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Application of the Sandmeyer Reaction Towards Synthesis of Selective
Toll-like Receptor 7 & 8 Antagonists
Rachel J. Anderson
Undergraduate Thesis
March 27th, 2018
University of Colorado Boulder
Thesis Advisor:
Hang Hubert Yin, Ph.D.: Department of Chemistry & Biochemistry
Committee Members:
Xuedong Liu, Ph.D.: Department of Chemistry & Biochemistry
Jeffrey Cameron, Ph.D.: Department of Chemistry & Biochemistry
Karolin Luger, Ph.D.: Department of Chemistry & Biochemistry
Pamela Harvey, Ph.D.: Molecular, Cellular & Developmental Biology
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ACKNOWLEDGEMENTS
I would like to begin by thanking Professor Hubert Yin and my lab mates in the Yin lab for putting up with my antics over the past two years and providing a great place to learn. This project would not have been possible without the extensive support and guidance of my direct mentor, Dr. Rosaura Padilla-Salinas, who has invested untold hours into my success. I would like to specifically thank Josh Kamps, Shafer Soars, and Dr. Adam Csakai for feedback, advice, and moral support. I would also like to thank my thesis committee: Xuedong Liu, Jeffrey Cameron,
Karolin Luger, and Pamela Harvey for their patience and assistance. Finally, I am grateful for financial support provided by the Undergraduate Research Opportunities Program (UROP) at the University of Colorado, Boulder which made eating possible. Anderson 3
TABLE OF CONTENTS
1. Abstract ...... 4 2. Introduction ...... 4 2-1. Overview of the immune system ...... 4 2-2. Toll-like receptors ...... 5 Historical context ...... 5 Function ...... 6 Structure and signaling ...... 7 Role in chronic inflammation & autoimmunity ...... 8 Summary and project justification ...... 10 3. Structure-activity relationship studies ...... 11 3-1. Introduction ...... 11 Previous work ...... 11 The Sandmeyer reaction ...... 12 Amide synthesis reactions ...... 15 Summary of biological assays ...... 17 3-2. Modifications of ring A ...... 17 3-3. Modifications of ring B ...... 23 3-4. Modifications of ring C ...... 25 3-5. Modifications of amide linker ...... 26 4. Discussion and future directions ...... 30 5. References ...... 31 6. Supporting Information ...... 35 6-1. Biological methods ...... 35 6-2. Chemical methods ...... 36 6-3. Reaction Protocols ...... 37 6-4. NMR Spectra ...... 69
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1. ABSTRACT
Toll-like receptors (TLRs) are an important part of the innate immune system responsible for detecting signs of microbial invasion and cell damage and initiating the immune response. Overactivation of TLRs, presumably by inappropriate detection of endogenous ligands, has been linked to chronic inflammation and autoimmunity. Current therapeutics are broad-spectrum and inhibit the overall function of the immune system. In this work, a library of dual-TLR7/8 antagonists were prepared with the goal of engineering TLR7 specificity, then tested against HEK 293 cells expressing either TLR 7 or 8. An antagonist with IC50 0.22 ± 0.33 μM against TLR 8 and greater than 50 μM against TLR 7 is presented, which may find use as a therapeutic or chemical probe. While this demonstrates that engineering specificity is possible, further work is required to locate a TLR7-selective antagonist.
2. INTRODUCTION
2-1. Overview of the immune system
The innate immune system is our first line of defense against microbial threats. Our protection begins with physical barriers such as skin and mucosal membranes. Once a threat breaches these barriers, it will rapidly proliferate unless dealt with. The innate immune system is non-specific and acts within hours to inhibit unchecked microbial growth, but often cannot fully clear an infection. Instead, it primes the adaptive immune system for a much stronger pathogen-specific response, which will both clear the infection and “remember” the pathogen in order to more rapidly eliminate the pathogen if it is re-encountered1. Anderson 5
2-2. Toll-like receptors
TLRs are pattern recognition receptors which bind conserved motifs of pathogen- associated molecular patterns (PAMPs) commonly expressed by invading microbes, or damage- associated molecular patterns (DAMPs) indicative of damaged and dying cells. The 10 human
TLRs are primarily expressed in antigen-presenting cells, such as macrophages, dendritic cells,
B-cells, and some epithelial cells2. TLRs are localized to either the plasma membrane (TLRs
1/2/4/5/10) or to the endosome (TLRs 3/7/8/9)3, and are thus positioned to sample the exterior environment of the cell.
Historical context
Despite the important role of TLRs in immunity, the toll gene was first identified in
Drosophila in 1980 for its role in embryonic development and it was not discovered until 1995 that toll played a second role in innate immunity4. By 1998, five toll homologues were known in humans, forming the human Toll-like receptor (hTLR or TLR) family4,5. At the time, it was unclear how the innate immune system functioned, especially considering that it must somehow communicate with the adaptive immune system. In the two decades since, extensive research has shown that although TLRs do function as a first line of defense, many other signaling pathways coexist with TLRs, such as the cGAS/STING pathway6, NOD-like receptors7, and RIG-like receptors8. Uniquely, the ligands of many TLRs can be of exogenous or endogenous origin, suggesting a role for inappropriately activated TLRs in autoimmunity. Anderson 6
Function
Figure 1. Overview of Toll-like receptors and their associated signaling pathways9.
TLRs localized to the plasma membrane primarily detect conserved bacterial and fungal motifs (Figure 1). In addition to directly activating an immune response, these TLRs also have been shown to upregulate pinocytosis10, presumably to promote a stronger immune response.
TLRs 1, 2, and 6 recognize a variety of lipopeptides, glucans, and teichoic acids commonly found in microbial membranes and cell walls2 and may indiscriminately dimerize (for example, dimers Anderson 7 of TLR 1/2 and 2/6 are known). TLR4 recognizes lipopolysaccharide, a common component of
Gram-negative bacterial cell walls3.
Although endosomal TLRs most commonly detect viral motifs, they may also detect endogenous ligands and thus they are thought to play a role in autoimmunity. TLR3 binds long stretches (40 or more bases) of double-stranded RNA11, which often signals invasion by enveloped viruses which pass through the endosome during their reproductive cycle. TLR9 detects unmethylated CpG double-stranded DNA3. As eukaryotic CpG DNA is commonly methylated, this indicates that the DNA may be of non-eukaryotic origin (i.e. bacterial or mitochondrial). TLRs 7 and 8 recognize single-stranded RNA and RNA degradation products
(monomers and small oligonucleotides such as UUG)12,13. RNA may enter the endosome from the exterior of the cell (through endocytosis) or from the interior of the cell through incorporation into autophagosomes14.
The role of TLR10 is poorly understood. Homology studies predicted TLR10 would bind lipopolysaccharides like TLR215, however, it has also been implicated as a down regulator of TLR activity16.
Structure and signaling
The 120k Da TLR monomers are roughly question mark shaped (Fig. 1), with an extracellular or endosomal domain containing numerous (14-26) leucine-rich repeats (LRRs, typically with sequence
LXXLXLXXN) used in ligand binding, a single-pass Figure 2. Structure of extracellular TLR4 (top) and TIR domain (bottom). PDB Molecule of the Month transmembrane domain, and an intracellular http://dx.doi.org/10.2210/rcsb_pdb/mom_2011_11 Anderson 8
Toll/Interleukin-1 Receptor (TIR) domain used in signal transduction17. TLRs must dimerize prior to signal transduction, however, it appears some TLRs exist as preformed dimers while other
TLRs have been shown to dimerize following ligand binding9. Furthermore, some TLRs form heterodimers, while others form homodimers.
In TLRs 7 and 8, following ligand binding, significant reorganization of the homodimer occurs, most notably with the C-termini decreasing in separation by 20 Å18. Presumably, this is somehow translated into a conformational change of the TIR domain, after which adaptor proteins possessing their own TIR domains such as MyD88 or TRIF are recruited to the TLR-TIR domain to assist in signaling17. Most TLRs, including 7 and 8, signal through MyD88 exclusively
(previously shown in Figure 1). Following a kinase cascade which is supplemented by ubiquitination, the nuclear factor NF-κB is released to enter the nucleus, resulting in the production of interleukins, pro-inflammatory cytokines, chemotaxis-stimulating chemokines, and the initiation of immune response9. For larger threats, i.e. those that the innate immune system cannot clear within 24-48 hours, TLR-rich dendritic cells will present portions of the
PAMPs or DAMPs to T-cells to stimulate the adaptive immune system19.
Role in chronic inflammation & autoimmunity
TLRs play a critical role in survival of microbial threats, however, their activity must closely correspond to the level of threat present. If TLRs are absent or defective, it seems obvious that the immune response would be weakened, which might lead to failure to respond to damage or infections and thus death. However, if TLRs are overexpressed or overactivated, the immune response would be excessive and chronic inflammation or autoimmunity might result. Anderson 9
Several TLRs are known to mistakenly recognize endogenous ligands, such as the causative role of TLRs 2 and 4 in septic and rheumatoid arthritis3, however the role of nucleic acid (NA)-sensing TLRs (7, 8, and 9) in autoimmunity is still being studied. It is thought that inappropriate detection of self-nucleic acids may lead to chronic inflammation and autoimmunity. In mouse models, both TLR7-/- and TLR7 knockdown mice are resistant to arthritis progression20. Moreover, TLR8-/- mice rapidly develop symptoms of autoimmunity20— even though mice do not signal through TLR8—suggesting that some method of crosstalk between TLRs 7 and 8 must exist. Transgenic mice with hTLR8 develop autoimmune issues directly correlated to level of expression21. Furthermore, a metagenomics study found single nucleotide polymorphisms in TLR 7 and 8 were associated with individuals suffering from celiac disease22. Finally, various works have linked the endogenous DNA-sensing TLR 9 with systemic lupus erythematosus14.
It is suspected that TLR 7 and 8 could similarly detect endogenous RNA and lead to autoimmunity, but demonstrating this has been hampered by lack of specific antagonists.
Similarly, as NA-sensing TLRs are clinically relevant targets that are implicated in autoimmunity, there have been previous efforts to modulate their activity therapeutically. Chloroquine and hydroxychloroquine have been used to treat various autoimmune diseases, however, these are toxic to the eye and cannot be used for long periods of time, making them inappropriate for treatment of chronic diseases23. Other works have focused inhibition of downstream targets from NA-sensing TLRs, such as IRAK4 or NF-κB24. Although these treatments may limit autoimmune response, they do so by inhibiting downstream targets common to all TLRs, thus Anderson 10 weakening the entire innate immune response and making patients more susceptible to any sort of pathogen threat.
Summary and project justification
Toll-like receptors 7 and 8 play a critical role in the defenses of the immune system, however, through various methods they may become overactivated. This is suspected to be causative of autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus. Although TLR 8-specific antagonists have recently been developed, there are no commercially available TLR 7-specific antagonists to be used as a chemical probe, nor are there therapeutic options to treat autoimmune diseases resulting from inappropriate TLR activation without affecting the overall innate immune response.
Because of the clinical relevance of TLRs 7 and 8 and the lack of selective antagonists to be used as therapeutics or chemical probes, a need for selective TLR-7 and -8 antagonists exist.
Accordingly, this project began with the hypothesis that a dual-TLR7/8 antagonist could be developed into a selective TLR 7 antagonist. This would be useful either as potential therapeutic for autoimmune diseases, or used as a chemical probe to further our understanding of Toll-like receptors.
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3. STRUCTURE-ACTIVITY RELATIONSHIP STUDIES
3-1. Introduction Previous work This project was spawned from prior work conducted in the Yin lab. During her graduate work, Dr.
Shuting Zhang screened a library of 14,400 compounds Figure 3. Structure of 4a, a dual TLR 7/8 antagonist. TLR 7 IC50 = 2.88 μM; TLR 8 IC50 = 1.64 μM. for small-molecule inhibitors of TLR 825 and located the lead hit 4a (Error! Reference source not found.), which was selected as it was the only compound inhibiting both TLR 7 and 8. Dr. Rosaura Padilla-Salinas, verified TLR selectivity and performed initial structure-activity relationship (SAR) studies on this scaffold. These first- generation SAR results suggested the amide linkage and A ring were both critical to TLR antagonism, as methylation of the amide resulted in a loss of TLR7 activity and removal of the halogens decreased TLR 7 and 8 activity. With the goal of engineering TLR 7 selectivity to develop a potential therapeutic and chemical probe, I have used a combination of synthetic organic chemistry and medicinal chemistry to expand upon the initial SAR by choosing targets that would provide useful information, designing synthetic routes to obtain them, characterizing them, and assaying their activity in vitro. Anderson 12
F R CF3 CF 3 H A N C B R O Cl F3C O R = EWG, EDG 4a bulky, H-bonding R = EWG, EDG, halogens, H, H-bonding H Het N N X Heterocycles O with H-bonding (pyridine), O O bulky (indazole), smaller (thiophene) Alternate linkers, X = CH2, C=S fused rings
Figure 4. Representative structure-activity relationship targets.
My SAR of 4a can be divided into four phases: one each for derivatives of the A, B, and C rings, and one where I explored alternative linkers between the A and B rings (Figure 4). After a brief introduction to two reactions I used extensively and the biological assays used to determine activity and selectivity, I will discuss the SAR in depth. Detailed reaction protocols and experimental information can be found in Supporting Information.
The Sandmeyer reaction
The Sandmeyer reaction was discovered on accident by Traugott Sandmeyer in 1884 as he attempted to react an aryldiazonium chloride and cuprous acetylide26. The classical
Sandmeyer reaction is a two-step procedure (Scheme 1). First, the aryldiazonium species is generated through addition of a nitrite species (commonly sodium nitrite or tert-butyl nitrite) and either a strong acid (often H2SO4 or HCl) or Lewis acid such as BF3OEt2 to some aniline derivative27. Next, the aryldiazonium species is reacted with some copper salt Cu+1/+2X, resulting in the loss of nitrogen gas and Ar-X27,28. Other proposed mechanisms exist, especially for reactions at high temperature (above 150 °C) which are thought to occur via aryl cations. Anderson 13
Scheme 1. Mechanism of the Sandmeyer reaction. a) General equation for Sandmeyer reaction. b) Formation of the aryl diazonium species. c) Radical dediazotization catalyzed by Cu+1.
A reoccurring theme in my synthetic efforts has been the Sandmeyer reaction, which I believe is an interesting and powerful method of introducing diversity. The Sandmeyer reaction has two main strengths: the immense variety of products that can be synthesized starting from a single aryl amine (Scheme 2), and the fact that ipso substitution of the amino group occurs without requiring directing groups elsewhere on the molecule. Ipso substitution allows access to products that would typically be challenging to otherwise synthesize. Anderson 14
3
4
+ Scheme 2. Selected reactions of aryldiazonium species. All direct reactions from Ar-N2 are Sandmeyer reactions (or Sandmeyer- like), while secondary reactions show how these Sandmeyer reactions may be used to introduce diversity. This list is non- exhaustive and only includes well-established chemistry27,29.
Although versatile, the Sandmeyer has many downsides which limit its use by modern synthetic organic chemists. Anecdotal experience says that even a well-executed Sandmeyer reaction will lose at least 20% of theoretical yield to “tars” of various products, including homocoupled, protodediazotized, and highly reactive aryne species. As I discovered during my
SAR, some substrates, most notably those with anthranilic acid (ortho-amino benzoic acid) moieties, may be extremely difficult to react under Sandmeyer-like conditions. The Sandmeyer Anderson 15 reaction has proved remarkably resistant to improvement over the original methods, although many people have tried (and achieved modest success) by using different metals such as Cu+2,
Fe+3, Co+3, or Zn+2 to increase yields and reduce the formation of side products27.
Amide synthesis reactions
My scaffold included an amide bond which was typically formed as the last step of the synthetic route for a compound, which means that amide synthesis was the most common reaction that I performed. I used three methods during my SAR: reaction of an amine and acyl chloride, HATU coupling, and phosphite coupling. Acyl coupling was the fastest and easiest route, however, not all my coupling partners were available as acid chlorides. HATU coupling
(so named for the reagent, hexafluorophosphate azabenzotriazole tetramethyl uronium) is a widely used method for amide bond formation from carboxylic acids (Scheme 3). Phosphite coupling was reserved for coupling partners with unprotected hydroxyl groups, as these can polymerize during HATU coupling. Anderson 16
Scheme 3. Mechanism of amide synthesis by HATU coupling. Typically, the carboxylic acid is stirred with Hünig's base (N,N- diisopropylethylamine) in a polar aprotic solvent for some time, after which the amine is added. This minimizes formation of guanidine byproducts.
HATU coupling is regarded as a near-ideal synthetic method due to its ease of use and excellent yields. However, tetramethylurea (TMU) is formed as a byproduct, and is coincidentally extremely difficult to separate from my final targets. TMU has very similar polarity to my final targets (such that it elutes at the same time on a column), will preferentially partition into any organic solvents except hexane, and is not UV-active. This combination of properties meant that removing TMU from my products was challenging and often required recrystallization, resulting in poor overall yields due to the small scale (0.5 – 1 mmol) of my reactions. Anderson 17
Summary of biological assays
To quantify TLR activity, a secreted embryonic alkaline phosphatase (SEAP) assay is used. Following activation, TLR 7 and 8 both signal through the same NF-κB-dependent pathway. The SEAP gene is linked to five NF-κB sites and is thus produced during incubation if the TLR is signaling. To assay TLR activity, a proprietary dye (Quanti-Blue, Invivogen) is added to the media. If present, SEAP will cleave the dye, causing a color change proportional to NF-κB activity which is observable at 620 nm. Compounds were tested separately against transgenic
TLRs 7 and 8 cell lines.
The WST-1 cell proliferation assay was used to determine cytotoxicity of tested compounds. WST-1 reagent contains a tetrazolium ring which is cleaved to yield a dye by cell- surface enzymes in a NAD(P)H dependent manner. This should only occur for metabolically active cells.
3-2. Modifications of ring A
CF3 CF3 NO2 a NO2
96-98% F3C OH F F3C O
1 b
77% R CF CF 3 H 3 N c, d, or e NH2 O F3C O 19-99% F3C O
4a-t 2
Scheme 4. Synthetic route for 4a and derivatives. (a) K2CO3, DMF, 90 °C; (b) H2, Pd/C, MeOH; (c) appropriate acyl chloride, Et3N or pyridine, CH2Cl2; (d) appropriate benzoic acid, HATU, iPr2NH, DMF; (e) appropriate benzoic acid, PCl3, chlorobenzene, reflux. Anderson 18
As my overall goal with this SAR was to engineer a selective antagonist, I hoped to see that modifying some functional group unequally affected TLR 7 and 8 antagonism. Ring A seemed an appealing place to introduce diversity, as I could access a variety of analogues late in the synthetic route by amide bond formation (Scheme 4). The chemistry was robust, allowing me to build up ample materials to couple: a SnAr reaction with 4-fluoronitrobenzene and 3,5- bis(trifluoromethyl)phenol afforded the nitro diaryl ether 1, which could readily be reduced to amine 2 and coupled with acid chlorides or carboxylic acids to afford amide derivatives 4a-t in high yields.
Scheme 5. Synthesis of carboxylic acid substrates. (a) nBuLi, THF, -78 °C, then CO2; (b) Oxone, NaOH, NaHCO3, EDTA, H2O, acetone; (c) BnBr, K2CO3, DMF, 70 °C, then LiOH, THF, H2O; (d) H2, Pd/C, MeOH; (e) Tf2O, CH2Cl2; (f) Ac2O, CH2Cl2.
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Many of the carboxylic acid coupling partners used to prepare 4a-t were available commercially, however, several had to be prepared (Scheme 5). 3a and 3b were not available commercially and were prepared by directed lithiation in acceptable yield. Carboxylic acids 4g,
4k, 4n, and 4p were protected with various groups to avoid side-products (self-coupling) during
HATU coupling. Finally, 4l was prepared by hydrogenation of benzyl-protected 4k.
Table 1. Non-heterocyclic derivatives synthesized for ring A.
(a) (a) Compound R1 TLR 8 IC50 (μM) TLR 7 IC50 (μM)
4a 6-Cl 1.64 ± 0.04(b) 2.88 ± 0.249(b) 4b 6-H 3 > 50 4c 6-Br 1 6 4d 6-I 0.22 ± 0.33(b) > 50 4e 6-CN n.d. (c) n.d. (c) 4f 6-OH n.d. (e) n.d. (e)
4g 6-NO2 > 50 > 50
4h 6-OCF3 > 50 > 50 4i 4-Me n.d. (d) n.d. (d) 4j 4-F n.d. (e) n.d. (e) 4k 4-OBn > 50 > 50 4l 4-OH ≈ 40 n.d. (e) 4m 4-I > 50 > 50
4n 4-NH2 > 50 > 50 4o 5-Cl 3 n.d. (e) (a) Average of results from two-replicate biological screen unless otherwise noted. (b) Results from three- replicate dose response curve. (c) Compound is unstable and decomposed before biological assays. (d)
Compound is too insoluble to test. (e) Two-replicate screen in progress. Anderson 20
My initial efforts were to prepare the series of derivatives 4a-d where the chlorine was replaced with hydrogen or other halogens. This yielded an early result where TLR 8 activity seemed to modestly increase with increasing halogen size, while TLR 7 activity decreased. With
4d (2-F, 6-I) the IC50 against TLR 8 increased by more than 5-fold, while TLR 7 activity was significantly reduced. While this validated my initial hypothesis that the original dual TLR7/8 inhibitor could be engineered for specificity against a single TLR, I had still not achieved my goal of designing a selective TLR 7 inhibitor.
From this, I outlined a set of derivatives continuing the 2,6-substitution pattern as this had shown it was capable of inducing selectivity. My hypothesis was that one of the following things must explain the activity of the halogen series: steric bulk of increasingly larger halogens, suggesting the TLR 7 binding site is smaller than TLR 8; electrostatic effects from electronegativity and polarizability of the halogens; or possibly, a σ-hole effect. σ-holes are a side effect of large, polarizable atoms. In halogens, especially aryl halogens due to the electron- withdrawing aryl ring, the lobe of the halogen opposite the aryl ring becomes increasingly electron deficient on the series F > Cl > Br > I (Figure 5)30.
Figure 5. Electrostatic potential map of the series Ar-H through Ar-I. Here, blue regions are electron poor and red regions are electron rich30. Anderson 21
This seemed to suggest that the halogen could be acting as an “X-bond acceptor”, that is, as a weak Lewis acid. I designed hydroxyl 4f, which could participate in hydrogen bonding, but have not completed biological screening of it. Finally, to test the idea that it could be an electrostatic effect, I synthesized electron deficient compounds 4e,g, and h. Cyano derivative
4e decomposed before I could do biological assays, while 4g and 4h both showed poor activity.
I was very interested in synthesizing a 2-F,6-CF3 derivative, as trifluoromethyl groups are bioisosteres for the larger halogens, but would be incapable of σ-hole bonding. As this compound was not available commercially, I screened several copper (I) catalyzed conversion of amino (Sandmeyer) or iodo groups to trifluoromethyl groups, but was unable to find conditions that worked. I believe this is related to the ortho-carboxylate moiety, which interferes with the metal mediated reaction.
I then probed the effect of substitution at the 4 (4i-n) position. These all resulted in poor activity relative to 4b (2-F, 6-H), suggesting that the binding pockets are not tolerant of substitution at these positions. I have only made one modification at the 5 position (2-F,5-Cl;
4o) which appears to be tolerated from preliminary data. This suggests 2,5,6 substitution might be allowed, and is a promising further target.
Finally, I purchased several common heterocyclic carboxylic acids and coupled them with 2 to yield derivatives 4p-t (Table 2). Although these heterocycles did not have the typical
2-F, 6-Cl motif present in the original hit, they could be compared to the mono and non- halogenated compounds to see if they improved on activity (data from Dr. Padilla-Salinas, not shown). If they had shown increased potency relative to the original, derivatized versions could have been synthesized. Anderson 22
Table 2. Hetercyclic derivatives of lead hit.
(a) (a) Compound R1 TLR 8 IC50 (μM) TLR 7 IC50 (μM)
4p > 50 > 50
4q > 50 n.d.(e)
4r 7 > 50
4s 3 > 50
4t > 50 n.d. (e)
(a) Average of results from two-replicate biological screen unless otherwise noted. (b) Results from three- replicate dose response curve. (c) Compound is unstable and decomposed before biological assays. (d)
Compound is too insoluble to test. (e) Two-replicate screen in progress.
Only thiophene 4s was interesting, as it showed a large decrease in TLR 7 activity with only minor reduction in TLR 8 activity, and perhaps more importantly, it was less toxic than other compounds. I proposed a synthetic route to obtain a dihalothiophene as these are completely unavailable commercially (Scheme 6). Anderson 23
Scheme 6. Proposed synthetic route to access dihalothiophene carboxylic acid substrate.
This relied on a Balz-Schiemann reaction (a Sandmeyer-like reaction) to install the fluorine at the 2-position. Unfortunately, I was not able to get the first reaction to work; although there is literature precedence for Sandmeyer reactions of 2-aminothiophenes, they require the 5-position to be blocked to avoid rapid polymerization. Other routes for accessing this compound would be lengthy and require significant use of protecting groups.
3-3. Modifications of ring B
Previous work had shown methylation of the amide nitrogen resulted in a loss of TLR 7 activity, suggesting hydrogen bonding was critical. As such, it seemed important to test a pyridine derivative which could act as an additional hydrogen bond acceptor (Scheme 7).
Scheme 7. Synthesis of pyridine derivative 7. (a) CuI, Cs2CO3, N,N-dimethylglycine, dioxane, 90 °C; (b) H2, Pd/C, MeOH; (c) 2- fluoro-6-chlorobenzoyl chloride, Et3N or pyridine, CH2Cl2, -78 or 0 °C; (d) NaOH, H2O, THF. Anderson 24
After attempting SnAr coupling to make 5 and finding the yield unacceptable (15%), I instead synthesized diaryl ether 5 through Ullmann coupling. This still suffered from mediocre yields and a side product which could not be removed prior to reduction to amine 6. Following this, I attempted to couple 6 with an acyl chloride. Regardless of conditions attempted, these reactions yielded a mixture of imide 8 with a small amount of amide 7, which were difficult to separate. I believe this is explained by resonance structures and inductive electron-withdrawing groups which act to lower the pKa of the amide proton and make it more nucleophilic by stabilizing the conjugate base. After numerous attempts, I hydrolyzed imide 8 to amide 7 under basic conditions. Both compounds showed no TLR 7 antagonism.
Next, I was interested in preparing a fused-ring derivative of the lead hit by fusing the A and B rings together. Fused ring systems are commonly used to “pre-pay” entropic costs required for binding. After retrosynthetic analysis (not shown), I decided the ideal option was to prepare the benzoxazole from a halogen on ring B (Scheme 8), as this would allow access to further derivatize ring B through Sandmeyer-type reactions.
Scheme 8. Synthesis of benzoxazole derivative. (a) K2CO3, DMF, 90 °C; (b) HBr, KNO2, DMSO; (c) Fe powder, AcOH, EtOH; (d) 2- chloro-6-fluorobenzoyl chloride, Et3N, CH2Cl2; (e) CuI, 1,10-phenanthroline, Cs2CO3, DME, 90 °C. Anderson 25
SnAr coupling yielded nitro amine 9 in moderate yield, followed by bromination by a
Sandmeyer reaction to afford aryl bromide 10. Following reduction of the nitro group, 11 was coupled with an acyl chloride resulting in an acceptable yield of amide 12, which will be converted to a benzoxazole in a future copper (I)-catalyzed reaction. Furthermore, amide 12 was assayed and showed poor activity (> 50 μM IC50) against both TLR 7 and 8, suggesting that ring B is not tolerant of large substituents at this position.
3-4. Modifications of ring C
Ring C was an interesting target due to the symmetrical 3,5-trifluoromethyl substitution pattern. I hypothesized that this substitution pattern was not needed, and that asymmetry could be introduced to increase binding and/or solubility. I proposed synthesizing a variety of asymmetric derivatives, including 3-CF3,5-H derivative to test if the second trifluoromethyl group was required, a halogen series (F, Cl, Br, I) to test trifluoromethyl bioisostere activity, and phenol which would test for H-bonding as well as electron donating effects (Scheme 9).
OH OH CF3 a b NO2 c 99% / 2 steps F3C NO2 F3C NH2 H2N O 36 - 82% 13 14 F CF3 CF3 H d, e N NO2 O Cl 40-44% / 2 steps R O R O
16a-c 15a-c
Scheme 9. Synthesis of unsymmetrical ring C derivatives via Sandmeyer reaction. (a) H2, Pd/C, MeOH; (b) 4-fluoronitrobenzene,
K2CO3, DMF, 90 °C; (c) various conditions, see appendix; (d) Fe powder, AcOH, EtOH; (e) 2-chloro-6-fluorobenzoyl chloride, Et3N,
CH2Cl2. Anderson 26
In my most recent work, I have prepared three asymmetric derivatives where R = H, F, or I, which are still being screened for TLR activity. Reactions involving R = Cl or Br have resulted in mixtures of the desired product contaminated with proto-dediazotization side-product
(where R = H), which is challenging to purify. Attempts to install a phenol have been unproductive thus far. Preliminary screening results suggest that removing the CF3 group (R =
H) is tolerated in both TLR 7 and 8, but further data is needed.
3-5. Modifications of amide linker
Of the common bioisosteres of amides, I was most interested in sulfonamides, amines, and thioamides. Esters are less biologically relevant as they are rapidly hydrolyzed, and other bioisosteres (including tetrazoles and oxadiazoles) are much more challenging to prepare.
I began with sulfonamides, as these are widely used in drug design. 2,6-substituted sulfonyl chlorides have limited commercial availability; 17 is available but at more than
$400/gram, I decided to synthesize it myself through a Sandmeyer-like reaction (Scheme 10).
Scheme 10. Synthesis of sulfonamides. (a) NaNO2, H2SO4, H2O, then potassium ethyl xanthogenate; (b) KOH, EtOH; (c) Oxone,
KCl, H2O; (d) pyridine, CH2Cl2. Anderson 27
Beginning with 2-chloro-6-fluoroaniline, the diazonium species was reacted with potassium ethyl xanthogenate (KEX). KEX is used to install sulfur groups without having to directly interact with H2S (a toxic, highly smelly gas), and avoids the possibility of thioether formation that would come from using sulfide (S-2) salts. Following hydrolysis, I obtained crude
17 in reasonable yield, followed by oxidation by Oxone to afford the mercifully non-odorous 18 in poor yield. After coupling both 18 and a commercially available sulfonyl chloride (as a backup plan, in the event the Sandmeyer reaction failed) with amine 2, I obtained sulfonamides 19a and 19b. These compounds both displayed poor activity against TLR 7 and 8 and were enormously cytotoxic.
Next, I attempted to prepare the amine bioisostere which would introduce additional flexibility into the molecule. Initially, I had imagined a simple lithium aluminum hydride (LiAlH4) reduction of amide 4a to the secondary amine (Scheme 11) under typical reflux conditions.
Scheme 11. Synthetic route for reduction of 4a. Various reduction conditions were screened.
However, this instead resulted in the cleavage of the C-N bond and production of aryl amine 2. I screened other reaction conditions, such as LiAlH4 at 0 °C and weaker reducing agents such as sodium borohydride with and without Lewis acids. In every case, I observed Anderson 28 either no reaction or cleavage of the C-N bond. I suspect this is due to the general electron deficient nature of this compound, similar to my issues with imide formation. Instead of searching for further optimized reduction conditions, I opted to prepare the amine through
Buchwald-Hartwig amination (Scheme 12), which will be completed soon.
Scheme 12. Synthesis of 2° amine through Sandmeyer-type iodination followed by Buchwald-Hartwig coupling. (a) KI, NaNO2,
TsOH, MeCN, H2O; (b) Pd2(dba)3, BINAP, NaOtBu, toluene.
Finally, recalling that methylation of the amide nitrogen nullified TLR 7 antagonism, I hoped to strengthen the hydrogen-bonding of the amide by making a thioamide derivative
(Scheme 13). Thioamides are more rotationally locked than amides and better hydrogen bond donors.
Scheme 13. Synthetic route for thioamide synthesis. Conditions varied, but Lawesson's reagent, toluene, reflux is representative.
Thionation by Lawesson’s reagent (LR) is often considered a trivial reaction as it “never fails”—however, it proved quite difficult for this substrate. LR reactions can often be run at low temperature, but I observed no product formation without forcing conditions (greater than 100
°C). Complicating matters, the reaction was sluggish and could not be pushed beyond 40-50% Anderson 29 conversion, and 3a and thioamide 21 were inseparable by flash chromatography, recrystallization, and HPLC. I screened conditions to force the reaction to completion (Table 3).
Table 3. Optimization of conditions for thionation using Lawesson's reagent (LR).
LR equivalent Solvent Temp (°C) Time (h) % Conversion 0.5 THF 50 2.5 0 0.5 Toluene 90 2.5 < 25 4 Toluene 120 120 40
4 Ph2O 145 43 0 (decomp) 4 Toluene 120 240 40-50 After several attempts, I monitored a 10-day reaction by 19F NMR (Figure 6).
Unexpectedly, the amount of product formed remains constant after two days and the reaction never goes to completion. This seems suggestive of an equilibrium, however, the thionation reaction is supposed to be favorable (ΔG ≪ 0) and irreversible due to the strong phosphorous- oxygen bonds formed31. It may not be possible to synethesize the thioamide via LR. I intend to attempt other routes such as the Willgerodt-Kindler thioamide synthesis in the future.
Figure 6. 19F NMR monitoring of thioamide synthesis by Lawesson's reagent. Data points shown are 66, 96, and 240 hours. The peak at -112.16 ppm is 4a, and -110.29 ppm is product. Relative integrations of these peaks are the same across all time points. Anderson 30
4. DISCUSSION AND FUTURE DIRECTIONS
> 30 targets prepared, F F CF assayed for potency CF 3 H 3 H N and selectivity N
O Cl O I F C O F C O 3 1) 7.5-fold increase 3 4a in TLR 8 potency 4d 2) Loss of TLR 7 activity
TLR 7 IC50 = 2.88 uM TLR 7 IC50 > 50 uM TLR 8 IC50 = 1.64 uM TLR 8 IC50 = 0.22 uM
Figure 7. Summary of major structure-activity relationship results.
The goal of this project was to take dual TLR7/8 antagonist 4a and engineer selectivity for TLR 7 and/or 8 by structural modifications. To date, more than 30 final targets were prepared, and compound 4d was identified as a potent TLR8-selective antagonist with a 7.5- fold increase in potency relative to 4a. This serves as a proof of concept for the project and may see use as a chemical probe. Additional work is on-going to locate a TLR7-specific antagonist, which could be useful as a therapeutic for autoimmune diseases or used as a chemical probe to further our understanding of the mechanisms of signaling in innate immunity.
In terms of chemistry, several interesting and low effort targets still exist on ring A. For example: synthesizing methoxy derivatives from the hydroxyl compounds 4f and 4l, and reducing nitro compound 4g to an amine. Furthermore, it would be interesting to test a tri- substituted ring, most likely with a 2,5,6 substitution pattern, as the efficacy of the 2,5- substituted 4o suggests this would be tolerated. I have presented preliminary data on introducing asymmetry to ring C which suggests one of the trifluoromethyl groups is unused in
TLR binding, but the binding pocket tolerates its existence. This is an exciting result, as it means it might be possible to gain new interactions in the binding pocket, increasing binding affinity Anderson 31 and perhaps specificity. Further derivatization is needed to explore these possibilities.
Additionally, if possible to synthesize, thioamide and retroamide linkers would provide additional information about the interactions of the molecule in the binding pocket.
For the biological side of the project, I would like to obtain a crystal structure of 4d bound to TLR 8. If these compounds bind in approximately the same location on both TLR 7 and
8, which is the current assumption, a crystal structure of TLR 8 with bound antagonist would clarify questions about the mechanism of action of this scaffold (e.g. does it affect the protein- protein interface?) and allow for docking studies to make rational design of a TLR 7-selective antagonist possible. Finally, I hope to probe the mechanism of antagonism in through ssRNA experiments and isothermal titration calorimetry.
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Anderson 35
6. SUPPORTING INFORMATION
6-1. Biological methods
HEK 293 cells
Commercially available Human embryonic kidney cells (HEK 293, Invivogen) stably co- transfected with either hTLR 7 or 8 gene and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used to assay potency and specificity of compounds.
The SEAP reporter is linked to a promoter fused to several NF-κB and AP-1 binding sites.
Activation of the TLR by natural ligand (ssRNA) or small molecule synthetic ligand (R848) stimulates NF-κB activation and induction of SEAP protein production.
During cell maintenance, DMEM growth media supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, as well as 10 μg/mL blasticidin and 100
μg/mL zeocin to maintain presence of transfected TLR and SEAP plasmids. For testing, DMEM media with 10% denatured fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin was used.
Experimental design
Cells in test media were plated between 75,000 and 100,000 cells per well in a 96-well plate. For antagonist studies, cells were treated with compound and 1 μg/mL R848 (TLR 7/8 agonist; Invivogen), then incubated 20-24 hours; agonist studies omitted R848. TLR signaling was assayed via NF-κB-linked SEAP reporter activity using a SEAP assay. Anderson 36
SEAP assay
Quanti-Blue (Invivogen) was used to quantify SEAP secretion by measuring absorbance at 620 nm, which is taken as a proxy for TLR-induced NF-κB activation. Data was normalized with untreated cells as negative control and R848-treated cells as positive control. All data is presented as average of at least two biological replicates, each containing three technical replicates. Exact IC50 values are calculated from a dosing curve generated from three biological replicates.
WST-1 assay
Toxicity of all compounds was determined using WST-1 cell proliferation assay (Sigma
Aldrich). Cells were treated with 1:10 diluted WST-1 reagent and incubated for 20-30 minutes.
Cell death was quantified by measuring absorbance at 450 nm, which is a proxy for formazan dye formation by metabolically active cells. Data was normalized with untreated cells as 100% survival and cells treated with 50% DMSO as 0% survival.
6-2. Chemical methods
Unless otherwise noted, reactions were performed in flame-dried glassware under normal atmosphere. Reagents were reagent grade and used without further purification. All chemicals were obtained from Sigma-Aldrich, Fisher Scientific, or CombiBlocks.
Flash chromatography was performed using SorbTech 60A silica gel, 40-63 μm. Thin layer chromatography (TLC) was performed using EMD Millipore F254 silica gel plates and visualized under UV light. Anderson 37
1H, 13C, and 19F NMR spectra were recorded on a Bruker Avance-III 400 MHz NMR
Spectrometer. For 1H and 13C, chemical shifts were reported relative to the solvent peak. 19F chemical shifts are uncorrected.
High resolution mass spectra were collected using Waters SYNAPT G2-Si HDMS (q-TOF). Unless noted, yields refer to isolated yields after drying under high vacuum for several hours. Product purity was quantified by 1H NMR spectroscopy, and purity was verified by high performance liquid chromatography (HPLC) prior to biological testing.
6-3. Reaction Protocols
General procedure A: amide coupling with HATU
Eur. J. Med. Chem., 2013, 61, 2-25.
To a flame-dried vial was added HATU (0.6 mmol), carboxylic acid (0.56 mmol), and DMF
(1 mL). The vial was cooled to 0 °C and diisopropylethylamine (0.6 mmol) was added, then the vial was removed from ice bath. After the vial stirred for at least 15 minutes the appropriate aniline (0.5 mmol) dissolved in DMF (1 mL) was added, and the reaction mixture was stirred overnight. After completion, the solvent was removed in vacuo and the crude product was partitioned between 5 mL each water and ethyl acetate. The aqueous layer was extracted 3x with 5 mL ethyl acetate, after which the combined organic layers were washed with 5 mL brine, dried over Na2SO4, and concentrated to dryness. Purification varied by compound, typically flash chromatography or trituration in cold hexane. Anderson 38
General procedure B: amide coupling with acyl chloride.
To a flame-dried flask containing substituted aniline (0.6 mmol) in CH2Cl2 (2 mL) was added acid chloride (0.72 mmol) dissolved in CH2Cl2 (2 mL). The flask was placed under nitrogen and cooled to 0 °C, after which triethylamine (0.1 mL, 0.72 mmol) was added dropwise. The reaction was allowed to return to rt and stirred overnight. The solvent was removed in vacuo and the resulting residue was partitioned between 10 mL each ethyl acetate and water. The aqueous layer was extracted 3x with 5 mL ethyl acetate, and the combined organic layers were sequentially washed with 5 mL 1M NaOH, 5 mL brine, dried over Na2SO4, and concentrated to dryness. Compounds were typically purified by flash chromatography.
General procedure C: SnAr with phenol and aryl fluoride
Med. Chem. Commun., 2015, 6, 671-676.
To a microwave reaction vial was added aryl fluoride (2.5 mmol), phenol (3.0 mmol),
K2CO3 (7.5 mmol), and DMF (12.5 mL). The vial was sealed with a crimp cap and heated at 90 °C for 24-76 hours, after which it was cooled and the mixture was diluted with water until cloudly
(approximately 10 mL). The reaction mixture was partitioned between water and 30 mL EtOAc, and the aqueous layer was extracted 3x with 10 mL EtOAc. The combined organic layers were washed 3x with 10 mL 5% LiCl, 1x with 10 mL 1 M NaOH, and finally 1x with 10 mL brine, dried over Na2SO4 and concentrated to dryness.
General procedure D: reduction of nitroarenes
ACS Med. Chem. Lett., 2013, 4, 647-650. Anderson 39
A flask was charged with nitroarene (9.6 mmol) and 10% Pd/C (2.9 mmol), then placed under vacuum and backfilled with N2. MeOH (80 mL) was added, and H2 was introduced via balloon. The reaction was monitored by TLC; at completion, the mixture was filtered through a pad of celite and concentrated to dryness. Purification method varied by compound, and was not required in all cases.
1; 1-(4-nitrophenoxy)-3,5-bis(trifluoromethyl)benzene
As general procedure C. Purified by flash chromatography (0-40% CH2Cl2 in hexane) to afford title compound as a yellow oil (3.37 g, 96%).
1H NMR (400 MHz, Chloroform-d) δ 8.32 – 8.27 (m, 2H), 7.73 (s, 1H), 7.52 (s, 2H), 7.16 –
13 7.11 (m, 2H); C NMR (101 MHz, CDCl3) δ 161.1, 156.4, 144.2, 134.5, 134.2, 133.8, 133.5, 126.8,
126.5, 124.1, 121.4, 120.20, 120.16, 120.12, 120.08, 118.7;
2; 4-[3,5-bis(trifluoromethyl)phenoxy]aniline
General procedure D was followed, yielding a light brown solid (3.01 g, 98%) which was used without further purification. This compound was observed to rapidly darken upon standing in air and storage under nitrogen at 0 °C is recommended. Anderson 40
1 H NMR (400 MHz, Methanol-d4) δ 7.56 (s, 1H), 7.36 (s, 2H), 6.93 – 6.86 (m, 2H), 6.86 –
13 6.79 (m, 2H). C NMR (101 MHz, CDCl3) δ 160.2, 146.6, 144.2, 133.5, 133.2, 132.9, 132.5,
124.5, 121.8, 116.8, 116.80, 116.77, 116.73, 116.65, 115.6, 115.41, 115.38, 115.34, 115.30,
19 115.26. F NMR (376 MHz, CDCl3) δ -63.00.
3a; 2-fluoro-6-(trifluoromethoxy)benzoic acid
Eur. J. Org. Chem. 2001, 3991-3997.
To a flame-dried flask containing THF (5 mL) was added lithium diisopropylamide (2M in
THF; 0.6 mL, 1.2 mmol) under nitrogen. After cooling to -78 °C, 3-
(trifluoromethyl)fluorobenzene (0.18 g, 1. mmol) was added and then stirred two hours at -78
°C. CO2 from a flask containing dry ice was bubbled through solution as it was allowed to warm to rt over one hour, then quenched by slow addition of H2O. Mixture was concentrated to dryness, then dissolved in 10 mL 1M NaOH and washed twice with 10 mL EtOAc. Following acidification with 3M HCl, the aqueous layer was extracted 3x with 10 mL EtOAc. Combined organic extracts were dried over Na2SO4 and concentrated to dryness to afford title compound as a white solid (0.126 g, 56%) which was used without further purification.
1H NMR (400 MHz, DMSO-d6) δ 14.14 (s, 1H), 7.66 (td, J = 8.5, 6.5 Hz, 1H), 7.43 (ddd, J =
13 9.3, 8.5, 0.9 Hz, 1H), 7.39 – 7.34 (m, 1H). C NMR (101 MHz, Acetone-d6) δ 162.7, 161.8, 159.3,
147.14, 147.11, 147.07, 147.05, 133.5, 133.4, 125.1, 122.5, 120.0, 118.5, 118.3, 118.04, 118.03, Anderson 41
118.01, 117.99, 117.98, 117.96, 117.4, 116.1, 115.9. 19F NMR (376 MHz, DMSO) δ -56.63, -
112.65.
3b; 2-cyano-6-fluorobenzoic acid
Prepared in the same manner as 3a. Used immediately without purification as rapid browning (decomposition) was observed. Crude spectral data are presented; carboxylic acid peak was not observed.
1H NMR (400 MHz, Chloroform-d) δ 7.70 – 7.63 (m, 2H), 7.49 – 7.43 (m, 1H). 19F NMR
(376 MHz, CDCl3) δ -105.04.
3c; 2-fluoro-6-nitrobenzoic acid
Tet. Lett. 1995, 36, 2377 – 2378.
NaOH (0.07 g, 1.8 mmol) was added to 12 mL H2O and cooled in an ice bath under vigorous stirring. 2-amino-6-fluorobenzoic acid (0.23 g, 1.5 mmol) and NaHCO3 (1.26 g, 15 mmol) were added in one portion. After stirring 15 minutes, two pre-chilled solutions were added simultaneously with rate of solution A roughly twice that of solution B. Solution A: Oxone
(2.8 g, 9 mmol), EDTA (15 mg), 12 mL H2O. Solution B: 6 mL H2O, 6 mL acetone. After 25 Anderson 42 minutes, the bright green mixture was removed from ice bath and stirred at rt for 6 hours, then quenched by addition of NaHSO3 and concentrated under reduced pressure. Residue was partitioned between 15 mL EtOAc and 15 mL 3M HCl, after which the aqueous layer was extracted 3x with 10 mL EtOAc. Combined organic layers were washed 1x with 10 mL brine, dried over Na2SO4 and concentrated to dryness to afford title compound as yellow crystals
(0.26 g, 95%).
1H NMR (400 MHz, Chloroform-d) δ 8.01 (dt, J = 8.2, 1.0 Hz, 1H), 7.65 (td, J = 8.3, 5.4 Hz,
19 1H), 7.52 (td, J = 8.4, 1.1 Hz, 1H). F NMR (376 MHz, CDCl3) δ -111.24.
3d; 4-(benzyloxy)-2-fluorobenzoic acid
Bioorg. Med. Chem. 2011, 19, 4953 – 4970.
To a flame-dried flask was added 2-fluoro-4-hydroxybenzoic acid (0.624 g, 4 mmol),
K2CO3 (1.66 g, 12 mmol), DMF (12 mL), and benzyl bromide (1.67 mL, 14 mmol). The mixture was heated at 70 °C for 12 hours, then cooled and partitioned between 10 mL ice wter and 10 mL EtOAc. The aqueous layer was extracted twice with 5 mL EtOAc, then combined organic layers were concentrated to dryness.
LiOH monohydrate (1.25 g, 30 mmol), THF (10 mL), and H2O (10 mL) were added and the mixture was heated at reflux 8 hours. After cooling, THF was removed under reduced pressure, then 10 mL EtOAc was added. The aqeous layer was extracted 3x with 10 mL EtOAc. Combined Anderson 43 organic layers were washed 3x with 5 mL 5% LiCl, once with 10 mL brine, then dried over
Na2SO4 and concentrated to dryness. The crude product was recrystallized from EtOH to obtain pure product as white crystals (0.514 g, 52%).
1H NMR (400 MHz, Chloroform-d) δ 7.98 (t, J = 8.7 Hz, 1H), 7.44 – 7.40 (m, 4H), 7.40 –
7.34 (m, 1H), 6.83 (ddd, J = 8.8, 2.5, 0.6 Hz, 1H), 6.74 (dd, J = 12.7, 2.4 Hz, 1H), 5.12 (s, 2H). 13C
NMR (101 MHz, Acetone) δ 165.6, 164.98, 164.95, 164.8, 164.7, 163.0, 137.3, 134.57, 134.55,
19 129.4, 129.0, 128.7, 112.00, 111.97, 111.86, 104.0, 103.8, 71.2. F NMR (376 MHz, CDCl3) δ -
104.79.
3e; 2-fluoro-4-(2,2,2-trifluoroacetamido)benzoic acid
Beilstein. J. Org. Chem. 2012, 8, 2085 – 2090.
To a flamedried flask was added trifluoroacetic anhydride (0.56 mL, 4 mmol) which was cooled over ice. 2-fluoro-4-aminobenzoic acid was added in portions, after which the flask was allowed to warm to rt and stirred for 2 hours. At completion, partitioned mixture between 20 mL ice water, 5 mL CH2Cl2. The aqueous layer was extracted twice with 5 mL CH2Cl2, then the combined organic layers were concentrated to afford semi-pure product (0.216 g, 86%) which was used without further purification. Anderson 44
1H NMR (400 MHz, Chloroform-d) δ 7.91 (td, J = 8.3, 5.3 Hz, 1H), 7.61 (dt, J = 8.2, 1.0 Hz,
19 1H), 7.39 (ddd, J = 9.5, 8.4, 1.0 Hz, 1H). F NMR (376 MHz, CDCl3) δ -72.61, -104.02.
3f; 1-acetyl-1H-indazole-6-carboxylic acid
Synth. Commun. 2006, 36, 3117 – 3123.
O CH3 O
N OH N
To a flame-dried vial was added 1-H-indazole-5-carboxylic acid (0.081 g, 0.5 mmol) and acetic anhydride (1 mL, 10.6 mmol). The vial was heated at 130 °C for 45 minutes, then cooled at 0 °C overnight. A brown solid was separated by filtration and washed with cold hexane to afford crude product as a mix of regioisomers (0.021 g, 21% crude) which was used without further purification.
4a; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-chloro-6-fluorobenzamide
As general procedure B. The crude product was purified by column chromatography (0-
80% CH2Cl2 in hexane) to yield the product as a white powder (0.236 g, 82%).
1H NMR (400 MHz, Chloroform-d) δ 7.76 – 7.67 (m, 2H, ArH), 7.58 (d, J = 1.6 Hz, 1H,
ArH), 7.49 (s, 1H, NH), 7.45 – 7.34 (m, 3H, ArH), 7.29 (dt, J = 8.1, 0.9 Hz, 1H, ArH), 7.17 – 7.06
(m, 3H, ArH). 19F NMR (376 MHz, Chloroform-d) δ -62.97, -112.27. Anderson 45
4b; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluorobenzamide
As general procedure B. The crude product was purified by column chromatography (0-
45% CH2Cl2 in hexane), then recrystallized from boiling methanol to afford the title compound as a white solid in 81% yield.
1H NMR (400 MHz, Chloroform-d) δ 8.52 (d, J = 16.0 Hz, 1H), 8.20 (td, J = 8.0, 1.9 Hz, 1H),
7.77 – 7.72 (m, 2H), 7.59 – 7.52 (m, 2H), 7.38 (s, 2H), 7.35 (td, J = 7.6, 1.1 Hz, 1H), 7.24 – 7.17
(m, 1H), 7.12 – 7.08 (m, 2H). 19F NMR (376 MHz, Chloroform-d) δ -62.97, -113.14.
4c; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-bromo-6-fluorobenzamide
Br CF 3 H N
O F F3C O
As general procedure A, except solvent was CH2Cl2. Purification by flash chromatography (0-60% CH2Cl2 in hexane) followed by trituration in cold hexane yielded a white-tan solid (76%).
1H NMR (400 MHz, Chloroform-d) δ 7.74 – 7.69 (m, 2H), 7.58 (tp, J = 1.4, 0.7 Hz, 1H),
7.52 (s, 1H), 7.45 (dt, J = 8.0, 0.9 Hz, 1H), 7.39 (dt, J = 1.7, 0.6 Hz, 2H), 7.32 (td, J = 8.3, 5.9 Hz,
1H), 7.15 (td, J = 8.5, 1.0 Hz, 1H), 7.12 – 7.08 (m, 2H); 13C NMR (101 MHz, Chloroform-d) δ
161.5, 160.9, 159.0, 158.3, 151.9, 134.5, 133.89, 133.6, 133.2, 132.9, 132.4, 132.2, 129.09, Anderson 46
129.06, 127.3, 127.1, 124.4, 122.5, 121.7, 120.9, 120.88, 120.83, 116.43, 116.39, 116.36, 115.5,
115.3; 19F NMR (376 MHz, Chloroform-d) δ -62.97, -111.60; HRMS (ES-) [M-H]- for
C21H10BrF7NO2 , m/z 519.9783, found 519.9781.
4d; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-6-iodobenzamide
Prepared by general method B. Purified by column chromatography (0-75% CH2Cl2 in hexane) to afford product as a white solid (94%).
1H NMR (400 MHz, Chloroform-d) δ 7.75 – 7.69 (m, 3H), 7.58 (s, 1H), 7.40 (s, 2H), 7.21 –
7.14 (m, 2H), 7.14 – 7.09 (m, 2H). 19F NMR (376 MHz, Chloroform-d) δ -62.94, -110.81. 13C NMR
(101 MHz, Chloroform-d) δ 158.8, 151.8, 135.44, 135.4, 134.3, 134.2, 133.4, 133.1, 132.6,
132.5, 122.4, 122.3, 120.8, 117.7, 116.3, 116.2, 115.9.
4e; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-cyano-6-fluorobenzamide
F CF 3 H N
O CN F3C O
Prepared by general method C. Purified by column chromatography (0-75% EtOAc in hexane) to afford semi-pure product. During recrystallization from hot MeOH, product decomposed. Characterization data for pure product is unavailable.
4f; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-6-hydroxybenzamide Anderson 47
To a flame-dried flask was added 2 (0.48g, 1.5 mmol), 2-fluoro-6-hydroxybenzoic acid
(0.23 g, 1.5 mmol), PCl3 (0.10 mL, 1.13 mmol), and chlorobenzene (7.5 mL). Mixture was heated at reflux for 4 hours. After cooling, solvent volume was removed in vacuo, then crude product was purified by column chromatography (0-75% CH2Cl2 in hexane) to afford pure product as a white solid (0.339g, 49%).
19F NMR (376 MHz, Chloroform-d) δ -62.98, -111.43.
4g; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-6-nitrobenzamide
Synthesized by general procedure A. Crude material was triturated in cold hexane, then recrystallized from hot MeOH to afford tan crystals (49%).
1H NMR (400 MHz, Chloroform-d) δ 8.03 (dt, J = 8.1, 1.0 Hz, 1H), 7.72 – 7.62 (m, 3H),
13 7.60 – 7.51 (m, 3H), 7.40 (s, 2H), 7.15 – 7.08 (m, 2H). C NMR (101 MHz, Acetone-d6) δ 161.31
160.4, 160.1, 160.0, 158.8, 152.2, 147.8, 137.0, 136.9, 134.2, 133.9, 133.6, 133.3, 132.9, 132.8,
128.9, 128.2, 125.5, 123.1, 122.9, 122.8, 122.7, 122.54, 122.45, 121.8, 121.50, 121.47, 120.36,
120.1, 118.89, 118.85, 116.98, 116.94, 116.90, 116.86, 116.82. 19F NMR (376 MHz, Chloroform- d) δ -62.94, -111.86. Anderson 48
4h; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-6-(trifluoromethoxy)benzamide
Prepared by general procedure A. Purified by column chromatography (0-60% CH2Cl2 in hexane), then recrystallized from hot MeOH to yield product as white crystals (21%).
1H NMR (400 MHz, Chloroform-d) δ 7.71 – 7.66 (m, 2H), 7.58 (s, 1H), 7.55 – 7.48 (m, 2H),
7.40 (s, 2H), 7.23 – 7.15 (m, 2H), 7.13 – 7.07 (m, 2H). 19F NMR (376 MHz, Chloroform-d) δ -
57.41, -62.96, -111.47. 13C NMR (101 MHz, Chloroform-d) δ 159.0, 158.8, 152.0, 134.4, 133.6,
133.2, 132.21, 132.11, 124.4, 122.5, 120.9, 117.1, 116.4, 115.2, 115.0.
4i; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-4-methylbenzamide
Prepared by general method A. Purified by column chromatography (0-60% CH2Cl2 in hexane) to obtain product as a tan solid (80%).
1H NMR (400 MHz, Chloroform-d) δ 8.51 (d, J = 16.7 Hz, 1H), 8.08 (t, J = 8.3 Hz, 1H), 7.77
– 7.71 (m, 2H), 7.56 (s, 1H), 7.38 (s, 2H), 7.14 (d, J = 8.0, 1.6, 0.8 Hz, 1H), 7.11 – 7.07 (m, 2H),
7.02 (d, 1H), 2.44 (s, 3H). 19F NMR (376 MHz, Chloroform-d) δ -62.97, -113.87. 13C NMR (101
MHz, Chloroform-d) δ 161.7, 161.6, 159.3, 159.2, 151.3, 145.8, 145.7, 135.2, 133.8, 133.5, Anderson 49
133.1, 132.8, 132.27, 132.25, 126.24, 126.21, 124.4, 122.7, 121.7, 120.9, 118.2, 118.1, 117.7,
117.6, 116.8, 116.6, 116.3, 116.23, 116.19, 21.6.
4j; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2,4-difluorobenzamide
Prepared by general procedure A. Purified by column chromatography (0-60% CH2Cl2 in hexane) to afford title product as tan solid (82%).
1H NMR (400 MHz, Chloroform-d) δ 8.41 (d, J = 15.4 Hz, 1H), 8.26 – 8.19 (m, 1H), 7.76 –
7.69 (m, 2H), 7.57 (s, 1H), 7.38 (s, 2H), 7.13 – 7.04 (m, 3H), 6.96 (ddd, J = 12.3, 8.3, 2.4 Hz, 1H).
19F NMR (376 MHz, Chloroform-d) δ -62.97, -102.55, -108.73.
4k; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-4-benzyloxybenzamide
Prepared by general method A. Recrystallized from hot EtOH to afford title compound as white crystals (63%).
1H NMR (400 MHz, Chloroform-d) δ 8.43 (d, J = 16.7 Hz, 1H), 8.15 (t, 1H), 7.78 – 7.69 (m,
2H), 7.56 (s, 1H), 7.48 – 7.31 (m, 7H), 7.14 – 7.05 (m, 2H), 6.93 (dd, J = 8.9, 2.4 Hz, 1H), 6.77 (dd,
J = 14.4, 2.4 Hz, 1H), 5.14 (s, 2H). 13C NMR (101 MHz, Acetone) δ 163.6, 163.5, 163.2, 162.8,
162.8, 160.7, 160.6, 151.62, 151.61, 137.6, 137.53, 137.47, 134.2, 133.9, 133.6, 133.2, 133.1, Anderson 50
133.0, 129.5, 129.1, 128.7, 128.2, 125.5, 122.9, 122.81, 122.78, 121.6, 120.1, 118.7, 118.6,
116.9, 116.83, 116.79, 116.75, 116.71, 116.67, 112.58, 112.55, 103.5, 103.2, 71.2.
19F NMR (376 MHz, Chloroform-d) δ -62.97, -110.24. 13C NMR (101 MHz, Chloroform-d)
δ 135.8, 135.3, 133.7, 129.0, 128.6, 127.67, 120.9, 117.7, 116.2, 112.0, 102.9, 102.7, 70.8.
4l; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-4-hydroxybenzamide
Benzyl protected 4k (0.30 g, 0.55 mmol), 10% Pd/C (0.016g, 0.15 mmol), and MeOH (15 mL) were added to a flask and stirred under a hydrogen balloon overnight. The crude mixture was filtered through Celite, then through a silica plug to yield pure compound as a tan powder
(0.247 g, 99%).
1H NMR (400 MHz, Chloroform-d) δ 8.43 (d, J = 16.8 Hz, 1H), 8.11 (t, 1H), 7.76 – 7.67 (m,
2H), 7.56 (s, 1H), 7.38 (s, 2H), 7.14 – 7.06 (m, 2H), 6.78 (dd, J = 8.7, 2.4 Hz, 1H), 6.69 (dd, J =
13.7, 2.4 Hz, 1H), 5.45 (s, 1H). 19F NMR (376 MHz, Chloroform-d) δ -62.97, -110.38.
4m; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluoro-4-iodobenzamide
Anderson 51
Prepared by general method A. Product was purified by column chromatography (0-
100% CH2Cl2 in hexane) followed by recrystallization from hot hexane to afford title compound as white crystals (19%).
1H NMR (400 MHz, Chloroform-d) δ 8.41 (d, J = 15.6 Hz, 1H), 7.90 (t, J = 8.4 Hz, 1H), 7.76
– 7.68 (m, 3H), 7.61 (dd, J = 11.4, 1.6 Hz, 1H), 7.57 (s, 1H), 7.38 (s, 2H), 7.15 – 7.06 (m, 2H). 19F
NMR (376 MHz, Chloroform-d) δ -62.97, -111.49.
4n; 4-amino-N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluorobenzamide
Prepared by general method A to obtain trifluoroacetamide protected compound.
Trifluoroacetamide (0.102 g, 0.18 mmol) was added to K2CO3 (0.14 g, 1 mmol) and MeOH (10 mL) and then heated at reflux for 4 hours. After cooling, the solvent was removed under reduced pressure and the resulting residue was partitioned between 5 mL each H2O and EtOAc.
The aqueous layer was extracted twice with 5 mL EtOAc, then combined organic layers were washed once with 5 mL H2O, once with 5 mL brine, and dried over Na2SO4. Crude product was purified by column chromatography (0-30% EtOAc in hexane) to afford title compound as a tan solid (32 mg, 39%/2 steps). Note: compound is oxidized rapidly on standing.
1H NMR (400 MHz, Chloroform-d) δ 8.41 (d, J = 17.5 Hz, 1H), 7.99 (t, J = 9.2, 8.6 Hz, 1H),
7.77 – 7.67 (m, 2H), 7.55 (s, 1H), 7.37 (s, 2H), 7.11 – 7.02 (m, 2H), 6.55 (dd, J = 8.6, 2.2 Hz, 1H),
6.39 (dd, J = 14.7, 2.2 Hz, 1H), 4.20 (s, 2H). 13C NMR (101 MHz, Chloroform-d) δ 163.4, 161.9, Anderson 52
161.8, 161.0, 159.3, 152.2, 152.1, 151.0, 135.6, 133.90, 133.86, 133.77, 133.4, 133.1, 132.8,
127.1, 124.4, 122.5, 121.7, 120.9, 119.0, 117.7, 117.6, 116.20, 116.16, 116.12, 116.08, 116.04,
111.39, 111.38, 110.3, 110.2, 101.1, 100.8. 19F NMR (376 MHz, Chloroform-d) δ -62.97, -111.81.
4o; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-5-chloro-2-fluorobenzamide
Prepared by general method A. Purified by column chromatography (0-100% CH2Cl2 in hexane) to yield pure product as white powder (75%).
1H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 15.6 Hz, 1H), 8.17 (dd, J = 6.7, 2.8 Hz,
1H), 7.78 – 7.69 (m, 2H), 7.57 (s, 1H), 7.50 (ddd, J = 8.8, 4.4, 2.8 Hz, 1H), 7.38 (s, 2H), 7.17 (dd, J
= 11.4, 8.8 Hz, 1H), 7.14 – 7.06 (m, 2H). 19F NMR (376 MHz, Chloroform-d) δ -62.97, -116.14.
4p; 1-acetyl-N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-1H-indazole-6-carboxamide
As general procedure A. Purified by trituration in cold hexane to afford title compound as a tan powder (18%). Anderson 53
1H NMR (400 MHz, Chloroform-d) δ 8.59 – 8.54 (m, 1H), 8.36 (dd, J = 1.7, 0.8 Hz, 1H),
8.26 (d, J = 0.8 Hz, 1H), 8.05 (dd, J = 8.7, 1.7 Hz, 1H), 7.91 (s, 1H), 7.78 – 7.72 (m, 2H), 7.58 (s,
13 1H), 7.39 (s, 2H), 7.15 – 7.09 (m, 2H), 2.84 (s, 3H). C NMR (101 MHz, CDCl3) δ 171.7, 165.88,
165.81, 160.6, 151.6, 141.3, 141.1, 141.0, 137.88, 137.79, 133.9, 133.5, 132.53, 132.50, 129.5,
127.3, 125.5, 123.0, 122.8, 122.4, 122.3, 121.6, 118.6, 115.8, 23.1.
19F NMR (376 MHz, Chloroform-d) δ -62.96.
4q; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-1H-benzimidazole-5-carboxamide
As general procedure A. Purification by flash chromatography (0-100% EtOAc in CH2Cl2, followed by 0-5% MeOH) followed by recrystallization from MeOH to give iridescent white solid
(28%).
1 H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H), 10.37 (s, 1H), 8.39 (s, 1H), 8.26 (s, 1H), 7.93
(d, J = 8.9 Hz, 2H), 7.85 (d, J = 13.2 Hz, 2H), 7.69 (s, 1H), 7.59 (s, 2H), 7.26 – 7.20 (m, 2H); 19F
NMR (376 MHz, DMSO) δ -61.47;
4r; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-3-fluoropyridine-2-carboxamide
F CF 3 H N N O F3C O Anderson 54
As general procedure A. Purification by flash chromatography (0-100% CH2Cl2 in hexane) followed by trituration in cold hexane afforded the title compound as a white-tan solid (82%).
1H NMR (400 MHz, Chloroform-d) δ 9.93 (s, 1H), 8.47 (s, 1H), 7.87 – 7.82 (m, 2H), 7.66 –
13 7.59 (m, 1H), 7.59 – 7.54 (m, 2H), 7.38 (s, 2H), 7.13 – 7.08 (m, 2H); C NMR (101 MHz, CDCl3) δ
160.1, 159.2, 151.2, 143.9, 143.8, 137.3, 135.1, 133.5, 133.2, 132.8, 128.8, 127.1, 126.9, 124.4,
19 121.7, 120.9, 117.71, 117.67, 116.3, 116.22, 116.18, 116.15; F NMR (376 MHz, CDCl3) δ -62.99,
- -118.07; HRMS (ES-) [M-H] for C20H10F7N2O2 , m/z 443.0630, found 443.0633.
4s; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-3-chlorothiophene-2-carboxamide
As general procedure A. Purified by trituration in cold hexane to afford the title compound as a pink solid (87%).
1H NMR (400 MHz, Chloroform-d) δ 8.81 (s, 1H), 7.74 – 7.69 (m, 2H), 7.56 (d, J = 5.3 Hz,
13 2H), 7.38 (s, 2H), 7.12 – 7.07 (m, 2H), 7.06 (d, J = 5.3 Hz, 1H); C NMR (101 MHz, CDCl3) δ 159.1,
158.4, 151.6, 134.6, 133.9, 133.5, 133.3, 133.2, 132.9, 130.5, 129.7, 127.1, 124.4, 123.2, 122.5,
121.7, 121.0, 119.0, 117.74, 117.70, 116.40, 116.36, 116.32, 116.29, 116.25; 19F NMR (376 MHz,
- CDCl3) δ -62.99; HRMS (ES-) [M-H] for C19H9ClF6NO2S , m/z 463.9947, found 463.9953.
4t; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-1H-indole-5-carboxamide
Anderson 55
As general procedure A. Purified by trituration in cold hexane, followed by flash chromatography (0-100% EtOAc in hexane) to afford the title compound as a white solid (67%).
1 H NMR (400 MHz, Methanol-d4) δ 8.27 (dd, J = 1.8, 0.7 Hz, 1H), 7.85 – 7.80 (m, 2H), 7.75
(dd, J = 8.6, 1.8 Hz, 1H), 7.66 (s, 1H), 7.52 – 7.46 (m, 3H), 7.35 (d, J = 3.2 Hz, 1H), 7.19 – 7.13 (m,
2H), 6.60 (dd, J = 3.2, 0.9 Hz, 1H); 13C NMR (101 MHz, MeOD) δ 170.5, 161.1, 152.3, 139.8,
137.9, 134.5, 134.2, 129.1, 127.4, 126.7, 125.8, 124.5, 123.1, 121.9, 121.8, 121.7, 118.6, 116.8,
116.78, 116.74, 112.2, 103.7; 19F NMR (376 MHz, MeOD) δ -64.57;
5; 5-[3,5-bis(trifluoromethyl)phenoxy]-2-nitropyridine
Org. Lett. 2003, 5, 3799 – 3802.
To a flame-dried vial was added 5-bromo-2-nitropyridine (0.406 g, 2 mmol), 3,5- bis(trifluoromethyl)phenol (0.45 mL, 3 mmol), copper (I) iodide (0.114 g, 0.6 mmol), N,N- dimethylglycine hydrochloride salt (0.084 g, 0.6 mmol), Cs2CO3 (1.3 g, 4 mmol), and dioxane (4 mL). The tube was sealed and placed under nitrogen atmosphere, then heated at 90 °C for 44 hours. The mixture was partitioned between 5 mL each EtOAc and H2O, and the aqueous layer extracted 3x with 5 mL EtOAc. The combined organic layers were washed once with 5 mL brine, once with 5 mL 2M NaOH, dried over Na2SO4, and concentrated to dryness. Column chromatography (0-100% CH2Cl2 in hexane) provided the title compound with residual Anderson 56 contamination (0.353 g, 50% semi-pure). Spectra data is presented from semi-pure product.
Note: One peak in 19F NMR is from impurity.
1 H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 2.7 Hz, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.06 (s,
3H), 7.89 (dd, J = 8.9, 2.9 Hz, 1H). 19F NMR (376 MHz, MeOD) δ -64.45, -64.69.
6; 5-[3,5-bis(trifluoromethyl)phenoxy]pyridin-2-amine
Prepared by general method D. Purified by column chromatography (0-40% EtOAc in hexane) to obtain product as tan solid (33%).
1 H NMR (400 MHz, Methanol-d4) δ 7.81 (d, J = 2.7 Hz, 1H), 7.63 (s, 1H), 7.42 (s, 2H), 7.35
(dd, J = 9.0, 2.9 Hz, 1H), 6.69 (dd, J = 9.0, 0.7 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 161.62,
159.13, 144.10, 140.60, 134.51, 134.18, 132.84, 125.79, 123.08, 117.72, 116.68, 116.64, 116.60,
111.34, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36. 19F NMR (376 MHz, MeOD) δ -64.63.
7; N-{5-[3,5-bis(trifluoromethyl)phenoxy]pyridin-2-yl}-2-chloro-6-fluorobenzamide
Crude mixture of 8 prepared from previous reactions (0.46 g, 0.74 mmol), 2 M NaOH (5 mL), EtOH (10 mL) and THF (20 mL) were stirred at rt for two days. The reaction mixture was concentrated, then partitioned between 20 mL EtOAc and H2O. Aqueous layer was extracted 3x Anderson 57 with 10 mL EtOAc, and the combined organic layers were washed once with 10 mL brine and dried over MgSO4 and concentrated to dryness. Product was purified by column chromatography (0-25% EtOAc in hexane) to obtain a white solid (0.153 g, 37%).
1 H NMR (400 MHz, Methanol-d4) δ 8.38 (dd, J = 9.0, 0.7 Hz, 1H), 8.25 (dd, J = 2.9, 0.7 Hz,
1H), 7.75 (s, 1H), 7.69 (dd, J = 9.0, 3.0 Hz, 1H), 7.59 (s, 2H), 7.50 (td, J = 8.3, 6.1 Hz, 1H), 7.37 (dt,
13 J = 8.2, 0.9 Hz, 1H), 7.24 (td, J = 8.7, 1.0 Hz, 1H). C NMR (101 MHz, CDCl3) δ 161.0, 160.8,
158.5, 158.4, 148.5, 148.2, 140.1, 134.2, 133.9, 133.5, 133.2, 132.7, 132.6, 132.0, 131.9, 130.5,
126.0, 125.9, 125.0, 124.8, 124.3, 121.5, 117.1, 117.1, 117.0, 115.9, 115.0, 114.7. 19F NMR (376
MHz, CDCl3) δ -62.95, -112.06.
8; 2-chloro-N-(2-chloro-6-fluorobenzoyl)-6-fluoro-N-(5-[3,5-bis(trifluoromethyl)] phenoxypyridin-2-yl)benzamide
Prepared by general method B, even when attempted at 0 °C or -78 °C. Product was not fully isolated; spectral data presented on semi-pure material.
1 H NMR (400 MHz, Methanol-d4) δ 8.25 (dd, J = 2.8, 0.8 Hz, 1H), 7.79 (tp, J = 1.5, 0.8 Hz,
1H), 7.69 (dd, J = 8.7, 0.6 Hz, 1H), 7.65 (dd, J = 8.7, 2.8 Hz, 1H), 7.47 (dt, J = 1.7, 0.6 Hz, 2H), 7.42 Anderson 58
(td, J = 8.3, 6.0 Hz, 2H), 7.26 (dt, J = 8.1, 0.8 Hz, 2H), 7.12 (td, J = 8.7, 0.9 Hz, 2H). 19F NMR (376
MHz, MeOD) δ -64.43, -113.35.
9; 5-[3,5-bis(trifluoromethyl)phenoxy]-2-nitroaniline
As general procedure B. Following flash chromatography (0-100% CH2Cl2 in hexane), the title compound was obtained as a vivid yellow solid (0.824g, 90%).
1H NMR (400 MHz, Chloroform-d) δ 8.19 (dd, J = 9.3, 0.4 Hz, 1H), 7.72 (s, 1H), 7.51 (s,
2H), 6.35 (dd, J = 9.3, 2.6 Hz, 1H), 6.32 (dd, J = 2.5, 0.4 Hz, 1H), 6.20 (s, 2H). 13C NMR (101 MHz,
Acetone) δ 162.8, 157.4, 148.9, 148.8, 134.5, 134.2, 133.8, 133.5, 129.6, 125.3, 122.6, 121.90,
121.86, 121.82, 121.79, 119.16, 119.12, 119.08, 119.04, 119.00, 118.96, 108.0, 106.4, 106.3. 19F
NMR (376 MHz, CDCl3) δ -62.97.
10; 4-[3,5-bis(trifluoromethyl)phenoxy]-2-bromo-1-nitrobenzene
Can. J. Chem. 2005, 83, 213- 219.
To a solution of 9 (0.73 g, 2 mmol), KNO2 (0.681 g, 8 mmol) dissolved in DMSO (5 mL) was added dropwise a mixture of 48% HBr (1 mL) and DMSO (5 mL). The mixture was stirred at
35 °C for 35 minutes, then poured into K2CO3 (2.5 g) in 50 mL ice water. The mixture was Anderson 59
extracted 3x with 10 mL Et2O, then combined organic layers were washed with 10 mL brine, dried over Na2SO4 and concentrated to dryness. Purified by column chromatography (0-40%
CH2Cl2 in hexane) to afford title product as a yellow solid (0.612 g, 71%).
1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 9.0 Hz, 1H), 7.76 (s, 1H), 7.52 (s, 2H),
13 7.38 (d, J = 2.6 Hz, 1H), 7.05 (dd, J = 9.0, 2.6 Hz, 1H). C NMR (101 MHz, CDCl3) δ 160.3, 157.5,
146.9, 134.8, 134.4, 134.1, 133.8, 129.99, 128.93, 128.9, 128.8, 128.1, 125.46, 125.43, 125.41,
19 125.35, 122.7, 122.0, 119.9, 119.6, 116.5. F NMR (376 MHz, CDCl3) δ -62.96.
11; 4-[3,5-bis(trifluoromethyl)phenoxy]-2-bromoaniline
Nitroarene 10 (0.43 g, 1 mmol), Fe powder (0.22 g, 4 mmol), AcOH (4 mL), EtOH (4 mL) were added to a flask and heated at reflux 70 minutes. After cooling, the mixture was quenched with saturated NaHCO3, then extracted 3x with 10 mL EtOAc. The combined organic layers were washed once with 10 mL saturated NaHCO3, once with 10 mL brine, dried over Na2SO4 and concentrated to dryness to afford title compound as a brown oil (0.374 g, 94%) which was used without further purification.
1H NMR (400 MHz, Chloroform-d) δ 7.53 (s, 1H), 7.31 (s, 2H), 7.20 (d, J = 2.6 Hz, 1H),
19 6.91 – 6.85 (m, 1H), 6.82 (d, J = 8.7 Hz, 1H), 4.19 – 4.03 (m, 2H). F NMR (376 MHz, CDCl3) δ -
13 62.96. C NMR (101 MHz, CDCl3) δ 159.8, 146.3, 142.2, 133.4, 133.1, 125.0, 124.5, 121.7, 121.0,
116.6, 115.94, 115.91, 109.4. Anderson 60
12; N-{4-[3,5-bis(trifluoromethyl)phenoxy]-2-bromophenyl}-2-chloro-6-fluorobenzamide
Prepared by general method B. Purified by trituration in cold hexane to afford title compound as a brown solid (0.212 g, 65%).
1H NMR (400 MHz, Chloroform-d) δ 8.58 (d, J = 9.0 Hz, 1H), 7.91 (s, 1H), 7.62 (s, 1H),
7.46 – 7.37 (m, 3H), 7.36 – 7.29 (m, 2H), 7.18 – 7.10 (m, 2H). 13C NMR (101 MHz, Acetone) δ
161.72, 161.68, 161.63, 159.5, 159.2, 154.0, 134.4, 134.1, 133.9, 133.77, 133.73, 133.4, 132.84,
132.78, 132.69, 128.1, 128.0, 126.8, 126.6, 126.5, 126.4, 125.5, 125.1, 122.8, 122.5, 121.8,
19 120.4, 120.0, 120.0, 119.9, 118.2, 118.1, 117.8, 115.6, 115.4. F NMR (376 MHz, CDCl3) δ -
62.93, -111.98.
13; 3-amino-5-(trifluoromethyl)phenol
Prepared by general method D in quantitative yield and used immediately without further purification. Spectral data was in agreement with literature.
1 H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 6.29 (s, 1H), 6.20 (s, 1H), 6.15 (s, 1H), 5.45
13 (s, 2H). C NMR (101 MHz, Acetone-d6) δ 163.0, 159.2, 159.0, 154.5, 151.1, 132.6, 132.3, 132.0,
131.7, 129.2, 126.5, 126.4, 123.8, 121.1, 115.4, 110.5, 107.97, 107.94, 107.89, 107.86, 107.12, Anderson 61
107.08, 107.04, 107.00, 104.5, 102.94, 102.90, 102.86, 102.82, 100.9, 100.83, 100.79, 100.75.
19F NMR (376 MHz, DMSO) δ -61.64.
14; 3-(4-nitrophenoxy)-5-(trifluoromethyl)aniline
Prepared by general method C. Purified by column chromatography to yield title compound as a yellow solid (2.98 g, 99%).
1H NMR (400 MHz, Chloroform-d) δ 8.27 – 8.19 (m, 2H), 7.09 – 7.02 (m, 2H), 6.76 (s, 1H),
13 6.68 (s, 1H), 6.52 (s, 1H), 4.00 (s, 2H). C NMR (101 MHz, Acetone-d6) δ 163.4, 157.26, 152.21,
152.16, 143.8, 133.7, 133.4, 133.1, 132.8, 128.9, 126.7, 126.3, 126.2, 123.5, 120.8, 118.6,
109.20, 109.19, 108.10, 108.06, 108.02, 107.98, 104.91, 104.87, 104.83, 104.79. 19F NMR (376
MHz, CDCl3) δ -63.14.
15a; 1-(4-nitrophenoxy)-3-(trifluoromethyl)benzene
Tett. Lett. 2001, 42, 5367 – 5369.
EtOH (10 mL), AcOH (3 mL) were added to a flask containing 14 (0.149g, 0.5 mmol).
NaNO2 (0.34 g, 5 mmol) in H2O (3 mL) was added, followed by NaHSO3 (0.52 g, 5 mmol) in 4 mL
H2O. Mixture was stirred at rt 8 hours, then concentrated under reduced pressure. The residue Anderson 62
was partitioned between 10 mL EtOAc and 10 mL H2O and the aqueous layer extracted twice with 10 mL EtOAc. Combined organic layers were rinsed twice with 10 mL 1M NaOH, once with
10 mL brine, dried over Na2SO4 and concentrated to dryness. Purified by column chromatography (0-40% CH2Cl2 in hexane) to afford title compound as a yellow oil (0.069 g,
49%).
1H NMR (400 MHz, Chloroform-d) δ 8.28 – 8.22 (m, 2H), 7.60 – 7.49 (m, 2H), 7.36 (s, 1H),
19 7.28 (d, 1H), 7.08 – 7.03 (m, 2H). F NMR (376 MHz, CDCl3) δ -62.75.
15b; 1-iodo-3-(4-nitrophenoxy)-5-(trifluoromethyl)benzene
Chem. Eur. J. 2014, 20, 12553 – 12553.
KI (0.332 g, 2 mmol), NaNO2 (0.172 g, 2.5 mmol) in H2O (3.5 mL) was added to a solution of TsOH monohydrate (0.86 g, 4.5 mmol) and 14 (0.298 g, 1 mmol) in MeCN (6 mL), then stirred at rt 3.5 hours. The reaction was quenched by addition of saturated NaHCO3, then extracted 4x with 10 mL EtOAc. Combined organic layers were washed twice with 10 mL 1M NaOH, twice with 10 mL 1M Na2S2O3, once with 10 mL brine, dried over Na2SO4 and concentrated to dryness. Purified by column chromatography (0-60% CH2Cl2 in hexane) to afford title compound as a yellow solid (0.334 g, 82%).
1H NMR (400 MHz, Chloroform-d) δ 8.31 – 8.24 (m, 2H), 7.82 (s, 1H), 7.60 (s, 1H), 7.31 (s,
13 1H), 7.11 – 7.05 (m, 2H). C NMR (101 MHz, CDCl3) δ 161.5, 155.9, 143.9, 134.7, 134.3, 134.0, Anderson 63
133.7, 132.3, 131.00, 130.96, 130.92, 130.9, 126.4, 123.7, 121.0, 118.3, 116.73, 116.69, 116.66,
19 116.62, 94.63. F NMR (376 MHz, CDCl3) δ -62.87.
15c; 1-fluoro-3-(4-nitrophenoxy)-5-(trifluoromethyl)benzene
J. Org. Chem. 1979, 44, 1572 – 1574.
To a flame-dried flask was added boron trifluoride ethyl etherate (0.15 mL, 1.2 mmol) under nitrogen, then cooled to -15 °C. 14 in CH2Cl2 (2 mL) was added, followed by dropwise addition of tert-butyl nitrite (0.11 mL, 0.9 mmol) in CH2Cl2 (0.5 mL). Mixture was stirred for 10 minutes at -15 °C, then 15 minutes at rt, then diluted with hexane and filtered to isolate the aryl diazonium tetrafluoroborate salt as a precipitate. After drying under a stream of air, the salt was mixed with sand to form a small cake which was placed into a small foil packet. The packet was placed into a beaker with an evaporating dish and ice setup on top, after which the beaker was placed on a hot plate set to maximum for 15 minutes, during which a solid appeared on the dish. The solid was collected and purified by column chromatography (0-35%
CH2Cl2 in hexane) to afford title compound as a pale yellow solid (0.081 g, 36%). Anderson 64
1H NMR (400 MHz, Chloroform-d) δ 8.33 – 8.24 (m, 2H), 7.21 (d, J = 8.1, 1.6, 0.8 Hz, 1H),
19 7.18 – 7.07 (m, 3H), 6.99 (dd, J = 9.0, 0.5 Hz, 1H). F NMR (376 MHz, CDCl3) δ -62.99, -106.50.
16a; 2-chloro-6-fluoro-N-{4-[3-(trifluoromethyl)phenoxy]phenyl}benzamide
A flask containing 15a (0.071 g, 0.25 mmol), Fe powder (0.060 g, 1 mmol), AcOH (1.5 mL), EtOH (1.5 mL) was heated at reflux for 90 minutes, then cooled and concentrated under reduced pressure. The residue was partitioned between 10 mL 1M NaOH and 10 mL EtOAc, and the aqueous layer extracted 3x with 10 mL EtOAc. Combined organic layers washed twice with
10 mL 1M NaOH, once with 10 mL brine, dried over Na2SO4 and concentrated to dryness.
Crude amine was added to CH2Cl2 (5 mL), Et3N (0.21 mL, 1.5 mmol), and 2- chloro-6-fluorobenzoyl chloride (0.15 g, 0.8 mmol) at 0 °C, then allowed to warm to rt while stirring overnight. Mixture was concentrated under reduced pressure, partitioned between 10 mL EtOAc and 10 mL H2O and the aqueous layer extracted 3x with 10 mL EtOAc. Combined organic layers were washed once with 10 mL brine, dried over Na2SO4 and concentrated to dryness. Purified by column chromatography (0-60% CH2Cl2 in hexane) to afford title compound as a white solid (0.040 g, 40%).
1H NMR (400 MHz, Chloroform-d) δ 7.70 – 7.65 (m, 2H), 7.53 (s, 1H), 7.49 – 7.44 (m, 1H),
7.43 – 7.35 (m, 2H), 7.30 (dt, J = 8.2, 0.9 Hz, 1H), 7.27 (s, 1H), 7.21 – 7.16 (m, 1H), 7.16 – 7.11
13 (m, 1H), 7.11 – 7.07 (m, 2H). C NMR (101 MHz, CDCl3) δ 161.0, 160.5, 158.5, 158.1, 153.1, Anderson 65
133.6, 132.70, 132.65, 132.55, 131.8, 131.7, 130.5, 127.0, 126.0, 125.3, 125.1, 122.3, 121.4,
120.4, 119.9, 119.81, 119.77, 119.73, 115.12, 115.08, 115.04, 115.0, 114.7. 19F NMR (376 MHz,
CDCl3) δ -62.66, -112.32.
16b; 2-chloro-6-fluoro-N-{4-[3-iodo-5-(trifluoromethyl)phenoxy]phenyl}benzamide
Prepared by same method as 16a. Purified by trituration in cold hexane, then trituration in cold CH2Cl2 to afford title compound as a white solid (0.118 g, 44%).
1 H NMR (400 MHz, Acetone-d6) δ 7.94 – 7.88 (m, 2H), 7.78 (s, 1H), 7.63 (s, 1H), 7.54 (td, J
= 8.3, 6.1 Hz, 1H), 7.39 (dt, J = 8.1, 0.9 Hz, 1H), 7.33 (s, 1H), 7.27 (td, J = 8.7, 1.0 Hz, 1H), 7.24 –
7.18 (m, 2H). 19F NMR (376 MHz, Acetone) δ -63.40, -115.04.
17; 2-chloro-6-fluorobenzene-1-thiol
2-chloro-6-fluoroaniline (0.332 mL, 3 mmol) was suspended in H2O (9 mL) and cooled to
0 °C. H2SO4 (0.4 mL, 7.2 mmol) in H2O (4.5 mL) was added, followed by dropwise addition of
NaNO2 (0.435 g, 6 mmol) in H2O (4.5 mL). The mixture was stirred at 0 °C for one hour, then added dropwise to a solution of potassium ethyl xanthogenate (3.97 g, 24.3 mmol) in H2O (9 mL) at 85 °C and the flask was rinsed with 1.5 mL MeCN. Mixture was stirred for 1.5 hours, then Anderson 66
cooled and extracted 4x with 10 mL CH2Cl2. Combined organic layers were washed with 10 mL brine, dried over Na2SO4 and concentrated to dryness. The residue was taken up in EtoH (12 mL) and solid KOH was added (0.643 g, 11.5 mmol) and heated at reflux for 14 hours. The solvent was removed under reduced pressure to yield a red-brown residue (0.3 g, 61%) which was used directly without further purification due to the immense odor of this compound.
Spectral data is presented for the impure mixture.
19 F NMR (376 MHz, CDCl3) δ -105.28, -116.00.
18; 2-chloro-6-fluorobenzene-1-sulfonyl chloride
Crude thiol 17 (0.14g, 0.86 mmol), Oxone (1.32 g, 2.15 mmol), KCl (0.07g, 0.9 mmol), and H2O (3 mL) were added to a flask and stirred at rt for 30 minutes. At completion, the mixture was partitioned between 10 mL H2O, 10 mL EtOAc. The aqueous layer was extracted 3x with 10 mL EtOAc and the combined organic layers were washed with 10 mL brine, dried over
Na2SO4, and concentrated to dryness. Product was isolated by column chromatography (0-
100% CH2Cl2 in hexane) in a semi-pure state as a red oil (0.038 g, 19%). Spectral data is presented for semi-pure compound.
1H NMR (400 MHz, Chloroform-d) δ 7.62 (td, J = 8.3, 5.2 Hz, 1H), 7.47 – 7.42 (m, 1H),
19 7.26 – 7.22 (m, 1H). F NMR (376 MHz, CDCl3) δ -101.69. Anderson 67
19a; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-chloro-6-fluorobenzene-1-sulfonamide
Bioorg. Med. Chem., 2017, 25, 2482-2490.
To a flame-dried vial was added 2-chloro-6-fluorobenzene-1-sulfonyl chloride (RA-i-122)
(0.038 g, 0.17 mmol), 4-[3,5-bis(trifluoromethyl)phenoxy]aniline (0.055 g, 0.17 mmol), CH2Cl2 (1 mL). After cooling to 0 °C, pyridine (0.5 mL, 6.2 mmol) was added dropwise. The reaction was stirred at rt for 5 hours, after which the solvent was removed in vacuo. The residue was partitioned between 10 mL each water and ethyl acetate, and the aqueous layer extracted 3x with 5 mL ethyl acetate. The combined organic layers were washed with 5 mL brine, dried over
Na2SO4, and concentrated to dryness. The crude product was purified by column chromatography (0-100% CH2Cl2 in hexanes, then 0-100% ethyl acetate) and finally recrystallized from hot hexane to give the product as tan crystals (0.037 g, 44%).
1H NMR (400 MHz, Chloroform-d) δ 7.56 (s, 1H), 7.44 (td, J = 8.2, 5.4 Hz, 1H), 7.34 (dt, J =
8.1, 1.2 Hz, 1H), 7.27 (s, 2H), 7.26 – 7.22 (m, 2H), 7.14 – 7.08 (m, 2H), 6.98 – 6.94 (m, 2H); 13C
NMR (101 MHz, CDCl3) δ 162.4, 159.8, 158.6, 153.2, 134.6, 134.5, 134.0, 133.6, 133.3, 132.6,
128.16, 128.12, 124.3, 124.1, 121.6, 121.1, 118.0, 116.8, 116.7, 116.5; 19F NMR (376 MHz,
- CDCl3) δ -63.04, -103.90. HRMS (ES-) [M-H] for C20H10ClF7NO3S, m/z 511.9958, found 511.9966.
19b; N-{4-[3,5-bis(trifluoromethyl)phenoxy]phenyl}-2-fluorobenzene-1-sulfonamide
Bioorg. Med. Chem., 2017, 25, 2482-2490. Anderson 68
To a flame-dried vial was added 2-fluorobenzene-1-sulfonyl chloride (0.146 g, 0.75 mmol), 4-[3,5-bis(trifluoromethyl)phenoxy]aniline (0.161 g, 0.5 mmol), CH2Cl2 (6 mL). After cooling to 0 °C, pyridine (2 mL, 25 mmol) was added dropwise. The reaction was stirred at rt for
4.5 hours, after which the solvent was removed in vacuo. The residue was partitioned between
10 mL each water and ethyl acetate, and the aqueous layer extracted 3x with 5 mL ethyl acetate. The combined organic layers were washed with 5 mL brine, dried over Na2SO4, and concentrated to dryness. The crude product was purified by column chromatography (0-80%
CH2Cl2 in hexanes) to afford the title compound as a white powder (0.186 g, 78%).
1 H NMR (400 MHz, Methanol-d4) δ 7.83 (ddd, J = 7.8, 7.2, 1.8 Hz, 1H), 7.63 (dddd, J = 8.3,
7.6, 5.0, 1.8 Hz, 2H), 7.36 – 7.31 (m, 2H), 7.30 – 7.22 (m, 4H), 7.03 – 6.98 (m, 2H); 13C NMR (101
MHz, MeOD) δ 160.77, 153.34, 136.83, 136.74, 135.84, 134.49, 134.16, 132.03, 128.45, 125.63,
125.59, 124.61, 122.10, 118.65, 1`118.10, 117.89, 116.96; 19F NMR (376 MHz, MeOD) δ -64.61, -
111.37;
20; 1-(4-iodophenoxy)-3,5-bis(trifluoromethyl)benzene
CF3 I
F3C O
Prepared as 15b. Purified by column chromatography to yield title compound as a colorless oil (0.644 g, 75%). Anderson 69
1H NMR (400 MHz, Chloroform-d) δ 7.75 – 7.70 (m, 2H), 7.60 (s, 1H), 7.39 (s, 2H), 6.86 – 6.81
19 (m, 2H). F NMR (376 MHz, CDCl3) δ -63.00.
6-4. NMR Spectra
Figure 8. 1H NMR for compound 1. Anderson 70
Figure 9. 13C NMR for compound 1.
Figure 10. 1H NMR for compound 2. Anderson 71
Figure 11. 13C NMR for compound 2.
Figure 12. 19F NMR for compound 2. Anderson 72
Figure 13. 1H NMR for compound 3a.
Figure 14. 13C NMR for compound 3a. Anderson 73
Figure 15. 19F NMR for compound 3a.
Figure 16. Crude 1H NMR for compound 3b. Anderson 74
Figure 17. Crude F19 NMR for compound 3b.
Figure 18. 1H NMR for compound 3c. Anderson 75
Figure 19. 19F NMR for compound 3c.
Figure 20. 1H NMR for compound 3d. Anderson 76
Figure 21. 13C NMR for compound 3d.
Figure 22. 19F NMR for compound 3d. Anderson 77
Figure 23. 1H NMR for compound 3e.
Figure 24. 19F NMR for compound 3e. Anderson 78
Figure 25. 1H NMR for compound 3f.
Figure 26. 1H NMR for compound 4a. Anderson 79
Figure 27. 19F NMR for compound 4a.
Figure 28. 1H NMR for compound 4b. Anderson 80
Figure 29. 19F NMR for compound 4b.
Figure 30. 1H NMR for compound 4c. Anderson 81
Figure 31. 13C NMR for compound 4c.
Figure 32. 19F NMR for compound 4c. Anderson 82
Figure 33. 1H NMR for compound 4d.
Figure 34. 19F NMR for compound 4d. Anderson 83
Figure 35. 19F NMR for compound 4f.
Figure 36. 1H NMR for compound 4f. Anderson 84
Figure 37. 19F NMR for compound 4f.
Figure 38. 1H NMR for compound 4g. Anderson 85
Figure 39. 13C NMR for compound 4g.
Figure 40. 19F NMR for compound 4g. Anderson 86
Figure 41. 1H NMR for compound 4h.
Figure 42. 19F NMR for compound 4h. Anderson 87
Figure 43. 13C NMR for compound 4h.
Figure 44. 1H NMR for compound 4i. Anderson 88
Figure 45. 19F NMR for compound 4i.
Figure 46. 13C NMR for compound 4i. Anderson 89
Figure 47. 1H NMR for compound 4j.
Figure 48. 19F NMR for compound 4j. Anderson 90
Figure 49. 13C NMR for compound 4j.
Figure 50. 1H NMR for compound 4k. Anderson 91
Figure 51. 13C NMR for compound 4k.
Figure 52. 19F NMR for compound 4k. Anderson 92
Figure 53. 1H NMR for compound 4l.
Figure 54. F19 NMR for compound 4l. Anderson 93
Figure 55. 1H NMR for compound 4m.
Figure 56. 19F NMR for compound 4m. Anderson 94
Figure 57. 13C NMR for compound 4m.
Figure 58. 1H NMR for compound 4n. Anderson 95
Figure 59. 19F NMR for compound 4n.
Figure 60. 13C NMR for compound 4n. Anderson 96
Figure 61. 1H NMR for compound 4o.
Figure 62. 19F NMR for compound 4o. Anderson 97
Figure 63. 1H NMR for compound 4p.
Figure 64. 13C NMR for compound 4p. Anderson 98
Figure 65. F19 NMR for compound 4p.
Figure 66. 1H NMR for compound 4q. Anderson 99
Figure 67. 19F NMR for compound 4q.
Figure 68. 1H NMR for compound 4r. Anderson 100
Figure 69. 13C NMR for compound 4r.
Figure 70. 19F NMR for compound 4r. Anderson 101
Figure 71. 1H NMR for compound 4s.
Figure 72. 13C NMR for compound 4s. Anderson 102
Figure 73. 19F NMR for compound 4s.
Figure 74. 1H NMR for compound 4t. Anderson 103
Figure 75. 13C NMR for compound 4t.
Figure 76. 19F NMR for compound 4t. Anderson 104
Figure 77. 1H NMR for compound 5.
Figure 78. F19 NMR for compound 5. Anderson 105
Figure 79. 1H NMR for compound 6.
Figure 80. 13C NMR for compound 6. Anderson 106
Figure 81. 19F NMR for compound 6.
Figure 82. 1H NMR for compound 7. Anderson 107
Figure 83. 13C NMR for compound 7.
Figure 84. 19F NMR for compound 7. Anderson 108
Figure 85. 1H NMR for impure compound 8.
Figure 86. F19 NMR for compound 8. Anderson 109
Figure 87. 1H NMR for compound 9.
Figure 88. 13C NMR for compound 9. Anderson 110
Figure 89. 19F NMR for compound 9.
Figure 90. 1H NMR for compound 10.
Anderson 111
Figure 91. 13C NMR for compound 10.
Figure 92. F19 NMR for compound 10. Anderson 112
Figure 93. 1H NMR for compound 11.
Figure 94. 13C NMR for compound 11. Anderson 113
Figure 95. F19 NMR for compound 11.
Figure 96. 1H NMR for compound 12. Anderson 114
Figure 97. 13C NMR for compound 12.
Figure 98. 19F NMR for compound 12. Anderson 115
Figure 99. 1H NMR for compound 13.
Figure 100. 13C NMR for compound 13. Anderson 116
Figure 101. 19F NMR for compound 13.
Figure 102. 1H NMR for compound 14. Anderson 117
Figure 103. 13C NMR for compound 14.
Figure 104. 19F NMR for compound 14. Anderson 118
Figure 105. 1H NMR for compound 15a.
Figure 106. 19F NMR for compound 15a. Anderson 119
Figure 107. 1H NMR for compound 15b.
Figure 108. 13C NMR for compound 15b.
Anderson 120
Figure 109. F19 NMR for compound 15b.
Figure 110. 1H NMR for compound 15c. Anderson 121
Figure 111. 19F NMR for compound 15c.
Figure 112. 1H NMR for compound 16a. Anderson 122
Figure 113. 13C NMR for compound 16a.
Figure 114. 19F NMR for compound 16a. Anderson 123
Figure 115. 1H NMR for compound 16b.
Figure 116. 19F NMR for compound 16b. Anderson 124
Figure 117. 19F NMR for highly impure compound 17.
Figure 118. 1H NMR for semi-pure compound 18. Anderson 125
Figure 119. 19F NMR for semi-pure compound 18.
Figure 120. 1H NMR for compound 19a. Anderson 126
Figure 121. 13C NMR for compound 19a.
Figure 122. 19F NMR for compound 19a. Anderson 127
Figure 123. 1H NMR for compound 19b.
Figure 124. 13C NMR for compound 19b. Anderson 128
Figure 125. 19F NMR for compound 19b.
.
Figure 126. 1H NMR for compound 20. Anderson 129
Figure 127. 19F NMR for compound 20.