THESIS
The Synthesis of Ligands for the Large Conductance Calcium Activated Potassium Channel
CONFIDENTIAL
In Partial Completion of the Degree of Doctor of Philosophy at University College London
Candidate: Eoin Power Supervisor: Prof. C.R.Ganellin ProQuest Number: U643003
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Dedication
I wish to dedicate the last three and a half years o f hard work and effort to my parents John and Bemie Power for their endless efforts on my behalf during the last twenty six. Acknowledgments
I would like to thank Professor Robin Ganellin for his assistance, advice and supervision. Thanks to Wasyl Tertuik for endless help and encouragement. In the Ganellin group Yong Jun Chen, Lejla Arifhodzic, Fabien Leurquin, Sanjeeda Samad, Patricia Zunzain, Ana Conejo Garcia, Salma Ishaq, Mario Sechi, Dang Lai Yang, and Henny Eggelte. The people on the fourth floor Sacha, Frieder, Linnea, Ruk, Andy, Gupreet, Mark, Mark (Junior), Soraya, Ying, Jorge, Alex, Alf, Kason, Chris, Prakash, and any that I may have overlooked. Alan and Jill in microanalysis, John and Steve in Mass Spec, Jorge and Abil in NMR. Thanks to all the lecturing and technical staff at the UCL Chemistry department. Thanks to Don Jenkinson, Guy Moss and Denis Flaylett in the UCL Pharmacology department for discussions on biology. Thanks also to David Benton in the UCL pharmacology department for testing the compounds and discussions on K-channel biology and electrophysiology. Thanks to my friends in Ireland and London. Benny, Caw, Claire, Cully, Donal, Eddie, Eamon, Caroline, Gav, Ger, Peter, Steve, Orla, Mike, Leigh, Caroline, Liz, Sian, Wes and Pauline for discussions on other equally pressing topics. Thanks above all to the Power’s Mum, Dad, Colm, Grainne and Maeve for endless love and encouragement. Also to Richard and Sinead my siblings other halves I don’t know how ye put up with us. Thanks to Eoin (Beag) for making me laugh last Sunday. Table of Contents
Dedication ...... 2 List of Abbreviations ...... 5 ABSTRACT OF THESIS...... 9 Schematic...... 10 1. Introduction ...... 11 1.1 The Kir family ...... 14 1.1.1 Inward rectifier K channels ...... 14 1.1.2 G-protein gated K-channel K^ch...... 15 1.1.3 KATp(Kir6)...... 16 1.2 6TM IP Channels...... 17 1.2.1 Voltage gated K-Channels ...... 17 1.2.2 Kv channels ...... 18 1.2.3 Eag channels...... 18 1.2.4 KCNQ Channels...... 19 1.3 Calcium activated potassium channels (K^a) ...... 20 1.3.1 Small conductance calcium activated potassium channels (SK) ...... 20 1.3.2 Intermediate conductance calcium activated potassium channels (IK) ...... 22 1.4 Large conductance calcium activated potassium channel (BKca) ...... 24 1.4.1 Structure of BK ...... 24 1.4.2 The Slo family ...... 25 1.4.3 Endogenous modification of BK ...... 26 1.4.4 Roles o f BK channels in vivo ...... 28 1.4.5 Pharmacology ...... 31 2. Selection o f compounds ...... 40 2.1 Series 1 ...... 40 2.2 Series 2 ...... 43 2.3 Series 3 ...... 44 2.4 Series 4 ...... 45 3 Synthesis ...... 47 3.1 The synthesis of ketoconazole analogues ...... 47 3.2. 2-Methyl-2-phenyl-4-phenoxy-1,3-dioxolane ...... 53 3.3 Non-dioxolane compounds ...... 62 3.4 Characterization o f Compounds ...... 68 3.4.1 Mass spectra 68 3.4.2 Assignment o f Isomerism by NMR 69 4. Molecular Modelling ...... 75 5. Biological testing ...... 78 6. Results and Discussion ...... 80 6.1 Series-1...... 80 6.2 Series 2 ...... 87 6.3 Series.3...... 92 6.4 Series.4...... 95 6.5 Summary ...... 101 6.6 Future work...... 102 7. Experimental ...... 103 7.1 General ...... 103 7.2 Syntheses ...... 105 8. Bibliography ...... 156 TABLE OF FIGURES
SCHEME 1 The synthesis of ketoconazole analogues ...... 47 SCHEME 2; Dioxolane ring formation ...... 48 SCHEME 3 Synthesis of phenol 54 ...... 49 SCHEME 4 Amide Hydrolysis ...... 50 SCHEME 5 Mechanism of Aryl Amination ...... 50 SCHEME 6 Synthesis of piperadine analogue ...... 51 SCHEME 7 Formation o f aniline ...... 52 SCHEME 9 Displacement of tosylate ...... 53 SCHEME 10 Formation of dioxolanyl Tosylate ...... 54 SCHEME 11 Cis and Trans isomers ...... 54 SCHEME 13 Displacement of tosylate ...... 55 SCHEME 14 Mitsunobu Mechanism ...... 56 SCHEME 15 Modified Mitsunobu ...... 57 SCHEME 16 Conventional Mitsunobu ...... 57 SCHEME 17. Salt formation ...... 58 SCHEME 18. Ring opening ...... 58 SCHEME 19 Combining useful compounds ...... 59 SCHEME 20 Synthesis of UCL-2273 ...... 59 SCHEME 21 Chlorophenyl Targets ...... 60 SCHEME 22 Synthesis of UCL-2333 ...... 60 SCHEME 23 Synthesis of UCL-2327 ...... 60 SCHEME 24 Synthesis of UCL-2335 ...... 61 SCHEME 25 Synthesis of 4-phenoxy analogues ...... 62 SCHEME 26 Glycerol targets ...... 62 SCHEME 34 Synthesis ofUCL-2304 ...... 66 SCHEME 36 Fragmentation of the Dioxolane ring ...... 68 SCHEME 37 Cis and Trans isomerism ...... 69 FIGURE 3.1 Cis Isomer ...... 70 FIGURE 3.2 Trans isomer ...... 70 MOLECULAR MODELLING RESULTS...... 76 FIGURE 1. Ketoconazole activity at the BKca channel ...... 80 CHART 1. Inactive partial Structures ...... 81 CHART 2. Investigating Piperazine...... 82 FIGURE 2 Graph comparing dose response o f UCL2242 and Ketoconazole ...... 83 FIGURE 3. Hydrogen bond acceptors ...... 84 FIGURE 4. UCL 2322...... 84 CHART3.UCL-2158 ...... 85 CHART 4. Parallel series ...... 85 FIGURE 5. Advantage of 2273 over 2158 and 224 2 ...... 86 CHART 5. Investigating the Dichlorophenyl moiety ...... 87 CHART 6. Further investigating the Dichlorophenyl m oiety ...... 88 FIGURE 6. Dose/Response for compounds in chart 6 ...... 89 FIGURE 7. Hypothetical binding pocket ...... 91 FIGURE 8. Comparison o f chloro with fluoro and methyl ...... 91 FIGURE 9. Cis and Trans Isomers ...... 92 CHART 7. Non Imidazole Blockers ...... 93 CHART 8. Non Imidazole Openers ...... 93 CHART 9. Acyclic Analogues ...... 96 FIGURE 10. Comparison of UCL-2334 with UCL-2273 ...... 96 FIGURE 11. Comparing effects of absolute stereochemistry ...... 97 FIGURE 12. Biphenyl BK chaimel openers ...... 98 CHART 10 Three atom chain dérivâtes...... 98 CHART 11 UCL-2304...... 99 CHART 12. UCL-2286 and UCL-2285 ...... 99 List of Abbreviations.
ADDP: Azodicarbonyl dipiperidine ADP: Adenosine diphosphate AHP: Afterhyperpolarisation (sAHP: slow afterhyperpolarisation) cAMP: cyclic adenosine monophosphate 4-AP: 4-Aminopyridine ATP: Adenosine triphosphate BK, BKCa: The large conductance calcium activated potassium channel BP ASM: Bovine pulmonary artery smooth muscle cells BPTI: Bovine pancreatic trypsin inhibitor CgTX: Conotoxin CTX: Charybdotoxin CNS: Central Nervous System DCM: Dichloromethane DHS: Dehydrosoyasaponin DM: Myotonic muscular dystrophy DMA: Dimethyl Acetamide DMEM: Dubelco’s Modified Eagle Medium DMF: Dimethyl Formamide DMSO: Dimethyl Sulfoxide Eag: Ether-a-gogo potassium channel 1-EBIO: Ethyl benzimidazolone
EC50: Concentration obtaining 50% of total optimal response EDHF: Endothelium derived hyperpolarising factor
EGTA: Ethylene Glycol-bis-(p-aminoethylether)
Ek: Equilibrium potential for potassium Elk: Ether-a-gogo like potassium channel Em: Cell membrane potential (at a given time) Erg: Ether-a-gogo related gene ES: Electrospray GDP: Guanidine diphosphate GIRK: G-Protein receptor coupled potassium channel G-Protein: Guanidine triphosphate binding protein GTP: Guanidine triphosphate H-bond: Hydrogen bond HEPES: (N-(2-Hydroxyethyl)-piperazine-N-[2-ethanesulfonic acid]) HPLC: High Performance Liquid Chromatography
IC50: Concentration that causes 50% inhibition o f response IK: Intermediate conductance K-channel
Kach- Acetylcholine gated potassium channel
K a t p - Adenosine triphosphate activated potassium channel Kir: Inwardly rectifying potassium channel Kca' Calcium activated potassium channel KcsA: Potassium channel from Streptomyces lividians KChiPs: Calcium sensor that associates with Kv4
KCNMB: Gene encoding the p-subunit of the BK potassium channel
KQT, KLQT, KCNQ: Potassium channels and genes linked to Long QT syndrome Ks: Potassium channel current in ventricle o f the heart Kv: Voltage gated potassium channel KvP: P-subunit associated with voltage gated channel
LQT: Long QT syndrome (cardiac arrhythmia) M-Current: Potassium channel modified by muscarine Mg ATP: Magnesium ATP complex MinK: Potassium channel protein associated with LQT MS: Mass Spectroscopy Ms: Methane sulfonyl NBF: Nucleotide binding fold NDP: Nucleotide diphosphate NMR: Nuclear magnetic resonance NOE: Nuclear Overhouser effect NOES Y : Spectra derived using NOE effect NS: NeuroSearch Inc PCR: Polymerase chain reaction QSAR: Quantitative Structure activity relationships Rf: Ratio of distance travelled by compound over distance travelled solvent front SHR: Spontaneously hypertensive rats SK, SKca: Small conductance calcium activated potassium channel Slack: Sequence like a calcium activated potassium channel Slo: Slowpoke gene, encoding the BK channel (e.g. mSlo; mouse slowpoke) Slob: Slowpoke binding protein Slip: Slowpoke interacting protein
Sn2: Bimolecular nucleophilic substitution SUR: Sulfonyl urea binding protein TEA: Tetraethylammonium THF: Tetrahydrofuran TLC: Thin Layer Chromatography TM: Transmembrane Section Ts: Toluene sulfonyl UCL: University College London UDP: Uracil diphosphate
Vi/2: Potential eliciting half maximal activation XED: extended Electronic Distribution ABSTRACT OF THESIS
Name of candidate: EOIN POWER
Title of thesis: SYNTHESIS OF LIGANDS FOR THE LARGE CONDUCTANCE
CALCIUM ACTIVATED POTASSIUM CHANNEL (BKca)
Large conductance calcium activated potassium ion channels are important in maintaining smooth muscle tone and a compound that increases this current could have potential therapeutic applications. The antihingal drug ketoconazole has been shown to increase current at these channels but only at high concentrations. Therefore partial structures o f ketoconazole were synthesised and tested in order to identify the pharmacophore
The results from this series led to the synthesis of 2-(dichlorophenyl)-2-methyl-4-
(phenoxymethyl)-1,3-dioxolane which was a strong blocker of the channel and 2 - (dichlorophenyl)-2-methyl-4-(4-aminophenoxymethyl)-1,3-dioxolane which was a strong opener. Variation of the substituents at the 4-position of the phenoxy moiety was investigated and found to be crucial in determining the type o f activity. Variation around the dichlorophenyl ring was performed but with no improvement for activity.
Molecular modelling was used in the search for a replacement of the dioxolane ring. It was found that l-(4-aminophenoxy)-2-hydroxy-3- (2,4-dichlorophenylmethyloxy)- propane folded in a similar fashion to the dioxolane. When synthesised and tested it was found to have and activity similar to ketoconazole.
These results will be discussed together with a review of potassium channel biology and pharmacology. Schematic
The action potential
Depohrbition Inflm: o f Ca or W à ions
Membrane potential Em
Influx o f K ions / Inward rectification Hyperpolarisation
Equilibiium potential Time -20mV for BK in smooth muscle
While at rest a cell is held at a negative potential Em that is detemiined by a combination of the equilibrium potentials for various ions. A biological stimulus causes an influx of Na^ or Ca^^ ions causing a depolarization of the membrane. This depolarization leads to an efflux of potassium ions, which carries the cell to hyperpolarised potentials. This hyperpolarisation is corrected back to the resting potential by an inwardly rectifying potassium current
10 1. Introduction
Potassium ions are concentrated in the interior of cells. The concentration of potassium inside the cell being ~200mM compared to an exterior concentration of ~2mM meaning that there is a concentration gradient. This gives rise to a potential difference which is mirrored and opposed by the concentrations of other ions such as sodium, calcium and chloride. These conflicting forces mean that the potassium current is central in controlling cell potential, which in turn is central to cell function. Potassium channels facilitate the moment of potassium through the lipid membrane. They are a large family of membrane bound proteins which are present in almost all cells and regulate a wide variety of functions. Since they are responsible for maintaining membrane potential in excitable cells they present a useful target for pharmacological intervention. All potassium channels can transport ions in and out of the cell. However this is determined by the position of the cell potential relative to the potassium potential. The potassium potential Ek is the potential at which there is no net current of ions. Where the membrane potential Em is more positive than Ek then ions will move out of the cell. This simple relationship is complicated by the fact that each type of channel contains a mechanism of activation (e.g. voltage, Ca^ concentration), that allows for the regulation of channel function in a particular cell. All potassium channels consist of 2 or 4 membrane spanning proteins (a-subunits) organized with a four-fold symmetry around a central pore region. They combine both specificity (10000:1, Na^) and high flux (10^ ions/see). Recently the crystal structure of a potassium channel (KcsA) from Streptomyces lividians has been solved by X-ray crystallography'. This illustrated several important points.
K Chàiuiel Cross section
K channel protein
Membrane Aqueous solution
Figure 1 Cross section of K-channel
11 The structure was a tetramer composed of eight a-helices fonning an inverted tee-pee, the top of which was closer to the intracellular matrix'. The amide oxygen atoms of the pore point into the pore and form the coordination sphere for the ion'. The pore region is closer to the extracellular matrix and the space between the pore region and the outer helices is an aqueous reservoir that stabilises ions at the centre of the structure. There are several ions in the pore at any one time so that a ion in the pore region is pushed out by the subsequent ion due to electrostatic repulsion. This allows for high flux along with high selectivity^. Although this was a bacterial K- channel the pore sequence is conserved well throughout the K-ehannel family and it is argued that the method of ion conduction is general'. K-channels represent a very wide range of proteins. In the past channels were classified according to function and/or phannacology. However molecular biology has provided a more fundamental classification for potassium channels as well as a deeper understanding of their properties^’^. These are classified according to numbers of transmembrane (TM) spanning regions and also to the number of pore regions in the a-subunit. A transmembrane section is a section of the protein that spans the cell membrane. Typically the subunit protein will contain several sections of protein that zigzag in and out of the cell; each defined as a transmembrane section. It will also contain a sequence that will fonn the selectivity filter of the channel called the pore section. There are three structural classes in mammals. The 2TM IP'^’ ^ '^(2 transmembrane, 1 pore), 4TM 2P" (4 transmembrane, 2 pore) and the 6TM IP ^"^(6 transmembrane, 1 pore).
2TM IP pore 4TM 2P 6TM IP
external
C T ransnienibraiie segment
Kiri-6 TWIK, TRASK. Kv. eag. TREK KLQT. Slo
12 O f these the 2TM IP and the 6 TM IP are the most significant in function. The 4TM 2P channels are thought to form the leak current in humans that determines the resting potential'\ These general structural classes are in turn subdivided into families based on sequence and or functional homology forming a large family of related proteins. These are summarized below.
6TM IP eag eag, elk, erg KQT KQTl, KQT2, KQT3 SK SKI, SK2, SK3, SK4 Slo SIol, Slo2, Slo3 Kv Kvl K v l.l, K vl.2 - K vl.7 Kv2 Kv2.1,Kv2.2 Kv3 Kv3.1,Kv3.2,Kv3.3,Kv3.4 Kv4 Kv4.1, Kv4.2, Kv4.3 Kv5 Kv5.1 Kv6 Kv6.1 Kv8 Kv8.1 Kv9 Kv9.1,Kv9.2,Kv9.3
4TM 2P TWIK, TREK, TASK, TRAAK
2TM IP Kir Kiri K irl.l Kir2 Kir2.1, Kir2.2, Kir2.3, Kir2.4 Kir3 Kir3.1, Kir3.2, Kir3.3, Kir3.4 Kir4 Kir4.1, Kir4.2 Kir5 KirS.l Kir6 Kir6.1, Kir6.2 Kir? KirT.l
The large number of cloned channels is greater than the number of observed native currents. It has been argued that this reflects continual tuning for optimal tissue specific expression". While the number of cloned channels is greater than the number o f native currents there are also a number o f other factors that modify the function of K-channels. 1. Firstly there is the existence of auxiliary subunits that modify or regulate channel function and pharmacology'^. 2. Secondly heteromultimer formation" (i.e. related but non-identical subunits forming a channel with mixed properties). 3. Thirdly alternative splicing, this is where parts of the sequence are omitted during the biosynthesis of the channel".
13 4. Finally postranslational modification, this includes phosphorylation^^ or modification by endogenous substances’^. These factors will be discussed in detail later where relevant to specific channels.
Much is now known about how the molecular biology and structure o f potassium channels determine the channels’ physical properties^. However the physiological role for this molecular diversity is not yet clear. The remainder of this discussion will only discuss the cloned channels that have been related to known currents, in particular the large conductance calcium activated potassium channel BKca-
The Kir family
Kir channels are channels with the general 2 transmembrane 1 pore subunits and were first cloned in 1993 There are currently 6 members of the Kir family (Kiri- 6 ). Kir genes are known to code three physiologically important currents. The inward rectifiers (Kir2) G-protein activated K-channels (Kir3) and ATP-sensitive K channels (Kir6 ) The other families Kiri, Kir4 & Kir5 express weak inward rectifiers, either homogeneously or as heteromultimers
Inward rectifier K channels.
Inward rectification describes the property that potassium ions can enter the cell at potentials negative to the potassium equilibrium potential (E k) more readily than it can leave the cell at positive potentials. This rectification of the K current has been inferred to play an important role in the long duration plateau o f the cardiac action potential Kiri and Kir2 both express K channels in vivo. Kirl^ expresses a weak rectifier whereas Kir2 expresses classical strong inward rectifiers’^. There are three subtypes known (Kir2.1, Kir2.2, and Kir2.3) that are widely expressed’^’ The cloned channels are indistinguishable from native channels in the heart^^ and glial cells^^. Kir channels do not have a voltage sensor. The voltage dependence of the current depends on Mg^^ ions^'’ or polyamines^^ blocking the channel internally at depolarized potentials. The binding of the blocking particles is also affected by high external [K^]^^. This blocking prevents the flow o f current in or out at positive potentials. At hyperpolarised potentials the ion or amine is no longer held in the pore and K^ ions are free to pass through. The endogenous amines thought to block the Kir channel are thought to be spermine and spermidine ’^. The block by spermine and
14 spermidine is stronger than that o f Mg^^. This explains why in situ inward rectification is steeper than would be predicted by Mg^^ block alone. Divalent cations have been shown to block other channels and weak rectification can be observed in
OC OA OQ many channels however polyamine block of Kir2 and Kach channels is responsible for strong inward rectification.
G-protein gated K-channel Kach
The Kir3 family encodes the G-protein regulated, inwardly reetifying K-ehannel (GIRK) There are four subtypes of GIRK (GIRKl -GIRK4) which are distributed throughout the heart, brain and pancreas^^. The cardiac channel Ikach is known as such due to its first identified agonist, acetylcholine. A range o f neurotransmitters activates the other GIRK channels^ \ Homologous expression o f GIRK subunits yields no current in the case o f GIRKl or currents that are not consistent with native channels for GIRK2, GIRK3 & GIRK4 iKACh is formed by a heteromultimer of GIRKl and GIRK4 subunits thought to associate on the order GIRKl-GIRKl- GIRK4-GIRK4 It is suggested that the association of GIRKl subunits with GIRK2, GIRK3 or GIRK4 may form native currents in other tissues The GIRK channel that has been studied in the most detail is Ikach since it plays a major role in the regulation o f the heart rate^"^. There are several currents implicated in the regulation of cardiac pacing, one of which is iKACh^^- This has been shown by experiments on knockout mice^^. Cardiac muscle only expresses GIRKl and GIRK4. Therefore GIRK4 knockout mice did not express I^ACh and could be compared to wild type mice. This showed that the Ikach branch of the parasympathetie cardiac regulatory system is required for rapid adjustments o f the heart rate even at rest. Also it was shown that IxACh is responsible for half the bradycardia (slow heart rate) induced by vagal stimulation and adenosine administration^^.
G-proteins are guanosine diphosphate binding proteins made up o f three subunits a, p and y. When agonist binds to the receptor GDP becomes GTP and the protein splits into a and py. In the case o f Ikack it has been shown that the Py subunit then interacts with the C-terminus of GIRK4 to activate the channel
15 K atp (K ir6)
ATP sensitive channels (K a tp) are weak inward rectifier channels that are an important link between cell metabolism and electrical activity^^. They are known to play a role in a variety of functions including insulin secretion"^^, excitability of skeletal muscle"^* and neurons"^^, recycling in renal epithelia"^^ and cytoprotection in cardiac and brain ischemia"^"^. Although they are called ATP sensitive channels the activity is possibly regulated by the ATP/ADP ratio. ATP binds to the channel causing it to close whereas Mg ADP opens the channef^. One of the most well
characterised roles for Kat? channels is in pancreatic p-cells where they are known to
mediate glucose mediated insulin release"^^’ Glucose enters the cell by active transport and its subsequent metabolism produces ATP. The increase in the ATP/ADP
ratio closes K atp channels, which in turn depolarizes the cell membrane and opens voltage dependent calcium channels. The increase in Ca^^ concentration triggers insulin secretion by granular exocytosis. There are several pharmacological agents
that can block or open K atp channels. Sulfonylurea drugs such as tolbutamide and glibenclamide block the channel. The channel is opened by drugs such chromakalim, 1 evchromakalim and diazoxide It was observed that the strength o f blocking or opening varies from tissue to tissue. This selectivity has now been given a molecular basis.
K a t p channels are formed from the co-assembly of Kir 6 and SUR proteins"^^. SUR is
called the sulfonylurea binding protein; it is the ATP binding section of K a t p- There are two Kir proteins (Kirb.l and Kir6.2) and three SUR subtypes (SURl, SUR2A and
SUR2B). Differing combinations of a Kir 6 subunit and a SUR subunit constitute K atp channels with different nucleotide sensitivities and pharmacological properties. Kir 6.2 and SURl form a weakly inwardly rectifying channel with a unitary conductance o f 76pS which is sensitive to ATP (Ki = lOpM)"^^. It is blocked by glibenclamide and opened by diazoxide. Therefore the currents reconstituted from SURl and Kir6.2
show features characteristic of the K a tp currents in pancreatic p-cells"^^’
Reconstitution o f Kir6.2 and SUR2A yielded a current that was similar to Kir6.2 and S U R l. The currents had similar conductance and were blocked by glibenclamide. However the burst duration was longer for Kir6.2 and SUR2A. Also the channel was not opened by diazoxide but was opened by chromakalim and glibenclamide.
Therefore this channel resembles the cardiac and skeletal muscle K atp channel
16 Reconstitution of Kir6.2 and SUR2B yields a channel with a conductance of 80pS but
is less sensitive to inactivation by ATP (Ki = 6 8 p,M). It is also more sensitive to block
by glibenclamide and tolbutamide. These pharmacological properties are similar to
the K atp channel in smooth muscle Co-expression of Kirb.l and SUR2B yielded a channel that was only sensitive to ATP at high concentrations (>0.1 mM) and was activated by UDP and GDP. Nicorandil and pinacidil also activated the channel. This
is similar to K n d p the nucleotide diphosphate dependent K-channel in vascular smooth muscle In summary;
Kir6.2 SURl Pancreatic P-cells
Kir6.2 SUR2A Cardiac and skeletal muscle Kir6.2 SUR2B Smooth muscle Kirb.l SUR2B Vascular smooth muscle
6TM IP Channels
Channels containing six transmembrane (S1-S6) and one pore (P) per subunit represent a diverse and important structural class o f potassium channels. These channels are formed by a tetramer o f subunits, with the pore section o f the subunits
lying between S5 and S 6 The putative voltage sensor is S4 There are several families within this structural class and for the purposes o f explanation they shall be split into voltage gated and calcium activated.
Voltage gated K-Channels.
There are three families of voltage gated K channels. The eag, KCNQ (KvLQT) and Kv channels. Each of these contains several members that in turn contain various subtypes. As has already been stated in this introduction that the role for this diversity is not fully understood. While the channels are classified according to their primary sequence it is worth noting that the former classification based on physical properties is still useful when describing channel function. These terms are the transient and delayed rectifier. Transient rectifiers (A-current) respond to depolarization rapidly but is rapidly inactivated even though the depolarization remains^^. A delayed rectifier remains activated as long as the depolarization is present but is more slowly activated^^.
17 Kv channels.
Kv channels were the first potassium ehannels to be cloned and sequenced^. Subsequent searches for similar proteins led to the discovery of many related genes making Kv the most diverse elass of K-channels. Kv channels encode outwardly rectifying K^ currents and play a central role in the function o f excitable as well as non-exeitable eells^^’ Several subtypes Kvl.x, Kv3.x and Kv4.x have been linked to K^ currents in cardiae and neuronal tissue^^’ However it is thought that Kv5,
Kv 6 , Kv 8 and Kv9 ean only form K-channels as heteromultimers with Kv2^^’ In the Kvl family the channels form slowly rectifying K-channels, they ean however
form A-type currents when they are expressed with the auxiliary K vpl^.
Physiological roles for several of the Kv channels have been suggested. The paranodal delayed rectifier K^ eurrent in mammals is made up of heteromultimers of Kvl.l and Kvl.2^^ and over aetivity resulting from demyelination of axons is implicated in diseases such as multiple sclerosis^^. K vl.3 is involved in the production and proliferation o f T lymphoeytes^^ and as such presents an interesting target for immunosuppresion. Transient currents in axon terminals are possibly due to K vl.4 and K vl .5 have been linked to the ultrarapid rectifying current in atrial myocytes^®. Kv3 channels are involved as heteromultimers in the repolarisation after short action potentials in fast spiking neurons. Kv4.2 forms a transient current in dendrites and somata in the CNS^^. Kv4.2 and Kv4.3 heteromultimers form the transient outward current in ventrieular myocytes^^. A family o f calcium sensors KChlPs modulates Kv4 channels in their inactivation kinetics^^. Kv channels are blocked to varying degrees by TEA^^ (tetraethylammonium salts), 4-AP^^ (4-aminopyridine), dendrotoxin^®, margatoxin^% verapamil and charybdotoxin^^.
Eag channels.
The eag (ether a go-go) phenotype has been known since 1969 on the basis of a mutation causing ether sensitive leg shaking in drosophilia^. This gene was isolated and cloned^. To test for similar related genes PCR screens were carried out in drosophilia and mammals. The genes discovered were erg (eag related gene) and elk (eag like gene)^"^. The eag product had many similarities to the cyclic nucleotide binding cation channel including a cNBF^^. Surprisingly then, when the channel was
18 expressed in Xenopus oocytes the current observed was voltage dependent, outwardly rectifying and highly selective for over Na^ Further features of the eag channel are sensitivity to block by external Mg^^ and They have been shown to interact
with the KvP subunit and co-precipitate with the slowpoke binding protein SloB^^.
Eag channels are also blocked when the activation of the muscarinic receptor releases internal Ca^^ Due to this feature, insensitivity to block by TEA or 4-AP and the lack of inactivation the rat eag current was proposed to be the molecular co-relate of the M-current in the pyramidal cells o f the neo-cortex^ \ However, this view has been disputed and other channels have been shown to better match the properties and tissue distribution of the wild channel^^. The erg channel is the only channel of this type to be related to a function in vivo. Mutations of erg have been linked to Romano-Ward syndrome, a form of long QT syndrome*^. Another K channel KvLQT 1 has been associated with the syndrome. The Q and T in question are peaks on the electrocardiogram (ECG). LQTS is potentially fatal cardiac arrhythmia, which is brought about by K-channel blockers, type III anti- arrhythmic drugs or antihistamines in susceptible patients. The outward delayed rectifier channel in ventricular myocytes is composed of two components Kr and Ks. The erg gene is known to encode a channel almost identical to Kr*"^. Biophysical properties are similar, inward rectification (12pS) at hyperpolarised potential and small (5pS) rapidly inactivating currents at negative potentials. Class III antiarrhythmics, (+)-sotalol, E-4031, MK-499 and dofletilide also block the channel. Even though the properties o f erg and Kr are very similar it is thought that the co assembly of erg with minK forms the wild current. Firstly this was shown by the diminished Ikt in a human cell line caused by minK antisense suppression*^. Secondly it has been shown that minK and Herg (human erg) form a stable complex*^. It was also shown that co-expression o f minK and Herg increase the number o f functional channels on the cell surface and so increases the macroscopic current*^. As yet the function of elk is unknown.
KCNQ Channels.
KCNQ is a family o f three proteins KCNQl (KvLQTl), KCNQ2 and KCNQ3**'^. They are closely related to the Kv family of potassium channel genes.
19 As with the Herg gene, mutations in KvLQTl are associated with LQT the disorder causing ventricular arrhythmia and sudden death. KvLQTl is known to be essential
for the slowly rectifying current Iks in ventricular myocytes^^. However the channel formed by KvLQTl alone does not have the conduction characteristics of the wildtype channel. The minK protein is required for KvLQTl to function as Ks^\ It has been shown that when co-expressed with minK, KvLQTl forms a channel with identical properties to Ks^^. Large increases in current amplitude with slow activation and deactivation. The two proteins have also been shown to co-immunoprecipitate giving direct evidence o f co-assembly^^. It has also been shown that minK is involved in forming the pore of the channel^^. The KCNQ2 and KCNQ3 genes have recently been shown to form a current, which is very similar to the M-current. The M-current is a slowly activating and deactivating current that plays a critical role in determining the sub-threshold activity o f neurons as well as the responsiveness to synaptic inputs^"^. The channel formed from a heteromultimer of KCNQ2+KCNQ3 not only had similar biophysical properties but the same sensitivity to block by XE991 and linopiridine. No eag or Kv channel had a similar sensitivity. The KCNQ2+3 channel also had low sensitivity to block by TEA, although KCNQ2 is blocked by much lower concentrations. Therefore the heteromultimer of KCNQ2+KCNQ3 is the most likely candidate so far for the M- current^^.
Calcium activated potassium channels (Kca)*
There are three general types of calcium activated potassium channel, which are classified according to conductance. The large conductance (BK), intermediate conductance (IK) and the small conductance (SK) calcium activated potassium channels.
Small conductance calcium activated potassium channels (SK).
SK channels play an important role in all excitable cells. They are particularly involved in the regulation of firing patterns in the nervous system. SK channels have been shown to operate in hippocampal neurons^^, vagal neurons^^ and cortical neurons^^ among others.
20 Dysfunction of SK channels has been implicated in several conditions including myotonic dystrophy (DM)^^ and bipolar disorder^^. Skeletal muscle from patients with DM contains apamin sensitive SK channels whereas normal skeletal muscle does not. Apamin is a potent peptidic blocker of SK isolated from bee venom. Apamin has also been shown to abolish myotonia in a patient DM while having no effect on a patient with mytonal congenita, which is a disorder due to defects in a chloride channel^^^. SK channels are also involved in maintaining circadian rhythm^and in processes of learning and memory SK channels are voltage insensitive and activated by the increase in calcium concentration after an action potential (conductance ~10pS). The association of the calcium binding protein calmodulin with the C-terminus in the SK protein possibly mediates the calcium sensitivity of the SK channelsThere are none o f the consensus Ca^^ binding motifs on the SK protein itself This activation causes a small outward current that lasts for as long as the increased calcium concentration is elevated, prolonging the period of hyperpolarisation. The current is called the slow afterhyperpolarisation (sAHP)'^^. Through this the SK channels regulate the firing pattern o f cells. There are two distinct types o f sAHP, apamin sensitive and apamin insensitive. These can often be found in the same cells’®^ although their tissue distribution does vary. In addition to differences in apamin binding these channels show differences in kinetic properties in vivo. The apamin sensitive channel activates more rapidly than the apamin insensitive channel and is sometimes referred to as the medium mAHP. It has also been shown in a cell containing both currents that mAHP can be activated by a single or a short burst o f action potentials whereas sAHP required long bursts of action potentials to evoke the current The differences in apamin sensitivity were partially explained when the SK channels were cloned'Thus far there are three members o f the SK family (SKI- SK3). SKI channels showed the apamin insensitivity and similar tissue distributions to the apamin insensitive channels. It also lacked aspartate and asparagine residues known to be essential for apamin binding. SK2 and SK3 were both apamin sensitive and are distributed similarly to the apamin sensitive AHP in vivo'®'^. All the cloned channels are similarly sensitive to calcium. It is possible that the differences in the sAHP gating may be due to differences in the rate of calcium exposure"''^' Apamin is a charged peptide and the potency of binding is dependent on this. Several other charged compounds are known to act as blockers of SK channels such as
21 scyllatoxin, tubocurarine and dequalinium’^^. QSAR studies on dequalinium led to the
discovery of bisquinolinium cyclophanes as potent blockers of the SK channels. One of these compounds, UCL-1684 was the first nonpeptidic blocker of the SK channel with nanomolar potency^ It was also selective for apamin sensitive over apamin insensitive SK subtypes.
Dequalinium UCL-1648 1-EBIO
HN NH
> =0
H,N
Several other quinolinium analogues have been shown to block both IK and SK channels The openers o f IK, chloezoxazone and 1-EBIO are known to open the SK2 channel^ It has also been shown that several neurotransmitters affect SK channels in the CNS**^.
Intermediate conductance calcium activated potassium channels (IK).
IK channels are voltage insensitive but calcium sensitive channels with conductance in the range 11- dOpS'^"^. Unlike the other calcium-activated channels (SK and BK) they are predominantly expressed in non-excitable tissues^ These include epithelial tissues in placenta and lung^^^ as well as T-lymphocytes^ ^^ and red blood cells^^^. Blocking IK in red blood cells would slow the dehydration seen in sickle cell anemia^ and has been suggested as a treatment for the disease. In T-lymphocytes IK channels are responsible for much o f the current subsequent to activation by antigens and it has been shown that blocking IK channels can affect T-cell proliferation^’^. IK may also be important in the secretion and absorption in salt transporting epithelia” ^. It has been shown that IK in combination with SK is involved in the action of EDHF (endothelium-derived hyperpolarizing factor). IK was the first type of calcium activated potassium channel to be described but the last to be cloned’^^"’^^. This was carried out using screens based on homology with the
22 SK channels. Three groups diseovered the IK gene independently at approximately the same time. It was initially deseribed as the SK4 gene’^°, however the homology with the SK ehannels was only ~ 40%. Thorough investigation of the tissue distribution, physical properties and pharmacology showed that the cloned channel matched the properties described for IK^^"^’ As yet only one IK gene has been discovered. The cloned channels are calcium activated and voltage insensitive. The pateh clamp current was inwardly reetifying in symmetric K^ with a conductance o f 9- 30pS^^\ This has also been observed in red blood eell IK channels^IK channels also have a
large range of toxins that modify their activity Charybdotoxin blocks IK with IC50 = 28nM. The channel is also bloeked by margatoxin (ICso^ 459nM) and Stichodactyla
toxin (IC 5o= 298nM). However apamin whieh bloeks SK2 and SK3 is inactive. It is not blocked by Iberiotoxin, which blocks the BK ehannels. Clotrimazole (ICso=
153nM), eeonazole (IC 5o= 2.4pM) and mieonazole (ICso^ 785nM) all block the ehannel whereas ketoeonazole is inaetive’^*^. The channel is bloeked by nitrendipine’^^
(ICso= 29nM) and cetiedil’^^ (ICso= 77pM) but opened by 1-EBIO” ^ (ECgo^ 76pM). It is also activated by chloroxazone. The cloned ehannels are similarly sensitive to wild type and the numbers given are from the cloned ehannel”'’. In addition to the cloned ehannel it has been shown that Slo subunits of BK channels can co-assemble with another K-channel subunit called Slaek to form ehannels with intermediate conduetance and some voltage sensitivity’^^. Sinee these subunits are present in exeitable tissue and in the nervous system they may be responsible for intermediate eonduetanee currents which have been observed in excitable cells where IK is not expressed’^*. As with SK ehannels it has been shown that the ealcium sensitivity of IK is due to the association of the C-terminus of IK with the calmodulin protein’^^. However IK is more sensitive to Ca^^ than SK, allowing aetivity even at rest or when an intracellular process liberates Ca 2+121
23 Large conductance calcium activated potassium channel (BKca) Structure of BK.
The BK channel
extra
lT intra
subunit « subunit
voltage pore unit calcium pocket
The large conductance calcium activated potassium channel is distinct from IKCa and SK duc to its voltage sensitivity and large conductance (240pS). It has a structure unique in potassium channels and it is worthwhile to note how the important structural features affect the aetivity of the channel. BK is made up of a tetramer of a- subunits that can also bind auxiliary p-subunits'^^. The a-subunit was originally isolated and cloned from the drosophila slowpoke gene^. It has also been cloned from mammalian and human sources and is widely expressed Slo is made up of seven transmembrane sections labeled S0-S6 (-300AA) and a long C-tenninal chain labeled S7-S10 (-600AA). The N-tenninus is located in the extra cellular matrix and the SO section is vital for the association of the p-subunit'^^. The S1-S6 section is similar to the voltage-gated channel, with the voltage sensor at S4 and the pore containing the Gly, Tyr, Gly signature lying between S5-S6 This sequence is the most common pore sequence in potassium channels with the tyrosine being especially important in contributing towards the specit'icity of the pore. The section from S7-S10 contains four hydrophobic sections that were originally thought to fonn transmembrane segments However it has subsequently been shown that all four sections are contained in the cytosolThe S7 and S8 section are poorly conserved between species but S9-S10 contains a region of negative charge called the
24 calcium bowl that is well-conserved This C-terminus has several points for splice variation, which means several gene products can come from the same gene'"^*^. Searches for genes similar to Slo has led to the discovery of two further members of the Slo family, Slo2 and Slo3 Comparing these to BK it is clear that Slo is a member o f a potassium channel family that links cell potential to a variety of intracellular factors.
The Slo family
Slol forms the large conductance calcium activated potassium channel and will be the topic for much of the discussion. However it is worthwhile to compare the properties o f Slol with Slo2 and Slo3. Slol forms channels that are activated by voltage and intracellular calcium^"^^. However at sufficiently depolarized potentials the channels can be activated even in the absence of calcium^'^'^’ The overall effect of calcium on the channel is to shift
the potential for half-maximal activation (V 1/2), to more negative potentials’"^^. The channel has a conductance of 240pS in symmetrical It is very sensitive to block by Iberiotoxin and Charybdotoxin. It is widely distributed in human tissue. In contrast Slo2 forms a channel which requires both Ca^^ and C f The binding of the ions is synergistic and Slo2 cannot be activated in the absence of either ion’'^. The current is voltage sensitive but surprisingly Slo2 does not contain the positive charges at S4 that are thought to form the voltage sensor” ’ It does not have an SO
section’"” and so can not therefore interact with the P-subunit. The S9-S10 section contains a series of positive charges that have been shown to effect the Cl" binding’"’^. Although the sequence for Slo2 was originally isolated from C.elegans^\ high levels of expression have been shown in human muscle and neurons” . However, while Slol has been linked to specific roles in these tissues the role for Slo2 is unknown. Slo2 channels have a conductance of I lOpS in symmetrical Slo3 also has a unique set of characteristics. Slo3 is insensitive to Ca^^ but sensitive to voltage and pH Almost no current is observed at pH 7.1, while maximal activation was seen at pH 8.0 Slo3 channels are located specifically in the spermatocytes and are thought to play a significant role in the cell cycle” ^. Although they do contain the SO section there is no Ca^^ bowl in S9-S10 and the pore sequence replaces Gly, Tyr, Gly with Gly, Phe, Gly. The replacement of the tyrosine with the phenylalanine leads
25 to a loss in specificity for over Na"^ Charybdotoxin or iberiotoxin do not block Slo3 channels. The unitary conductanee in symmetrical K"^ is 90pS In addition to Slo2 and Slo3 the Slack channel is similar to Slo (slack = sequence like a calcium activated potassium channel). It has been suggested as the mammalian homologue of Slo2, however the sequence homology between slack and Slo2 is -40% compared to -60% between drosophila Slo^^'^ and human Slo^^^ In addition to this the physical properties of the channels are distinct. Slack forms a channel with a unitary outward conductance of 25-65pS that is inhibited by increases in intracellular calcium^^^. The channel contains the long intracellular C-terminus of Slo but it lacks the S4 voltage sensor and the SO p-subunit-binding domain. Heteromultimers of
Slack and Slo have been shown to form charybdotoxin resistant channels resembling a neural IK channelGiven that slack is widely expressed in the nervous system and IK is not, then it is possible that heteromultimer of Slack and Slo form some of the IK channels described in the nervous system. Slo2, Slo3 and Slack are poorly characterised in comparison to Slol, which has been extensively studied. One area that has received a large amount of attention is the modification of BK channels by endogenous factors. These can have a large effect on channel function and pharmacology. Therefore they will be an important consideration for the design o f new pharmacological agents in the future.
Endogenous modification of BK
BK channels are modified directly and indirectly by a large number of factors. Phosphorylation and dephosphorylation mediate indirect modulation of BK^"^^. The activation of a protein kinase or by a ligand causes the enzyme to directly phosphorylate the channel. The channel is then dephosphorylated by protein phosphatase 2A^^®. There are several sites for phosphorylation on BK*^\ Phosphorylation can either activate or inhibit the current^A possible explanation for the differing effects was given when cAMP dependent protein kinase was applied to cloned channels In channels with a-subunits only the phosphorylation increased the Popen^"*^ (opening probability). However in channels of a-subunits coupled to P-subunits the P q pen was decreasedExamples o f substances that act indirectly on BK by activation of protein kinase or otherwise are stress hormonessomatostatin and arachidonic acid^^^. NO directly activates the
26 channeland it has also been shown that hSlo expressed in Xenopus oocytes are modulated by redox processes. The application of an oxidizing agent reduces the channel activity. Reducing agents enhance and stabilize the activity of Of the endogenous substances that directly modify BK activity the most closely
associated are the P-subunitsThey are widely expressed in vivo^^^'^^* and
extensively modulate the activity of p-Subunits contain two
transmembrane sections connected by an extra-cellular loop^^"^, which bind to the SO
segment of Slo^^^. It has been shown that p-subunits can markedly change the
physiology*and the pharmacology*^^’ *"*^ o f the channel. For example the
soyasaponin activators of BK can only act when the p-subunit is present and the
potency of charybdotoxin is modulated depending on the subtype*^^’*^**. So far four
separate p-subunits have been isolated and sequenced (KCNMBl-4). These subunits
all interact with Slol when co-expressed, however they modulate channel behavior in different ways and are expressed in different tissues.
pi is only expressed in smooth muscle and is not expressed in neural tissue. Co
expression of pi with Slol increases the apparent Ca^^ sensitivity, slows activation kinetics, deactivation kinetics and increases charybdotoxin binding.
P2 have a similar sequence to P 1. It is expressed at high levels in the ovaries and at lower levels throughout the body. When combined with Slol it slows activation, increases apparent Ca^^ sensitivity and increases charybdotoxin binding *^*. However
it differs from the other P-subunits in that the N-terminus can block the channel
causing rapid inactivation o f the current *^^. A P-subunit may be the basis for the rapid
inactivation o f BK in some tissues *^^'*^"*.
p3 have limited effects on Slol currents when a and P subunits are co-expressed*^*.
However they do associate with the channel and cause a modest increase in activation
rates*^*. It is worth noting that P3 are mainly expressed in the testis where Slo3 is also
exclusively expressed*"*^. The relation between P3 and Slo3 has not been investigated but it is possible that they may associate in vivo.
p4 are widely expressed in the nervous system*^*. When expressed with Slol P4 varies the behavior broadly at different Ca^^ concentrations. They radically slowed the onset of action and totally removed inactivation at high Ca^^ concentrations, but show similar decreases in activation to p 1 and p2 at low Ca^^ concentrations. Also calcium sensitivity is increased at high Ca^^ levels and decreased at low Ca^^ levels*^*. This
27 causes unusual shifts in the current voltage curve suggesting that P4 can modulate the activity of BK differently in different concentrations^^\ This suggests an important role for p4 in tuning the electrical activity of BK in neurons, a + p4 BK channels are insensitive to charybdotoxin and iberiotoxin^^^.
P-Subunits interact with the BK channel in an “all or nothing” fashion. This means that two distinct sets o f properties are expressed rather than a mixture o f the two. This was demonstrated by experiments where 4 splice variants of Slol were co-expressed with a p-subunit from quail cochlea. Co-expression gave rise to 8 currents with separate properties
Other proteins that interact with Slo in addition to the P-subunit are Slob and Slip.
Slob (slowpoke binding protein) was discovered in drosophila''^. In heterologous expression systems it has been shown to co-localize with dSlo into large intracellular doughnut structures removing the Slo protein from the cell membrane and may therefore be important in the processing of Slo^^. It was also shown to increase the current in cell excised patches containing Slo channels^^. This increased current may be due to Slob acting as an adapter to bring signaling proteins into close proximity to Slo. Slob does not interact with hSlo (human) or mSlo (mouse) but does interact with Eag^^. The precise purpose of Slob in vivo is unknown. Slip (Slowpoke interacting protein) was also discovered in Drosophila but does interact with hSlo and mSlo^^^. It was shown to reduce the macroscopic current when co-expressed in Xenopus oocytes^^^. Since it was shown that it does not change the conduction characteristics o f the channel it is probable that Slip reduces the current by reducing the number o f BK channels in the cell membrane^It has been shown that processing of BK channels subsequent to protein synthesis is important in the nervous system and it is possible that Slob and Slip (or homologues) are important in this process.
Roles of BK channels in vivo.
BK channels are widely expressed and are found in all excitable cells. Three of the most important functional roles for BK in vivo are maintaining smooth muscle tone^^^, forming the fast phase of afterhyperpolarization and regulating neurotransmitter release^^^. Smooth muscle cells depolarize and contract in response to increased pressure. This constriction, which is independent of any external signals, is called myogenic tone.
28 This tone is brought about by the action of voltage dependent calcium channels. It can be abolished by removing Ca^^ from the external solution or by treating the tissue with calcium channel blockers. Since intracellular calcium and depolarisation activate BK channels they are ideal candidates to regulate potential and maintain a constant smooth muscle tone^^^’^^^. They are also widely expressed in smooth muscle^^^. While there are other channels that function in smooth muscle it is clear that BK plays an important role in maintaining myogenic tone^^^'^^\ BK was first shown to maintain tone in the rabbit cerebral artery where the application o f charybdotoxin and iberiotoxin caused the loss of myogenic tone^^^. Subsequent to this a similar effect was shown on uterine tissue. More selective experiments on porcine coronary artery showed that under resting conditions charybdotoxin caused little vasoconstriction but 4-AP caused significant vasoconstriction^^^. This shows that in resting conditions the tone is controlled by Kv. However when the tissue was stimulated to mimic hypertensive tissue charybdotoxin caused a significant depolarization of the membrane. Further proof that BK channels are activated in hypertension was given by experiments with spontaneously hypertensive rats^^V These were compared to Wistar- Kyoto rats. In SHR (spontaneously hypertensive rats) BK was responsible for much of the myogenic tone in the femoral artery as shown by treatment with charybdotoxin. In Wystar-Kyoto rats BK channel blockers had little effect^T hese results mean that under hypertensive conditions BK acts to minimize the depolarization and therefore the contraction of the tissue. Therefore an activator of BK channels could have applications as an antihypertensive. However given the broad expression of Slo it would have to be highly selective. BK channels are also involved in bradykinin induced epithelium dependent relaxation in the guinea-pig bronchus. NO release and subsequent activation of the BK channel mediates this process^^^. They may also inhibit the release of neurotransmitters at the neuromuscular junction thereby preventing inflammation^Therefore in airway tissue a BK channel opener provides a possible mechanism for treatment o f asthma. However the design o f tissue specific BK channel openers presents a significant challenge. BK channels in smooth muscle are composed mainly of a+pi subunitsw hich increase the channel sensitivity to calcium and charybdotoxin. The pi subunit also yields the channel sensitive to soyasaponins, which activate the current. Deletion o f the pi gene in arterial smooth muscle cells leads to an increase in muscle tone and blood pressure^^^.
29 The BK channel is also known to regulate repolarization after an action potential in skeletal muscle. It has also been shown that BK channels from mouse skeletal muscle can be activated by stretch in excised cell patches over a range of calcium concentrations BK channels are widely expressed in the nervous system^^^. In the neo-cortex, olfactory system, habenula striatium, hippocam pusand purkinje cells. This expression pattern is consistent with its targeting into a presynaptic compartment^*^. Cross sections of the frog neuromuscular junction revealed that BK channels are clustered in the presynaptic membrane facing the postsynaptic membrane. Also co-
CgTX (conotoxin, a calcium channel blocker) blocked BK currents and transmitter release during activation therefore Ca^^ and BK channels are co-localised at presynaptic active zones This suggests that they may play an important role in neurotransmission. There are two main phenotypes of BK, type I and type II. The rapidly inactivating BK channel found in chromaffin cells or insulinoma cells ^^\type II) and the more typical non-inactivating BK channels (type I). This inactivation is removed by the application o f trypsin. The inactivating channels are insensitive to charybdotoxin and the inactivation is dependent on internal calcium. Since BK channels have an extracellular N-terminus the mode of activation is unknown. It has been suggested that inactivation is brought about by the co-assembly of Slo subunits with inactivating subunits Since a-subunits cloned from chromaffin cells show no evidence of inactivationone explanation is that inactivation is brought about by the action of a p-subunit on the channels. An inactivating p-subunit does exist and it is expressed in chromaffin cells Therefore this seems to be the more likely explanation. BK channels are activated by Ca^^ entry during the rising phase of action potential. This rapid response is due to the proximity of calcium channels and BK channels in neurons. The opening of clustered Ca^^ channels produces high local calcium concentrations in submembrane domains and BK channels located within these domains are activated^**’ There is evidence that BK channels couple to specific types of calcium channel However this varies depending on the type of cell. In chromaffin cells BK is coupled to Q-type Ca^^ channelsin hippocampal neurons N-type channelsin ciliary ganglion neurons L-type channels and any type in sympathetic neuronsThe close relationship between calcium influx and BK
30 channels implies an important role for BK in the regulation o f neural activity. In neural tissues BK channels act as regulators o f repetitive firing patterns or as limiters of neurosecretion. In hippocampal neurons BK is responsible for the falling phase of the action potential and the generation o f the fast afterhyperpolarisation'^^. In dorsal root ganglion cells BK causes suppression of the action potential and a prolonged refi-actory period between firing^^®. BK channels are also known to be important in the electrical tuning of cochlear hair cells^®*’ Electrical resonance acts as an electrical filter maximising a hair cell’s response to sounds of frequencies near the resonant frequency and attenuating the responses at other fi*equencies^°'^. This is possible because the differential splicing of BK in the cochlea provides different
phenotypes^^^'^^^. This combined with a non-uniform distribution of p-subunits gives
a range o f properties needed to describe the tonotopic organization of the cochlea^®^. Calcium influx potentiates the release of neurotransmitters, this influx is inhibited by the activation of BK and therefore it back regulates neurotransmitter release^^^. Blocking BK at the neuromuscular junction is known to cause an increase of neurotransmitter levels. It has been suggested that, since some neuro-degenerative diseases are marked by an increase in intracellular calcium, blocking BK is a possible treatment. However this requires caution since indole diterpene blockers of BK, which are very potent and selective, are toxic in animals^^^. The treatment of ruminants with indole alkaloids causes tremors, hyperexcitability, convulsions, ataxia and tetanic seizures. Openers of BK channels however would reduce the levels of neurotransmitter release and so could possibly be useful in the treatment of any dysfunction that results in excess neurotransmitter release. The BK channel also presents a promising avenue in the treatment of dysfunctions such as hypertension or genitourinary malfunction. The diversity of BK phenotypes brought about by splice
variation, p-subunit co-expression and phosphorylation suggests that the specific
opening of BK in certain tissues may be possible. However given the ubiquitous nature of the BK gene the challenge may be formidable.
Pharmacology
There are three main classes of compounds that modify the activity of the BK channel. These are the peptidic blockers, natural products and synthetic molecules. TEA^"^ and Ba^^ also block BK at the external face of the channel at submicromolar
31 concentrations. Although TEA has been used extensively and the kinetics of Ba^^ block is known in detaiP^, these agents are non-specific. Therefore their use is limited, as the results obtained are open to ambiguity. Of the agents that modify BK the best characterized are the peptidic blockers. The peptides all inhibit the channel activity, however the mode of action is not identical in all cases. The scorpion toxins such as charybdotoxin^and iberiotoxin^^ ^ block the pore of the channel preventing the conduction of K^. BPTI (bovine pancreatic trypsin inhibitor) binds to the calcium bowl region and possibly prevents the conformational changes necessary for op en in g^ T h ere are several toxins that are known to inhibit BK such as kaliotoxin^*^, charybdotoxin^and iberiotoxin^^ % which have been extensively used in the characterization of BK. Examples include the isolation of functional c h a n n e ls^ in the discovery o f natural products that affect BK^^^ and in defining the pore structure of BK^^^. The activity of the peptide toxins is modified by
the presence of the p-subunit in BK. Charybdotoxin (CTX) is a 37 amino acid peptide originally isolated from the scorpion leiurus quinestriatus and found to block the channel in a simple bimolecular fashion with a Kd of lOnM^’^. It has been used extensively to investigate BK but also blocks IK and Kvl.3. Much of the interaction
with K vl.3 arises from interactions between first 6 amino acids o f CTX and the channel^CTX sits in the external pore o f the channel and is a globular structure containing 3-disulfide bonds^*^. The structure o f CTX has been used to map the probable pore section of BK. This showed a pore region similar to the crystallised bacterial channel KcsA^^^'^^^. Iberiotoxin (IbTX) is also a 37 amino acid peptide and
has a 6 8 % homology with CTX^^\ It is more potent than CTX and is specific for BK. The peptide binds to the channel pore with longer blocked times than CTX^^\ Due to increased specificity it is more useful than CTX as a probe for investigating BK channels. Kaliotoxin is a 33 amino acid chain with 44% homology with CTX and IbTX^^^. Although this seems quite low it contains three disulfide bridges similar to the other toxins and so may have a similar structure. It has a dissociation constant o f 20nM and is specific for BK. Another classes o f peptides that interact with BK are dendrotoxin-1, chicken ovoinhibitor and bovine pancreatic trypsin inhibitor^These compounds are serine protease inhibitors, which interact with the C-terminus of BK to produce a flickering block of the channel. Kd for BPTI is 2-7pM depending on the Slo expressed and the
32 rate of association is highly dependent on ionic strength of the solution and the positive charge of the peptide^^^. The mutagenesis studies of BPTI have shown that the BPTI binding section of BK resembles serine protease enzymes and since it
contains many negative charges it is a putative binding site for calcium^^^’ While peptides are useful in the characterisation of BK they are not useful candidates for drugs as they are rapidly metabolized and poorly bio-available. Therefore much effort has been expended on the discovery of non-peptide BK channel blockers and openers. The most potent non-peptide modulators of BK channels activity currently available have been isolated from natural sources. These compounds were screened according to their ability to increase or inhibit [*^^I]CTX binding to BK channels in bovine smooth muscle sarcolemmal membranes. Compounds that affected CTX binding were more fully characterized by electrophysiology^^^' This has led to the discovery of openers and blockers of the channel. Tremorogenic indole alkaloids are known to cause neurological disorders in ruminants and increases in neurotransmitter release. Studies using ligand binding and electrophysiology revealed that these are the most potent non-peptide inhibitors o f BK currently available^®^. All the compounds blocked BK activity but the effect on CTX varied. Some of the compounds (paxilline, verrculogen and paspilicine) increased the binding of CTX to the BK channel whereas others (paspilatrem A, paspilitrem C, alfatrem, paspilinine & penitrem A) inhibited CTXbinding^®^
OH
OH
Paxilline
Paxilline has a Ki = 1.1 nM for CTX binding and is specific for BK^^^. It binds to the
internal face of the channel on the a-subunit and this binding allosterically affects the binding of CTX to the channel. The binding is highly dependent on Ca 2+ concentration, the block being lowest at high Ca^^. The channel conductance is
33 reduced and the voltage dependence is shifted +20mV to less depolarized potentials^^^. OMe MeO
OMe
NH
Tetrandine
Tetrandine is an isoquinoline alkaloid that is used in China to treat arrhythmias and hypertension. It blocks both L-type Ca^^ channels with an IC 5o= ImM and type II BK channels from the extracellular face with an ICso^ 0.2 ImM^^*. It is the only blocker of type II channels that are insensitive to CTX and therefore may be useful in their characterisation. However a question arises as to how tetrandine can act as a muscle relaxant when it blocks BK channels more potently than Ca^^ channels. The likely explanation is that tetrandine blocks the type II channel in neural tissue but is inactive against BK in smooth muscle, allowing the drug to interact solely as a calcium channel blocker. Soyasaponins were isolated fi*om the medicinal herb Desmodium adscendens, which is used as a treatment for asthma in Ghana. The most potent compound dehydrosoyasaponin (DHS) was found to inhibit the binding of CTX to bovine smooth muscle membrane with a Ki of 120nM^^^.
R= trisachharide OR
Dihydrosoyasaponin
34 DHS is inactive when applied to the external face of the channel and is thought to inhibit CTX binding by an allosteric interaction. At lOOnM it caused an 80-fold increase in the opening probability of BK channels in lipid bilayers and still had a marked effect at lOnM. However DHS is only effective on channels where the a+p subunits are co-expressed and is inactive on a subunits alone^^^. This demonstrates that tissue specific BK channel opening is a possibility and therefore BK channels openers may be a viable therapeutic approach. However DHS is a large molecule and not an ideal candidate for optimization. Using the same screen two small molecule compounds were discovered that inhibit CTX binding in the smooth muscle membrane binding assay. MaxiKdiol^^^ and CAF603^^^ both compounds activate the
BK channel when applied intracellularly and show clear effects between 3-lOpM.
HO
HO OH OH
MaxiKdiol CAP 603
These compounds are potential leads for QSAR studies but were discovered in 1993 by scientists at Merck and therefore it was thought that they might have been previously investigated. Phloretin is a fiavonoid compound that activates BK when applied to the external face of the channel. It was shown that SOpM phloretin shifted the potential for half-maximal activation to -69mV. The onset of action and washout were prolonged^^^. Due to the non-specific nature and lack of potency phloretin does not present a good candidate for investigation however it does have features in common with other activators of BK that will subsequently be discussed. There are several synthetic compounds that have been shown to modulate the activity o f BK. The blockers of BK include 1 -(4-methoxyphenyl)-indole^^ \ which is a modest potent blocker of BK (20pM) that was proposed in a patent as a treatment for
Alzheimer’s Disease. Clotrimazole also blocks the BK channel in ferret portal vein at sub-micromolar levels^^^. A metabolite of clotrimazole lacking imidazole was also similarly active^^^. Clotrimazole also blocks BK in GH 3 pyramidal cells in rat with an
IC50 o f 2 . 3 Also local anesthetics such as lignocaine and QX-314 at ImM
35 reduced the BK current in rat hippocampal neurons when applied from the interior face of the channel.
1 (Methoxyphenyl)-indole Clotrimazole Phloretin
O OH
Most of these are compounds have other biological activities but were also found to activate or block BK. Exceptions to this statement are the compounds produced by the Neurosearch Company, l-(4-Methoxyphenyl)-indole, NS004, NS1619 and NS1608. NS004 was the first reported compound and was found to activate the BK channel in the micromolar range by shifting the voltage dependence by 20-3 OmV toward negative potentials^^^’ This potency is modest and unfortunately it is not a specific compound, blocking Kv and K a tp in smooth muscle, calcium currents in cardiac muscle and opening the CFTR Cl" channeP^^. NS1619 was the next generation of the compound. It was more potent, shifting the voltage dependence by 5OmV but still in the micromolar range (3-30mM). It was also more selective, not affecting Cl" or Ca^^ channels and many K^ channels. However NS1619 was found to substantially inhibit Kv and K atp from smooth muscle^^^. By opening the imidazolone ring in NS004 to obtain the biphenylurea NS1608 the potency was significantly improved^^^. NS1608 also appears to be more specific than NS004 and NS1619 239
OH
OH OH
NS004 NS1619 NS1608
36 As yet NS1608 is the best small molecule opener of BK shifting the Ek by 74mV to more negative concentrations at the maximal concentration and displaying an EC 50 o f
2.1pM Other biphenyls have been tested for BK activity such as phloretin, which has already been discussed and analogues of niflumic acid. These were found to increase the current when applied to the external face o f the channel^"^^. The potency of the niflumic acids is modest, lOOpM causing the opening. They also block calcium activated Cl" channels so are nonspecific^'^^. They are interesting however because they resemble NS1608 in some respects.
COpH COpH
OF; OF; Niflumic acidFlufenamic acid MCM54
The pyridyl amine MCI-154 is a known vasodilator and was found to directly activate
BK in cultured smooth muscle cells at concentrations of lOOjuM K atp channel openers, chromakalim and lemakalim also activate BK. Increasing the opening probability o f BK channels from rabbit aorta in lipid bilayers by 56% at 50nM chromakalim^'^^. However patch clamp recordings o f BK currents from calf aorta cells showed no enhancement even at lOOpM lemakalim and therefore the previous results must be viewed with caution^"^^. Calcium channel antagonists nitrendipine and niguldipine have also been shown to enhance the BK current, in vascular smooth muscle cells^'^'^’
Nitrendipine (+)-Chromakalim (-)-Lemakalim
O2N EtOgC pH^
\ N
IVIeOgC CH3
37 Few o f the compounds that open BK are very potent and comparing one opener with another is difficult. This can be because either the dose response curve will not reach a maximum or there is a different maximum for each compound. Therefore the descriptions of activity vary. For example in the case of DHS (see page 24) the equilibrium constant for displacement of CTX from muscle membrane was given, along with increases in current from electrophysiology experiments at given
concentrations. In the case of NS1608 the EC 50 could be measured but this can’t compare with the Ki value quoted for DHS. In addition to this DHS can only be used
where the channel is co-expressed with p-subunits. It is uncertain given the non
specific nature of NS 1619 and NS004 whether NS1608 is fully selective for BK. There is also a lack o f literature describing the structure activity relationships o f any of these compounds. Given that these are the most potent compounds available it is clear that there is much room for improvement in the field of BK channel activators. In addition to this the most potent small molecule blocker of BK is clotrimazole. Given that this also blocks the IK it is not ideal as a probe for BK activity. Therefore a potent and selective blocker of BK would be of significant value as a pharmacological probe.
Ketoconazole
Ketoconazole is known to open BK channels in ferret portal vein at micromolar
concentrations, lOpM increasing the opening probability by 545% by increasing the mean open time 495%. At 30pM it has been shown to shift the voltage of activation by 2OmV in bovine aorta smooth muscle^^^. Interestingly it has also been shown to block BK in rat GH3 cells^^^. Given the need for new openers of this channel and an interest in investigating the structure activity relationship it was decided to synthesize and test analogues of ketoconazole. The aims were 4-fold. Firstly to identify the pharmacophore for BK activity, secondly to increase the activity at BK, thirdly to
38 remove the monooxygenase inhibition from the molecule and finally to improve the physical properties o f the drug.
39 Selection of compounds Series 1
In order to identify the pharmacophore for BK activity fragments of ketoconazole were systematically removed from the molecule. To perform this partial structures of ketoconazole were synthesized and tested. The schematic drawing below shows the relationship between the fragments removed on paper and the corresponding molecules synthesized. These compounds were synthesized to investigate the phenyl piperazine section.
No.1 No.2
C> 0
No.3 No.4
, 0 ^ 0 Cl ,______K / ^ T ^ o Cl
No.5 No.6
This set of compounds was designed to investigate the role of hydrogen bonding on the piperazine ring. The amide group, which can accept a H-bond, was removed to yield a basic amine group that can donate or accept a H-bond. This was then replaced by a lipophilic methylene to leave only the tertiary amine (H-bond acceptor). The piperidine was removed to unmask the primary amino group (H-bond donor/acceptor). The removal of this group was followed by the removal of the phenyl moiety to investigate the significance of this group. To investigate the
40 significance of the imidazole ring it was decided to remove the moiety entirely sinee there was only one available H-bonding group on this imidazole ring.
Cl Cl
p p P Oi° \
No.1 No.7
The first eompound tested was a partial structure based on a simple biseetion o f the moleeule in order to determine whether both sides were neeessary for funetion.
Cl
O M e 0 ‘ N N \ No.1 N o.8
In addition to these eompounds there were 3 synthetic intermediates that it was considered worthwhile to test. There were also two partial structures o f ketoconazole where imidazole is substituted by triazole, whieh were tested for comparison.
Cl Cl Q 6' X N o .9 N o.10 N o.11
Cl
Cl
OH
N o .1 2 o N o .1 3
41 Modification of the dioxolane ring was deferred at this point as replacement with a carbacycle or THF ring would require a longer synthesis and may have delayed the project. It was considered that this could be dealt with subsequently.
42 Series 2
From the first series it was clear that the imidazole was unnecessary for function. Also that the nature of the substituent at the 4 position of the phenoxy moiety was critical. However little was known about the role of the 2,4-dichlorophenyl group. In order to obtain a clear picture of the role of the 2,4-dichlorophenyl group it was decided to assemble the acetal group by group.
HO OH cis and trans N0.16&17 ^ O R No.14 No.15 OR OR
No.18 OR No.7 OR No.19 OR
R=
The cyclohexylidene acetal analogue N o .20 was also synthesised and tested, as the alcohol was commercially available. 2,4-Dichlorophenyl was by far the most potent, therefore from this initial series a further set of compounds was synthesized to investigate positional effects; 2,4-difluorophenyl and 2,4-dimethylphenyl were synthesized in order to determine the specificity for chloride substituents. No.21 X= 4-Chlorophenyl, No.22 X= 2,4-Dichlorophenyl, \ / No.23 X= 2,5-Dichlorophenyl, \ No.24 X= 3,4-Dichlorophenyl, No.25 X= 2,4-Difluorophenyl, R= 4-Aminophenyl No.26 X= 2,4-Dimethylphenyl.
Compounds 24 & 25 synthesized by Ana Conejo Garcia.
43 Series 3
From series one it was clear that the phenoxy moiety is crucial in determining the activity of the eompounds. This was investigated with a range of hydrogen bond acceptors, donor/acceptors and lipophilic substituents at the 4-phenoxy position. Again imidazole was omitted from the structure. The hydrogen and amino substituents were the first to be synthesised in an attempt to draw a parallel between eompounds 4, 5 and 7. These eompounds are placed among the other compounds shown below. In as many eases as possible the isomers were isolated and tested separately. These eompounds were assigned as eis or trans based on the orientation of the 4-substituent relative to the 2-methyl substituent. This was done to maintain continuity with the imidazole containing eompounds of series 1. Cl
O
r-2-Methyl-c-4-phenoxymethyl r-2-Methyl-t-4-phenoxymethyl
Substituent Property Isomerism Number X-H- Cis 27 X =H - Trans 28 X= Methyl Lipophilic Mainly Cis 2 9 - X= 3,4 indole Lipophilic Mixture 3 0 # X= Chlorine Lipophilic Mainly trans 31 - X= Cyano Acceptor Cis 32 X= Cyano Acceptor Trans 33 X= Acetyl Acceptor Cis 3 4 - X= Acetyl Acceptor Trans 3 5 - X= Methoxy Acceptor Mixture 3 6 - X= Trifluoromethyl Acceptor Mixture 3 7 - X= Nitro Acceptor Cis 3 8 - X= Amido Donor/ Acceptor Cis 39 X= Amido Donor/ Acceptor Mainly trans 40
X - Amino Donor/ Acceptor Mixture 2 2 X= Acetamido Donor/ Acceptor Mainly Cis 41 #
Made by S.Ishaq, # Made by A.Conejo-Garcia
44 Series 4
The dioxolane ring system is highly labile to acid hydrolysis and many of the compounds were viscous oils. In addition to this the compounds were insoluble at high micromolar concentrations, which made the results difficult to compare. Separation of both isomers could be difficult and time consuming; thus the synthesis of the enantiomers would have been problematic. Therefore replacing the dioxolane ring was considered to be a necessity. Two distinct approaches were taken in varying the dioxolane core of the molecule. The first approach placed strict requirements on the properties of the replacement molecule. The global minimum of the molecule needed to show a similar folding pattern as the dioxolane compounds when modelled in XED. It had to be less labile to acid and more soluble in water. In order to investigate the effect of absolute configuration the molecule would ideally have one chiral center accessible from enantiopure starting materials. The compounds chosen are shown below.
OH p H Cl
No.42 NHo M0.4JNo.43 NH
In contrast to this approach the other compounds were based on the possibility that the dioxolane moiety is merely a spacer between two phenyl groups. If this was the case then the length of spacer may be critical. In the case of NS004 and NS1608 the imidazolone ring was opened to yield a 3-atom linker with a consequent increase in activity. Niflumic acid and its analogues have similar (acidic) substituents but with a 1-atom linker and are less active. Phloretin also has a 3-atom chain and some activity at the BK channel. Since NS1608 is the most potent compound in the literature it was possible that this length o f chain is optimal and that our compounds were part o f a class o f compounds that bind to a biphenyl receptor on BK. The compounds 44, 45 and 46 were synthesized to evaluate this idea. Compound 46 was intended as the monosubstituted aniline compound but was obtained as disubstituted and tested, so it is included here.
45 C l o Cl Cl O
'o O Cl O Cl NH, Cl NH2 Cl No.44 No.45 No.46 Cl No.46 prepared by A.Conejo-Garcia
Ether oxygens are known to bind to inorganic cations. Therefore was it possible to draw a parallel between the ethyleneglycol units of crown ethers and the glycerol oxygens of our ketal molecules. It was tempting to envisage the phenyl moieties of the molecule binding to the channel with the ether oxygens pointing towards the pore region or in the calcium pocket. To investigate such an idea synthetically would take a huge effort. Instead it was decided to use two commercially available crown ethers to test the validity of the idea.
No.46 selective No.47, Ca 2+ selective
O
O O
Finally the dioxolane ring was replaced by furan in order to examine whether rigidification of the ring would increase activity.
No.49
O N N \ /
46 3 Synthesis
This chapter is concerned with four areas.
• The synthesis of ketoconazole analogues.
• 2-Methyl-2-phenyl-4-phenoxy-1,3 -dioxolanes.
• Non-dioxolane compounds.
• Characterisation of compounds.
3.1 The synthesis of ketoconazole analogues
The synthesis of ketoconazole has been published^"^^ and is shown below.
SCHEME 1 The synthesis of ketoconazole analogues.
O Cl OH
No.51 No.52
01
OH
No.9 No . 6
01
VII P O
OMs
No.53 No.1
i. Glycerol, TsOH, Benzene/Butanol, Dean-Stark, Reflux; ii. Br 2 , RT; iii. BzCl, pyridine, 0°C; iv. Imidazole, DMA, reflux; v. NaOH, dioxane/water, reflux; vi. MsCl, pyridine, 0°C; vii. NaH, 1-Acetyl- 4(4-hydroxyphenyl)-piperazine, DMSG, 80°C. In our work steps i to iv (scheme 1) were used for series one with little modification. Heating 2,4-dichloroacetophenone and glycerol to reflux in benzene/butanol with
47 toluene sulfonic acid monohydrate effected acid catalysed formation of the ketal alcohol.
SCHEME 2; Dioxolane ring formation.
+ H+ ROM
OH.+
+ ROH p
-H'
The formation of this ring is reversible. Dioxolane rings can also be opened under acidic conditions but in our case the reaction was carried to completion with a Dean- Stark water trap. This was cooled, and treated in situ with Bri to produce 51 in 78% yield. After an aqueous work up the crude compound was reacted with benzoyl chloride in pyridine. Recrystallisation of the crude compound from ethanol yielded the cis isomer 52 in approximately 50% yield. After drying under vacuum overnight to remove residual ethanol and water, 52 and imidazole were heated to reflux in dimethylacetamide for 4 days to produce 9 in a yield of 55%. The moderate yield and forcing conditions probably reflect the low reactivity of the neo-pentyl bromide and starting material could be isolated after the precipitation of 9 with nitric acid in ether. In cases where starting material was not frilly dried the product was not isolated and complex mixtures arose. The benzoyl group was then hydrolysed by NaOH in dioxane/water and the crude product recrystallised from EtOAc to give 6 in 78% yield. In the case of triazole derivatives the reaction of the bromide 52 with triazole in
48 the presence of base in DMF was followed by careful purification by flash chromatography yielded both benzoyl 12 and hydroxy 13 analogues. At this point the hydroxyl group in compound 6 is converted to a leaving group by treatment with methane sulfonyl chloride. Toluene sulfonyl chloride has also been used for this purpose. In our hands there was little difference in terms of yield at the tosylation/mesylation stage or at the substitution stage. The tosyl derivative 10 (see chapter 2) was favoured due to the fact that it recrystallised cleanly from DCM/hexane rather than benzene in the case of the mesyl derivative. The yield for mesylation/tosylation was typically -70% after recrystallisation. The HCl salt of 10
was tested for activity. The phenoxy moiety was introduced by means of a Sn2 substitution of the mesylate 9 or the tosylate 10. The yield of the substitution varied for each phenol. Preforming the phenoxide in DMF or DMSO at 0°C followed by addition o f the tosylate and heating was the general method used. It was found that the dry DMF was a more practical solvent for this reaction and it was used in most cases. DMF is also the solvent of choice in recent literature dealing with the synthesis of ketoconazole analogues^"^^’ The l-acetyl-4-(4-hydroxyphenyl)-piperazine (54, scheme.3) was synthesized in a manner similar to the published method^'^^. However it was simplified by the commercial availability of l-(4-hydroxyphenyl)-piperazine. This was diacetylated by treatment with acetic anhydride to give 53 in 75% yield. The ester moiety was hydrolysed with ammonia solution in methanol to yield the phenol 54 in 89%.
SCHEME 3 Synthesis of phenol 54.
NH AC2O, NEt3 ^
\—I DCM, 0“C y \—/ \
Aq NH 3 / = \ /— \ O
This phenol was then used in the synthesis of 2 (scheme 4) as well as several other compounds. Ketoconazole that had been synthesised according to scheme 1 was heated to reflux with potassium hydroxide in isopropanol to produce 2 in 68% yield^"^^.
49 SCHEME 4 Amide Hydrolysis. Cl
KOH Cl Isopropanol, reflux
No.1 No.2
In the synthesis o f the target compound 3 (scheme 6 ) was interesting in that the necessary phenol, although a simple molecule, was not commercially available. Also the literature procedures seemed either overcomplicated or unsuitable^^^’ This led us to consider two options, firstly to prepare the phenol by an alternative method or to form the aryl piperidine linkage as a final step. Whichever route chosen it was decided that the methodology to be used would be the palladium catalyzed aminations of aryl halides. These reactions were developed in the early/mid 1990s by the groups of Hartwig^^^ and Buchwald^^^. Scheme 5 shows the proposed mechanism for this reaction. The main side reaction results from the proton on the secondary amine replacing the halide rather than the secondary amine. This is known as the reduced side product. Initially the technique was limited as strong bases were used and these conditions are replicated here but weaker bases such as cesium carbonate are now used.
SCHEME 5 Mechanism of Aryl Amination.
P d L z
NR Br R
P d -L
L R- ■Pd NR. X
B ase.H B r HNR2 + Base
50 Initially they have been reported as coupling secondary amines to aryl bromides but the method has been widened to include alcohol groups and aryl chlorides^^"^. Given the occurrence o f aryl amines and ethers in several important compounds such as vancomycin or the azoic antifungals they represent a useful reaction for medicinal and organic chemists. In our case it was thought that reacting the aryl bromide with piperidine represented a cleaner method than forming the primary amine and reacting it with a dihalide. The aromatic nucleophilic substitution of l-fluoro-4-nitrobenzene with piperidine followed by diazotization was considered but not attempted as the catalytic route was found to yield the product^^.
SCHEME 6 Synthesis of piperidine analogue.
Cl
Piperidine, Pd 2 (dba ) 3
LiN(SiMe3 )2 , 90°C No.11 Toluene, P(oTol ) 3 No.3
Piperidine, Pd2 (dba > 3 MeO—' '— Br ------► MeO— /)—N
LiN(SiMe3 )2 , 90°C ^ ^ Toluene, P(oTol ) 3 No.55
BBr-> /—\ / \ NaH, 10 No.3 DOM, 0°C \ -----f dMF, 0-80°C No.56
The coupling of 4-bromoanisole and piperidine had already been reported^^^ and it was thought that once this was achieved then the removal of the methoxy group should be straightforward. This was also used as a trial reaction for the direct amination of 11 (scheme 6 ), which was seen as desirable as it would allow further modification at this site if necessary. Attempts to perform this reaction with 11 using the conditions above failed. It is likely that this reaction could be optimized with the correct base, ligand or by adding the correct inorganic salt^^"^. However it was considered more efficient to produce the phenol 56 (scheme 6 ) and react it with the tosylate 10 (scheme 7). The Pd catalyzed coupling of piperidine and bromoanisole did not proceed with the same yield as the literature (37% vs 96%). The catalyst used for this reaction in the literature was PdCl 2(P(o-T 01)3)2, which is not available
51 commercially^^^. Instead commercially available Pd 2(dba)3 and P(o-Tol )3 were used and the difference in yield possibly reflects the quality of the catalyst. Also the literature method gave little detail on procedure, in our case it was found necessary to mix catalyst and ligand with heating before adding the reactants in the order bromide, amine and base. Column chromatography yielded a mixture of product and ligand since they had identical Rf values. The pure compound was isolated by forming the oxalate salt. BBr 3 effected the removal of the methoxy group in dichloromethane at - 78°C and after aqueous work up the compound was isolated as the oxalate salt in 61%. Formation of 3 proceeded smoothly in DMF at 80°C overnight and yielded a glassy solid after purification in 60%. However when it was attempted to convert the compound into the oxalate salt the compound formed a hygroscopic syrup on filtering. This was collected and passed through preparative HPLC to ensure purity. The synthesis of 4 (scheme 7) involved the formation of a 4-aminophenoxy moiety. The first approach towards this was to react 4-nitrophenol with the tosylate 10 and reduce the nitro group. Surprisingly the substitution reaction failed in DMSO and an alternative method was chosen.
SCHEME 7 Formation of aniline
,01 Cl ^ 0 !
Cl ► O XCl /•'/ \ /OTs NaH, DMSO, 80°C \
No.10 N ^ NHg Ph
N2H4.H2O
Ethanol, reflux Scheme .5 i ^NH2
N0.4
By reacting 4-(benzylidenamino)-phenol (scheme 7 & 8) with 10 (scheme 9) in DMSO and treating the product with hydrazine hydrate in ethanol 4 was obtained in 21% yield. When isolating the imine product by flash chromatography, it was noticed by TLC that the product seemed to be decomposing on the column. This lower spot was isolated with the imine fractions and when the mixture was treated with
52 hydrazine in ethanol the imine spot disappeared to give only the lower spot, which was the primary amine 4. 4-(Benzylimino)-phenol was obtained in 72% yield by adding benzaldehyde to 4-aminophenol in methanoP^^.
SCHEME 8 Formation of iminophenol.
Benzaldehyde No.57 HO HO— ^—NH2 M eOH, 0°C
To produce compound 5, tosylate 10 (scheme 9) was reacted with phenoxide in DMSO to produce 5 in 70% yield.
SCHEME 9 Displacement of tosylate
.0!
Phenol, NaH Cl DMSO, 80°C rr' p O Ts
N0.5
The formation of the phenyl alkyl ether was only achieved by displacement of a tosyl or mesyl leaving group in the case of the imidazole containing compounds. Attempts to perform Mitsunobu reactions with DEAD and triphenylphosphine^^^ did not yield product even with simple phenols. This contrasts with the non-imidazole compounds that will be discussed subsequently.
3.2. 2-Methyl-2-phenyl-4-phenoxymethyl-l,3-dioxolane derivatives.
The non-imidazole containing compounds are still partial structures o f ketoconazole, but without the imidazole moiety they have different chemical and physical properties. The first compound of this type to be made was the dichlorophenyl derivative UCL-2158 from series one with other similar compounds synthesised by the same route. The synthesis was similar to that of ketoconazole, simplified by the fact that the extra steps were not used to separate the isomers and that the 2-methyl group did not require substitution.
53 SCHEME 10 Formation of dioxolanyl tosylate
TsOH
Benzene/butanol Pyridine, 0 C O reflux. Dean-Stark
58: X-| —H, % 2 —H, X3 —H 62: X-( =H, X2 —H, X3 =H cis 59: Xi =CI,X2=H,X3=H 63: X-|=H, X 2 =H, X3 =H trans 60: X-| —Cl, X2 —H, X3 =01 64: X-j =01, X 2 —H, X3 =H 61 : X-| =01, X2 —01, X3=0I 65: X-j =01, X2 —H, X3 =01 6 6 : X-( =0 1 , X2 —0 1 , X3 =0 I cis 67: X-( =01, X2 =01, X3 =0 I trans Formation of the dioxolane ring followed by aqueous work up yielded the product in (in 80% yield) without the need for distillation or column purification as long as the compound was evaporated with chloroform or carbon tetrachloride to effect the removal of the n-butanol. This is essential to prevent a side reaction during the tosylation step. Tosylation (scheme 10) was preferred to mesylation due to the difficulty in obtaining pure mesylated compound. This was perhaps due to the persistence of the n-butanol solvent and its subsequent side reaction. Tosylation in pyridine, aqueous work up and column purification was the standard method for obtaining the product in good (70-80%) yields. In the case of the non-chlorinated compounds the tosylates were white crystalline solids becoming viscous liquids in the chloroaryl cases. The phenyl derivative can be recrystallised from ethanol to give the cis and trans isomers^^* (scheme 11). We found this separation to be improved by the addition of several drops of toluene into the ethanol solution. Although it was a viscous liquid it was also possible to isolate the cis and trans isomers o f the trichlorophenyl derivative by recrystallising from methanol/toluene. The unambiguous assignment o f each isomer as cis or trans was performed by NMR using either ID or 2D NOESY, which shall be discussed subsequently.
SCHEME 11 Cis and Trans isomers
X
X
OTs OTs 62: X =H 63: X =H 66: X =CI 67: X =01
54 For the dimethyl and cyclohexyl acetals the alcohols are commercially available, so
the tosylates 6 8 and 69 (scheme 12) were produced in one step^^^’ In both cases the compounds could be recrystallised after work up without chromatography.
SCHEME 12 Dimethyl and cyclohexylidene isomers.
Rl TsCI 6 8 : Ri=R2=H, Yield = 45% Pyridine, 0°C O' O 69: Ri=R2=(CH2)3 , Yield= 70%
OH OTs
To synthesize the compounds for testing, the tosylates were reacted with the phenoxide anion of 54 in DMSO or DMF. In general the reaction required heating overnight at 80°C. An exception is the dimethyl derivative 15(UCL-2245), the reaction for which proceeded at room temperature (scheme 13).
SCHEME 13 Displacement of tosylate
o o 0 O N R O -OTs NaH, DMF or DMSO, 80oC
Compound No. From Tosylate R1 R2 % Yield (Solvent)
16 (UCL-2254) 62 CH3 Phenyl 58% (DMF)
17 (UCL-2264) 63 CH3 Phenyl 49% (DMF)
18 (UCL-2254) 64 CH3 2 - Chlorophenyl 58% (DMF)
7 (UCL-2158) 65 CH3 2,4 - Dichlorophenyl 45% (DMSO)
19 (UCL-2253) 6 6 CH3 2,3,4 -Trichlorophenyl 24% (DMSO)
15 (UCL-2245) 69 CH3 CH3 51% (DMSO) 20 (UCL-2280) 69 (CH2)5 --- 52% (DMF)
In general, DMF was seen to be a give slightly better yields than similar reactions in DMSO, it was also easier to remove and so DMF was favoured from this point on. It is worth stating that the difference in yields may reflect the quality of an individual bottle of DMSO rather than a real difference in efficacy between the two solvents. Initial attempts to form the ether by the Mitsunobu reaction with the alcohol 60 and phenol 54 (scheme 10) were unsuccessful. This was similar to the result for the imidazole containing compounds and was initially thought to be for reasons of steric hindrance. The fact that dimethyl tosylate 6 8 (scheme 12) was substituted at room
55 temp seemed to raise the possibility that the substitutions at the 2 position of the ring raise the activation energy for the substitution reaction. Subsequent to this however, the fact that 54 (scheme 10) may not be acidic enough to react under normal Mitsunobu conditions was considered. For the reaction to be successful the phenol needs a pKa of less than 11. A simplfied mechanism is shown in scheme below.
SCHEME 14 Mitsunobu Mechanism
Ph Ph^ u .P h ROM EtO OEt
P P h
A O
It has been shown that using ADDP and tributylphosphine in benzene that nucleophiles with pKa higher than 11 could be coupled with alcohols^^\ With this in mind we attempted to produce UCL-2158 (scheme 10) using this methodology (Scheme 15).
56 SCHEME 15 Modified Mitsunobu
A D D P, PBu;
V Benzene, 0°C-RT
OH
O A D D P = o
The reaction was successful, albeit in moderate yield (37%) but this raised a question as to whether the standard Mitsunobu (scheme 16) could be used with this type of alcohol. To test this we chose to couple 2-nitrophenol and 3-nitrophenol with 60.
SCHEME 16 Conventional Mitsunobu
Cl
NO •^^2 d e a d , PPh3
HO-Y^ THF, 0°C // No.70 60% yield
01
02N DEAD,PPh3
HO \ / THF,0°C No.71 40% yield
This shows that the failure to react 54 with 60 (scheme 15) was based on the acidity of the phenol 54 not on the nature of the alcohol 60. It is difficult to compare the yield for the D E A D /P P h s conditions with the ADDP/PBU 3 conditions given the different solvents and differing reactivities of the phenols. However comparing the yields of 70 and 71 is valid. The yield of the 3-nitrophenyl was greater than the yield of the 2- nitrophenyl derivative, which may point to a lower reactivity, based on steric effects.
This is given more validity given that D E A D /P P hg did not couple phenol with the imidazole derivative 6 but the reaction proceeded with the non-imidazole derivative
60. In addition to these observations the dimethyl tosylate 6 8 could be substituted at room temperature compared to 80°C for 2-methyl-2-phenyl derivatives. It seems that substitution of the 4-(hydroxymethyl)dioxolanes is sensitive to steric effects.
57 One characteristic of the non-imidazole compounds is their acid sensitivity. Since the remaining amine in the molecule is donating electron density to an aryl ring it is less basic. Therefore it does not form a salt as readily and the opening o f the acetal in acidic conditions is made more likely. It was possible to form the oxalate salt of 7 (UCL-2158) but not of 18 (UCL-2254) or 15 (UCL-2245, scheme 10). 18 and 15 were converted to the diol. The simplest explanation for this is that all the compounds become equilibrium mixtures of diol, acetal, hemiacetal and ketone when placed in acidic ethanol solutions. For UCL-2158 (scheme 17) the salt of the dichlorophenyl derivative 7 was insoluble in the ethanol solution and therefore precipitated, shifting the equilibrium towards the desired product.
SCHEME 17. Salt formation
Cl 01
NH
UCL-2158 UCL-2158 oxalate
In the case of the 2,2-dimethyl 69 (UCL-2245) and 2-methyl-2-chlorophenyl 64 (UCL 2254, scheme 18) derivatives it was likely that the attempts to extract the free base fi-om solution caused the ring opening. Since the ring opening is formally an acid catalyzed hydrolysis, placing the compounds in solution effectively shifted the equilibrium to the right.
SCHEME 18. Ring opening
y — ' ^— ' '—' ^ several steps O HO--V UCL-2245 & UCL-2254 UCL-2238 R= Methyl, 2-Chlorophenyl
Therefore when the organic component was re-extraeted from basic solution it was found to be exclusively diol 14 (UCL-2238). The salt of 7 (UCL-2158) was examined by NMR and found to be stable in DMSO after several weeks. Therefore the salt of the compound is stable, but the free base in acidic solution is not. For example it was also observed that the compounds became discoloured in deuteriated chloroform over several days. However due to the possible pit falls associated with producing the salt
58 the remainder of the compounds were tested as free bases. The assay is performed in physiological saline solution (pH=7.4), and so acid hydrolysis would not occur. As the compounds were tested it was seen as interesting to combine the properties of several structures from series 1 that exhibited interesting biological properties. The first combination was o f 4 (UCL-2242) and compound 7 (UCL-2158) to give UCL- 2273 (scheme 19).
SCHEME 19 Combining the characteristics of useful compounds
Cl
0 p— N O 2242 2273
The route shown below was used to synthesize this compound.
SCHEME 20 Synthesis of UCL-2273
NaH, No.57 + Ethanol. 0°C DMF,0-80oC
OTs No.65 Ph NHz NHz 22, UCL-2273 Mixture
Tosylate 65 (scheme 10) was synthesized from 2,4-dichloroacetophenone (Scheme 12). Reaction with 4-(benzylimino)-phenol (57, scheme 8) in DMF gave a mixture of the protected product and de-protected product after colunm chromatography. This mixture was treated with hydrazine hydrate in ethanol at 0°C. The yield for the two steps was 22%. This poor yield was mainly accounted for by inefficient substitution of the tosylate. UCL-2273 (scheme 19&20) was used as the template for a further set o f compounds that further examined the substitution pattern o f the dichlorophenyl moiety. This set of compounds was synthesized via the modified Mitsunobu reaction with the alcohols synthesized as before. It is more useful to discuss these individually as the method differed in each case.
59 SCHEME 21 Chlorophenyl Targets
ÇI 0 1
21, UCL-2327 24, UCL-2333 23, UCL-2335
In the case of 3,4-dichlorophenyl derivative (24, scheme 21) a mixture of protected and deprotected compound was isolated by colunm chromatography. It was not possible to crystallize the compound at this point and so it was reacted with hydrazine hydrate as a mixture. The fully deprotected product was isolated by column but since it could not be crystallized it was tested as a waxy solid. The yield for the two steps was 32% (scheme 22).
SCHEME 22 Synthesis of UCL-2333.
,CI 24, UCL-2333 ,0 'C l ______► Mixture of imine Ethanol A D D P . PBU3 , B enzene NH;
N 0.72
For the 4-chlorophenyl compounds it was possible to isolate the cis isomer of the protected product by crystallizing it from the crude mixture in methanol solution in 11% yield. The remaining mixture o f trans imine and aniline o f 74 were treated with hydrazine hydrate to obtain the trans product. Unfortunately this was not obtained in sufficient amount and purity to characterize or submit for testing. The cis isomer 74 was also treated with hydrazine in ethanol and recrystalised from methanol/hexane to obtain the pure compound 21 in 78% yield (scheme 23).
SCHEME 23 Synthesis of UCL-2327. 01 01
fTT^' 21, UCL-2327 Phenol, ADDP. PBug NgH^ HzO
Recrystalise MeOH ^'^Ph Ethanol.0°O ^^2
N0 .7 3
The protected 2,5-dichlorophenyl compound (scheme 24) crystallized from methanol after isolation by column chromatography as a mixture of isomers in 21% yield. This
60 was treated with hydrazine hydrate in ethanol and purified by flash chromatography to yield a mixture of isomers in 6 6 % yield. The trans isomer 77 (scheme 24) was recrystallised fi*om methanol and the mother liquor evaporated in vacuo. Recrystallisation of the resulting oil in dichloromethane/hexane yielded the cis isomer in 28%. While both isomers were isolated only the cis isomer was obtained in the quantity necessary for testing.
SCHEME 24 Synthesis of UCL-2335
23, UCL-2335 Phenol, ADDP, PBug
Recrystalise MeOH \_J ^ph Ethanol,0°C OH No.76 and trans No.77 No.75
The inability to test both isomers did not present us with a large problem. Several sets of isomers had been tested at the point that these compounds were being synthesized and there was little difference between them in most cases. One problem with this route however is the low yields in the ADDP/PBus conditions used to obtain the product. In all examples we found that the small amounts of solvent quoted in the original reference were completely insufficient. In order for the mixture to remain stirring it was necessary to continuously add more solvent (up to 30ml); possibly due to the insolubility of the intermediates. Standard Mitsunobu conditions were subsequently attempted but it proved problematic to remove the dihydroDEAD from the products. The low yields may be the result of the low nucleophilicity of the phenol. A better yield would probably be obtained by obtaining the nitro compound and reducing with an appropriate procedure to the amine. Attempts to reduce nitro intermediates 70 and 71 (scheme 16) by hydrogenation in a Parr apparatus provided mixtures of products. Therefore since the compounds were obtained using the imine intermediate, the route was used generally. A second series of compounds, which closely resemble the compounds already discussed, was synthesized. This was in order to elucidate the role of the basic centre of the molecule. In addition to the analogues synthesized by myself, a number of analogues were also prepared by project students Salma Ishaq and Ana Conejo Garcia, which will be included in the discussion of results.
61 SCHEME 25 Synthesis of 4-phenoxy analogues
HO Chromatography
Crystalisation NaH,DMF, 0-80°C
OTs
UCL-2292, X=H, cis UCL-2302, X=H, trans UCL-2270, X=CN, cis UCL-2303, X=CN, trans
UCL-2298, X=C 0 NH 2 , cis
Trans UCL-2319, X=C0 NH 2 , trans
Formation o f the tosylate was carried out as described previously (scheme 12). The substitution reactions generally proceeded in yields of 70% or above. Yields of individual isomers are distorted by the large amount of column chromatography required. In some cases it was possible to crystallize the cis isomer from methanol after flash chromatography. The Mitsunobu chemistry was carried out subsequent to the synthesis of this series therefore the compounds were synthesized by tosylation and substitution.
3.3 Non-dioxolane compounds.
Several compounds were synthesized and tested that did not contain the dioxolane including 42 and 43 (scheme 26). This pair of enantiomers was of interest for several reasons. Firstly to investigate absolute configuration, secondly to test ideas generated from molecular modeling studies and to make more water soluble compounds.
SCHEME 26 Glycerol targets
OH p H
01 01
No.42 NH, No.43 NH, 01 01
62 Thankfully there are many chiral glycerol analogues commercially available. In our case we chose a route using both enantiomers of glycidyl tosylate. It has been shown that the epoxide ring of glycidyl tosylate can be opened cleanly and regiospecifically with catalytic amounts o f BFg.OEt: by benzyl alcohol in dichloromethane^^^. This reaction followed by tosyl displacement to form a new epoxide provided a rapid route to the target compounds without any recourse to protecting the alcohol groups.
SCHEME 27 Epoxide Opening.
2,4-dichlorobenzylalcohol OTs 0 7 s BF3 .OEt2 .3 A mol Sieves DOM, 0°C No.78 & 79
In our hands the attack of the dichlorobenzyl alcohol under acid catalyzed conditions proceeded in 50-60% yield of the desired regio-isomer. Purification of compound R-(- )-78 (scheme 27) was performed by chromatography rather than distilling out the benzyl alcohol because the boiling point of the dichlorobenzyl alcohol is higher and the product may have been affected. This separation required care to obtain the pure compound and it may have resulted in lower yields. The yield of the S-(+)-79 was
62% compared to 54% for the R-(-)-78 and they had opposing ao values. Products arising from attack at the secondary position of the epoxide were not isolated as only starting materials and product were observed by TLC.
SCHEME 28. Forming the epoxide.
K2 CO3
No.78 & 79 N0 .8 O & 81
Formation of the epoxides 80 and 81 was effected (scheme 28) as in the literature by intramolecular displacement of the tosyl group in methanol solution. Stronger base is often used for similar reactions but in this case K 2CO3 was sufficient. Filtration through silica followed by aqueous work up yielded (-)-80 in a 50% yield. However a simplified procedure for the (+)-81 yielded the product in 72%. It was found that the
63 epoxide decomposed over time even when stored at 0°C. Therefore 80 and 81 were used rapidly after isolation.
SCHEME 29 Reopening the Epoxide.
N o.80 & 81 N o.42 & 4 3
i. 4-Benzyliminophenol, NaH, DMF,0-80°C; ii N 2 H4 .H2 O, EtOH, 0°C
Although the epoxide was unstable when stored it was surprisingly resistant to opening by the phenoxide anion in DMF. Stirring the reactants overnight in DMF at room temperature yielded no product whereas heating to 80°C was successful. As before, substitution by 4-benzylidenaminophenol followed by chromatography yielded a mixture of imine and amine products. This mixture was stirred with hydrazine hydrate in ethanol to yield the amine. In the case of 42 (scheme 29) the substitution proceeded smoothly but the overall yield was lowered by an aqueous work-up subsequent to the imine removal. For 43 (scheme 29) the substitution reaction was did not proceed well but the reaction mixture for the hydrazine reaction was purified by column directly with an improved yield. The net result of this was that the overall yields o f 42 and 43 were identical (23% from the epoxide). Both compounds were characterized and tested as the oxalate salts. Another target from the medicinal chemistry standpoint was to use our optimal aryl substituents with the three atom linkers seen in NS1608 and phloretin. Two compounds were chosen.
SCHEME 30 Three-atom chain targets.
N . .N
N0 .4 4 N0.45
Compound 44 (UCL-2347) was produced from 4-aminobenzoic acid and 2,4- dichlorobenzyl alcohol in Mitsunobu conditions. The reaction proceeded smoothly
64 yielding the compound in a 60% yield (scheme 31), but it was important to dry the starting materials before use by evaporating with toluene for azeotropic removal of water.
SCHEME 31 Synthesis of UCL-2347.
Cl ÇI o /=\ PPhg, DEAD , OH + ^— (( ))—NH2 ------► ^ HO THF, 0°C A J or ^ or ^ NH2
Compound 45 (UCL-2355) was synthesized from 2,4-dichlorophenylisocyanate and 4-nitroaniline. The compounds reacted smoothly in dry toluene to yield the 1,3- biphenylurea 82 in reasonable yield (78%, scheme 32). These compounds were insoluble in most solvents apart from DMSO and most recrystallisations of the compound would also precipitate the impurities. Therefore to ensure a clean product it was important to dry all starting materials thoroughly before reaction. When the conditions were dry the pure product was obtained simply by filtering the reaction mixture and rinsing the product with dichloromethane (scheme 32).
SCHEME 32.
Toluene 01 N = C = 0 + HoN NO; Reflux
No.82
SnCl2 No.82 ------EtOH, Reflux
The reduction of the nitroarene 82 to the arylamine was attempted in ethanol with tin (II) chloride. This mild technique reduced the nitro group in good yields (82%) however care had to be taken to ensure that the reaction went to completion. The oxidation of Sn (II) is a two electron process therefore three equivalents should reduce
ArN0 2 to ArNHi. In our case four equivalents were used as the quality o f the SnCl 2 was unclear. Hydrazine hydrate with palladium on carbon also reduced the nitroarene. However the reaction time of 1 hour was not long enough and a mixture of products was obtained.
65 SCHEME 33 UCL-2304.
UCL-2304
The furan-containing compound 49 (UCL-2304, scheme 33) was synthesized before it
was clear that the dichlorophenyl moiety was essential and therefore a ( 2 - chlorophenyl)-furfural was used as the starting material. It was planned to reduce to the alcohol, prepare the tosylate and perform substitution but this proved impossible. The difficulty seems to arise fi'om the nature o f the alcohol. Reduction o f the aldehyde with sodium borohydride followed by acidic workup led to a complex mixture. Since the reaction mixture showed a clean conversion by TLC it was thought that the acidic work-up was responsible. When the reaction mixture was quenched with NaOH solution the alcohol was obtained as a clear yellow oil. It was noticed that the alcohol discoloured and solidified over days to form a black solid. Since the acid sensitivity of the furfuryl alcohol was probably due to the elimination of the alcohol group it seemed unwise to proceed via a route that would make the alcohol into a more effective leaving group. Therefore the route chosen involved the reduction and modified Mitsunobu coupling with the phenol 54 (scheme 2) as one procedure without isolation of the alcohol. This proceeded in a yield of 39%, which compares reasonably with the other examples of this reaction that were carried out on the dioxolane analogues.
SCHEME 34 Synthesis of UCL-2304
U C L -2304
1. NaBH 4 , MeOH O 2. 54, ADDP. PBus N N B en zen e
The final compound to be discussed was the first compound to be prepared. To obtain the partial structure 8 (UCL-2112, scheme 35) commercially available 4-(4-
66 methoxyphenyl)-piperazine was acetylated by acetic anhydride to yield 8 in 89% yield.
SCHEME 35 Synthesis of UCL-2112.
/ \ AC2O / \ /9 MeO- N NH MeQ- \ /
UCL-2112
67 Characterization of Compounds
3.4.1 Mass spectra
In the majority of cases atmospheric pressure chemical ionization or electrospray were used to ionize the compounds. For compounds containing a basic centre this normally produced the parent ion in 100% relative intensity. This was true for imidazole, primary amine and piperazine compounds. Electrospray generally yielded a MNa"^ ion in addition to the ion. Fast Atom Bombardment was generally less useful for these compounds than electrospray and APCI, which was a serious disadvantage as it was the only mild ionization source suitable for high resolution spectra available to us. In compounds without a basic amine the molecule would fragment to provide two different peaks. For the 2-methyl-2-phenyl-4-hydroxymethyl-1,3 -dioxolanes the compounds generally fragmented to yield the acetophenone as the most abundant ion. This ion was also observed in the compounds where a phenyl or tosyl group replaced the hydroxy group. However for these compounds the molecular ion would fragment to give the 1-substituted glycerol as the most abundant ion. This may be due to the decomposition of the compound in solution before it reaches the ion beam, as the process requires a water molecule to attack the cationic center of the molecule. Or they may represent the ionization of trace impurities within the sample that are more stable and therefore more abundant on the mass spectra.
SCHEME 36 Fragmentation of the Dioxolane ring
2 H2O + H3O" O
Possible sources of ions in compounds without basic centers
68 Since most of the compounds contain chlorine atoms isotopic splitting is observed in
spectra and is listed in the experimental section.
3.4.2 Assignment of Isomers by NMR
Interpreting the spectra of the 2-methyl-2-phenyl-4-phenoxymethyl-1,3-dioxolanes
presented some difficulties. While almost all of the imidazole containing compounds are known and the sepæ*ation o f cis and trans isomers is unambiguous. The assignment of peaks in mixtures of the novel non-imidazole compounds was non trivial. Even in cases where the isomers were separated, assigning the peaks based on chemical shifi and coupling constants was difficult. However it was clear that the two isomers had a distinct pattern particularly in the methylene region. There were also differences in the phenyl and methyl groups. The scheme below shows a 3- dimensional representation o f the cis and trans isomers.
SCHEME 37 Cis and Trans isomers.
x o XO rO
H
Cis Trans r-Methyl-c-oxymethyl-1,3-dioxolane r-Methyl-t-oxymethyl-1,3-dioxolane
Although the phenyl and methyl signals showed some variations between cis and trans isomers the most diagnostic signals were for the dioxolane and oxymethylene regions. Previous attempts to assign the isomerism of dioxolane used arguments based on the deshielding effects of the phenyl ring and the oxymethyl substituents^^^. Other methods relied on comparing the shifts in the dioxolane ring with analogues of known conformation and avoided the complex splitting patterns of the CH protons^"^^. Since there are two discernible patterns for each isomer our approach was to assign the relative stereochemistry unambiguously using NOE spectroscopy. Since most compounds had very similar spectra (apart from the aromatic region) it is more instructive to describe the spectra for a given pair o f isomers.
69 SCHEME 38 Labeling of protons for NMR Assignment.
Cl
HbX-OPh Hd H e
T r a n s
Below we can see the spectra of UCL-2335 and its trans isomer. These are very
typical spectra for compounds of this type. The CH 2 methylene groups contain two non-equivalent protons and so we see four double doublets.
Figure 3.1 Cis Isomer
V V
Figure 3.2 Trans isomer
70 As we can see the cis isomer UCL-2335 has a methylene region between 3.80ppm and 4.30ppm. The trans isomer has a methylene region between 3.70ppm and 4.60ppm. The signals at 4,30 and 4.60 can be assigned to the He (scheme 38) proton for both isomers. In the case of the trans isomer the Hy (scheme 38) proton is also shifted dramatically downfield to 4.30ppm. It has been argued that this process is due to the alignment of the aromatic moiety at the 2-position. The coupling constants are useful in assigning the spectra since the geminal JHa-Hb= 8.5Hz and JHd-He= 9.5Hz are similar in most cases apart from the alcohol where Jnd-He^ 12.0Hz. The coupling constants for Ha-Hc and Hb-Hc are 6 .8 Hz and 4.7Hz respectively. The coupling for Hd-Hc and He-Hc are 6.5 and 4.6 respectively. Although there is one large and one small vicinal coupling it is not consistent with an anti conformation. The average coupling with He consists of two similar values 6.0Hz and 5.0Hz with Hd and He. Similarly with He and Ha/Hb (7.0 and 5.0Hz). The dioxolane ring has an envelope structure and it is likely to be in constant flux at room temperature and too rapid for the NMR time frame. Therefore the couplings may represent an average o f several conformations. The two spectra shown are assigned as cis or trans based on the evidence o f NOESY spectroscopy. Although this was not performed for every compound the results were unambiguous enough to assign other compounds as cis or trans isomers with some confidence. The exact technique used was NOESY spectroscopy and both 1-D and 2- D were used for individual cases. NOE spectra are based on the nuclear-Overhouser- effect. When irradiated the angular spin momentum o f the nucleus is typically rotated by 90°. The signal is obtained by recording the relaxation back towards the ground state and the free induction decay of all the nuclei is typically then converted to the spectra by Fourier transform. The nucleus returns to the ground state by donating energy to the surrounding atoms that have the same magnetic spin. This relaxation occurs through space and so an excited nucleus can donate energy to atoms in the molecule, which are close in space but not within a certain number o f bonds. In NOE spectra we irradiate the molecule at the resonance frequency o f a particular proton as well as obtaining the standard NMR. Therefore signals for protons in close proximity to the irradiated proton will have enhanced signals. The NOESY spectra in 1-D is effectively the NOE spectra minus the IH NMR. Therefore the ID-NOESY only shows the enhancements in the signals and
71 reduces ambiguity. 2D-N0ESY is a series of ID-NOESY spectra performed at each frequency and combined to give the NOE interactions within ~4A for each proton on the moleeule. This makes it an extremely powerful analytical technique. The example shown below is for the tosylate of the trichlorophenyl compound. Firstly the cis isomer. In both of the speetra the shift (ppm) values quoted in the text are larger than the values on the spectra. This is to correct for the error due to the ineorrect reference value assigned to chloroform (7.13).
Figure 3.3 Cis Isomer
Figure 3.4 Trans Isomer
r-g a
"-----
Scheme 39 below shows the interaetions that occur and the section of spectra chosen shows all of the relevant signals.
72 SCHEME 39 NOESY Interactions.
Cl
Cl O p Cl A—^Hc
Ho Hm
Trans NOE interactions between protons The NOESY spectra provide experimental basis on which to assign individual peaks in the NMR. Firstly the interaction of the 2-methyl group with the methylene protons. This group interacts with three of the five methylene protons shown in scheme 39. Since Hb and He are on the opposite side of the ring the remaining protons are Ha, He and Hf. He (4.03ppm) and Hf (4.10ppm) had higher shifts than Ha (3.88ppm) and Hb (3.70ppm). The methylene peaks are well separated due to the strongly electron withdrawing nature o f the tosyl group. Often two peaks (Ha/He) will merge for example when the substituent at the oxymethyl position is electron rich. The ortho proton of the trichlorophenyl group (Hg) has NOE interactions with both He and He proving that for this compound that it is the cis isomer. This proton also interacts with the methyl group, which shows that the ring can rotate fully. On the tosyl ring the ortho protons interact with He and Hf but not with any of the protons on the ring. For the trans isomer the interaction o f methyl (l.TOppm) with Hb (4.30ppm) and He (4.60) confirms the structure. This is augmented by the interaction o f the trichlorophenyl proton Hg with Ha and He. It is surprising that the He/Hf do not both interact and it may show that the conformation is fixed. The trichlorophenyl ring can rotate but the interaction between Hg and the methyl group does not seem as strong as in the cis isomer. The shifts for the Ha proton decreased from 3.8ppm to 3.6ppm whereas Hb increased from 3.70 to 4.3. Hf and He were more shielded moving from 4.0 & 4.1 to 3.9 and 3.8. This is the pattern that is replicated for many sets o f isomers. NOE is a sensitive technique and signal noise can be a problem however the signals
73 discussed here are representative and consistent with the NOESY spectra for other sets o f isomers.
74 4. Molecular Modelling
The basic premise of molecular mechanics is that classical physical calculations can be used to approximate the forces between atoms. The most straightforward model would treat all atoms as being identical and that a ball and spring model can approximate the molecule. This means that all steric forces can be treated using Hooke’s law and potential energy can be estimated by an equation such as the one below.
V= Sl/2Kfc(b-bof + El/2Ke(0-9o)^
+S1/2K4(Ç-^o)'
+21/2IC|,(<|)-5) +Sl/2[C,2(ij)/r,j‘^-C6(i,j)/r/]
+Zqiqj/(47rGoer)
Where b= bond length, 0= dihedral angle, a fixed dihedral (i.e= aromatic), torsional angle, Cij= Van der Waals interactions, q= charge This equation is illustrative of the overall technique but is oversimplified. Modem techniques use many different atom environments and better estimations of electronic contributions to obtain more accurate results. For this project molecular modeling was performed using the XED program (extended Electronic Distribution), a program which combines electronic and steric factors when calculating the minimum energy of a molecule. Minimization of the conformation is performed using a Monte-Carlo simulation. This means that the program will move the molecule about its rotatable bonds until it finds the local energy minimum. It then picks a random conformation of higher energy and repeats the process. This continues until the calculations reach a consistent minimum value or a series of minima. The lowest of these values is called the global minimum. The XED program then adds factors to allow for electronic effects and performs further calculation. In our calculations the method was as follows. Molecules were drawn using the package and then minimised to ensure that bond lengths and angles were correct and aromatic rings were fiat. This structure was then used in the conformation hunt, which is the Monte-Carlo simulation described above. In order to mimic the conformation of the molecule at the channel the temperature was set at 36°C and the dielectric constant was set at s=2. This value is low in order to mimic the lipophilic environment of a
75 channel bound to the cell membrane but may not be true for individual sites on the channel. The range of energies allowed above the local minimum was large (ISkcal) to ensure that conformations did not become caught in an energy trough. After the minimization was finished the set was restricted to values Skcalmol’^ above the energy minimum because conformations above this energy are unlikely at ambient temperature. The global minima obtained do not correlate to any experimental values however obtaining similar values for similar molecules would suggest that the program was finding the global minima. The global minima calculated for several sets o f compounds are shown below
Molecular Modeling results Cl
UCL2292; X= H, E= 11.04 UCL2302; X= H, E= 7.94 UCL2270; X= CN, E=11.56 UCL2303; X= CN, E=8.61 UCL2298; X= CONHg, E=12.24
Cl
L UCL2202: X=H, E=23.50 UCL2281 : X=Br, E=21.51 Cl UCL2242: X=NH2, E=18.15 Q O- X UCL2297; X=piperazine, E=24.63 Ketoconazole; E=21.71
- 7 ^ 0 0, o
UCL2255; X=Phenyl, E=24.32 UCL2255; X=Phenyl, E=22.47 UCL2254cis; X=Chlorophenyl, E=23.42 UCL2254trans: X=Chlorophenyl, E=20.61 UGL2158cis; X=Dichiorophenyl, E=17.55 UCL2158trans; X=Dichlorophenyl, E=21.01 UCL2253: X=trichlorophenyl, E=21.90
Molecular modeling and orbital calculations are often used to gain an insight into the QSAR o f a series o f compounds or to dock a drug into a virtual binding site. In our case it would have been unwise to use either of these approaches for the following
76 reason. Even when the sets o f conformations are limited to within S.Okcal o f the global minimum there was still a wide range of possible conformations and there is no indication which one is the active conformation. Creating a picture of fhe binding site based on the compounds tested would therefore become arbitrary. There is no crystal structure of the BK channel and if there was it may not be useful as there are many open and closed states. So docking simulation would not be useful. Also it is unlikely that it could be used to interpret the QSAR o f the compounds given that the results obtained have large errors due to the difficulty of the experiments and the variability of the cell’s behaviour. Also the openers of the channel do not reach an optimal
response and so the results can not be expressed as EC 50. It was decided not to use modelling as a predictive tool but rather to give an insight as to how the compound may look in 3-D. It was also thought interesting to compare openers and blockers of the channel. When a number of the compounds had been minimized it was clear that there were no great differences between the global minima of blockers and openers. This is unsurprising and consistent with results that shall be discussed subsequently but consistently the global minima of the compounds showed a folded structure in which the phenyl rings stacked over each other. Even in cases where sets of isomers were minimized, both cis and trans isomers folded to stack the phenyl rings on top of each other. This was observed for a range of phenyl ring substituents. This led us to consider replacements for the dioxolane. One option was to open the ring to obtain a glycerol analogue, which was desirable for several reasons. The conformation of the molecule (41) was calculated as before. The compound folded in a very similar fashion to the equivalent dioxolane (UCL-2273) compound and this provided the impetus for the synthesis of 41 and 42. If the compounds proved to be active at the channel it would not prove that the folded conformation binds to the channel or even that the folded conformation is the most stable. However it does demonstrate that molecular modelling is a useful tool for generating and testing ideas. For an approximate visual representation of UCL-2273 in folded and open forms see appendix II.
77 5. Biological testing
Ketoconazole and UCL-compounds were tested using smooth muscle cells cultured from bovine pulmonary artery.
Culture of Bovine Pulmonary Artery Smooth Muscle (BPASM) Cells A sample of BPASM cells was kindly provided by Dr. R.Corder, William Harvey Institute, at passage 10. Stocks o f cells were frozen in liquid at passage 12. Cells used in the study were from passage 13-19. Cells were cultured in T25 culture flasks (Nunc) in DMEM (Life Technologies) supplemented with 10 % foetal calf serum, 2 mM L-glutamine, 100 units’ ml'* penicillin and 100 pg ml'* streptomycin. For electrophysiological recording cells were plated into 35mm tissue culture dishes and used within 1-3 days.
Electrophysiological recording BK currents were recorded under whole cell voltage clamp using an EPC7 patch
clamp amplifier controlled by pClamp 6 software that was also used for data acquisition and analysis. All experiments were conducted at room temperature (23-25 ""C). For recording the cells were placed on the stage of an inverted microscope (Nikon Diaphot) and continuously superfused at 3-5 ml min'* with a solution containing (in mM) NaCl 136, KCl 6 , M gS0 4 , 1, CaCb 2, glucose 10, HEPES 10. pH was adjusted to 7.4 with 1 M NaOH. The ‘internal’ solution used for filling patch pipettes contained (in mM) KCl 136, MgS 0 4 1, CaCL 1, HEPES 10, and EGTA 2. The pH was adjusted to 7.2 with 1 M KOH. Free Mg^^ and Ca^^ were calculated to be 150 nM. Patch pipettes were fabricated firom 1.5-mm borosilicate capillaries (Clarke ElectroMedical). The pipettes were fire polished and coated with Sylgard Resin and had resistances of 3-5 MQ when filled with internal solution. Routinely cells were clamped at a holding potential of -20 mV. Voltage dependent activation of BK currents could be elicited by applying 100 ms voltage jumps to more positive potentials. The effect of test compounds was assessed by measuring the change in current activated on stepping from the holding potential to +80 mV.
78 Stock solutions of test compounds were prepared in DMSO at a concentration of 2x10'^ M and diluted in bath solution to give the desired final coneentration. The concentration of DMSO did not exceed 0.5 % v/v and at this concentration had no visible effect on the current. Drugs were applied by means of a manifold system that allowed the control solution to be replaced by solution containing test compound. Each concentration of tested compound was applied to at least three different cells and the effect is reported as the mean change in current at +80 mV ± s.e.m. As an internal control the effectiveness of ketoconazole was tested on every cell included in the study. Materials All tissue culture reagents were obtained from Life Technologies. HEPES and EGTA were from Sigma, all other reagents were of ‘Analar’ quality and obtained from Merck.
79 Results and Discussion
In the chapter dealing with the selection of compounds, the compounds were grouped based on a rationale for synthesizing the set or sets of compounds within a series. For
example series -1 was to locate the pharmacophore of the channel and series -2 was to investigate the role of lipophilicity at the dichlorophenyl ring. This reasoning will be discussed in more detail along with the results from the biological testing. In addition, several key compounds that link the series together shall be discussed where appropriate. The overall aim of this chapter is to reflect how the project evolved and what it has shown. Therefore the discussion will use the framework of series-1 to series-4 from chapter 2 more loosely, as they were formalisms to introduce the content rather than explanations o f it.
Series-1
In order to investigate the activity of ketoconazole our approach was to ask, which parts of the molecule are necessary for BK activity. In an attempt to answer this
question we synthesized partial structures o f ketoconazole as indicated in figure 1
r
Ketoconazole. 60% increase in current at lOpM, 125% increase at SOpM, 400% increase at lOOfiM, Current obtained by stepping between -20 and 80mV 30mM Ketoconazole causes 20mV shift in activation potential towards less polarised potentials
The curved arrows represent sections o f the molecule being removed to locate the pharmacophore Figure 1. Ketoconazole activity at the BKca channel
80 The first results to be discussed here are the compounds that had little activity. These are shown in Chart 1. However some negative results can provide useful information.
CHART 1. Inactive partial Structures
N N“ - ° - \ \ // Me
HO BzO
UCL-2112 UCL-2135 UC L-2134
Cl 01
c,
TsO BzO HO
UCL-2250 UCL-2203 UCL-2220
UCLNO Cone fiM % Activation % block UCL-2112 100 None None UC L-2134 30 N on e 40 U C L-2135 100 50 N one UCL-2203 10 None None UCL-2220 100 None None UCL-2250 10 N on e 8%
% Activation/Inhibition of BK current after stepping the potential from -20 to 80mV. As we can see from chart 1 the alcohol-2135 has some opening activity showing that it may contain part of the pharmacophore. Interestingly, benzoyl compound (UCL- 2134) displayed modest blocking activity but the tosyl analogue (UCL-2250) was inactive. Piperazine (UCL-2112) was inactive. The lack of activity for the triazole derivatives 2203 and 2220 compared to the imidazole compounds could be due to a lack of basicity or low tolerance for the more hydrophilic triazole at this position. A later compound showed that removing substituents at this position improved activity. M ost o f these compounds are not partial structures o f ketoconazole and show that more o f the structure must be included for BK activity.
81 Biological activity became more interesting when the phenoxy moiety was introduced and the role of hydrogen bonding to the piperazine ring was investigated, in the compounds shown in Chart 2.
C H A R T 2. Investigating Piperazine
NHc UCL-2242
/■ RN UCL-2322 V
X /—\ RN NH UCL-2927 \ /
X=H UCL-2202 / \ O RN N Ketoconazole X=Br UCL-2281
% inhibition of current % in crea se in BK current C one pM UCL-2202 U C L-2322 UCL-2281 C one pM UCL-2242 UCL-2297 Ketocon 3 40 — — 10 175 — ------10 60 79 ------30 580 310 175 30 73 ------95 100 200 998 350
There were two striking features in chart 2. The increased opening activity of 2242 and 2297 over ketoconazole being the first. Secondly the fact that 2202 and 2322 blocked the channel. The IC 50 being 5pM in the case of 2202. It should be noted that in all cases for the openers of BK that an EC 50 measurement was impossible since a maximal activity was not reached even at concentrations of lOOpM. Above these concentrations the compounds were insoluble. In the case of UCL-2242 the dose response curve showed unusual behavior at high concentrations. As we can see from figure 2 the activity peaks at 30pM, and is similar at lOpM and lOOpM.
82 Figure 2 Graph comparing dose response of UCL2242 and Ketoconazole
2242 Vs ketoconazole UCL-2242 ketocon
800
600
% £ 400 2 = o u 200
1 .OOE-05 1.00E -04
C o n e I m o la r
The fall of in activity at high concentrations cannot be explained easily. For example 2242 (chart 2) may affect a separate site on the channel, or damage the cell in some way. If the compound acts by stabilizing an open state of the channel then it could possibly stabilize a closed state of the channel at a higher concentration. There are many possibilities, however the true cause of this behavior could only be elucidated by further biological evaluation. This result as it stood allowed for two opposing interpretations on which to develop the project. Firstly the mixed behavior of the amine could be seen to show that this is an unsuitable group at this position. Contrarily the large increase in BK current compared to ketoconazole could be seen as a sign that the amino group is optimal and the activity at high concentration was unique to this compound. Of the two options the second was the least limiting and could be tested easily with further compounds. Therefore the primary amine was included in several more compounds. Looking at Chart 2 as a whole the most significant result was the blocking of the channel by UCL-2202 and UCL-2322. This points to the four position of the phenoxy moiety as a detenninant for biological activity. It is important to note that due to difficulties in the synthesis, 2322 was not available at the time that a further series investigating this position was synthesized, although 2322 had been a target from the start of the project. Thankfully the subsequent results are consistent with the result for 2322. Analysis of the piperazine ring provides an interesting picture of the hydrogen- bonding interaction between the molecule and the protein. When the compound has an unsubstituted phenoxy group (UCL-2202) it is a blocker of the channel. Replacing
83 piperazine with a lipophilic group such as bromine (UCL-2281) increased the blocking activity but this was only tested at one concentration. Placing an amino group at the 4 position in place of piperazine not only changed the activity to opening the channel but increased the activity compared to Ketoconazole.
Figure 3. Hydrogen bond acceptors
B lo ck er O p en er O p e n e r O :0RO He * RO—(f '^N N-^ M/ Hydrogen bond Donor/acceptor a c c e p to r s
The amine can donate or accept a hydrogen bond, but since ketoconazole can only accept a hydrogen bond at this position it was thought that a hydrogen bond acceptor at this position was the determining factor in opening the channel. UCL-2322 blocking the channel contradicted this idea and raised the interesting possibility that the lead compound contains a functional group that may work to antagonize the overall effect of the compound. This may be the molecular correlate of the blocking
activity of ketoconazole in GH 3 cells compared to opening BK in smooth muscle.
Figure 4. Comparative hydrogen bonding properties of 2242, 2322 and ketoconazole
Opener Blocker Opener
O : > 0- N o- N
Hydrogen bond Donor/acceptor Hydrogen bond acceptor a ccep tors
Ketoconazole and UCL-2297 (see chart 2) have two possible hydrogen bonding groups on the piperazine ring. Since 2322 shows that the tertiary amine on the phenyl ring is at best redundant, then the amide or amine is hydrogen bonding with the channel. This is a useful observation because it shows that the hydrogen bonding at this position could be with a hydrogen-bond donor/acceptor or acceptor whereas the interaction beside the phenyl ring could only be with a donor or donor/acceptor. This shows that the hydrogen-bond interaction seen with the primary amine is new since it could not occur with the lead compound. This was home out by subsequent investigations.
84 The imidazole ring also contains a basic nitrogen group and as we have seen replacing it with a triazole caused reduced activity. When the imidazole group was removed in 2158 (see chart 3) the BK opening activity increased 2-fold. This shows that imidazole was not only unnecessary for BK activity but has an adverse effect. It was decided not to investigate other more lipophilic groups since UCL-2158 was
insoluble at lOOpM. Interestingly the removal of imidazole also yielded an acitve
compound in the case of clotrimazole, another antifungal which blocks the BK channel.
CHART 3. UCL-2158
Cl UCL-2158 1 UCL-2 1 58] ketocon” ' O 1 L Micromoles % increase in current O 1 10 120 60 1 30 225 98 i 100 325
At the end of series-1 two large groups have been removed from the molecule and increased the activity at BK in both cases. We have also seen that the substituent at the 4-position of the phenoxy ring was crucial. Therefore it was important to combine both effects in order to test whether they are additive. The compounds are part of series three but they included here as they were synthesized specifically to address this question. This led to the parallel series shown below in chart 4.
CHART 4. Parallel series
,CI Cl Cl
Cl Cl Q Q Q
Ketoconazole UCL-2242 UCL-2202 65% a t 10|aM 150% increase IC50 5nM at lOfiM NHc 72% block at 30pM ro
UCL-2273, U C L 2 1 5 8 , ■ UCL-2303, UCL-2297 245% Increase at lO^M 125% in c re a s e at lOfiM 92% b lock a t 30plVI. ' f Both isomers tested
85 Comparing the activity of 2273 to 2242 as seen in chart 4 is difficult, as the shape of the dose response curve for 2242 was erratic. At lOpM 2273 was more potent than
2242 as we would expect, however at 30pM 2242 was more active than 2273. At
lOOpM the value for 2273 was far larger than for 2242. This means that we cannot say that the increases in activity are additive although this seems to be the case at the top and bottom of the concentration range. Unfortunately a full dose response curve was not available for 2303 and 2292 so we can only say that at 30pM 2292 and 2302 were more active than 2202. Therefore at this concentration the effects of removing the piperazine and imidazole seem to be additive for this case. One problem in comparing results is the large difference in the response of the cells to ketoconazole in different experiments. The graph below has used the values for ketoconazole from the same set of experiments as 2273 to adjust the data for 2158 and 2242. This was used rather than an average value since the increase in current is comparative to the control as measured in the same set of experiments and this varied considerably.
Figure 5. Advantage of 2273 over 2158 and 2242
. U CL-2273 Advantage of 2273 . UCL-2242 UCL-2158
800
c 600 j 0(/}) TO 2 400 O2 3 - - 200
0 1 .OOE-05 1 .OOE-04
C o n e m o la r
This comparison in figure 5 gives a more correct picture than the absolute figures given above and it shows that 2273 is approximately equipotent to 2158. It also shows that it is less potent than 2242 at 30pM, which means that the increases in activity are not additive. However we can say that removing the imidazole from 2242 abolished it’s unusual behavior at high concentrations and that removing the piperazine group of 2158 increased the solubility, therefore this is a useful compound.
86 Series 2
The next target for investigation was the dichlorophenyl moiety. The aim was to gain a detailed picture of the role for each part the group. The first series used UCL-2158 as a template keeping the l-acetyl-4-(oxyphenyl)-piperazine section, with the remainder of the compound being viewed as the sum of three parts. The acetal, phenyl ring and chlorine atoms were added in turn. The acetal would simply increase the lipophilicity and two alkyl compounds deriving fi’om cyclohexanone and acetone were tested. Phenyl analogues would enhance any lipophilic binding and open the possibilities of electronic interactions. The chlorine atoms change the behavior of the molecule by increasing the lipophilicity and changing the electronic nature of the phenyl ring. The first set o f compounds investigating this is shown below in Chart 5.
CHART 5. Investigating the Dichlorophenyl moiety I ^ HQ OR OR I 1 ^ 0 OR ? o OR
H O ^ o - V o i y — ^
UCL-2238 UCL-2245 UCL-2280 UCL-2255
I ^
O OR U o OR J^O OR /
UCL-2264 UCL-2254 UCL-2158 UCL-2253
87 R / = \ / — \ P R - P h en yl Inactive at SOjxM ' 'Q P—^ ^—N N- o^y R = 2-Chlorophenyl 42% increase at 30)iM
2238 2245 & 2280' Inactive ^ ~ 2,4-Dichlorophen 125% at lOpM, 225% at 30pM
R = 2,3,4-Trichlorophe 15% increase at lOpM
It is clear that the 2,4-dichlorophenyl moiety is essential for activity. Adding or removing chlorine atoms to the phenyl ring dramatically affects the biological activity. This series does not give us an insight into the role of the dioxolane ring. The dioxolane ring does not seem to play a role in binding, since the acetal 2 2 4 5 is inactive. However it may play an important structural role which could only be elucidated by further investigation. The 2,4-dichlorophenyl substitution may be essential for several reasons. The lipophilicity of the phenyl ring will increase with each new chloride group. It is possible that the dichlorophenyl ring has the optimum lipophilicity for the binding site, the monochloro and trichloro lying at either side o f the maximum. In the case o f the trichlorophenyl it was insoluble above 30pM and may be near the limit of it’s solubility at 1 OpM
The chloride group is electron withdrawing and may tune the electronic nature of the ring. Also the 2,4-dichloro may be the optimal substitution pattern. For example the substituent at the 3 position may cause a negative steric interaction. There may be a mixture of lipophilic, electronic and substitutional effects contributing to the binding of the 2,4-dichlorophenyl moiety. Therefore 5 further compounds were synthesized to investigate positional effects and electronic effects.
CHAJRT 6 . Further investigating the Dichlorophenyl moiety UCL-2327 X= 4-Chlorophenyl, UCL-2273 X= 2,4-Dichlorophenyl, UCL-2335 X= 2,5-Dichlorophenyl, UCL-2333 X= 3,4-Dichlorophenyl, OR UCL-2348 X= 2,4-Difluorophenyl, R= 4-Aminophenyl UCL-2349 X= 2,4-Dimethylphenyl.
The graph below presents the data comparing chlorinated compounds of the type shown below. The data has been adjusted to allow for the differing potencies of ketoconazole in the experiments. Figure 6 . Dose/Response for compounds in chart 6
Modification of dichlorophenyl
900
800 c g 700 3 600 Ü m 500
400 - UCL-2273 2,4-dichlor 0) w B UCL-2327 4-Chloro (TJ 300 - 2 UCL-2333 3,4-dichlor Ü 200 ^ -X— UCL-2335 2,5-dichlor C 100 ^ — ketocon
1.00E-05 1 OOE-04
C o n c /m o la r
As we can see from figure 6 the 2,4-dichlorophenyl is the most potent compound in this set. These results allow for a more detailed interpretation than the previous set of compounds. One interesting perspective is to fit each substitution pattern into a hypothetical binding pocket.
The 4-chlorophenyl compound U C L -2 3 2 7 displays much of the activity of the dichloro analogue, unlike the 2-ehlorophenyl. This indicates the 4-position as being important for binding, which raises the question of the role played by the 2 - substituent. Does it simply raise the lipophillicity of the compound or does it play a role in the positioning of the 4-substituent. The fact that the 3,4-dichlorophenyl
(UCL-2333) seems to be approximately equipotent with U C L -2 3 2 7 would suggest that the 2-substituent might place the 4-substituent in the correct space for binding. The 2,5-dichlorophenyl analogue at lOpM would seem to be equipotent with 3,4- dichlorophenyl and 4-chlorophenyl, which could fit with the description given above. At 30pM it would seem that the potency of 2,5-dichlorophenyl is intermediate between U C L -2 2 7 3 and U C L -2 3 3 3 . Given the errors in these results it is difficult to determine whether the 2,5-diehlorophenyl provides greater binding than 3,4- dichlorophenyl or 4-ehlorophenyl. If the argument above is true then the 2,3,4- triehlorophenyl should have been active. Its lack of activity can be explained in two ways. Firstly that the substitution pattern is tolerated in binding but the higher
89 lipophilicity of the ring is not. Secondly that substitution at the 3-postion is not tolerated at the binding site. The first explanation will allow us to retain the argument unchanged; however the second interpretation requires us to rethink the argument. If the 3-position were detrimental to activity then one would expect the 3,4- dichlorophenyl compound to be less active than the 4-chlorophenyl since the benefit o f the 4-substituent would be negated by the negative interactions of the 3-substituent. In fact they have approximately the same activity, as does 2,5-dichlorophenyl compound. It is clear that the 3 and 5 positions may be identical. How therefore can 2,5-dichloro and 3,4-dichloro be so different to the 2,3,4-trichloro? The explanation that the trichloro moiety is too lipophilic may be valid. However we can also explain this in terms of binding pockets for the phenyl substituent. The 2-chloro substituent will limit the rotation of the ring and maximize the positive interaction at the 4-position so 2,4-dichloro is the most potent compound. The 4- chloro can freely rotate and so there will be an unfavorable entropy change in orientation of the ring, hence the loss in potency. For the 2,5-dichloro the ring is held in the correct position by the 2-substituent and the 5-chloro substituent is well tolerated at the binding site. 2,3,4-trichlorophenyl is orientated by the 2-chloro substituent but a steric clash occurs at the 3-position preventing tight binding. This steric clash is avoided on the 3,4-dichloro ring because the ring is not held in place by the 2-substituent and so can rotate to appear as a 4,5-dichloro. In short the binding site can distinguish between the 3 and 5 position when the phenyl ring contains a 2- substituent. This is displayed graphically below in figure 7.
90 Figure 7. Hypothetical binding pocket
Possible schematic to explain the substitutional effects on the phenyl ring 2-substituents locks the ring into position, positive interaction at 4, steric clash at 3. 5-substituent tolerated
While this description may be true and could be easily tested by synthesizing 2,4,5- trichlorophenyl and 2,3-dichlorophenyl analogues, it is also based on a limited number of compounds and an assay that contains large margins of error. Therefore it is only one of several arguments that could be made to fit the data. For example the electronic effect of the chloride groups may be significant. Therefore two further compounds were synthesized and tested. Both were 2,4-disubstituted and the substituent groups chosen were methyl and fluoro. Methyl is slightly less lipophilic than the chloro-substituent and is electron donating, while fluoride is electron withdrawing and less lipophilic. It was found that the tluoro analogue UCL- 2348
(Chart 6 ) was less active whereas the methyl substituents in UCL-2349 (Chart 6 ) were well tolerated and approximately as potent as the chloro analogues.
Figure 8. Comparison of chloro with fluoro and methyl.
UCL2273 UCL-2349 Comparing 2,4 Subst UCL-2348 ketocon 1000 -, 800 c 600 - 3t O 400 , c 0) 200 % 2 Ü c 1.00E-05 1.00E-04
C o n c /M o la r
This shows that the binding is lipophilic and supports the argument that the substituent effects are the result of steric and lipophilic effects rather than tuning the electronic nature of the phenyl ring.
91 6.3 Series.3
In series 1 we saw that the hydrogen bonding properties of the piperazine ring were crucial in determining the activity of the compounds at BK. Particularly interesting was the nature of the group attached to the phenoxy moiety. Given the parallel series
of imidazole and non-imidazole compounds and that some of the results from series -2 were available to us at this time we decided to use compounds of the type shown below to investigate the hydrogen bonding properties further. It was also decided to separate the isomers as often as possible, which led to the separation of four sets of cis and trans isomers. The compounds were cis or trans based on naming the methyl
substituent at the 2 -position of the 1,3-dioxolane as the reference substituent and assigning the 4-substituent accordingly. In most cases it was only possible to test an enriched mixture of the trans isomers, as they were more difficult to isolate and purify.
Figure 9. Cis and Trans Isomers
X
Cis: r-Methyl-c-phenoxymethyl Trans: r-methyl-c-phenoxymethyi
The first attempts to obtain openers of the BK channel apart from the piperazine and amino substituents were based on the mistaken conclusion that the nature of the amino and piperazine interactions would be similar. Therefore a series of compounds were synthesized with hydrogen bond acceptors at this position. For comparison methyl and chloro-analogues were also synthesised and tested. The unsubstituted compound is included here for comparison.
92 Chart 7. Non Imidazole Blockers
Response is % block of channel current elicited by stepping from -2 0 to 80mV
UGL-No Substituent Properties Isomerism C one pM R esp o n se Error UCL-2292 X=H C is 30 91% 10% UCL-2302 X=H Trans 30 80% 13% UCL-2270 X=CN Acceptor C is 30 64% 8% UCL-2303 X=CN Acceptor Trans 30 71% 22% U C L-2307 X=Acetyl A cceptor C is Not tested UCL-2315 X=Acetyl Acceptor Trans 3 52% 19% UCL-2316 X=CF3 Acceptor Cis 3 49% 13% UCL-2326 X=N02 Acceptor Cis 3 76% 6% UCL-2306 X=OMe Acceptor C is 3 74% 13% UCL-2331 X=CH3 Lipophilic Mixture 3 87% 2% UCL-2321 X=CI Lipophilic Trans 10 71% 14%
As we can see none of the hydrogen bond acceptors open the channel. The pairs of isomers tested seem to have similar activities. However it would be erroneous to state that both isomers were equipotent given that the dose response curves have not been obtained for these compounds. This is also a problem when comparing the activities of the various substituents. It is significant that electron withdrawing, electron donating, lipophilic, and hydrophilic substituents have all been shown to block the channel. Also hydrogen bond acceptors at the 4-position block the channel, which is consistent with the results from series-1. The results from this set of compounds corroborated the results from series 1 namely that unless the compound contains the correct hydrogen-bonding group at the 4-position it will block the channel. Blocking activity is seemingly less sensitive than opening to changes in the substitution with groups as different as nitro and methyl displaying very similar activities. Given these results it seemed clear that hydrogen bond donors or donor/acceptors should open the channel. The compounds tested and the results obtained are shown below.
CHART 8. Non Imidazole Openers
Hydrogen bond donating compounds 01
O O Cl Q
— NHz QNH )==0 )=0 HgN HgN
U CL-2273 UCL-2350 UCL-2298 UCL-2319 UCL-2351
93 UCL-No Cone pM % Increase Error Reference Cone pM % Increase Error UCL-2273 10 2 2 3 59 Ketocon 30 247 95 30 424 163 100 682 — UCL-2350 10 333 187Ketocon 30 120 21 30 1850 1150 UCL-2351 10 117 45 Ketocon 30 120 21 UCL-2319 30 210 45Ketocon 30 393 133 UCL-2298 ————— — —
% Increase refers to the increase in BK current in smooth muscle aorta cells stepping from -20 to 80mV at a given concentration.
2350 Vs 2273
2 5 0 0
0) Keto (0 03 UCL-2273 2 =3 1000 Ü . UCL-2350 c 500
1.00E-05 1.00E-04
Cone / molar
This set of compounds is consistent with the conclusion that a hydrogen bond donating group is a necessity for BK activation for this type of compound. The most potent compound U C L -2 3 5 0 being 15-fold more potent than ketoconazole. The indole analogue U C L -2 3 5 1 demonstrates that a hydrogen bond donor is the minimum for BK activation since the nitrogen lone pair is deloealised in the aromatic ring. It is not clear whether the hydrogen bond acceptor qualities of the other compounds also play a role in the interaction. It would also be interesting to test compounds of lower pKa such as phenol and sulfonamide. The cis and trans amide compounds were in the same range of potency and so no difference can be seen between the isomers in this ease.
94 Series.4
This series comprises a diverse range of compounds that were synthesized with a view to investigating or replacing the dioxolane ring. Replacement of the dioxolane ring is important for several reasons. The compounds are acid sensitive, displaying instability even in deuteriated chloroform. Some of the compounds displayed poor
solubilities even at 1 OpM. The ring contains two stereo centres and separation often
required extensive chromatography or experimentation to determine the correct recrystallisation conditions. Any asymmetric synthesis would have to separate the diastereomers at some point and the published procedures for ketoconazole are cumbersome for this reason. Therefore addressing absolute stereochemistry would be difficult with dioxolane compounds. Since most o f the data thus far pointed to the importance o f the phenyl rings for binding, the question remained as to whether the dioxolane ring was necessary. It was decided to investigate the molecular mechanics of the compounds using the XED program. The most important feature o f this work was the similarity in the stmctures o f compounds that had various activities. In all cases the global minimum conformation folded to leave the phenyl rings stacked (either face to face or edge to face). If this picture is correct then it is consistent with the biological data, which shows the phenyl ring substitution to be the cmcial factor in determining the activity. Since molecules of similar conformation but different substitution patterns had different activities, the question remaining was whether the dioxolane performs a structural role in the orientation o f the phenyl rings or simply a spacer group between them. It was decided to test the hypothesis that the compound was folded by using an acyclic analogue with a similar skeleton to the dioxolane compound UCL-2273. This was modelled and shown to fold in a similar manner to the cyclic compounds. The reasoning for using an acyclic analogue was as follows. Since it was acyclic there are a greater number of conformations possible around the global minimum. Therefore if the folding is significant then the acyclic compound should have a lower activity than the cyclic compounds. If the folded conformation is not important however then the acyclic compound should be more active for the same reason.
95 The compounds are shown below with a graph comparing the activity of the R- enantiomer with UCL-2273 and UCL-2327.
CHART 9. Acyclic Analogues
Cl OH
R-UCL-2334 S-UCL-2356 NH,
Figure 10. Comparison of UCL-2334 with UCL-2273
Comparing 2334
g 800
^ 600 » ketocon , UCL-2273 “ 400 _gg_ UCL-2334 % 300 UCL-2327 0)
.OOE-05 1.00E-04 Cone I molar
As we can see the activity of the acyclic compound is less than for the dioxolane compound UCL-2273 and therefore based on the argument above the folding of the compound is significant in determining the activity of the compound. The difference seems approximately the same as removing the 2 -chlorogroup when we compare it to UCL-2327. This would be quite neat since the difference between 2273 and 2327 was argued to be due to the lack of the 2-chloro group in orientating the 4-chloro group. It seems clear that absolute stereochemistry does not affect the activity greatly since both enantiomers have similar potencies. However for more potent compounds this may not be the case. It may also be significant that the activities at 30pM and lOOpM were the same for UCL-2334 although this was not the case for UCL-2360. Although
96 the margin of error is large at 100|aM it is the first compound which seems to have reached a maximal activity, even though it is plainly less active as a BK opener than 2273. This raises a difficult point, even if a maximal activity were reached for each activator
o f BK the EC 50 value would be a meaningless point of comparison since it would be based on half maximal activity which would be different for each compound. Few
papers mention EC 50 values with regard to BK channel activation and there are no publications discussing QSAR o f BK channel activators. So it is possible that this is a problem encountered with other classes of BK activators. Comparing the activity of the enantiomers UCL-2334 and UCL-2360 it is clear that the enantiomers are approximately equipotent although at lower concentrations it could be argued that 2334 is more potent. As before it is difficult to argue that one compound is more potent than the other since the slope of the graphs are rather shallow and there is a large error at each data point. With more potent compounds it may be the case that one enantiomer is favored.
Figure 11. Comparing effects of absolute stereochemistry
2334 Vs 2360
400 c 350 t Ü3 300 bd 250 J m Ç 200 150 I o 100 < c qn i
1 .OOE-05 1 .OOE-04
C o n e I M o la r ketocon UCL-2334 UCL-2360
We have shown that when modelled the dioxolane compounds fold and used an acyclic analogue to support this argument. However this argument was dangerous because it ignores the fact that the five-membered dioxolane ring is flexible and that the sets o f conformations were very large. It also assumes implicitly that the difference between the dioxolane and the glycerol can be accounted for by the loss of
97 pre-organization in the glycerol leading to a loss in binding energy. Therefore the hypothesis that was proposed for the role of the dioxolane ring is not proved but it can be argued that it is supported by the activities of the glycerol analogues. Perhaps more importantly molecular modelling has provided the impetus for the synthesis of a compound, which is more soluble, acid stable and contains one accessible stereo- center while retaining BK activity. Although the synthesis of UCL-2334 and UCL- 2360 could have been justified for any of these reasons it was synthesized to test the validity of the modelling. The glycerol analogues can be argued to show that the molecules fold at the global minimum and that this is important for activity. However given the weaknesses in this argument that have been discussed it seemed worthwhile to take a contrary approach. The next compounds were based on analogy with other BK channel activators. The phenyl rings are the most important moieties for BK activity on ketoconazole and this is also true for a range of activators of BK. Specifically NS1608, phloretin and the nifiumic acids
Figure 12. Biphenyl BK channel openers
Phloretin UCL2273 OH F:,C T ÎJ OH O O NH;
01 HO' OHOH
Analogy between these compounds and UCL-2273 led to the synthesis of the compounds shown below.
CHART 10 Three atom chain derivatives
Cl o Cl Cl
o Cl Cl NHo Cl NH, Cl UCL-2355 UCL-2347 UCL-2352
UCLNo Cone pM % Increase Error UCL-2347 30 18 21 UCL-2355 30 99N/A 100 260 149 keto 30 120 18 UCL-2352 10 -11 6
98 It would be surprising to see such a large difference in potency between 2347 and 2355 if the nature of the linker was unimportant. Given the similarity between the skeleton o f UCL-2355 and NS1608 it may be true that UCL-2355 binds to a different site entirely to the other UCL compounds. Comparing the activity of 2334 and 2355 we see that 2334 is definitely more potent but 2355 is not inactive. Since 2355 is almost certainly a more fiat molecule than the dioxolane it does dent the notion that the phenyl rings necessarily stack flat upon each other at the binding site. It also shows that there may be a variety o f linkers that would increase the activity. The furan analogue shown below for example was too lipophilic to use as a basis for more
compounds but showed the same activity at 3pM as the equivalent dioxalane
compound at 30| liM. This represents a nominal 10-fold increase in activity. However
it is dangerous to read much into modest increases in current given the difficulty in the experiments.
CHART 11 UCL-2304
O
N r \ -^1 / = \ ^ p
UCL-2304 48% increase at 3|iM UCL-2254 42% increase at SOfiM
One m otif that all o f these compounds have in common is that the aryl rings are separated by linkers with centrally located electron rich oxygen atoms. Therefore is it possible that the electron density may be pointing into the pore of the channel. Two crown ethers were purchased to test the idea.
CHART 12. UCL-2286 and UCL-2285
99 2 + UCL-2286 K"’selective UCL-2285 Ca selective 55% increase at SOjiM 7.8% block at 30|iM
The results are modest for the concentration but it is interesting that the Ca^^ selective compound is inactive whereas the selective crown obtained a modest increase in the current.
100 6.5 Summary
Several series of compounds deriving from the antifungal drug Ketoconazole have been synthesized and tested by whole cell voltage clamp recordings on bovine aortic smooth muscle cells for BK opening activity. The initial partial structure search led to the removal of the piperazine and imidazole from the molecule. The properties of the phenoxy moiety were found to be very significant in determining the activity. It was shown that some analogues could block the channel. The effects of removing piperazine and imidazole were seen to be complementary. The blocking activity was shown to be due to the nature o f the substituent at the 4-position o f the phenoxy ring. Hydrogen bond donors are essential for opening and by extension the lead compound contains a functional group that may antagonize the overall effect of the compound. The most potent substituent in opening the channel was 4-acetamido while the most potent in blocking the channel was 4-methyl. The dichlorophenyl moiety was found to be crucial for binding. The dioxolane ring may fold to stack the phenyl rings upon one another in conditions of low dielectric constant but it is not clear whether this is important for the biological activity. Glycerol or urea can replace the dioxolane ring with some loss of activity. No discernible difference in activity could be seen for pairs of enantiomers or diastereomers that were tested based on the data obtained.
101 Future work
Given the preliminary nature of the work there is a large potential for modification. They can however be split into three sections. Phenyl ring A (dichlorophenyl). Spacer and Phenyl ring B (phenoxy). For phenyl ring-A the range of modifications is limited by the preference for 2,4- disubstitution. However a wide variety of groups could be located in these positions. For phenyl ring B the four position could be further investigated with a variety of functional groups as shown below along with investigating the 2 and 3 positions.
Possible variations of Phenyl group B
RO-^^^^S-NHR 0-<^^^^)^C0NHR
NH2 H2N
\N // RO
The greatest opportunity for further diversity arises from variation of the central spacer unit of the molecule. Some potential examples are shown below that all contain a centrally located oxygen or lone pair.
Possible variations of the spacer
Ph
N ^ o Q CH2)n CH2)n H2NPhO \ /
HO OH O HgNPhO
102 7. Experimental
7.1 General
• Starting materials and solvents purchased from Aldrich, Lancaster or Merck. THF was distilled from sodium under a diy atmosphere immediately before use. Dried toluene, benzene, DMF and DMSO were purchased in Sureseal® containers and handled according to manufacturer’s instructions
• Sodium Hydride was purchased as a 60% suspension in mineral oil and used without modification
• Flash Chromatography was carried out using Fluka silica gel 60. TLC using
Merck Kiselgel 60 F254 glass plates. Visualisation with UV (254nm) was followed by staining with iodine, potassium iodoplatinate (KIP) or potassium permanganate. The retention factor (Rf) is the ratio of the distance from the baseline to the centre of the spot and the distance of baseline from the solvent front.
• Melting points were determined on an Electrothermal® electrically heated copper block apparatus using an open capillary and are uncorrected.
• Infrared spectra (IR) were recorded on a Perkin-Elmer 1605 FT-IR spectrophotometer using KBr discs for solids and NaCl plates for oils and liquids. The wave number is given in cm'\
• Analytical High Performance Liquid Chromatography (HPLC) was carried out on
a Shimadzu or a Gilson HPLC apparatus fitted with a Kromasil Cig 5pM reversed
phase column (250 x 4.6 mm) at a flow rate of Iml/min and detected at X= 254nm.
Preparative HPLC was performed carried out on a Gilson HPLC apparatus fitted
with a Kromasil Cig 5pm reversed phase column (250 x 22mm) at a flow rate of
18ml/min detected at À,= 215 & 254 nm. In all cases the mobile phase contains a
mixture of water (A) and methanol (B). Both containing 0.1% trifluoroacetic acid (TEA). The ratio A/B is indicated. The retention time (Rt) is given in minutes and decimal subdivisions.
► Electron impact (El), fast atom bombardment (FAB), electrospray (ES) and atmospheric pressure chemical ionisation (APCl) mass spectra were performed by the UCL mass spectrometry service. The instruments used were a VG 7070 (El), a
103 VG ZAB-SE double focusing (FAB) and a Micromass Quattro LC (ES and APCI) mass spectrometer. The values given refer to mass to charge ratio (m/z) and relative abundance (%) of the ions. In most cases isotopic splitting was observed and the relative intensities are quoted.
Proton Nuclear Magnetic Resonance (*H NMR) spectra were recorded using a Varian VXR 400 or a Brucker DRX 500 with deuteriated dimethylsulphoxide
(DMSO dô) or deuteriated chloroform (CDCI 3) as solvent. The chemical shift Ô in
parts per-million (ppm) was measured relative to the residual protic solvent in
CDCI3 (7.24ppm) or DMSO (2.49ppm). The COSY and NOES Y (1-D and 2-D) spectra were performed by the UCL NMR spectrometry service. In the assignments “s” denotes a singlet, “d” a doublet, “t” a triplet, “dd” a double doublet and “bs” a single broad peak. In mixtures o f cis and trans isomers the integration is in comparison to the total number of protons and therefore some protons do not give full integer values.
Elemental analyses were carried out by the departmental microanalysis service on a Perkin Elmer 2400 CHN elemental analyser. Compounds were dried under vacuum (0.1-1 torr) at room temperature for a minimum o f 48 hours prior to submission for elemental analysis.
104 7.2 Syntheses
No.l l-Acetyl-4-{4-[(2-(2,4-dichlorophenyl)-r-2-(lH-imidazol-l-ylmethyl)-l^- dioxalan-c-4-yl)methyloxy]phenyl}piperazine Ketoconazole, EP711A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (0.324g, 1.45mmol) and sodium hydride (0.058g, L45mmol) were stirred in dry DMF (6ml) at 0°C for 1 hour. Mesylate 53a (0.5g, 1.22mmol) in dry DMF (6ml) was added dropwise and the mixture was heated to 80°C for 12 hours. It was then cooled, diluted with IN NaOH (50ml) and extracted with dichloromethane (60ml x 3). The organic layer was then
washed with water (50ml x 3), dried (MgS 0 4 ), filtered and evaporated in vacuo. This was purified by flash chromatography (dichloromethane: methanol, 20:1) to yield an oil which solidified on evaporation with ether (0.445g, 0.83mmol, 69%) Rf: 0.2 (dichloromethane: methanol, 20:1) Melting point: 145-148°C (lit 146°C^'"^ MS: ES: MH 531 (100%), 533 (70%)
NMR: 500MHz, CDCI3 , ô ppm; 2.11(3H, s. Me amide); 3.00(4H, m, CHzNPh); 3.24(1H, dd, J= 9.5, 7.0Hz, CHzOPh); 3.59(2H, t, J= 5.0Hz, CHzNCOMe); 3.67-3.75(4H, m, 2CH2NCOMe,
ICH2CH, lCH20Ph); 4.32(1H, m, CH2ÇH); 4.45(2H, ABq,
J=14.7Hz, ÇH2N); 6.75(2H, d, J= 8.9Hz, OC6H4N H3 & H5); 6.86(2H, d, J=8.9Hz, OPhN H2 & H6); 6.94(1H, s, imidazol), 6.97(1H, s, imidazol); 7.23(1H, dd, J= 8.3, 2.1Hz, dichlorophen H5); 7.44(1 H, d, J= 2.0Hz, dichlorophen H3); 7.50(1 H, s, imidazol); 7.58(1 H, d, J=8.3Hz, dichlorophen H6)
N0 . 2 l-{4[(2-(2,4-Dichlorophenyl)-r-2-(lH-imidazoI-l-ylmethyl)-l,3-dioxolan-c- 4-yI)methyloxy]phenyl}piperazine Hemihydrate, UCL2297, EP793A Ketoconazole 1 (150mg, 0.28mmol) and potassium hydroxide (200mg, excess) in isopropanol/water (7ml/2ml) was heated to reflux for 24 hours. The reaction mixture was then evaporated in vacuo and taken up in water (30ml). This aqueous suspension was extracted with dichloromethane (3 x 30ml). The organic layer was dried
(M gS0 4 ), filtered and evaporated in vacuo. The crude material was purified by fiash chromatography (dichloromethane/methanol, 5:1). It was then taken up in isopropanol
105 and filtered. Evaporation yielded a clear oil that solidified to white powder under vacuum 2 (93mg, 0.019mmol, 67.9%) Rf: 0.07 (dichloromethane/methanol, 5:1) Melting point: Discolored above 150 melted at 167-170°C (lit = 171^C^'*^) MS: MH 489.2 m/z & 491m/z, 100% and 70% RI
NMR: 500MHz, CDCI3, Ô ppm; 3.02(8H, s, piperazine); 3.28(1H, dd,
J= 9.5, 6.7Hz, CHzOPh); 3.70(2H, m, CH2CH & CHzOPh);
3.84(1H, dd, J - 8.4, 6.5Hz, CH 2CH); 4.30(1H, m, CH2ÇH);
4.44(2H, ABq, J= 14.7Hz, CH2N); 6.73( 2H, d, J= 9.0Hz,
OC6H4N); 6.85(2H, d, J= 9.0Hz, O C ^ N ); 6.93(1H, s, imidazol); 6.96(1 H, s, imidazol); 7.22(1 H, dd, J= 8.4, 2.1Hz, dichlorophen H5); 7.43(1H, d, J= 2.1Hz, dichlorophen H3);
7.47(1 H, s, imidazol); 7.54(1H, d, J= 8 .3Hz, dichlorophen H 6 )
IR: KBr disc, cm '; (3424, H2O); (3289, NH); (2954, CH); (2823, CH); (1511, C=C aromatic); (1200, C-O); (1045, C-O) HPLC: (45:55, A/B) Rt = 7.12min, 100% Microanalysis C24H26CI2N4O3.O.5H2O calculated C:57.84 H:5.46 N: 11.24 found C: 57.71 H: 5.31 N: 11.00
No.3 2-(2,4-DichIorophenyI)-r-2-(lH-imidazol-l-yImethyI)-c-4-((4- piperidinylphenyl)oxymethyl)-l,3-dioxoIane trifluoroacetate hydrate, UCL-2322-
F3, EP909B 1-(4-Hydroxyphenyl)-piperidine oxalate 56 was converted to it’s free base by
disolving in Na 2C0 3 soln followed by extraction into dichloromethane solution. It was
then dried (Na2S0 4 ), filtered and evaporated in to give the free base (23mg, 0.12mmol). The free base was stirred with sodium hydride (5mg, 0.12mmol) in dry DMF (3ml) for 1 hour at 0°C. Tosylate 10 (61 mg, 0.12mmol) in dry DMF (2ml) was added and the mixture was heated to 80°C overnight. The mixture was then cooled, diluted with brine (15ml) and extracted with DCM (15ml x 3). The organic layer was
washed with water (40ml x 2), dried (Na 2S0 4 ) and evaporated in vacuo. Purification by flash chromatography (EtOAc 100%) yielded a clear wax (37mg, 0.075mmol, 60%, 98% pure). This was dissolved in ethanol and a solution of oxalic acid in
106 ethanol was added. It was then evaporated and purified by preparative HPLC (50:50, A/B) to yield a white orange solid 3 (19mg) Rf: 0.05 (100% EtOAc) for ffeebase Melting point: Hygroseopie solid hydrates rapidly in air. MS: ES; MH, 488 (100%) 490 (70%)
NMR: 500MHz, CDCI3 , 5 ppm; 1.56(2H, bs, piper); 1.69(4H, bs,
piper); 3.17(4H, bs, piper); 3.69(2H, m, CH 2 diox); 3.86(1H, m,
CH2 diox); 3.89(1H, dd, J= 8.3, 7.0Hz, CH 2 CH diox); 4.35(1H,
m, CH2ÇH); 4.80(2H, ABq, J= 14.6Hz, CH2N); 6.83(2H, bs,
OC6 H4 N); 7.05(2H, bs, OC 6 H4N); 7.51(1H, dd, 8.5, 2.1Hz, diehloro H5); 7.58(2H, s, imidazol); 7.62(1 H, d, J= 8.5Hz,
diehloro H 6 ); 7.73(1H, d, J= 2.1Hz, diehloro H3); 8.99(1H, s, imidazol) HPLC: (50:50, A/B) Rt = 10.5min, 97.3%
Mieroanalysis C2 5 H2 7 Cl2 N 3 O3 .3 CF3 CO2 H.H2 O found C: 43.83 H:3.81 N: 4.63 calculated C: 43.88 H: 3.80 N: 4.95
N0 . 4 2(2,4-DichlorophenyI)-r-2-(lH-imidazoI-l-ylmethyi)-c-4-((4- amlnophenyl)oxymethyl)-l,3-dioxoIane, UCL2242, EP477A 4(Benzylidenemino)-phenol 57 (0.197g, Immol) and sodium hydride (44mg, 1.1 m m ol) were stirred in dry DMSO (5ml) for 1 hour at 0°C. Tosylate 10 (0.483g, 1 .Ommol) in dry DMSO (5ml) was then added and the mixture was heated to 80°C and stirred overnight. The mixture was then cooled, dissolved in water (60ml) and extracted with dichloromethane (60ml x 3). The organic layer was dried (MgS 0 4 ), filtered and evaporated in vacuo. The crude material was purified by fiash chromatography (100% EtOAc, Rf 0.05) to give a mixture of amino and imine products (0.128g). This mixture was dissolved in ethanol ( 8 ml) and treated with hydrazine hydrate (0.051ml). This solution was heated at reflux for 4 hours. The ethanol was then evaporated in vacuo and the crude purified by fiash chromatography (20:1, dichloromethane: MeOH) to give an oil which was recrystalised fi*om methanol to give brown crystals 4 (0.088g, 0.2Immol, 21%) Rf: 0.45 (20:1, diehloromethane:MeOH)
107 Melting point: 160-162°C MS: FAB; MH 420 (100%) 422, (65%)
NMR: 500MHz, DMSO dg, ô ppm; 3.47(1H, dd, 10.2 & 5.3 Hz,
CHzOPh); 3.60(2H, m, CHzCH & CHiOPh); 3.85(1H, dd, 8.3
& 6.7 Hz, CH 2CH); 4.28(1H, m, CH 2ÇH); 4.50(2H, ABq, 14.7
Hz, CH2N); 6.40(2H, d, 8.7Hz, phenoxy); 6.50(2H, d, 8.7Hz, phenoxy); 6.80(1 H, s, imidazol); 6.99(1 H, s, imidazol); 7.42(1H, dd, 8.5 & 2.0Hz, dichlorophen H5); 7.45(1 H, s,
imidazol); 7.54(1H, d, 8.7Hz, dichlorophen H 6 ); 7.65(1H, d, 2.0Hz, dichlorophen H3) IR: KBr disc, cm '; (3500 & 3300, NH); (3000-2900, CH); (1500, C=C); (1250, CO) HPLC: (50:50, A/B) Rt =10.05min, 98.7%
Microanalysis: O20H 19CI2N 3O3 calculated C: 57.16 H: 4.56 N: 10.0 found C: 56.93 H: 4.61 N: 9.70
No.5 2-(2,4-Dichlorophenyl)-r-2-(lH-imidazol-l-ylmethyl)-c-4-(phenoxymethyl)- 1,3-dioxolane nitrate, UCL-2202, EP291A Phenol (0.083g, 0.86mmol) and sodium hydride (22mg, 0.86mmol) were stirred in dry
DM SO (6 ml) for 1 hour at 0°C. A solution of tosylate 10 (0.390mg, 0.8mmol) in dry DMSO was added dropwise and the mixture was heated to 80°C for 4 hours. This was then dissolved in DCM (80ml) and washed with brine (80ml x 3). It was then dried
(M gS0 4 ) evaporated in vacuo and purified by fiash chromatography (2:1, EtOAc: Pet spirit) to yield a waxy solid (0.224g, 0.55mmol, 75%). This was dissolved in ether and precipitated with several drops of concentrated nitric acid (65%) to give the nitrate salt 5. Rf: 0.05 (2:1, EtOAc: Pet Spirits) for free base M elting point: 178-181T MS: APCI; MH 405 (100%), 407 (70%) isotopic
NMR: 400 MHz, DMSO dô, ô ppm; 3.65(1 H, dd, J= 5.8 & 1.5Hz,
ÇH2CH); 3.67(1H, d, J= 5.7Hz, CHgCH); 3.80(1H, dd, J= 10.3
& 3.9Hz, CH2 0 Ph); 3.90(1H, t, J= 8.9Hz, CH2 0 Ph); 4.37(1H,
108 m, CH2ÇH); 4.80(1H, ABq, 14.5Hz, ÇH 2N); 6.85(2H, d, J=7.8Hz, orthophenoxy); 6.96( IH, t, J= 7.33, paraphenoxy);
1.2% 2H, dd, 8 .6 & 7.4 Hz, meta phenoxy); 7.54(1 H, dd, 8.4 & 2.1 Hz, dichlorophenyl H-5); 7.61(2H, s, imidazol); 7.65(1H, d,
8.4Hz, dichlorophen H- 6 ); 7.74(1 H, d, 2.1Hz, dichlorophen H- 3); 9.06( IH, s, imidazol) IR: KBr disc, cm‘^ (3055, C-H aromatic); (1584, C=C aromatic); (1383, C-O); (1046, C-O) HPLC: (40:60, A/B), Rt =25 min, 100%
Microanalysis: C20H 18CI2N 2O3.HNO3 calculated C: 51.30 H: 4.09 N: 8.97 found C: 51.17 H: 3.88 N: 8.79
6^ 2-(2,4-Dichlorophenyl)-r-2-(lH-imidazol-l-ylmethyl)-c-4-(hydroxymethyl)-l,3- dioxolane UCL2135, EP89A A solution of 9 (4.02g, 8.3mmol) with NaOH (1.44g, 0.36mol) was heated to reflux in
a dioxane (30ml) / water ( 6ml) solution for 12 hours. It was then diluted with water (80ml) and extracted with dichloromethane (80ml x 3). The organic layer was dried
(MgS0 4 ), filtered and evaporated in vacuo to give a clear oil. Evaporating with ether provided a white solid, which was recrystallised from ethyl acetate to give the pure
solid 6 (2.08g, 6.3mmol, 78%)
Melting point: 138-140°C (lit 140°C) MS: APCI; MH+ 329(100%), 331 (60%) isotopic splitting
NMR: 400MHz, DMSO de, ô ppm, 3.03(1H, dd, J=11.2 & 6.1Hz,
CH2 0 Ph); 3.29(1H, dd, J=11.2 & 5.9Hz, CH2 0 Ph); 3.53(1H,
dd, J= 8.15 & 5.6Hz, CH2CH); 3.77(1H, t, J=7.8Hz, CH 2CH);
4.00 (IH, m, CH2ÇH); 6.8(1H, s, imidazol H 5 ); 7.0(1H, s,
imidazol H4); 7.41 (IH, dd, J=6.4 & 0.8Hz, dichlorophenyl H5);
7.45(1H, s, imidazol H 2); 7.50(1H, d, J = 6.4Hz, dichlorophen
Hô); 7.67(1 H, s, J= 0.9Hz, dichlorophen H3). IR: KBr cm ' (3400, OH); (3100 & 2833, CH); (1513, C=C aromatic); (1057, C-O). HPLC: (50:50, A/B), Rt 8.9 min, 100% pure
109 Mieroanalysis. C14H14CI2N2O3 O.25H2O found C: 50.51 H: 4.20 N: 8.26 calculated C: 50.37 H: 4.19 N: 8.39
N0 . 7 2(2,4- DichIorophenyl)-2-methyl-4-(4-(4-acetylpiperazinyl)phenoxy)methyl- 1,3-dioxolane oxalate, UCL-2158, EP143A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (0.996g, 4.7nunol) and sodium hydride (0.19g, 4.8mmol) were stirred in dry DMSO (20ml) for 1 hour at 0°C . Tosylate 65 (1.72g, 4.7mmol) in dry DMSO (20ml) was then added and the solution stirred overnight at 80°C. The mixture was then cooled and the DMSO removed by vacuum distillation. The remaining mixture was dissolved in water (60ml) and extracted with dichloromethane (60ml x 3). The organic layer was dried (MgS 0 4 ), filtered and evaporated in vacuo. The crude material was purified by flash chromatography (3:2, EtOAc/dichloromethane) to give a clear foam (0.892g, 1.9Immol, 45%). This foam was then dissolved in ethanol and treated with a solution o f oxalic acid in ethanol. The solution was evaporated in vacuo, dissolved in dichloromethane and precipitated with hexane. This was then recrystalised (x3) by dissolving in the minimum ethanol and precipitating with ether to give the pure oxalate (0.190g) Rf: 0.2 (5:2, EtOAc/dichloromethane) Melting point: 101-102°C MS: APCI; MH 465 (100%) 467m/z (70%) isotope
NMR: 400MHz, DMSO dô, ô ppm; 1.67(1.5H, s, methyl trans); 1.74(1.5H, s, methyl cis); 2.02(3H, s, acetyl); 2.94(2H, m,
CH2NCOMe); 3.00(2H, m, CH2NCOMe); 3.55(4H, m,
CH2NPh); 3.77(1.5H, m, CH 2); 3.84(0.5H, dd, 10.5 & 4.0Hz,
CH2 0 Ph); 3.90(0.5H, dd, 8.5 & 5.2Hz, CH 2CH); 4.00( IH, d,
5.3Hz, CH2); 4.26( IH, m, CH2 & CH cis); 4.53(0.5H, m, CH trans); 6.71(1H, d, 9.1Hz, phenoxy trans); 6.86(1H, d, 9.1Hz, phenoxy trans); 6.90(2H, ABq, 9.37 , phenoxy cis); 7.38(0.5H, dd, 8.5 & 2.1Hz, dichlorophen H5); 7.43(0.5H, dd, 8.5& 2.1, dichlorophen H5); 7.56(0.5H, d, 2.1Hz, dichlorophen H3); 7.60(0.5H, d, 2.1Hz, dichorophen H3); 7.63(1 H, t, 8.5Hz, dichlorophen H3)
110 IR: KBr disc cm ' (2935, C-H); (2525, NH*), (1647, C=0); (1512, C=C); (1035, C-O) HPLC: (25:75, A/B), Rt = 9.40, 11.65 min (43.2, 55.8%)
Mieroanalysis O23H25CI2N2O4.C2H2O4 calculated C: 54.06 H: 5.08 N: 5.04 found 0:54.10 H: 5.03 N: 5.05
No.7a 2(2,4- Dichlorophenyl)-2-methyI-4-(4-(4- acetylpiperazinyI)phenoxy)methyl-l,3-dioxolane (alternative method), EP837A
2-(2,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl- 1,3-dioxolane (0.467g, 1 .Vmmol) was stirred in dry benzene ( 8 ml) at 0°C. Tributylphosphine (0.643ml, 1.9mmol), ADD? (0.655g, 1.9mmol) and l-acetyl-4-(4-hydroxyphenyl)-piperazine (0.572g, 1.9mmol) were added in this order. The mixture was stirred for 24 hours warming to room temperature. It was then diluted with 2ml hexane and filtered. The filtrate was dissolved in ether (50ml) washed with 10% NaOH (50ml) and water (50ml x 2). This was dried (MgS0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (EtOAc) yielded a clear viscous oil (286mg, 0.62mmol, 36%). Rf: 0.1 (EtOAc) Mixture: 80:20 (cis: trans) MS: APCI; MH 465 (100%); 467 (65%) isotope
NMR: 400MHz, CDCI3, Ô ppm; 1.78(3H, s, Me diox); 2.11(3H,
MeCO); 2.99-3.05(4H, m, CH2NPh); 3.68 -3.76(2.2H, m,
CHzNCOMe & CH2 diox trans); 3.82(0.8H, dd, J= 8.5, 6.9Hz,
CH2CH cis); 3.87-3.95(1.2H, m, CH 2 0 Ph & 2 CH2 trans);
3.98(0.8H, dd, J= 8.5, 4.5Hz, CH 2CH cis); 4.07(0.8H, dd, J=
9.4, 5.0Hz, CH2 0 Ph cis); 4.29(1H, m, CH2ÇH cis & CH2
trans); 4.57(0.2H, m, CH 2CH trans); 6.68(0.4H, d, J= 9.1Hz,
OC6H4N H3 & H5); 6.80-6.89(3.6H, m, OÇ 6H4N cis & trans); 7.I6(0.2H, dd, J= 8.4, 2.1Hz, dichlorophen H5 trans); 7.19(0.8H, dd, J= 8.4, 2.1Hz, dichlorophen H5 cis); 7.34(0.2H, d, J= 2.1Hz, dichlorophen H3 trans); 7.38(0.2H, d, J= 2.1Hz, dichlorophen H3 cis); 7.58(0.8H, d, J= 8.4Hz, dichlorophen
H6 ); 7.60(0.2H, d, J= 8.4Hz, dichlorophen H 6)
111 No.8 l-Acetyl-4(4-methoxyphenyl)-piperazine UCL2112A, EPIOA l(4-Methoxyphenyl)-piperazine hydrochloride (0.5g, 2.2mmol) was dissolved in O.IM NaOH (30ml) and extracted with dichloromethane (3x30ml). This was dried
(MgS0 4 ), filtered and evaporated in vacuo. The free base was then heated to reflux for 3 hours in acetic anhydride (7ml). It was slowly diluted with cold water (40ml) then heated to 50°C for 30 minutes. This was neutralized (2N NaOH) and extracted
with dichloromethane (3 x 50ml). The organic layer was dried (MgS 0 4 ), filtered and evaporated under reduced pressure. The crude product was recrystalised from dichloromethane / hexane and converted to the oxalate salt by mixing ethanol solutions of the free base and oxalic acid. Concentration to dryness and
recrystalisation from ethanol yielded the oxalate salt 8 (644mg, 91%). Melting point : 158-164°C MS: APCI; MH 235 m/z, 100% R.I.
NMR: CDCI3, 400MHz, ô ppm; 2.0(3H, s, amide Me); 2.8(4H, m, piper); 4.5(2H, t, J=5Hz, 2H piper); 4.7(5H, m, piper & methoxy); 6.7(4H, q, J = 9.3Hz, 4H, aryl) IR: KBr disc (3000, CH); (1636, C=0 amide and oxalate), (1513, C=C Aromatic); (1249, C-O) HPLC: (70:30,A/B), Rt = 6.18min, 99.5%.
Mieroanalysis: Ci 3HigN2 0 2 .(C0 2 H)2 Calculated: C: 55.55 H: 6.22 N: 8.64 Found: C: 55.18 H: 6.23 N: 8.76
N0 . 9 2-(2,4-Dichlorophenyl)-r-2-(lH-imidazoI-l-yImethyl)-c-4- (benzoyIoxyniethyl)-l,3-dioxolane nitrate UCL-2134, EP95A A solution of bromide 52 (lOg, 0.022mol) and imidazole (4.641 g, 0.068mol) were heated to reflux in dry DMA (20ml) for four days. The mixture was then cooled, diluted with water (100ml) and extracted with ether (3x100ml). The organic layer was dried (M gS0 4 ), filtered and acidified with HNO 3 (65%). The precipitate was allowed to solidify, filtered and recrystallised from ethanol/ether to give a white solid (6.023g, 0.012, 54.2%).
112 Melting point: 168-170°C (lit 170°C) MS: APCI; MH^ 433 (100%) 435 (65%) isotopic splitting
NMR: 400MHz, CDCI3, Ô ppm; 3.76(1H, dd, J = 8 .6 , 5.35Hz,
CH2CH); 3.92(2H, m, CH2CH & CH2OCOPI1); 4.18(1H, dd, J
= 11.8, 3.8Hz, CH 2OCOPI1); 4.43(1H, m, CH2ÇH); 4.84(2H,
ABq, J= 14.3Hz, ÇH2N); 7.53-7.60(4H, m, Ar); 7.68-7.75(5H, m, Ar); 7.93-7.95(2H, m, Bz ortho); 9.12(1H, s, Imidaz H2) IR: KBr disc, cm'% (3150-3016, CH aromatic); (2967-2700, CH aliphatic); (2467, N^-H stretch); (1725, benzoate CO); (1391, 1313, C-O benzoate); (1060, C-O benzoate) HPLC: (40:60, A/B) Rt: 12.57min, 97.6% pure
Mieroanalysis: C21H18CI2N2O4.HNO3 Calculated C: 50.81 H: 3.62 N: 8.46 Cl: 14.31 Found C: 50.91 H: 3.89 N: 8.27 Cl: 14.39
No.lO 2-(2,4-DichlorophenyI)-r-2-(lH-imidazoIyl-l-yImethyI)-c-4- (toIuenesuIfonyIoxymethyl)-l,3-dioxalane EP339A Toluenesulfonyl chloride (1.43g, 7.5mmol) in pyridine (5ml) was added to a solution
of the alcohol 6 (1.90g, 5.8mmol) in pyridine (10ml) that was stirring at 0°C. The mixture was stirred for a further three hours at 0°C. It was then diluted with
dichloromethane (60ml), washed with brine (60ml) and distilled water (60ml x 2 ).
The organic layer was dried (MgS 0 4 ), filtered and evaporated in vacuo. After the crude solidified under vacuum it was recrystalised from dichloromethane/hexane to yield a white solid 10 (2.01g, 4.2mmol, 71.8%) Melting point: 108-110°C (lit ^"^117°C from toluene) MS: APCI, MH 483 (100%) 485 (65 %)
NMR: 500MHz, CDCI3, Ô ppm; 2.43(3H, s, tosyl); 3.44(1H, dd, J=
10.4, 6.2Hz, CH2OTS); 3.57(1H, dd, J= 8.7, 4.8Hz, CH 2CH)
3.73-3.78(lH, m, CH 2CH & CH2OTS); 4.22(1 H, m, CH2ÇH)
4.37(2H, ABq, J= 14.7Hz, CH 2N); 6.84(1 H, s, imidazol) 6.89(1H, s, imidazol); 7.20(1H, dd, J= 8.4, 2.0Hz, dichlorophen H5), 7.37(2H, d, J= 8.0Hz, Ts); 7.42(3H, m, 2h Ts &
113 dichlorophen H3); 7.75(2H, d, J= 8.2Hz, dichlorophen H 6 & imidazol)
No.lOa 2-(2,4-Dichlorophenyl)-r-2-(lH-imidazol-l-ylmethyl)-c-4- (toluenesulfonyloxymethyl)-l ^-dioxolane hydrochloride, UCL-2250, EP339A A saturated solution of HCl in ethanol (3 drops) was added to a solution of the free base 10 (30mg) in ether (5ml). The solid was filtered and washed with ether. This was recrystallised from ethanol / ether to yield a white solid (32mg, quantitative). melting point: 152-156°C MS: APCI; MH 483 (100%), 485 (65%)
NMR: 400MHz, DMSO de, ô ppm; 2.45(3H, s, CH3 toi); 3.54(lH,dd,
J= 8.5, 5.7Hz, CH 2CH); 3.68(1H, dd, J= 11.1, 5.8Hz, CHzOTs);
3.77(1H, t, J= 8.5Hz, CH 2CH); 3.92(1H, dd, J=11.3, 2.9Hz,
ÇH2OTS); 4.24(1 H, m, CH2ÇH); 4.67(2H, ABq, J= 14.6Hz,
CH2N); 7.53(6H, m, ArClz & Ts); 7.73(1H, s, H3 ArCh); 7.77(1H, s, imidazole); 7.79(1H, s, imidazole); 8.99(1H, s, imidazole). IR: KBr disc cm ' (3000, CH); (2500, N*-H); (1500, C=C); (1400- 1200, S=0). HPLC: (30:70, A/B) Rt = 15min, 100%
Mieroanalysis: C21H20CI2N 2OS.HCI calculated C: 48.61 H: 3.78 N: 5.27 found C: 48.52 H: 4.07 N: 5.39
N o.ll 2-(2,4-DichIorophenyl)-r-2-(lH-imidazol-l-yImethyl)-c-4-(4- bromophenoxymethyl)-l,3-dioxoIane Nitrate, UCL2281, EP623B 4-Bromophenol (0.128g, 0.68mmol) and sodium hydride (30mg) were stirred in dry DMF (5ml) at 0°C for 1 hour. Tosylate 10 (31 mg, 0.66mol) in dry DMF (5ml) was then added dropwise and the mixture was heated to 80°C for fifteen hours. It was then cooled and diluted with IN NaOH (50ml). This was extracted with dichloromethane
(60ml X 3). The organic layer was washed with water (40ml x 3), dried (MgS 0 4 ), filtered and evaporated in vacuo. The crude mixture was purified by fiash chromatography (EtOAc 100%) to give a white solid (0.191g, 0.4mmol, 63%). The
114 solid was dissolved in ether and several drops of cone nitric acid were added to precipitate the nitrate salt. Rf: 0.1 (EtOAc 100%) for free base Melting point: 150-154T MS: APCI; MH 482 (60%), 484 (100%), 486 (45%) isotopic
NMR: 500MHz, DMSO ô ppm; 3.68(2H, m, CHz); 3.88(2H, m,
CHz); 4.40(1H, m, CH); 4.81(2H, ABq, J =14.7Hz, CHzN);
6.84(2H, d, J=9.0, OC6H4Br); 7.47(2H, d, 8.9, OC 6H4Br); 7.54(1H, dd, J=8.4,2.1Hz, dichlorophen H5); 7.62(2H, s,
imidazol); 7.66(1H, d, J=8.4Hz, dichlorophen H 6 ); 7.74(1H, d, J=2.1Hz, dichlorophen H3); 8.88(1H, s, imidazol) IR: KBr disc, cm '; (3104, CH); (1538,0=0); (1384, NO); (1242, 0-0); (1037, 0-0) HPLC: (25:75, A/B) Rt =11.2min, 100% Mieroanalysis O20Hi7BrOl2N2O3.HNO3 calculated C: 43.90 H: 3.32 N: 7.68 found C: 44.00 H: 3.22 N: 7.65
No.l2 2-(2,4-DichIorophenyl)-r-2-(lH-l,2,4-triazoI-l-ylmethyI)-c-4- (benzoyloxymethyI)-l,3-dioxolane, UCL2203, EP229A 1,2,4-Triazole (2.5g, 0.036mmol), NaH (1.4g, 0.036mol) and r-2-(bromomethyl)-2- (2,4-dichlorophenyl)-c-4(benzoyloxylmethyl)-1,3-dioxolane 52 (5.4g, 0.012mmol) were heated to reflux in dry DMF (50ml) for four hours. The reaction mixture was cooled, diluted with brine (150ml) and extracted with ether (150ml x 3). The ethereal layer was dried (MgSO#), evaporated in vacuo and purified by flash chromatography (2:1, Ethyl Acetate: Pet spirit) to give a waxy solid (0.535g, 1.2mmol, 10.2%). This was disolved in ethanol and mixed with a solution of oxalic acid in ethanol. Recrystalisation of the salt from ethanol/ ether provided the oxalate salt
Rf: 0.05 (2:1, ethyl acetate: pet spirits) Melting point: 129-13rc MS: APCI; MH 434 (100%), 436 (70%)
NMR: 400MHz, CDCI3 Ô ppm; 3.80(1H, dd, J= 8.5 & 5.6Hz,
CH2CH); 3.88(1H, dd, J= 8.6 & 6 .8 Hz, CH2CH); 4.05(1H, dd.
115 J=1L7& 6.0Hz, CHsOCOPh); 4.2(1H, dd, J= 11.8 & 4.1,
CHzOCOPh); 4.34(1H, m, CHzCH); 4.79(2H, s, ÇH 2N);
7.40(1 H, dd, J= 8.4, 2.03Hz, H 5 ArCli); 7.48(1H, d, J= 8.4Hz,
H6 ArCL); 7.51(2H, t, J= 7.9Hz, metabenzoyl); 7.66(1 H, d, J= 1.9Hz, H3 ArCb); 7.68(1H, t, J= 7.5Hz, para benzoyl); 7.79(1 H, s, triazol); 7.96(2H, d, J= 7.1Hz, ortho benzoyl); 8.38(1 H, s, triazol) IR: KBr disc, cm ' (3000, C-H); (1720,0=0); (1452, 0=0); (1269- 1 1 0 3 ,0 -0 ) HPLC: (60:40, A/B) Rt = 17.0 min, 100% pure
Mieroanalysis C20H 17CI2N 3O4 calculated C: 50.40 H: 3.65 N: 8.01 found C: 50.08 H: 3.47 N: 7.87
No.13 2(2,4- Dichlorophenyl)-r-2-(lH-l,2,4-triazol-l-yImethyl)-c-4- hydroxymethyl-l,3-dioxolane UCL2220, EP329B
1,2,4-Triazole (1.82g, 0.26mol), Na 2C0 3 (2.83, 0.026mol) and 52 (3.94g, 8 .8 mmol) were heated to reflux in dry DMF for 3 days. The reaction mixture was then cooled and diluted with a saturated brine solution (80ml). This was extracted with ether
(100ml X 3). The organic layer was dried (MgS 0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (2:1, EtOAc: pet spirit to 100% EtOAc) followed by recrystalisation from EtOAc provided a white solid (0.309g, 0.8mmol, 10.3%) Rf: 0.05 (EtOAc) Melting point: 137-139T (lit 138°C) MS: APCI; MH 330 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 3.24(1H, dd, J= 12.1, 4.5 Hz,
CH2OH); 3.62(1H, dd, J= 12.1, 3.5Hz, CH2OH); 3.67(1H, dd,
8.0, 6.3Hz, CH 2CH); 3.82(1H, dd, 7.7, 7.3Hz, ÇH 2CH);
4.12(1H, m, CH2ÇH); 4.73(2H, s, CH 2N); 7.21(1H, dd, 8.5, 2.0Hz, dichlorophen H5); 7.44(1 H, d, J= 2.0Hz, dichlorophen
H3); 7.54(1H, d, J= 8.5Hz, dichlorophen H 6 ); 7.91(1H, s, triazole); 8.11(1 H, s, triazole)
116 IR: KBr, cm-1, (3300, O-H stretch); (2986, CH stretches); (1550, C=C stretch); (1030, C-O stretch) HPLC: (60:40, A/B) Rt: 14.6min, 99.7%
Mieroanalysis: C13H13CI2N3O3 calculated C: 47.29 H: 3.97 N: 12.73 found C: 47.32 H: 3.78 N: 12.82
No.14 l-Acetyl-4-(4-(2,3-dihydroxypropyH-oxy)phenyl)-piperazine 251 UCL- 2245, EP487A, UCL-2238, (also EP441B) l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (200mg, 0.9Immol) and sodium hydride (36mg, 0.9mmol) were stirred in dry DMSO (5ml) for 1 hour at 0°C. 2(2- Chlorophenyl)-2-methyl-4-(toluenesulfonyl)oxymethyl-l ,3-dioxolane 64 (347mg, 1.Ommol) in dry DMSO (5ml) was added and the mixture heated to 80°C overnight. The reaction mixture was then diluted with water (50ml) and extracted with
dichloromethane (50ml x 3). This was dried (MgS 0 4 ), filtered, evaporated in vacuo and purified by flash chromatography to yield a clear oil (490mg, Rf= 0.15 EtOAc). It was then dissolved in ethanol and treated with oxalic acid in ethanol. On adding ether the salt quickly precipitated and was filtered forming a gum on the sinter funnel. The material was transfered to a round bottomed flask and converted to the free base in aqueous solution (50ml) with NaOH. This was extracted into dichloromethane. The organic layer was dried, filtered and evaporated in vacuum to give a white solid which was recrystalised from dichloromethane/hexane to give a white solid 14 (49mg, 0.17mmol, 18%). Rf: 0.0 EtOAc Melting point: 120-122°C (lit = 116-117^^') MS: APCI; MH 295 (100%)
NMR: 500MHz, CDCI3 Ô ppm; 2.08(1 H, t, 6.3Hz, primary OH);
2.41(3H, s. Amide Me); 2.67(1 H, d, 4.7Hz, secondary OH); 3.16(4H, m, piperazine CHiNPh); 3.72(2H, t, 5.1Hz, piperazine CHzNCOMe); 3.87(3H, m, CHz piper & CHzOPh); 3.92(1H,
dd, 6.4 & 3.9Hz,CHzCH); 4.12(2H, m, CH2CH & CH2 0 Ph);
4.20(1 H, m, CH2ÇH); 6.98(4H, ABq, 9.2Hz, OÇ6H4N)
117 IR: KBr disc, cm'^; (3300, OH); (2985, CH); (1685, CO amide); (1531, C=C); (1200 & 1035, C-O) HPLC: (85:15, A/B) R t= 10.41, 100%
Mieroanalysis C 15H22N 2O4.0 .1H2O calculated C: 60.84 H: 7.56 N: 9.46 found C: 60.68 H: 7.49 N: 9.33
No.15 l-Acetyl-4-(4-(2,2-dimethyl-l,3-dioxolan-4-yI)methoxy)phenyl)piperazine UCL2245, EP487A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (200mg, 0.9mmol) and sodium hydride (39mg, 0.9mmol) were stirred in dry DMSO (5ml) at room temp for 1 hour. 2,2-
Dimethyl-4-tosyloxymethyl-1,3-dioxolane 6 8 (259mg, 0.8mmol) in dry DMSO (5ml) was added and the mixture was stirred overnight at room temperature. The mixture was diluted with water (50ml) and extracted with dichloromethane (100ml x 3). The organic layer was dried, filtered and evaporated in vacuo. Purification by fiash chromatography (100% EtOAc) yielded a white solid that was recrystallised from dichloromethane/hexane to yield a white solid 15 (154mg, 0.45, 51%) Melting point: 87-89°C (lit^^® 73-75) MS: APCI; 335 (100%)
NMR: 500MHz, CDCI3, 5 ppm; 1.51(3H, s, dimethyl); 1.56(3H, s,
dimethyl); 2.24(3H, s, amide Me); 3.16(4H, m, CH 2NPh);
3.72(2H, s, CH 2NCOMe); 3.87(2H, s, CH 2NCOMe); 4.00(2H,
m, CH2CH & CH2 0 Ph); 4.13(1H, dd, J=9.5 & 5.3Hz,
CH2 0 Ph); 4.26(1H, dd, J= 8.4 & 6.4Hz, ÇH2CH); 4.55(1H, m,
CH2ÇH); 6.98(4H, m, OC 6H4N). IR: KBr disc, cm '; (2990, CH); (1685, C=0 amide); (1500, C=C aromatic); (1200, C-O) HPLC: (50:50, A/B) Rt = 5.51min, 100%
Mieroanalysis C18H26N 2O4 calculated C: 64.65 H: 7.84 N: 8.38 found C: 64.40 H: 7.80 N: 8.37
118 No.l6 l-Acetyl-4-(4(r-2-methyl-2-phenyl-l,3-dioxolan-c-4-yl)inethoxyphenyl)- piperazine, UCL2255, EP549A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (350mg, 1.6mmol) and sodium hydride (69mg, 1.6mmol) were stirred in dry DMF (10ml) for 1 hour at 0®C. Tosylate 62 (500mg, 1.4mmol) in dry DMF was then added and the mixture was heated to 80^C overnight. It was then diluted with IN NaOH (100ml) and extracted with dichloromethane (3x 100ml). The organic layer was washed with water (2 x 50ml), dried, filtered and evaporated in vacuo. Purification by flash chromatography (100% EtOAc) yielded a white solid which was recrystallised from dichloromethane/hexane to yield 16 (336mg, 0.084mmol, 58%). Rf: 0.1 (EtOAc) Melting point: 99-lOfC MS: APCI; 397 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.67(3H, s, dioxolane Me); 2.11(3H,
s, amide Me); 3.02(4H, m, CH2NPh); 3.59(2H, t, J= 5.0Hz, CHzNCOMe); 3.74(2H, t, J= 5.0Hz, CHzNCOMe); 3.83(1H,
dd, J= 8.0 & 7.0Hz, CH 2CH); 3.92(2H, m, CH2CH &
CHzOPh); 3.93(1H, dd, J= 9.4 & 5.0Hz, CH2 0 Ph); 4.30(1H, m,
CH2ÇH); 6.84(4H, s, OC 6H4N); 7.33(3H, m, mPh & pPh); 7.48(2H, d, J= 7.4Hz, o-Ph) IR: KBr disc, cm'*; (2990 & 2855, CH); (1649, C=0 amide); (1509, C=C); (1249 & 1049, C-O) HPLC: (40:60, A/B) Rt = 15.0 (100%)
Mieroanalysis C23H28N 2O4 calculated C: 69.68 H: 7.12 N: 7.07 found C: 69.50 H: 6.95 N: 7.14
No.17 I - AcetyI-4“(4 (r-2-methyl-2-phenyI-1,3-dioxolan-t-4-yl )methoxyphenyi)piperazine, UCL2264, EP553A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (350mg, 1.6mmol) and sodium hydride (69mg, 1.6mmol) were stirred in dry DMF (10ml) for 1 hour at 0°C. Tosylate 63 (500mg, 1.4mmol) in dry DMF (10ml) was then added and the mixture was heated to 80°C overnight. It was then diluted with IN NaOH (100ml) and extracted with
119 dichloromethane (3x 100ml). This was dried, filtered and evaporated in vacuo. Purification by flash chromatography (100% EtOAc) yielded a clear oil 17 (285mg, O.Vmmol, 49%) Rf: 0.1 (EtOAc) Melting point: clear oil MS: APCI; 397 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.58(3H, s, Me dioxolan); 2.03(3H, s,
MeCO); 2.93(4H, m, CHzNPh); 3.50(2H, t, J= 5.1Hz,
CHzNCOMe); 3.60(2H, m, CH2CH & CHiOPh); 3.66(2H, t, J=
5 . 1Hz,CH2NCOMe); 3.85(1H, dd, J= 9.4 & 5.9Hz, CH2 0 Ph)
4.21(1H, dd, J= 8.4 & 6 .6 Hz, CH2CH); 4.51(1H, m, CH2ÇH)
6.70(4H, ABq, J= 12.0Hz, OÇ6H4N); 7.23(3H, s, mPh & pPh) 7.43(2H, d, J=12.8Hz, oPh). Other peaks correspond to cis isomer - 15% IR: NaCl plate, cm '; (3463, OH water); (2929, CH); (1649, C=0 amide); (1511, C=C aromatic); (1246 & 1042, C-O) HPLC: (40:60,A/B) Rt =10.4 (83.5%), Rt =12.56 (15.5%) cis isomer overall purity 99.0 %
Mieroanalysis C23H28N 2 0 g .O.2 H2O calculated C: 69.05 H: 7.15 N: 7.00 found C: 69.14 H: 7.07 N: 6.95
N 0 .I 8 l-Acetyl-4-(4-(2-(2-chlorophenyl)-2-methyl-l,3-dioxoIan-4- yl)methoxyphenyl)piperazme, UCL2254, EP529A 1-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (312mg, 1.4mmol) and sodium hydride
(62mg, 1.4mmol) were stirred in dry DMF ( 8 ml) for 1 hour at 0°C. Tosylate 64
(500mg, 1.2mmol) in dry DMF ( 8 ml) was then added and the mixture was heated to 80°C overnight. It was then diluted with IN NaOH (100ml) and extracted with dichloromethane (3 x 1 00ml). The organic layer was dried, filtered and evaporated in vacuo. Purification by flash chromatography (100% EtOAc) yielded a pink oil 18 (322mg, 0.74mmol, 58%). Rf: 0.1 (EtOAc) Melting point: Pink oil
120 MS: APCI; 431 (100%)
NMR: 400MHz, CDCI3, 5 ppm; 2.14(0.75H, s, trans dioxolane Me);
2.16(2.25H, s, cis dioxolan); 2.48(3H, s, amide Me); 3.39(4H, m, CHzNPh); 3.96(2H, m, CHiNCOMe); 4.02(0.5H, m,
CH2CH & CH2OPI1 trans); 4.11(2H, m, CH2NCOMG);
4.20(0.75H, dd, J= 8.2 & 6.9Hz, CH2CH cis); 4.33(1.75H, m,
CH2); 4.47(0.75H, dd, J= 9.5 & 5.1Hz, CH2OPI1 cis ); 4.69(1 H,
m, CH2ÇH cis & CH2 trans); 4.96(0.25H, m, CH2CH trans);
7.1(1H, ABq, J= 8.9Hz, OÇ6H4N trans); 7.23(3H, s, OÇ 6H4N cis); 7.60(2H, m, H4,H5 chlorophen); 7.76(1 H, m, H3
Chlorophen); 8.00(1 H, m, H 6 chlorophen) IR: KBr disc, cm '; (3446, OH water); (2992, CH); (1628,0=0 amide); (1512, C=C aromatic); (1230, C-O); (1035, C-O) HPLC: (25:75, A/B) Rt=6.34 (24.9%), Rt=7.23 (74.5%) 99.4%
Mieroanalysis C23H28N 2O4.O.2 5 H2O Calculated C: 63.44 H: 6.37 N: 6.43 Found C: 63.48 H: 6.29 N: 6.33
No.l9 l-Acetyl-4-(4(r-2-methyl-2-(2,3,4-trichlorophenyI)-l,3-dioxolan-c-4- yl)methoxyphenyl)piperazine, UCL-2254, EP529A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (242mg, 1.1 mmol) and sodium hydride
(48mg, 1.1 mmol) were stirred in dry DMSO (5ml) at 0°C for 30 minutes. Tosylate 6 6 (500mg, 1.Ommol) in DMSO (5ml) was then added and the solution heated to 80°C for five hours. It was then diluted with water (100ml) and extracted with dichloromethane (120ml x 3). The organic layer was dried, filtered and evaporated in vacuo. Purification by fiash chromatography (100% EtOAc) gave a yellow solid which was recrystallised from dichloromethane/ hexane to yield a white solid 19 (135mg, 0.24mmol, 24%). Rf: 0.1 (EtOAc) Melting point: 137-139T MS: ES; MH 499 (97%); 501 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 1.80(3H, s, dioxolane Me); 2.11(3H,
s, Amide Me); 3.03(4H, m, CH2NPh); 3.59(2H, t, J= 5.2Hz,
121 CHzNCOMe); 3.75(2H, t, J= 5.2Hz, CHzNCOMe); 3.81(1H, dd, J= 8.4 & 7.0 Hz, CHzCH); 3.93(1H, dd, J= 9.5 & 6.3 Hz,
CHzOPh); 3.98(1H, dd, J= 8.5 & 4.6 Hz, CH2CH); 4.07(1H, dd,
J= 9.5 & 5.0, CH20Ph); 4.27(1H, m, CH2ÇH); 6.85(4H, ABq,
J= 9.5Hz, 0 _C6H4N); 7.34(1H, d, J= 8 .6 Hz, trichlorophen H5);
7.55(1H, d, J= 8.4, trichlorophen H 6 ) IR: KBr disc, cm'^; (2985, CH); (1650, CO amide); (1500, C=C);
(1200 & 1035, C-O) HPLC: (25:75, A/B) Rt = 22.78 (100%)
Mieroanalysis C 18H26N2O4.O.2 5 H2O calculated C: 54.78 H: 5.10 N: 5.55 found C: 54.76 H: 5.06 N: 5.28
N 0 .2 O R-(+)-Dioxaspiro-2-((4-acetylpiperazyI)phenoxy)methyl [4.5] decane,UCL- 2280, EP643A l-Acetyl-4-(4-hydroxyphenyl)-piperazine 54 (400mg, 1.8mmol) and sodium hydride (72mg, 1.8mmol) were stirred in dry DMF (5ml) for 1 hour at 0°C. Tosylate 69 (500mg, 1.6mmol) in dry DMF (5ml) was then added and the mixture was heated to 80°C overnight. It was then diluted with IN NaOH (100ml) and extracted with
dichloromethane (3 x 100ml). This was washed (3 x 50ml), dried (MgS 0 4 ), filtered and evaporated in vacuo. Purification by fiash chromatography (100% EtOAc)
yielded a solid which was recrystallised from methanol to give white needles 2 0 (304mg, 0.8Immol, 52%) Rf: 0.4 EtOAc
[a]o (CHCI3): +60° (0.5g/10ml, 20°C) Melting point: 109-112°C MS: FAB; 374 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 1.35(1H, m, decane H 8 ); 1.41(1H, m,
decane H 8 ); 1.60(8H, m, decane H 6 , 7, 9, 10); 3.01 (4H, m,
CH2NPh piper); 3.58(2H, t, J=5.1Hz, CH2NC0 Me piper);
3.74(2H, t, J= 5.2Hz, CH2NCOMe piper); 3.86(2H, m, CH 2
decane); 3.98(1 H, dd, J= 9.5, 5.21 Hz, CH2 0 Ph); 4.12(1H, dd,
122 J= 8.4, 6.3 Hz, CH2CH); 4.42(1 H, m, CH2ÇH); 6.84(4H, d, J=
6.5Hz, OC6H4N) IR: KBr disc, cm '; (2932, CH); (1646, 0=0), (1516, C=C); (1246,
C-O); (1096, C-O) HPLC: (30:70, A/B) Rt = 5.8 (99.5%)
Mieroanalysis O2JH30N2O4 Calculated C: 67.36 H: 8.07 N: 7.48 Found C: 67.28 H: 8.07 N: 7.45
No.21 2-(4-Chlorophenyl)-r-2-methyl-c-4-(4-aminophenoxymethyl)-l,3- dioxolane, UCL2327, EP961C Hydrazine hydrate (0.018ml) was added to a suspension of the imine 74 (lOOmg, 0.24mmol) in ethanol (15ml). This was stirred at 0°C for two hours. It was then evaporated in vacuo and dissolved in methanol. The methanolic solution was cooled and filtered to remove starting material. The filtrate was evaporated in vacuo, purified by flash chromatography (1:1, EtOAc; pet spirit) and recrystallised from methanol/hexane to give a pure white solid (61 mg, 0.19mmol, 78%) Melting point: 78''C MS: APCI; MH 320 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 1.62(3H, s. Me); 3.47(0.7H, s,
MeOH); 3.81(1H, dd, J= 8.4, 7.0Hz, CH 2CH); 3.92(2H, m,
CH2CH & CH2 0 Ph); 4.05(1H, dd, J= 9.6, 5.4Hz, CH2 0 Ph)
4.26(1 H, m, CH2CH); 6.80(2H, d, J= 8 .8 Hz, OÇ6H4N)
6.85(2H, d, J=8.3Hz, OÇ 6H4N); 7.30(2H, d, 8.5Hz, ClPh)
7.41 (2H, d, J= 8 .6 Hz, ClPh) IR: KBr disc, cm '; (3454, NH); (3370, NH); (2998, CH); (1512 C=C); (1200, C-O); (1041, C-O) HPLC: (30:70, A/B) Rt = 7.10 (97.9%) Mieroanalysis CnH[gClNO3.0.25MeOH calculated C: 63.21 H: 5.84 N: 4.27 found C: 63.42 H: 5.70 N: 4.24
123 No.22 2(2,4-DichIorophenyl)-2-methyl-4-((4-aminophenoxy)methyl)-l,3- dioxolane, UCL-2273, EP601A 4(Benzylidenamino)-phenol 57 (329mg, 1.6mmol) and sodium hydride (67mg, 1.6mmol) were stirred in dry DMF (5ml) for 1 hour at 0°C. Tosylate 65 (0.48 Ig, 1.4mmol) in dry DMF (5ml) was then added and the mixture was heated to 80°C and stirred overnight. The mixture was then dissolved in IN NaOH (60ml) and extracted with dichloromethane (60ml x 3). The organic layer was washed with water (2 x
60ml), dried (MgS0 4 ), filtered and evaporated in vacuo. The crude material was purified by flash chromatography (5:2 pet spirit/EtOAc, Rf 0.6) to give a mixture of
amino and imine products (0.168g). This was dissolved in ethanol ( 8 ml) and treated with hydrazine hydrate (0.103ml) and heated to reflux for 3 hours. It was then evaporated in vacuo and purified by flash chromatography (5:1, pet spirits/EtOAc) to give a yellow oil 22 (0.106g, 0.29mmol, 22%) Rf: 0.35 (5:2, pet spirits/EtOAc) Melting point: yellow oil. MS: APCI; 354 (100%), 355 (70%)
NMR: 500MHz, CDCI3, Ô ppm; 1.80(1H, s, trans Me); 1.85(2H, s, cis
Me); 3.40(2H, bs, NHz); 3.72(0.7H, m, CH 2 trans); 3.85(0.65H,
dd, J= 8.3, 6.9 Hz, CH2CH cis); 3.95(1H, m, CH2 cis & trans);
4.02(0.65H, dd, J= 9.5, 6.5 Hz, CH2 0 Ph cis); 4.10(0.65H, dd,
J=9.5, 5.1 Hz, CH2 0 Ph cis); 4.33(1H, m, CH2 trans & CH cis);
4.60(0.35, m, CH2ÇH); 6.63(1.4H, d, 1.7 Hz, OC 6H4N trans);
6.67(1.3H, d, J= 6.5, OC6H4N cis); 6.78(1.3H, d, J- 6.5Hz,
OC6H4N cis); 7.25(1 H, m, dichlorophen H5); 7.40(0.35H, d, J= 2.1Hz, dichlorophen H3 trans ); 7.43( 0.65H, d, J= 2.1Hz,
dichlorophen H3 cis ); 7.65( IH, m, dichlorophen H 6 ) IR: KBr disc, cm'; (3360, NH); (2933, CH); (1511, C=C aromatic); (1233, C-O); (1037, C-O). HPLC: (25:75, A/B) Rt = 5.8 & 7.2 (35.1 & 64.9%) 100% pure
Mieroanalysis C17H 17CI2N 2O3 calculated C: 57.64 H: 4.84 N: 3.95 found C: 57.72 H: 4.81 N: 3.89
124 No.23 2-(2,5-Dichlorophenyl)-r-2“methyl-c-4-(4-ammophenyl)oxymethyl)-l,3- dioxolane hemihydrate, UCL2335, EP1019C Hydrazine hydrate (0.026ml) was added to a suspension of imine 76 (220mg, O.Smmol) in ethanol at 0°C. This solution was stirred in ethanol at 0°C for 1 hour. The ethanol was then evaporated in vacuo. Purification by flash chromatography (1:1, pet
spirit/EtOAc) yielded a yellow oil (llVmg, 6 6 %). Recrystalisation from methanol yielded the trans (see below) isomer. This was filtered and the filtrate was evaporated in vacuo to give an oil, which was recrystallised from dichloromethane/hexane to give a pale orange solid 23 (50mg, O.Mmmol, 28.4%) Melting point: 119-12rC MS: APCI; MR 354 (100%), 356 (65%)
NMR: 500MHz, CDCI3, Ô ppm; 1.79(3H, s. Me); 3.83(1H, dd, J= 8.5,
6.9Hz, CH2CH); 3.91(1H, dd, J= 9.5, 6.5Hz, CHzOPh);
3.98(1H, dd, J= 8.5, 4.6Hz, CH2CH); 4.05(1H, dd, J= 9.5,
4.9Hz, CH2 0 Ph); 4.31(1H, m, CH2ÇH); 6.68(2H, d, J= ll.lH z ,
OÇ6H4N); 6.75(2H, d, J=ll.lHz, OC6H4N); 7.18(1H, dd, J= 10.5, 3.2Hz, dichlorophen H4); 7.30(1H, d, J= 10.5Hz,
dichlorophen H3); 7.64(1 H, d, J= 3.2Hz, dichlorophen H 6 ) IR: KBr disc, cm-1; (3354, NH); (2985, CH); (1514, C=C); (1240, C-O); (1035, C-O) HPLC: (30:70, A/B) Rt = 5.85 & 6.93 (3.5 & 96.5%) 3.5% trans isomer by HPLC, 100% purity
Microanalysis C 17 H 17 CI2 NO 3 .O.5 H2 O Calculated C: 56.21 H: 4.99 N: 3.86 Found C: 56.05 H: 4.82 N: 3.76
No.24 2-(3,4-Dichlorophenyl)-2-methyl-4-(4-aminophenyI)oxymethyl-l,3- dioxolane, UCL2333, EP993B 2-(3,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl-l ,3-dioxolane 72 (0.500g, 1.9mmol) was stirred in dry benzene (20ml) at room temperature. Tributylphosphine (565mg, 689ml, 2.1 mmol), 4-(benzylideneamino)phenol 57 (538mg, 2.1 mmol) and ADDP (706mg, 2.1 mmol) were added in this order. The mixture was stirred for 12 hours at which point further benzene (10ml) was added to maintain the stirring. It was
125 then stirred for a fiirther 3 hours. The mixture was diluted with ether (40ml), filtered and washed through with ether (80ml). The organic layer was washed with 10%
NaOH (80ml), brine (80ml) and water (80ml). It was dried (Na 2S0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (3:1, pet spirit/EtOAc) yielded a waxy solid (322mg) which was stirred with hydrazine hydrate (0.046ml) in ethanol for three hours at 0°C. The ethanol was evaporated in vacuo and the crude material purified by flash chromatography (3:1, pet spirit/EtOAc) to give a viscous brown oil 24 (219mg, 0.6mmol, 32%) Rf: 0.1 ( 1:1, pet Spirit/EtOAc) Melting point: brown oil MS: APCI; MR 354 & 356.1, (100 & 60 %)
NMR: 500MHz, CDCI3, Ô ppm; 1.60(0.9H, s. Me trans); 1.63(2.1H, s.
Me cis); 3.11(2H, bs, NH2), 3.65(0.6H, m, CH2 trans);
3.79(0.7H, dd, J= 10.5, 8 .8 Hz, CH2 cis); 3.88(1H, m, CH 2 cis
& trans); 3.92(0.7H, dd, J= 10.5, 5.9Hz, CH2 cis); 4.02(0.7H,
dd, J= 12.0, 6.7Hz, CH2cis); 4.27(1 H, m, CH 2 trans & CH cis);
4.55(0.3H, m, CH trans); 6.61(2.6H, m, OC6H4N cis & trans);
6.74(1.4H, d, J= 10.9Hz, OÇ6H4N); 7.30(1H, dd, J- 10.4, 2.3Hz, dichlorophen H5); 7.37(0.3H, d, J= 10.4Hz,
dichlorophen H 6 trans); 7.40(0.7H, d, J= 10.4Hz, dichlorophen
H6 cis); 7.57(0.7H, d, J= 2.5Hz, dichloro H2 cis); 7.58(0.3H, d, J=2.5Hz, dichloro H2 cis) IR: NaCl plate, cm-1; (3361, NH); (2980, CH); (1514, C=C); (1240, C-O); (1035, C-O) HPLC: (30:70, A/B) Rt = 6.75 & 8.71(27.6% & 69.7%) 97.3%
Microanalysis: C17H 18CINO3.O.2 5 H2O Calculated C: 56.92 H: 4.62 N: 3.90 Found C: 57.10 H: 4.79 N: 3.77
No,27 2-(2,4-DichlorophenyI)-r-2-methyl-c-4-(phenyIoxymethyI)-l,3-dioxoIane, UCL-2292, EP751A Phenol (131mg, 2.1 mmol) and sodium hydride (57mg, 2.1 mmol) were stirred in dry DMF (5ml) for 1 hour at 0°C. Tosylate 65 (500mg, 1.9mmol) in dry DMF (5ml) was
126 then added and the mixture was heated to 80®C overnight. The reaction mixture was then diluted with IN NaOH (60ml) and extracted with dichloromethane (3 x 60ml). The organic layer was dried, filtered and evaporated in vacuo. Purification by flash chromatography (8:1 to 5:1, pet spirit/EtOAc) followed by collection of fractions containing only cis isomer by TLC yielded a clear oil 27 (1 13mg, 0.3mmol, 28%) Rf: 0.53 (5:1, pet spirit/ EtOAc) Melting point: Clear oil MS: APCI; 338 (100%); 340 (70%)
NMR: 500MHz, CDCI3, Ô ppm; 1.80(3H, s. Me); 3.83(1H, dd, J= 8.4,
6.9Hz, CH2CH); 3.99(2H, m, CH2CH & CH2 0 Ph); 4.I2(1H,
dd, J= 9.5, 5.0Hz, CH2 0 Ph); 4.32(1H, m, CH2ÇH); 6.90(2H, d,
J= 8 .8 Hz, o-Ph); 6.95(1 H, t, J= 7.4Hz, p-Ph); 7.25(3H, m, m- OPh & dichlorophen H5); 7.39(IH, d, J= 2.1Hz, dichlorophen
H3); 7.60(1 H, d, J= 8.4Hz, dichlorophen H 6 ) IR: NaCl plate, cm *; (2934, CH); (1591, C=C aromatic); (1242, C- O); (1038, C-O) HPLC: (20:80, A/B) Rt = 30.0min (100%)
Microanalysis C 17H16CI2O3 calculated C: 60,19 H: 4.75 N: 0.0 found C: 60.37 H: 4.52 N: 0.0
No.28 2(2,4-DichIorophenyI)-r-2-methyl-t-4-(phenoxymethyl)-l,3-dioxolane, UCL2303, EP751B After isolation of the cis isomer (see 27) the remaining mixture was repurified twice by column isolating fractions mainly containing trans isomer by TLC. This yielded a clear oil 28 (98mg, 24%). Cis isomer 27 present approx 15% Rf: 0.47 (5:1, pet spirit/EtOAc) Melting point: clear oil. MS: APCI; 338 (100%), 340 (70%)
NMR: 500MHz, CDCI3, Ô ppm; 1.79(3H, s. Me); 3.73(2H, m, CH 2CH
& CH2 0 Ph); 3.96(1H, dd, J= 9.6, 6.2Hz, CH2 0 Ph); 4.29(1H,
dd, J= 8 .6 , 6.4Hz, ÇH2CH); 4.60(1H, m, CH2ÇH); 6.75(2H, d, J= 8.7Hz, o-OPh); 6.93(1H, t, J= 7.3Hz, p-OPh); 7.17(1H, dd, J
127 = 8.4, 2.1Hz, dichlorophen H5); 7.21(2H, m, m-OPh); 7.36(1H, d, J= 2.1Hz, dichlorophen H3); 7.60(1 H, d, J= 8.4Hz,
dichlorophen H 6 ) IR: NaCl plate, cm'*; (2934, CH); (1591, C=C ar); (1495, C=C ar), (1242, C-O); (1038, C-O) HPLC: (20:80, A/B) Rt = 38.92 (81.7%), Rt = 44.64 (16.6%), (98.3%
pure, 6:1 trans: cis),
Microanalysis C17H 16CI2O3 Calculated C: 60.19 H: 4.75 N: 0.0 Found C: 60.41 H: 4.66 N: 0.0
No.32 2(2,4-Dichlorophenyl)-r-2-methyl-c-4-((4-cyanophenoxy)methyl)-l,3- dioxolane, UCL2270, EP611A 4-Cyanophenol (207mg, 2.0mmol) and sodium hydride (76mg, 2.0mmol) were stirred in dry DMF (5ml) for 1 hour at 0°C. Tosylate 65 (500mg, 1.9mmol) in dry DMF (5ml) was then added and the mixture was heated to 80°C overnight. The reaction mixture was then diluted with IN NaOH (80ml) and extracted with dichloromethane (3 X 100ml). The organic layer was washed with (3 x 50ml), dried, filtered and evaporated in vacuo. Purification of the crude material by flash chromatography (5:1, pet spirit/EtOAc) followed by collection of fractions containing only cis isomer by TLC yielded a white solid 32 (143mg, 3.9mmol, 37%) Rf: 0.1 (5:1, EtOAc: pet spirit) Melting point: 91-92°C MS: FAB; 364 (100%), 366 (65%)
NMR: 500MHz, CDCI3, Ô ppm; 1.83(3H, s, Me); 3.89(1H, dd, J=8.5,
6.9Hz, Π2CH); 4.05(1H, dd, J=8.5, 4.4Hz, CH2CH); 4.10(1H,
dd, J=9.5, 5.9Hz, CH2pPh); 4.20(1 H, dd, J=9.5,5.0Hz,
CH2 0 Ph); 4.38(1H, m, CH 2ÇH); 7.17(2H, d, J=8.9Hz,
OC6H4CN meta); 7.26(1 H, dd, J=8.5, 2.1Hz, dichlorophen H5);
7.44(1 H, d, J=2.1Hz, dichlorophen H3); 7.34(3H, m, OC 6H4N
ortho & Dichlorophen H 6 ) IR: KBr disc, cm'^; (2943,CH); (2233, CN); (1511, C=C aromatic); (1265,C-0); (1039,C-O)
128 HPLC: (30:70, A/B) Rt = 5.1 (98.6%)
Microanalysis C 18H 15CI2NO 3.O.2 5 H2O Calculated C: 58.63 H: 4.24 N: 3.80 Found C: 58.69 H: 4.09 N: 3.68
N0 . 3 3 2(2,4-Dichlorophenyl)-r-2-methyl-t-4-((4-cyanophenyl)oxymethyl)-l,3- dioxolane, UCL2303, EP611B After isolation of the cis isomer (see 32) the remaining mixture was repurified three times by flash chromatography (5:1, pet spirit/EtOAc) isolating fractions mainly containing trans isomer by TLC. This yielded a clear oil 33 (53mg, 10%) Rf: 0.06 (5:1, pet spirits/EtOAc) Melting point: clear oil MS: FAB; MH 364 (100%), 366 (65%) isotopic splitting
NMR: 500MHz, CDCI3, 6 ppm; 1.78(3H, s, Me); 3.49(1H, dd, J=8 .6 ,
7.1Hz, CH 2CH); 3.83(1H, dd, J=9.7, 5.4Hz, CH2 0 Ph);
3.97(1H, dd, J= 9.7, 4.5Hz, CH2 0 Ph); 4.30(1H, dd, J= 8 .6 ,
6.4Hz, CH2CH); 4.60(1H, m, CH2CH); 6.77(2H, d, J= 8.9Hz,
OÇ6H4CN); 7.17(1H, dd, J= 8.4, 2.1Hz, dichlorophen H5); 7.34(1 H, d, J= 2.1Hz, dichlorophen H3); 7.52(2H, d, J=9.0,
OC6H4N); 7.57(1 H, d, J= 2.1Hz, dichlorophen H 6 ) IR: KBr disc, cm-1; (2936, CH); (2224, CN); (1608, C=C); (1508, C=C); (1259, C-O); (1036, C-O) HPLC: (25:75, A/B) Rt = 24.84 (98.2%)
Microanalysis C]gH,5Cl2N03 calculated C: 59.36 H: 4.15 N: 3.85 found C: 58.98 H: 4.21 N: 3.54
No.39 2(2,4-Dichlorophenyl)-r-2-methyl-c-4-((4-carbamoylphenyl)oxymethyl)- 1,3-dioxolane, UCL-2298, EP809A 4-Hydroxybenzamide (180mg, 2.0mmol) and sodium hydride (52mg, 2.0mmol) were stirred in dry DMF (5ml) for 1 hour at 0°C. Tosylate 64 (497mg, 1.9mmol) in dry DMF (5ml) was then added and the mixture heated to 80°C overnight. The reaction mixture was then diluted with brine (50ml) and extracted (3 x 70ml) with
129 dichloromethane. The organic layer was dried (MgS 0 4 ), filtered and evaporated in vacuo to give a solid, which was recrystallised from methanol to give a white solid 39 (136mg, 0.35mmol, 19.0%) Melting point: 173-175T MS: APCI; MH 382 & 384 (100% & 70%) isotopic
NMR: 500MHz, CDCI3 , Ô ppm; 1.88(3H, s. Me); 3.89(1H, dd, J= 8.4,
6.9Hz, CH2 CH); 4.05(1H, dd, J= 8.5, 4.5Hz, ÇH2 CH); 4.09(1 H,
dd, J= 9.6, 6.1Hz, CH2 0 Ph); 4.21(1H, dd, J= 9.6, 5.0Hz,
CH2 0 Ph); 4.38(1H, m, CH 2 ÇH); 6.99(1H, d, J= 8 .8 Hz,
OC6 H4 CONH2 ); 7.26(1H, dd, J= 8.3, 2.1Hz, dichlorophen H5); 7.44(1 H, d, J= 2.1Hz, dichlorophen H3); 7.63(1H, d, J= 8.3Hz,
dichlorophen H 6 ); 7.80(1 H, d, J= 8 .8 Hz, OC6 H4 CONH 2 ) IR: KBr disc, cm '; (3177, NH); (2904, CH); (1647, 0=0); (1255, C-O); (1044, C-O) HPLC: (25:75, A/B) Rt = 14.65min (99.0 %)
Microanalysis C 18H17CI2NO4 Calculated C: 56.56 H: 4.58 N: 3.66 Found C: 56.80 H: 4.62 N: 3.44
No,40 2(2,4-Dlchlorophenyl)-r-2-methyl-t-4((4-carbamoylphenyl)oxymethyl)-l,3- dioxolane, UCL 2319, EP809B After isolating the cis isomer (see 39) by crystalisation the supernatant liquid was evaporated in vacuo and purified by flash chromatography (EtOAc). This material was then further purified by flash chromatography (diethyl ether). Fractions containing mainly the trans isomer by HPLC were evaporated in vacuo to give a white solid 40 (30mg, 6 %) Mixture 7:3, trans:cis Rf: 0.3 (diethyl ether) Melting point: 92-95°C MS: ES; MH 382 & 384, (100% & 70%) isotopic MNa 404 & 406 (80% & 50%) isotopic
NMR: 500MHz, CDCI3, 8 ppm; 1.76(2.1H, s. Me trans); 1.79(0.9H, s.
Me cis); 3.75(0.7H, dd, J=8.5, 7.3Hz, CH 2 CH trans); 3.82(1H,
130 m, CH2OPI1 trans & CH2 cis); 3.98(1.3H, m, CH 2OPI1 trans &
CH2 cis); 4.10(0.3H, dd, J= 9.5, 4.7Hz, CH2OPI1 cis); 4.30(1H,
m, CH2CH trans & CH2ÇH cis); 4.61(0.7H, m, CH 2ÇH);
5.70(2H, bs, CONH 2); 6.75(1.4H, d, J= 8 .8 Hz, OÇ6H4CONH2);
6.95(0.6H, d, J= 8 .8 Hz, OC6H4CONH2); 7.18(0.7H, dd, J= 8.4, 2.1Hz, dichloro H5 trans); 7.22(0.3H, dd, J= 8.4, 2.1Hz, dichloro H5 cis); 7.35(0.7H, d, J= 2.1Hz, dichloro H3 trans); 7.39(0.3H, d, J= 2.1Hz, dichloro H3 cis); 7.58(1H, m, dichloro
H6 ); 7.71(1.4H, d, J= 8 .8 Hz, OÇ6H4CONH2 trans); 7.77(0.6H,
d, J= 8 .8 Hz, OC6H4CONH2 cis) IR: KBr disc, cm'^; (3420, NH); (2933, CH); (1658, C=0); (1257, C-O); (1036, C-O) HPLC: (20:80, A/B) Rt= 7.83 & 9.58 (70.8 & 28.4 %) 99.2% pure,
Microanalysis C 18H17NO4 calculated C: 56.56 H: 4.58 N: 3.66 found C: 56.81 H: 4.61 N: 3.30
No.42 R-l-(4-Aminophenoxy)-2-hydroxy-3(2,4-dichlorobenzyloxy)-propane oxalate, UCL-2334, EP1013A 4-(Benzylidenamino)-phenol 57 (114mg, 0.6mmol) and sodium hydride (24mg, 0.6) were stirred in dry DMF (1ml) for 1 hour at 0°C. Epoxide 80 (0.119g, 0.49mmol) in dry DMF (1ml) was then added and the mixture was heated to 80°C and stirred overnight. The mixture was then diluted with brine (50ml) and extracted with dichloromethane (60ml x 3). The organic layer was washed with 10% NaOH (1
X 100ml), dried (Na2S0 4 ), filtered and evaporated in vacuo. The crude material was purified by flash chromatography (1:1 pet spirit/EtOAc, Rf 0.7) to give a mixture of amino and imine products (0.145g). This was dissolved in ethanol (10ml) and treated with hydrazine hydrate (0.011ml) for 30 minutes at 0°C. The solution was evaporated in vacuo, dissolved in ether (30ml) and washed with water (3 x 20ml). The ether was dried (Na 2S0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (1:1, pet spirits/EtOAc) yielded to give a yellow solid 42 (39mg, 0.12mmol, 23%). This solid was converted to the oxalate salt and recrystallised in ethanol/ether Melting point: 144-146°C
131 [a]o (MeOH) -150°(10mg/10ml, 17T) MS: ES; MH 342 (30%); MH - dichorobenzyl 183 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 3.52(1H, dd, J= 9.8, 4.3Hz, CHzOPh);
3.58(1H, dd, J= 9.8, 5.1Hz, CHjOPh); 3.78(1H, dd, J= 9.7, 5.9Hz, CHiOBn); 3.85(1H, dd, J= 9.7, 4.9Hz, CHiOBn);
3.94(1H, m, CH2ÇHCH2); 4.56(2H, s, CH2 benzyl); 6.32(2H, d,
J= 8.7Hz, OC 6H4N); 6.71(2H, d, 8.9Hz, OC 6H4N); 7.41(1H, dd, J= 8.3, 2.1Hz, dichloro H5); 7.52(1 H, d, J= 8.3Hz, dichloro
H6 ); 7.58(1H, d, J= 2.1Hz, dichloro H3) IR: KBr disc, cm '; (3411, OH & NH); (2875, CH); (1600, C=0 oxalic acid); (1510, C=C) HPLC: (40:60, A/B) Rt = 8.03min, (100%) Microanalysis C16H17CI2NO3.C2H2O4.O.25H2O calculated C: 49.60 H: 4.50 N: 3.24 found C: 49.55 H: 4.42 N: 3.09
N 0 .4 3 S-l-(4-Amlnophenoxy)-2-hydroxy-3(2,4-dichlorobenzyloxy)-propane oxalate, UCL2360, EP1133A 4-(Benzylidenamino)-phenol 57 (114mg, 0.6mmol) and sodium hydride (24mg) were stirred in dry DMF (1ml) for 1 hour at 0°C. Epoxide 81 (0.123g, 0.5mmol) in dry DMF (1ml) was added and the mixture was heated to 80°C and stirred overnight. The mixture was diluted with brine (50ml) and extracted with dichloromethane (60ml x 3).
The DCM was washed with 10% NaOH (1 xlOOml), dried (Na 2S0 4 ), filtered and evaporated in vacuo. The crude material was purified by flash chromatography (1:1 pet Spirit/EtOAc, R f 0.7) to give a mixture o f amino and imine products (0.145g). This was dissolved in ethanol (10ml) and treated with hydrazine hydrate (0.011ml) for 30 minutes at 0°C. This solution was then evaporated in vacuo, and purified by flash chromatography (1:1, pet spirits/EtOAc) yielded to give a yellow solid (39mg, 23%). This was converted to the oxalate salt and recrystalised from ethanol. Melting point: 144-146°C
[a]o (MeOH): +109° (15mg/l 0ml, 1TC)
MS: ES; MH 342 & 344 (100% & 70%)
132 NMR: 500MHz, CDCI3, Ô ppm; 3.52(1H, dd, J= 9.8,4.3Hz, CH2OPI1);
3.58(1H, dd, J= 9.8, 5.1Hz, CHzOPh); 3.78(1H, dd, J= 9.7,
5.9Hz, CH2 0 Bn); 3.85(1H, dd, J= 9.7, 4.9Hz, CHzOBn);
3.94(1H, m, CH2ÇHCH2); 4.56(2H, s, CH2 benzyl); 6.32(2H, d,
J= 8.7Hz, O Q H 4N ortho); 6.71 (2H, d, 8.9Hz, OC^HLN); 7.41(1H, dd, J= 8.3,2.1Hz, dichloro H5); 7.52(1 H, d, J= 8.3Hz,
dichloro H 6 ); 7.58(1H, d, J= 2.1Hz, dichloro H3) IR; KBr disc, cm '; (3411, OH & NH); (2875, CH); (1600, C O oxalic acid); (1510, C=C) HPLC: (40:60, A/B) Rt = 8.03min, 97.3%
Microanalysis C16H17CI2NO3.3C2H2O4 calculated C: 43.15 H: 3.79 N: 2.29 found C: 42.95 H:4.12 N: 2.13
N 0 .4 4 l-(4-Amiiiophenyl)-3-(2,4-dichIorophenyl)-urea, UCL-2355, EP1103A Nitro compound 82 (200mg, 0.6mmol) and tin(II) chloride (427mg, 3eq) were heated to reflux in ethanol. It was then cooled, evaporated and stirred in IN NaOH (30ml). This was extracted with ethyl acetate. This was dried, filtered and evaporated in vacuo to give a white solid. This was recrystalised from ethanol to give a white solid 44 (146mg, 0.5mmol, 82.2%) Melting point: decomposed above 235°C Rf: 0.8 (EtOAc) MS: APCI; MH 296 (100%)
NMR: 400MHz, dô-DMSO + D2O, ô ppm; 6.51(2H, d, J= 8.7Hz,
NC6H4N H3 & H5); 7.05(2H, d, J= 8.7Hz, NC 6H4N H2 & H6); 7.33(1H, dd, J= 8.9, 2.5Hz, dichlorophen H5), 7.55(1H, d, J= 2.5Hz, dichlorophen H3); 7.61 (IH, d, J= 8.9Hz, dichlorophen H6) IR: KBr disc, cm '; (3301, NH stretch); (1633, C=0); (1532, C=C); (1300, C-N) HPLC: (40:60, A/B) Rt = 9.7min, 97.7%
Microanalysis C13H11CI2N 3O.O.2 5 H2O Calculated C: 51.93 H: 3.86 N: 13.98
133 Found C: 52.17 H: 3.56 N: 13.63
No.45 (2,4-Dichlorobenzyl)-4-aminobenzoate, UCL-2347, EP1091 2,4-Dichlorobenzyl alcohol (873mg, 5.4mmol), 4-aminobenzoic acid (1.00g, 5.9mmol) and triphenylphosphine (2.58g, 6.5mmol) were stirred in dry THF (30ml) at 0°C. DEAD (1.84 ml, 6.5mmol) in THF (20ml) was added over 1 hour. The solution was then stirred for a further 24 hours warming to room temperature. It was diluted with water (80ml) and ether (50ml). The ether was washed with water (2 x 60ml), dried, filtered and evaporated. The crude product was purified by fiash chromatography (3:1, pet spirit/EtOAc) and recrystalised from methanol to give white needles (889mg, 3.0mmol, 61.3%). Rf: 0.75 (1:1, pet spirit/EtOAc) Melting point: 173-175°C MS: APCI: MH 296 (100%)
NMR: 500MHz, CDCI3, 6 ppm; 5.27(2H, s, CH2 benzyl); 6.01 (0.8H,
bs, NH2); 6.55(2H, d, J= 8.7Hz, Ç 6H4N H3 & H5); 7.46(1 H, dd, J= 8.3, 2.1Hz, dichlorophen H5); 7.56(1 H, d, J= 8.3Hz,
dichlorophen H6); 7.67(3H, m, C 6H4N & dichlorophen H3) IR: KBr disc, cm ' (3426-3333, NH); (3214, CH); (1694, C=0 benzoate); (1514, C=C ar); (1283,1123, CO) HPLC: (20:80, A/B) Rt = 9.8 (99.3%)
Microanalysis C 14H11CI2NO2 calculated C: 56.78 H: 3.74 N: 4.73 found C: 56.53 H: 3.70 N: 4.63
N 0 .4 9 l-AcetyI-4-(4-(2-(2-chlorophenyl)furan-5-yl)methoxy)phenyl)piperazme, UCL-2304, EP849A
Sodium borohydride was added portionwise to a solution of 5-(2-chlorophenyl)- fiirfural (84mg, 0.4mmol) in methanol (5ml). This was stirred for 20 minutes at 0®C. It was then diluted with 10% NaOH (30ml) and stirred for a further 10 minutes. This was extracted with dichloromethane (3 x 30ml) which was dried (Na 2S0 4 ), filtered and evaporated in vacuo to give a yellow oil (81 mg). The dichloromethane was dissolved in dry benzene (3ml) and stirred at 0°C. Tributylphosphine (0.113ml,
134 0.4Immol) was added and the reaction stirred for 5 minutes. This was followed by 1- acetyl-4-(4-hydroxyphenyl)-piperazine 54 (128mg, 0.41 mmol) and ADDP (146mg, 0.41 mmol) at intervals of five minutes. It was then allowed to warm to room temperature and stirred for 14 hours. It was then diluted with hexane (2ml) and filtered. The filtrate was taken up in ether (30ml), washed with 10% NaOH (20ml)
and water (2 x 20ml). It was then dried (MgS 0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography followed by recrystalisation Jfrom dichloromethane/hexane yielded a white solid (69mg, O.lTmmol, 39%) Melting point: 119-120°C MS: APCI; MH 411 (100%); 413 (35%) isotope
NMR: 500MHz, CDCI3, Ô ppm; 2.12(3H, s. Me); 2.96(4H, m,
CHzNPh); 3.61(2H, bs, CHiNCOMe); 3.76(2H, bs,
CHzNCOMe); 5.03(2H, s, CH2 benzyl); 6.51(1H, d, J= 3.4Hz,
furan); 6.95(4H, bs, OC6H4N, restricted rotation); 7.07(1 H, d, J= 3.4Hz, furan); 7.19(1H, m, ArCl H4); 7.27(1 H, m, ArCl
H5); 7.41(1H, dd, J=8.0, 1.3Hz, ArCl H 6 ); 7.84(1 H, dd, J= 7.9, 1.7Hz, ArCl H3)
IR: KBr disc, cm '; (3448, H 2O); (2826, CH); (1620, CO amide); (1513, C=C aromatic); (1249, C-O); (1002, C-O) HPLC: (25:75, A/B) Rt = 8.58min, (99.5%)
Microanalysis C23H23CIN2O3.O.2 5 H2O Calculated C: 66.50 H: 5.70 N: 6.74 Found C: 66.32 H: 5.54 N: 6.62
No.51 2-(Bromomethyl)-2-(2,4-dichIorophenyl)-4(hydroxymethyl)-l,3-dioxolane EP17A Glycerol (lOg, 0.1 mol), 2,4-dichloroacetophenone (18.9g, 0.1 mol) and toluene sulphonic acid monohydrate (0.6g, 3mol%) were heated to reflux in a mixture of benzene (40ml) and n-butanol (20ml) with azeotropic removal of water for 24 hours. After cooling to room temperature bromine (19.2g, O.lmol) was added over a period o f 2 hours and stirred for a further 2 hours. The mixture was then concentrated in vacuo. The residue was dissolved in dichloromethane, washed with 6N NaOH
135 solution (20ml) and water (2x20ml). The organic layer was then dried (MgSO#), filtered and concentrated to dryness to give a pale yellow oil 50 (35g, 95%). Mixture: 3:2, cis: trans MS: ES; MNa 363, 365, 367 (65%, 100% & 45%)
NMR: 400MHz, CDCI3, Ô ppm; 3.46(0.4H, dd, J= 12.0, 5.01Hz,
CH2OH trans); 3.67-3.77(1.6H, m, CH2); 3.86-3.95(3H, m,
CH2); 4.10(0.6H, dd, J= 8.3, 5.9Hz, CH2CH cis); 4.18(0.6H, m,
CH2CH cis); 4.30(0.4H, dd, J=8.3, 6.4Hz, CH2CH trans);
4.54(0.4H, m, CH2CH trans); 7.24(1 H, dd, J= 8.5, 2.2Hz, dichlorophen H5); 7.40(1H, d, J=2.2Hz, dichlorophen H3),
7.59(0.6H, d, J= 8.5Hz, dichlorophen H 6 cis); 7.62(0.4H, d, J=
8.5Hz, dichlorophen H 6 trans)
No.52 r-2-Bromomethyl-2-(2,4-dlchlorophenyI)-c-4-(benzoyloxymethyl)-l,3- dioxolane EP23B Benzoyl chloride (12ml, 14.4g, 0.102mol) was added dropwise to a solution of 50 (27.2g, 0.074mol) in dry pyridine (60ml) at 0°C over 1 hour. The reaction mixture was stirred for a further 4 hours and then it was quenched with water (100ml) the solution was extracted with CHCI3 (3x150ml). The organic layer was washed with 6 N
HCL (250ml), dried with MgS0 4 and evaporated in vacuo. Stirring with methanol yielded a white solid that was recrystalised in ethanol to give the cis isomer as a white solid (17.49g, 0.037mol, 50.16%). M elting point: 117-118°C (lit 118.3^'“^) Mass Spectra: ES: MNa+: 467 (38%) 469 (60.47%) 471 (27.11%); MH"": 445,
2.5%, 447 (3.94%), 449(1.41%); loss ofBiCH 2COPhCl2: 179, ( 100%)
'H NMR; 400MHz, CDCI3 5 ppm; 3.87(2H, ABq, J = 11.3Hz, CH;Br);
3.99(1H, t, J = 8.15Hz, CH2CH); 4.33(1H, t, J = 8.17Hz,
CH2CH); 4.40(1H, m, CH2ÇH); 4.52( 2H, dd, J =
4.97&1.91Hz); 7.23(1H, dd, J = 8.40, 1.9Hz, ArCl2 C5 proton);
7.39(1 H, d, J = 2Hz, ArCl2 C3 proton); 7.42(2H, t, J = 7.9Hz, benzoyl meta); 7.55(2H, dt, J = 8.3, IHz, benzoyl para);
136 7.60(1H, d, J = 8.49Hz, A iC h H6 ); 8.40(2H, dd, J = 8.0, IHz, benzoyl ortho)
No.53 2-(2,4-Dichlorophenyl)-r-2-(lH-imidazol-l-ylmethyl)-c-4- (methansulfonyl)oxymethyI-l,3-dioxolane EP699A Methanesulfonylchloride (0.76ml, O.Olmol) was added over 20 minutes to a solution
of the alcohol 6 (2.7 Ig, 8.2mmol) stirring in dry pyridine (10ml) at 0°C. The reaction mixture was stirred for a further hour and was then diluted with water (20ml). The solution was filtered and the solid was recrystalised from benzene to give a white solid 53 (2.28g, 5.6mmol, 68.1%) Melting point: 109-110% (lit MS: FAB; MH 407 (100%), 409 (70%)
NMR: 500MHz, CDCI3, Ô ppm; 3.05(3H, s. Me); 3.68(1H, dd, J=
10.7, 5.5 Hz, ÇH 2OMS); 3.72(1 H, dd, J= 8.7, 4.7 Hz, CH 2CH)
3.87(2H, m, CH 2CH & CH2OMS); 4.33(1H, m, CH2ÇH)
4.47(2H, ABq, J= 14.8Hz, CH 2N); 7.02(1 H, s, imidazol) 7.05(1H, s, imidazol); 7.28(1H, dd, J=8.3, 2.1Hz, H5 dichlorophen); 7.49(1 H, d, J= 2.1Hz, H3 dichlorophen);
7.54(1 H, s, imidazol); 7.56(1H, d, J= 8.3Hz, H 6 dichlorophen)
No.53 l-Acetyl-4-(4-acetoxyphenyl)-piperazine EP77A 1 (4-Hydroxyphenyl)-piperazine (8.0g, 0.045mol), acetic anhydride (40ml) and dry pyridine (9ml) were stirred at room temperature under nitrogen overnight. The mixture was then placed in water (80ml) and stirred at 0°C. The solution was carefully neutralised with 5N NaOH and extracted with chloroform (120ml x 3). The organic
layer was dried (MgS 0 4 ), filtered and evaporated in vacuo. The crude material was purified by flash chromatography to give a white solid 53 (8.71g, 0.033mmol, 74.3%). Rf: 0.1 (EtOAc 100%) Melting point: 88-9rC MS: APCI; MH 263 (100%)
NMR: 400MHz, CDCI3, 5 ppm; 2.11(3H, s, MeCON); 2.24(3H,s,
MeC0 2 ); 3.06-3.12(4H, m, CH2 H3 & H5 piper); 3.58(2H, t, J=
5.1Hz, CH2 H2 & H6 piper); 3.74(2H, t, J= 5.2 Hz, CH2 H2 &
137 H6 piper); 6.89(2H, d, J=9.1Hz, OÇ6H4N H2 & H6); 6.97(2H,
d, J= 9.1Hz, OC6H4N H3 & H5)
N0 . 5 4 l-Acetyl-4-(4-hydroxyphenyl)-piperazine EP83A l-Acetyl-4-(4-acetoxyphenyl)-piperazine 53 (8.0g, 0.03mol) and 0.33% ammonia solution (6ml) were stirred in methanol (50ml) for 24 hours at room temperature. The reaction mixture was then filtered and the mother liquor evaporated in vacuo. Both solids were combined and recrystalised from ethanol to yield the phenol as brown crystalline solid 54 (5.8g, 0.026mmol, 89%) Rf: 0.05 (EtOAc 100%) Melting point: 179°C ( lit 178^"*^) MS: APCI; MH 221 (100%)
NMR: 400MHz, CDCI3, ô ppm; 2.12(3H, s. Me); 2.96(4H, m, CH2
piper H3 & H5); 3.59(2H, t, J= 5.1Hz, CH2 piper H2 & H6);
3.75(2H, t, J= 5.1Hz, CH2 piper H2 & H6 ); 6.76(2H, d, J=
8.9Hz, OÇ 6H4N H3 & H5); 6.83(2H, d, J= 8.9Hz, OC6H4N H2
&H 6 )
N0 . 5 5 l-(4-MethoxyphenyI)-piperadinium oxalate EP565A Palladium dibenzylidenylacetone (18.3mg, lmol%) and tri-o-tolylphosphine (12.2mg, 4mol%) were placed in a dry round bottomed flask under argon. Dry degassed toluene (10ml) was added and the mixture was heated to 60°C for 10 minutes. It was the cooled. p-Bromoanisole (187mg, 0.271ml, Immol) was added to the mixture followed by piperidine (95mg, 0.01ml, 1.2mmol) and lithium hexamethyldisilizane solution (1.3ml, IM in THF). This was heated to 100°C overnight. The mixture was cooled and dissolved in ether (40ml). The organic layer was washed with 10% NaOH (30ml
X 1) and water (30ml x 2). This was dried, filtered, evaporated in vacuo and purified by flash chromatography (13:1, Pet : EtOAc) to give a clear oil. This was dissolved in ethanol and treated with a solution of oxalic acid in ethanol. Ether was added and the product collected by filtration (105mg, 0.37mmol, 37%). Rf: 0.45 (10:1, Pet spirit/EtOAc) Melting point: 134°C MS: APCI; MH 192 (100%)
138 NMR: 500MHz, DMSO dé, ô ppm; 1.51(2H, m, piper H4); 1.66(4H,
m, piper H3 & H5); 3.04(4H, t, J= 5.2Hz, piper H2 & H6 );
6.83(2H, d, J= 8.6Hz, OÇ 6H4); 6.98(2H, d, J= 8 .6 Hz, Ç6H4N)
No.56 l(4-Hydroxyphenyl)-piperidinium oxalate EP901A A solution of boron tribromide (0.942ml, IM in dichloromethane) was added to a solution of l(4-methoxyphenyl)-piperidine 55 (55mg, 0.3mmol) which was stirring in dry dichloromethane (20ml) at -78°C. This solution was allowed to warm to room temperature overnight. It was extracted with 10%NaOH (10ml x 3). The organic layer was disguarded. The aqueous layer was neutralised with NaHCOs and extracted with
dichloromethane (30ml x 3). The organic layer was dried MgS 0 4 filtered and evaporated in vacuo to yield an off white solid. This was dissolved in ethanol and treated with an ethanolic solution of oxalic acid. Recrystallisation from ethanol/ether gave the oxalate salt as a white solid 56 (46mg, 0.2mmol, 61%). Melting point: 207-210°C MS: FAB; MH 178 (100%)
NMR: 500MHz, DMSO dg, ô ppm; 1.48(2H, m, H4 piper); 1.64(4H, m, H3 & H5 piper); 3.00(4H, m, H2 & H6 piper); 6.66(2H, d,
J= 8.7Hz, OÇ 6H4); 6.88(2H, bs, Ç 6H4N)
N0 . 5 7 4-(Benzylidenamino)-phenol EP367A
Benzaldehyde ( 6 .6 ml, 0.05mol) was added to a solution of 4-aminophenol (5g, O.OSmol) in dry methanol (30ml) at 0®C. This was stirred for a further 3 hours. It was then filtered and the solid recrystalised from methanol to give pale green crystals 57 (6.11g, 0.04mol, 72%) Melting point: 189°C (lit 189°C^^^) MS: APCI; 198(100%)
NMR: 400MHz, DMSO dé, ô ppm; 6.78(2H, d, J= 8 .6 Hz, OÇ6H4N H3
& H5); 7.19(2H, d, J= 8 .6 Hz, OC6H4N H2 & H6 ); 7.47(3H, m, NCHPh Hm & Hp); 7.87(2H, m, NCHPh Ho); 8.59(1H, s, NCHPh); 9.50(0.8H, s, OH)
No.58 2-Methyl-2-phenyl-4-hydroxymethyl-l,3-dioxolane EP537A
139 Acetophenone (lOg, O.OSmol), glycerol (8.4g, 0.091 mol) and toluene sulfonic acid monohydrate (0.78g, 4.Immol) were heated to reflux overnight in benzene/ n-butanol (40ml/20ml) with a Dean Stark water trap. Upon completion, the mixture was concentrated in vacuo. It was then taken up in dichloromethane (80ml) washed with
brine (80ml x 3) and dried (MgS 0 4 ). Filtration, concentration in vacuo followed by distillation under reduced pressure yielded a clear liquid 58 (11.23g, 0.057mmol, 70%) Mixture: 3:2 cis: trans Boiling point: 136-140®C, 8mmHg MS: FAB; MH 195 (100%)
NMR: 400MHz, CDCI3, ô ppm; 1.63(0.75H, s. Me diox);
1.67(2.25H, s. Me diox); 3.43(0.4H, dd, J=11.8, 5.5Hz,
CH2OTS); 3.57- 3.62(1.4H, m, CH 2 cis & CH2 trans x
2); 3.75(1.2H, m, CH 2 cis x 2); 3.84(0.6H, dd, J= 8.0,
5.3Hz, CH2CH cis); 4.06(0.6H, m, CH2ÇH cis);
4.15(0.4H, dd, J= 8.3, 6.5Hz, CH2CH trans); 4.36(0.4H,
m, CH2CH trans); 7.25-7.35(3H, m, Ar H m&p ); 7.43- 7.49(2H, m, Ar Ho)
N0 . 5 9 2-(2-Chlorophenyl)-2-methyl-4-hydroxymethyl-l,3-dioxoIane, EP425A 2-Chloroacetophenone (3g, 0.019mol), glycerol (1.96g, 0.021 mol) and toluene sulfonic acid monohydrate (0.18g, Immol) were heated to reflux in benzene/n-butanol (40ml/20ml) for six hours with a Dean-Stark water trap. The reaction mixture was then cooled, evaporated in vacuo and dissolved in chloroform (80ml). The organic
layer was then washed with brine solution (50ml x 3), dried (MgSÛ 4), filtered and evaporated in vacuo. Purification by flash chromatography (5:1, pet spirit/ EtOAc) yield a clear oil 59 (3.45g, 0.015, 82%) Mixture 7:3 cis: trans Rf: 0.1 (5:1, pet spirit / EtOAc) MS: APCI; MH 229(100% )
NMR: 500MHz, CDC13, ô ppm; 1.81(1H, s. Me trans); 1.85(2H, s. Me
cis); 3.40(0.3H, m, CH2 trans); 3.57(0.3H, m, CH 2 trans);
3.63(1.4H, m, CH2 cis); 3.81(1H, m, CH 2 cis & trans);
140 3.92(0.7H, dd, J= 8.1, 5.3 Hz, CH2CH cis); 4.11(0.7H, m,
CH2ÇH cis); 4.21 (0.3H, dd, J= 8.5, 6.2Hz, CH2CH trans);
4.21(0.3H, m, CH2ÇH trans); 7.26(2H, m, Ç 6H4CI H4&5);
7.39(1H, m, C 6H4CI H3); 7.67(1H, m, C 6H4CI Hé)
N0 .6 O 2-(2,4-DichIorophenyl)-2-methyl-4-hydroxymethyl-l,3-dioxolane, EP67A 2.4-Dichloroacetophenone (14.6g, 0.07mol), glycerol (7.84g, 0.085mol) and toluene sulfonic acid monohydrate (0.76g, 4.0mmol) were heated to reflux in benzene/n- butanol (40ml/20ml) with a Dean-Stark water trap for four hours. The reaction mixture was then cooled and evaporated in vacuo. Dichloromethane was added (100ml) and the organic solution was washed with brine (50ml x 3). It was dried
(MgS0 4 ), filtered and azeotroped several times with chloroform. Evaporation to dryness yielded a clear syrup 60 (16.6g, O.Obmol, 82%). MS: FAB: MH 263, (12%); (CgH6Cl20)H 189 (100%) NMR: 400MHz, CDCI3, 6 ppm; 1.69(1H, s. Me diox); 1.76(2H, s. Me
diox); 3.39(0.3H, dd, J=11.8, 5.9Hz, CH 2OH trans); 3.57-
3.71(1.3H, m, CH 2 cis & CH2 trans x 2); 3.75(1.4H, m, CH 2 cis
X 2); 3.86(0.7H, dd, J-8.1, 5.4Hz, CH 2CH cis); 4.02(0.7, m,
CH2ÇH cis); 4.17(0.3H, dd, J= 8.3, 6.5Hz, CH 2CH trans);
4.36(0.3H, m, CH2CH trans); 7.19(1H, m, dichlorophen H5);
7.36(1H, m, dichlorophen H3); 7.52(1H, m, dichlorophen H 6 )
N0 .6 I 2-(2,3,4-Trichlorophenyl)-2-methyl-4-hydroxyinethyl-l,3-dioxoIane, EP401A 2.3.4-Trichloroacetophenone (2.1g, 9.4mmol), glycerol (0.877g, 9.5mmol) and toluene sulfonic acid monohydrate (0.054g, 0.28mmol) were heated to reflux overnight in benzene/n-butanol (30ml/10ml) with a Dean-Stark water trap. The reaction mixture was evaporated in vacuo, diluted with dichloromethane (50ml) and washed with brine (50 x 3ml). This was dried filtered, evaporated in vacuo and purified by flash chromatography (5:1, pet spirit/EtOAc to give a yellow oil 61 (2.1g, 7 .Immol, 75%). Mixture: cis: trans, 3:2 Rf: 0.15 (3:1, pet spirits/EtOAc)
141 MS: FAB; MH 297 (50%); (C8H5Cl30)H 225 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 1.73(1H, s, Me trans); 1.78(2H, s, Me
cis); 3.40(0.4H, dd, J= 11.7, 5.6Hz, CH2OH); 3.56-3.64(1.2H,
m, CH2 cis X 2); 3.73-3.79(1.4H, m, CH 2 cis & CH2 trans x 2);
3.88(0.6H, dd, J= 8.2, 5.4Hz, CH 2CH); 4.05(0.6H, m, CH2ÇH
cis); 4.17(0.4H, dd, J=8.4, 6.4Hz, CH 2CH trans); 4.37(0.4H, m,
CH2CH trans); 7.34(1 H, d, J= 8.5Hz, H5 trichlorophen);
7.50(1 H, d, J= 8.5Hz, H 6 trichlorophen) Acc MS FAB 297.98108 (error 14.8ppm)
No.62 r-2-Methyl-2-phenyl-c-4-(toIuenesulphonyl)oxymethyl-l,3-dioxolane EP541A 2-Methyl-2-phenyl-4-hydroxymethyl-1,3-dioxolane 58 (8.0g, 0.04mol) was stirred in dry pyridine (10ml) at 0°C. A solution of tosyl chloride (9.4g, 0.048mol) in dry pyridine (10ml) was added over 30 minutes and the mixture stirred for a further 5 hours at 0°C. The reaction mixture was then diluted with water (150ml) and extracted with dichloromethane (150ml x 3). The organic layer was washed with water (2 x
50ml), dried (MgS0 4 ), filtered and evaporated in vacuo. The crude material was purified by column (pet spirit: ether, 4:1). Fractions containing mainly the cis isomer were combined and recrystalised from ethanol/toluene. Product which had crystalised in column test tubes was combined with this and the solid further recrystallised from ethanol/toluene to give white needles 62 (3.1g, 8 .8 mmol, 27%). The remaining mixture of isomers was a white solid (6.9g, 61%). Rf: 0.5 (9:2, pet spirit/ EtOAc) Melting point 86.5-87T (lit 85-87°C) MS: FAB; MH 349 m/z, 100% R.I.
NMR: 300MHz, CDCI3, ô ppm; 1.62(3H, s. Me dioxalan);
2.50(3H, s, Me tosyl); 3.76(1H, dd, J= 8 .6 , 6.8 Hz,
CH2CH); 3.85(1H, dd, J=8 .6 , 4.0Hz, CH2CH); 4.10(2H,
m, CH2OTS); 4.20(1H, m, CH2ÇH); 7.30-7.46(7H, m,
Ph & Ts); 7.85(2H, d, 8.3Hz, Ts)
142 No.63 r-2-Methyl-2-phenyl-t-4-(toluenesulphonyl)oxymethyl-l,3-dioxolane EP545 A mixture of cis and trans (6.9g, 0.019mol) from the isolation of the cis isomer 62 (see above) was purified by flash chromatography (4:1, pet/EtOAc). The fractions containing the trans isomer by TLC were combined and evaporated in vacuo to give a white solid 63 (0.956g, 2.7mmol, 8.4%). Rf: 0.47 (9:2, pet spirit/EtOAc) Melting point 67°C (lit ^^*65°C) MS: FAB; MH 349 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.57(3H, s, Me diox); 2.43(3H, s, Me
Ts); 3.50(1H, dd, J= 8.7, 6 .8 Hz, CH2CH); 3.74(1H, dd, J= 10.2, 6.1 Hz, CHzOTs); 3.90(1H, dd, J= 10.2, 6.1 Hz, CHzOTs);
4.15(1H, dd, J= 8.7, 6.3Hz, CH 2CH); 4.41(1H, m, CH2ÇH); 7.23-7.35(7H, m, Ph & Ts); 7.69(2H, d, J- 8.3Hz, Ts) 1-D NOEsy: Irradiation at dioxolane methyl showed NOE signals at (4.15
CH2CH) and (4.41, CH2ÇH)
No.64 2-(2-ChlorophenyI)-2-methyl-4-(toluenesulphonyl)oxymethyI-l,3- dioxolane EP429A Tosyl chloride (3.43g, 0.017mol) and 2-(2-chlorophenyl)-2-methyl-4-hydroxymethyl- 1,3-dioxalane 59 (3.45g, 0.015mol) were stirred in dry pyridine for 3 hours. The mixture was then taken up in dichloromethane (100ml). The organic layer was washed with dilute HCl solution (2 x 100ml) and water (1x100ml). It was then dried
(M gS0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (5:1 to 4:1, pet Spirit/EtOAc) yielded clear oil (4.9Ig, 0.012, 81%). Mixture 7:3 cis: trans Rf: 0.45 (4:1, pet spirits/EtOAc) MS: APCI; MH 383 (20%); MH - Ts 228.9 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.61(3H, s. Me diox); 2.34(3H, s. Me
tosyl); 3.45(0.3H, dd, J= 8.7, 6.9Hz, CH 2CH trans); 3.63(1H,
m, CH2CH cis & trans); 3.73(0.7H, dd, J= 8 .8 , 4.0Hz, CH2CH);
3.81(0.3H, dd, J=10.2, 5.8Hz, CH 2OTS); 3.99(1H, m, CH2 cis
& trans); 4.08(0.7H, m, CH 2ÇH); 4.32(0.3H, m, CH2ÇH); 7.04-
143 7.26(5H, m, Ts & ÇôHÆI); 7.40-7.45(1 H, m, Ç 6H4CI);
7.59(0.6H, d, J= 8.3Hz, C6H4CI H3&6 trans); 7.70(1.4H, d, J=
8.3Hz, C 6H4CI H3&6 cis)
No.65 2-(2,4-Dichlorophenyl)-2-metliyI-4-(toluenesulphonyI)oxymethyl-ly3- dioxolane EP117A 2-(2,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl-l ,3-dioxalane 60 (2.0g, 0.0Immol) and tosyl chloride (1.8g, 8.7mmol) were stirred in dry pyridine for 3 hours at 0°C then stirred at room temperature overnight. It was then diluted with water (80ml) and extracted with dichloromethane (80ml x 3). This was then washed with
brine (50ml x 3), dried (MgS 0 4 ) and evaporated in vacuo. Purification by fiash chromatography (4:1, pet spirit/EtOAc) gave a clear syrup 60 (1.97g, 4.6mmol, 60%). Rf: 0.7 (2:1, pet spirits/ EtOAc) both isomers visible MS: APCI; MH 417 (30%) 419(20%); MH - CgHéCliO (229 m/z, 100% R.I.)
NMR: 400MHz, CDCI3, Ô ppm; 1.67(3H, s. Me d iox); 2.43(3H, s, Me
tosyl); 3.53(0.4H, dd, J= 8 .8 , 6.9Hz, CH2CH trans); 3.68(0.6H,
dd, J= 8 .8 , 6 .8 Hz, CH2CH cis); 3.76(0.4H, dd, J=10.3, 5.6Hz,
CH2OTS trans); 3.83(0.6H, dd, J=8 .8 , 4.0Hz, CH2CH cis);
3.87(0.4H, dd, J= 10.3, 5.6Hz, CH 2OTS trans); 4.01 (0.6H, dd,
J=10.2, 5.2Hz, CH2OTS cis); 4.06(0.6H, dd, J=10.3, 5.2Hz,
CH2OTS cis); 4.17(1H, m, CH 2CH trans & CH2ÇH cis);
4.42(0.4H, m, CH2CH trans); 7.07(0.4H, dd, J= 8.4, 2.1Hz, dichloro H5 trans); 7.17(0.6H, dd, J= 8.4, 2.1Hz, dichloro H5
cis); 7.31(3H, m, Ts H 3 & 5 & dichlor H3); 7.43(0.4H, d, J=
8.5Hz, dichloro H 6 trans); 7.51(0.6H, d, J=8.5Hz, dichlor H 6 cis); 7.67(0.8H, d, 8.3Hz, Ts H2&6 trans); 7.77(1.2H, d, J=
8.3Hz, Ts H2 & H 6 cis)
N0 . 6 6 2-(2,3,4-Trichlorophenyl)-r-2-methyl-c-4-(toluenesuIphonyI)oxymethyl- 1,3-dioxolane, EP461A 2(2,3,4-Trichlorophenyl)-2-methyl-4-hydroxymethyl-l,3-dioxalane 61 (2.1g, 7mmol) and tosyl chloride (1.48g, 7.7mmol) were stirred in dry pyridine (9ml) at 0°C at for 3
144 hours then overnight warming to room temp. The reaction mixture was diluted with dichloromethane (50ml). The organic layer was washed with dil HCl solution (30ml),
brine (30ml) and water (30ml). This was then dried (MgS 0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (5:1, pet spirit/EtOAc) yielded a yellow oil (2.6g, 5.6mmol, 82% yield). This was recrystallised from ethanol to yield
the cis isomer (1.18g, 37%) as a white solid 6 6 . Melting point: 87-88.5°C Rf: 0.7(1:1, EtOAc/pet spirit) MS: FAB; MH 451 (10%)
NMR: 400MHz, CDCI3, Ô ppm; 1.68(3H, s. Me diox); 2.44(3H, s. Me
Ts); 3.69(1H, dd, J= 8 .8 , 6 .8 Hz, CH2CH); 3.85(1H, dd, J= 8 .8 ,
4.0Hz, CH2CH); 4.01(1H, dd, J= 10.2, 6.1Hz, CH2OTS);
4.08(1 H, dd, J= 10.2, 5.1Hz, ÇH2OTS); 4.15(1H, m, CH2ÇH);
7.33(3H, t, J= 8.4Hz, H 6 ArClg & H3, H5 Tosyl); 7.42(1H, d,
J= 8.5Hz, H5 ArCl]); 7.79(2H, d, J= 8.3Hz, H2 & H 6 tosyl)
No.67 2-(2,3,4-Trichlorophenyl)-r-2-methyl-t-4-(toluenesuIphonyl)oxymethyl-l,3- dioxolane, EP461B
Procedure as above for cis isomer 6 6 . After crystallizing the cis isomer the filtrate was evaporated in vacuo to yield a waxy yellow solid 67 (1.1 Og, 2.4mmol, 34%) 20% cis isomer by NMR MS: APCI: MH 451 (5 %); MH- CgHgClsO 229 (100%)
NMR: 400MHz, CDCI3, ô ppm; 1.67(3H, s. Me diox); 2.37(3H, s. Me
Ts); 3.51(1H, dd, J= 8.7, 7.0Hz, CH 2CH); 3.76(1H, dd, 10.3,
5.3Hz, CH2OTS); 3.84(1H, dd, J= 10.3, 5.3Hz, CH2OTS);
4.12(1H, dd, J= 8.7, 6.4Hz, ÇH 2CH); 4.37(1H, m, CH 2ÇH), 7.18(1H, d, J= 8.5Hz, ArCl] H5); 7.24(2H, d, J= 8.1Hz, Ts H3
& H5), 7.42(1H, d, J= 8.5, ArCl] H 6 ); 7.58(2H, d, J= 8.3Hz, Ts
H2 & H6) A cc MS 450.99399 (error 0.1 ppm)
N 0 .6 8 2,2-Dimethyl-4-(toluenesulfonyl)oxymethyl-l,3-dioxolane^^^, EP351A
145 2,2-Dimethyl-4-hydroxymethyl-l,3-dioxolane (8.45g, 0.061 mol) was added dropwise to a solution of tosyl chloride (13.34g, 0.068mol) in pyridine (16ml) stirring at 0°C. The mixture was stirred overnight while warming to room temperature and was then dissolved in dichloromethane (100ml). The organic layer was washed with brine
(100ml) and distilled water (100ml x 2). This was dried (MgS 0 4 ) filtered and evaporated in vacuo to yield a brown oil which solidified under vacuum. This was
recrystalised fi’om ethanol to yield white crystals 6 8 (7.62g, 0.026mol, 41%) Melting point: 40-43°C MS: APCI; MH 287 (40%); MH - acetone 229 (100%)
NMR: 300MHz, CDCI3, Ô ppm; 1.30(3H, s, Me diox); 1.31 (3H, s, Me
diox); 2.50(3H, s, Me tosyl); 3.77(1 H, dd, J=9.5 & 4.0Hz,
ÇH2CH); 3.95-4.10(3H, m, CH2CH & CH2OTS); 4.25(1 H, m,
CH2ÇH); 7.35(2H, d, J= 10.9Hz , Ts H3 & H5); 7.80(2H, d, J=
11.0 Hz, Ts H2 & H6 )
No.69 R-(+)-Dioxaspiro-2-(toluenesulphonyl)oxymethyl[4.5]decane^^®, EP615A R-(-f-)-Dioxaspiro-2-hydroxymethyl[4.5]decane (0.755g, 4.6mmol) and tosyl chloride (l.OOg, 5 .Immol) were stirred in dry pyridine (4ml) at 0°C to room temperature overnight. It was then diluted with brine solution (20ml) and extracted with dichloromethane (3 x 20ml). The organic layer was washed with distilled water (2 x 30ml), dried, filtered and evaporated in vacuo. Purification by flash chromatography (5:1, pet Spirit/EtOAc) yielded a white solid 69 (0.995g, 3.0mmol, 70%) Rf: 0.3 (5:1, pet spirit/EtOAc) Melting point: 48.5- 49°C (lit ^“48.5-49)
ao (CHCI3): +17.4°(0.5g/10ml, 20°C)
MS: FAB; MH 327 (65%); MH -OTs 154 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.40(2H, m, CH2 hexyl); 1.50(8H, m,
CH2 hexyl); 2.43(3H, s. Me tosyl); 3.73(1 H, dd, J= 8 .8 , 4.9Hz,
CH2CH dioxo); 3.93(1H, dd, J= 9.9, 6.3Hz, CH2OTS); 3.99(2H,
m, CH2 dioxo); 4.24(1 H, m, CH 2ÇH dioxo); 7.34(2H, d, J= 8.2Hz, Tosyl); 7.77(2H, d, J= 8.2Hz, Tosyl)
146 No,70 2-(2,4-Dichlorophenyl)-r-2-methyl-c-4-(3-iiitrophenyl)oxymethyl-l,3- dioxolane, EP1023B 2-(2,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl-l ,3-dioxolane 60 (890mg, 3.38mmoI), 2-nitrophenoI (705mg, 3.72mmol) and triphenylphosphine (1.32g, 3.8mmol) were stirred in dry THF (10ml) at 0°C. DEAD (0.790ml, 3.8mmol, 882mg) in THF (10ml) was added over 1 hour. The solution was then stirred for a further 24 hours while warming to room temperature. The reaction mixture was evaporated in vacuo and dissolved in dichloromethane. This was washed with 10% NaOH (30ml) and water (2 x 30ml). The organic layer was dried filtered and evaporated in vacuo.
Purification by flash chromatography yielded a white solid (856mg, 2.3mmol, 6 6 %). The cis isomer was reciystallised from dichloromethane/hexane 70 (390mg, 30%). Melting point: 134-136°C MS: APCI: MH 384 (5%); (CgH6Cl20)H 189 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.79(3H, s. Me); 3.85(1H, dd, J=8.5,
7.0Hz, CH 2CH); 4.02(1 H, dd, J=8.5, 4.5Hz, CHzCH); 4.09(1H, dd, J= 9.5, 5.7Hz, CHzOPh); 4.17(1H, dd, J= 9.5, 5.1Hz,
CHzOPh); 4.34(1H, m, CH2ÇH); 7.22(1H, dd, J= 8.4, 2.1Hz,
dichlorophen H5); 7.26(1 H, m, C 6H4NO2 H6 ); 7.40(1 H, d, J=
2.1Hz, dichlorophen H3); 7.43(1 H, t, J= 8.2Hz, C 6H4NO2);
7.57(1H, d, J= 8.4Hz, dichlorophen H 6 ); 7.76(1H, t, J= 2.4,
C6H4NO2 HI); 7.82(1H, m, Ç 6H4NO2 H5)
No.70a 2-(2,4-DichlorophenyI)-r-2-methyI-t-4-(3-nitrophenyl)oxymethyl-l,3- dioxolane, EP1023D 2-(2,4-Dichlorophenyl)-2-methyl-4-hydroxym ethyl-1,3-dioxolane 60 (890mg, 3.4mmol), 2-nitrophenol (705mg, 3.7mmol) and triphenylphosphine (1.32g, 4.0mmol) were stirred in dry THF (10ml) at 0°C. DEAD (0.790ml, 882mg, 4.0mmol) in THF (10ml) was added over 1 hour. The solution was then stirred for a further 24 hours warming to room temperature. It was then evaporated in vacuo and dissolved in dichloromethane. This was washed with 10% NaOH (30ml) and water (2 x 30ml). This was dried filtered and evaporated in vacuo. Purification by flash chromatography yielded a white solid (856mg, 2.3mmol, 6 6 %). The cis isomer was recrystallised from dichloromethane/hexane (390mg, 0.1 mmol, 30%). The supernatant liquid was
147 evaporated and recrystallised from methanol. The solid was filtered and the filtrate
evaporated in vacuo to yield the trans isomer 70a (99mg, 8 %). Melting point: 115-120T
MS: APCI: MH 384 ( 6 %); (CgH6Cl20)H 189 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.76(3H, s, Me); 3.76(1H, dd, J=8.5,
7.5Hz, CH 2CH); 3.90(1H, dd, J= 9.7, 5.6Hz, CHzOPh); 3.99(1H, dd, J=9.8, 5.3Hz, CHzOPh); 4.33(1H, dd, 8.5, 6.5Hz,
CH2CH); 4.61(1H, m, CH2ÇH); 7.02-7.05(lH, m, PhN 0 2 ); 7.21(1H, dd, J= 8.4, 2.1Hz, dichlorophen H5); 7.34(1H, d,
J=2.1Hz, dichlorophen H3); 7.37(1 H, t, J= 8.2Hz, C 6H4NO2
H5), 7.55(1H, t, J= 2.1Hz, Ç6H4NO2 HI); 7.59(1H, d, J= 8.4Hz,
dichlorophen H 6); 7.79-7.81 (IH, m, C 6H4NO2 H6 )
No.71 2-(2,4-Dichlorophenyl)-2-methyl-4-(2-nitrophenyl)oxymethyl-l,3- dioxolane, EP1033A 2-(2,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl-l ,3-dioxalane 60 (890mg, 3.4mmol), 2-nitrophenol (705mg, 3.7mmol) and triphenylphosphine (1.32g, 4.0mmol) were stirred in dry THF (10ml) at OoC. DEAD (0.790ml, 882mg, 4.0mmol) in THF (10ml) was added over 1 hour. The solution was then stirred for a further 24 hours warming to room temperature. It was then diluted with ether (40ml) and washed with 10% NaOH (50ml) and water (2 x 50ml). It was then dried, filtered and evaporated in vacuo. Purification by flash chromatography (3:1, pet spirit/ EtOAc) gave a white solid 71 (784mg, 2.0mmol, 40%). Rf: 0.6 (3:1, pet spirit/ EtOAc) Melting point: 82-85°C MS: APCI; MH 386 (38%); MH - CgHôCbO 196 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.74(1.2H, s, CH 3 trans); 1.77(1.8H,
s, CH3 cis); 3.83-3.88(1.6H, m, 2 CH2 cis + ICH2 trans); 4.06-
4.15(1.4H, m, ICH2 cis + 2 CH2 trans); 4.26(0.6H, dd, J= 9.3,
4.6Hz, CH2 0 Ph cis); 4.31(1H, m, CH2ÇH cis + CH2 trans);
4.62(0.4, m, CH2ÇH trans); 7.03-7.12(2H, m, C 6H4NO2); 7.12(0.4H, dd, J=8.4, 2.1Hz, dichlorophen H5 trans); 7.22(0.6H, dd, J= 8.4, 2.1Hz, dichlorophen H5 cis); 7.32(0.4H,
148 d, J= 2.1Hz, dichlorophen H3); 7.38(0.6H, d, J= 2.1Hz,
dichlorophen H3 cis); 7.45-7.54(lH, m, C 6H4NO 2); 7.56(1 H, d,
J= 8.4Hz, dichlorophen H 6 ); 7.76-7.84(lH, m, C 6H4NO2) Acc Mass; FAB; MH = 384.04029 (error = 0.6ppm)
No.72 2-(3,4-Dichlorophenyl)-2-methyl-4-hydroxymethyl-1,3-dioxolane, EP941A 3,4-Dichloroacetophenone (18.9g, O.lmol), glycerol (11.0g, 0.14mol) and toluene sulphonic acid monohydrate (0.57g, 3.0mmol) were heated to reflux in benzene/butanol (40ml/20ml) with a Dean-Stark water trap for 10 hours. The mixture was then cooled, evaporated and diluted with dichloromethane (80ml) and washed
with water (80ml x 3). This was dried (Na 2S0 4 ) filtered and evaporated in vacuo. It was then purified by flash chromatography (3:1, pet spirit/EtOAc) to give a clear oil 72 (20.7g, 0.078mol, 78%) Mixture: 3:1, cis:trans Rf: 0.05 (3:1, pet spirit / EtOAc)
MS: APCI; MH 263 (8 %); (CgH6Cl20)H 189 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.58(0.75H, s. Me trans); 1.61(2.25H,
s. Me cis); 3.38(0.25H, dd, J= 11.7, 5.6Hz, CH 2CH trans);
3.60(1.25H, m, CH2 cis & CH2 trans x 2); 3.75(1.5H, m, CH2
cis X 2); 3.85(0.75H, dd, J=8.2, 5.4Hz, CH 2CH cis);
4.05(0.75H, m, CH 2CH); 4.15(0.25H, dd, J=8.4,6.4Hz, CH2CH
trans); 4.35(0.25H, m, CH2ÇH); 7.29(1 H, td, J= 8.3, 2.0, dichlorophen H5 cis & trans); 7.39(1 H, d, J=2.0Hz,
dichlorophen H 6 ); 7.54(0.75, d, J=2.0Hz, dichlorophen, HI cis); 7.57(0.25H, d, J=2.0Hz, dichlorophen HI trans)
Acc mass: FAB; 263.02105 (error = 11. 8 ppm)
N0 . 7 3 2-(4-ChIorophenyl)-2-methyl-4-hydroxymethyI-l,3-dioxolane EP929A 4-Chloroacetophenone (15.4g, O.lmol), glycerol (1 Ig, 11 mol) and toluene sulphonic acid monohydrate (0.570g, 3.0mmol) were heated to reflux in benzene/n-butanol (40ml/20ml) with a Dean-Stark water trap. The reaction mixture was then cooled, evaporated in vacuo and taken up in dichloromethane (150ml). The organic layer was washed with water (100ml x 3), dried (Na 2S0 4 ), filtered and evaporated in vacuo. The
149 crude material was then purified by flash chromatography (3:1, pet/EtOAc) to yield a clear oil 73 (13.1g, 0.057mol, 57%) Mixture 4:1 cis:trans Rf: 0.15 (3:1, pet spirit/EtOAc) MS: APCI; MH 229 (10%); (CgH7C10)H 155 (100%)
NMR: 400MHz, CDCI3, Ô ppm; 1.53(0.6H, s. Me trans); 1.56(2.4H, s.
Me trans); 3.34(0.2H, dd, J=11.7, 5.6Hz, CH2OH trans);
3.56(1.2H, m, CH2OH cis & CH2 trans); 3.69(1.6H, m, CH2CH
cis & CH2OH cis); 3.79(0.8H, m, J= 8.1, 2.3Hz, CH2CH cis);
3.97(0.8H, m, CH 2ÇH cis); 4.09(0.2H, dd, J= 8.3, 6.5Hz,
CH2CH trans); 4.29(0.2H, m, CH2CH trans); 7.23(2H, d, J=
8.5Hz, ArCl H3 & H5); 7.33(2H, d, J= 8.7Hz, ArCl H2 & H 6 )
N0 . 7 4 2-(4-Chlorophenyl)-r-2-methyl-c-4-(4--benzylidenaminophenyl)oxymethyl- 1,3'dioxolane, EP945A 2-(4-Chlorophenyl)-2-methyl-4-(hydroxymethyl)-l ,3-dioxalane 73 (560mg, 2.45mmol) was stirred in dry benzene at 0°C. Tributylphosphine (1.2ml, 3.7mmol) was then added followed by 4-(benzylidenamino)-phenol 57 (926mg, 3.7mmol). This solution was stirred for 10 minutes at which point ADD? (1.2g, 4.0mmol) was added. It was stirred for a further twelve hours warming to room température. At this point a
solid cake had formed which was broken up and stirred with dichloromethane ( 2 0 ml). The mixture was then filtered. The filter cake was washed through with dichloromethane (30ml). The organic layers were then washed with water (50ml x 3),
dried (Na 2S0 4 ) filtered and evaporated in vacuo. The crude product was purified by flash chromatography (3:1, pet spirit/ EtOAc) and recrystalised from methanol to give the cis isomer 74 (114mg, 0.27mmol, 11%) Melting point: 125.5-127°C Rf: 0.8 (1:1, pet spirit/EtOAc) MS: APCI; MH 408 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 1.66(3H, s. Me); 3.84(1H, dd, J= 8.3,
7.0Hz, ÇH 2CH); 3.97(1H, dd, 8.4, 4.6Hz, CH 2CH); 4.00(1 H, dd, J= 9.6, 6.1Hz, CHzOPh); 4.14(1H, dd, J= 9.5, 5.3Hz,
CH2 0 Ph); 4.31(1H, m, CH2ÇH); 6.96(2H, d, J= 8.9Hz,
150 OCôEUN); 7.27-7.32(4H, m, OÇ 6H4N & Ç6H4CI); 7.41-
7.47(5H, m, 3 NCHPh & 2 Ç 6H4CI); 7.93(2H, bs, NCHPh ortho); 8.46(1 H, s, NCHPh) Acc mass: FAB; 408.13596 (error 1.6ppm)
N0 . 7 5 2-(2,5-Dichlorophenyl)-2-methyl-4-hydroxymethyH,3-dioxolane, EP933A 2,5-Dichloroacetophenone (10g, 0.053mol), glycerol (5.8g, 0.06mol) and toluene sulphonic acid monohydrate (0.3g, 1.53mmol) were heated to reflux in benzene/n- butanol (20ml/10ml) with Dean-Stark apparatus for four hours. The mixture was cooled, evaporated in vacuo and the residue dissolved in dichloromethane (80ml). The
dichloromethane solution was then washed with water (80ml x 3), dried (MgS 0 4 ) and evaporated to give a yellow oil. This was evaporated several times with chloroform and dried under vacuum to yield a yellow syrup 75 (11.Ig, 0.042mol, 80%) Mixture: 70:30 cis: trans. MS: APCI; MH 263 (5%); (CgH6Cl20)H 189 (100%)
NMR: 400MHz, CDCI3, ô ppm; 1.72(0.9H, s. Me diox trans); 1.76(2.1H, s. Me diox cis); 3.41(0.3H, dd, J= 11.8, 5.8Hz,
CH2OH trans); 3.54-3.63(1.IH, m, CH2 cis & trans); 3.75-
3.78(1.2H, m, CH 2 cis & trans); 3.87(0.7H, dd, J= 8.2, 5.4Hz,
CH2CH cis); 4.08(0.7H, m, CH 2ÇH cis); 4.16(0.3H, dd, J= 8.4,
6.5Hz, CH2CH trans); 4.38(0.3H, m, CH 2ÇH trans); 7.18(1H, dt, J= 8.5, 2.5Hz, dichlorophen H4 trans & cis); 7.29(1 H, dd, J= 8.5Hz, dichlorophen H3 cis & trans); 7.59(0.7H, d, J= 2.5Hz, dichlorophen H6 cis); 7.61(0.3H, d, J= 2.5Hz, dichlorophen H6 trans). Acc mass: FAB; 263.02392 (error Ippm)
No.76 2-(2,5-Dichlorophenyl)-2-methyI-4-(4-benzylideiiiininophenyl)oxymethyl- 1,3-dioxalane, EP997A 2-(2,5-Dichlorophenyl)-2-methyl-4-hydroxymethyl-l ,3-dioxolane 75 (l.OOg, 3.8mmol) was stirred in dry toluene (20ml) at room temperature. Tributylphosphine (1.15g, 1.40ml, 5.7mmol), 4-benzyliminophenol 57 (1.07g, 5.4mmol) and ADDP (1.43g, 5.7mmol) were added in this order. The mixture was stirred for 12 hours at
151 which point further toluene (15ml) was added to maintain the stirring. The suspension was then stirred for a further 2 days at room temperature. The mixture was then diluted with ether (40ml), filtered and washed through with ether (100ml). This was washed with 10% NaOH (80ml), brine (80ml) and water (80ml). It was then dried
(Na 2S0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography followed by recrystalisation from methanol yielded a white solid 76 (350mg,
0 .8 mmol, 2 1 %) Melting point: 77-79°C Rf: 0.7 (1:1, pet Spirit/EtOAc) MS: APCI; MH 442 (100%)
NMR: 500MHz, CDCI3, 5 ppm; 1.78(0.9H, s. Me trans); 1.81(2.1H, s.
Me cis); 3.78(0.3H, dd, J= 8.5, 7.3Hz, CH 2CH trans);
3.82(0.3H, dd, J= 9.7, 5.9Hz, CH2 0 Ph); 3.88(0.7H, dd, J= 8.5,
6.9Hz, CH2CH cis); 3.98-4.05(1.7H, m, CH 2 2 cis & 1 trans);
4.14(0.7H, dd, J= 9.5, 5.0Hz, CH2pPh); 4.31(0.3H, dd, J= 8 .6 ,
6.4Hz, CH2CH trans); 4.37(0.7H, m, CH 2CH cis); 4.61 (0.3H,
m, CH2ÇH cis); 6.78(0.6H, d, J= 8.9Hz, OC 6H4N 2H ortho
trans); 6.95(1.4H, d, J= 8.9Hz, OC6H4N 2H ortho cis); 7.14- 7.21(3H, m, OPhN meta 2H & dichlorophen IH); 7.27- 7.31(1 H, m, dichlorophen H3); 7.45(3H, m, NCHPh);
7.65(0.7H, d, J= 2.6Hz, dichlorophen H 6 cis); 7.69(0.3H, d, J=
2.6Hz, dichlorophen H 6 trans); 7.90(2H, m, NCHPh ortho); 8.43(0.3H, s, NCHPh trans); 8.46(0.7H, s, NCHPh cis) Acc mass: FAB; 442.09716 (error Ippm)
N 0 .7 7 2-(2,5-Dichlorophenyl)-r-2-methyl-t-4-(4-aminophenyl)oxymethyl-l,3- dioxolane, EP1019B Hydrazine hydrate (0.026ml) was added to a suspension of imine 76 (220mg, 0.5mmol) in ethanol at 0°C. This was stirred in ethanol at 0°C for 1 hour then evaporated in vacuo. Purification by flash chromatography (1:1, pet spirit/EtOAc) yielded a yellow oil (117mg, 0.33mmol, 6 6 %). Recrystalisation from methanol yielded the trans isomer 77 (48mg, 0.14mmol, 27%). Rf: 0.25 (1:1, pet spirit/EtOAc)
152 Melting point: 103°C MS: APCI; MH 354 & 356 (100% & 60%)
NMR: 500MHz, CDCI3, Ô ppm; 1.76(3H, s, Me); 3.70(2H, m, CH 2);
3.91(1H, dd, J= 9.7, 5.2Hz, CHzOPh); 4.27(1H, dd, J= 8 .6 ,
6.4Hz, CH2CH); 4.56(1 H, m, CH2ÇH); 6.48(4H, m, OÇ 6H4N); 7.17(1H, dd, J= 10.5, 3.2Hz, dichlorophen H4); 7.27(1H, d, J= 10.5Hz, dichlorophen H3); 7.67(1 H, d, J= 3.2Hz, dichlorophen
H6 ) HPLC: (30:70 A/B) Rt= 5.8min (100%) Microanalysis C17H18CINO3 calculated C: 57.78 H: 4.85 N: 3.97 found C: 57.36 H: 4.72 N: 3.82
No.78 (i?7-(-)--l-(2,4-Dichlorobenzyloxy)-2-hydroxy-3-(toluenesulphonyloxy)- propane, EP873A (T^-(-)-Glycidyl tosyate (Ig, 4.1 mmol), 2,4-dichlorobenzylalcohol (1.55g, 8.1 mmol), 3 A molecular sieves (lOOmg) and boron trifluoride etherate (0.03Ig, 0.035ml,
0.25mmol) were stirred in dry dichloromethane (10ml) for 6 hours at 0°C. This was
followed by stirring at room temperature for a further 8 hours. The solution was then filtered through cellite and diluted with further dichloromethane (80ml). This was washed with 15% Na 2C0 3 (80ml), brine (80ml) and water (80ml). It was then dried
(Na 2S0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (3:1, pet spirit/ EtOAc) yielded a clear oil 78 (0.957g, 2.4mmol, 54%). Rf: 0.6 (1:1, pet spirit/EtOAc)
[a]D (CHCI3): -31.4° (O.lg/lOml, 16°C) MS: APCI; MH 405 (10%); dichlorobenzyl cation 159 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 2.45(3H, s. Me); 3.56(2H, dd, J= 4.9,
2.5Hz, CH2 0 bn); 4.02(2H, m, ÇHOH); 4.06 - 4.16(2H, m,
CH2OTS); 4.52(2H, s, CH2Ph); 7.21(1H, dd, J = 8.2, 2.0Hz, H5
dichlorophen); 7.30(1 H, d, J = 8.2Hz, H 6 -dichloro); 7.32(2H, d, J= 7.8 Hz, OTs); 7.35(1H, d, J= 2.2Hz, H3 dichloro); 7.77(2H, d, J= 8.3Hz, OTs)
153 No.79 (5^-(+)-l-(2,4-Dichlorobenzyloxy)-2-hydroxy-3-(toluenesulphonyloxy)- propane, E P llllA fiS)-(+)-Glycidyl tosyate (Ig, 4.1 mmol), 2,4-dichlorobenzyl alcohol (1.55g, 8.1 mmol), 3 A molecular sieves (lOOmg) and boron tri floride etherate (0.03 Ig, 0.035ml,
0.25mmol) were stirred in dry dichloromethane (30ml) for 6 hours at 0°C. The solution was then stirred at room temperature overnight. It was then filtered through cellite and diluted with further dichloromethane (80ml). This was washed with 15%
Na2C0 3 (80ml), brine (80ml) and water (80ml). It was then dried (Na 2S0 4 ), filtered and evaporated in vacuo. Purification by flash chromatography (3:1, pet spirit/ EtOAc) gave a clear oil 79 (1.114g, 2.75mmol, 62%). Rf: 0.6 (1:1, pet spirits/EtOAc)
[a]o (CHCI 3) = +24.0° (0.19g/10ml, I6 0 C)
MS: APCI; MH 405 (10%); dichlorobenzyl cation 159 (100%)
NMR: 500MHz, CDCI3, ô ppm; 2.45(3H, s. Me); 3.56(2H, dd, J= 4.9,
2.5Hz, CH2pbn); 4.02(2H, m, CHOH); 4.06 - 4.16(2H, m,
ÇH2OTS); 4.52(2H, s, CH2Ph); 7.21(1H, dd, J = 8.2, 2.0Hz, H5
dichlorophen); 7.30(1 H, d, J = 8.2Hz, H 6 -dichloro); 7.32(2H, d, J= 7.8 Hz, OTs); 7.35(1H, d, J= 2.2Hz, H3 dichloro); 7.77(2H, d, J= 8.3Hz, OTs)
N0 .8 O 2?-(-)-l(2,4-Dichlorobenzloxy)-propane-2,3-epoxide, EP905A (7^-Tosylate 78 (504mg, 1.2mmol) and potassium carbonate (345mg, 3.6mmol) were stirred in methanol (15ml) for 3 hours at 0°C. The solution was then filtered through silica and washed through with ether (50ml). This was washed with sodium carbonate solution (50ml) and water (2 x 50ml). The dichloromethane solution was then dried
(Na2S p 4), filtered and evaporated in vacuo to yield a clear liquid 80 (207mg, 0.9mmol, 72%) Rf: 0.8 (1:1, pet spirit/ EtOAc)
[a]o (CHCI 3): -107.5° (O.lg/lOml, 16°C) MS: FAB; MH 233 (5%); dichlorobenzyl cation 159 (100%)
NMR: 500MHz, CDCI3, Ô ppm; 2.63(1H, dd, J= 5.0, 2.7Hz,
CH2 0 Bn); 2.80(1H, dd, J= 5.0, 4.2, CHzOBn); 3.19(1H, m,
CH); 3.48(1H, dd, J= 11.5, 5.9Hz, ÇH2 epox); 3.85(1H, dd, J=
154 11.5, 2.4Hz, CH2 epox); 7.24(1H, dd, J= 8.3, 2.1Hz, H5 dichlorophen); 7.35(1 H, d, J= 2.1Hz, H3 dichlorophen);
7.42(1 H, d, J= 8.3Hz, H 6 dichlorophen)
No.81 (*S)-(+)-l(2,4-Dichlorobenzyloxy)-propane-2,3-epoxide, EP1115A (^-Tosylate 79 (931 mg, 2.3mmol) and potassium carbonate (345mg) were stirred in dry methanol (15ml) for 2 hours at 0°C. The solution was then evaporated in vacuo and purified by flash chromatography (1:1, pet spirit/ EtOAc) to yield a clear liquid 81 (420mg, 1.8mmol, 72%)
[a]D (CHCI3): +104.4° (O.lg/lOml, 16°C) MS: FAB; MH 233 (5%); dichlorobenzyl cation 159 (100%)
NMR: 500MHz, CDCI3, 6 ppm; 2.63(1H, dd, J= 5.0, 2.7Hz,
CHzOBn); 2.80(1H, dd, J= 5.0, 4.2, CHzOBn); 3.19(1H, m,
CHOH); 3.48(1H, dd, J= 11.5, 5.9Hz, ÇH2 epox); 3.85(1H, dd,
J= 11.5, 2.4Hz, CH2 epox); 7.24(1H, dd, J= 8.3, 2.1Hz, H5 dichlorophen); 7.35(1H, d, J= 2.1Hz, H3 dichlorophen);
7.42(1 H, d, J= 8.3Hz, H 6 dichlorophen)
No.82 l-(2,4-dichlorophenyl)-3-(4-nitrophenyI)-urea, EP1099A 2,4-Dichlorophenylisocyanate (500mg, 2.7mmol) and 4-nitroaniline (367mg, 2.7mmol) were heated to reflux in dry toluene overnight. The reaction mixture was then cooled to room temperature, diluted with dichloromethane ( 2 0 ml) and filtered to yield a yellow solid 51 (675mg, 2.1 mmol, 78%). Melting point: 290-29 r c MS: El; M + 325 (10%)
NMR: 400MHz, DMSO, Ô ppm; 7.40(1H, dd, J= 8.9, 2.4Hz,
dichlorophen H5); 7.65(1 H, d, J= 2.4Hz, dichlorophen H3); 7.68(2H, d, J= 9.0Hz, nitrophen H2 & H5); 8.15(1H, d, J=
8.9Hz, dichlorophen H 6 ); 8.20(2H, d, J= 9.0Hz, nitrophen H3
& H5); 8.58(1H, s, NHC 6H4CI2); 10.00(1H, s, NHC6H4NQ2)
Microanalysis C 13H9CI2N 3O 3 calculated C: 47.88 H: 2.78 N: 12.88 found C: 47.85 H: 2.62 N: 12.83
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188 Apendix. 1 list of compounds with UCL numbers.
Number UCL-number Number UCL-number 1 Ketoconazole 29 UCL-2331 2 UCL-2297 30 UCL-2351 3 UCL-2322 31 UCL-2321 4 UCL-2242 32 UCL-2270 5 UCL-2202 33 UCL-2303 6 UCL-2135 34 UCL-2307 7 UCL-2158 35 UCL-2315 8 UCL-2112 36 UCL-2306 9 UCL-2134 37 UCL-2316 10 UCL-2250 38 UCL-2326 11 UCL-2281 39 UCL-2298 12 UCL-2203 40 UCL-2319 13 UCL-2220 41 UCL-2350 14 UCL-2238 42 UCL-2334 15 UCL-2245 43 UCL-2360 16 UCL-2255 44 UCL-2347 17 UCL-2264 45 UCL-2355 18 UCL-2254 46 UCL-2352 19 UCL-2253 47 UCL-2286 20 UCL-2280 48 UCL-2285 21 UCL-2327 49 UCL-2304 22 UCL-2273 23 UCL-2335 24 UCL-2333 27 UCL-2292 28 UCL-2303
189 Appendix II
3-D representation UCL-2273 in folded and unfolded conformations
190