Inhibition of amyloidogenesis by non-steroidal anti-inflammatory drugs and their hybrid nitrates

Abstract

Poor blood-brain barrier penetration of non-steroidal anti-inflammatory drugs (NSAIDs) has been blamed for the failure of the selective amyloid lowering agent (SALA) R-flurbiprofen in phase 3 clinical trials for Alzheimer’s disease (AD). NO-donor NSAIDs (NO-NSAIDs) provide an alternative, gastric-sparing approach to NSAID SALAs, which may improve bioavailability. NSAID analogs were studied for anti-inflammatory activity and for SALA activity in N2a neuronal cells transfected with human amyloid precursor protein (APP). Flurbiprofen (1) analogs were obtained with enhanced anti-inflammatory and anti-amyloidogenic properties compared to 1, however, esterification led to elevated Aβ_1–42 levels. Hybrid nitrate prodrugs possessed superior anti-inflammatory activity and reduced toxicity relative to the parent NSAIDs, including clinical candidate, CHF5074. Although hybrid nitrates elevated Aβ_1–42 at higher concentration, SALA activity was observed at low concentrations (≤ 1 µM): both Aβ_1–42 and the ratio of Aβ_1–42/Aβ_1–40 were lowered. This biphasic SALA activity was attributed to the intact nitrate drug. For several compounds the selective modulation of amyloidogenesis was tested using an immunoprecipitation MALDI-TOF approach. These data support the development of NO-NSAIDs as an alternative approach towards a clinically useful SALA.

Introduction

Alzheimer’s disease (AD), the major cause of dementia in the elderly population, is a neurodegenerative disorder characterized by the abnormal accumulation of amyloid plaques and neurofibrillary tangles.^{1, 2} According to the amyloid cascade hypothesis, beta-amyloid (Aβ) peptide, the main constituent of amyloid plaques, is the cause of the observed neurodegeneration in AD patients.³ In 2001, Weggen et al. discovered that certain NSAIDs were able to selectively lower the levels of Aβ_1–42 without affecting levels of Aβ_1–40; these drugs were referred to as SALAs.⁴ The importance of this discovery lay in evidence that the 42 amino acid fragment, Aβ_1–42, aggregates more rapidly and is a much more potent neurotoxin than the more abundant Aβ_1–40.^{3, 5} Aβ_1–42 is widely viewed as the causative molecule in AD. The enzymes responsible for the production of Aβ, namely β- and γ- secretase, remain primary drug targets for the treatment of AD.⁶ Changes in the levels of Aβ elicited by NSAID SALAs in vitro and in vivo have been attributed to modulation of Aβ production,^{4, 7–11} via: modulation of γ–secretase activity;^{4, 7} downregulation of β-secretase;¹² or Rho kinase inhibition.^{13, 14} In addition, NSAIDs including flurbiprofen (1) have been reported to inhibit amyloidosis by stabilizing transthyretin.¹⁵ The N2a neuronal cell line stably transfected with human βAPP695 bearing the Swedish mutation (N2a.hAPP) provides a model for amyloidogenesis, producing Aβ peptides associated with human pathophysiology. Studies on this cell line demonstrated that NSAID SALAs stimulate metalloprotease degradation of Aβ_1–40 and Aβ_1–42.¹⁶ The enhanced proteolysis of Aβ is currently seen as a preferred drug discovery strategy.^{17, 18}

The effect of NSAID SALAs on Aβ levels was shown previously to be independent of the cyclooxygenase (COX) inhibitory activity, a dissociation of actions considered crucial for drug development, since NSAID gastrotoxicity is associated with COX inhibition.^{10, 11} Supported by epidemiological studies that reported a decreased risk of developing AD after chronic treatment with NSAIDs,^19–21 the COX-inactive R-flurbiprofen was studied in phase 3 clinical trials as a gastric-sparing therapeutic agent for AD (Scheme 1).⁷ In 2008, this trial was reported as a failure due to lack of efficacy, possibly linked to poor bioavailability.^{22, 23} Nevertheless, CHF5074 (11d), a new COX-inactive analog of flurbiprofen (1) appears to be on course for clinical development.²⁴ An alternative approach to developing a gastric-sparing NSAID has been to incorporate a gastroprotective organic nitrate moiety in a so-called NO-donating NSAID (NO-NSAID) to counteract the NSAID-induced inhibition of prostaglandin synthesis.²⁵ HCT-1026 (2),²⁶ an NO-NSAID prodrug of 1, has been reported: i) to reduce Aβ load in an amyloid transgenic mouse model;²⁷ ii) to reverse cognitive deficits in mice under cholinergic blockade;²⁸ and, iii) to reduce inflammatory markers, including iNOS, in adult rats injected with Aβ_1–42.²⁹

Scheme 1.

The purpose of this study was to assay libraries of indomethacin (3), sulindac (4), and flurbiprofen analogs, including novel compounds and preclinical agents (e.g. CHF5022, 11c;³⁰ and 11d), as a basis for development of new NSAID hybrid nitrates as SALAs. Anti-inflammatory activity and toxicity were assayed in cell culture and for selected compounds inhibition of amyloidogenesis was assayed in neuronal cell cultures. Based upon these data, prototype hybrid nitrate analogs of 1 were synthesized and studied for SALA activity, revealing further insight into the mechanism of SALA activity associated with NO-NSAIDs.

Results and Discussion

Synthesis

Synthesis was directed at the generation of a library of compounds possessing variations at the terminal phenyl ring (R₁), α-carbon (R₂ and R₂’), and isosteric replacement of the carboxylic acid (R₃) (Scheme 1).

Synthesis began with allylic bromination of the commercially available 4-bromo-3-fluorotoluene 6 with N-bromosuccinimide using a catalytic amount of dibenzoyl peroxide to yield 7, which was then reacted with potassium cyanide to give 8. Exposure to either iodoethane or 1,2 dibromoethane yielded alkylated products 9a and 9b that were then oxidized to the carboxylic acid followed by Suzuki coupling to the appropriate boronic acids to give biarylcarboxylic acids 11a–d in moderate yields (60–70 %). Quantitative yield was attained through direct coupling of the cyano analogs 8 and 9b, yielding the α-cyano-biaryls 12a and 12b, along with 13a and 13b. Fluorination of 13a and 13b was accomplished via reaction with t-BuLi followed by fluorination with N-fluorobenzenesulfonimide (NFSi) to give 14a–b and 15a–b. While base catalyzed oxidation of 14a, 14b, 15a, and 15b gave the corresponding biaryl fluoro-amides 16a, 16b, 17a and 17b, the desired biaryl-fluorinated carboxylic acids were not isolated. Acid catalyzed hydrolysis of 14a and 14b yielded 18a & 18b as products that were also evaluated for inhibition of amyloidogenesis and anti-inflammatory activity. Base and acid catalyzed oxidation of the diethyl derivative 12a was hindered by solubility issues, therefore the corresponding acid was synthesized by first oxidizing 13a to give the acid 19 which was then reacted with iodoethane in aqueous NaOH and toluene to give 20, followed by hydrolysis with LiOH to yield the desired acid 21 (Scheme 2).

Scheme 2^a.

^aReagents and Conditions: (i) NBS, CCl₄; (ii) KCN, EtOH; (iii) 1,2 dibromoethane for 9a or CH₃CH₂I for 9b, NaOH, toluene-H₂O; (iv) NaOH, MeOH, 100 °C; (v) R₁B(OH)₂, Pd(OAc)₂, Na₂CO₃, TBAB, H₂O, 120 °C; (vi) NFSi, t-BuLi, THF, −78 °C; (vii) K₂CO₃, DMSO, H₂O₂, 0 °C yields 16a–b & 17a–b, or H₂SO₄ (49 %) 90 °C to give 18a–b; viii) K₂CO₃, DMSO, H₂O₂, 0 °C; ix) CH₃CH₂Br, NaOH, toluene-H₂O; x) LiOH, THF/H₂O/MeOH, 0 °C.

To synthesize the biaryl mono carboxylic acids 23a and 23b, a synthetic pathway was adopted in which 1 or 19 were reacted with t-butyldimethylsilyl chloride (TBSCl) and lithium bis(trimethylsilyl)amide (LiHMDS) at 0 °C to give the desired bis-ketenes 22a and 22b, which were then reacted with Selectfluor followed by acid-base workup to give the desired fluorinated acids, 23a and 23b (Scheme 3). Flurbiprofen derivative and analogs were synthesized from 1, 11a, 11c and 11d through alkylation with 1,2-dibromoethane for 11a, 11c and 11d to give intermediates 25a–c, or via alkylation of 1 with 1,4-dibromobutane to yield 24a. The bromides were then transformed to the corresponding nitrates with AgNO₃ to yield 2, and 26a–c. In a similar manner, the amide 27 was synthesized by coupling of 1 with 4-aminobutanol followed by treatment with tetrabromomethane to give 24b and subsequent nitration with AgNO₃ to afford the nitrate 27 (Scheme 4).

Scheme 3^a.

^aReagents and Conditions: i) TBSCl, LiHMDS, THF 0 °C, 12 h; ii) Selectfluor, CH₃CN, < 50 °C.

Scheme 4^a.

^aReagents and Conditions: i) NaOCH₃, CH₃OH; ii) Br(CH₃)₄Br for 24a; Br(CH₃)₂Br for 25a–c; or 4-aminobutanol followed by PPh₃ with CBr₄ for 24b; DMF; iii) AgNO₃, CH₃CN, reflux.

Activity

Three structural approaches to analogs of 1 were developed: 1) variations at the terminal phenyl ring (R₁); 2) modification of the α-carbon (R₂ and R₂’); and 3) isosteric replacement of the carboxylic acid (R₃) (Scheme 1). Derivatization of the α-carbon is known to be a subtle modification resulting in reduction or ablation of COX activity, whereas replacement of the carboxylate group is expected to be important in enhancing blood brain barrier penetration. For these modifications to be successful, enhanced SALA activity was desired without loss of NSAID anti-inflammatory activity. Mouse macrophage-like RAW 264.7 cells treated with lipopolysaccharide (LPS) bacterial endotoxin, undergo induction of COX-2, inducible NO synthase (iNOS), and other inflammatory mediators.³¹ Thus, the RAW 264.7 cell line is routinely used to examine the ability of agents to inhibit cellular inflammatory response. iNOS activity is readily assessed by measuring the levels of inorganic nitrite, the major product of NO oxidative metabolism, quantified using the Griess assay.³¹ Flurbiprofen (1) itself was a modest inhibitor of iNOS induction in RAW cells treated with LPS and no evidence of toxicity was seen at the lower concentration tested (Table 1).^{28, 32, 33}

Table 1.

Anti-inflammatory effects of selected 1 analogues (10 µM)

Compound	R1	R2, R2′	R3	NO2− as %a	Cell viability as %b
DMSO	-	-	-	100 ± 0.8	100 ± 2.3
1	Phenyl	H, CH3	COOH	80.7 ± 3.6	102 ± 2.7
11b	4-F-Phenyl	Cyclopropyl	COOH	86.4 ± 3.5	102 ± 1.7
12b	Napthyl	Ethyl, Ethyl	CN	91.3 ± 4.6	107 ± 1.1
14b	4-F-Phenyl	H, F	CN	65.3 ± 9.0	84.5 ± 1.2
15b	4-F-Phenyl	F, F	CN	75.7 ± 9.1	98.2 ± 3.3
16b	4-F-Phenyl	H, F	CONH2	74.7 ± 7.2	90.6 ± 1.1
17a	Phenyl	F, F	CONH2	82.2 ± 3.9	100 ± 0.4
17b	4-F-Phenyl	F, F	CONH2	84.1 ± 5.3	98.8 ± 1
18a	Phenyl	H, OH	COOH	59 ± 3.4	89.1 ± 3.5
18b	4-F-Phenyl	H, OH	COOH	76.3 ± 4.9	102 ± 1
23a	Phenyl	F, CH3	COOH	80.8 ± 3.4	99.7 ± 3.6
23b	Phenyl	F, H	COOH	81.5 ± 3.9	92.7 ± 4.1

^aGriess assay.

^bMTT assay.

In comparison with 1, several analogs, gave a significantly greater anti-inflammatory response, although, in the case of 14b and 18a, this was associated with somewhat increased toxicity. These data demonstrate that the three structural modifications can be accommodated whilst maintaining or enhancing the anti-inflammatory activity of 1 towards iNOS induction. At higher concentration, the anti-inflammatory activity of 1 was increased, again without toxicity (Table 2). Cyclopropyl analogs 11a and 11c gave similar responses; however, 11d was highly toxic towards RAW cells. Comparison with other test compounds showed that toxicity was not a feature associated with one of the 3 structural motifs (R₁, R₂, R₃), but a property of the molecule as a whole.

Table 2.

Anti-inflammatory effects of 1 analogues (100 µM)

Compound	R1	R2, R2′	R3	NO2− as %a	Cell viability as %b
DMSO	-	-	-	100 ± 0.8	100 ± 2.3
1	Phenyl	H, CH3	COOH	69.1 ± 3.2	109 ± 2.7
2	Phenyl	H, CH3	COO(CH2)4ONO2	49.8 ± 2.5	108 ± 3.4
11a	Phenyl	Cyclopropyl	COOH	60.7 ± 1.3	101 ± 3.1
11c	4-CF3-Phenyl	Cyclopropyl	COOH	62.4 ± 3.4	102 ± 4.6
11d	3,4 Dichlorophenyl	Cyclopropyl	COOH	-	19.1 ± 2.8
25a	Phenyl	Cyclopropyl	COO(CH2)2Br	18.9 ± 1.0	109 ± 4.0
25b	4-CF3-Phenyl	Cyclopropyl	COO(CH2)2Br	14.7 ± 3.2	113 ± 2.1
25c	3,4 Dichlorophenyl	Cyclopropyl	COO(CH2)2Br	27.8 ± 2.0	114 ± 3.7
26a	Phenyl	Cyclopropyl	COO(CH2)2ONO2	47.3 ± 1.5	113 ± 6.5
26b	4-CF3-Phenyl	Cyclopropyl	COO(CH2)2ONO2	42.8 ± 2.1	128 ± 1.8
26c	3,4 Dichlorophenyl	Cyclopropyl	COO(CH2)2ONO2	40 ± 2.4	135 ± 2.9

^aGriess assay.

^bMTT assay.

We have previously reported use of the Griess assay in measurement of the anti-inflammatory activity of nitrates towards LPS-induced RAW cells.³¹ There is a potential pitfall with this approach, because metabolic denitration of nitrate drugs directly yields NO₂⁻.³⁴ However, a complete analysis of denitration in induced and non-induced RAW cells demonstrated that simple aliphatic mononitrates such as isosorbide mononitrate (ISMN) and 2, release < 3 % of the theoretical yield of NO₂⁻ after 24 h.³¹ We previously reported that 2 was significantly more potent than 1 in attenuating induction of iNOS activity, whereas the classical mononitrate, ISMN, showed no significant inhibition of iNOS activity, and in combination with 1 was no more efficacious than 1 alone.²⁸ These observations extend to the hybrid nitrate derivatives, 26a–c, which were observed to be significantly more potent than their parent flurbiprofen analog (Table 2). Interestingly, conversion of the cytotoxic 11d to a hybrid nitrate (26c) completely ablated toxicity. However, neither the anti-inflammatory activity nor the ablated toxicity could be ascribed to the nitrate group, since the precursor bromo-derivatives, 25a–c, were the most potent anti-inflammatory agents, reducing NO₂⁻. production fourfold relative to the parent carboxylic acids, in the absence of any cytotoxicity.

Approaches to the design of improved analogs of 1 as anti-amyloidogenic agents have been published previously. Modification of the α-carbon through the incorporation of a cyclopropyl group¹¹ or a dimethyl group¹⁰ were reported to lead to increased potency for lowering Aβ while eliminating COX activity. Previous work published by Peretto et al. focused mainly on substituting the terminal phenyl ring (R₁) of the cyclopropyl analogue of 2, leading to compound 11d.¹¹ The murine N2a.hAPP cell line has previously been employed by various investigators to assay SALAs using ELISA to measure Aβ_1–40 and/or Aβ_1–42.^{16, 28, 35} In this assay, confluent N2a/APPsw cells were treated with drug for 24 h, following which the supernatant was collected and Aβ_1–42 measured by ELISA. Compound 11d, observed to be toxic in RAW cells at higher concentration (100 µM), was not toxic towards N2a cells at lower concentration (1 µM). However, in contrast to reports of the activity of this compound tested at higher concentrations (i.e. 50–500 µM),^{30, 36} at the low concentration tested herein (1 µM), 11d did not reduce levels of Aβ_1–42 (Table 3). Carboxylate analogs possessing a phenyl R₁ substituent uniformly lowered Aβ_1–42, with the exception of the diethyl analog 21.

Table 3.

Effects of 1 analogues (1 µM) on Aβ_1–42 levels

Compound	R1	R2, R2′	R3	Aβ1–42 as %	Cell viability as %a
DMSO	-	-	-	100 ± 3.2	100 ± 0.9
1	Phenyl	H, CH3	COOH	86.6 ± 0.9	101 ± 0.9
11a	Phenyl	Cyclopropyl	COOH	69.5 ± 2.0	103 ± 1.9
11b	4-F-Phenyl	Cyclopropyl	COOH	90.4 ± 1.3	102 ± 1.3
11c	4-CF3-Phenyl	Cyclopropyl	COOH	99.3 ± 10.7	97.2 ± 3.1
11d	3,4 Dichlorophenyl	Cyclopropyl	COOH	125 ± 5.0	92.9 ± 0.6
12a	Phenyl	Ethyl, Ethyl	CN	125 ± 5.1	97.3 ± 0.7
12b	Napthyl	Ethyl, Ethyl	CN	105 ± 3.3	91.6 ± 1.4
14a	Phenyl	F, H	CN	65.6 ± 1.5	93.1 ± 1.0
14b	4-F-Phenyl	F, H	CN	-	69.4 ± 0.3
15b	4-F-Phenyl	F, F	CN	125 ± 5	98.2 ± 0.6
16a	Phenyl	F, H	CONH2	101 ± 0.9	102.8 ± 1.0
16b	4-F- Phenyl	F, H	CONH2	-	69.1 ± 3.0
17a	Phenyl	F, F	CONH2	115 ± 3.5	102 ± 1.3
17b	4-F- Phenyl	F, F	CONH2	109 ± 5.8	95.9 ± 0.4
18a	Phenyl	OH, H	COOH	82.5 ± 1.6	101 ± 0.3
18b	4-F- Phenyl	OH, H	COOH	106 ± 1.3	99.7 ± 1.4
21	Phenyl	Ethyl, Ethyl	COOH	124 ± 7.1	95.5 ± 0.3
23a	Phenyl	F, CH3	COOH	98.8 ± 2.1	101 ± 1.8
23b	Phenyl	F, H	COOH	91.8 ± 1.3	98.7 ± 1.3

^aMTT assay.

A number of derivatives (i.e. 11b, 18a, and 23b) demonstrated anti-amyloidogenic activity, comparable to 1, in the absence of substantial toxicity. These compounds all contain the NSAID carboxylate group, but are otherwise structurally diverse. Two compounds stood out as superior to 1, 11a and 14a, both bearing an unsubstituted phenyl R₁ substituent. The activity of the nitrile 14a was of interest, because the observation of anti-amyloidogenic activity shows that the carboxylate group is not an absolute requirement. This was the most interesting of the novel α-fluoro analogs, but obviously was not suitable for further study as a hybrid nitrate.

Given the ability of at least one analog, lacking a carboxylate group, to lower Aβ_1–42, and several such examples inhibiting iNOS activity, it was of interest to assay analogs of 3 and 4, which contained examples with amides in place of carboxylic acids. The derivatives of 3 provided the expected anti-inflammatory activity and the expected anti-amyloid activity for 3, itself, at high concentration (Table 4). Other activity of note was the amyloid raising activity of the amide derivative, 34, which was of the same magnitude as seen for so-called selective amyloid raising agents (SARAs), such as fenofibrate (39). Derivatives of 4 (28–33) provided one compound with a good profile of anti-inflammatory and anti-amyloidogenic activity without cytotoxicity, the desmethylated derivative 28. Amide derivatives did not reduce Aβ_1–42, and were comparable to 4 (Table 5).

Table 4.

Aβ_1–42 inhibitory activity and anti-inflammatory activity of 3 analogues

Compound	R1	R2	Aβ1–42 as %a	NO2− as %b	Cell viability as %c
DMSO	-	-	100 ± 3	100 ± 7.4	100 ± 7.4
3	CH3	COOH	108 ± 6	70.9 ± 7.4	98.7 ± 1
3 (100 µM)	CH3	COOH	76.7 ± 2.9	39.8 ± 10.5	92.0 ± 7.3
34	CH3	CONH-4-chlorophenyl	295 ± 27.2	68.6 ± 6.3	92.2 ± 5.3
35	H	COOH	102 ± 5.3	65.9 ± 0.4	97.9 ± 1.1
36	CH3	NHCH2CH2OH	118 ± 9.4	52.5 ± 2.3	100 ± 1.6
37	CH3	CONHCH2CH2-3-pyridine	135 ± 8.7	100 ± 7.4	95.6 ± 2.6

^a1 µM.

^bGriess assay.

^cMTT assay in RAW cells.

^{b & c}10 µM unless otherwise stated.

Table 5.

Anti-amyloidogenic activity and anti-inflammatory activity of 4 analogues.

Compound	R1	R2	R3	Aβ1–42 as %a	NO2− as %b	Cell viability as %c
DMSO	-	-	-	100 ± 3.8	100 ± 11.4	100 ± 1.6
4	SOCH3	CH3	COOH	100 ± 0.1	85.7 ± 9.3	104 ± 1.2
5	SCH3	CH3	COOH	82.6 ± 2.2	-	-
28	SCH3	H	COOH	80.8 ± 4.3	47.5 ± 7.2	100 ± 1.6
29	SOCH3	H	COOH	95 ± 5	91.6 ± 11	104 ± 1.2
30	SCH3	H	CONHCH2Ph	94.5 ± 1.8	58.4 ± 12.5	100 ± 1.6
31	SCH3	H	CONHCH2CH3	95.7 ± 1.8	80.4 ± 11.1	104 ± 1.2
32	CF3	H	COOH	91 ± 1.5	29.2 ± 8	100 ± 1.6
33	SCF3	H	COOH	99.4 ± 0.4	1.7 ± 6.3	104 ± 1.2

^a1 µM.

^bGriess assay at 10 µM.

^cMTT assay in RAW cells at 10 µM.

For selected agents, the dependence of anti-amyloidogenesis on concentration was tested further, again using N2a.hAPP cell cultures treated with agents for 24 h, followed by ELISA assay of the supernatant. The flurbiprofen analog 11a was compared to 1 itself and shown to be a more potent SALA, lowering Aβ_1–42 by 40 % at the highest concentration studied, with no significant toxicity (Fig. 1A). In contrast, the hybrid nitrate derivative 2, as we have reported previously,²⁸ showed biphasic activity. The full concentration-response data for 2 showed SALA activity more potent than 1 and similar to 11a at low concentrations, and in contrast, SARA activity at > 10 µM (Fig. 1B).

Figure 1.

Fluorbiprofen and derivatives showed varied SALA and SARA activity in neuronal cell incubations. Levels of Aβ_1–42 from N2a/APPsw cell supernatants were measured by ELISA and normalized to DMSO vehicle treated control after incubation with different concentrations of compounds 1 and 11a (A); or, 1 and 2 (B). The average level of Aβ_1–42 in the DMSO treated groups was ~500 pg/ml. The data show mean and s.e.m. from at least 4 separate experiments performed using separate cell passages.

It was important to determine whether this biphasic activity was peculiar to compound 2, or if indeed, this was common to related NSAID hybrid nitrates. Therefore, nitrate 26a was studied over a range of concentrations, revealing a similar response, albeit with less efficacy as a SALA than 2 (Fig. 2A). To determine if the nitrate group was relevant to the observed biphasic activity of these NSAID hybrid nitrates, the anti-amyloidogenic activity of the nitrate 2 was compared with that of the simple ethyl ester 38 in N2a.hAPP cell culture incubations (Fig. 2B). The ester behaved as a typical SARA agent increasing Aβ_1–42 2.5-fold at 10 µM, indicating the importance of the nitrate for SALA activity at low concentrations. However, this SALA activity of hybrid nitrate NSAIDs towards Aβ_1–42 modulation is unlikely to be associated with NO activity delivered by the nitrate functionality, since we have previously noted that the classical nitrate ISMN is without activity in this assay.²⁸ The alkyl ester linker of the hybrid nitrates, 2 and 26a, is expected to be labile towards non-specific esterase activity; therefore it was important to test a hybrid nitrate with a stable linker. The amide linked nitrate, 27, was synthesized expressly to test if the more labile ester linker of 2 and 26a was essential for the observed anti-amyloidogenic activity. The anti-amyloidogenic activity at low concentration (0.5 µM) was retained compared to 26a, indicating that the intact nitrate is required for SALA activity (Fig. 2C). This appears to be only the second example where biological activity can be attributed to the intact NO-NSAID, rather than to the NO-NSAID acting as a prodrug releasing NSAID, aliphatic nitrate and/or NO.³⁷

Figure 2.

Hybrid nitrate flurbiprofen derivatives caused biphasic regulation of Aβ_1–42 in neuronal cell incubations, acting as SALAs at lower concentration. Levels of Aβ_1–42 from N2a/APPsw cell supernatants were measured by ELISA and normalized to DMSO vehicle treated control after incubation with different concentrations of compounds 2 and 26a (A); 2 and 38 (B); or, 26a (0.5 µM) and 27 (0.5 µM) (C). The average level of Aβ_1–42 in DMSO treated group was ~500 pg/ml. The data show mean and s.e.m. from at least 4 separate experiments performed using separate cell passages.

The NO-flurbiprofens, NCX-2216 and HCT-1026 (2), have been reported to lower Aβ in mouse models, however, NCX-2216 was not detected in brain tissue,³⁸ and both plasma and brain levels of HCT-1026 were reported to be below detection limits.³⁹ Hydrolysis of HCT-1026 yields 4-hydroxybutyl nitrate, and it is possible that this metabolite has brain bioavailaibility since animal studies have shown that the level of inorganic nitrite in the brain increases after oral administration of HCT-1026.^{29, 40} It was of interest both to confirm the increased stability of the amide linked hybrid nitrate 27 relative to the ester linked homolog, 2, and to determine if unlike 2, the NO-flurbiprofen, 27, was detectable in the brain. Therefore, 27 (10 mg/kg) was delivered by i.p. injection to male C75/BL6 mice (25g) and brain and plasma levels detected by LC-MS/MS at 30 min and 120 min after injection. The brain/plasma ratio of 27 was 37% and 13%, at 30 min and 120 min, respectively (Figure 3). Most importantly, 27 was present in detectable amounts both in the brain and plasma 2 h after administration, in contrast to reports on 2.

Figure 3.

Hybrid nitrate 27 has brain bioavailaibility and in vivo stability after administration to mice (10 mg/kg i.p.) 30 min or 120 min prior to sacrifice. Quantitation of 27 by LC-MS/MS used MRM analysis of the m/z transition 361 → 200. A. Representative MRM chromatogram of 27 from mouse brain sample 120 min after administration, showing fragment observed as protonated cation (m/z = 200) in MRM. B. Quantitative analysis of brain and plasma levels at 30 min and 120 min using LC-MS/MS detection. Data (N=4) show mean and s.e.m.

The SARA activity of 1 derivatives 2, 26a, and 38 at higher concentrations has precedent in SARA agents including NSAIDs such as celecoxib, and other drugs, such as 39 that have been reported to elevate Aβ_1–42 levels while lowering the level of other fragments including Aβ_1–38.^41–43 In order to study the effect of agents on amyloidogenesis in more detail, we determined the profile of Aβ fragment production from N2a.hAPP cells for selected compounds using an immunoprecipitation MALDI-TOF technique introduced in 2009.¹⁶ This technique quantifies the amyloid fragments Aβ_1–37, Aβ_1–38, Aβ_1–40, and Aβ_1–42, using Aβ_1–43 as an internal standard, since this fragment is not a product of amyloidogenesis. In the N2a.hAPP amyloidogenesis assay, we observed that the known SARA 39 (200 µM) increased Aβ_1–42 levels threefold but had modest effects on Aβ_1–37 and Aβ_1–38, although lowering the level of the latter fragment (Fig. 4). In contrast, fibric acid, the free acid form of 39 was reported to lower the levels of Aβ_1–42.¹⁶ A similar observation on Aβ_1–42 modulation was made herein with 1 and its ester, 38. Whereas, the ester acted as a SARA, the free acid, 1 at 100 µM, selectively lowered Aβ_1–42 and elevated Aβ_1–37 relative to the vehicle control (Fig. 4). Indeed, the amyloid peptide product profiles for the 3 carboxylate esters, 2, 25c, and 39 are very similar at high concentrations; all act as SARAs, significantly and selectively increasing Aβ_1–40, Aβ_1–42, and the ratio Aβ_1–42/Aβ_1–40. In contrast, the hybrid nitrate ester, 2, behaves quite differently; at low concentrations 2 lowers total Aβ generation with similar efficacy to the 1 analog, 11a, although with less selectivity for Aβ_1–42.

Figure 4.

Quantitative analysis of Aβ_1–37, Aβ_1–38, Aβ_1–40, and Aβ_1–42 in neuronal cell supernatant after drug treatment. For each individual experiment, peak heights from IP-MALDI-TOF spectra were normalized to the peak height of the Aβ_1–43 standard after incubation of drugs with N2a/APPsw cells. These normalized values were then expressed relative to the DMSO vehicle control in each set of experiments. See ref 16 for full details. The data show mean and s.e.m. from at least 4 separate experiments.

Sulindac (4) is known to act as an NSAID SALA. The expected selective lowering of Aβ_1–42 and raising of both Aβ_1–37, and Aβ_1–38 levels was observed (Fig. 4).⁴⁴ Flurbiprofen derivative 11a was a more potent SALA than both 1 and 4 (Figs 1, 4), moreover, the profile of Aβ modulation by 11a was very different from that of 4. The concept that NSAID SALAs act as γ-secretase modulators was initially rationalized by a shift in secretase cleavage from producing Aβ_1–42 to production of Aβ_1–37, however, current opinion is that SALAs do not operate universally via this synchronized shift from Aβ_1–42 production to Aβ_1–38 production.⁴³ As we have seen, the well-established SALA and SARA activity of several drugs is recapitulated in the N2a.hAPP model system. However, a simple “control experiment” has shown that in this system, the SALA and SARA activity is not related to γ-secretase modulation. In this experiment, wild-type N2a cells were treated with drug or vehicle for 24 h, at which time, conditioned media (CM) was removed from the culture dish and added to media containing a freshly made solution of Aβ_1–42 and Aβ_1–40. CM delivered the same modulation of Aβ_1–42, Aβ_1–40, and Aβ_1–42/Aβ_1–40 observed when drugs were incubated in N2a.hAPP cell cultures. Further experiments showed that the modulation of Aβ_1–42 and Aβ_1–40 levels by SARA and SALA drugs was mediated by expression of metalloprotease activity induced by drug treatment of cells.¹⁶ Levels of Aβ_1–42 and Aβ_1–40 were measured by the MALDI-TOF method after treatment of N2a cells with drugs for 24 h and transfer of the CM to a solution of Aβ_1–42 and Aβ_1–40 (Fig. 5). The results of this experiment confirm that 1, 2 and 28 are able to act as SALAs by increasing degradation of Aβ_1–42 and Aβ_1–40 and decreasing the ratio of Aβ_1–42/Aβ_1–40.

Figure 5.

SALA activity was observed in mixtures of Aβ_1–40 + Aβ_1–42 treated with conditioned media (CM) from drug treated N2a/WT cells. Cells were treated with 1 (100 µM), 2 (1 µM), and 28 (10 µM) for 24 h before the collection of the CM. CM was added to a mixture of Aβ_1–40 + Aβ_1–42 and incubated for 8 h. CM from cell free wells (media) was used as the control. Aβ was quantified by IP-MALDI-TOF and normalized to “media” control. Data show mean and s.d. from 2 separate cell cultures.

Conclusions

The poor blood-brain barrier penetration common to most NSAIDs has been blamed for the failure of the SALA R-flurbiprofen in Phase 3 clinical trial for AD. Hybrid nitrate NO-NSAIDs provide an alternative, gastric-sparing approach to NSAID SALAs of use in AD. Since these compounds contain an ester linkage in place of the carboxylate anion, improved CNS bioavailability is expected. In this work, analogs of indomethacin and sulindac did not show a significantly improved profile of anti-inflammatory activity nor Aβ_1–42 lowering. However, analogs of flurbiprofen, 1, were prepared with superior anti-inflammatory and anti-amyloidogenic activity to the parent NSAID. A number of examples were converted to hybrid nitrate ester prodrugs, including a prodrug of the AD drug candidate, 11d.²⁴ The hybrid nitrates possessed superior anti-inflammatory activity to the parent NSAID. Esterification of 1 led to elevated Aβ_1–42, however, this SARA activity was observed only at high concentrations for the hybrid nitrates, whereas at low concentrations (< 1 µM), Aβ_1–42 and the ratio of Aβ_1–42/Aβ_1–40 were lowered. Interestingly, the intact nitrate group was found to be necessary for the SALA activity of these NO-NSAIDs. An amide-linked NO-NSAID SALA, 27, was shown to have brain bioavailability and to be detectable in brain and plasma at 120 min. These data support the optimization of hybrid nitrate SALAs, based upon modified flurbiprofen scaffolds that retain anti-inflammatory activity, and provide enhanced brain bioavailability.

Experimental

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. Murine biotinylated anti-human Aβ 4G8 and 6E10 antibodies produced by Signet were purchased from Covance Research Products, Inc. (Dedham, MA). All cell culture supplies, human Aβ ELISA kit, and streptavidin Dynabeads T1 were purchased from Invitrogen (Carlsbad, CA).

Cell culture and treatments

The murine neuroblastoma N2a cells obtained from American Type Culture Collection (Manassas, VA) were cultured in 1:1 Dulbecco's Modified Eagle's medium (DMEM) and OPTI-MEM supplemented with 5 % fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. N2a cells stably transfected with the Swedish mutant of human APP (N2a/APPsw were kind gift of Dr. Gopal Thinakaran, University of Chicago) were additionally supplemented with 200 µg/ml G418 but omitted during all experiments. RAW 264.7 mouse macrophage-like cells, provided by Dr. J. Cook (University of Illinois at Chicago, Chicago, IL), were maintained in DMEM, supplemented with 1 % penicillin-streptomycin and 10 % fetal bovine serum. Cells were maintained at 37 °C and 5 % CO₂.

Griess Assay

RAW cells were plated at a concentration of 25 × 10⁴ cells/well in a 24-well plate and incubated at 37 °C for 24 h. The medium was changed, and the cells were drug treated followed 30 min later by the addition of lipopolysaccharide (LPS) from Escherichia coli 055:B5 (LPS; Sigma-Aldrich). 24 h later, 100 µl of the supernatant was removed and incubated with the Griess reagent [100 µl; 0.5 % sulfanilamide, 0.05 % (N-1-naphthyl)ethylenediamine dihydrochloride, 2.5 % H₃PO₄, and 97 % H₂O by weight] for 30 min at rt in the dark. The absorbance was measured at 530 nm on a Dynex MRX II microplate spectrophotometer and calibrated using a standard curve constructed with sodium nitrite to yield nitrite concentration.

Amyloid beta measurement from N2a/APPsw cells supernatant

N2a/APPsw cells were plated 24 h before the experiment at a density of 25 × 10⁴ cells/well in a 24-well plate. Cells were washed with phosphate buffered saline (PBS; 50 mM, pH 7.4) before the addition of 500 µL of DMEM supplemented with 0.2 % fetal bovine serum (FBS) followed by drug treatment. Cells were incubated for a period of 24 h. CM was collected, followed by the addition of NaN₃ (0.01 % final concentration) and a mixture of protease inhibitors (10 mM phenanthroline and P2714 protease inhibitor cocktail from Sigma containing 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin, E-64, leupeptin, and ethylenediaminetetraacetate (EDTA)), and centrifuged for 2 min at a speed of 10,000 g. Aβ_1–42 levels were determined by sandwich ELISA using human Aβ_1–42 ELISA kit and following the supplied protocol. For immunoprecipitation, cells were treated as described above, but plated in a 6-well plate at a density of 100 × 10⁴ cells/well in 2 mL media. 24 h after treatment, 1 mL of the CM is collected, followed by the addition of NaN₃ and protease inhibitors and spiked with Aβ_1–43 at approximately 1 ng/mL final concentration as an internal standard before performing immunoprecipitation. A stock of 200 ng/mL Aβ_1–43 was prepared in sodium bicarbonate (50 mM, pH 9.0) and stored in aliquots in 1.5 mL low binding eppendorf tubes at −80 °C, once defrosted any remaining Aβ_1–43 stock was discarded. Following immunoprecipitation the samples were analyzed in a MALDI-TOF instrument as described below for the purpose of measuring the relative abundance of Aβ peptides. In all experiments, Aβ levels were assayed relative to vehicle controls by treating all the groups with the identical sample of Aβ_1–43 internal standard.

Aβ degradation associated with N2a/WT cells

N2a/WT cells were plated 24 h before the experiments at a density of 100 × 10⁴ cells/well in 6-well plates. Cells were washed with PBS before the addition of 2 mL DMEM supplemented with 0.2 % FBS followed by drug treatment. 18 h after drug treatment, CM was collected and treated with NaN₃ and centrifuged at 10,000 g; the mixture of Aβ_1–40 and Aβ_1–42 was added and further incubated for 8 h at 37 °C. The CM was then treated with the protease inhibitor mixture and spiked with Aβ_1–43 as an internal standard followed by immunoprecipitation and MALDI-TOF analysis.

Immunoprecipitation and MALDI/TOF analysis

Immunoprecipitation of Aβ was performed by the addition of a mixture of 0.25 µl biotinylated 4G8 antibody and 0.25 µl biotinylated 6E10 antibody, samples were then rotated at room temperature for 4 h followed by the addition of 4 µL T1 streptavidin coated magnabeads and rotated for an additional 2 h. Beads were washed three times with NH₄CO₃ (10 mM, pH 8.0) and the Aβ peptides were eluted using 4 µL of a mixture of 75 % acetonitrile and 0.5 % TFA in H₂O followed by the addition of 3 µL of a saturated matrix solution (alpha-cyano-4-hydroxycinnamic acid in 1:1 acetonitrile and H₂O). 1 µL of this mixture was spotted onto a MALDI plate and analyzed using a MALDI-TOF instrument (Applied Biosystems). Mass spectra were acquired automatically in a linear positive mode at 1950 shot per spectrum. For each individual experiment, peak heights were normalized to the peak height of the Aβ_1–43 standard; these normalized values were then expressed relative to the DMSO vehicle control in each set of experiments.

MTT assay

The MTT assay was performed by incubating the cells in a culture media containing 0.5 mg/ml solution of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h, the cells were then washed with PBS and the formed purple formazan crystals were solubilized in DMSO. The plate was shaken on a plate rocker for 30 min then the absorbance was measured at 570 nm using 630nm as a reference wave length on a Dynex MRX II microplate spectrophotometer.

Bioavailability

Use of animals was approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. Nitrate 27 was dissolved in DMSO/H₂O (1:1 v/v) and administrated at 10mg/kg via i.p. injection to male C57BL/6 mice (25g). Animals were sacrificed at 30 and 120 min and both blood and whole brain were collected for analysis. Two equal portions of each brain or blood sample were processed for analysis. Brain tissue was homogenized in methanol manually and then centrifuged at 4 °C, 13000 rpm for 10 min. The remaining pellet was washed with methanol (0.3 mL) and supernatants combined. After concentration under N₂, reconstitution in MeOH/H₂O (1:1, v/v), and centrifugation at 4 °C, 13000 rpm for 15 min, samples were analyzed by LC-MS/MS. Plasma was extracted in a similar way prior to analysis by LC-MS/MS, except acetonitrile was used instead of methanol. LC separation was achieved using a Waters Xbridge C18 3.0×100 mm, 3.5 µm column and a linear gradient mobile phase from 55 % to 95 % MeOH in 0.1 % formic acid over 21 min at a flow rate of 350 µL/min. MS/MS analysis was performed on a Sciex API 3000 triple-quad instrument monitoring the transition m/z=361 → m/z=200 in positive ion mode. Quantitative extraction from plasma was estimated by spiking untreated samples. Concentrations were interpolated from a standard curve constructed from analyte in MeOH/H₂O (4 – 500 ng/mL).

General Methods

Unless stated otherwise, all reactions were carried out under an atmosphere of dry argon in oven dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25 °C. CH₂Cl₂ was distilled over CaH₂, and THF distilled over Na(s)/benzophenone. All other solvents were of anhydrous quality purchased from Aldrich Chemical Co. and used as received. Commercially available starting materials and reagents were purchased from Aldrich, TCI and Fisher Scientific and were used as received unless specified otherwise. Indomethacin and sulindac libraries were provided by Prof. Larry Marnett of Vanderbilt University. Analytical thin layer chromatography (TLC) was performed with Sorbent Silica glass backed TLC plates (20 × 20 cm, 60 Å, 250 µm) with UV detection at 254 nm. ¹H- and ¹³C NMR spectra were recorded on either a Bruker Avance 400 MHz spectrometer or a Bruker DPX 400 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as internal standard ([CDCl₃ 7.27 ppm, 77.23 ppm] [DMSO-d₆ 2.5 ppm, 39.51 ppm] and [MeOD-d₄ 4.78, 49.0] for ¹H, ¹³C respectively). Data are reported as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, bs = broad singlet, m = multiplet), and coupling constants. Compounds submitted for biological testing were found to be > 95 % pure via analytical HPLC analysis using Shimadzu HPLC. High resolution mass spectral data was collected in-house using Shimadzu LCMS IT-TOF.

1-Bromo-4-bromomethyl-2-fluorobenzene (7).⁴⁵

N-bromosuccinimide (11.39 g, 640 mmol) was added to a solution of 1-bromo-2-fluoro-4-methylbenzene 6 (5.36 ml, 8.01 g, 42 mmol), in CCl₄ (100 mL). The mixture was heated to 80 °C, then dibenzoyl peroxide (0.27 g, 32 mmol) was added. The mixture was stirred at this temperature for 1 h then cooled to rt and washed in sequence with H₂O (2 × 80 ml) and brine (2 × 50 ml), dried over Na₂SO₄ and concentrated to afford red oil (~14 g) which was purified by column chromatography (hexane/ethyl acetate [10:1]) to give 7 as a yellow crystalline solid (12.2 g, yield 75.6 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.53 (t, 1H); 7.19-7.17 (dd, 1H); 7.08-7.06 (dd, 1H); 4.42 (s, 2H).

4-Bromo-3-fluorophenyl)acetonitrile (8)

A solution of 7 (12.0 g, 41 mmol) and KCN (2 g, 0.04 mol) in ethanol (100 mL) was heated to 80 °C for 2 h, then cooled to rt and concentrated under vacuo. The residue was suspended in H₂O (200 ml) and extracted with ethyl acetate (3 × 150 ml). Combined organic extracts were washed with brine (2 × 50 ml), dried over Na₂SO₄ and concentrated to afford a brown oil. Column chromatography (hexane/ethyl acetate [7:1]) yielded 8 as a light yellow solid (5.0 g, yield 52 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.57 (dd, 1H); 7.13 (dd, 1H); 7.02 (dd, 1H); 3.72 (s, 2H).

1-(4-Bromo-3-fluorophenyl)cyclopropanecarbonitrile (9a)

To a solution of 8 (5.0 g, 23 mmol) and 1,2-dibromoethane (3 mL, 35 mmol) in toluene (20 mL), 50 % NaOH (20 mL) and TBAB (1.6 g, 5 mmol) were added. The mixture was vigorously stirred at rt for 4 h, then diluted with H₂O (20 ml) and extracted with ethyl acetate (3 × 150 ml). The organic layer was washed with 1 M HCl (25 ml), then with brine (2 × 15 ml), dried over Na₂SO₄ and concentrated to afford a brown solid (5.7 g). Column chromatography (hexane/ethyl acetate [5:1]) afforded 9a as yellow solid. ¹H NMR (CDCl₃, 400 MHz): δ 7.54-7.50 (t, 1H); 7.05-7.03 (dd, 1H, J = 7.48 Hz); 7.00-6.98 (dd, 1H, J = 7.48 Hz); 1.80-1.77 (q, 2H); 1.43-1.40 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 160.42 (d, J = 240 Hz), 138.01 (d, J = 6.9 Hz); 133.94, 122.54 (d, J = 3.5 Hz); 121.49, 114.07 (d, J = 8.4 Hz); 108.23 (d, 20.8 Hz); 18.71, 13.50.

1-(4-Bromo-3-fluorophenyl)diethylcarbonitrile (9b)

To a solution of 8 (235 mg, 1.1 mmol) and bromoethane (240 mg, 2.2 mmol) in toluene (20 mL), 50 % NaOH (20 mL) and TBAB (420 mg, 2.8 mmol) were added. The mixture was vigorously stirred at rt for 4 h, then diluted with H₂O (20 ml) and extracted with ethyl acetate (3 × 100 ml). The organic solution was washed with 1 M HCl, then with brine, dried over Na₂SO₄ and concentrated to afford a brown solid (950 mg). Column chromatography (hexane/ethyl acetate [5:1]) afforded 9b as white solid (278 mg, yield 93.5 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.73-7.70 (t, 1H); 7.18-7.15 (dd, 1H, J = 7.48 Hz); 7.12-7.09 (dd, 1H, J = 7.48 Hz); 2.10-2.01 (m, 2H); 1.92-1.83 (m, 2H); 0.95-0.91 (t, 6H). ¹³C NMR (CDCl₃, 100 MHz): 160.11 (d, J = 240 Hz); 139.82 (d, J = 6.9 Hz); 133.46, 122.83 (d, J = 3.5 Hz); 120.97, 114.28 (d, J = 2.4 Hz); 107.96 (d, 21.1 Hz); 49.19, 33.33, 9.82.

1-(4-Bromo-3-fluorophenyl)cyclopropanecarboxylic Acid (10)

To a solution of 9a (5.1 g, 21 mmol) in methanol (10 mL), 35 % NaOH (40 mL) was added and the mixture was heated to 100 °C for 8 h. After cooling to rt, the mixture was acidified (pH 2) with 2 M HCl, the precipitate was filtered off, washed with H₂O and redissolved in 5 % NaHCO₃. Insoluble materials were filtered off and the solution acidified with 2 M HCl. The precipitate was filtered, washed with H₂O and dried under reduced pressure to afford 10 as a white solid (4.0 g, 72.5 %). ¹H NMR (CDCl₃, 400 MHz): δ 11.82 (bs, 1H); 7.51-7.47 (t, 1H); 7.14-7.12 (dd, 1H, J = 9.39 Hz); 7.05-7.03 (dd, 1H, J = 9.39 Hz); 1.73-1.70 (q, 2H); 1.29-1.22 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 180.50, 159.83 (d, J = 246.1 Hz); 140.25 (d, J = 7.0 Hz); 133.15, 127.28 (d, J = 3.4 Hz); 118.78 (d, J = 22.2 Hz); 107.20 (d, J = 20.7 Hz); 28.30, 17.54.

General procedure for biaryl Suzuki coupling

A suspension of the appropriate aryl bromide (1 eq), arylboronic acid (2 eq), TBAB (1.1 eq) and Pd(OAc)₂ (catalytic amount) in 2 M Na₂CO₃ (5–10 ml) was heated to 120 °C in a sealed tube for a period of 1–4 h. After cooling to rt, 2 M HCl (3 ml) and ethyl acetate (10 ml) were added and the mixture filtered through a celite pad. The organic layer was separated, washed with brine (2 × 30 ml), dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by flash chromatography (hexane/ethyl acetate [1:1]) to afford the corresponding biaryl compounds.

1-(2-Fluorobiphenyl-4-yl)cyclopropanecarboxylic acid (11a)

The general procedure using: 10 (259 mg, 1 mmol); phenylboronic acid (244 mg, 2 mmol); TBAB (340 mg, 1 mmol); Pd(OAc)₂ (30 mg); afforded the title compound as a white solid (yield 73.7 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.63-7.55 (t, 4H); 7.50-7.36 (m, 2H); 7.24-7.16 (q, 2H); 1.76-1.73 (q, 2H); 1.35-1.33 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 178.68, 160.63 (J = 243.2 Hz), 140.60 (d, J = 8.0 Hz), 135.70, 130.56, 129.15, 129.13, 128.61, 128.21, 128.08, 127.84, 126.56 (d, J = 3.0 Hz), 118.45 (d, J = 23.0 Hz), 28.01, 17.90. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₆H₁₃FO₂, 255.0826; observed, 255.0832.

1-(2,4'-Difluorobiphenyl-4-yl)cyclopropanecarboxylic acid (11b)

The general procedure using: 10 (150 mg, 0.58 mmol); 4-F-phenylboronic acid (162 mg, 1.16 mmol); TBAB (206 mg, 0.64 mmol); Pd(OAc)₂ (20 mg); afforded the title compound as a white solid (yield 75.5 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.70-7.33 (m, 5H); 7.24-7.17 (q, 2H); 1.76-1.70 (q, 2H); 1.34-1.32 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 179.96, 140.99, 132.57, 131.95, 131.32, 130.69, 130.08 (d, J = 3.6 Hz), 128.90, 128.21 (d, J = 3.4 Hz) 127.16, 126.54, 127.61, 118.51 (d, J = 22.8 Hz), 28.35, 17.54. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₆H₁₂F₂O₂, 273.0733; observed, 273.0746.

1-(2-Fluoro-4'-(trifluoromethyl)biphenyl-4-yl)cyclopropanecarboxylic acid (11c)

The general procedure: 10 (259 mg, 1.0 mmol); 4-CF₃-phenylboronic acid (228 mg, 1.2 mmol); TBAB (322 mg, 1.0 mmol); Pd(OAc)₂ (40 mg); afforded the title compound as a white solid (yield 67.8 %). ¹H NMR (CDCl₃, 400 MHz): δ 11.91 (bs, 1H); 7.72-7.65 (q, 4H); 7.42-7.38 (t, 1H); 7.26-7.19 (q, 2H); 1.76 (s, 2H); 1.35 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz): 180.29, 160.06, 157.58, 140.68 (d, J = 3.2 Hz), 138.65, 129.95, 129.55, 129.22, 128.89 (d, J = 1.3 Hz), 126.47 (d, J = 13.3Hz), 126.21 (d, 2.9 Hz), 125.02 (d, J = 3.8 Hz), 118.13, 117.90, 28.04, 17.20. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₇H₁₂F₄O₂, 323.0701; observed, 323.0710.

1-(3',4'-Dichloro-2-fluorobiphenyl-4-yl)cyclopropanecarboxylic acid (11d)

The general procedure using: 10 (259 mg, 1.0 mmol); 3,4-dichlorophenylboronic acid (190 mg, 1.2 mmol); TBAB (322 mg, 1.0 mmol); Pd(OAc)₂ (25 mg); afforded the title compound as a white solid (yield 72.9 %) ¹H NMR (CDCl₃, 400 MHz): δ 7.52-7.49 (t, 2H); 7.37-7.33 (t, 1H); 7.23-7.01 (m, 4H); 1.75-1.72 (q, 2H); 1.35-1.32 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 179.96, 160.10 (d, J = 246.5 Hz), 140.99 (d, 9.2 Hz), 132.57, 130.69, 130.06, 129.55, 129.22, 128.90, 128.19, 127.61, 126.54, 118.45 (d, J = 22.8 Hz), 28.35, 17.54. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₆H₁₁Cl₂FO₂, 323.0047; observed, 323.0050.

2-Ethyl-2-(2-fluorobiphenyl-4-yl)butanenitrile (12a)

The general procedure using: 9b (150 mg, 0.56 mmol); phenylboronic acid (177.3 mg, 0.83 mmol); TBAB (206 mg, 0.64 mmol); Pd(OAc)₂ (20 mg); afforded the title compound as a white solid (140 mg, yield 94.3 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.56-7.54 (d, 2H); 7.47-7.43 (m, 3H); 7.40-7.36 (t, 1H); 7.27-7.25 (d, 1H); 7.25-7.19 (d, 1H); 2.12-2.03 (m, 2H); 1.97-1.88 (m, 2H); 0.98-0.90 (t, 6H). ¹³C NMR (CDCl₃, 100 MHz): 160.05 (d, J = 242.8 Hz), 139.36 (d, J = 7.0 Hz), 135.42, 131.44, 129.35 (d, J = 4.0 Hz), 129.59, 128.94, 127.52, 122.50, 121.91, 118.5 (d, J = 22.8 Hz) 113.79, 113.55, 51.17, 33.38, 10.51.

2-Ethyl-2-(3-fluoro-4-(naphthalen-1-yl)phenyl)butanenitrile (12b)

The general procedure using: 9b (51 mg, 0.19 mmol); 2-napthylboronic acid (120 mg, 0.70 mmol); TBAB (125 mg, 0.21 mmol); Pd(OAc)₂ (20 mg); afforded the title compound as a yellow solid (45 mg, yield 75.1 %). ¹H NMR (CDCl₃, 400 MHz): δ 8.03 (s, 1H); 7.93-7.88 (m, 3H); 7.58-7.50 (m, 4H); 7.31-7.24 (q, 2H); 2.15-2.05 (m, 2H); 2.03-1.88 (m, 2H); 0.98-0.89 (t, 6H). ¹³C NMR (CDCl₃, 100 MHz): 162.09 (d, J = 246.2 Hz), 140.29 (d, J = 9.8 Hz), 133.75, 133.17, 133.87, 131.69 (d, J = 5.5 Hz), 128.80, 128.65, 128.50, 128.07, 127.18 (d, J = 4.1 Hz), 126.80 (d, J = 3.7 Hz), 124.22, 122.81 (d, J = 4.7 Hz), 122.21, 121.15, 115.60, 50.01, 34.21, 10.16.

2-(2-Fluorobiphenyl-4-yl)acetonitrile (13a)

The general procedure using: 8 (428 mg, 2.0 mmol); phenylboronic acid (490 mg, 4.0 mmol); TBAB (710 mg, 2.2 mmol); Pd(OAc)₂ (67 mg); afforded the title compound as a yellow solid (350 mg, yield 82.8 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.42 (m, 6H), 7.18-7.10 (m, 2H), 3.75 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz): 159.40 (d, J = 249.7 Hz), 134.52, 131.10 (d, J = 3.8 Hz), 130.61 (d, J = 8.0 Hz), 129.26, 128.58, 128.55, 128.21, 127.93, 127.67, 123.56 (d, J = 3.4 Hz), 116.93, 115.53 (d, J = 24.5 Hz), 22.76.

2-(2,4'-Difluorobiphenyl-4-yl)acetonitrile (13b)

The general procedure using: 8 (500 mg, 2.1 mmol); 4-F-phenylboronic acid (560 mg, 4.0 mmol); TBAB (710 mg, 2.2 mmol); Pd(OAc)₂ (45 mg); afforded the title compound as a yellow solid (480 mg, yield 90 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.51-7.39 (m, 3H), 7.19-7.11 (m, 4H), 3.81 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz): 162.21 (d, J = 247.6 Hz), 158.04, 130.91 (d, J = 3.6 Hz), 130.70 (d, J = 8.0 Hz), 130.44, 130.29 (d, J = 2.9 Hz), 130.20 (d, J = 2.9 Hz), 127.59 (d, J = 13.5 Hz), 124.80, 123.61 (d, J = 3.5 Hz), 116.84, 115.57 (d, J = 24.6 Hz), 115.18 (d, J = 21.7 Hz), 22.75.

Preparation of fluoromethylnitriles (14a, 14b, 15a, 15b).⁴⁶

To a solution of the appropriate benzylic nitrile in anhydrous THF (approximately 5–10 mL of THF/ 1 mmol of nitrile) at −78 °C, t-BuLi (2.2 equiv for difluorination, 1.1 equiv for monofluorination) was added over a period of 2 min. The resulting orange to dark red solution was stirred for 1 h at −78 °C. A solution of NFSi (2.5 equiv for difluorination, 1.3 equiv for monofluorination) in anhydrous THF (approximately 2–4 mL of THF/ mmol of NFSi) was added over 2 min. After this addition, the solution was stirred for 3 h at −78 °C. The reaction was quenched with 0.01 M HCl, and the resulting solution was extracted with CH₂Cl₂ (2 × 100 ml). The combined organic layers were washed with 5 % NaHCO₃ and brine, dried over Na₂SO₄, and concentrated in vacuo to give a yellow residue. Purification was achieved using silica gel flash chromatography (hexane/ethyl acetate [5:1]).

2-Fluoro-2-(2-fluorobiphenyl-4-yl)acetonitrile (14a)

The general procedure using: 13a (192 mg, 0.91 mmol); t-BuLi (1.2 ml, 2.0 mmol); NFSi (710 mg, 2.4 mmol); yielding after purification a mixture of 14a (yellow solid, 42 mg, yield 20.1 %) and 15a (yellow solid, 111 mg, yield 49.4 %). 14a: ¹H NMR (CDCl₃, 400 MHz): δ 7.59-7.38 (m, 8H), 6.11-6.09 (d, 1H, J = 11.8 Hz). ¹³C NMR (CDCl₃, 100 MHz): 159.73 (d, J = 250.5 Hz), 134.34, 132.15 (d, J = 6.7 Hz), 132.11, 128.98, 128.79, 128.65, 128.49, 127.92, 123.25 (d, J = 3.9 Hz), 115.39 (d, J = 25.2 Hz), 114.77 (d, J = 33.3 Hz), 79.18 (d, J = 182.8 Hz), 60.37. APCI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₉F₂N, 228.0630; observed, 228.0625.

2,2-Difluoro-2-(2-fluorobiphenyl-4-yl)acetonitrile (15a)

¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.32 (m, 8H). ¹³C NMR (CDCl₃, 100 MHz): 159.58 (d, J = 251.0 Hz), 133.79, 133.74 (d, J = 13.6 Hz), 131.93 (d, J = 3.8 Hz), 131.76 (d, J = 7.8 Hz), 129.00 (d, J = 3.0 Hz), 128.78, 128.34, 124.93, 121.33 (q, J = 5.0 Hz), 116.82, 113.58 (d, J = 26.6 Hz), 112.19 (t, J = 47.9 Hz), 107.92 (t, J = 245.0 Hz). APCI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₈F₃N, 246.0536; observed, 246.0515.

2-(2,4'-Difluorobiphenyl-4-yl)-2-fluoroacetonitrile (14b)

Synthesized via the general procedure using: 13b (115 mg, 0.5 mmol); t-BuLi (0.65 ml, 1.1 mmol); NFSi (394 mg, 1.5 mmol); yielding after purification a mixture of 14b (yellow solid, 56 mg, yield 42.1 %) and 15b (yellow solid, 32 mg, yield 32 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.54-7.30 (m, 5H), 7.15 (t, 2H, J = 8.4 Hz), 6.08 (d, 1H, J = 46.8 Hz). ¹³C NMR (CDCl₃, 100 MHz): 162.50 (d, J = 248.6 Hz), 159.25 (d, J = 250.9 Hz), 131.82 (dd, J = 21.0 Hz, 7.7 Hz), 131.29, 130.74 (dd, J = 13.5 Hz, 2.9 Hz), 130.43 (d, J = 2.7 Hz), 130.34 (d, J = 2.9 Hz), 128.84, 122.96 (t, J = 3.8 Hz), 115.45, 115.24, 115.06 (dd, J = 25.4 Hz, 4.4 Hz), 114.41 (d, J = 33.2 Hz), 78.88 (d, J = 183.0 Hz). APCI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₈F₃N, 246.0536; observed, 246.0511.

2-(2,4'-Difluorobiphenyl-4-yl)-2,2-difluoroacetonitrile (15b)

¹H NMR (CDCl₃, 400 MHz): δ 7.52- 7.37 (m, 5H), 7.09 (t, 2H, J = 8.0 Hz). ¹³C NMR (CDCl₃, 100 MHz): 163.04 (d, J = 249.2 Hz), 159.45 (d, J = 251.7 Hz), 132.68 (d, J = 13.2 Hz), 131.86 (d, J = 8.0 Hz), 131.73 (d, J = 3.6 Hz), 130.83 (d, J = 3.0 Hz), 130.75 (d, J = 3.0 Hz), 129.92, 129.88, 121.40 (q, J = 4.9 Hz), 115.83 (d, J = 21.6 Hz), 113.64 (dt, J = 26.6 Hz, 5.1 Hz), 112.12 (d, J = 48.3 Hz), 107.79 (t, J = 244.3 Hz). APCI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₇F₄N, 264.0442; observed, 264.0460.

General procedure fluoronitrile (14a,b & 15a,b) to fluoroamide (16a,b & 17a,b)

To a stirred solution of fluoronitrile in DMSO (30 mg / 1.5 ml) was added K₂CO₃ and the mixture was cooled to 0 °C. To this mixture was added dropwise H₂O₂ at the same temperature and the reaction mixture was stirred for 45 min at rt under N₂. The reaction was quenched by adding cold H₂O and the mixture was extracted three times with ethyl acetate (3 × 75 ml). The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated. The residue was purified by flash column chromatography (CH₂Cl₂/MeOH [95:5]) to give corresponding fluoroamides.

2-Fluoro-2-(2-fluorobiphenyl-4-yl)acetamide (16a)

(white solid, yield 75.4 %) ¹H NMR (CDCl₃, 400 MHz): δ 7.59-7.38 (m, 8H), 6.11-6.09 (d, 1H, J = 11.8 Hz). ¹³C NMR (CDCl₃, 100 MHz): 171.10, 159.73 (d, J = 250.5 Hz), 136.52, 134.34, 132.15 (d, J = 6.7 Hz), 132.11, 128.98, 128.79, 128.65, 128.49, 123.25 (d, J = 3.9 Hz), 115.39 (d, J = 25.2 Hz), 114.77 (d, J = 33.3 Hz), 79.18 (d, J = 182.8 Hz). ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₄H₁₁F₂NO, 270.0701; observed, 270.0709.

2-(2,4'-Difluorobiphenyl-4-yl)-2-fluoroacetamide (16b)

(white solid, yield 82.4 %): ¹H NMR (400 MHz, CDCl₃): δ 7.54-7.30 (m, 5H), 7.15-7.13 (t, 2H, J = 8.4 Hz), 6.08-6.02 (d, 1H, J = 46.8 Hz,). ¹³C NMR (CDCl₃, 100 MHz): 171.52, 162.50 (d, J = 248.6 Hz), 159.25 (d, J = 250.9 Hz), 131.82 (dd, J = 21.0 Hz, 7.7 Hz), 131.29, 130.74 (dd, J = 13.5 Hz, 2.9 Hz), 130.43 (d, J = 2.7 Hz), 130.34 (d, J = 2.9 Hz), 128.84, 122.96 (t, J = 3.8 Hz), 115.45, 115.06 (dd, J = 25.4 Hz, 4.4 Hz), 114.41 (d, J = 33.2 Hz), 78.88 (d, J = 183.0 Hz). ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₄H₁₀F₃NO, 288.0607; observed, 288.0609.

2,2-Difluoro-2-(2-fluorobiphenyl-4-yl)acetamide (17a)

(white solid, yield 81.0 %): ¹H NMR (DMSO-d₆, 400 MHz): δ 8.43 (s, 1H), 8.11 (s, 1H), 7.71-7.69 (t, 1H, J = 8.0 Hz), 7.76-7.46 (m, 7H). ¹³C NMR (DMSO-d₆, 400 MHz): 171.60, 160.63 (d, J = 242.5 Hz), 134.58, 131.22 (d, 3.8 Hz), 128.99 (d, J = 2.9 Hz), 128.58, 128.32, 127.90, 127.63, 125.63, 121.55 (d, J = 3.9 Hz), 117.59, 114.09 (d, J = 10.06 Hz), 113.96 (d, J = 19.5 Hz). ESI-HRMS (m/z): [M+H]⁺ calcd. for C₁₄H₁₀F₃NO, 266.0787; observed, 266.0797.

2-(2,4'-Difluorobiphenyl-4-yl)-2,2-difluoroacetamide (17b)

(white solid, yield 87.9 %). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.42 (s, 1H), 8.10 (s, 1H), 7.71-7.62 (m, 3H), 7.53-7.49 (m, 2H), 7.34 (t, 2H, J = 8.8 Hz). ¹³C NMR (DMSO-d₆, 100 MHz): 164.77 (t, J = 30.7 Hz), 162.20 (d, J = 245.8 Hz), 158.61 (d, J = 247.4 Hz), 134.62 (td, J = 25.7 Hz, 8.1 Hz), 131.50 (d, J = 3.2 Hz), 131.12 (d, J = 2.7 Hz), 131.03 (d, J = 3.0 Hz), 130.36, 129.78 (d, J = 13.0 Hz), 127.64, 121.78, 115.71 (d, J = 21.5 Hz), 113.81 (t, J = 254.0 Hz), 113.82 (dt, J = 25.8 Hz, 6.4 Hz). ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₉F₄NO, 282.0547; observed, 282.0544.

General procedure for acid hydrolysis of fluoromethylnitriles (16a, 16b) to fluoromethylacetic acids (18a, 18b)

A vigorously stirred emulsion of the fluoromethylnitrile in a 49 % aqueous solution of H₂SO₄ (5 mL: per 100 mg of substrate) was heated at 80 °C for 3 h. After being cooled to rt, the aqueous phase was extracted with diethyl ether (3 × 50 ml). Acids were washed out from the combined organic phases with sat. NaHCO₃. The aqueous phase was acidified to pH 2 and extracted with Et₂O (3 × 50 ml). The combined extracts were dried over Na₂SO₄ and concentrated. The residue was recrystallized from chloroform: petroleum ether.

2-(2-Fluorobiphenyl-4-yl)-2-hydroxyacetic acid (18a)

(white solid, yield 56.0 %). ¹H NMR (MeOD-d₄, 400 MHz): δ 7.54-7.33 (m, 8H), 5.21 (s, 1H). ¹³C NMR (MeOD-d₄, 100 MHz): 175.72, 165.27, 162.83, 159.73, 143.03 (d, J = 7.8 Hz), 142.95, 133.13, 132.12, 131.84 (d, J = 3.3 Hz), 129.08 (d, J = 13.6 Hz), 122.20 (d, J = 3.1 Hz), 116.54 (d, J = 21.8 Hz), 115.58 (d, J = 24.3 Hz), 73.42. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₁₁FO₃, 245.0609; observed, 245.0639.

2-(2,4'-Difluorobiphenyl-4-yl)-2-hydroxyacetic acid (18b)

(white solid, yield 41.5 %). ¹H NMR (MeOD-d₄, 400 MHz): δ 7.56-7.54 (t, J = 6.8 Hz, 1H), 7.46-7.44 (t, 1H, J = 8.0 Hz), 7.39-7.31 (m, 3H), 7.18-7.16 (t, 2H, J = 8.8 Hz), 5.21 (s, 1H). ¹³C NMR (MeOD-d₄, 100 MHz): 175.72, 164.05 (d, J = 245.7 Hz), 160.95, 160.18, 142.99 (d, J = 7.8 Hz), 133.13, 132.11 (d, J = 3.2 Hz), 132.04, 131.82 (d, J = 3.3 Hz), 129.01 (d, J = 13.7 Hz), 124.17 (d, J = 3.1 Hz), 116.43 (d, J = 21.8 Hz), 115.46 (d, J = 24.3 Hz), 73.42. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₁₀F₂O₃, 263.0525; observed, 263.0575.

2-(2-Fluorobiphenyl-4-yl)acetonitrile (19)

The acid catalyzed hydrolysis procedure used in the preparation of 18a, starting with 13a (510 mg, 2.41 mmol) in the presence of H₂SO₄ (100 ml) afforded the product as a white solid (480 mg, yield 86.3 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.37 (m, 6H); 7.21-7.15 (m, 2H); 3.80 (s, 2H).

Preparation of the biaryl-diethyl ester (20)

To a solution of 19 (350 mg, 1.5 mmol) and bromoethane (500 mg, 5 mmol) in toluene (20 mL), 50 % NaOH (20 mL) and TBAB (700 mg, 5 mmol) were added. The mixture was vigorously stirred at rt for 8 h, then diluted with H₂O (25 ml) and extracted with ethyl acetate (3 × 50 ml). The organic solution was washed with 1 M HCl (1 × 50 ml), then with brine, dried over Na₂SO₄ and concentrated to afford a brown solid. Purification was achieved using silica gel flash chromatography to give the product as a white solid (300 mg, 70.3 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.50-7.36 (m, 7H); 7.05-7.01 (m, 1H); 4.34-4.32 (q, 2H); 2.12-2.02 (m, 2H); 1.99-1.90 (m, 2H); 1.01-0.98 (t, 6H); 0.71-0.70 (t, 3H). ¹³C NMR (CDCl₃, 100 MHz): 174.30, 165.40, 140.05, 130.69, 130.28, 129.61, 128.93, 128.40, 127.58, 125.48, 122.69 (d, J = 4.82 Hz), 117.42, 113.56, 61.33, 52.09, 26.67, 14.30, 8.43.

Preparation of diaryl-diethyl acid (21)

The ester 20 (200 mg, 0.5 mmol) was dissolved in CH₃CN (15 ml) with a few drops of H₂O. TEA (0.2 ml, 1.5 mmol) was then added followed by LiBr (430 mg, 5 mmol) and the reaction vigorously stirred at reflux for 6 h. The reaction was cooled to rt and extracted with diethyl ether (3 × 50 ml). The aqueous layer was then acidified to pH ~ 2 with 1 M HCl, extracted with ethyl acetate (3 × 50 ml). Combined organic extracts were washed with brine (2 × 30 ml) and H₂O (1 × 25 ml), dried over Na₂SO₄ and concentrated to give the product as a white solid (56 mg, 39 % conversion). ¹H NMR (CDCl₃, 400 MHz): δ 7.59-7.41 (m, 6H) 7.29-7.19 (m, 2H); 2.14-2.07 (m, 2H); 1.99-1.92 (m, 2H); 1.01-0.94 (t, 6H). ¹³C NMR (CDCl₃, 100 MHz): 178.25, 160.67 (d, J = 245.6 Hz), 139.39 (d, J = 7.4 Hz), 134.62, 130.65 (d, J = 3.9 Hz), 129.52, 128.55 (d, J = 2.8 Hz); 127.99, 127.54, 121.92 (d, J = 3.3 Hz); 121.42, 117.20, 113.82 (d, J = 24.8 Hz); 49.19, 33.38, 9.35. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₈H₁₉FO₂, 285.1296; observed, 285.1273.

Preparation of bis-TBS ketene acetals (22a & 22b)

The appropriate biaryl acid (1 eq) and TBDSCl (2.3 eq) were dissolved in THF at rt. LiHMDS (2.2 eq) was introduced dropwise at 0 °C for 15 min. The resulting reaction mixture was allowed to warm to rt and stirred overnight before being concentrated under vacuum. The residual oil was redissolved in hexanes, filtered and concentrated. After confirmation of the olefinic proton and TBDS protection by ¹H NMR, the product was used in the next step without further purification.

General procedure for fluorination of the alpha carbon using Selectfluor.⁴⁷

The bis-TBS ketene acetal (1.0 eq) in MECN (10 ml) was added dropwise to Selectfluor (1.5 eq) in MECN for 15 min while maintain reaction < 50 °C. The reaction was allowed to warm to rt and stirred for an additional 15 min. The reaction was then diluted with 1 M HCl (25 ml) and extracted with ethyl acetate (3 × 50 ml). Combined organic layer was extracted with 1 M NaOH (3 × 50 ml). Aqueous layer was washed with ethyl acetate (3 × 50 ml), followed by acidification of the aqueous layer to pH ~1 with 3 M HCl. The acidic aqueous layer was extracted with ethyl acetate (3 × 75 ml), combined organic extracts were dried over Na₂SO₄ and concentrated.

2-Fluoro-2-(2-fluorobiphenyl-4-yl)propanoic acid (23a)

Synthesized using fluorination general procedure using the following values: 22a (232 mg, 0.491 mmol); Selectfluor (226 mg, 0.638 mmol); yielding 23a as a white solid (42 mg, yield 32.6 %). ¹H NMR (CDCl₃, 400 MHz): δ 10.15 (bs, 1H); 7.44-7.27 (m, 6H); 7.14-7.05 (m, 2H), 1.52 (d, 3H, J = 7.18 Hz). ¹³C NMR (CDCl₃, 100 MHz): 171.90, 161.52 (d, J = 242.2 Hz), 141.98 (d, J = 7.4 Hz), 134.80, 132.21, 129.84, 129.60, 128.97, 128.94, 128.46, 127.98, 123.60, 115.42 (d, J = 24.8 Hz), 81.81, 19.02. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₅H₁₂F₂O₂, 261.0733; observed, 261.0706.

2-Fluoro-2-(2-fluorobiphenyl-4-yl)acetic acid (23b)

Synthesized using fluorination general procedure using the following values: 22b (58 mg, 0.13 mmol); Selectfluor (40 mg, 0.10 mmol); yielding 23b as a white solid (12 mg, yield 38.2 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.52 (m, 2H), 7.43-7.32 (m, 4H), 7.14-7.09 (m, 2H), 5.40-4.28 (d, 1H). ¹³C NMR (CDCl₃, 100 MHz): 174.9, 161.0 (d, J = 242.2 Hz), 141.9 (d, J = 7.4 Hz), 135.50, 132.05, 130.9 (d, J = 3.2 Hz), 129.52, 129.03 (d, J = 13.7 Hz), 128.51, 128.0, 127.83, 123.62, 115.40 (d, J = 24.8 Hz), 72.60. ESI-HRMS (m/z): [M-H]⁺ calcd. for C₁₄H₁₀F₂O₂, 247.0576; observed, 247.0601.

4-Bromobutyl R-(−)-2-fluoro-a-methyl-4-diphenylacetate (24a)

Flurbiprofen (1.0 g, 4.09 mmol) was dissolved in methanol (10 mL) and then 0.5 M sodium methoxide in methanol (8.18 mL, 4.09 mmol) was added. The reaction mixture was stirred at rt for 10 min, and then the solvent was evaporated under reduced pressure, yielding the sodium salt (1.08 g, 4.09 mmol) which was suspended in dimethylformide (10 mL) and stirred for 10 min, followed by dropwise addition of 1,4-dibromobutane (1.5 mL, 2.74 g, 12.6 mmol). The reaction mixture was stirred at rt for an additional 48 h. The reaction mixture was poured into H₂O (100 mL) and extracted with ethyl acetate (3 × 50 ml). The organic layers were washed with H₂O (3 × 30 ml), brine (3 × 50 ml), dried over Na₂SO₄, and evaporated under reduced pressure. The resulting residue was purified by column chromatography (hexane/CH₂Cl₂ [1/1]) to give the product as yellow oil (1.35 g, 87.5 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.53-7.50 (m, 2H), 7.45-7.34 (m, 4H), 7.14-7.07 (m, 2H), 4.11-4.09 (t, 2H, J = 6.15 Hz), 3.73-3.71 (q, 1H, J = 7.15 Hz), 3.34-3.32 (t, 2H, J = 6.28 Hz), 1.86-1.73 (m, 4H), 1.52-1.50 (d, 3H, J = 7.17 Hz).

N-(4-Bromobutyl)-2-(2-fluorobiphenyl-4-yl)propanamide (24b)

A solution of (R)-2-(2-fluorobiphenyl-4-yl)propanoic acid (0.60 g, 2.4 mmol) in SOCl₂ (4.4 g, 36.9 mmol) was heated at reflux. After 2 h the reaction mixture was allowed to cool to rt and the excess of SOCl₂ removed under vacuum. Both a solution of the residual material in CH₂Cl₂ (2 mL) and a solution of NaOH (98.3 mg, 2.40 mmol) in H₂O (0.32 mL) were simultaneously added to a cooled (−20 °C) solution of 4-amino-1-butanol (438.2 mg, 4.92 mmol) in CH₂Cl₂ (2 mL) under vigorously stirring. The reaction was kept at −20 °C for 2 h and allowed to warm to rt overnight. The organic layer was washed with H₂O, NaHCO3, brine, and concentrated to afford the intermediate alcohol (50 % yield) as an oil which was used in the subsequent procedure without further purification. The intermediate alcohol (390 mg, 1.24 mmol) and PPh₃ (356.3 mg, 1.36 mmol) in CH₂Cl₂ (8 mL) were cooled to 5 °C and a solution of tetrabromomethane (493 mg, 1.48 mmol) in CH₂Cl₂ (4 ml) was added dropwise over 10 min. The resulting clear solution was stirred at rt for 4 h, before removal of solvent under vacuum. The concentrate was purified by flash chromatography (hexane/ethyl acetate [20:80]) to yield pure 24b (64 % yield) as an oil. ¹H NMR (CDCl₃, 400 MHz): 7.56-7.53 (m, 2H); 7.47-7.35 (m, 4H); 7.16-7.11 (m, 2H); 3.57-3.54 (q, 1H); 3.39-3.37 (t, 2H); 3.31-3.24 (m, 2H); 1.87-1.78 (m, 2H); 1.67-1.60 (m, 2H); 1.55-1.53 (d, 3H, J = 6.9 Hz).

General procedure for alkylation of biaryl cyclopropyl acids (11a, 11c, 11d)

To a solution of the appropriate carboxylic acid (1 eq) in CH₂Cl₂ was added oxalyl chloride (5 eq, 2.0 M in CH₂Cl₂) and the mixture stirred at rt for 5 h. The mixture was concentrated, redissolved in CH₂Cl₂ containing TEA (2.5 eq), followed by the dropwise addition of 2-bromoethanol (1.0–1.5 eq). After stirring overnight the solution was concentrated and purified by column chromatography.

2-Bromoethyl-1-(2-fluorobiphenyl-4-yl)cyclopropanecarboxylate (25a)

Synthesized using general procedure with the following values: 11a (130 mg, 0.50 mmol); oxalyl chloride (1.25 ml, 2.5 mmol); 2-bromoethanol (0.1 ml, 0.8 mmol); TEA (0.5 ml); afforded 25a as colorless oil (160 mg, yield 86.8 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.59-7.39 (m, 6H); 7.25-7.21 (qs, 2H); 4.41-4.38 (t, 2H); 3.50-3.46 (t, 2H); 1.75-1.71 (q, 2H); 1.33-1.29 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 173.68, 159.8 (d, J = 246.6 Hz), 140.57 (d, J = 8.0 Hz), 135.70, 130.56, 129.15, 129.13, 128.61, 128.21, 128.08, 127.84, 126.56, 126.53, 118.32 (d, J = 23.1 Hz), 64.39, 28.87, 17.19. ESI-HRMS (m/z): [M+H]⁺ calcd. for C₁₈H₁₆BrFO₂, 363.0390; observed, 363.0396.

2-Bromoethyl-1-(2-fluoro-4'-(trifluoromethyl)biphenyl-4-yl)cyclopropanecarboxylate (25b)

Synthesized using general procedure with the following values: 11c (270 mg, 0.83 mmol); oxalyl chloride (2.1 ml, 4.15 mmol); 2-bromoethanol (0.3 ml, 2.4 mmol); TEA (1.0 ml); afforded 25b as colorless oil (280 mg, yield 78 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.73-7.67 (q, 4H); 7.43-7.39 (t, 1H); 7.28-7.21 (qs, 2H); 4.42-4.39 (t, 2H); 3.50-3.47 (t, 2H); 1.80-1.73 (q, 2H); 1.33-1.26 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 173.31, 160.44 (d, 246.6 Hz), 141.55, 139.11, 130.25, 130.21, 129.86, 129.27, 129.24, 126.62, 126.59, 125.34, 125.30, 122.80, 118.46, 64.26, 28.60, 16.99. ESI-HRMS (m/z): [M+H]⁺ calcd. for C₁₉H₁₅BrF₄O₂, 431.0264; observed, 431.0264.

2-Bromoethyl-1-(3',4'-dichloro-2-fluorobiphenyl-4-yl)cyclopropanecarboxylate (25c)

Synthesized using general procedure with the following values: 11d (260 mg, 0.80 mmol); oxalyl chloride (2.0 ml, 4.0 mmol); 2-bromoethanol (0.1 ml, 0.8 mmol); TEA (0.5 ml); afforded 25c as a white solid (300 mg, yield 87.8 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.69 (s, 1H); 7.65-7.59 (d, 1H); 7.54-7.18 (m, 4H); 4.40-4.37 (t, 2H); 4.14-4.12 (t, 2H); 1.73-1.70 (q, 2H); 1.29-1.24 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 172.92, 159.92 (d, J = 242.0 Hz), 141.14 (d, J = 8.0 Hz), 135.04, 132.17, 131.50, 130.35, 129.72, 129.61, 129.57, 127.84, 126.25, 125.12, 118.12 (d, J = 23.0 Hz), 63.89, 28.32, 16.66. ESI-HRMS (m/z): [M]⁺ calcd. for C₁₈H₁₄BrCl₂FO₂, 430.9538; observed, 430.9611.

General procedure for nitration of the ethyl bromide esters (25a–c)

To a solution of the appropriate bromide (1 eq) in anhydrous MECN was added AgNO₃ (2–5 eq) and the reaction stirred at reflux for 2 h. The mixture was filtered through celite, the filtrate concentrated, and the resulting crude oil purified by column chromatography (hexane/ethyl acetate [3:1]) to give the desired nitrates characterized below.

2-(Nitrooxy)ethyl 1-(2-fluorobiphenyl-4-yl)cyclopropanecarboxylate (26a)

Synthesized using general nitration procedure with the following values: 25a (120 mg, 0.33 mmol); AgNO₃ (112 mg, 0.66 mmol); afforded 26a as yellow oil (85 mg, yield 74.5 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.58-7.38 (m, 6H); 7.27-7.14 (m, 2H); 4.64-4.63 (t, 2H); 4.36-4.35 (t, 2H); 1.70-1.68 (m, 2H); 1.32-1.30 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz): 173.35, 159.90 (d, J = 242.0 Hz), 141.12(d, J = 8.0 Hz), 139.83, 135.11, 130.07, 129.53, 128.59, 128.06, 127.31, 125.93, 117.79, 117.56, 69.67, 60.75, 28.13, 16.76. ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₈H₁₆FNO₅, 368.0905; observed, 368.0911.

2-(Nitrooxy)ethyl 1-(2-fluoro-4'-(trifluoromethyl)biphenyl-4-yl)cyclopropanecarboxylate (26b)

Synthesized using general nitration procedure with the following values: 25b (220 mg, 0.49 mmol); AgNO₃ (415 mg, 2.44 mmol); afforded 26b as colorless oil (190 mg, yield 90.1 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.73-7.67 (q, 4H); 7.43-7.39 (t, 1H); 7.25-7.18 (qs, 2H); 4.67-4.64 (t, 2H); 4.38-4.36 (t, 2H); 1.73-1.70 (q, 2H); 1.33-1.31 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 173.54, 160.46 (d, J = 240.2 Hz), 141.33(d, J = 8.0 Hz), 139.10, 130.36 (d, J = 3.6 Hz); 129.85, 129.29 (d, J = 3.1 Hz); 128.23, 126.58 (d, J = 3.3 Hz); 125.38 (q, J = 11.2 Hz); 124.10, 122.83, 118.35, 118.12, 70.00, 61.20, 28.54, 17.09. ESI-HRMS (m/z): [M-NO₃]⁺ calcd. for C₁₈H₁₆FNO₅, : 351.1008; observed, 351.1029.

2-(Nitrooxy)ethyl 1-(3',4'-dichloro-2-fluorobiphenyl-4-yl)cyclopropanecarboxylate (26c)

Synthesized using general nitration procedure with the following values: 25c (300 mg, 0.69 mmol); AgNO₃ (589 mg, 3.47 mmol); afforded 26c as colorless oil (210 mg, yield 73.0 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.65-7.16 (m, 6H); 4.66-4.64 (t, 2H); 4.38-4.35 (t, 2H); 1.75-1.68 (q, 2H); 1.35-1.28 (q, 2H). ¹³C NMR (CDCl₃, 100 MHz): 172.92, 159.92 (d, J = 242.0 Hz), 141.14 (d, J = 8.0 Hz), 135.04, 132.17, 131.50, 130.35 (d, J = 1.8 Hz), 130.01, 129.72, 129.61, 127.88 (d, J = 4.0 Hz), 126.28 (d, J = 3.8 Hz), 125.26, 118.12 (d, J = 23.0 Hz), 69.59, 60.84, 28.16, 16.76. ESI-HRMS (m/z): [M-NO₃]⁺ calcd. for C₁₈H₁₄Cl₂FNO₅, 351.0349; observed 351.0536.

4-(Nitrooxy)butyl 2-(2-fluorobiphenyl-4-yl)propanoate (2)

The bromide 24a (1.34 g, 3.58 mmol) was dissolved in anhydrous MeCN (20 mL) and then AgNO₃ (1.84 g, 10.74 mmol) was added. The suspension was stirred at rt for 1 h and then at reflux 14 h. The solid was filtered through celite, and the solvent was removed under vacuum. Flash column chromatography of the residue (hexane/ethyl acetate [7:3]) afforded the desired product as yellow oil (1.11 g, 85.9 %). ¹H NMR (CDCl₃, 400 MHz): δ 7.53-7.50 (m, 2H), 7.43-7.32 (m, 4H), 7.14-7.09 (m, 2H), 4.37 (t, 2H, J = 6.28 Hz), 4.11 (t, 2H, J = 6.15 Hz), 3.73 (q, 1H, J = 7.15 Hz), 1.86-1.73 (m, 4H), 1.52 (d, 3H, J = 7.17 Hz). ¹³C NMR (CDCl₃, 100 MHz): 173.9, 161.0, 158.5, 141.9, 135.5, 132.18, 130.9, 129.0, 128.5, 127.83, 123.6, 115.4, 115.1, 72.6, 64.1, 45.1, 25.0, 23.6, 18.3. ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₉H₂₀FNO₅, 384.1218; observed, 384.1227.

4-[[2-(2-Fluorobiphenyl-4-yl)propanoyl]amino]butyl nitrate (27)

The bromide 24b (0.24 mmol) was dissolved in CH₃CN and AgNO₃ (82.39 mg, 0.48 mmol) is added. The suspension is stirred at rt for 14 h then at reflux for 2 h. The solid was filtered through celite pad and the solvent removed under vacuum. The resulting residue was purified by column chromatography (hexane/ethyl acetate [60:40]) to afford the product as yellow oil (63 % yield). ¹H NMR (CDCl₃, 400 MHz): δ 7.55-7.52 (m, 2H), 7.47-7.34 (m, 4H); 7.15-7.09 (m, 2H); 4.43 (t, 2H); 3.56 (q, 1H); 3.30-3.24 (m, 2H); 1.77-1.53 (m, 7H). ¹³C NMR (CDCl₃, 100 MHz): 173.85, 161.00, 158.53, 124.85, 135.33, 131.02, 128.90 128.50, 127.90, 127.76, 123.55, 115.32, 115.09, 73.98, 47.58, 40.37, 26.05, 24.13, 18.49. ESI-HRMS (m/z): [M+H]⁺ calcd. for C₁₉H₂₁FN₂O₄, 361.1558; observed, 361.1568.

Abbreviations

SALA

selective amyloid lowering agent

AD

Alzheimer’s disease;

APP

amyloid precursor protein

Aβ

beta-amyloid peptide

NSAID

non-steroidal anti-inflammatory drug

LPS

lipopolysaccharide

iNOS

inducible NO synthase

ISMN

isosorbide mononitrate

SARA

selective amyloid raising agent

CM

conditioned media

DMEM

dulbecco's modified eagle's medium

PBS

phosphate buffered saline

FBS

fetal bovine serum

EDTA

ethylenediaminetetraacetate

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NFSI

N-fluorobenzenesulfonimide

TBSCl

tert-butyldimethylsilyl chloride

LiHMDS

lithium bis(trimethylsilyl)amide