Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function

ABSTRACT:

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit ³H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1–FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Graphical Abstract

INTRODUCTION

Native polyamines 1–3 (Figure 1) play diverse roles in cells including key roles in translation, transcription, chromatin remodeling, autophagy, growth, eIF-5A formation, and immune response.¹ Therefore, targeting polyamine metabolism provides the opportunity to inhibit cancers, which are often addicted to polyamine growth factors.¹ One approach to inhibit cell growth is to starve cells of their polyamine components by inhibiting polyamine biosynthesis.^2,3 This can be accomplished by the use of the ornithine decarboxylase (ODC) inhibitor (difluorome-thylornithine, DFMO, 4). However, cancers often circumvent DFMO by upregulating polyamine import. This escape route can be blocked via a combination therapy of DFMO and a polyamine transport inhibitor (PTI; trimer44NMe 5).⁴ Indeed, the DFMO + PTI or “polyamine blocking therapy” (PBT) approach has shown broad application across numerous cancer types,^5–7 including gemcitabine-resistant pancreatic cancer.⁸

Figure 1.

Structures of native polyamines 1–3, ornithine decarboxylase inhibitor DFMO (4), the polyamine transport inhibitor, trimer44NMe (5), and fluorescent anthracenyl polyamine Ant44 (6).

Even though polyamines are an established antiproliferative target, strategies to deplete polyamine pools often fail. Indeed, efforts to inhibit the polyamine biosynthetic enzymes ODC, spermidine synthase (SRM), and spermine synthase (SMS) in human cancers often induce intracellular polyamine depletion (Figure 2) but have limited effect on growth unless the total polyamine pools are decreased dramatically.² Moreover, polyamine import, homeostasis, and catabolic processes allow for the import and interconversion of the native polyamines making it difficult to fully suppress intracellular polyamine pools. For example, in pancreatic ductal adenocarcinoma (PDAC) cells (L3.6pl cells), one needed to reduce the intracellular polyamine pools below 80% of the untreated control to affect growth in cell culture.⁴ In this regard, cancers like PDAC often have excess supplies of polyamines to survive any temporary reduction of polyamine resources and have polyamine transport proteins like ATP13A3 to import polyamines as needed.⁹

Figure 2.

Polyamine metabolism and the DFMO + PTI intervention. Abbreviations: AZIN1: antizyme inhibitor 1; DFMO: difluoromethylornithine; diAc-Spm: N¹,N¹²-diacetylspermine; eIF-5A: eukaryotic initiation factor 5A; FUBP1: far upstream element binding protein 1; N1Ac-Spd: N¹-acetylspermidine; N-Ac-Spm: N¹-acetylspermine; OAZ1: antizyme 1; ODC: ornithine decarboxylase; PAO: polyamine oxidase; PTI: polyamine transport inhibitor; SAT1: spermidine/spermine acetyl transferase; SMOX: spermine oxidase; SMS: spermine synthase; Spd: spermidine; Spm: spermine; SRM: spermidine synthase; FUSE: far upstream element; FUBP1: FUSE binding protein 1; TFIIH: transcription factor II Human; RNA Pol II: RNA polymerase II.

In this report, we investigated other ways to effectively reduce intracellular polyamines beyond the DFMO + PTI approach. We focused our efforts upstream of polyamine metabolism to c-Myc and its master regulator, the far upstream binding protein 1 (FUBP1). FUBP1 is an attractive anticancer target because it controls a specific panel of far upstream element (FUSE)-controlled genes that facilitate tumor survival like c-Myc (upregulated by FUBP1) and p21 (downregulated by FUBP1). Few FUBP1 inhibitors have been described and most have low potency with IC₅₀ values > 300 μM.¹⁰ This target is especially interesting because a FUBP1 inhibitor should be able to alter the expression pattern of specific FUSE-controlled genes to reprogram cells to an antitumor phenotype. The possibility of specific gene targeting stems from the unique DNA–protein interaction of the FUSE DNA sequence on the noncoding strand and the four K-homology (KH) domains of the FUBP1 protein (i.e., KH1, KH2, KH3, and KH4). Of these four, KH3 and KH4 appear to be the most critical for DNA recognition.¹¹

A FUBP1 inhibitor should decrease c-Myc expression and increase p21 expression. Since c-Myc is a transcriptional activator of ODC (the rate-limiting enzyme in polyamine biosynthesis), inhibition of c-Myc expression should lead to intracellular polyamine depletion; an outcome that should synergize with DFMO (Figure 2). In this regard, a FUBP1 inhibitor should lead to decreased ODC expression (via decreased c-Myc) and work synergistically with the ODC inhibitor (DFMO) to inhibit growth.

To discover new DFMO adjunct agents that limit cell growth, we developed two orthogonal screens. Both screens monitored changes in cell growth based on polyamine import of either a toxic polyamine Ant44 (Assay 1, 48 h CHO-K1 IC₅₀ = 2 μM) or spermidine rescue of DFMO-treated cells (Assay 2).^12,13 A PTI should inhibit uptake of the toxic Ant44 probe and rescue growth in Assay 1 as well as inhibit spermidine uptake and decrease relative growth in DFMO-treated cells in Assay 2. In contrast, a FUBP1 inhibitor is only expected to have activity in Assay 2. In this regard, we envisioned segregating the dual hits in Assays 1 and 2 as potential PTI candidates and the Assay 2-only hits as novel DFMO adjunct agents (and potential FUBP1 inhibitors). While these assays were originally designed to identify new PTIs, this two-assay approach has also identified agents that affect polyamine pools and cell growth by novel mechanisms including LAT-1 efflux agonism.¹⁴ In this report, we describe the serendipitous discovery of novel anthranilic acid-based FUBP1 inhibitors from our Assay 2 hits.

To support a large screening effort, we miniaturized our 96-well plate assays¹² to a 1536-well plate format and screened the large Sanford Burnham compound library containing over 300,000 compounds. Although our overall hit rate was low, the screening results identified several anthranilic acid derivatives as positive hits in Assay 2. We then synthesized select hit compounds and demonstrated that these compounds alone and in combination with DFMO reduced the growth of pancreatic cancer cells. Further work confirmed that the top performing anthranilic acid derivative inhibited FUBP1–FUSE interactions as measured by gel shift and ChIP assays, decreased the expression of c-Myc mRNA and protein and increased the expression of p21 mRNA and protein in a dose-dependent manner and depleted polyamine pools. Moreover, a head-to-head comparison of our lead compound to the most potent known FUBP1 inhibitor described by Hauck et al.¹⁵ demonstrated the increased potency of our design. These exciting findings now open the door to future structure–activity relationship (SAR) studies of this molecular class.

RESULTS AND DISCUSSION

The assays were optimized for high-throughput screening (HTS) at the Sanford Burnham Conrad Prebys Center for Chemical Genomics at the Sanford Burnham Medical Research Institute (Orlando, Florida) as part of the Florida Translational Research Program. Both L3.6pl metastatic human pancreatic cancer cells¹⁶ and Chinese hamster ovary (CHO) cells gave excellent responses to the presence of PTI 5 as reflected by changes in their relative growth in both assays. However, CHO cells gave superior performance in the HTS format (1536-well plates, z′ score of 0.62) and were selected for the large-scale screening effort. Hits from the HTS were then repurchased as powders and screened again in a 384-well plate format (Table 1). Selected hits were then rescreened again in 96-well format in both CHO and L3.6pl cells.

Table 1.

Structure–Activity Study Results in CHO-K1 cells in a 384-well format^a

					Comprehensive SAR
Compd #		SBI-code	R1		R2		R3	DFMO Assay 2 EC50 (μM)	Ant44 Assay 1 EC50 (μM)	Cytotoxicity MTC (μM)
8		0800682	2-CI-Ph		2-CO2H		4-OMe-Ph	27	>40	28
9		0754479	2-Me-5-CI-Ph		2-CO2H		4-OMe-Ph	8	>40	13
20		0801722	2-Me-5-CI-Ph		2-CO2Me		4-OMe-Ph	>40	>40	>40
21		0800660	4-OMe-Ph		3-CN		cHexyl	23	>40	17
22		0800692	CO2Et		2-CO2H		4-OMe-Ph	>40	>40	34
23		0800690	CH2CONEt2		2-CO2H		4-OMe-Ph	>40	>40	28
24		0800689	CH2CONH(CH2)2OMe		2-CO2H		4-OMe-Ph	>40	ND	>40
25		0800685	cHexyl		2-CO2H		4-OMe-Ph	>40	ND	>40
26		0800684	3-CI-Ph		2-CO2H		4-OMe-Ph	17	ND	20
27		0800683	2-OEt-Ph		2-CO2H		4-OMe-Ph	20	>40	37
28		0800681	2-F-Ph		2-CO2H		4-OMe-Ph	33	>40	25
29		0800680	4-OMe-Ph		2-CO2H		4-OMe-Ph	>40	>40	>40
30		0800679	Ph		2-CO2H		4-OMe-Ph	15	>40	17
31 *		0800678	2-OMe-Ph		2-CO2H		4-OMe-Ph	26	9	>40
32		0800677	cHexyl		2-CO2H		4-F-Ph	>40	ND	>40
33 *		0800676	2,3-di-Me-Ph		2-CO2H		2-OMe-Ph	23	2	28
34		0800673	2,3-di-Me-Ph		2-CO2H		3-CI-Ph	8	ND	10
35		0800672	2,3-di-Me-Ph		2-CO2H		2-CI-Ph	22	ND	26
36 *		0800675	2-CI-Ph		2-CO2H		CH2-(4-Me-Ph)	32	16	>40
37		0800674	4-Ac-Ph		2-CO2H		4-OMe-Ph	21	>40	20
38		0800671	3-Me-Ph		2-CO2H		4-OMe-Ph	16	>40	22
39 *		0800670	2-OMe-Ph		2-CO2H		2-CI-Ph	26	16	36
40		0800668	2-Me-5-CI-Ph		3-CO2H		2-Me-Ph	>40	>40	>40
41		0800667	2,5-di-Me-Ph		3-CO2H		4-CI-Ph	>40	ND	>40
42		0800666	2-OMe-Ph		3-CO2H		2-thienyl	27	>40	>40
43		0800665	2-F-Ph		3-CO2H		4-CI-Ph	>40	ND	>40
44		0800664	2-OMe-Ph		3-CO2H		Ph	>40	ND	>40
45		0800669	2-Me-5-CI-Ph		3-CO2H		4-Me-Ph	5	>40	12
46		0754480	2-Me-5-CI-Ph		2-CO2H		CH2(4-OMe-Ph)	25	32	24
47		0800662	2-Me-5-CI-Ph		3-CO2H		4–0Me-Ph	5	>40	14
48		0801324	2-Me-5-CI-Ph		2-CO2H		2,4-di-OMe-Ph	>40	>40	>40
49		0814425	2-Me-Bz		H		t-Bu	2	>40	2
50		0814419	2-pyridyl		2-CO2H		2-f uran	>40	>40	>40
51		0814416	(CH2) 3-Ph		2-CO2H		2-OMe-Ph	>40	>40	>40
52		0814414	Bn		2-CO2H		2-OMe-Ph	>40	>40	>40
53		0814424	CO-(2-pyridyl)		2-CO2H		2-OMe-Ph	>40	>40	>40
54		0814421	CH2-(2-pyridyl)		2-CO2H		2-OMe-Ph	10	>40	>40
55		0801308	2-pyridyl		2-CO2H		2-OMe-Ph	>40	4	21
56		0814417	2-pyridyl		H		2-OMe-Ph	>40	>40	14
57		0801723	2-Me-5-CI-Ph		2-CO2Me		2,4-di-OMe-Ph	>40	>40	>40
58		0801724	2-Me-5-CI-Ph		2-CO2Me		2-OMe-Ph	>40	>40	>40
59		0351221	2-Me-5-CI-Ph		2-CO2H		2-OMe-Ph	21	2	19
60		0814423						>40	>40	>40
61		0814422						>20	>20	>20
62 *		0814418						21	18	>40
63 *		0814420						19	32	>40
64		0800691						>40	12	>40
65		0372958						27	ND	32
66		0800661						30	>40	27
67		0800663						>40	ND	>40
68		0800687						>40	>40	>40
69		0800693						>40	>40	22
70		0800694						>40	ND	>40

^aTypical errors were ±10% (n = 3) and entries highlighted in red provided activity in Assay 2 below their MTC

^*denoted pan inhibitor activity where the compound showed activity in both assays below the compound’s MTC value.

Assay 1 (“Life Assay”) was originally designed to identify PTI compounds, which target polyamine uptake. Ant44 (6 in Figure 1) was used as a cytotoxic polyamine probe, which enters and kills cells via the polyamine transport system.^17–19 The negative and positive controls in this assay were the Ant44-only control (2 μM, 50% relative growth) and untreated CHO-K1 cells (100% relative growth), respectively. Using the 48 h IC₅₀ dose of Ant44 (2 μM), we monitored changes in CHO-K1 cell growth to identify new compounds that rescue cells from Ant44 toxicity. Positive hits in Assay 1 provided increased relative cell growth in the presence of an IC₅₀ dose of Ant44. A EC₅₀ value was then determined and represents the concentration of the hit molecule necessary to give a relative growth percentage halfway between the two controls (i.e., 75% growth in the presence of an IC₅₀ dose of Ant44). This approach facilitated the ranking of hits via EC₅₀ values, where the lower EC₅₀ value indicated a more potent compound. There were clear caveats with using this approach as a single screening tool because one could identify compounds that mitigate Ant44 toxicity via polyamine-independent mechanisms. Moreover, false negatives could occur if the compound was too toxic at the concentration tested. For this reason, we also investigated the hits using the orthogonal assay, Assay 2.

In developing Assay 2, we took advantage of the fact that DFMO-treated cells compensate for ODC inhibition via obligate polyamine import.⁴ We also noted that both CHO-K1 and human L3.6pl pancreatic cancer cells responded similarly in these assays. For example, L3.6pl pancreatic cancer cells incubated with DFMO (8 mM; 48 h) gave 64% relative growth (compared to an untreated control) and had no detectable intracellular putrescine, 2.1 nmol spermidine/mg protein, and 18.6 nmol spermine/mg protein.⁴ In contrast, L3.6pl cells incubated with DFMO (8 mM; 48 h) in the presence of a rescuing dose of spermidine (1 μM) gave 103% relative growth (compared to an untreated control), no detectable intracellular putrescine, 6.4 nmol spermidine/mg protein, and 17.4 nmol spermine/mg protein.⁴ Therefore, extracellular spermidine can be used to increase intracellular spermidine pools (from 2.1 to 6.4 nmol/mg protein) and to increase relative cell growth in the presence of DFMO.⁴

Assay 2 (Death Assay) was designed to identify compounds that blocked the ability of spermidine (Spd, 1 μM) to rescue CHO-K1 cells treated with a 48 h IC₅₀ dose of DFMO (4.2 mM in CHO cells). The positive and negative controls in Assay 2 were DFMO-only (50% relative cell growth) and DFMO + spermidine (100% relative cell growth), respectively. A hit compound in Assay 2 should provide a percentage relative growth close to the DFMO-only control (e.g., 50% relative growth) in the presence of DFMO + Spd. An EC₅₀ value was then assigned in Assay 2 and represents the concentration of compound required to observe a percentage relative growth halfway between the DFMO-only control (50%) and the DFMO + Spd control (100%), i.e., 75% relative growth in the presence of an IC₅₀ dose of DFMO and the rescuing dose of Spd. Using the midpoint of the available spermidine rescue response, we sought to address expected minor variations in the controls on each plate during the HTS campaign. The EC₅₀ values from Assay 2 also allowed us to rank and identify potent compounds, where the lower EC₅₀ value denoted the more potent hit compound.

Since innate toxicity of each hit compound was important for our interpretation of the datasets in both Assays 1 and 2, a parallel screen was performed to determine the cytotoxicity profile of each candidate compound. In this manner, we could determine the maximum tolerated concentration (MTC) of each candidate, where essentially no toxicity was observed (≥90% relative growth was observed compared to an untreated control). The MTC values were determined for each hit compound in both CHO and L3.6pl cells. This information was critical as the MTC was the highest dose one could examine in Assays 1 and 2 without overtly biasing the assay results.

Understanding each compound’s toxicity was of prime importance because a compound screened at a toxic dose in Assay 1 would result in lower growth and be discarded as a false negative. A compound screened at a toxic dose in Assay 2 would result in decreased cell growth and appear as a positive hit. Thus, it was critical especially in Assay 2 to rule out false positives using concentrations ≤ MTC of the candidate compound to determine the EC_x values. Since both L3.6pl cells and Chinese hamster ovary (CHO) cells responded similarly in these assays, the hits from the CHO HTS were repeated in 96-well format with L3.6pl cells. As such, this search ultimately resulted in compounds that were active in highly metastatic L3.6pl human pancreatic cancer cells.¹⁶

Using the two orthogonal assays (Assays 1 and 2) in HTS, we screened 322,716 compounds in the Sanford Burnham molecular library for their ability at a single dose to block the 48 h toxicity of a cytotoxic polyamine probe (Ant44, 48 h CHO-K1 IC₅₀ = 2 μM) in CHO-K1 cells. Assay 1 was designed to identify compounds that blocked Ant44 uptake (or its toxicity) as measured by an increase in relative cell growth in the presence of an IC₅₀ dose of Ant44. The final tested compound concentration in each well was either 20 μM or 10 μg/mL (depending upon the compound source). Toxic compounds in Assay 1 were excluded from the promotion to Assay 2. The data from each assay was only considered valid if the compound, when tested alone at the same original concentration, gave a relative percentage growth of >90% (i.e., was nontoxic). Using these selection criteria, only 1028 compounds initially met the criteria in Assay 1 for promotion to Assay 2 (e.g., a 0.3% hit rate). However, when these were retested in the primary assay (Assay 1), again only 362 reconfirmed as hits (0.1% hit rate). These reconfirmed 362 compounds were then promoted to Assay 2.

Assay 2 measured changes in relative cell growth and evaluated the ability of the candidate to function as a DFMO adjunctive agent and limit cell growth in the presence of a rescuing dose of spermidine (Spd, 1 μM) and DFMO-treated CHO-K1 cells (after 48 h). In Assay 2, DFMO was dosed at its 48 h IC₅₀ concentration (4.2 mM in CHO cells) with and without the rescuing dose of spermidine. DFMO alone gave 50% relative cell growth, whereas the DFMO + Spd control gave ∼100% relative growth (compared to an untreated control). As stated earlier, a hit in Assay 2 was expected to give a relative growth percentage somewhere between the DFMO and DFMO + Spd controls. Of the 362 compounds tested in quadruplicate, 138 compounds were active in this assay. These structures were then triaged by medicinal chemists to provide 80 compounds of interest. Of these 80 compounds, 53 compounds were available from commercial sources and were ordered or synthesized for further testing.

The 53 compounds were tested again via dose–response experiments in both assays in CHO-K1 cells in a 384-well format and the data are summarized in Table 1. As a result of this large screening effort, substituted anthranilic acid derivatives (predicated upon scaffold A, Figure 3) were found to be consistently active hits in Assay 2. We next developed synthetic methods to access this structural class and determined the respective EC₅₀ values for selected compounds in L3.6pl human pancreatic cancer cells in Table 2.

Figure 3.

Scaffold A and retrosynthesis to B–F.

Table 2.

Compounds Screened in Both Assay 1 (Ant44) and Assay 2 (DFMO/Spd) with L3.6pl Human PDAC Cells^a

		Performance in L3.6p1 PDAC cell line (72h)
Compd # (SBI-code)	Structure	MTC (μM)	IC50 (μM)	DFMO/Spd ECx (at MTC)	Ant44 ECx (at MTC)
8 0800682		90	124.8	EC2	EC27
9 0754479		8	16.8	EC49	Not active
27 0800683		10	>20	EC36	Not active
31* 0800678		6	13.3	EC8	EC25
33 0800676		8	22.9	EC38	Not active
48 0801324		40	61.6	EC23	Not active
49 0814425		10	>10	EC15	EC15
50 0814419		12	>50	EC31	EC41
51 0814416		10	>10	EC16	Not active
52 0814414		10	>10	EC26	Not active
53 0814424		10	>10	EC33	Not active
54 0814421		10	>100	EC23	Not active
55 0801308		2	>100	EC9	Not active
56 0814417		10	>10	EC20	Not active
59 0351221		7	29.1	EC28	Not active
60 0814423		10	>10	EC19	Not active
62 0814418 ChemDiv 0884		9	46.3	EC43	Not active
63* 0814420		10	38.6	EC45	EC35
64 0800691		25	>30	EC42	Not active
68 0800687		25	39.9	EC22	Not active

^aThe MTC column lists the maximum tolerated concentration of inhibitor where the relative cell growth is still >90%. The IC₅₀ column denotes the concentration of inhibitor that inhibits 50% relative growth of L3.6pl cells compared to an untreated control. None of the compounds reached the EC₅₀ value and thus are listed as their EC_X value as defined in the text. The higher the EC_x value, the more efficacious the compound performed in Assay 1 or Assay 2. Using the arbitrary cutoff of EC₃₀, the red text indicates high activity in the DFMO/Spd rescue Assay 2 vs Assay 1, and the purple text indicates activity in both Assays 1 and 2 (i.e., pan activity). Data is the average of two or more experiments performed in triplicate (n ≥ 6) and the typical standard deviation was ±5%.

Synthesis of this structural class of anthranilic acids was dependent upon the availability of the N-substituted piperazine starting material B needed to couple to methyl 5-fluoro-2-nitrobenzoate (C: R = Me). In some cases, these piperazines were acquired via Pd-catalyzed coupling of bromo-aryl derivatives and piperazine directly²⁰ or via commercial vendors (e.g., Sigma-Aldrich). In cases where appreciable steric hindrance blocked Pd-mediated coupling, it was necessary to build the desired piperazine derivative B from its respective aniline precursor E and the nitrogen mustard F. Examples of our synthetic approaches to methyl esters like 7 and carboxylic acids like 8 and 9 are summarized in Schemes 1 and 2, respectively.

Scheme 1.

^aReagents: (a) Pd₂(dba)₃, RuPhos, t-BuONa, toluene, reflux (47% yield); (b) methyl 5-fluoro-2-nitrobenzoate, Et₃N, dioxane, reflux (80%); (c) zinc, acetone/water, NH₄Cl, 60 °C (22%); (d) 4-methoxybenzoyl chloride, aq. Na₂CO₃, dichloromethane (DCM), rt (79%); (e) 1 M aq. NaOH, MeOH, DCM, rt, then 0.1 M HCl (92%).

Scheme 2.

^aReagents: (a) diethylene glycol methyl ether, under N₂ gas, 150 °C, then MeOH, Et₂O, Na₂CO₃(aq), EtOAc (40% yield); (b) methyl 5-fluoro-2-nitrobenzoate, Et₃N, dioxane, reflux (61%); (c) zinc, acetone/water, NH₄Cl, 60 °C (100%); (d) 4-methoxybenzoyl chloride, aq. Na₂CO₃, DCM, rt (89%); (e) 1 M aq. NaOH, MeOH, DCM, rt, then 0.1 M HCl (87%).

As shown in Scheme 1, compounds 7 and 8 were synthesized from 2-chloro-1-bromobenzene 10 and piperazine 11 in the presence of Pd₂(dba)₃ and RuPhos²⁰ to give the N-phenylpiperazine derivative 12 (47% yield), which was then coupled to methyl 5-fluoro-2-nitrobenzoate to give the substituted 2-nitrobenzoate 13 (80%). The nitro group of 13 was selectively reduced using zinc to give the aniline derivative 14 (22%). Compound 7 was then generated via acylation of 14 using 4-methoxybenzoyl chloride (79%). Lastly, carboxylic acid 8 was obtained in 92% yield after hydrolysis using aq. NaOH in MeOH. Later experiments found that anilines like 14 were unstable as their free base form and that their yields and storage could be improved via generation as their HCl salt form.

Efforts to couple piperazine to bromobenzene derivatives with bulky ortho substituents gave very poor yields using the Pd₂(dba)₃ and RuPhos reagents.²⁰ In this regard, we developed an alternative route from aniline starting materials to generate N-phenylpiperazines.

As shown in Scheme 2, compound 9 was synthesized from the commercial aniline derivative 15. The desired N-phenylpiperazine 17 was obtained in 40% yield via the reaction of 15 and nitrogen mustard 16. Compounds 18 (61% yield), 19 (100%), and 20 (89%) were obtained via similar synthetic steps as described for 8 in Scheme 1. Carboxylic acid 9 was then generated in 87% yield via hydrolysis. In this regard, a general synthetic path was developed to provide the desired anthranilic acid derivatives in good overall yield from either N-phenylpiperazine or aniline starting materials (e.g., 19% overall yield of 9 from 15).

The remaining compounds were either purchased from ChemDiv (compounds 21–32, 36–47, 67–70), Aurora Fine Chemicals (64), AKos (compounds 33–35, 49, 56, 65, 66), or synthesized with similar synthetic methods (compounds 48, 50, and 59–63).

Anthranilic Acid Compound SAR.

The top hits from Assays 1 and 2 in CHO-K1 cells are shown in Table 1 in a 384-well format. To demonstrate the utility of the hit compounds in pancreatic cancer cells, we also determined the EC₅₀ value in each assay, the maximum tolerated concentration (MTC) and the IC₅₀ value for select candidates in L3.6pl human pancreatic cancer cells. These L3.6pl cells were chosen to compare the results with prior experiments in this cell line.^4,12,13 The L3.6pl results are shown in Table 2 and the EC_x values determined in each assay were assigned according to the rubric shown in Figure 4.

Figure 4.

Illustrations of Assays 1 and 2 results and how EC values are defined. In Assay 1, the EC₅₀ value was defined as the concentration of compound needed to obtain a percentage relative growth halfway (e.g., 75% relative growth) between the Ant44 IC₅₀ dose (50% relative growth) and the untreated control (100% relative growth). In Assay 2, the EC₅₀ value was defined as the concentration of compound needed to obtain a relative growth percentage halfway (e.g., 75% relative growth) between the DFMO IC₅₀ dose (50% relative growth) and the DFMO + Spd control (100% relative growth). Note: the direction of the EC value scale in Assay 2 (right y-axis) is opposite of that in Assay 1. This is because the assays are orthogonal and the respective EC₅₀ values are based on the success of the compound in inhibiting the action of assay reagents that are either toxic (Ant44) or nontoxic (spermidine, Spd).

As shown in Table 1, numerous entries (highlighted in red) showed activity in Assay 2 when tested below their MTC and some were active in both assays (i.e., were pan inhibitors). This investigation led to a better understanding of the SAR of this class of molecules. For example, comparison of 9 (4-OMe-Ph), 48 (2,4-di-OMe-Ph), and 59 (2-OMe-Ph) revealed that the cytotoxicity and potency in Assay 1 could be readily modified by alterations to the R₃ substituent in Table 1. The presence of the (4-methoxy)-2-phenylacetamide group in 46 (compared to phenylcarboxamide 9) imparted decreased cytotoxicity but also loss of potency in Assay 2 (DFMO/Spd). Moreover, a comparison of 34 (3-Cl-Ph) and 35 (2-Cl-Ph) indicated that introduction of a 3-chloro group in the phenylcarboxamide substituent increased the cytotoxicity and potency in Assay 2.

Assay 1 Hits.

While not the focus of this paper, we noted that comparison of 8 (2-Cl-Ph), 27 (2-OEt-Ph), 28 (2-F-Ph), 30 (Ph), and 31 (2-OMe-Ph) revealed that the 2-methoxy substituent on the terminal aniline ring (i.e., R₁ in Table 1) significantly improved potency in the Ant44 assay (Assay 1 EC₅₀ 31: 9 μM), whereas the other compounds (8, 27, 28, and 30) had Assay 1 EC₅₀ values > 40 μM. Other substituents in R₁ (see Table 1) such as 29 (4-MeO-Ph), 37 (4-Ac-Ph), 38 (3-MePh), and 47 (2-Me-5-Cl-Ph) were found to be ineffective in Assay 1. Thus, within this series, the 2-methoxyphenyl substituent in R₁ was important for activity in Assay 1 and was less toxic than the phenyl parent 30. These hits may provide future tools to study intracellular polyamine trafficking but were not the focus of our study.

Assay 2 Hits.

Instead, we focused on compounds like 9 that were not effective in rescuing cells from Ant44 in Assay 1 but showed good activity in Assay 2 (DFMO/Spd, Assay 2 EC₅₀ for 9 = 8 μM). The related compound 20 (methyl ester of 9) was inactive in both Assays 1 and 2 (and nontoxic), indicating the importance of the free carboxylic acid for inhibition. Insertion of a methylene unit via 46 decreased cytotoxicity but also decreased potency in Assay 2 compared to 9. Thus, 2-phenylacetamide substituents (46) as R₃ in Table 1 were not as effective in Assay 2 as phenylcarboxamides (e.g., 9).

Remarkably, the 2-pyridyl compounds 54 (R₁ = CH₂-(2-pyridyl)) and 55 (R₁ = 2-pyridyl) had nearly equal and opposite activities in Assays 1 and 2. A derivative of 55 without a carboxylic acid (i.e., 56: R₁ = 2-pyridyl, R₂ = H, Table 1) demonstrated that the carboxylic acid (2-COOH at R₂) did not significantly influence compound cytotoxicity as 55 and 56 had similar cytotoxicity values of 21 and 14 μM, respectively.

Comparison of the constitutional isomers 9 (R₂ = 2-CO₂H) and 47 (R₂ = 3-CO₂H) suggested that the location of the carboxylic acid was flexible to maintain activity in Assay 2. However, later synthetic experience demonstrated enhanced instability of the aniline intermediate used to make 47, suggesting materials like 9 may have fewer synthetic liabilities. Interestingly, compounds 53 (CO-2-pyridyl, Assay 2 EC₅₀ > 40 μM) and 54 (R₁ = CH₂-(2-pyridyl), Assay 2 EC₅₀ 10 μM) implied that a flexible linker was preferred in the DFMO Assay 2. Overall, compound performance in Assay 1 (55 vs 60) and Assay 2 (53 vs 54) showed sensitivity to the placement of the flexible methylene linker.

A comparison of 2-methoxycarboxamides (33, 62, and 63) revealed that various hydrophobic substituents at R₁ gave similar potency in Assay 2 (Assay 2 (DFMO/Spd), EC₅₀ values ∼ 20 μM). We also noted that compound 49 was the most potent in Assay 2 (EC₅₀ = 2 μM) but 49 was also the most cytotoxic (MTC = 2 μM). Compounds 45 (4-Me-Ph) and 47 (4-OMe-Ph) contained the 3-CO₂H group at R₂ and were potent in Assay 2 (with EC₅₀ values = 5 μM, Table 1). Both compounds contained para substituents on the phenylcarboxamide at R₃ (Table 1) and the same 2-methyl 5-chlorophenyl group at R₁. However, both compounds were relatively toxic as well with MTC values of 12 and 14 μM, respectively. In a similar fashion, compounds 9 (EC₅₀ = 8 μM) and 34 (EC₅₀ = 8 μM) gave MTC values in Assay 2 of 13 and 10 μM, respectively. In terms of balancing performance in Assay 2 with low cytotoxicity, the 2-pyridyl group in compound 54 was advantageous and provided an Assay 2 EC₅₀ value for 54 of 10 μM and a MTC > 40 μM. Thus, our top hits in Assay 2 from the CHO screen conducted in a 384-well format were compounds 9, 34, 45, 47, 49, and 54.

Pan Actives.

In terms of compounds displaying pan activity (i.e., activity in both Assays 1 and 2), we noted that six compounds (31, 33, 36, 39, 62, and 63) had activity in both assays below their respective MTC values. Four of the six compounds (31, 33, 36, and 39) had an ortho substituent on R₁ (e.g., R₁ = 2-OMe-Ph, 2,3-diMe-Ph, or 2-Cl-Ph) and the other two (62 and 63) were predicated upon different scaffolds. Thus, the ortho substituent at R₁ is likely a favorable aspect of the design, e.g., see Table 1: 29 (R₁ = 4-OMe-Ph, inactive) vs 31 (R₁ = 2-OMe-Ph, pan activity). We speculated that this substituent may cause the R₁ phenyl group to twist out of plane to avoid steric interactions with the nearby piperazine ring.

Interestingly, compounds 31 and 36 had para-substituted phenyl rings at R₃, whereas 33 and 39 had ortho substituents at this position (Table 1) suggesting some variability of substituents was tolerated for the pan actives in the phenylcarboxamide ring in R₃. Comparison of 9 (R₃ = 4-OMe-Ph, EC₅₀ 8 μM in Assay 2), 48 (R₃ = 2,4-di-OMe-Ph; no activity in CHO cells), and 59 (R₃ = 2-OMe-Ph, EC₅₀ 2 μM in Assay 1) suggested that ortho or para methoxy substitutions at R₃ provide enhanced selectivity in Assays 1 and 2, respectively. In terms of overall potency, Table 1 suggested that the pan active compounds 31 (R₁ = 2-OMe-Ph, R₃ = 4-OMe-Ph, MTC > 40 μM) and 33 (R₁ = 2,3-diMe-Ph, R₃ = 2-OMe-Ph, MTC = 28 μM) had the best balance of properties in terms of potency in Assays 1 and 2 and relatively high MTC values.

We noted in many cases that compounds appeared to function as hits in Assay 2 near their maximum tolerated concentration (MTC, e.g., especially compounds 8, 26, 30, 33, 34, 35, 38, 45, 47, 49, and 65). This was concerning because Assay 2 is sensitive to false positives, which are cytotoxic and provide readouts of decreased growth. Therefore, when these hits were reevaluated in L3.6pl cells, we again controlled for this potential bias by independently determining each compound’s MTC value and dosing each compound up to its MTC to determine the EC₅₀ value. The top hits from the CHO screens in Table 1 were repeated in 96-well format in L3.6pl human pancreatic cancer cells and the results are highlighted in Table 2. Additional compounds were also screened and are listed in Table 2 to better interpret the SAR observed.

Interestingly, none of the compounds tested in L3.6pl cells had sufficient activity to determine an EC₅₀ value and the EC_x values ranged from 0 to 49. When a compound did not reach a formal EC₅₀ value at its MTC, the EC_x value is listed in Table 2, where x denotes the percentage inhibition observed in the specific assay at the compound’s MTC. The MTC provided a convenient dosing ceiling in these studies to investigate each compound’s maximum effect because the results listed are the best one can obtain for each compound without introducing a toxicity bias in each assay that happens when one increases the inhibitor concentration beyond the MTC. In this regard, a compound like 9 with a higher x value (e.g., EC₄₉, where x = 49) at a lower concentration (MTC = 8 μM) is deemed more potent than a related compound 54 with a lower x value at a higher concentration, e.g., EC₂₃ at an MTC of 10 μM.

Table 2 provided interesting activity profiles where compounds 9, 27, 33, 52, 53, 59, 62, and 64 showed selective activity in DFMO/Spd Assay 2 compared to Ant44 Assay 1. In addition, two compounds 50 and 63 showed pan activity and had EC_x values ≥ 25 in both Assays 1 and 2. Since our goal was to identify DFMO adjunct agents, we focused on the most efficacious compound in Assay 2 when dosed at its MTC, i.e., compound 9 (EC₄₉ at 8 μM). This lead compound significantly reduced cell growth in DFMO/Spd/compound treatment compared to DFMO/Spd control (Figure 5). Interestingly, nearly all of the other top performing compounds in Assay 2 (27, 33, 52, 53, 59, 62, and 64) have either ortho (33, 52, 53, 59, or 62) or para (27 and 64) methoxy substituents on their benzamide ring with the lone exception of pan inhibitor 50, which contained a furan substituent. This suggested an important role for the ortho and para groups in binding to the putative target.

Figure 5.

Compound 9 toxicity and performance in L3.6pl cell growth assays. (A) Compound 9 toxicity curve in L3.6pl cells over 72 h. (B, C) Compound 9 performance in Assays 1 and 2 in L3.6pl cells over 72 h. Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, and ****p < 0.0001. Note: the 24 h cytotoxicity curve for compound 9 in L3.6pl cells can be found in the Supporting Information.

Having shown that the anthranilic acid scaffold provided DFMO adjunct agents in vitro, we performed additional experiments on compound 9 to gain insight into its mechanism of action.

Polyamine Import.

First, we needed to rule out inhibition of polyamine import as the target of 9. We recognized that polyamine transport is a multistep process that involves initial recognition and uptake (import) and then multiple intracellular trafficking steps for polyamines to reach their putative intracellular target(s). Polyamine-based PTIs like compound 5 act as competitive inhibitors of polyamine import²¹ and use their appended polyamine motifs to outcompete the native polyamines for cell surface receptors (like heparan sulfate proteoglycans on glypican-1).²²

We conducted a radiolabeled spermidine (³H-Spd) uptake study to see if compound 9 blocked the basal uptake of exogenous polyamines. As shown in Figure 6A, the polyamine-based PTI 5 blocked significant spermidine uptake. In contrast, the non-polyamine-based compound 9 did not block the uptake of ³H-Spd into L3.6pl cells. Since cell growth data (Figure 5C) indicated that compound 9 performed in combination with DFMO, we also tested ³H-Spd levels in L3.6pl cells treated with combination therapy (DFMO + 9; Figure 6B). The presence of both DFMO and compound 9 did not significantly inhibit ³H-Spd import compared to DFMO-only, whereas PTI 5 showed a statistically significant reduction in ³H-Spd import. This data confirmed that compound 9 does not inhibit ³H-Spd uptake into cells under either basal or obligate polyamine import conditions. Therefore, inhibition of spermidine uptake was ruled out as a mechanism to explain the ability of compound 9 (at a nontoxic dose) to potentiate inhibition of cell growth in the presence of DFMO.

Figure 6.

Inhibition of radiolabeled spermidine (³H-Spd) uptake in L3.6pl cells. (A) Intracellular ³H-Spd levels upon 24 h treatment with a non-polyamine-based compound 9 versus polyamine-based PTI (5) prior to ³H-Spd addition for 15 min. (B) Intracellular ³H-Spd levels after 72 h incubation with DFMO alone or a combination of DFMO/compound prior to ³H-Spd addition for 15 min. Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, and ****p < 0.0001. This confirmed that compound 9 inhibited neither basal nor obligate (DFMO-inspired) polyamine import.

In our efforts to understand the mechanism by which the Assay 2 hits like 9 affect cell growth, we performed an unbiased search for all known biological targets of the anthranilic acid parent compound and discovered a lone paper describing a NMR study, where anthranilic acid was found to bind to the far upstream element (FUSE) binding protein 1 (FUBP1) albeit with very low affinity.¹⁰ Curious about this possibility, we explored FUBP1 as potential target of the Assay 2 hits. As discussed previously, FUBP1 was an interesting target to consider because it is a known transcriptional regulator of c-Myc, which itself is a known transcriptional activator of ODC, the rate determining enzyme in polyamine biosynthesis (Figure 2).

To explore the ability of compound 9 to inhibit FUBP1, we investigated known FUBP1 targets. FUBP1 regulates the expression of several important genes via a FUSE-controlled process. During DNA transcription, the DNA double helix is unwound to expose single-stranded DNA sequences. Certain sequences like the FUSE sequence on the noncoding strand prefer an extended conformation, which facilitates the docking of DNA binding proteins like FUBP1. Once bound, FUBP1 recruits other proteins like the helicase TFIIH, which then releases a stalled RNA polymerase to initiate transcription of a downstream gene. The FUSE-controlled process can also block transcription of a gene.

To validate the putative FUBP1 target, we searched for genes that were positively or negatively regulated by FUBP1/FUSE. A recent paper by Debaize described FUBP1 as a master regulator of c-Myc transcription.²³ The c-Myc oncogene is known to regulate cell metabolism and proliferation and, thus, is a desirable target in cancer research.²⁴ Other FUBP1 targets included ubiquitin-specific peptidase 29 (USP29), oncogene c-KIT, cell cycle inhibitor p21 (CDKN1A), cyclin D2 (CCND2), and the proapoptotic protein BIK.²³ From these FUBP1 targets, we selected two target genes that are FUSE-controlled in an opposite manner: FUBP1 facilitates c-Myc transcription, whereas p21 transcription is suppressed by FUBP1. These two genes provided a predicted “orthogonal” readout for FUBP1 inhibition, wherein c-Myc expression should decrease and p21 expression should increase in the presence of 9.

L3.6pl cells were treated with increasing concentrations of compound 9 and changes in c-Myc and p21 protein and mRNA levels were assessed. We noted that higher concentrations of 9 (e.g., 40–60 μM) were needed to clearly demonstrate these effects at the 24 h point compared to the 72 h whole cell assay experiments performed in Table 2 (9: MTC = 8 μM). While compound 9 did not affect FUBP1 protein levels, it reduced c-Myc protein expression and increased p21 protein expression in a dose-dependent manner that is consistent with FUBP1 being the target of compound 9 (Figure 7A). We also performed quantitative real-time polymerase chain reaction (qRT-PCR) of L3.6pl pancreatic cancer cells treated with 9. In agreement with the protein results, the mRNA of FUBP1 targeted genes was significantly decreased for c-Myc and increased for p21 compared to the matched vehicle controls (Figure 7B). We also tested the related compound 63 for its effect on c-Myc and p21 protein expression. Compound 63-treated L3.6pl cells also had reduced c-Myc and increased p21 protein levels (Figure S1); however, the reduction of c-Myc was not as significant as compared to compound 9 treatment. Thus, we promoted compound 9 as a putative FUBP1 inhibitor.

Figure 7.

Protein and mRNA studies consistent with the inhibition of FUBP1/FUSE interaction by 9 in L3.6pl cells. (A) Representative Western blot images for c-Myc, p21, and FUBP1 protein levels. GAPDH was used as protein loading control. At least three independent experiments were performed. (B) mRNA levels of p21 and c-Myc genes after 24 h treatment with compound 9. Expression levels of p21 and c-Myc were normalized to β-actin. * denotes significant mRNA expression difference between the experimental treatment and its matched dimethyl sulfoxide (DMSO) control. Data are presented as log mean ± SD (n = 2). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

We then conducted a chromatin immunoprecipitation (ChIP) assay as a direct way to show FUBP1 protein inhibition by compound 9. The ChIP assay works by temporarily crosslinking the putative DNA-bound protein (FUBP1) to its corresponding DNA sequence (FUSE region) and then shearing the unbound DNA apart. The intact FUBP1–DNA complex is then immunoprecipitated using a FUBP1 antibody. The crosslink is then reversed and the liberated DNA strand is amplified using specific primers for the precise FUSE sequence associated with the gene in question (e.g., c-Myc) and is quantified via q-PCR. Untreated cells should give higher levels of FUSE DNA in the ChIP assay because the FUBP1/FUSE DNA complex will be pulled down via the FUBP1 antibody and give rise to basal levels of FUSE DNA via q-PCR. In contrast, the presence of a FUBP1 inhibitor should block the association between FUBP1 and FUSE DNA and cause less FUSE DNA to be bound and pulled down via the FUBP1 antibody, which in turn would give rise to lower levels of FUSE DNA after q-PCR.

L3.6pl cells were treated with or without compound 9 (60 μM) over 24 h. FUBP1 antibody was used to pull down the complex of FUBP1 protein and the specific FUSE DNA sequence located upstream of the c-Myc gene promoter. ChIP primers for the FUSE c-Myc were designed according to Venturutti et al.²⁵ As anticipated, the untreated sample had higher levels of the FUSE c-Myc DNA indicating a higher association of FUBP1 and FUSE c-Myc region compared to cells treated with compound 9 (Figure 8). The reduced levels of c-Myc FUSE DNA in cells treated with 9 were consistent with its inhibition of the FUBP1–FUSE DNA interaction as implied by Figure 7. In sum, the ChIP assay results also supported FUBP1 as a target for compound 9.

Figure 8.

Quantitative real-time PCR amplification of ChIP DNA samples indicated the relative binding of FUBP1 protein to the FUSE c-myc promoter region in L3.6pl cells in the absence and presence of compound 9 (24 h). Data are presented as mean fold change ± S.E.M. (n = 2).

Next, we sought to further corroborate this finding via a gel shift assay technique, wherein affinity-purified GST-FUBP1 (KH3-KH4), i.e., a FUBP1 protein containing the key KH3/KH4 domains, was challenged to bind an oligonucleotide sequence targeted by the FUBP1 KH4 domain (Oligo-KH4) in the presence and absence of escalating concentrations of compound 9. As shown in Figure 9A and quantified from four replicates in panel B, compound 9 provided a dose-dependent decrease in the FUBP1 (KH3-KH4) protein–FUSE DNA-bound complex compared to the relevant DMSO control. Figure 9C demonstrates the specificity of this FUBP1–FUSE DNA interaction when the same FUBP1 (KH3/KH4) protein is challenged to form a bound complex with Oligo-1 vs the preferred oligomer normally targeted by the KH4 domain (Oligo-KH4). As shown in Figure 9C (left side third lane), very little to no complex is formed with Oligo-1 compared to Oilgo-KH4 (rightmost lane).

Figure 9.

Gel shift assay. Effect of compound 9 on FUBP1 binding to DNA. (A) A total of 1 pmol of 16-mer single-stranded FUSE oligo was incubated with 5 pmol of affinity-purified GST-FUBP1 (KH3-KH4) in the presence of indicated amounts of compound 9, and the respective DMSO loading as a control. The experiment was conducted four times and a representative image is shown. (B) Quantification of bound complex with FUBP1(KH3-KH4) in the presence of 9 (at 30 or 60 μM). Error bar representing the mean with standard error of the mean (SEM) from four independent experiments. Statistical analysis (one-way analysis of variance (ANOVA) nonparametric) was performed using GraphPad Prism Software. P value < 0.05 was considered as significant. * ≤0.05, ** ≤0.01, ns—not significant. Note: the four gels used to compile panel (B) are shown in Figure S2. (C) EMSA gel showing the specificity of FUBP1 (KH3-KH4) protein binding to the KH4 targeted FUSE DNA sequence (oligo-KH4) vs Oligo-1 that contains a different DNA sequence.

Having confirmed 9 as a FUBP1 inhibitor by ChIP assay, gel shift assay, mRNA expression changes, and Western blot, we then compared its inhibitory activity against a known FUBP1 inhibitor (Figure 10). There have been few reports describing other FUBP1 inhibitors that affect FUBP1 targeted genes.^10,15,26,27 Of those in the literature, the pyrazolo[1,5a] pyrimidine (Hauck inhibitor, Figure 10) was reported to be a potent FUBP1 inhibitor in the liver-derived Hep3B cell line.¹⁵ Thus, we tested the Hauck FUBP1 inhibitor in our Western blot and qRT-PCR assays alongside compound 9. Interestingly, the Hauck inhibitor did not perform well in our system using L3.6pl pancreatic cancer cells and only slightly increased p21 protein levels, while p21 mRNA levels were not significantly reduced compared to the control (Figure 10). The Hauck inhibitor had a modest effect on c-Myc protein levels, and c-Myc mRNA levels were not significantly reduced compared to the control. Of these two inhibitors, compound 9 was superior in terms of inducing p21 (e.g., Figure 10B) in the human PDAC cell line.

Figure 10.

Protein and mRNA levels of FUBP1 targets in L3.6pl cells treated with Hauck inhibitor. (A) Structure of Hauck inhibitor, (B, C) Protein levels of p21 and c-Myc expression in L3.6pl cells treated with Hauck inhibitor or compound 9 and its matched DMSO loading over 24 h vs GAPDH. (D) mRNA levels of p21 and c-Myc in cells treated with Hauck inhibitor over 24 h. Data are presented as log mean ± SD (n = 2).

Having shown compound 9 to be a FUBP1 inhibitor that affects c-Myc expression, we wanted to also investigate its effect on the polyamine pathway downstream of c-Myc to link its mechanism of action to its role in DFMO adjunctive therapy.

The c-Myc gene is known to regulate many progrowth genes, including ODC (Figure 2).²⁸ In this regard, c-Myc provides a direct connection between the putative target FUBP1 and polyamines. A FUBP1 inhibitor would be expected to inhibit the FUBP1–FUSE c-Myc interaction, and thus decrease c-Myc transcription along with other FUSE-controlled genes associated with tumor survival.²³ Reduced c-Myc transcription would in turn lead to lower c-Myc protein, reduced ODC transcription, and lower polyamine levels. To test the link between FUBP1 inhibition and the downstream effect on polyamine levels, we treated L3.6pl cells with compound 9 and measured intracellular polyamine levels. As anticipated, L3.6pl cells treated with 9 had significantly lower polyamine levels when compared to the vehicle control (Figure 11). We noted that compound 9 lowered intracellular polyamine levels while maintaining similar ratios between the native polyamines 1–3. In contrast, the ODC inhibitor DFMO typically causes a complete depletion of putrescine and a decreased spermidine pool as noted earlier.⁴ This suggested that compound 9 limits c-Myc signaling but does not shut it off under these conditions. This ability to reduce downstream c-Myc signaling could have diverse applications in the treatment of human cancers as implied by Levens et al.²⁹

Figure 11.

Native polyamine levels inside of L3.6pl pancreatic cancer cells after incubation for 24 h at 37 °C. Cell were incubated with compound 9 (60 μM) or 0.6% DMSO (vehicle). Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Having evidence to support compound 9 as a FUBP1 inhibitor, we turned to molecular modeling to better understand how compound 9 prefers to bind to FUBP1.

Molecular Modeling.

We started by determining the relative binding affinity of the lead compounds in Assay 2 for FUBP1 using in silico methods. The FUBP1 protein contains four K-homology (KH) DNA binding domains, where domains KH3 and KH4 were critical for FUBP1’s ability to bind the single-stranded DNA FUSE sequence and regulate c-Myc transcription.^11,30 Using the available structure of FUBP1 bound to single-stranded DNA (PDB code: IJ4W; Figure 12A),¹¹ we removed the bound ssDNA in silico and used a ChemDraw/Chem3D/PyRx/PyMol workflow to draw, energy minimize, dock, rank, and render the top hits in terms of their ability to bind FUBP1. Rewardingly, anthranilic acid compounds specifically bound to the same groove of the KH4 domain of FUBP1 even though the KH3 domain was also available. This finding was particularly rewarding as the gel shift assay in Figure 9 used the FUSE sequence associated with KH4 domain–FUSE interactions and demonstrated that compound 9 interferes with FUBP1–FUSE complex formation.

Figure 12.

In silico modeling of interactions between KH4 domain of FUBP1 and ssDNA or compounds 9, 59, and 63 and their binding affinities (PDB code: IJ4W). (A) Interaction of FUSE ssDNA with the groove domain of KH4 on FUBP1; (B) compound 9 prefers to bind to the groove domain of KH4 on FUBP1 that is consistent with the gel shift assay with 9 (see Figure 9); and (C and D) respective anthranilic acid compounds 59 and 63 also target the groove domain of KH4 on FUBP1.

For example, as shown in Figure 12, compounds 9, 59, and 63 all prefer to bind to the main groove of KH4 and not the KH3 domain of FUBP1. Interestingly, the COOH group of the active compounds 9, 59, and 63 orients each molecule in the groove normally used to bind ssDNA. The anthranilic amido NH group twists out of plane to interact with the FUBP1 KH4 top “red cleft” region. We speculate that the adjacent ortho carboxylic acid facilitates this twist. In the case of 9, the amido NH of 9 hydrogen bonds to the available Ser-130 hydroxyl group in the top red cleft region of KH4 (Figures 12B and S3). The twist-out-of-plane amide orientation of 9, 59, and 63 all point their amido carbonyl away from the FUBP1 target precisely into the region of space where ssDNA crosses over the KH4 groove (Figure 12A). These collective observations provide a satisfying target-based explanation for the earlier SAR findings and a rationale for why the anthranilic acid motif is required, as it enables key docking events needed to correctly orient the molecule into the FUBP1 KH4 target. While clearly more work is necessary here to be conclusive (e.g., future cocrystallization experiments), these early modeling insights provide a possible explanation of the SAR we observed in Table 2.

CONCLUSIONS

In summary, using a HTS approach, we screened over 300,000 compounds to discover novel anthranilic acid constructs, which can function as FUBP1 inhibitors. Our data suggests that they do not function as formal inhibitors of polyamine import at the cell surface, even though they provide positive hits in Assay 2. Both a ChIP assay and gel shift assay were used to show that compound 9 inhibited FUBP1 and disrupted the FUBP1–FUSE interaction. In silico modeling suggested that the Assay 2 hits likely target the K-homology domain 4 of FUBP1, a target that nicely explained the observed SAR and gel shift data. FUBP1 targeting was also indicated via the expected inverse outcomes on the FUSE-controlled genes, c-Myc and p21. Success in these development efforts could provide important new adjunct agents for clinical use in the treatment of human cancers as c-Myc modulators or in combination with DFMO.

In closing, we envision these materials may find applications in oncology as well as other proliferative diseases, which rely upon polyamines for growth.³¹ Future work will describe our detailed investigation of the FUBP1 target with other anthranilic acid derivatives.

EXPERIMENTAL SECTION

Materials.

CHO-K1 cells were purchased from ATCC and L3.6pl cells were obtained from Dr. Isaiah Fidler at MD Anderson Cancer Center (Houston, TX). DFMO, aminoguanidine, and spermidine were purchased from Sigma-Aldrich. The 0.25% trypsin, 100× penicillin/streptomycin (P/S), 100× antibiotic–antimycotic, 100× l-glutamine, RPMI, and phenol-free RPMI were obtained from GIBCO. Dulbecco’s phosphate-buffered saline (PBS) was purchased from Corning. Heat-inactivated fetal bovine serum (FBS) was acquired from Hyclone. ATPLite was obtained from PerkinElmer. White plates (384-well) and 1536-well white plates were purchased from Greiner and Aurora, respectively. ³H-Spd was obtained from PerkinElmer. Ant44 was synthesized by the Phanstiel lab (UCF).³² Compound purity is ≥95% with the exception of compounds 52 and 54 that were tested at 94 and 93% purity, respectively. Purity was determined by elemental analysis or high-performance liquid chromatography (HPLC) (see the Supporting Information). Note: descriptions of the assays in 96- and 384-well plate formats are in the Supporting Information.

Methods (1536-Well Assays, HTS Phase of the Project).

Assay 1 (Primary Screening Assay with Ant44 6).

CHO-K1 cells were grown in growth medium (RPMI + 10% FBS + 1× Pen–Strep + 1% l-glutamine) to 70% confluency, washed in PBS, and trypsinized. Briefly, cells were plated in assay medium (serum-free RPMI + 2% FBS + 1× l-glutamine + 1× Pen/Strep + 300 μM aminoguanidine) at a density of 50,000 cells/mL (200 cells per well in 4 μL) using a Thermo Multidrop Combi into a 1536-well white Aurora plate. Note: Aminoguanidine (AG) is added to the cell culture media to inhibit the activity of polyamine oxidase (PAO) present in fetal bovine serum.¹⁹ This is important as polyamine-based agents like Ant44 (Assay 1) and spermidine (in Assay 2) can be degraded by PAO. We found that AG is not toxic to most human pancreatic cancer cell lines at 300 μM and is not toxic to CHO cells even at 1 mM.¹⁹ Cells were spun at 100g for 1 min and incubated at 37 °C in a humidified CO₂ incubator for 24 h. Each test compound (10 mM, 10 nL) was then dispensed with a Labcyte Echo 555 acoustic dispenser for a final assay concentration (FAC) of 20 μM after the followup addition of 1 μL of 10 μM Ant44 (6, FAC 2 μM) in assay medium added with a Beckman BioRAPTR FRD dispenser or 1 μL of assay medium (positive control). The plates were spun at 200g for 1 min and incubated at 37 °C in a humidified 5% CO₂ incubator for 48 h. Cellular ATP content (cell viability) was then assayed by adding 3 μL of ATPLite with a Beckman BioRAPTR FRD dispenser and incubated for 10 min at room temperature. Plates were spun at 200g for 1 min and read on a PerkinElmer Viewlux multimode plate reader using luminescence mode. In the primary HTS assay, compounds were run as singletons with an average z′ of 0.62. In the reconfirmation assay, compounds were run in quadruplicate with an average z′ of 0.56.

Assay 2 (DFMO/Spermidine).

CHO-K1 cells were grown in growth medium to 70% confluency and washed in PBS and trypsinized. Cells were plated in assay medium (containing aminoguanidine as in Assay 1) at 15,000 cells/mL (60 cells per well) using a Thermo Multidrop Combi in a 4 μL volume into a 1536-well white Aurora plate. Cells were spun at 100g for 1 min and incubated at 37 °C in a humidified CO₂ incubator for 24 h. The test compound (10 mM, 10 nL) was then dispensed with a Labcyte Echo 555 acoustic dispenser (for a final assay concentration of 20 μM) followed by 1 μL of 5 mM DFMO and 5 μM spermidine (FAC: 5 mM DFMO and 1 μM spermidine) added in assay medium with a Beckman BioRAPTR FRD dispenser or 5 mM DFMO (1 μL) was added in assay medium as a positive control. The plates were spun at 200g for 1 min and incubated at 37 °C in a humidified 5% CO₂ incubator for 48 h. Cellular ATP content (cell viability) was then assayed by adding 3 μL of ATPLite with a Beckman BioRAPTR FRD dispenser and incubated for 10 min at room temperature. The plates were spun at 200g for 1 min and read on a PerkinElmer Viewlux multimode plate reader using luminescence mode. In the reconfirmation assay, compounds were run in quadruplicate. Note: z′ score for this assay in 96-well format is 0.43.

Cytotoxicity Assay (1536-Well Format).

CHO-K1 cells were grown in growth medium to 70% confluency and washed in PBS and trypsinized. Cells were plated in assay medium at 15,000 cells/mL (60 cells per well) using a Thermo Multidrop Combi in 4 μL into a 1536-well white plate (Aurora). Cells were spun at 100g for 1 min and incubated at 37 °C in a humidified CO₂ incubator for 24 h. The test compound (10 mM, 10 nL) was then dispensed with a Labcyte Echo 555 acoustic dispenser (for a final assay concentration of 20 μM) followed by adding 1 μL of assay medium with a Beckman BioRAPTR FRD dispenser, spun at 200g for 1 min, and incubated at 37 °C in a humidified 5% CO₂ incubator for 48 h. Cellular ATP content was then assayed by adding 3 μL of ATPLite with a Beckman BioRAPTR FRD dispenser and incubated for 10 min at rt. Plates were spun at 200g for 1 min and read on a PerkinElmer Viewlux multimode plate reader using luminescence mode. This assay was used for testing dose–response of dry powders. Note: ATP is present in all metabolically active cells and is a good marker for cell viability as it indicates cytostatic, cytotoxic, and proliferation changes. ATPLite is an ATP monitoring system sold by PerkinElmer and is based on firefly luciferase (Phontinus pyralis). Note: the protocols for running these assays in 96-well and 384-well format are in the Supporting Information.

Radiolabeled Spermidine Uptake Assay.

To determine if a top compound was a competitive polyamine transport inhibitor (PTI), we tested its ability to block radiolabeled spermidine (³H-Spd) uptake in CHO-K1 or L3.6pl cells according to our published protocol.¹² The amount of protein was determined using the Pierce BCA protein assay kit (Pierce, Rockfold, IL) from the remaining lysate volume to normalize the radioactive counts obtained. All of the experimental conditions were performed in triplicate. The data was reported in Figure 6 as pmol ³H-Spd/μg protein ± standard deviation.

Gel Shift Assay.

5′-Fluorescent (IRDye700)-tagged single-stranded oligonucleotide (TGTATATTCCCTCGGG) representing the FUSE sequence of c-Myc was incubated with the recombinant FUBP1 (KH3-KH4 domain) protein and different concentrations of compound 9 (30 and 60 μM). The oligonucleotide (Oligo-KH4) is the preferred FUSE DNA sequence for FUBP1 KH4 domain binding. The reaction was performed in binding buffer (20 mM Tris-HCl, pH 8.0; 1 mM EDTA; 100 mM KCl; 0.5 mg/mL BSA; 5% glycerol and 1 mM DTT) at 25 °C for 30 min. The resulting reaction was then electrophoresed in a native polyacrylamide gel (4–12% TBE gel, Invitrogen, EC62352BOX) in 0.5× TBE running buffer (Novex, LC6675) at 10 V/cm. The DNA–protein complexes were detected using the Odyssey infrared imaging (LI-COR) System. The ratio of the intensity of FUBP1–FUSE complex compared to free oligonucleotide was used to assess the degree of complex formation in the presence and absence of the FUBP1 inhibitor. Note: the assay was run in quadruplicate and the four individual gels used to compile the bar graphs in Figure 9 are shown in Figure S2.

Intracellular Polyamine Quantitation Using HPLC.

For this experiment, we seeded L3.6pl cells at 100,000 cells/mL in a 10 cm dish (Corning, #430293) containing RPMI 1640 medium and aminoguanidine (250 μM) and incubated the cells overnight at 37 °C with 5% CO₂ atmosphere. The next day, cells were dosed with compound 9 (60 μM). Since the compound was dissolved in DMSO, DMSO was added into each relevant dish so that the solvent composition (0.6% DMSO) was identical in each experimental arm. Cells were incubated for 24 h (Figure 11) and samples were processed and quantified by a published polyamine N-dansylation protocol using authentic N-dansylated polyamine standards.^4,12 The final methanol solution containing dansylated polyamines was analyzed by HPLC to determine intracellular polyamine levels and the data was normalized over protein. The amount of protein was determined using the Pierce BCA protein assay kit (Pierce, Rockfold, IL). The data was reported as means of nmol polyamine/mg protein ± standard deviation.

Molecular Modeling.

Each small molecule was constructed in ChemDraw (PerkinElmer) using ACS 1996 settings and imported into Chem3D, where the structure was energy minimized using MM2 to generate the lowest energy conformer. The minimized ligand was imported into the PyRx program (as a ligand with FUBP1 being the docking macromolecule). Note: the pdb file for FUBP1 (pdb IJ4W) contained both the KH3 and KH4 domains of FUBP1 along with bound single-stranded DNA as a complex. The ssDNA was removed in silico and the remaining DNA-free FUBP1 surface was docked with each compound. Remarkably, there was a strong in silico binding preference of these compounds to bind to KH4, which was consistent with the gel shift assay indicating that compound 9 inhibits the ability of FUBP1 to bind to the preferred KH4 oligonucleotide sequence (Figure 9). PyRx was used to generate and rank 9 orientations of each ligand with the highest binding affinity toward FUBP1. The conformer with the highest binding affinity is shown aligned in the preferred docking site, i.e., the KH4 groove. The top conformer was bound to FUBP1 KH4 domain and was rendered using PyMol (Schrödinger, Inc.) to better visualize the interactions between each inhibitor and FUBP1. Visual representation of the available electrochemical gradients on the FUBP1 surface were rendered with blue being relatively positive in charge and red being relatively negative in charge. Relative ligand efficacy was rationalized by comparing its FUBP1 binding location, orientation, distance to available hydrogen bond partners, and electrochemical favorability within the FUBP1 KH4 groove domain (see Figure S3).

Western Blot.

A L3.6pl cell suspension (400,000 cells in 2 mL) in RPMI 1640 medium (Gibco) with 10% FBS and 1% antibiotic–antimycotic (Gibco) was seeded in a 6-well cell culture dish (MidSci) followed by overnight incubation at 37 °C with 5% CO₂. The next day, the cells were dosed with appropriate compounds (DMSO or compound 9) and incubated at 37 °C with 5% CO₂. After 24 h incubation, cells were washed with cold PBS three times and lysed using lysis buffer (Pierce RIPA buffer, ThermoScientific, #89900) containing protease inhibitors (cOmplete Tablet, Mini EDTA-free, EASYpack, Roche) and phosphatase inhibitors (PhosSTOP EASYpack, Roche). Note: lysis buffer is prepared using 10 mL of RIPA buffer with 1 tablet of proteases inhibitors and 1 tablet of proteases inhibitors. The collected lysates were centrifuged, and the protein concentration was determined using the Pierce BCA kit. Equal amounts of protein were loaded for each sample into sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel that was then transferred onto a nitrocellulose membrane (BioRad, #1620115). The membrane was blocked using 5% skim milk in 1× PBST (0.1% Tween-20 in PBS) for 1 h at rt, followed by the overnight incubation with primary antibody at 4 °C. The next day, the membrane was washed three times with 1× PBST and incubated with secondary antibody for 1 h at rt, followed by another three times wash with 1× PBST. Finally, the membrane was treated with a chemiluminescent substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, #34577) and exposed to an X-ray film. Antibodies used for WB: c-Myc (ab32072, dilution 1:1000) and FUBP1 (ab192867, dilution 1:1000) antibodies were purchased from Abcam, p21 antibody (2947S, dilution 1:100) was obtained from Cell Signaling, and GAPDH (G8795, dilution 1:40,000) antibody was purchased from Sigma-Aldrich. Experiments were repeated at least three times.

qRT-PCR.

A L3.6pl cell suspension (400,000 cells) in RPMI 1640 medium (Gibco) with 10% FBS and 1% antibiotic–antimycotic (Gibco) was seeded in a 6-well cell culture dish (MidSci) followed by overnight incubation at 37 °C with 5% CO₂. The next day, the cells were dosed with appropriate compounds (DMSO or compound 9) and incubated at 37 °C with 5% CO₂. After 24 h incubation, cells were washed with ice-cold PBS three times and the cell pellets were collected. Total RNA was extracted from cells using Qiagen RNeasy kit (#74104). Reverse transcription kit (QuantiTect, #205310) was used to synthesize cDNA using 500 ng of RNA. qRT-PCR analysis was performed using the SYBR Green PCR kit (QuantiFast, #204054). All signals were normalized to β-actin and the comparative C_t (2-ΔΔCt) method was used to quantify the fold change.³³ Primers for qRT-PCR were ordered from QuantiTech: c-myc (QT00035406), p21 (QT00062090), and β-actin (QT00095431). The experiments were repeated at least two times and qRT-PCR was performed in duplicate.

Experimental Procedure for Chromatin Immunoprecipitation (ChIP) Studies.

A L3.6pl cell suspension (300,000 cells/1 mL) in RPMI 1640 medium (Gibco) with 10% FBS and 1% antibiotic–antimycotic (Gibco) was seeded in a 10 cm cell culture dish followed by overnight incubation at 37 °C with 5% CO₂. The next day, the cells were dosed with the appropriate compounds (0.6% DMSO vehicle or compound 9 at 60 μM) and incubated at 37 °C with 5% CO₂. After 24 h incubation, cells were fixed using a final concentration of 1% formalin for 10 min at 37 °C with 5% CO₂. Cells were then washed with ice-cold PBS two times, and the cells were collected by scraping in ice-cold PBS containing protease inhibitors (10 mL PBS + 1 tablet of cOmplete Tablet, Mini EDTA-free, EASYpack, Roche). ChIP assay was performed according to the Millipore ChIP Assay kit (cat. #17–295) protocol with slight modification. Briefly, the lysed cells were sonicated on ice using Sonic Dismembrator (Fisher Scientific, Model 100, Setting “5”) using a series of short pulses, 7 rounds of 20 1-s pulses with 2 min rest in between rounds. A sample of the sonicated chromatin (50 μL) was de-crosslinked and DNA was quantified for each sample. Equal amounts (20–25 μg) of sonicated chromatin were aliquoted and diluted 5-fold with ChIP dilution buffer (10 mL of ChIP dilution buffer containing 1 tablet of proteases inhibitors) to give a final volume of diluted sample equal to 500 μL. Further, the samples were precleared from nonspecific binding using 50 μL of Protein A/Salmon Sperm DNA beads (Millipore, #16–157). The diluted, precleared DNA supernatant was then collected and immunoprecipitated overnight at 4 °C on a rotating platform using 5 μL of FUBP1 antibody (ab192867), 5 μL of H4Ac antibody (Millipore #06–866) as a positive control, or 3 μL of rabbit IgG antibody (Millipore #PP64B) as a negative control. The immunoprecipitated DNA–protein–antibody complex was collected using 65 μL of Protein A/Salmon Sperm DNA beads, which were washed with the Millipore ChIP Assay kit provided buffers. Next, DNA was eluted from the beads, and de-crosslinked overnight at 65 °C on a shaking platform. DNA was then purified using Qiagen QlAquick extraction kit (#28704) followed by q-PCR. q-PCR analysis was performed using the SYBR Green PCR kit (QuantiFast, #204054). All signals were normalized to H4Ac control, and the fold enrichment method (2^∧ΔΔCt) was used to quantify the fold change differences. ChIP primers for the FUSE c-myc region were designed according to the published sequences in Venturutti et al.²⁵ Experiments were repeated at least two times and qRT-PCR was performed in duplicate.

Statistical Analysis.

Experimental data was managed in Excel 2019, and GraphPad Prism version 9.0.0 was used to perform data analysis. Two-way ANOVA was used to calculate the statistical significance for HPLC and qRT-PCR experiments, and one-way ANOVA was used to evaluate MTS cell growth assay and [³H]-Spd uptake assay. The p value was set to <0.05 to show the statistical significance in the data.

Synthetic Procedures to Access Other Compounds (and Their Synthetic Intermediates/Precursors) Shown in Table 1.

5-[4-(2-Chloro-phenyl)-piperazin-1-yl]-2-(4-methoxy-benzoylamino)-benzoic Acid Methyl Ester (7).

To a solution of 14 (34.3 mg, 0.099 mmol) in dichloromethane (2 mL) was added saturated sodium carbonate (2 mL) and stirred at 0 °C. 4-Methoxy-benzoyl chloride (34 mg, 0.2 mmol) in dichloromethane (1 mL) was added dropwise while stirring and the solution was allowed to warm to rt and stirred for 2 days. The reaction mixture was evaporated in vacuo to provide a crude residue and the residue was further purified by column chromatography (100% dichloromethane) to yield ester 7 (37 mg, 79%, >95% pure by HPLC). 1H NMR (400 MHz, CDCl₃): δ 11.71 (s, 1H), 8.85 (d, 1H, J = 9.2 Hz), 8.01 (m, 2H), 7.65 (d, 1H, J = 3.1 Hz), 7.39 (dd, 1H, J = 7.9, 1.5 Hz), 7.26 (m, 2H), 7.09 (dd, 1H, J = 8.1, 1.5 Hz), 7.00 (m, 3H), 3.97 (s, 3H), 3.87 (s, 3H), 3.36 (m, 4H), 3.23 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 169.4, 165.3, 162.8, 149.4, 146.8, 135.6, 131.1, 129.5, 129.2, 128.0, 127.8, 124.3, 123.9, 122.0, 120.8, 118.0, 116.1, 114.3, 55.8, 52.82, 51.6, 50.2. HRMS m/z calcd C₂₆H₂₆ClN₃O₄ (M + H)⁺ theory: 479.1612, found: 479.1590. HPLC (>95% pure).

5-[4-(2-Chloro-phenyl)-piperazin-1-yl]-2-(4-methoxy-benzoylamino)-benzoic Acid (8).

Methyl ester 7 (35.0 mg, 0.073 mmol) was dissolved in a minimum amount of dichloromethane (3 mL). To the solution was added methanol (2 mL) at room temperature, and 1 M NaOH (200 μL, 0.20 mmol) was added slowly at 0 °C and let warm to room temperature. The reaction mixture was allowed to stir overnight and concentrated to a crude residue. To the crude residue, 0.1 M HCl (3 mL) was added and checked for acidity. A precipitate was formed and ethyl acetate was added to the mixture and an extraction was performed. The organic layer was dried with sodium sulfate, filtered, and evaporated in vacuo to yield 8 (31 mg, 92%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.80 (s, 1H), 8.58 (d, 1H, J = 9.1 Hz), 7.90 (d, 2H, J = 8.7 Hz), 7.59 (d, 1H, J = 2.9 Hz), 7.43 (dd, 1H, J = 7.9, 1.3 Hz), 7.34 (m, 2H), 7.20 (d, 1H, J = 7.0 Hz), 7.07 (m, 3H), 3.84 (s, 3H), 3.30 (m, 4H), 3.14 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ 170.1, 163.7, 162.1, 148.8, 146.2, 133.7, 130.4, 128.8, 128.1, 127.7, 126.9, 124.1, 121.9, 121.2, 120.9, 117.4, 116.9, 114.2, 55.5, 50.8, 48.8. HRMS m/z calcd C₂₅H₂₄ClN₃O₄ (M + H)⁺ theory: 465.1455, found: 465.1470. Anal. Chem. CHN.

5-[4-(5-Chloro-2-methyl-phenyl)-piperazin-1-yl]-2-(4-methoxybenzoylamino)-benzoic Acid (9).

Methyl ester 20 (100 mg, 0.20 mmol) was dissolved in a minimum amount of dichloromethane (10 mL). To the solution was added methanol (6 mL) at room temperature, and 1 M NaOH (600 μL, 0.60 mmol) was added slowly at 0 °C and let warm to room temperature. The reaction mixture was allowed to stir for 2 days and was reduced in vacuo. To the resulting residue, 0.1 M HCl (2 mL) was added and the mixture was checked for acidity. The precipitate that formed was extracted into ethyl acetate and the organic layer was evaporated in vacuo to yield 9 (83 mg, 87%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.78 (s, 1H), 8.58 (d, 1H, J = 9.2 Hz), 7.90 (d, 2H, J = 8.9 Hz), 7.59 (d, 1H, J = 2.9 Hz), 7.37 (dd, 1H, J = 9.2, 2.9 Hz), 7.09 (m, 5H), 3.85 (s, 3H), 3.29 (m, 4H), 3.02 (m, 4H), 2.26 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 170.0, 163.6, 162.1, 152.4, 146.2, 133.7, 132.2, 130.7, 130.6, 128.8, 126.9, 122.6, 121.9, 121.2, 118.9, 117.4, 116.8, 114.1, 55.5, 50.9, 48.9, 17.2. HRMS m/z calcd C₂₆H₂₆ClN₃O₄ (M + H)⁺ theory: 479.1612, found: 479.1630. Anal. Chem. CHN.

1-(2-Chloro-phenyl)-piperazine (12).

1-Bromo-2-chlorobenzene 10 (500 mg, 303 μL, 2.61 mmol, d = 1.649), piperazine 11 (899 mg, 10.44 mg), sodium tert-butoxide (376 mg, 3.92 mmol), Pd₂(dba)₃ (23.9 mg, 2.61 × 10⁻² mmol), RuPhos (24.4 mg, 5.22 × 10⁻² mmol), and toluene (10 mL) were added to a round-bottom flask and stirred at reflux for 3 days. The reaction was monitored by thin-layer chromatography (TLC) (100% hexane) for the loss of 1-bromo-2-chlorobenzene. The reaction mixture was cooled and filtered and the filtrate was evaporated in vacuo to provide a crude residue. This residue was partitioned between diethyl ether and aqueous sodium carbonate and the organic layer was dried with sodium sulfate, filtered, and evaporated in vacuo. The resulting residue was further purified by column chromatography (93% dichloromethane, 6% methanol, 1% NH₄OH) to yield 12 (244 mg, 47%). ¹H NMR (400 MHz, CDCl₃): δ 7.36 (m, 1H), 7.22 (m, 1H), 7.04 (dd, 1H, J = 8, 1.5 Hz), 6.97 (m, 1H), 3.04 (m, 8H). ¹³C NMR (100 MHz, CDCl₃): δ 149.6, 130.6, 128.8, 127.5, 123.6, 120.4, 52.5, 46.2. HRMS m/z calcd C₁₀H₁₃ClN₂ (M + H)⁺ theory: 196.0767, found: 196.0760. Anal. Chem. CHN.

5-[4-(2-Chloro-phenyl)-piperazin-1-yl]-2-nitro-benzoic Acid Methyl Ester (13).

A solution of 12 (204.4 mg, 1.04 mmol), methyl 5-fluoro-2-nitrobenzoate 71 (213.1 mg, 1.07 mmol), and triethylamine (154.6 μL, 1.10 mmol; d = 0.72) in dioxane (3.5 mL) was stirred at reflux for 2 days. Loss of starting material was observed by TLC (100% dichloromethane) and the crude mixture was evaporated in vacuo. The resulting residue was further purified via column chromatography (100% DCM) to yield 13 (310 mg, 80%). ¹H NMR (400 MHz, CDCl₃): δ 8.05 (m, 1H), 7.40 (dd, 1H, J = 7.9, 1.5 Hz), 7.26 (m, 1H), 7.04 (m, 2H), 6.91 (m, 2H), 3.94 (s, 3H), 3.61 (m, 4H), 3.20 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 167.8, 153.9, 148.4, 136.1, 131.8, 130.8, 128.9, 127.7, 126.9, 124.4, 120.4, 113.6, 112.5, 53.23, 50.7, 47.2. HRMS m/z calcd C₁₈H₁₈ClN₃O₄ (M + H)⁺ theory: 375.0986, found: 375.1010. Anal. Chem. CHN.

2-Amino-5-[4-(2-chloro-phenyl)-piperazin-1-yl]-benzoic Acid Methyl Ester (14).

To 13 (272 mg, 0.723 mmol) in 4:1 acetone/water (15 mL) was added zinc (0.236 mg, 0.0036 mmol) and NH₄Cl (387 mg, 7.23 mmol). Note: later experiments used excess zinc and gave higher yields. The mixture was allowed to stir overnight at 60 °C. The reaction was filtered with acetone to remove zinc and the filtrate was evaporated in vacuo. The resulting crude residue was further purified via column chromatography (100% dichloromethane → 93% dichloromethane, 7% methanol) to yield 14 (55 mg, 22% yield). Crude ester 14 was used in the next step without further purification.

Precursors to compounds 9, 20, 46, 48, and 57–59:^a

^aReagents: (i) methyl 2-nitro-5-fluoro benzoate, Et₃N, 1,4-dioxane, reflux; (ii) Zn, HOAc.

1-(5-Chloro-2-methyl-phenyl)-piperazine (17).

A mixture of 5-chloro-2-methylaniline 15 (708 mg, 5.0 mmol), bis-(2-chloroethyl)-amine hydrochloride 16 (892 mg, 5.0 mmol), and diethyl glycol methyl ether (3.00 mL) was stirred and bubbled with N₂ gas for 15 min and then sealed and heated at 150 °C overnight. After being cooled to room temperature, the mixture was dissolved in MeOH (25 mL) followed by the addition of Et₂O (550 mL). The precipitate was filtered off and washed with Et₂O to provide HCl salt. The HCl salt was further converted to free amine by treatment with aqueous Na₂CO₃ solution and extracted with EtOAc (2×). The combined organic layers were concentrated in vacuo. The resulting residue was further purified by column chromatography (94% dichloromethane, 5% methanol, 1% NH₄OH) to yield 17 (426 mg, 40%). ¹H NMR (400 MHz, CDCl₃): δ 7.06 (m, 1H), 6.93 (m, 2H), 3.00 (m, 4H), 2.83 (m, 4H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 152.9, 131.8, 131.5, 130.7, 122.7, 119.3, 52.9, 46.3, 17.3. HRMS m/z calcd C₁₀H₁₅ClN₂ (M + H)⁺ theory: 210.0924, found: 210.0930. Anal. Chem. CHN.

5-[4-(5-Chloro-2-methyl-phenyl)-piperazin-1-yl]-2-nitro-benzoic Acid Methyl Ester (18).

A solution of 17 (197.4 mg, 0.94 mmol), methyl 5-fluoro-2-nitrobenzoate 71 (191.2 mg, 0.96 mmol), and triethylamine (141 μL, 1 mmol, MW = 101.19 g/mol, d = 0.72) in dioxane (5 mL) was stirred at reflux for 2 days. Loss of starting material was seen by TLC (100% dichloromethane) and the crude mixture was evaporated in vacuo. The resulting residue was further purified via column chromatography (100% DCM) to yield 18 (222 mg, 61%). ¹H NMR (400 MHz, CDCl₃): δ 8.05 (m, 1H), 7.13 (d, 1H, J = 8.0 Hz), 7.01 (m, 2H), 6.92 (m, 2H), 3.94 (s, 3H), 3.58 (m, 4H), 3.05 (m, 4H), 2.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 167.8, 153.9, 151.6, 136.3, 132.1, 131.9, 131.8, 126.9, 123.8, 119.6, 113.7, 112.6, 53.3, 51.2, 47.4, 17.4. HRMS m/z calcd C₁₉H₂₀ClN₃O₄ (M + H)⁺ theory: 389.1142, found: 389.1150. Anal. Chem. CHN.

Methyl 2-Amino-5-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-benzoate (19).

A solution of methyl 5-(4-(5-chloro-2-methylphenyl)-piperazin-1-yl)-2-nitrobenzoate 18 (2.0 g, 5.13 mmol) in acetic acid (80 mL) was treated with powdered zinc (2.1 g, 32 mmol) portionwise over 10 min to control the exothermic reaction. The mixture was stirred for 1 h, filtered, and evaporated to provide an oil that was partitioned with ethyl acetate and aqueous sodium bicarbonate. The organic phase was evaporated to a brown oil that was used without further purification (1.74 g, 86%). ¹H NMR (500 MHz, chloroform-d) δ 7.50 (d, J = 2.9 Hz, 1H), 7.13 (t, J = 7.2 Hz, 2H), 7.04 (d, J = 2.2 Hz, 1H), 7.02–6.97 (m, 1H), 6.70 (dd, J = 8.8, 1.7 Hz, 1H), 5.50 (s, 2H), 3.91 (d, J = 1.8 Hz, 3H), 3.21 (t, J = 4.5 Hz, 4H), 3.09 (d, J = 4.6 Hz, 4H), 2.31 (d, J = 1.6 Hz, 3H). LRMS (ESI+ve): calcd for C₁₉H₂₂ClN₃O₂, [M + H] = 360.15, observed [M + H] = 360.46.

5-[4-(5-Chloro-2-methyl-phenyl)-piperazin-1-yl]-2-(4-methoxybenzoylamino)-benzoic Acid Methyl Ester (20).

To a solution of crude 19 (90 mg, 0.25 mmol) in dichloromethane (10 mL) was added saturated sodium carbonate (10 mL) and stirred at 0 °C. 4-Methoxybenzoyl chloride (59.7 mg, 0.35 mmol) in dichloromethane (2 mL) was added dropwise to the stirring solution, which was allowed to come to rt and stir overnight. It was monitored for the loss of the starting amine by TLC (100% dichloromethane). The reaction solution was reduced in vacuo and the resulting residue was further purified by column chromatography (100% dichloromethane) to yield ester 20 (109 mg, 89%, >95% pure by HPLC). ¹H NMR (400 MHz, CDCl₃): δ 11.70 (s, 1H), 8.84 (d, 1H, J = 9.2 Hz), 8.00 (m, 2H), 7.63 (d, 1H, J = 3.1 Hz), 7.25 (m, 1H), 7.10 (dd, 1H, J = 8.0, 0.3 Hz), 6.99 (m, 4H), 3.96 (s, 3H), 3.86 (s, 3H), 3.30 (m, 4H), 3.05 (m, 4H), 2.28 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 169.3, 165.1, 162.7, 152.6, 146.6, 135.5, 132.3, 132.1, 131.2, 129.4, 127.7, 123.6, 123.5, 121.8, 119.8, 117.8, 116.0, 114.2, 55.7, 52.7, 51.8, 50.2, 17.8. HRMS m/z calcd C₂₇H₂₈ClN₃O₄ (M + H)⁺ theory: 493.1768, found: 493.1760.

5-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-2-(2,4-dimethoxybenzamido)benzoic Acid (48).

A solution of methyl 5-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-2-(2,4-dimethoxybenzamido)benzoate 57 (58 mg, 0.11 mmol) in dioxane (1.5 mL) was stirred with 0.5 M aqueous lithium hydroxide solution (0.5 mL, 0.25 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 0–10% methanol in dichloromethane (40 mg, 71%). ¹H NMR (500 MHz, DMSO-d₆) δ 12.2 (s, 1H), 8.69–8.63 (m, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 3.9 Hz, 1H), 7.23 (m, 2H), 7.08–7.00 (m, 2H), 6.68 (d, J = 13.3 Hz, 2H), 3.99 (s, 3H), 3.85 (s, 3H), 3.28 (d, J = 5.6 Hz, 4H), 3.02 (d, J = 5.6 Hz, 4H), 2.26 (s, 3H). LRMS (ESI+ve): calcd for C₂₇H₂₈ClN₃O₅, [M + H] = 510.18, observed [M + H] = 510.28.

2-(Furan-2-carboxamido)-5-(4-(pyridin-2-yl)piperazin-1-yl)-benzoic Acid (50).

A solution of furan-2-carboxylic acid (16.8 mg, 0.15 mmol), triethylamine (0.044 mL, 0.32 mmol), and HATU (58 mg, 0.15 mmol) in acetonitrile (1.5 mL) was stirred 20 min before methyl 2-amino-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate (32 mg, 0.10 mmol) was added. After stirring overnight, the reaction was concentrated, the residue partitioned with ethyl acetate and aqueous sodium bicarbonate, and then the organic phase was evaporated. The residue was then dissolved in dichloromethane and captured on a 1 g SCX-2 SPE column. The column was flushed with dichloromethane and ethyl acetate prior to eluting the desired product with 10% triethylamine in ethyl acetate. The solvent was evaporated to give the methyl ester as a residue that was used without further purification (33 mg, 79%). ¹H NMR (500 MHz, chloroform-d) δ 11.77 (s, 1H), 8.80 (d, J = 9.1 Hz, 1H), 8.25 (dd, J = 5.0, 1.8 Hz, 1H), 7.66 (d, J = 2.9 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 7.54 (ddd, J = 8.7, 7.2, 1.9 Hz, 1H), 7.27 (d, J = 3.1 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 6.74 (d, J = 8.6 Hz, 1H), 6.69 (dd, J = 7.1, 4.9 Hz, 1H), 6.57 (dd, J = 3.4, 1.7 Hz, 1H), 4.01 (s, 3H), 3.81–3.68 (m, 4H), 3.40–3.29 (m, 4H). The ester (33 mg, 0.08 mmol) in dioxane (3 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.35 mL, 0.18 mmol) and water (0.3 mL) overnight. The reaction was concentrated and then partitioned with ethyl acetate, acetonitrile, and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on reverse-phase HPLC (20 mg, 63%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.84 (s, 1H), 8.55 (d, J = 9.2 Hz, 1H), 8.12 (d, J = 5.2 Hz, 1H), 7.96 (d, J = 1.6 Hz, 1H), 7.68 (s, 1H), 7.58 (d, J = 3.0 Hz, 1H), 7.38 (dd, J = 9.2, 3.0 Hz, 1H), 7.23 (d, J = 3.5 Hz, 1H), 7.03 (s, 1H), 6.73 (dd, J = 3.5, 1.7 Hz, 2jH), 3.70 (t, J = 5.0 Hz, 4H), 3.28 (d, J = 5.2 Hz, 4H). HRMS (ESI+ve): calcd for C₂₁H₂₀N₄O₄ = 392.1485, observed = 392.1492.

2-(2-Methoxybenzamido)-5-(4-(3-phenylpropyl)piperazin-1-yl)-benzoic Acid (51).

A solution of methyl 2-(2-methoxybenzamido)-5-(piperazin-1-yl)-benzoate (35 mg, 0.094 mmol) in acetonitrile (1 mL) was treated with potassium carbonate (15 mg, 0.11 mmol) and (3-bromopropyl)-benzene (0.016 mL, 0.11 mmol) and then stirred at 60 °C overnight. The reaction was concentrated and then partitioned with ethyl acetate and water. The organic phase was evaporated and the crude product purified on silica gel eluting with 40% ethyl acetate in hexanes (42 mg, 91%). ¹H NMR (500 MHz, chloroform-d) δ 11.97 (s, 1H), 8.82 (d, J = 9.3 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 3.0 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.35–7.29 (m, 2H), 7.26–7.17 (m, 4H), 7.10 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 4.10 (d, J = 1.9 Hz, 3H), 3.94 (d, J = 2.0 Hz, 3H), 3.28–3.17 (m, 4H), 2.70 (t, J = 7.8 Hz, 2H), 2.64 (t, J = 4.8 Hz, 4H), 2.47 (t, J = 7.6 Hz, 2H), 1.90 (p, J = 7.7 Hz, 2H). The ester (42 mg, 0.086 mmol) in dioxane (1 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.43 mL, 0.22 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 60% ethyl acetate in hexanes (35 mg, 86%). ¹H NMR (500 MHz, chloroform-d) δ 12.14 (s, 1H), 8.70 (d, J = 9.1 Hz, 1H), 8.08 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 3.0 Hz, 1H), 7.47–7.40 (m, 1H), 7.28 (m, 2H), 7.18 (d, J = 7.5 Hz, 3H), 7.13 (dd, J = 9.1, 3.1 Hz, 1H), 7.09–7.01 (m, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.38 (p, J = 1.6 Hz, 4H), 2.88 (m, 4H), 2.68 (t, J = 7.5 Hz, 2H), 2.57 (m, 2H), 2.02 (t, J = 7.9 Hz, 2H). HRMS (ESI+ve): calcd for C₂₈H₃₁N₃O₄ = 473.2315, observed = 473.2340.

5-(4-Benzylpiperazin-1-yl)-2-(2-methoxybenzamido)benzoic Acid (52).

A solution of methyl 2-(2-methoxybenzamido)-5-(piperazin-1-yl)-benzoate (35 mg, 0.094 mmol) in acetonitrile (1 mL) was treated with potassium carbonate (15 mg, 0.11 mmol) and benzyl chloride (0.014 mL, 0.12 mmol) and then stirred at 60 °C overnight. The reaction was concentrated and then partitioned with ethyl acetate and water. The organic phase was evaporated and the crude product purified on silica gel eluting with 40% ethyl acetate in hexanes. Yield = 32 mg. (74%). The ester (35 mg, 0.076 mmol) in dioxane (1 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.38 mL, 0.19 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 60% ethyl acetate in hexanes (29 mg, 85%). ¹H NMR (500 MHz, chloroform-d) δ 12.02 (s, 1H), 8.73 (d, J = 9.1 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.45 (d, J = 7.1 Hz, 3H), 7.36 (dt, J = 12.0, 7.0 Hz, 3H), 7.14 (dd, J = 9.2, 2.9 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.85 (s, 2H), 3.32 (d, J = 4.7 Hz, 4H), 2.87 (s, 4H). HRMS (ESI+ve): calcd for C₂₆H₂₇N₃O₄ = 445.2002, observed = 445.2009.

2-(2-Methoxybenzamido)-5-(4-picolinoylpiperazin-1-yl)benzoic Acid (53).

A solution of picolinic acid (22 mg, 0.18 mmol), triethylamine (0.05 mL, 0.36 mmol), and HATU (68 mg, 0.18 mmol) in acetonitrile (2 mL) was stirred 30 min prior to the addition of methyl 2-amino-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate (50 mg, 0.14 mmol). After stirring overnight, the reaction was concentrated and the residue partitioned with ethyl acetate and water. The organic phase was evaporated and the residue was purified on silica gel eluting with 0–5% methanol in dichloromethane (44 mg, 69%). ¹H NMR (500 MHz, chloroform-d) δ 12.01 (s, 1H), 8.85 (dd, J = 9.1, 1.7 Hz, 1H), 8.64 (d, J = 4.8 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.85 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 2.6 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.39 (t, J = 6.4 Hz, 1H), 7.25–7.18 (m, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 4.11 (d, J = 1.8 Hz, 3H), 4.02 (t, J = 4.9 Hz, 2H), 3.94 (d, J = 1.7 Hz, 3H), 3.84 (t, J = 4.8 Hz, 2H), 3.32 (t, J = 5.0 Hz, 2H), 3.22 (d, J = 5.0 Hz, 2H). The ester (38 mg, 0.08 mmol) in dioxane (1.5 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.27 mL, 0.14 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 0–3% methanol in dichloromethane (7 mg, 19%). 1H NMR (500 MHz, chloroform-d) δ 12.02 (s, 1H), 8.81 (d, J = 9.1 Hz, 1H), 8.71 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 7.88 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.62 (s, 1H), 7.53–7.36 (m, 2H), 7.19 (s, 1H), 7.09 (d, J = 7.9 Hz, 1H), 7.03–6.92 (m, 1H), 3.99 (s, 5H), 3.73 (s, 2H), 3.28 (s, 2H), 3.19 (s, 2H). HRMS (ESI+ve): calcd for C₂₅H₂₄N₄O₅ = 460.1747, observed = 460.1773.

2-(2-Methoxybenzamido)-5-(4-(pyridin-2-ylmethyl)piperazin-1-yl)benzoic Acid (54).

A solution of methyl 2-(2-methoxybenzamido)-5-(piperazin-1-yl)-benzoate (32 mg, 0.087 mmol) in acetonitrile (2 mL) was treated with potassium carbonate (30 mg, 0.22 mmol) and 2-(bromomethyl)-pyridine hydrobromide (23 mg, 0.091 mmol) and then stirred at 60 °C overnight. The reaction was concentrated and then partitioned with ethyl acetate and water. The organic phase was evaporated and the crude product was used without further purification (36 mg, 90%). ¹H NMR (500 MHz, chloroform-d) δ 11.96 (s, 1H), 8.78 (d, J = 9.3 Hz, 1H), 8.58 (d, J = 4.8 Hz, 1H), 8.19 (dd, J = 7.8, 1.9 Hz, 1H), 7.70 (td, J = 7.6, 1.8 Hz, 1H), 7.56 (d, J = 3.0 Hz, 1H), 7.48 (dq, J = 7.4, 3.7, 2.9 Hz, 2H), 7.20 (ddd, J = 12.2, 8.3, 4.0 Hz, 2H), 7.09 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 4.08 (s, 3H), 3.92 (s, 3H), 3.75 (s, 2H), 3.24 (t, J = 4.9 Hz, 4H), 2.71 (t, J = 4.8 Hz, 4H). The ester (36 mg, 0.078 mmol) in dioxane (1.2 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.33 mL, 0.17 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on reverse-phase HPLC (13.5 mg, 39%). ¹H NMR (500 MHz, chloroform-d) δ 12.03 (s, 1H), 8.75 (d, J = 9.2 Hz, 1H), 8.59 (d, J = 4.9 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.06 (s, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.16 (dd, J = 9.2, 2.9 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.96 (s, 2H), 3.30 (t, J = 4.9 Hz, 4H), 2.94 (t, J = 4.7 Hz, 4H). HRMS (ESI+ve): calcd for C₂₅H₂₆N₄O₄ = 446.1954, observed = 446.1956.

Compound 55 via its methyl ester precursor:

2-(2-Methoxybenzamido)-5-(4-(pyridin-2-yl)piperazin-1-yl)-benzoic Acid (55).

A solution of crude methyl 2-amino-5-(4-(pyridine-2-yl)piperazin-1-yl)benzoate 72 (25 mg, 0.08 mmol) and triethylamine (0.022 mL, 0.16 mmol) in dry dichloromethane (2 mL) was treated with o-anisoyl chloride (0.014 mL, 0.096 mmol) and stirred at room temperature overnight. The reaction was partitioned between water and dichloromethane and then the volatile organics were evaporated in vacuo to afford crude methyl 2-(2-methoxybenzamido)-5-(4-(pyridine-2-yl)piperazin-1-yl)benzoate (22 mg, 62%). ¹H NMR (500 MHz, CDCl₃): δ 11.99 (s, 1H), 8.81 (d, J = 9.3 Hz, 1H), 8.21 (dd, J = 11.1, 5.9 Hz, 2H), 7.61 (d, J = 3.2 Hz, 1H), 7.50 (dt, J = 21.8, 7.0 Hz, 2H), 7.25 (d, J = 9.2 Hz, 1H), 7.09 (dd, J = 8.7, 5.6 Hz, 1H), 7.04 (t, J = 5.7 Hz, 1H), 6.77–6.62 (m, 2H), 4.09 (d, J = 4.1 Hz, 3H), 3.94 (d, J = 3.9 Hz, 3H), 3.73 (d, J = 5.2 Hz, 4H), 3.31 (d, J = 5.2 Hz, 4H). LRMS (ESI +ve): calcd for C₂₅H₂₆N₄O₄, [M + H] = 447.2, observed [M + H] = 447.35.

Crude methyl 2-(2-methoxybenzamido)-5-(4-(pyridin-2-yl)-piperazin-1-yl)benzoate (max 22 mg, 0.049 mmol) and LiOH (11.8 mg, 0.49 mmol) were charged into a vial followed by the addition of methanol (1 mL) and water (4 mL) and the mixture heated at 60 °C overnight. Liquid chromatography-mass spectrometry (LCMS) analysis of aliquot indicated only partial conversion to product. The reaction mix was cooled to room temperature and volatiles evaporated in vacuo overnight. The residue was resuspended in 1,4-dioxane (1 mL) and water (4 mL) and heated at 90 °C. LCMS analysis of aliquot indicated complete conversion to product. The reaction mixture was neutralized with dil. HCl solution. The precipitate formed was collected by filtration, washed exhaustively with water, and dried to afford 2-(2-methoxybenzamido)-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoic acid 55 as a yellow solid (15 mg, 70%). ¹H NMR (400 MHz, methanol-d₄) δ 8.70 (d, J = 9.2 Hz, 1H), 8.11–8.01 (m, 2H), 7.97 (d, J = 6.4 Hz, 1H), 7.70 (d, J = 2.9 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.45 (d, J = 9.3 Hz, 1H), 7.30 (dd, J = 9.3, 2.8 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 6.7 Hz, 1H), 4.05 (s, 3H), 3.89 (t, J = 5.2 Hz, 4H), 3.44 (t, J = 5.2 Hz, 4H). LRMS (ESI+ve): calcd for C₂₄H₂₄N₄O₄, [M + H] = 433.19, observed [M + H] = 433.34. HRMS (ESI+ve): calcd for C₂₄H₂₄N₄O₄ = 432.1798, observed = 432.1805.

2-Methoxy-N-(4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)-benzamide (56).

A solution of 4-(4-(pyridin-2-yl)piperazin-1-yl)aniline 73 (30 mg, 0.12 mmol), triethylamine (0.018 mL, 0.13 mmol), 2-methoxybenzoyl chloride (0.017 mL, 0.13 mmol), and a catalytic amount of 4-dimethylaminopyridine (DMAP) in dichloromethane (1 mL) was stirred for 1 h at which time LCMS indicated the reaction was complete. Methanol (0.3 mL) was added to convert the unreacted acid chloride to ester to simplify purification. The solvent was removed in vacuo and the residue partitioned with ethyl acetate and aqueous sodium bicarbonate. The organic phase was evaporated and the crude product purified on silica gel eluting with 5% methanol in dichloromethane (34 mg, 74%). ¹H NMR (500 MHz, chloroform-d) δ 9.70 (s, 1H), 8.32 (dd, J = 7.9, 1.8 Hz, 1H), 8.25 (dd, J = 5.0, 1.8 Hz, 1H), 7.67–7.57 (m, 2H), 7.52 (dddd, J = 12.1, 8.3, 7.1, 1.9 Hz, 2H), 7.20–7.11 (m, 1H), 7.05 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 8.9 Hz, 2H), 6.73 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 7.1, 4.9 Hz, 1H), 4.07 (s, 3H), 3.77–3.70 (m, 4H), 3.34–3.26 (m, 4H). HRMS (ESI+ve): calcd for C₂₃H₂₄N₄O₂ = 388.1899, observed = 388.1922.

Methyl 5-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-2-(2,4-dimethoxy-benzamido)-benzoate (57).

A solution of methyl 2-amino-5-(4-(5-chloro-2-methylphenyl)-piperazin-1-yl)benzoate 19 (65 mg, 0.18 mmol), triethylamine (0.03 mL, 0.22 mmol), 2,4-dimethoxybenzoyl chloride (0.04 mL, 0.2 mmol), and a catalytic amount of DMAP in dichloromethane (1 mL) was stirred overnight. The reaction was diluted with dichloromethane, washed with water, and evaporated to a residue that was purified on silica gel eluting with 5–20% ethyl acetate in hexanes (60 mg, 63%). ¹H NMR (400 MHz, chloroform-d) δ 11.95 (s, 1H), 8.86 (d, J = 9.2 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.25 (m, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 7.01 (dd, J = 8.0, 2.1 Hz, 1H), 6.64 (dd, J = 8.8, 2.4 Hz, 1H), 6.56 (d, J = 2.3 Hz, 1H), 4.10 (s, 3H), 3.96 (s, 3H), 3.90 (s, 3H), 3.36 (s, 4H), 3.11 (s, 4H), 2.32 (s, 3H). LRMS (ESI+ve): calcd for C₂₈H₃₀ClN₃O₅, [M + H] = 524.20, observed [M + H] = 524.31.

Methyl 5-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-2-(2-methoxybenzamido)-benzoate (58).

A solution of methyl 2-amino-5-(4-(5-chloro-2-methylphenyl)-piperazin-1-yl)benzoate 19 (65 mg, 0.18 mmol), triethylamine (0.03 mL, 0.22 mmol), 2-methoxybenzoyl chloride (0.02 mL, 0.22 mmol), and a catalytic amount of DMAP in dichloromethane (1 mL) was stirred overnight. The reaction was diluted with dichloromethane, washed with water, and evaporated to a residue that was purified on silica gel eluting with 5–20% ethyl acetate in hexanes (75 mg, 84%). ¹H NMR (400 MHz, chloroform-d) δ 12.02 (s, 1H), 8.87 (d, J = 9.2 Hz, 1H), 8.23 (dd, J = 7.8, 1.8 Hz, 1H), 7.66 (s, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.12 (q, J = 7.7 Hz, 2H), 7.08–6.95 (m, 4H), 4.11 (d, J = 1.2 Hz, 3H), 3.96 (d, J = 1.2 Hz, 3H), 3.36 (d, J = 5.0 Hz, 4H), 3.11 (s, 4H), 2.32 (s, 3H). LRMS (ESI+ve): calcd for C₂₇H₂₈ClN₃O₄, [M + H] = 494.19, observed [M + H] = 494.07.

5-(4-(5-Chloro-2-methylphenyl)piperazin-1-yl)-2-(2-methoxybenzamido)benzoic Acid (59).

A solution of methyl 5-(4-(5-chloro-2-methylphenyl)piperazin-1-yl)-2-(2-methoxybenzamido)-benzoate 58 (72 mg, 0.15 mmol) in dioxane (1.5 mL) was stirred with 0.5 M aqueous lithium hydroxide solution (0.5 mL, 0.25 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 0–10% methanol in dichloromethane (40 mg, 57%). ¹H NMR (500 MHz, chloroform-d) δ 11.86 (s, 1H), 8.86 (d, J = 9.6 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.71 (s, 1H), 7.56–7.46 (m, 1H), 7.32 (d, J = 9.5 Hz, 1H), 7.18–7.09 (m, 2H), 7.09–6.97 (m, 3H), 4.03 (d, J = 3.0 Hz, 3H), 3.37 (d, J = 5.3 Hz, 4H), 3.11 (d, J = 5.4 Hz, 4H), 2.32 (t, J = 2.3 Hz, 3H). LRMS (ESI+ve): calcd for C₂₆H₂₆ClN₃O₄, [M + H] = 480.17, observed [M + H] = 480.26.

2-(2-Methoxybenzamido)-5-((4-(pyridin-2-yl)piperazin-1-yl)-methyl)benzoic Acid (60).

A solution of methyl 2-(2-methoxybenzamido)-5-((4-(pyridin-2-yl)piperazin-1-yl)methyl)benzoate 61 (28 mg, 0.061 mmol) in dioxane (1 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.24 mL, 0.12 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 5% methanol in dichloromethane (11.5 mg, 42%). ¹H NMR (500 MHz, chloroform-d) δ 12.85 (s, 1H), 8.85 (d, J = 8.4 Hz, 1H), 8.55 (s, 1H), 8.17 (dd, J = 5.0, 1.9 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.50 (td, J = 7.0, 3.5 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.73–6.61 (m, 2H), 3.88 (d, J = 6.2 Hz, 5H), 2.99 (s, 4H), 2.48 (s, 4H). HRMS (ESI+ve): calcd for C₂₅H₂₆N₄O₄ = 446.1954, observed = 446.1976.

Methyl 2-(2-Methoxybenzamido)-5-((4-(pyridin-2-yl)piperazin-1-yl)methyl)benzoate (61).

A solution of methyl 5-(bromomethyl)-2-(2-methoxybenzamido)benzoate 74 (44 mg, 0.12 mmol) and 1-(pyridine-2-yl)piperazine (0.019 mL, 0.13 mmol) in acetonitrile (2 mL) was stirred at 60 °C 1 h before the solvent was removed in vacuo. The residue was partitioned with ethyl acetate and saturated aqueous potassium carbonate, the organic phase was concentrated, and the residue was purified on silica gel eluting with 0–5% methanol in dichloromethane to give methyl ester 61 (32 mg, 60%). ¹H NMR (500 MHz, chloroform-d) δ 12.18 (s, 1H), 8.91 (d, J = 8.6 Hz, 1H), 8.21 (t, J = 6.2 Hz, 2H), 8.02 (s, 1H), 7.63–7.56 (m, 1H), 7.54–7.45 (m, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.67–6.61 (m, 2H), 4.11 (d, J = 1.4 Hz, 3H), 3.95 (d, J = 1.4 Hz, 3H), 3.58 (d, J = 7.1 Hz, 6H), 2.59 (t, J = 5.1 Hz, 4H). LRMS (ESI+ve): calcd for C₂₆H₂₈N₄O₄, [M + H] = 461.22, observed [M + H] = 461.56.

2-(2-Methoxybenzamido)-5-(4-phenylpiperidin-1-yl)benzoic Acid (62).

A solution of methyl 2-amino-5-(4-phenylpiperidin-1-yl)benzoate 75 (40 mg, 0.13 mmol), triethylamine (0.02 mL, 0.14 mmol), 2-methoxybenzoyl chloride (0.02 mL, 0.15 mmol), and a catalytic amount of DMAP in dichloromethane (2 mL) was stirred overnight. The reaction was concentrated and then partitioned with ethyl acetate and water. The organic phase was washed with aqueous potassium carbonate, evaporated, and purified on silica gel eluting with 30% ethyl acetate in hexanes (54 mg, 94%). ¹H NMR (500 MHz, chloroform-d) δ 11.98 (s, 1H), 8.83 (d, J = 9.2 Hz, 1H), 8.23 (dd, J = 7.8, 1.7 Hz, 1H), 7.64 (d, J = 3.0 Hz, 1H), 7.50 (td, J = 7.8, 1.8 Hz, 1H), 7.36 (t, J = 7.5 Hz, 2H), 7.30 (m, 2H), 7.28–7.23 (m, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 4.11 (s, 3H), 3.95 (s, 3H), 3.86–3.77 (m, 2H), 2.86 (td, J = 12.0, 2.9 Hz, 2H), 2.68 (tt, J = 11.8, 4.1 Hz, 1H), 2.05–1.88 (m, 4H). The ester (54 mg, 0.12 mmol) in dioxane (6 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.6 mL, 0.3 mmol) and water (0.5 mL) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 0–5% methanol in dichloromethane (45 mg, 86%). ¹H NMR (500 MHz, chloroform-d) δ 11.87 (s, 1H), 8.84 (d, J = 9.2 Hz, 1H), 8.25 (dd, J = 7.8, 1.7 Hz, 1H), 7.75 (d, J = 3.0 Hz, 1H), 7.50 (td, J = 7.8, 1.8 Hz, 1H), 7.38–7.31 (m, 3H), 7.30 (m, 2H), 7.27–7.23 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H), 3.83 (dd, J = 12.5, 3.4 Hz, 2H), 2.88 (td, J = 11.9, 3.3 Hz, 2H), 2.69 (tt, J = 11.5, 4.3 Hz, 1H), 2.00 (qd, J = 12.6, 3.6 Hz, 4H). HRMS (ESI+ve): calcd for C₂₆H₂₆N₂O₄ = 430.1893, observed = 430.1932.

2-(2-Methoxybenzamido)-5-(4-phenoxypiperidin-1-yl)benzoic Acid (63).

A solution of methyl 2-amino-5-(4-phenoxypiperidin-1-yl)benzoate 76 (57 mg, 0.18 mmol), triethylamine (0.05 mL, 0.36 mmol), 2-methoxybenzoyl chloride (0.025 mL, 0.18 mmol), and a catalytic amount of DMAP in dichloromethane (2 mL) was stirred overnight. The reaction was diluted with dichloromethane, washed with water, and evaporated to a residue that was purified on silica gel eluting with 20% ethyl acetate in hexanes (32 mg, 40%). ¹H NMR (500 MHz, chloroform-d) δ 11.98 (s, 1H), 8.82 (d, J = 9.2 Hz, 1H), 8.23 (dt, J = 7.8, 1.4 Hz, 1H), 7.62 (d, J = 2.9 Hz, 1H), 7.51–7.46 (m, 1H), 7.31 (d, J = 7.9 Hz, 2H), 7.24 (dd, J = 9.2, 3.0 Hz, 1H), 7.11 (t, J = 7.5 Hz, 2H), 7.05 (d, J = 8.3 Hz, 1H), 7.01–6.91 (m, 3H), 4.51 (tt, J = 7.4, 3.7 Hz, 1H), 4.11 (d, J = 1.0 Hz, 3H), 3.94 (d, J = 1.0 Hz, 3H), 3.52 (ddd, J = 11.6, 7.3, 3.6 Hz, 2H), 3.14 (ddd, J = 12.0, 8.0, 3.5 Hz, 2H), 2.15 (ddd, J = 11.4, 7.9, 3.7 Hz, 2H), 2.00 (dtd, J = 12.0, 7.6, 3.6 Hz, 2H). The ester (32 mg, 0.069 mmol) in dioxane (2 mL) was hydrolyzed with 0.5 M aqueous lithium hydroxide solution (0.2 mL, 0.1 mmol) overnight. The reaction was concentrated and then partitioned with ethyl acetate and aqueous ammonium chloride. The organic phase was evaporated and the crude product purified on silica gel eluting with 0–3% methanol in dichloromethane (8 mg, 26%). ¹H NMR (500 MHz, DMSO-d₆) δ 12.23 (s, 1H), 8.64 (d, J = 9.2 Hz, 1H), 7.99–7.92 (m, 1H), 7.58–7.50 (m, 2H), 7.29 (dd, J = 8.6, 7.3 Hz, 2H), 7.25 (dd, J = 9.2, 2.9 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.1 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 4.57 (dt, J = 8.3, 4.3 Hz, 1H), 3.98 (s, 3H), 3.58–3.45 (m, 2H), 3.05 (ddd, J = 12.5, 9.1, 3.2 Hz, 2H), 2.12–2.00 (m, 2H), 1.75 (dtd, J = 12.6, 8.7, 3.6 Hz, 2H). HRMS (ESI+ve): calcd for C₂₆H₂₆N₂O₅ = 446.1842, observed = 446.1876.

Compound 64 was purchased from Aurora Fine Chemicals (San Diego, CA, 95% purity) and used without further purification.

Synthesis of compound 72 as a precursor to 55:^a

^aReagents: (i) methyl 2-nitro-5-fluoro benzoate, Et₃N, 1,4-dioxane, reflux; (ii) Zn, HOAc.

Methyl 2-Nitro-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate (72a).

A solution of 1-(pyridin-2-yl)piperazine 81 (1 g, 6.1 mmol), methyl 5-fluoro-2-nitrobenzoate 71 (1.28 g, 6.4 mmol), and triethylamine (1.71 mL, 12.3 mmol) in 1,4-dioxane (20 mL) was stirred at room temperature for 5 min followed by heating at 95 °C overnight. LCMS of the aliquot showed complete conversion of the starting material. The solvent was evaporated in vacuo and residue partitioned between water and ethyl acetate. The water layer was discarded and organic layer evaporated in vacuo to afford methyl 2-nitro-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate as an orange solid (2.05 g, 98%). ¹H NMR (500 MHz, chloroform-d) δ 8.24 (dd, J = 5.0, 2.1 Hz, 1H), 8.12–8.01 (m, 1H), 7.56 (t, J = 7.8 Hz, 1H), 6.95–6.85 (m, 2H), 6.77–6.65 (m, 2H), 3.96 (d, J = 1.7 Hz, 3H), 3.80–3.75 (m, 4H), 3.64–3.60 (m, 4H). LRMS (ESI+ve): calcd for C₁₇H₁₈N₄O₄, [M + H] = 343.14, observed [M + H] = 343.25.

Methyl 2-Amino-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate (72)

A solution of crude methyl 2-nitro-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate 72a (2.05 g, 6.1 mmol) in glacial acetic acid (30 mL) was treated with Zn dust (2.41 g, 36.8 mmol) at a rate to avoid setting up an uncontrolled exothermic reaction. The reaction mixture was allowed to stir overnight and quenched by pouring slowly into aq. K₂CO₃ solution. After adjusting the final pH to ∼12.0, the mixture was extracted with ethyl acetate and the solvent was evaporated in vacuo to afford crude methyl 2-amino-5-(4-(pyridin-2-yl)piperazin-1-yl)benzoate as a brown solid (1.6 g, 83%). ¹H NMR (500 MHz, chloroform-d) δ 8.34–8.18 (m, 1H), 7.59–7.43 (m, 2H), 7.19–7.03 (m, 1H), 6.78–6.59 (m, 3H), 5.08 (broad s, 2H), 3.90 (s, 3H), 3.76–3.66 (m, 4H), 3.18–3.12 (m, 4H). LRMS (ESI+ve): calcd for C₁₇H₂₀N₄O₂, [M + H] = 313.17, observed [M + H] = 313.5.

Compound 73 as a precursor to 56:^a

^aReagents: (i) 4-fluoro nitrobenzene, Et₃N, 1,4-dioxane, reflux; (ii) Zn, HOAc.

1-(4-Nitrophenyl)-4-(pyridin-2-yl)piperazine (73a).

A solution of 1-(pyridin-2-yl)piperazine 81 (900 mg, 6.8 mmol), 1-fluoro-4-nitrobenzene (960 mg, 6.8 mmol), and potassium carbonate (1.24 g, 6.8 mmol) in acetonitrile (30 mL) was stirred at reflux overnight. The solvent was removed in vacuo and the residue was triturated with 10% methanol in dichloromethane. The insolubles were filtered and the solvent evaporated to provide a yellow solid that was then triturated with hexanes. The product was collected by filtration (1.2 g, 76%). ¹H NMR (500 MHz, chloroform-d) δ 8.27–8.22 (m, 1H), 8.18 (d, J = 9.4 Hz, 2H), 7.55 (ddd, J = 8.8, 7.2, 2.1 Hz, 1H), 6.91–6.84 (m, 2H), 6.74–6.67 (m, 2H), 3.81–3.75 (m, 4H), 3.62 (td, J = 5.1, 2.4 Hz, 4H). LRMS (ESI+ve): calcd for C₁₅H₁₆N₄O₂, [M + H] = 285.14, observed [M + H] = 285.07.

4-(4-(Pyridin-2-yl)piperazin-1-yl)aniline (73).

A solution of 1-(4-nitrophenyl)-4-(pyridin-2-yl)piperazine 73a (1.2 g, 1.2 mmol) in acetic acid (15 mL) was treated with powdered zinc (1.1 g, 16.8 mmol) portionwise over 10 min to control the exothermic reaction. The mixture was stirred for 1 h, filtered, and evaporated to provide an oil that was partitioned with ethyl acetate and aqueous potassium carbonate. The organic phase was evaporated to provide a residue that was used without further purification (1.05 g, 98%). ¹H NMR (500 MHz, chloroform-d) δ 8.24 (dd, J = 4.9, 2.1 Hz, 1H), 7.55–7.49 (m, 1H), 6.92–6.85 (m, 2H), 6.69 (ddd, J = 16.9, 12.6, 8.0 Hz, 4H), 3.71 (t, J = 5.0 Hz, 4H), 3.48 (s, 2H), 3.17 (t, J = 5.0 Hz, 4H). LRMS (ESI+ve): calcd for C₁₅H₁₈N₄, [M + H] = 255.16, observed [M + H] = 255.14.

Compounds 83 and 74 as precursors to 60 and 61:

Methyl 2-(2-methoxybenzamido)-5-methylbenzoate, Compound (74a).

A solution of methyl 2-amino-5-methylbenzoate 82 (330 mg, 2.0 mmol), triethylamine (0.30 mL, 2.15 mmol), and 2-methoxybenzoyl chloride (0.27 mL, 2.0 mmol) and a catalytic amount of DMAP in dichloromethane (20 mL) was stirred overnight. The reaction was diluted with dichloromethane, washed with water and aqueous sodium bicarbonate, and then evaporated to a residue that was purified on silica gel eluting with 15–30% ethyl acetate in hexanes (510 mg, 85%). ¹H NMR (500 MHz, chloroform-d) δ 12.11 (s, 1H), 8.84 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.53–7.45 (m, 1H), 7.40 (dd, J = 8.7, 2.1 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 4.10 (d, J = 1.6 Hz, 3H), 3.95–3.90 (m, 3H), 2.37 (s, 3H). LRMS (ESI+ve): calcd for C₁₇H₁₇NO₄, [M + H] = 300.12, observed [M + H] = 300.37.

Methyl 5-(Bromomethyl)-2-(2-methoxybenzamido)benzoate (74).

A mixture of methyl 2-(2-methoxybenzamido)-5-methylbenzoate 74a (139 mg, 0.46 mmol), N-bromosuccinimide (NBS) (92 mg, 0.51 mmol), and a catalytic amount of azobisisobutyronitrile (AIBN) in carbon tetrachloride (5 mL) was stirred at reflux 3 h. The reaction was partitioned with dichloromethane and water and then the organic phase evaporated and purified on silica gel eluting with 15% ethyl acetate in hexanes (88 mg, 50%). ¹H NMR (500 MHz, chloroform-d) δ 8.97 (dd, J = 8.9, 1.5 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.65–7.58 (m, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.15–7.09 (m, 1H), 7.06 (d, J = 8.4 Hz, 1H), 4.53 (d, J = 1.5 Hz, 2H), 4.10 (d, J = 1.5 Hz, 4H), 3.96 (d, J = 1.6 Hz, 3H). LRMS (ESI+ve): calcd for C₁₇H₁₆BrNO₄, [M + H] = 378.03, observed [M + H] = 378.36.

Compounds 75a and 75 as precursors to 62:^a

^aReagents: (i) methyl 2-nitro-5-fluoro benzoate, K₂CO₃, MeCN, reflux; (ii) Zn, HOAc.

Methyl 2-Nitro-5-(4-phenylpiperidin-1-yl)benzoate (75a).

A solution of 4-phenylpiperidine 83 (196 mg, 1.21 mmol), methyl 5-fluoro-2-nitrobenzoate 71 (242 mg, 1.21 mmol), and potassium carbonate (170 mg, 1.23 mmol) in acetonitrile (12 mL) was stirred at reflux overnight. The solvent was removed in vacuo and the residue was triturated with dichloromethane. The insolubles were filtered and the solvent evaporated to provide a yellow solid that was used without further purification (410 mg, 99%). ¹H NMR (500 MHz, chloroform-d) δ 8.09–8.03 (m, 1H), 7.39–7.32 (m, 2H), 7.31–7.21 (m, 3H), 6.91 (d, J = 10.5 Hz, 2H), 4.16–4.07 (m, 2H), 3.96 (d, J = 2.1 Hz, 3H), 3.18–3.07 (m, 2H), 2.83 (tt, J = 12.2, 3.5 Hz, 1H), 2.07–1.96 (m, 2H), 1.82 (qd, J = 12.8, 3.8 Hz, 2H). LRMS (ESI+ve): calcd for C₁₉H₂₀N₂O₄, [M + H] = 341.15, observed [M + H] = 341.45.

Methyl 2-Amino-5-(4-phenylpiperidin-1-yl)benzoate (75).

A solution of methyl 2-nitro-5-(4-phenylpiperidin-1-yl)benzoate 75a (410 mg, 1.2 mmol) in acetic acid (10 mL) was treated with powdered zinc (400 mg, 6.1 mmol) portionwise over 10 min to control the exothermic reaction. The mixture was stirred for 2 h, filtered, and evaporated to provide an oil that was partitioned with ethyl acetate and aqueous potassium carbonate. The organic phase was evaporated to a residue that was used without further purification (369 mg, 98%). ¹H NMR (500 MHz, chloroform-d) δ 7.50 (d, J = 2.9 Hz, 1H), 7.35 (dd, J = 7.9, 7.1 Hz, 2H), 7.30 (dd, J = 7.4, 1.6 Hz, 2H), 7.27–7.21 (m, 1H), 7.14 (dd, J = 8.8, 2.9 Hz, 1H), 6.68 (d, J = 8.9 Hz, 1H), 5.45 (s, 2H), 3.90 (s, 3H), 3.59 (d, J = 11.9 Hz, 2H), 2.75 (td, J = 11.5, 3.9 Hz, 2H), 2.63 (tt, J = 10.4, 5.0 Hz, 1H), 1.98 (dp, J = 11.7, 4.3 Hz, 4H). LRMS (ESI+ve): calcd for C₁₉H₂₂N₂O₂, [M + H] = 311.18, observed [M + H] = 311.48.

Synthesis of 76 as a precursor to 63:^a

^aReagents: (i) 4-piperidinone hydrochloride, Et₃N, 1,4-dioxane, reflux; (ii) NaBH₄, MeOH; (iii) phenol, P(Ph) ₃, DIAD; (iv) Zn, HOAc.

Methyl 2-Amino-5-(4-phenoxypiperidin-1-yl)benzoate (76).

A solution of methyl 2-nitro-5-(4-phenoxypiperidin-1-yl)benzoate 86 (114 mg, 0.32 mmol) in acetic acid (4 mL) was treated with powdered zinc (100 mg, 1.52 mmol) portionwise over 10 min to control the exothermic reaction. The mixture was stirred for 30–60 min, filtered, and evaporated to provide an oil that was partitioned with ethyl acetate and aqueous potassium carbonate. The organic phase was evaporated to provide an orange oil that was used without further purification (88 mg, 84%). ¹H NMR (500 MHz, chloroform-d) δ 7.48 (d, J = 2.8 Hz, 1H), 7.34–7.29 (m, 2H), 7.11 (dt, J = 8.9, 1.9 Hz, 1H), 7.00–6.93 (m, 3H), 6.67 (dd, J = 8.9, 1.1 Hz, 1H), 5.45 (s, 2H), 4.46 (tt, J = 7.5, 3.7 Hz, 1H), 3.89 (d, J = 1.2 Hz, 3H), 3.34 (ddd, J = 11.3, 7.1, 3.6 Hz, 2H), 2.96 (ddd, J = 11.9, 8.2, 3.4 Hz, 2H), 2.14 (dq, J = 11.0, 3.6 Hz, 2H), 1.99 (dtd, J = 12.0, 7.9, 3.5 Hz, 2H). LRMS (ESI+ve): calcd for C₁₉H₂₂N₂O₃, [M + H] = 327.17, observed [M + H] = 327.48.

Compounds 77–80 as precursors to 8, 51–54, and 58:^a

^aReagents: (i) 1-Boc-piperazine, Et₃N, 1,4-dioxane, reflux; (ii) Zn, HOAc; (iii) 4-methoxybenzoyl chloride, Et₃N, DMAP, 1,4-dioxane then trifluoroacetic acid (TFA); (iv) 2-methoxybenzoyl chloride, Et₃N, DMAP, 1,4-dioxane then TFA.

tert-Butyl 4-(3-(Methoxycarbonyl)-4-nitrophenyl)piperazine-1-carboxylate (77).

A solution of methyl 5-fluoro-2-nitrobenzoate 71 (1.09 g, 5.48 mmol), tert-butyl piperazine-1-carboxylate (1.02 g, 5.48 mmol), and triethylamine (0.78 mL, 5.6 mmol) in dioxane (30 mL) was stirred at reflux overnight. The solvent was removed in vacuo and the residue partitioned with ethyl acetate and water. The organic phase was evaporated to provide an oil that was used without further purification (1.96 g, 98%). ¹H NMR (500 MHz, chloroform-d): δ 8.05 (d, J = 9.9 Hz, 1H), 6.90–6.82 (m, 2H), 3.95 (s, 3H), 3.62 (dd, J = 6.6, 4.0 Hz, 4H), 3.45 (dd, J = 6.5, 4.1 Hz, 4H), 1.51 (s, 9H). LRMS (ESI+ve): calcd for C₁₇H₂₃N₃O₆, [M + H] = 366.17, observed [M + H] = 366.31.

tert-Butyl 4-(4-Amino-3-(methoxycarbonyl)phenyl)piperazine-1-carboxylate (78).

A solution of tert-butyl 4-(3-(methoxycarbonyl)-4-nitrophenyl)piperazine-1-carboxylate 77 (1.6 g, 4.38 mmol) in acetic acid (30 mL) was treated with powdered zinc (1.0 g, 15.3 mmol) portionwise over 10 min to control the exothermic reaction. The mixture was let stir overnight, filtered, and evaporated to provide an oil that was partitioned with ethyl acetate and aqueous potassium carbonate. The organic phase was evaporated and the crude product purified on silica gel eluting with 50% ethyl acetate in hexanes (1.1 g, 75%). ¹H NMR (500 MHz, chloroform-d) δ 7.57–7.44 (m, 1H), 7.14 (s, 1H), 6.68 (d, J = 8.8 Hz, 1H), 3.89 (s, 3H), 3.65 (s, 4H), 3.02 (s, 4H), 1.50 (s, 9H). LRMS (ESI+ve): calcd for C₁₇H₂₅N₃O₄, [M + H] = 336.19, observed [M + H] = 336.21.

Methyl 2-(4-Methoxybenzamido)-5-(piperazin-1-yl)benzoate (79).

A solution of tert-butyl 4-(4-amino-3-(methoxycarbonyl)phenyl)-piperazine-1-carboxylate 78 (410 mg, 1.22 mmol), 4-methoxybenzoyl chloride (0.19 mL, 1.38 mmol), triethylamine (0.21 mL, 1.51 mmol), and catalytic DMAP in dioxane (10 mL) was stirred for 30–60 min at which time LCMS indicated the limiting reagent was consumed. The solvent was removed in vacuo and the residue was purified on silica gel eluting with 20–50% ethyl acetate in hexanes. Yield = 400 mg. (70%). 1H NMR (500 MHz, chloroform-d) δ 11.73 (s, 1H), 8.86 (d, J = 9.2 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.65 (s, 1H), 7.26 (d, J = 10.0 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 3.98 (s, 3H), 3.90 (s, 3H), 3.64 (t, J = 5.1 Hz, 4H), 3.15 (t, J = 5.0 Hz, 4H), 1.51 (s, 9H). LRMS (ESI+ve): calcd for C₂₅H₃₁N₃O₆, [M + H] = 470.23, observed [M + H] = 470.07. The Boc protecting group was removed with 20% trifluoroacetic acid in dichloromethane at ambient temperature over 2.5 h. The solvent was removed in vacuo and the residue partitioned with ethyl acetate and aqueous sodium bicarbonate. The precipitated product was collected by filtration and was used without further purification (300 mg, 95%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.17 (s, 1H), 8.36 (d, J = 9.1 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 3.0 Hz, 1H), 7.30 (dd, J = 9.1, 3.1 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.04 (t, J = 4.9 Hz, 4H), 2.84 (t, J = 4.9 Hz, 4H). LRMS (ESI+ve): calcd for C₂₀H₂₃N₃O₄, [M + H] = 370.18, observed [M + H] = 370.57.

Methyl 2-(2-Methoxybenzamido)-5-(piperazin-1-yl)benzoate (80).

A solution of tert-butyl 4-(4-amino-3-(methoxycarbonyl)phenyl)-piperazine-1-carboxylate 78 (290 mg, 0.86 mmol), 2-methoxybenzoyl chloride (0.12 mL, 0.88 mmol), triethylamine (0.14 mL, 1.0 mmol), and catalytic DMAP in dioxane (10 mL) was stirred overnight. The solvent was removed in vacuo and the residue was purified on silica gel eluting with 30–60% ethyl acetate in hexanes (362 mg, 89%). ¹H NMR (500 MHz, chloroform-d) δ 12.00 (s, 1H), 8.84 (d, J = 9.2 Hz, 1H), 8.22 (dd, J = 7.8, 1.8 Hz, 1H), 7.59 (s, 1H), 7.53–7.46 (m, 1H), 7.21 (d, J = 9.7 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.11 (s, 3H), 3.94 (s, 3H), 3.64 (d, J = 5.0 Hz, 4H), 3.19–3.09 (m, 4H), 1.51 (s, 9H). LRMS (ESI+ve): calcd for C₂₅H₃₁N₃O₆, [M + H] = 470.23, observed [M + H] = 470.03. The Boc protecting group was removed with 20% trifluoroacetic acid in dichloromethane at ambient temperature over 2.5 h. The solvent was removed in vacuo and the residue partitioned with ethyl acetate and aqueous potassium carbonate. The organic phase was evaporated to provide a yellow solid that was used without further purification (270 mg, 95%). ¹H NMR (500 MHz, chloroform-d) δ 12.00 (s, 1H), 8.82 (d, J = 9.2 Hz, 1H), 8.21 (dd, J = 7.8, 2.0 Hz, 1H), 7.56 (d, J = 3.0 Hz, 1H), 7.49 (ddd, J = 8.6, 7.2, 1.7 Hz, 1H), 7.17 (dd, J = 9.3, 3.0 Hz, 1H), 7.10 (td, J = 7.6, 1.0 Hz, 1H), 7.06–7.00 (m, 1H), 4.66 (s, 1H), 4.09 (d, J = 1.2 Hz, 3H), 3.93 (d, J = 1.9 Hz, 3H), 3.28–3.21 (m, 4H), 3.22–3.13 (m, 4H). LRMS (ESI+ve): calcd for C₂₀H₂₃N₃O₄, [M + H] = 370.18, observed [M + H] = 370.27.

Methyl 2-Nitro-5-(4-oxopiperidin-1-yl)benzoate (84).

A solution of methyl 5-fluoro-2-nitrobenzoate 71 (500 mg, 2.5 mmol), piperidin-4-one hydrochloride (400 mg, 2.6 mmol), and triethylamine (0.8 mL, 5.7 mmol) in acetonitrile (15 mL) was stirred at reflux overnight. The solvent was removed in vacuo and the residue partitioned with ethyl acetate and water. The organic phase was evaporated to a yellow solid that was used without further purification (647 mg, 93%). ¹H NMR (500 MHz, chloroform-d) δ 8.12–8.07 (m, 1H), 6.91 (d, J = 2.9 Hz, 1H), 6.89 (s, 1H), 3.96 (s, 3H), 3.84 (t, J = 6.2 Hz, 4H), 2.66 (t, J = 6.2 Hz, 4H). LRMS (ESI+ve): calcd for C₁₃H₁₄N₂O₅, [M + H] = 279.10, observed [M + H] = 279.31.

Methyl 5-(4-Hydroxypiperidin-1-yl)-2-nitrobenzoate (85).

A solution of methyl 2-nitro-5-(4-oxopiperidin-1-yl)benzoate 84 (106 mg, 0.38 mmol) in methanol (2 mL) was treated with sodium borohydride (38 mg, 1 mmol) and stirred overnight. Excess reducing agent was quenched with the addition of acetone (1 mL) before the solvents were removed in vacuo. The residue was partitioned with ethyl acetate and aqueous sodium bicarbonate and the organic phase evaporated to provide a yellow oil (105 mg, 99%). ¹H NMR (500 MHz, chloroform-d) δ 8.06–8.01 (m, 1H), 6.87 (d, J = 2.9 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 4.03 (tt, J = 7.8, 3.8 Hz, 1H), 3.95 (s, 3H), 3.79 (ddd, J = 13.3, 6.8, 3.9 Hz, 2H), 3.29 (ddd, J = 13.0, 8.7, 3.5 Hz, 2H), 2.01 (ddd, J = 13.4, 7.0, 3.6 Hz, 2H), 1.67 (dtd, J = 12.6, 8.3, 3.8 Hz, 2H), 1.60 (s, 2H). LRMS (ESI+ve): calcd for C₁₃H₁₆N₂O₅, [M + H] = 281.11, observed [M + H] = 281.38.

Methyl 2-Nitro-5-(4-phenoxypiperidin-1-yl)benzoate (86).

A solution of methyl 5-(4-hydroxypiperidin-1-yl)-2-nitrobenzoate 85 (105 mg, 0.38 mmol), triphenylphosphine (100 mg, 0.38 mmol), and phenol (37 mg, 0.39 mmol) in DMF (6 mL) was treated with DIAD (0.75 mL, 0.38 mmol). The reaction was stirred overnight and was then evaporated to a residue that was purified on silica gel eluting with dichloromethane (114 mg, 84%). ¹H NMR (500 MHz, chloroform-d) δ 8.05–8.01 (m, 1H), 7.32 (dd, J = 8.6, 7.3 Hz, 2H), 7.00 (td, J = 7.3, 1.1 Hz, 1H), 6.97–6.93 (m, 2H), 6.90–6.84 (m, 2H), 4.62 (tt, J = 6.5, 3.5 Hz, 1H), 3.94 (s, 3H), 3.71 (ddd, J = 12.8, 8.6, 3.6 Hz, 2H), 3.49 (ddd, J = 13.3, 6.7, 4.0 Hz, 2H), 2.06 (ddt, J = 12.6, 8.1, 3.8 Hz, 2H), 1.97 (dtd, J = 13.2, 6.4, 3.7 Hz, 2H). LRMS (ESI+ve): calcd for C₁₉H₂₀N₂O₅, [M + H] = 357.15, observed [M + H] = 357.32.

ABBREVIATIONS USED

AG

aminoguanidine

Ant44

N¹-(9-anthracenylmethyl)-homospermidine; arginine

AZIN1

antizyme inhibitor 1

ChIP

chromatin immunoprecipitation

CHO

Chinese hamster ovary

DFMO

difluoromethylornithine

diAc-Spm

N¹,N¹²-diacetylspermine

EC₅₀ Assay 1

effective concentration to inhibit 50% of Ant44 uptake as evidenced by attaining a percentage growth halfway between the Ant44 and untreated controls

EC₅₀ Assay 2

effective concentration to inhibit 50% of Spd uptake as evidenced by attaining a percentage growth halfway between the DFMO and DFMO + Spd controls

eIF-5A

eukaryotic initiation factor 5A

FBS

fetal bovine serum

FUBP1

far upstream binding protein 1

HPLC

high-performance liquid chromatograph

HTS

high-throughput screening

MTC

maximum tolerated concentration

N¹Ac-Spd

N¹-acetylspermidine

N-Ac-Spm

N¹-acetylspermine

OAZ1

antizyme 1

ODC

ornithine decarboxylase

PAO

polyamine oxidase

PDAC

pancreatic ductal adenocarcinoma

PTI

polyamine transport inhibitor

SAT1

spermidine/spermine acetyl transferase

SMOX

spermine oxidase

SMS

spermine synthase

Spd

spermidine

SRM

spermidine synthase