BRD4 protein, it therefore represents a reasonable starting point for further optimization.

Our previous SAR data of compound 6 and its analogues demonstrated that targeting the “WPF” hydrophobic pocket in BET proteins is a very effective way of improving the binding affinities of BET inhibitors.³³ Overlay of our modeled structure of 7 in a complex with BRD4 BD2 and the co-crystal structure⁴ of compound 5 (I-BET151) complexed with BRD4 BD2 led to the design of 8 (Table 1), which contains the same hydrophobic group as was used in 5 to target the “WPF” hydrophobic pocket. Compound 8 binds to BRD4 BD1 and BD2 with K_i = 12.0 nM and 10.4 nM (Table 1) and is thus >30-times more potent than 7, further supporting the precept that targeting the WPF binding pocket can indeed significantly improve the binding affinity of BET inhibitors. In comparison, compound 8 is as potent as 1 and 6 in binding to both BDR4 BD1 and BD2 proteins.

In our previous study, we found that substituted 5-membered aromatic rings such as that in compound 6 can be used to effectively target the “WPF” pocket in BET proteins.^{7,10–17,28,33} Accordingly, we next further explored the SAR resulting from targeting the “WPF” pocket using different 5-membered aromatic rings (Table 2). Among compounds 9–13, which contain an unsubstituted 5-membered ring, compounds 11 and 12 containing a furan and a pyrrole respectively, have the highest binding affinities and are both >5-times more potent than 7 in binding to both BRD4 BD1 and BD2 proteins.

Table 2.

Design of BET inhibitors targeting the WPF binding pocket using five-membered aromatic rings

Compound	BRD4 BD1		BRD4 BD2
	IC50 (nM)	Ki (nM)	IC50 (nM)	Ki (nM)
9	2060±420	655±269	1882±406	641±200
10	393±127	131±50	199±81	57.0±17.3
11	155±47	44.3±13.9	180±37	56.4±11.7
12	165±50	51.9±18.6	108±37	30.6±9.0
13	1456±441	456±150	1139±198	335±25
14	102±40	41.2±16.9	164±44	68.7±21.4
15	51.7±15.0	19.7±8.2	63.5±16.4	20.2±9.2
16	40.9±14.2	18.5±4.0	89.7±18.5	31.2±11.8
17	24.1±5.5	7.0±3.3	11.7±2.0	2.7±0.5

In the co-crystal structure of compound 6 complexed with BRD4 BD2, the cyclopropyl group is inserted deeply into the WPF hydrophobic pocket,³³ suggesting that additional hydrophobic substituent(s) on the 5-membered aromatic ring may further improve binding affinity to the BRD4 protein. Indeed, 14 and 15 with dimethyl substituents on the 3-isoxazole and 3-(1H-pyrazole) groups are both more potent than the unsubstituted compounds 9 and 10. Compound 14 is 9–10 times more potent than 9, and 15 is 3–7 times more potent than 10 in binding to both the BD1 and BD2 proteins from BRD4. In particular, 15 has K_i values of 19.7 and 20.2 nM to the BD1 and BD2 proteins, and is therefore a fairly potent BRD4 inhibitor.

Encouraged by the good binding affinities of 15 to BRD4 BD1 and BD2 proteins, we further modified 15 to obtain 16 and 17. Compound 16 with an additional methyl substituent on the 5-membered 1H-pyrazole ring of 15 has similar binding affinities to BRD4 BD1 and BD2 proteins, as compared to 15, but 17, which targets the WPF pocket with the same pyrazole group as compound 6, binds to BRD4 BD1 and BD2 with K_i values of 7.0 nM and 2.7 nM, respectively and is thus more potent than compound 6. It is surprising to observe such potency gap between 6 and 17 given these two molecules differ by only one atom, i.e. a carbon to a nitrogen atom.

BET inhibitors such as compound 1 and 6 have been shown to be very effective in inhibition of cell growth in MV4;11 and MOLM-13 acute leukemia cell lines harboring MLL1 fusions.^3,33 Indeed, 17 potently inhibits cell growth in both the MV4;11 and MOLM-13 cell lines with IC₅₀ values of 6 and 36 nM, respectively (Table 3). In direct comparison, 17 is more potent than 1, 5 and 6 in inhibition of cell growth in these two acute leukemia cell lines, and is a very potent BET inhibitor.

Table 3.

Inhibition of cell growth by BET inhibitors in MV4;11 and MOLM-13 cell lines.

Compound	IC50 (nM)		Compound	IC50 (nM)
	MV4;11	MOLM-13		MV4;11	MOLM-13
1	24±19	56±24	24	29	380
4	93±45	241±58	25	15	262
5	162±112	228±52	26	13±8	139±24
6	20±9	66±14	27	>10000	>10000
11	2863	>10000	28	>10000	>10000
12	253±123	612±97	29	2±1	20±4.6
13	1062	9271	30	17±2	69±34
14	182±91	709±116	31	26±5	53±11
15	99±36	233±13	32	28±13	76±45
16	95±30	193±21	33	99±43	220±101
17	6±3	36±2	34	47±11	124±78
18	57	205	35	295±48	656±240
19	190±89	489±9	36	>1000	1140
20	76±31	374±52	37	334	NT
21	6.6±1.6	65±11	38	77	971
22	5.1 ±2.8	51±6	39	69±13	243±23
23	13±6	127±21	40	423	716

We evaluated compound 17 in a quick pharmacokinetic study with three time points to assess its oral exposure in mice. Compound 17 achieves drug concentrations of 2200 ± 331, 872 ± 351 and 72 ± 46 ng/ml, respectively, at 1, 3 and 6 h time-points with 25 mg/kg oral administration, indicating that 17 has a reasonable oral exposure in mice.

We next turned our attention to modifications of 12, which contains a pyrrole ring. Compound 12 has reasonable binding affinities to both BRD4 BD1 and BD2 with K_i values of 30.6 nM and 51.9 nM, respectively and sub-micromolar potencies in cell growth inhibition in the MV4;11 and MOML-13 cell lines (Table 3). Our computational modeling suggested that a fused [5,6] bicyclic system can effectively target the WPF pocket and we therefore synthesized a series of compounds containing a bicyclic [5,6] ring system and whose activities are summarized in Table 4.

Table 4:

BET inhibitors containing [5,6] fused aromatic rings targeting the WPF binding pocket.

Compd	BRD4 BD2		BRD4 BD1
	IC50 (nM)	Ki (nM)	IC50 (nM)	Ki (nM)
12	165±50	51.9±18.6	108±37	30.6±9.0
18	20.3±4.9	4.9±2.2	5.5±2.4	1.1±0.7
19	11.6±3.6	7.0±2.4	22.7±7.3	7.1±2.2
20	12.2±4.2	2.6±0.8	2.2±1.0	< 0.5
21	5.0±1.7	0.7±0.1	1.1±0.5	< 0.5
22	5.6±1.9	0.9±0.3	2.3±1.1	< 0.5
23	10.8±3.0	4.2±1.1	25.9±11.8	9.1±4.3
24	32.4±10.2	9.3±3.8	12.2±4.4	2.8±1.1
25	29.4±.6	9.9±3.3	9.4±2.7	2.8±1.1
26	48.7±17.3	28.0±3.2	26.5±6.1	6.5±0.6
27	147±85	85.1±12.5	74.9±20.7	22.5±6.3
28	> 1000	> 1000	> 1000	> 1000

Compound 18, containing an indole group, binds to BRD4 BD1 and BD2 with K_i values of 4.9 nM and 1.1 nM, respectively and is therefore 10-times more potent than 12 in binding to both the BD1 and BD2 domains of BRD4. Replacement of the indole in 18 with 1H-pyrrolo[2,3-c]pyridine resulted in 19, which has a binding affinity to BRD4 BD1 similar to that of 18 but is >5-times less potent than 18 in binding to BDR4 BD2. Installation of a fluorine atom at C4 of the indole in 18 yielded 20, which binds to BRD4 BD1 and BD2 proteins with very high affinities (K_i = 2.6 and 1 nM, respectively). Addition of a methyl group at C2 of 18 led to compound 21, which also achieves a very high binding affinity to both BRD4 BD1 and BD2 (K_i < 1 nM). Combination of the fluorine and methyl substitutions in compounds 20 and 21 yielded 22, which has a high binding affinity to both BRD4 BD1 and BD2 proteins (K_i < 1 nM). Replacing C6 of the indole group in 21 by a nitrogen atom led to 23, which is 10-times less potent than 21. The weaker binding affinity of 23 than 21 is consistent with the hydrophobic nature of the WPF binding pocket.

Encouraged with the high binding affinities of these indole-containing compounds, we synthesized a series of compounds containing [5,6] bicyclic, non-indole groups. Compound 24, with a 2-methylbenzofuran and 25 containing a benzoimidazole bind to BRD4 BD1 and BD2 with high affinities but are less potent than 18. Compound 26, containing a pyrazolo[1,5-a]pyridine is 4-times less potent than 18 and compound 27, containing a 1-methyl-1H-indazole is >10-times less potent than 18. Compound 28, with a 1H-benzo[d][1,2,3]triazole fails to show any appreciable binding to BRD4 BD1 and BD2 proteins at 1 μM. These data thus show that not only the shape but also the electronic properties of the bicyclic aromatic rings at this site play an important role in the binding to BRD4 BD1 and BD2 proteins.

We evaluated these compounds for their ability to inhibit cell growth in the MV4;11 and MOML-13 cell lines, and the results are summarized in Table 3. Consistent with their very high binding affinities to BRD4 BD1 and BD2 proteins, compounds 21 and 22 display very potent cell growth inhibitory activity in both the MV4;11 and MOLM-13 cell lines. Compound 21 has IC₅₀ values of 6.6 nM and 65 nM, respectively, in the MV4;11 and MOLM-13 cell lines, while 22 has IC₅₀ values of 5.1 nM and 51 nM, respectively in these two leukemia cell lines. Compounds with weaker binding affinities to BRD4 BD1 and BD2 proteins also display weaker cell growth inhibitory activity in both the MV4;11 and MOLM-13 cell lines.

Based upon its high binding affinities to BRD4 BD1 and BD2 proteins and potent cell growth inhibitory activity, we evaluated 21 for its oral bioavailability in rats. The pharmacokinetic (PK) data show that 21, orally administered to rats at 25 mg/kg can achieve a reasonable plasma exposure and achieves a Cmax of 1470 ng/ml, or approximately 3 μM (Table 5).

Table 5.

Pharmacokinetics of compounds 21 and 31 in rats and mice

Compound	Species	Route (Dose)	t1/2 (h)	Cmax (ng/mL)	AUC0-t (ngh/mL)	Vss (L/kg)	CL (L/h/kg)	F (%)
21	Rat	PO (25 mg/kg)	1.7	1396.7±277.4	13444.7±933.4	-	1.9
31	Rat	IV (5 mg/kg)	2.4	-	6343	2.0	0.79	-
	Rat	PO (25 mg/kg)	2.9	7333	29547	-	0.88	93%
	Mouse	IV (5 mg/kg)	0.5	-	3688.8	2.75	1.36	-
	Mouse	PO (25 mg/kg)	1.60	983.1	9135	-	-	50%

A shortcoming we discovered in 21 is that although it has a good microsomal stability in rat microsomes (T_1/2 > 60 min), it has a poor microsomal stability in mouse and human microsomes with T_1/2 of 4 and 8 minutes, respectively (Table 6). Our investigation of 21 in human liver microsomes showed that oxidation of the indole group is the major metabolic weak spot and consequently, we next focused our modifications on the indole group in 21 with the objective of improving its metabolic stability and oral pharmacokinetics.

Table 6.

Microsomal stability of selected BET inhibitors.

Compound	Microsomal Stability T1/2 (min)
	Mouse	Rat	Human
21	4	>60	8
29	>60	>60	9
30	>60	>60	>60
31	>60	>60	>60

First, we introduced a methyl group onto the tricyclic core structure in 21, generating compound 29 (Table 7) which binds to BRD4 BD1 and BD2 proteins with very high affinities (K_i < 1 nM). Consistent with its high binding affinity, 29 potently inhibits cell growth in the MV4;11 and MOML-13 cell lines achieving IC₅₀ values of 2 and 20 nM, respectively (Table 3), and is therefore 3-times more potent than 21. Compound 29 has an excellent microsomal stability in mouse and rat microsomes (T_1/2 > 60 min) but has a poor microsomal stability in human microsomes (T_1/2 = 9 min) (Table 6).

Table 7.

BET Inhibitors containing indole or quinoline to target the WPF pocket.

Compound	BRD4 BD2		BRD4 BD1
	IC50 (nM)	Ki (nM)	IC50 (nM)	Ki (nM)
29	1.9±0.7	< 0.5	6.1±1.4	0.8±0.1
30	5.2±1.4	1.6±0.5	19.7±7.5	5.7±3.6
31	7.2±1.9	1.4±0.4	28.2±4.4	8.2±1.2
32	15.5±4.7	3.1±1.0	30.9±7.6	10.1±4.0
33	36.3±6.9	10.5±1.9	93.8±15.8	38.4±10.5
34	12.7±5.9	3.8±0.9	35.6±8.8	9.5±2.4
35	436±219	146±56	451±128	139±32
36	167±55	52.3±17.1	316±86	78.6±12.6
37	193±53	58.2±12.6	412±126	129±24
38	92.7±36.7	26.9±12.6	393±115	122±34
39	14.6±6.3	4.2±1.4	73.1±12.9	31.8±5.9
40	128±27	42.6±6.9	397±47	136±12

We next explored if replacement of the [5,6] bicyclic indole ring system with [6,6] bicyclic ring systems could lead to potent BET inhibitors with improved metabolic stability. Another issue we have found for compound 21 was its poor aqueous solubility. We have synthesized a series of compounds containing a quinoline ring system, which has a basic nitrogen in the bicyclic aromatic system and should have an improved solubility over compound 21 containing a less basic indole aromatic group. The test data from these compounds are summarized in Table 7.

Compound 31 (CD161) containing a quinolin-4-yl ring, binds to BRD4 BD1 and BD2 with K_i values of 8.2 nM and 1.4 nM, respectively (Table 7) and potently inhibits cell growth in the MV4;11 and MOLM-13 cell lines with IC₅₀ values of 26 and 53 nM, respectively (Table 3). Significantly, 31 has excellent microsomal stability in mouse, rat and human microsomes (Table 6). These data show that replacing the [5,6] bicyclic indole ring system with a [6,6] bicyclic quinoline ring system can maintain high binding affinities to BRD4 BD1 and BD2, and at the same time achieve potent cell growth inhibitory activity and excellent microsomal stability.

Based upon these encouraging results for 31, we synthesized a series of compounds containing quinoline ring systems in which the nitrogen in the quinoline ring is located in different positions. Compound 34 containing a quinolin-3-yl system is slightly less potent than 31 in both binding affinities to BRD4 BD1 and BD2 and also in inhibition of cell growth in the MV4;11 and MOLM-13 cell lines. Similarly, 32 with a quinolin-5-yl system is slightly less potent in its binding to BRD4 BD1 and BD2 proteins than 31 and is 2-times less potent than 31 in inhibition of cell growth in MV4;11 and MOLM-13 cell lines (Table 3). Compound 35, 36 and 37 containing a nitrogen in the 5, 6 and 7-positions respectively, are much less potent than 31 in both their binding affinities to BRD4 BD1 and BD2 proteins, as well as in inhibition of cell growth. Hence, among these compounds containing a quinolinyl ring system, 31 has the best binding affinities and cellular potencies.

We evaluated 31 for its pharmacokinetics in rats with the data provided in Table 5. At 25 mg/kg, orally administered, compound 31 achieves a Cmax of 7333 ng/mL, 6-times higher than that for 21 and an oral bioavailability of 93%. Compound 31 was also found to have much better solubility than 21; while 31 was readily soluble in the dosing vehicle (5% DMSO+10% Cremophor+85% saline), 21 was difficult to dissolve in the vehicle. One possible reason for the superior oral pharmacokinetics of 31 over 21 may be due to the improved solubility for 31. We further determined the pharmacokinetics of 31 in mice (Table 5). Compound 31 achieves a Cmax of 983.1 ng/mL with 25 mg/kg, oral administration and an oral bioavailability of 50% (Table 5). Hence, 31 has excellent oral pharmacokinetics in rats and moderate oral pharmacokinetics in mice.

To gain an understanding of its target selectivity, we evaluated compound 31 for its binding affinities to BET family proteins (BRD2–4 and BRDT) and other sub-families of bromodomain-containing proteins in the BROMOScan platform by DiscoverX³⁴ and the data are summarized in Table 8.

Table 8.

Binding affinities of 31 to different BET proteins and selectivity over other bromodomain-containing proteins, as determined using the DiscoverX BROMO Scan platform

Protein	Kd (nM)	Protein	Kd (nM)	Protein	Kd (nM)
BRD2 (BD1)	4.5	CREBBP	520	BRPF1	>4000
BRD2 (BD2)	0.9	CECR2	1600	BRPF3	>4000
BRD2 (BD1-BD2)	1.1	EP300	550	FALZ	>4000
BRD3 (BD1)	2.0	ATAD2A	>4000	GCN5L2	>4000
BRD3 (BD2)	0.69	ATAD2B	>4000	PBRM1(2)	>4000
BRD3 (BD1-BD2)	0.73	BAZ2A	>4000	PBRM1(5)	>4000
BRD4 (BD1)	8.4	BAZ2B	>4000	PCAF	>4000
BRD4 (BD2)	2.0	BRD1	>4000	SMARCA2	>4000
BRD4 (BD1-BD2)	1.8	BRD7	>4000	SMARCA4	>4000
BRDT (BD1)	15	BRD8 (BD1)	>4000	TAF1 (BD2)	>4000
BRDT (BD2)	5.5	BRD8 (BD2)	>4000	TRIM24	>4000
BRDT (BD1-BD2)	9.7	BRD9	>4000	WDR9 (BD2)	>4000

Compound 31 has K_d values of 4.5, 0.9, 2, 0.69, 8.4, 2, 15 and 5.5 nM, to BRD2 BD1, BRD2 BD2, BRD3 BD1, BRD3 BD2, BRD4 BD1, BRD4 BD2, BRDT BD1 and BRDT BD2 proteins, respectively. It binds to CREBBP, EP300 and CECR2 with K_d values of 520, 550 and 1600 nM, respectively, and has K_d values > 4,000 nM against other 21 bromodomain-containing proteins tested. Hence, 31 is a highly potent BET inhibitor and demonstrates a high selectivity over the other bromodomain-containing proteins.

Compound 31 was further tested for its potential inhibitory activity against 372 human kinases in the KINOMEscan platform by DiscoverX. The kinase screening data showed that 31 only has a modest inhibitory activity against PLK4 with an IC₅₀ value of 3.9 μM and has IC₅₀ values >50 μM against all other 371 kinases.

We next tested compound 31 for its in vivo efficacy in the MV4;11 xenograft model, with compound 5 used as a control (Figure 2). The efficacy data showed that both 5 and 31 have similar strong antitumor activity with a tumor growth inhibition of >80%, and both compounds did not cause weight loss or other signs of toxicity in mice (Figure 2).

Figure 2.

Antitumor activity of 31 in the MV4;11 acute leukemia xenograft model, with compound 5 included as a control.

In addition to acute leukemia cell lines, we evaluated the cell growth inhibitory activity of 31 in a panel of 19 human breast cancer cell lines. The resulting data, provided in Table 9 show that 31 potently inhibits cell growth in 9 breast cancer cell lines with IC₅₀ values < 1 μM, displays IC₅₀ values between 1–2 μM in 6 other cell lines and has IC₅₀ values > 2 μM in the remaining 4 cell lines. Therefore, while 31 is less potent against breast cancer cell lines than against acute leukemia MV4;11 and MOLM-13 cell lines carrying MLL1 fusion, it nevertheless potently inhibits cell growth against nearly 50% of 19 breast cancer cell lines tested with IC₅₀ values of 1 μM or better.

Table 9.

Inhibition of cell growth by compound 31 in a panel of breast cancer cell lines

Cell Line	Subtype	IC50 (nM)	Cell Line	Subtype	IC50 (nM)
MDA-MB-157	Basal B	39	HCC1954	Basal A	1604
MDA-MB-453	Luminal	188	HCC70	Basal A	1623
MDA-MB-361	Luminal	189	MDA-MB-436	Basal B	1698
MDA-MB-231	Basal B	244	MDA-MB-468	Basal A	1018
HCC38	Basal B	285	BT-20	Basal A	1713
HCC1428	Luminal	319	SUM-159PT	Basal B	>2000
BT-474	Luminal	434	HBL100	Basal-B	>2000
BT-549	Basal B	410	HCC1143	Basal A	>2000
SUM-149PT	Basal B	601	HCC1937	Basal A	>2000
HCC1395	Basal B	1233

To further evaluate its therapeutic potential, we evaluated 31 for its antitumor activity in the MDA-MB-231 xenograft model in mice (Figure 3). The data show that 31 is very effective in inhibition of tumor growth in the MDA-MB-231 xenograft model. During the treatment period, 31 at 40 mg/kg dosing daily achieved essentially complete tumor growth inhibition. Mice treated with 31 did not experience weight loss or other signs of toxicity throughout the entire experiment.

Figure 3.

Antitumor activity of 31 in the MDA-MB-231 breast cancer xenograft in mice

Previous studies have shown that BET inhibitors can effectively down-regulate c-Myc and upregulate p21 in tumor cells.⁴ Accordingly, we examined the effect of 31 in the MV4;11 cells on c-Myc and p21 levels (Figure 4). The resulting data showed that 31 is very effective in inducing rapid down-regulation of c-Myc at as early as the 1 h time-point and in a dose-dependent manner (Figure 4A). Furthermore, compound 31 is effective in inducing upregulation of the cell cycle regulator p21 and cleavage of PARP, a biochemical marker of apoptosis, in a dose-dependent manner (Figure 4B). Hence, our western blotting data further showed that compound 31 functions as a potent BET inhibitor in cells.

Figure 4.

Western blot analysis of the effect of compound 31 on c-Myc, PARP and p21 in the MV4;11 leukemia cells. (A) MV4;11 cells were treated with different concentrations of 31 for 1 h and c-Myc protein was probed by a specific antibody. (B) MV4;11 cells were treated with compound 31 for 24 h, and PARP, cleaved PARP (CL-PAPR) and p21 proteins were probed by specific antibodies. β-Actin was used as the loading control.

To understand the structural basis for the high binding affinity of 31 to BRD4, we successfully determined the co-crystal structure of 31 complexed with BRD4 BD2 (Figure 5). Overall, the co-crystal structure shows 31 binding to BRD4 BD2 with a binding mode similar to that assumed by compound 6. The dimethylisoxazole group of 31 penetrates deeply into the binding pocket and forms an extensive hydrogen bonding network with Asn433 and two conserved water molecules. One of the methyl groups in the dimethylisoxazole group binds to a hydrophobic pocket formed by residues Tyr332, Val380, Leu387 and Tyr390, and the other methyl group has hydrophobic contacts with Pro375, Phe376 and Val439. The tricyclic ring binds to the lysine channel. As envisaged in our design, the quinolinyl group inserts into the “WPF” pocket with the nitrogen atom exposed to solvent and the fused phenyl group in the hydrophobic pocket formed by Trp374, Pro375, Val439, Met442 and the hydrophobic portion of Glu438. The quinolinyl group of 31 has more extensive interactions than the cyclopropyl group of 6, consistent with the higher binding potency of 31 to BRD4. This co-crystal structure provides a solid structural basis for the high binding affinity of 31 to BRD4 BD2 and for further structure-based optimization.

Figure 5.

Crystal structure of BRD4 BD2 bound to 31 determined at 2.5 Å resolution. (A) BRD4 BD2 is shown as a grey cartoon with its sidechains interacting with 31 shown as sticks (carbon atoms in grey, nitrogens in blue, oxygens in red and sulfurs in gold. 31 is shown as sticks in the same coloring as the protein except carbon atoms are in yellow. (B). The omit Fo-Fc electron density map for compound 31 shown as a green grid contoured at 3σ. (C) Previously determined crystal structure of BRD4 BD2 complexed with 6 overlaid onto the co-crystal structure of BRD4 BD2 complexed with 31. Panel C is rotating 90° about the x-axis with respect to panel A. Compound 6 is shown as sticks with carbon atoms in cyan.

CHEMISTRY

The synthesis of 9H-pyrimido[4,5-b]indole-containing intermediate 7 is shown in Scheme 1.

Reaction conditions: a. Pd(PPh₃)₄, DME-H₂O, reflux overnight, >80% yield; b. NaH, anhydrous DMF, overnight, 53% yield; c. Zn, HOAc, 85 °C, 3 h, 47% yield; d, Ammonium formate 2.0 equiv, formamide, 175 °C, 16 h, 55% yield; e. POCl₃, 90 °C, 5 h, 66% yield

Suzuki coupling of a previously reported substituted bromobenzene (S1) and its regioisomer 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (ratio 2:1)³⁵ and S2 furnished S3 as a mixture of two isomers in >80% yield. SN_Ar substitution of the fluorine of S3 by the cynoacetate carbanion afforded S5, which was obtained as a single isomer after flash column chromatography purification. S5 was reduced, giving S6 by treatment with Zn-HOAc at 85 °C.³⁶ Condensation of a mixture of S6 and ammonium formate in formamide at 175 °C yielded S7 in 55% yield. Finally, chlorination of S7 provided the final key intermediate (7) in 66% yield.

The key 9H-pyrimido[4,5-b]indole-containing intermediate S10 was synthesized from S6 following the synthetic route depicted in Scheme 2.³⁶

Reaction conditions: a. S6 in dry MeCN, bubble HCl gas for 30 min, then reflux 2.5 h; b. S9 in EtOH, 10% NaOH aq., reflux 6 h, 78% over two steps; b. POCl₃, 90 °C, 5 h, 75% yield

To a dry solution of S6 in MeCN, excess hydrogen chloride gas was bubbled and the mixture was heated at reflux for 2.5 h. Removal of the volatile components furnished the crude intermediate S8, which cyclized into S9 upon reflux in basic aqueous EtOH. Chlorination of S9 provided the key intermediate S10 in 75% yield.

Compound 8 in Table 1 was synthesized following the synthetic route depicted in Scheme 3.

Scheme 3:

Synthesis of 8

Reaction conditions: a. 1-methylpyrrolidin-2-one (NMP), 140 °C, overnight.

An anhydrous solution in NMP of 7 and the corresponding amine was heated at 140 °C in a sealed tube overnight. The final products (8) were purified by reverse phase HPLC and obtained in the form of the trifluoroacetic acid salt. Compounds 13 and 25 were synthesized in a similar manner.

Compounds 9–12, 14–24, 26, 27, 29–40 in Tables 2–4 and 7 were synthesized from key intermediates 7 or S10 as shown in Scheme 4.

Scheme 4:

General Suzuki coupling reaction conditions

Reaction conditions: a. Pd(PPh₃)₄, DME-H₂O, K₂CO₃ 5.0 equiv., reflux overnight; or Pd(dppf)₂Cl₂-CH₂Cl₂, DME-Na₂CO₃ 2 M, reflux overnight.

Most boronic acids or pinacolboronic esters are commercially available. Others were synthesized according to known methods reported previously.^37–39 Suzuki coupling^37,40,41 of the corresponding boronic acids or pinacolboronic esters and 7 or S10 followed by reverse phase HPLC purification furnished pure final products as salts of trifluoroacetic acid.

Compound 28 was synthesized from 7 and 1H-benzo[d][1,2,3]triazole following a literature reported method with modifications.⁴²

CONCLUSIONS

In this study, we have designed and synthesized a class of 9H-pyrimido[4,5-b]indole-containing compounds with the goal of obtaining potent and orally bioavailable BET bromodomain inhibitors. These efforts have led to the discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31), which binds to BET family proteins with low nanomolar affinities and demonstrates high selectivity over non-BET bromodomain-containing proteins. Compound 31 potently inhibits cell growth in acute leukemia cell lines and breast cancer cell lines. Significantly, it achieves excellent oral bioavailability and is metabolically stable. It also displays potent antitumor activity in MV4;11 acute leukemia and MDA-MB-231 breast cancer xenograft models in mice at well-tolerated dose-schedules. Compound 31 is a potent, selective, orally bioavailable and efficacious BET inhibitor.

EXPERIMENTAL SECTION

Chemistry

General Methods:

All reactions were conducted in a round-bottom flask equipped with a Teflon-coated magnet stirring bar. Experiments involving components sensitive to moisture or air were performed under a nitrogen atmosphere. Commercially available reagents and anhydrous solvents were used without further purification. The crude reaction product was purified by flash column chromatography packed with silica gel. Further purification was performed on a preparative HPLC (Waters 2545) with a C18 reverse phase column. The mobile phase was a gradient flow of solvent A (water, 0.1% of TFA) and solvent B (CH₃CN, 0.1% of TFA) at a flow rate of 30 mL/min. Proton nuclear magnetic resonance (¹H NMR) and carbon nuclear magnetic resonance (¹³C NMR) spectroscopy were performed in Bruker Advance 300 NMR spectrometers. Low resolution ESI mass spectrum analysis was performed on a Thermo-Scientific LCQ Fleet mass spectrometer. The analytical UPLC model was Waters ACQUITY H class (UV detection at 230 nm and 254 nm wave lengths) and the reverse phase column used was the ACQUITY UPLC® BEH (C18–1.7 μm, 2.1 × 50 mm). All final compounds were purified to ⩾ 95% purity as determined by analytical UPLC analysis.

1. Procedures for the synthesis of 9H-pyrimido[4,5-b]indole-containing cores 7 and S10

4-(4-Fluoro-2-methoxy-5-nitrophenyl)-3,5-dimethylisoxazole (S3)

1-Bromo-4-fluoro-2-methoxy-5-nitrobenzene³⁵ and 1-bromo-2-fluoro-4-methoxy-5-nitrobenzene (3 g, 12 mmol, 2:1 ratio), 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (5.35 g, 24 mmol), and K₂CO₃ (5.0 g, 36 mmol) were added to a round-bottom flask. DME (50 mL) and water (30 mL) were added at room temperature. The solution was degassed before addition of Pd(PPh₃)₄ (700 mg, 0.6 mmol) in one portion. The solution was degassed and was then heated at reflux for 16 h. The aqueous layer was extracted with EtOAc and combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The volatile components were removed on a rotary evaporator and the residue was purified by flash column chromatogram. The desired product (S3) and its regioisomer were isolated in > 80% yield (3.38 g). S3: ¹H NMR (CDCl₃, 300 MHz): 8.03 (d, J = 8.47 Hz, 1H), 6.93 (d, J = 12.56 Hz, 1H), 4.00 (s, 3H), 2.40 (s, 3H), 2.24 (s, 3H).

Ethyl 2-cyano-2-(4-(3,5-dimethylisoxazol-4-yl)-5-methoxy-2-nitrophenyl)acetate (S5).

NaH (4.32 g, 60% in mineral oil, 100 mmol, 2.0 equiv) was placed in round-bottom flask and dry DMF (200 mL) was added. Ethyl cyanoacetate (7.35 g, 65 mmol, 1.2 equiv) was added dropwise at 0 °C via a syringe. The solution was stirred at room temperature for 30 min. The mixture was cooled to 0 °C, anhydrous DMF solution (30 mL) of S3 and its regioisomer (14.26 g, 54 mmol, 1.0 equiv) was added via a syringe. The reaction mixture was allowed to warm up to room temperature and was then stirred for 16 h. The reaction was quenched by 0.5 N HCl, and the aqueous layer was extracted with EtOAc, the combined organic layers were washed with brine, then dried over anhydrous Na₂SO₄. The volatile components were removed on a rotary evaporator and the residue was purified by flash column chromatogram. Pure S5 was isolated in 53% yield (6.86 g, 19.1 mmol, based on 66% correct isomer). ¹H NMR (CDCl₃, 300 MHz): 8.10 (s, 1H), 7.27 (s, 1H), 5.78 (s, 1H), 4.35 (q, J = 7.12 Hz, 2H), 3.99 (s, 3H), 2.33 (s, 3H), 2.18 (s, 3H), 1.37 (t, J = 7.14 Hz, 3H).¹³C NMR (CDCl₃, 75 MHz): 167.2, 163.6, 161.6, 159.2, 140.0, 129.7, 128.0, 121.6, 114.7, 112.8, 110.7, 64.0, 56.7, 42.0, 14.0, 11.7, 10.8. ESI-MS calculated for C₁₇H₁₈N₃O₆ [M+H]⁺ = 360.12, Observed: 360.58

Ethyl 2-amino-6-(3,5-dimethylisoxazol-4-yl)-5-methoxy-1H-indole-3-carboxylate (S6).

To a solution of S5 (6.86 g, 19.1 mmol, 1.0 equiv) in AcOH (50 mL) at 85 °C, Zn powder (2.6 g, 40 mmol, 2.0 equiv) were added in small portions. The mixture was stirred at 85 °C for 1 h, a further 2.6 g Zn powder (40 mmol, 2.0 equiv) was added, and the reaction was kept at the same temperature for 2 h. The reaction was cooled, filtered, and washed with AcOH. The AcOH solution was combined and the volatile components were removed on a rotary evaporator. Purification by flash column chromatogram furnished the desired product S6 (2.62 g, ca. 47% yield based on recovering 13% starting material). ¹H NMR (CDCl₃, 300 MHz): 8.01 (br, s, 1H), 7.44 (s, 1H), 6.78 (s, 1H), 5.73 (br, s, 2H), 4.40 (q, J = 7.08 Hz, 2H), 3.82 (s, 3H), 2.29 (s, 3H), 2.15 (s, 3H), 1.45 (t, J = 7.08 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): 167.2, 165.8, 160.6, 153.7, 153.1, 128.1, 126.7, 114.5, 112.0, 110.6, 101.8, 86.0, 55.6, 53.5, 14.7, 11.5, 10.6. ESI-MS calculated for C₁₇H₂₀N₃O₄ [M+H]⁺ = 330.15, Obtained: 330.25

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-4-ol (S7).

S6 (0.45 g, 1.4 mmol), ammonium formate (1.06 g, 17 mmol), and formamide (16 mL) were heated at 175 °C for 16 h. The reaction mixture was cooled to room temperature and water was added. Filtration of the mixture yielded S7 as a brown solid (0.24 g, 0.77 mmol, 55% yield). ¹H NMR (DMSO-d6, 300 MHz): 8.09 (s, 1H), 7.57 (s, 1H), 7.24 (s, 1H), 3.81 (s, 3H), 3.30 (s, 1H), 2.62 (s, 3H), 2.06 (s, 3H), ¹³C NMR (DMSO, 75 MHz): 165.3, 162.9, 159.2, 158.3, 153.9, 152.5, 129.6, 122.7, 115.6, 114.1, 113.6, 102.0, 100.2, 55.5, 11.2, 10.3. ESI-MS calculated for C₁₆H₁₅N₄O₃ [M+H]⁺: 311.11, Obtained: 311.75

4-(4-Chloro-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (7, CD55)

S7 (0.95 g, 3.05mmol) was dissolved in POCl₃ (15 mL) and the mixture was heated at 90 °C for 5 h. The mixture was cooled to room temperature and the volatile components were removed on a rotary evaporator. The POCl₃ solution was poured into EtOAc (40 mL), followed by saturated NaHCO₃ (60 mL) and water (100 mL). The mixture was quickly filtered and the desired product (7) was collected as a brown solid (0.3 g). The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The volatile components were removed on a rotary evaporator affording a brown solid (0.36 g). The total yield of 7 is 66% (0.66 g). ¹H NMR (DMSO-d6, 300 MHz): 8.74 (s, 1H), 7.84 (s, 1H), 7.45 (s, 1H). 3.89 (s, 3H), 3.31 (br, s, 1H), 2.29 (s, 3H), 2.09 (s, 3H) ¹³C NMR (DMSO-d6, 75MHz): 167.8, 161.2, 155.8, 122.2, 120.3, 117.0, 115.2, 113.1, 105.8, 57.8, 13.4, 12.4. ESI-MS calculated for C₁₆H₁₄³⁵ ClN₄O₂ [M+H]⁺ = 329.08, Obtained: 329.67

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-4-ol (S9).

To a round-bottom flask, S6 (0.37 g, 1.1 mmol) and MeCN (20 mL) were added at room temperature. Dry HCl was bubbled through MeCN for 30 min and the reaction mixture was heated at reflux for 2.5 h. The reaction was then cooled to room temperature and the volatile components were removed on a rotary evaporator. To this crude mixture, 10% NaOH aqueous solution (20 mL) and EtOH (50 mL) were added and the solution was heated at reflux for 6 h. The volatile components were then removed on a rotary evaporator and the aqueous residue was acidified with 2N HCl aqueous solution. The product S9 was allowed to precipitate at 0 °C. Filtration of the mixture furnished S9 in 0.278 g (78% yield, 2 steps). ¹H NMR (DMSO-d6, 300 MHz): 7.57 (s, 1H), 7.20 (s, 1H), 3.81 (s, 3H), 2.37 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ¹³C NMR (DMSO-d6, 75 MHz): 165.2, 160.4, 159.4, 157.7, 155.4, 152.2, 130.0, 123.2, 114.5, 114.0, 113.8, 101.9, 97.9, 55.6, 21.9, 11.3, 10.4.

4-(4-Chloro-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (S10)

S9 (0.278 g, 0.8 mmol) and POCl₃ (8 mL) were added to a round-bottom flask. The mixture was heated at 90 °C for 6 h. The reaction mixture was cooled to room temperature and the volatile components were removed on a rotary evaporator. Water (20 mL) and EtOAc (20 mL) were added and the pH was adjusted to 8 using a large excess of NaHCO₃ saturated aqueous solution. Rapid filtration of the mixture furnished S10 as a brown solid in 0.208 g (75% yield). ¹H NMR (DMSO-d6, 300 MHz): 7.81 (s, 1H), 7.43 (s, 1H), 3.89 (s, 3H), 2.69 (s, 3H), 2.31 (s, 3H), 2.11(s, 3H). ¹³C NMR (DMSO-d6, 75 MHz): 165.8, 163.6, 159.2, 156.9, 152.5, 151.1, 132.5, 119.5, 118.3, 114.7, 113.2, 108.5, 103.6, 55.8, 25.5, 11.3, 10.4. ESI-MS calculated for C₁₇H₁₆³⁵ ClN₄O₂ [M+H]⁺ = 343.10, Obtained: 343.58.

2. Synthesis of Final Products

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N-((R)-1-(pyridin-2-yl)ethyl)-9H-pyrimido[4,5-b]indol-4-amine (8).

7 (80 mg, 0.3 mmol), (R)-1-(pyridin-2-yl)ethanamine (122 mg, 1 mmol), and EtN(i-Pr)₂ (0.3 mL, 1.5 mmol) were added to a round-bottomed flask. NMP (3 mL) was added at room temperature. The solution was heated at 140 °C for 16 h. The reaction mixture was quenched by water (1 mL). The mixture was purified by reverse phase HPLC. The desired product 8 was isolated as a salt of CF₃CO₂H. (16 mg, 20%). ¹H NMR (MeOD-d4, 300 MHz): 8.81 (d, J = 5.58 Hz, 1H), 8.54 (s, 1H), 8.56–8.47 (m, 1H), 8.23–8.17 (m, 1H), 8.19 (s, 1H), 7.92 (t, J = 6.39 Hz, 1H), 7.50 (s, 1H), 6.00 (q, J = 7.11 Hz, 1H), 4.02 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H), 2.00 (d, J = 7.11 Hz, 3H). ESI-MS calculated for C₂₃H₂₃N₆O₂ [M+H]⁺ = 415.19, Obtained: 415.92

4-(4-(Isoxazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (9)

Method I using 7 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole: 13% yield. ¹H NMR (300 MHz, MeOD-d4): 9.73 (s, 1H), 9.22 (s, 1H), 9.07 (s, 1H), 7.57 (s, 1H), 7.55 (s, 1H), 3.87 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). ESI-MS calculated for C₁₉H₁₆N₅O₃ [M+H]⁺= 362.13, Obtained: 362.58.

4-(6-Methoxy-4-(1H-pyrazol-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (10),

Method I using 7 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole: 41% yield. ¹H NMR (300 MHz, MeOD-d4): 9.09 (s, 1H), 8.61 (s, 2H), 7.79 (s, 1H), 7.60 (s, 1H), 3.87 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESI-MS calculated for C₁₉H₁₇N₆O₂ [M+H]⁺= 361.14, Obtained: 361.50.

Method A: Synthesis of 4-(4-(Furan-3-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (11), using Suzuki coupling catalyzed by Pd(PPh₃)₄: 7 (33 mg, 0.1 mmol, 1.0 equiv), 2-(furan-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (60 mg, 0.3 mmol, 3.0 equiv), and K₂CO₃ (56 mg, 0.4 mmol, 4.0 equiv) were added to a round-bottom flask. DME (6 mL) and H₂O (4 mL) were added at room temperature. The solution was degassed before Pd(PPh₃)₄ (10–15 mg, 0.008–0.012 mmol, ca. 0.1 equiv.) was added in one portion. The solution was degassed again and then was heated at reflux for 16 h. The aqueous layer was extracted with EtOAc and combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The volatile components were removed on a rotary evaporator and the residue was treated with TFA (0.5 mL) and then purified by reverse phase HPLC. 11 was isolated as a colorless solid in the form of TFA salt (14 mg, 39% yield). ¹H NMR (MeOD-d4, 300 MHz): 9.10 (s, 1H), 8.61 (dd, J = 1.52, 0.88 Hz, 1H), 8.04 (t, J = 1.52 Hz, 1H), 7.78 (s, 1H), 7.59 (s, 1H), 7.28 (dd, J = 1.91, 0.88 Hz, 1H), 3.86 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H). ESI-MS calculated for C₂₀H₁₇N₄O₃ [M+H]⁺= 361.13, Obtained: 361.33

4-(6-Methoxy-4-(1H-pyrrol-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (12).

Method A using N-TIPS pyrrole-3-boronic acid pinacol ester: 21% yield. ¹H NMR (MeOD-d4, 300 MHz): 8.95 (s, 1H), 8.08 (s, 1H), 7.93 (t, J = 1.65 Hz, 1H), 7.57 (s, 1H), 7.23 (dd, J = 2.91, 1.81 Hz, 1H), 7.03 (dd, J = 2.91, 1.62 Hz, 1H), 3.87 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H). ¹³C NMR (MeOD-d4, 75MHz): 168.2, 161.2, 157.6, 155.7, 150.4, 148.4, 135.4, 124.6, 123.5, 123.0, 122.0, 117.0, 115.1, 114.7, 110.6, 110.1, 105.4, 56.5, 11.7, 10.8. ESI-MS calculated for C₂₀H₁₈N₅O₂ [M+H]⁺= 360.15, Obtained: 360.25

4-(4-(1H-Imidazol-1-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (13)

7 (26 mg, 0.08 mmol), imidazole (60 mg, 0.88 mmol), and anhydrous NMP (10 mL) were heated at 170 °C in sealed tube for 24 h. HPLC purification of the reaction mixture provided the title compound in 27% yield (10 mg). ¹H NMR (MeOD-d4, 300 MHz): 9.73 (s, 1H), 8.95 (s, 1H), 8.46 (s, 1H), 7.93 (s, 1H), 7.53 (s, 1H), 7.47 (s, 1H), 3.85 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). ESI-MS calculated for C₁₉H₁₇N₆O₂ [M+H]⁺ =361.14, Obtained: 361.33

4,4′-(6-Methoxy-9H-pyrimido[4,5-b]indole-4,7-diyl)bis(3,5-dimethylisoxazole) (14)

Method A using 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole ester: 41% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.15 (s, 1H), 7.58 (s, 1H), 7.08 (s, 1H), 3.79 (s, 3H), 2.51 (s, 3H), 2.32 (s, 3H), 2.31 (s, 3H), 2.15 (s, 3H). ESI-MS calculated for C₂₁H₂₀N₅O₃ [M+H]⁺ = 390.16, Obtained: 390.42.

4-(4-(3,5-Dimethyl-1H-pyrazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (15).

Method A using 3,5-dimethylpyrazole-4-boronic acid pinacol ester: 67% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.17 (s, 1H), 7.62 (s, 1H), 7.12 (s, 1H), 3.77 (s, 3H), 2.34 (s, 6H), 2.32 (s, 3H), 2.15 (s, 3H), ¹³C NMR (MeOD-d4, 75MHz): 168.3, 161.1, 157.7, 155.8, 150.3, 148.3, 146.2, 135.7, 123.8, 121.4, 117.2, 114.6, 110.9, 104.9, 56.4, 11.7, 11.6, 10.8. ESI-MS calculated for C₂₁H₂₁N₆O₂ [M+H]⁺ = 389.17, Obtained: 389.83

4-(6-Methoxy-4-(1,3,5-trimethyl-1H-pyrazol-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (16).

Method A using 1,3,5-trimethyl-1H-pyrazole-4-boronic acid pinacol ester: 42% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.20 (s, 1H), 7.65 (s, 1H), 7.16 (s, 1H), 3.97 (s, 3H), 3.81 (s, 3H), 2.40 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H), 2.17 (s, 3H). ESI-MS calculated for C₂₂H₂₃N₆O₂ [M+H]⁺ = 403.19, Obtained: 403.50

4-(4-(3-cyclopropyl-5-methyl-1H-pyrazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (17)

Method A using tert-butyl 3-cyclopropyl-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate: 65% yield; ¹H NMR (MeOD-d4, 300 MHz): 9.13 (s, 1H), 7.58 (s, 1H), 7.30 (s, 1H), 3.83 (s, 3H), 2.38 (s, 3H), 2.37 (s, 3H), 2.19 (s, 3H), 1.90–1.70 (m, 1H), 1.10–1.00 (m, 1H), 1.00–0.80 (m, 3H).

4-(4-(1H-Indol-3-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (18)

Method A using (1-(tert-butoxycarbonyl)-1H-indol-3-yl)boronic acid: 40% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.06 (s, 1H), 8.27 (s, 1H), 7.71 (d, J = 8.20 Hz, 1H), 7.57 (s, 1H), 7.50 (d, J = 7.96 Hz, 1H), 7.41 (ddd, J = 8.24, 7.21, 1.13 Hz, 1H), 7.29 (ddd, J = 8.03, 7.08, 0.97 Hz, 1H), 7.15 (s, 1H), 3.42 (s, 1H), 2.32 (s, 1H), 2.13 (s, 1H). ESI-MS calculated for C₂₄H₂₀N₅O₂ [M+H]⁺ = 410.16, Obtained: 410.33

4-(6-Methoxy-4-(1H-pyrrolo[2,3-c]pyridin-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (19).

Method A using 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-c]pyridine: 37% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.40 (s, 1H), 9.16 (s, 1H), 9.09 (s, 1H), 8.46 (d, J = 6.54 Hz, 1H), 8.25 (d, J = 6.54 Hz, 1H), 7.58 (s, 1H), 7.19 (s, 1H), 3.58 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H). ¹³C NMR (MeOD-d4, 300 MHz): 168.0, 161.2, 158.1, 154.5, 141.4, 137.1, 135.1, 134.0, 130.9, 130.2, 122.3, 120.9, 119.4, 116.3, 114.9, 113.2, 105.6, 56.3, 11.7, 10.8. ESI-MS calculated for C₂₃H₁₉N₆O₂ [M+H]⁺= 411.16, Obtained: 411.42

4-(4-(6-Fluoro-1H-indol-3-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (20).

Method A using tert-butyl 6-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate: 44% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.10 (s, 1H), 8.38 (s, 1H), 7.80–7.70 (m, 1H), 7.62 (s, 1H), 7.30–7.10 (m, 3H), 3.52 (s, 3H), 2.35 (s, 3H), 2.16 (s, 3H). ESI-MS calculated for C₂₄H₁₉FN₅O₂ [M+H]⁺= 428.15, Obtained: 428.25

4-(6-Methoxy-4-(2-methyl-1H-indol-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (21).

Method A using tert-butyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate: 26% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.12 (s, 1H), 7.61–7.58 (m, 1H), 7.58 (s, 1H), 7.37–7.15 (m, 3H), 6.82 (s, 1H), 3.32 (s, 3H), 2.67 (s, 3H), 2.31 (s, 3H), 2.12 (s, 3H). ESI-MS calculated for C₂₅H₂₂N₅O₂ [M+H]⁺= 424.18, Obtained: 424.42

4-(4-(6-Fluoro-2-methyl-1H-indol-3-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl isoxazole (22)

Method A using tert-butyl 6-fluoro-2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate: 52% yield. ¹H NMR (MeOD-d4, 300 MHz): 12.04 (NH), 9.16 (s, 1H), 7.62 (s, 1H), 7.34 (dd, J = 9.33, 2.16 Hz, 1H), 7.27 (dd, J = 8.74, 5.06 Hz, 1H), 7.01 (ddd, J = 3.42 (s, 3H), 2.67 (s, 3H), 2.33 (s, 3H), 2.14 (s, 3H). ¹³C NMR (MeOD-d4, 75 MHz): 168.2, 161.7 (d, J_C-F = 238.7 Hz), 161.1, 157.7, 155.3, 149.3, 148.7, 141.5, 138.0 (d, J_C-F = 12.8 Hz), 135.5, 123.7, 123.2, 121.8 (d, J_C-F = 9.8 Hz), 121.5, 116.7, 114.6, 113.4, 110.7 (d, J_C-F = 24.9 Hz), 106.8, 105.1, 99.6 (d, J_C-F = 26.4 Hz), 55.8, 13.0, 11.7, 10.8. ESI-MS calculated for C₂₅H₂₁FN₅O₂ [M+H]⁺ = 442.17; Obtained: 442.50

4-(6-Methoxy-4-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (23)

Method A using tert-butyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-c]pyridine-1-carboxylate: 61% yield.¹H NMR (MeOD-d4, 300 MHz): 9.28 (s, 1H), 9.24 (s, 1H), 8.37 (d, J = 6.53 Hz, 1H), 7.87 (d, J = 6.53 Hz, 1H), 7.63 (s, 1H), 6.77 (s, 1H), 3.47 (s, 3H), 2.85 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H) ESI-MS calculated for C₂₄H₂₁N₆O₂ [M+H]⁺ = 425.17, Obtained: 425.50

4-(6-Methoxy-4-(2-methylbenzofuran-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (24).

Method A using 4,4,5,5-tetramethyl-2-(2-methylbenzofuran-3-yl)-1,3,2-dioxaborolane: 31% yield.¹H NMR (MeOD-d4, 300 MHz): 9.22 (s, 1H), 7.73 (d, J = 8.32 Hz, 1H), 7.60 (s, 1H), 7.55–7.45 (m, 1H), 7.40–7.30 (m, 2H), 6.85 (s, 1H), 3.37 (s, 3H), 2.68 (s, 3H), 2.30 (s, 3H), 2.12 (s, 3H), ESI-MS calculated for C₂₅H₂₁N₄O₃ [M+H]⁺ = 425.16; Obtained: 425.50

4-(4-(1H-Benzo[d]imidazol-1-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (25).

7 (20 mg, 0.06 mmol), benzimidazole (72 mg, 0.6 mmol), and EtN(i-Pr)₂ (0.1 mL) were dissolved in anhydrous DMSO (4 mL). The solution was heated at 170 °C for 16 h. HPLC purification of the reaction mixture provided 25 in 32% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.45 (s, 1H), 9.01 (s, 1H), 8.00 (d, J = 8.09 Hz, 1H), 7.67–7.51 (m, 3H), 7.51 (s, 1H), 6.78 (s, 1H), 3.44 (s, 3H), 2.31 (s, 3H), 2.12 (s, 3H). ESI-MS Calculated for C₂₃H₁₉N₆O₂ [M+H]⁺ = 411.16, Found: 411.75

4-(8-Methoxy-1-(pyrazolo[1,5-a]pyridin-3-yl)-5H-pyrido[4,3-b]indol-7-yl)-3,5-dimethylisoxazole (26).

Method A using pyrazolo[1,5-a]pyridin-3-yl-boronic acid pinacol ester: 29% yield. ¹H NMR (MeOD-d4, 300 MHz): 9.05 (s, 1H), 8.89 (d, J = 7.00 Hz, 1H), 8.76 (s, 1H), 7.76 (d, J = 7.85 Hz, 1H), 7.66–7.58 (m, 1H), 7.56 (s, 1H), 7.25 (dd, J = 6.89, 6.92 Hz, 1H), 7.19 (s, 1H),3.60 (s, 3H), 2.32 (s, 3H), 2.14 (s, 3H). ESI-MS calculated for C₂₄H₂₀N₅O₂ [M+H]⁺ = 410.16, Obtained: 410.12

4-(6-Methoxy-4-(1-methyl-1H-indazol-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (27)

Optimized Suzuki coupling conditions³⁷ were used to synthesize 27. 7 (34 mg, 0.1 mmol), 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (75 mg, 0.3 mmol), and Na₂CO₃ (2M aqueous solution, 50 mg) were mixed in round-bottom flask. To this flask, MeOH (4 mL), PhMe (4 mL), and water (1 mL) were added and the system was degassed and refilled with pure N₂. Pd(PPh₃)₄ (20 mg) was then added. The system was degassed and refilled again with pure N₂. The mixture was heated at reflux for 12 h. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The volatile components were removed on a rotary evaporator and the residue was purified by reverse phase HPLC. The desired product 27 was isolated as TFA salt in 23% yield. ¹H NMR (DMSO-d6, 300 MHz): 12.36 (s, 1H), 9.31 (s, 1H), 9.05 (s, 1H), 8.82 (d, J = 8.18 Hz, 1H), 7.85 (d, J = 8.48 Hz, 1H), 7.60–7.50 (m, 1H), 7.39 (s, 1H), 7.40–7.32 (m, 1H), 4.36 (s, 3H), 4.00 (s, 3H), 3.37 (s, NH), 2.32 (s, 3H), 2.13 (s, 3H). ¹³C NMR (DMSO-d6, 75 MHz): 165.7, 159.3, 156.9, 153.9, 152.9, 151.7, 141.0, 132.8, 127.0, 123.8, 123.1, 122.4, 119.9, 119.0, 113.4, 110.4, 109.3, 108.1, 55.5, 36.3, 11.4, 10.5. ESI-MS Calculated for C₂₄H₂₁N₆O₂ [M+H]⁺ = 425.17, Found: 425.83

4-(4-(1H-Benzo[d][1,2,3]triazol-1-yl)-6-methoxy-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (28)

A modified reported method was followed.⁴² 7 (34 mg, 0.1 mmol), benzotriazole (14 mg, 0.12 mmol), and K₃PO₄ (42 mg, 0.2 mmol) were added into a round-bottom flask. The round-bottom flask was degassed and refilled with pure N₂ gas. In a second round-bottom flask, Pd₂(dba)₃ (9 mg, 0.01 mmol) and (±)-BINAP (15 mg, 0.024 mmol) were added. The round-bottom flask was degassed and refilled with pure N₂. To this flask, anhydrous toluene (10 mL) was added and the solution was heated at 120 °C for 3 min to generate the active catalyst. The active catalyst was transferred into the first flask and the reaction mixture was heat at reflux for 12 h. The mixture was then diluted with EtOAc and washed with water and brine. The organic layer was collected, the volatile components were removed on a rotary evaporator, and the residue was purified by reverse phase HPLC. The desired product 28 was isolated as a TFA salt in 29% yield. ¹H NMR (DMSO-d6, 300 MHz): 12.88 (s, 1H), 9.04 (s, 1H), 8.61 (s, 1H), 8.26–8.18 (m, 2H), 7.68–7.60 (m, 2H), 7.48 (s, 1H), 3.87 (s, 3H), 3.30 (s, 3H), 3.15 (s, NH), 2.31 (s, 3H), 2.11 (s, 3H). ¹³C NMR (DMSO-d6, 75 MHz): 167.6, 160.8, 160.3, 155.3, 153.8, 146.7, 135.3, 130.8, 122.3, 120.5, 119.3, 119.0, 115.9, 114.8, 109.0, 105.8, 57.0, 13.1, 12.1. ESI-MS Calculated for C₂₂H₁₈N₇O₂ [M+H]⁺ = 412.15, Found: 412.42

4-(6-Methoxy-2-methyl-4-(2-methyl-1H-indol-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (29)

Method A using S10 and tert-butyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate afforded a mixture of 29 and a Boc group protected 29. Treatment of the crude mixture with TFA in DCM followed by HPLC purification yield 29 in 27% yield. ¹H NMR (MeOD-d4, 300 MHz): 11.94 (NH), 7.62 (d, J = 8.13 Hz, 1H), 7.57 (s, 1H), 7.35 (ddd, J = 8.17, 6.82, 1.30, 1H), 7.32–7.18 (m, 2H), 6.80 (s, 1H), 3.00 (s, 3H), 2.70 (s, 3H), 2.34 (s, 3H), 2.15 (s, 3H). ¹³C NMR (MeOD-d4, 75 MHz):168.1, 161.1, 160.1, 158.2, 155.3, 148.8, 141.0, 137.8, 135.5, 127.2, 124.3, 122.7, 122.5, 121.6, 120.7, 116.6, 114.6, 113.4, 111.7, 107.0, 104.5, 55.8, 22.4, 12.9, 11.7, 10.8. ESI-MS calculated for C₂₆H₂₄N₅O₂[M+H]⁺ = 438.19, Obtained: 438.42.

Method I: Synthesis of 4-(6-Methoxy-2-methyl-4-(2-methylquinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (30), using Suzuki coupling catalyzed by Pd(dppf)Cl₂-CH₂Cl₂ complex: S10 (728 mg, 2.1 mmol, 1.0 equiv) and 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (1.674g, 6.22 mmol, 3.0 equiv) were placed in a round-bottom flask. DME (60 mL) and Na₂CO₃ (2 M aq solution, 20 mL) were added subsequently. The system was degassed to remove oxygen. Pd(dppf)Cl₂-CH₂Cl₂ complex (257 mg, 0.31 mmol, > 0.1 equiv) was added in one portion. The system was degassed again and refilled with N₂. The reaction was heated at reflux for 16 h before quenching with H₂O. The reaction was then diluted with H₂O, the aqueous layer was extracted with EtOAc (100 mL × 3) and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed in vacuum and the residue was purified by flash column chromatography to yield 30 in 0.64 g (63% yield). Further purification was aided by reverse phase HPLC to yield the corresponding products 30 as a salt of CF₃CO₂H. ¹H NMR (300 MHz, MeOD-d4): 8.32 (d, J = 8.47 Hz, 1H), 8.13 (s, 1H), 8.10 (ddd, J = 8.44, 7.03, 1.26 Hz, 1H), 7.93 (d, J = 7.86 Hz, 1H), 7.75 (t, J = 7.71 Hz, 1H), 7.49 (s, 1H), 6.30 (s, 1H), 3.25 (s, 3H), 3.04 (s, 3H), 2.95 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for C₂₇H₂₄N₅O₂ [M+H]⁺ = 450.19, Obtained: 450.42

4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31).

Method A using S10 and quinoline-4-boronic acid: 57% yield; ¹H NMR (MeOD-d4, 300 MHz): 9.31 (d, J = 4.59 Hz, 1H), 8.38 (d, J = 8.50 Hz, 1H), 8.08 (d, J = 4.63 Hz, 1H), 8.12–8.00 (m, 1H), 7.88 (d, J = 7.76 Hz, 1H), 7.78–7.70 (m, 1H), 7.53 (s, 1H), 6.21 (s, 1H), 3.21 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ¹³C NMR (MeOD-d4, 75 MHz): 168.2, 162.1, 161.0, 158.6, 155.3, 151.0, 150.3, 148.3, 141.3, 136.1, 133.3, 130.8, 129.9, 126.7, 126.2, 123.7, 123.6, 120.4, 117.0, 114.5, 112.9, 105.3, 55.9, 23.6, 11.6, 10.7. ESI-MS Calculated for C₂₆H₂₂N₅O₂ [M+H]⁺ = 436.18, Found: 436.50.

4-(6-Methoxy-2-methyl-4-(quinolin-5-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (32).

Method A using S10 and quinoline-5-boronic acid: 27%. ¹H NMR (300 MHz, MeOD-d4): 9.15(dd, J = 4.39, 1.39 Hz, 1H), 8.61–8.53 (m, 1H), 8.41 (d, J = 8.60 Hz, 1H), 8.25 (d, J = 1.33 Hz, 1H), 8.24 (s, 1H), 7.72 (dd, J = 7.74, 4.42 Hz, 1H), 7.57 (s, 1H), 6.30 (s, 1H), 3.28 (s, 3H), 3.03 (3H), 2.29 (s, 3H), 2.10 (s, 3H). ESI-MS calculated for C₂₆H₂₂N₅O₂ [M+H]⁺ = 436.18, Obtained: 436.33.

4-(6-Methoxy-2-methyl-4-(6-methylquinolin-5-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethyl-isoxazole (33).

Method A using S10 and 3-methylquinoline-4-boronic acid: 36%. ¹H NMR (300 MHz, MeOD-d4): 9.08 (d, J = 4.56 Hz, 1H), 8.46 (J = 8.81 Hz, 1H), 8.22 (d, J = 8.56 Hz, 1H), 8.17 (d, J = 8.86 Hz, 1H), 7.69 (dd, J = 8.63, 4.64 Hz, 1H), 7.55 (s, 1H), 6.14 (s, 1H), 3.28 (s, 3H), 3.01 (s, 3H), 2.47 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H). ESI-MS calculated for C₂₇H₂₄N₅O₂ [M+H]⁺ = 450.19, Obtained: 450.50.

4-(4-(Isoquinolin-4-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (34).

Method A using S10 and isoquinoline-4-boronic acid: 54%. ¹H NMR (300 MHz, MeOD-d4): 9.78 (s, 1H), 8.98 (s, 1H), 8.53–8.46 (m, 1H), 8.04–7.92 (m, 2H), 7.90–7.82 (m, 1H), 7.55 (s, 1H), 6.23 (s, 1H), 3.20 (s, 3H), 3.01 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for C₂₆H₂₂N₅O₂ [M+H]⁺ = 436.18, Obtained: 436.50.

4-(4-(Isoquinolin-5-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (35).

Method A using S10 and isoquinoline-5-boronic acid: 29%. ¹H NMR (300 MHz, MeOD-d4): 9.85 (s, broad, 1H), 8.80 (d, J = 8.32 Hz, 1H), 8.75–8.50 (broad, 1H), 8.56 (dd, J = 7.24, 1.11 Hz), 8.26 (dd, J = 8.29, 7.30 Hz, 1H), 8.04 (d, J = 6.08 Hz, 1H), 6.32 (s, 1H), 3.29 (s, 3H), 3.00 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ¹³C NMR (75 MHz, MeOD-d4): 168.1, 162.2, 161.0, 158.7, 155.2, 153.2, 151.8, 141.8, 136.6, 136.0, 135.6, 134.1, 130.6, 130.1, 123.4, 121.1, 120.7, 112.0, 114.5, 113.1, 105.1, 55.9, 23.6, 11.6, 10.7. ESI-MS calculated for C26H₂₂N₅O₂ [M+H]⁺= 436.18, Obtained: 436.68.

4-(4-(Isoquinolin-8-yl)-6-methoxy-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (36)

Method A using S10 and isoquinoline-8-boronic acid: 35%. ¹H NMR (300 MHz, MeOD-d4):9.54 (s, 1H), 8.74 (s, 1H), 8.61 (dd, J = 7.75, 1.22 Hz, 1H), 8.52 (d, J = 6.18 Hz, 1H), 8.47–8.35 (m, 2H), 7.54 (s, 1H), 6.42 (s, 1H), 3.32 (s, 3H), 2.99 (s, 3H), 2.26 (s, 3H), 2.08 (s, 3H). ¹³C NMR (75 MHz, MeOD-d4): 168.1, 162.8, 161.1, 158.8, 155.1, 152.5, 149.7, 140.6, 139.4, 135.9, 134.2, 133.6, 132.8, 132.0, 126.8, 125.0, 123.2, 120.7, 116.9, 114.6, 113.1, 104.9, 56.0, 24.1, 11.6, 10.7. ESI-MS calculated for C₂₆H₂₂N₅O₂ [M+H]⁺= 436.18, Obtained: 436.56.

4-(6-Methoxy-2-methyl-4-(quinolin-8-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (37).

Method A using S10 and quinoline-8-boronic acid: 18%. ¹H NMR (300 MHz, MeOD-d4): 8.89 (dd, J = 4.28, 1.72 Hz, 1H), 8.65 (dd, J = 8.41, 1.68 Hz, 1H), 8.47 (dd, J = 8.28, 1.31 Hz, 1H), 8.37 (dd, J = 7.18, 1.36 Hz, 1H), 8.02 (dd, J = 8.20, 7.26 Hz, 1H), 7.74 (dd, J = 8.38, 4.28 Hz, 1H), 7.54 (s, 1H), 6.43 (s, 1H), 3.33 (s, 3H), 2.99 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H). ESI-MS calculated for C₂₆H₂₂N₅O₂ [M+H]⁺ =436.18, Obtained: 436.83.

4-(6-Methoxy-2-methyl-4-(2-(trifluoromethyl)quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (38).

Method I using S10 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethyl)quinoline: 10% yield. ¹H NMR (300 MHz, MeOD-d4): 8.44 (d, J = 8.61 Hz, 1H), 8.28 (s, 1H), 8.04 (t, J = 7.71 Hz, 1H), 7.90 (d, J = 8.19 Hz, 1H), 7.76 (t, J = 8.03 Hz, 1H), 7.45 (s, 1H), 6.20 (s, 1H), 3.21 (s, 3H), 2.93 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for C₂₇H₂₁F₃N₅O₂ [M+H]⁺= 504.16, Obtained: 504.58.

4-(6-Methoxy-4-(2-methoxyquinolin-4-yl)-2-methyl-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (39).

Method I using S10 and 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline: 35% yield.¹H NMR (300 MHz, MeOD-d4): 8.11 (d, J = 8.44 Hz, 1H), 7.83 (ddd, J = 8.39, 7.05, 1.30 Hz, 1H), 7.63 (d, J = 8.29 Hz, 1H), 7.54 (s, 1H), 7.49 (s, 1H), 7.44 (t, J = 7.63 Hz, 1H), 6.27 (s, 1H), 4.20 (s, 3H), 3.21 (s, 3H), 3.00 (s, 3H), 2.26 (s, 3H), 2.07 (s, 3H). ESI-MS calculated for C₂₇H₂₄N₅O₃ [M+H]⁺ = 466.19, Obtained: 466.68.

4-(6-Methoxy-2-methyl-4-(quinolin-3-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (40)

Method A using S10 and quinoline-3-boronic acid: 22% yield. ¹H NMR (300 MHz, MeOD-d4): 9.47 (d, J = 2.01 Hz, 1H), 9.21 (d, J = 2.03 Hz, 1H), 8.29 (t, J = 7.52 Hz, 2H), 8.08 (ddd, J = 8.50, 6.99, 1.40 Hz, 1H), 7.88 (ddd, J = 8.05, 7.14, 1.00 Hz, 1H), 7.57 (s, 1H), 7.29 (s, 1H), 3.60 (s, 3H), 3.00 (s, 3H), 2.31 (s, 3H), 2.13 (s, 3H). ESI-MS calculated for C₂₆H₂₂N₅O₂ [M+H]⁺ = 436.18, Obtained: 436.83. The procedures for FP-Based Protein Binding Assay, Cell Growth Inhibition Assay, and Western Blotting have been reported in our previous paper and please refer that paper for experimental details.³³

Determination of Biochemical Binding Affinities to BET Proteins

Recombinant human proteins corresponding to BRD2 BD1 (residues 72–205), BRD2 BD2 (residues 349–460), BRD3 BD1 (residues 24–144), BRD3 BD2 (residues 306–417), BRD4 BD1 (residues 44–168), BRD4 BD2 (residues 333–460) were used in the biochemical binding assays. Binding affinities of BET inhibitors to BRD2 (BD1 and BD2 proteins), BRD3 (BD1 and BD2 proteins), BRD4 (BD1 and BD2 proteins) were determined using our established fluorescence-polarization binding assays as described previously.³³

Determination of the co-crystal structure of compound 31 in complex with BRD4 BD2 protein

Prior to crystallization, BRD4 BD2 concentrated to 7.1 mg/mL in 25 mM Tris-HCl pH 7.5, 150 mM NaCl and 1 mM TCEP was incubated with a 2-fold molar excess of compound 31 for 1 hr then incubated with 0.5% β-mercaptoethanol for an additional 2 hr at room temperature. Crystals were grown at 20 °C from drops containing equal volumes of protein complex and well solution (50% polyethylene glycol 400, 10 mM imidazole pH 8.0, and 0.5 mM ATP). For data collection, the crystals were mounted directly from the drop and flash frozen in liquid nitrogen. Data were collected at the Advance Photon Source at Argonne National Lab on the LS-CAT beamlines 21-ID-G. The complex crystallized in space group P2₁2₁2 and contained 1 molecule per asymmetric unit. Data were processed with HKL2000⁴³ and the structure was solved by molecular replacement with Phaser⁴⁴ using an in-house structure as a model. The structure was refined with BUSTER⁴⁵ and electron density maps fit with COOT⁴⁶. Coordinates and restraints for the compounds were developed using Grade with the mogul+qm option⁴⁵. Structures were validated using Molprobity⁴⁷ Ligand statistics were obtained from the wwPDB Validation Server. Data refinement and statistics are given in Supporting Information Table S1. The coordinates were deposited in the PDB with the following ID: 5UOO.

Cell Growth Inhibition and Western Blotting

The SUM-149PT human breast cancer cell line was developed at the University of Michigan Comprehensive Cancer Center.⁴⁸ All the other human cancer cell lines were purchased from the American Type Culture Collection. Cells were used within one month after thaw and were cultured as recommended.

In cell growth assay, cells were seeded in 96-well cell culture plates at 10,000 cells/well for leukemia cells and 3,000 cells/well for breast cancer cells in 75 μL of culture medium. Compounds were serially diluted in culture medium, and 75 μL of the diluted compounds was added to the plates. The cells were then cultured for 4 days. Cell growth was evaluated by the dehydrogenases-based WST-8 assay (Dojindo Molecular Technologies). The WST-8 reagent was added to the plate at a final concentration of 10% (v/v), incubated for 1–3h, and read at 450 nm using a Tecan Infinite M1000 multimode microplate reader (Tecan, Morrisville, NC). The readings were normalized to the DMSO-treated cells and the IC₅₀ was calculated by nonlinear regression analysis using GraphPad Prism 6 software.

For Western blot analysis, cells were treated with compounds at indicated conditions. Cells were collected and lysed in RIPA buffer containing protease inhibitors. Whole cell lysates were separated by 4–20% SDS- PAGE gels and blotted into PVDF membranes. For immunoblotting, rabbit polyclonal antibodies for BRD2, BRD3 and BRD4 were from Bethyl Laboratories (Montgomery, TX, USA), mouse monoclonal antibody for c-Myc was from Cell Signaling Technology (Danvers, MA, USA) and GAPDH was from Santa Cruz Biotechnologies (Dallas, TX, USA).

Microsomal Stability Studies

The metabolic stability was assessed using pooled CD-1 mouse liver microsomes, Pooled SD rat liver microsmes and pooled human liver microsome (purchased from Xeno Tech). Briefly, 1 μM of each compound (21, 29, 30 and 31) was incubated with 0.5 mg/mL microsomes and 1.7 mM cofactor β-NADPH in 0.1 M phosphate buffer (pH = 7.4) containing 3.3 mM MgCl₂ at 37 °C. The DMSO concentration was less than 0.1% in the final incubation system. At 5, 10, 15, 30, 45, and 60 min of incubation, the 40 μL of reaction mixture were taken out, and the reaction is stopped immediately by adding 3-fold excess of cold acetonitrile containing 100 ng/mL of internal standard for quantification. The collected fractions were centrifuged at 15000rpm for 10 min to collect the supernatant for LC–MS/MS analysis, from which the amount of compound remaining was determined. The natural log of the amount of compound remaining was plotted against time to determine the disappearance rate and the half-life of tested compounds.

Pharmacokinetic Studies in Rats and Mice

All animal experiments were approved by the University of Michigan Committee on Use and Care of Animals and Unit for Laboratory Animal Medicine under the approved protocol (PRO00005315, P.I. Shaomeng Wang).

The pharmacokinetics of compound 21 and 31 was determined in Sprague Dawley rats following intravenous (iv) dosing at 5 mg/kg or oral (PO) dosing at 25 mg/kg. The quick pharmacokinetics of compound 17 was determined in SCID Mice following PO dosing at 25 mg/kg.

A tested compound was dissolved in the vehicle containing 20% (v/v) PCP and 70% (v/v) PBS. Blood samples (100 μL) were collected from rats with a catheter at different time points (0, 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 6hr, 8 hr and 24 hr) after the treatment of drugs. The blood samples were centrifuged at 15 000 rpm for 10 min, then the supernatant plasma was saved at −80 °C until analysis.

Plasma concentrations of the compounds were determined by the LC–MS/MS method developed and validated for this study. The LC–MS/MS method consisted of a Shimadzu HPLC system, and chromatographic separation of tested compound was achieved using a Waters XBridge-C18 column (5 cm × 2.1 mm, 3.5 μm). An AB Sciex QTrap 4500 mass spectrometer equipped with an electrospray ionization source (Applied Biosystems, Toronto, Canada) in the positive-ion multiple reaction monitoring (MRM) mode was used for detection. The mobile phases were 0.1% formic acid in purified water (A) and 0.1% formic acid in acetonitrile (B). The gradient (B) was held at 10% (0–0.3 min), increased to 95% at 0.7 min, then stayed at isocratic 95% B for 2.3 min, and then immediately stepped back down to 10% for 2 min re-equilibration. Flow rate was set at 0.4 mL/min. All pharmacokinetic parameters were calculated by noncompartmental methods using WinNonlin, version 3.2 (Pharsight Corporation, Mountain View, CA, USA).

Efficacy Studies in the MV4;11 and MDA-MB-231 Xenograft Models in Mice

All efficacy experiments were done under the guidelines of the University of Michigan Committee for Use and Care of Animals and using an approved animal protocol (PRO00005315, P.I. Shaomeng Wang).

To develop xenograft tumors, 5 × 10⁶ MV4;11 or MDA-MB-231 cells with 50% Matrigel were injected subcutaneously on the dorsal side of severe combined immunodeficient (SCID) mice, obtained from Charles River, one tumor per mouse. When tumors reached ~100 mm³, mice were randomly assigned to treatment and vehicle control groups. Animals were monitored daily for any signs of toxicity and weighed 2–3 times per week during the treatment and weighed at least weekly after the treatment was ended. Tumor size was measured 2–3 times per week by electronic calipers during the treatment period and at least weekly after the treatment was ended. Tumor volume was calculated as V = L × W²/2, where L is the length and W is the width of the tumor.

ABBREVIATIONS USED

DME

1,2-Dimethoxyethane

DMF

Dimethylformamide

NMP

1-Methylpyrrolidin-2-one

Cmax

The peak plasma concentration of a drug after administration

Vss

The apparent volume in which a drug is distributed at steady state

CL

The volume of plasma cleared of the drug per unit time

PARP

Poly (ADP-ribose) polymerase

CL-PARP

cleaved PAPR