29817882.Pdf

STRUCTURE - ACTIVITY RELATIONSHIPS

IN SOME HERBICIDAL FLUORENE DERIVATIVES

MICHAEL WILLIAM HARTNELL B.Sc, C. Chem. ,M.R. I. C.

Ph.D,._T}ffiSIS^

This thesis is submitted in partial fulfilment of the imjj i requirements for j^the degree of/Doctor of .^Philosophy of the Council for Na'tioharAcademic Awards...

Sponsored by:' In collaboration with:

The School of Environmental Chesterford Park Research Sciences Station Plymouth Polytechnic Fisons Limited Drake Circus Agrochemicals Division PLYMOUTH Nr.SAFFRON WALDEN Devon PL4 8AA Essex CBIO IXL

January 1979 PLYMOUTH POLYtECHHIC LEARHiHB BESOURCES CEHTRE

ACCIi. No.

CLASS No. CONTENTS

1. INTRODUCTION 6 1.1 Morphactins 6 1.2 Effects of Morphactins 7 1.3 Chemistry of Morphactins 9 1.4 Objects of Research 11

2. THEORETICAL 13

2.1 Fluorene 13 2.2 Synthetic Methods 13 2.3 Structure 19 2.4 Properties 20

2.4.1 Oxidation 21 2.4.2 Hydrogenation 23 2.4.3 Electrophilic Substitution in the Benzenoid Rings 23 2.4.4 Substitution at the 9-Position 26

DISCUSSION 33 3.1 Chemistry of 9,9-Bishydroxymethylfluorene 33 3.2 Hydroxyl Modifications 37 3.2.1 Esters 37 3.2.2 Chloromethyl Derivatives 37 3.2.3 Miscellaneous Reactions 39 3.3 Ring Substituted Derivatives of 9,9-Bishydroxymethylfluorene 40 3.3.1 2-Nitro-9,9-bishydroxymethylfluorene 40 3.3.2 Halogen Substituents 42 3.3.3 Acetyl Substituents 42 3.3.4 4-Substituted Derivatives 43 3.4 Methyl 2-Chloro-9-hydroxymethylfluorene- 9-carboxylate 43 3.5 Biological Screening of 9,9-Bishydroxy- methylfluorene Derivatives 43 XH20H

(21) Rl = R2 = R3 = H

Similar hydroxymethylations are theoretically possible using a variety of substituted fluorenes. Consequently, although the level of

(HCHO) ft- DMSO

(11) (21)

Scheme 6 activity of 9,9 - bishydroxymethylfluorene(21) was in all cases lower than that of Chlorflurecol Methyl(9) it v;as considered worthwhile to explore the structure-activity relationships in a series of hydroxymethylated fluorenes with the object of producing a novel compound possessing enhanced morphactin-like activity-

The objects of the work described in this thesis can therefore be stated as:

1) The synthesis of a series of derivatives of 9,9 - bishydroxy methylfluorene(21) containing ring substituents and/or modified hydroxyl groups.

11) A study of the herbicidal activities of such compounds in relation to their structure.

12 PLYMOUTH POLYTECHNIC

"STRUCTURE - ACTIVITY RELATIONSHIPS

IN SOf^E HERBICIDAL FLUORENE DERIVATIVES"

MICHAEL WILLIAM HARTNELL> B.Sc, C.Chem., M.R.I.C

ABSTRACT OF THESIS

Synthetic plant-growth regulators derived from fluorene-9-carbo)0'lic acid ('morphactins') are introduced and the literature on them is reviewed. Similar herbicidal activity has been found with 9,9-bishydroxymethylfluorene, a compound which can be obtained directly from the hydrocarbon fluorene. There follows a detailed review of the chemistry of fluorene and 9,9- bishydro)^methylfluorene.

9-Substituted and ring-substituted derivatives of this diol have been synthesised and their chemistry and biological activity are discussed. The hydro)^methylation of substituted fluorenes is possible on a small scale in suitable solvents which do not stabilise the corresponding radical anions. This radical anion formation, when it occurs, seriously reduces the yield of hydroxymethylated product. Acetonitrile as solvent and aqueous benzyltrimethyl ammonium hydroxide as base were found to be a particularly useful system for these reactions.

Methyl 2-chloro-9-hydro)0'methylfluorene-9-carboxylate was found to be the most active compound synthesised. It was concluded that a 9-carbo>:*'lic acid group is required for high activity. None of the compounds examined were found to be more active than 'chlorflurecoT, the commercial product.

! /i 1 i CONCLUSION 47

EXPERIMENTAL A9

5.1 General Procedures 49

5.2 Hydroxyl Modifications 49

5.3 Ring Substituted Derivatives 61

5.4 Methyl 2-chloro-9-hydroxymethylfluorene- 9-carboxylate 68

REFERENCES 70

APPENDICES 75

1. Biological Screening Results 75

2. Herbicide Screening Methods 78 ACKNOWLEDGetENTS

I wish to record my thanks to ray supervisors, Dr. R.W. Hanson and Dr. G.T. Newbold for their help throughout this project. Thanks are also due to Dr. R. Adams and Dr. Wendy Vincent for help v;ith the e.s.r. investigations. Finally,! would like to thank the tyuists, particularly Mrs. I.K. East, for their efforts. ABSTRACT . ^. , _

Synthetic plant-growth regulators derived from fluorene-9- cerboxyli'c acid ('morphfictins') are introduced and the literature on

them is reviewed. Similar herbicidal activity has been found with

9,9-bishydroxymcthii1 fluorene, a compound which can be obtained directly

from the hydrocarbon fluorene. There follows a detailed review of

the chemistry of fluorene and 9,9-bishydroxyraethylfluorene.

9-Substituted and ring-substituted derivatives of this diol

have been synthesiscd-and their chemistry and biological activity are

discussed. The hydroxymethylation of substituted fluorenes is possible

on a srr.all scale in suitable solvents which do not stabilise tho

corresponding radical anions. This radical anion formation,when it

occurs,seriously reduces the yield of hydroxymethylated product.

Acetonitrile as solvent and aqueous benzyltriraethylammonium hydroxide

as base were found to be a particularly useful system for these

reactions.

Methyl 2-chloro-9-hydroxymethylfluorene-9-carboxyiate

was found to be the most active compound synthesised. It was concluc'cd

that a 9-carboxylic acid group is required for high activity. None of

the compounds examined v^re found to be more active than 'chlorflurecol',

the commercial product. 1. INTRODUCTION

1.1 MORPHACTINS

The different factors contributing to the growth and development of plants are controlled by a group of naturally occurring chemicals collectively called the phytohormones Some or all of these hormones act together in regulating the various growth processes. The natural growth control mechanisms can be interfered with chemically by several different types of synthetic growth regulator, one class of which is derived from fluorene - 9 • 2 carboxylic acid(l) .

(1) = R^ = H, R^ = CO^H = R2 = R3 = H •^1 (11) (2) = CI, R^ = OH.R^ = CO^H (14) = H, R^ = OH.R^ = CO^H ^1 h (6) = R2 = H, R^ = CN (15) = H,R2= CI, R3 = CO^Cl ^1

(7) = H, R^ = OHjR^ = CN (18) = H,R2/R3 = 0 \ ^1 (8) = R^ = H.R^ = ONO^ (19) = R2 = H.R^ = CONH^

(9) = CljR^ = OHjR^ = CO^Hc (20) = R^ = H,R3 = CI

(10) = HjR^ = OHjR^ = CO^Bu"

In 1953 Wain reported that fluorene - 9 - carboxylic acid(l) was active in the tomato leaf epinasty test (a downward curvature of the leaf petiole) and Went's pea test (the curvature of pieces of pea stem slit longitudinally). A year later,Jones, Metcalfe and Sexton showed that this compound inhibited the geotropism of various seedlings (downward curvature of the roots under the influence of gravity). Furthermore they discovered that certain derivatives of this acid, particularly 2 - chloro - 9 - hydroxyfluorene - 9 - carboxylic acid(2), were effective at very much lower concentrations. Similar effects were shown by N - 1 - naphthylphthalamic acid

(Naptalam, 3) and 2, 3, 5 - tri-iodobenzoic acid(TIBA, 4) Their hypothesis was that since growth was not inhibited all of CH2C02^ NHCO

CO2H (5) these antigeotropic substances competed with the phytohormone auxin or indole - 3 - acetic acid (lAA, 5) in its lateral transport mechanism, a process which occurs in plants influenced by gravity. Whilst these compounds have been shown to interfere with the internal movement of auxin, it seems unlikely, because of their structural differences, that

they would compete for any hypothetical indoleacetic acid transporting

site.

It was subsequently shown, in 1960, by workers at the Merck 2 6 7 Biological Research Laboratories, ' ' ' that derivatives of fluorene - 9- carboxylic acid{l) designated as raorphactins, exerted a pov/erful effect

upon the growth and development of higher plants. Further research led to the discovery of other related active compounds, particularly 9 -

cyanofluorene, 9 - cyano - 9 - hydroxyfluorene and 9 - fluorenyl nitrate

(6,7,8). Two morphactins, methyl 2 - chloro - 9 - hydroxyfluorene - 9 - carboxylateCChlorflurecol Methyl, 9) and n - butyl 9 - hydroxyfluorene - 9

carboxylate(Flurecol Butyl, 10) were marketed as commercial products in 9 1965 ^.

1.2 EFFECTS OF MORPHACTINS

The effects of morphactins on plants have been reviewed in detail

by Schneider^^ and Ziegler^^. These active compounds are absorbed into

the plant via the leaves and the roots to be subsequently translocated

throughout the plant system. They become active in the Uegetative apex and -2 -7 meristematic tissues over a very wide range of concentration(10 to 10

mol.dm ) without any toxic side-effects . It is a feature of the

morphactins that they have very little effect on organs which at the time

of application are already developing. Their major effects are seen on

new growth v/hich has developed after treatment. The appearance of symptoms is slow but progressive. Development of the plant in general is slowed down, growth of the main, axis is suppressed and branching occurs. However, the lateral shoots which are produced are stunted as well. The root system of the plant is similarly affected. The normal geotropism of roots and phototropism of shoots are affected too. When applied at the correct stage of growth, morphactins inhibit the development of flowers or cause premature shedding of the buds. In certain species, they can induce parthenocarplc fruit development i.e. fruit development without pollination. It was because of this fundamental action on the morphogenesis of plants that these substances were given the name morphactins^'^(morphologically active substances).

The basic molecular skeleton of all effective derivatives is the morphogenetically inactive fluorene residue(ll) which only becomes active by the introduction of specific groups, especially carboxyl, f 12 into the - 9 - position . A substantial Increase in effectiveness is achieves by substitution of the second hydrogen atom in position 9 - by hydroxy or chloro groups. A further increase in activity occurs when halogen, especially chlorine, is substituted into the 2 - position of the fluorene skeleton. In addition to the free acids, their esters, amides and salts are also particularly active.

Another feature of the morphactins is their remarkably low toxicity to warm blooded and other animal species (e.g. Chlorflurecol 9 Methyl(9) LD^Q oral, acute (7 days) 5g/kg. for dogs ). An important practical advantage is their lack of persistence. They are rapidly metabolised in plants within a matter of days or weeks, so that actual residue problems are minimal. In soil, degradation takes place within a matter of weeks with the participation of microbes . This lack of persistence differentiates the morphactins from the majority of known synthetic growth regulators, the use of which are often associated with residue problems. Morphactins have two major applications , One is in chemically retarding growth, especially in combination with maleic hydrazide(i2). Chlorflurecol Methyl(9) has a broad spectrum CH2CO2H

of activity at higher dose rates, especially in the earlier stages of growth and will control the broad leaved plants whilst maleic hydrazide attacks the grasses , This is particularly useful where complete eradication of vegetation would cause erosion. The second use for morphactins, when mixed with phenoxyacetic acid derivatives (e.g. 2,4 - dichlorophenoxyacetic acid(13))is in combating weeds in corn and grassland . By means of appropriate dosages, the weeds can either be killed quickly or merely retarded so that they are overgrown by the crop and destroyed by lack of light. In the early stages of crop production they will protect the soil from drying out and erosion In combination with these phenoxy growth regulators, morphactins show synergistic effects. The phenoxy growth regulators are thought to act as stimulants to cell division and elongation, increasing the plants susceptability to the morphactin. This synergism often results in accelerated herbicide toxicity within the range of action of the 9 phenoxy growth regulator ,

1.3 CHEMISTRY OF MORPHACTINS 2 8 Morphactins can be prepared by well known procedures ' . Esters and amides of 9 - hydroxyfluorene - 9 - carboxylic acid(14) can be obtained either directly from the parent acid or via the intermediate 9 - chlorofluorene - 9 - carboxylic acid chloride(15) (Scheme 1). AgQH

p-toluene suVpWu octA

—^ (14) Scheme 1

The 9 - hydroxy - 9 - carboxylic acid(14) can itself be used as a starting material in order to make ring substituted derivatives from it directly (e.g. by halogenation or nitration). Substituted phenanthraquinones(16) offer an alternative route. These compounds will undergo a benzilic acid-type ot rearrangement on treatment v/ith alkali to give the corresponding 9 - hydroxyfluorene - 9 - carboxylic acid(17) (Scheme 2).

1) alkali, 10%KOH

2) dLtUteacli

(16) (17)

Scheme 2

Other fluorene derivatives are obtained from fluorene - 9 • carboxylic acid(l) or from fluoren - 9 - one (18) (for preparations see Chapter 2). The 9 - cyanofluorenes(6) are prepared via the 9 - carboxylic acid(l) by conversion to the amide(19) follo^i^ed by dehydration (Scheme 3). The nitro ester grouping is introduced into the 9 - position of fluorene(ll) via the 9 - chloro derivative (Scheme 4)-

10 Scheme 3

For example, reacting 9 - chlorofluorene(20) with silver nitrate in ethylene glycol solution at 40*^0 gives 9 - fluorenyl nitrate(8). 9 - cyano - 9 - hydroxy compounds of fluorene(7) can be obtained from

AgNO AO'

(20) Scheme 4

the corresponding substituted fluorenones(18) by reaction with hydrogen cyanide or cyanide salts (Scheme 5).

DMF NaCN

Scheme 5

1.4 OBJECTS OF RESEARCH 13 In 1971 it was found'""' that 9,9- bishydroxymethylfluorene(21) exhibited plant growth regulating activity similar to that of Chlorflurecol Methyl(9) (Tables 2 & 3, pages 75 & 76). The diol, which 14 was first reported by Stevens and Winch in 1958, can easily be obtained in high yield from the base catalysed reaction between fluorene(ll) and formaldehyde in dimethyl sulphoxidc"^'(Scheme 6).

11 2. THEORETICAL 2,1 FLUORENE

There have been few general surveys of the chemistry of fluorene(ll 17 18 After Everest's early review Rieveschl Jnr. and Ray published a very comprehensive survey of the chemistry of fluorene(ll) in 1938, Since 19 then, summaries have been published by Badger & Cook in Rodd's 20 'Chemistry of Carbon Compounds' and by Clar in'Polycyclic Hydrocarbons'. More recently, the reactivity of fluorene has been discussed by Moskowitz 21 and Mloque in 1968.

Fluorene(Cj^^H^^,ll) is a polycyclic hydrocarbon(colourless leaflets, mp 116°, bp 293 - 295**) which was discovered by Marcellin

(1) Rl CO^H (18) Rl = H,R2/R3 = O (11) Rl = R2 (23) Rl = R2 = H,R3 = Na

22 Berthelot in 1876 . He Isolated it from the anthracene oil fraction of coal tar(boillng range 270 - 310**). Berthelot was so impressed by the beautiful violet fluorescence of this compound that he named it "fluorene". (This fluorescence was later shown to be due to .naphthacene(22) 23 present as an impurity; ) The hydrocarbon is isolated commercially from coal tar by means of its sodio-derivatlve(23) which is formed on heating the tar with sodium at 300** or sodium amide at 120-150**, and separated 24 by filtration. Treatment of 9 - fluorenyl sodium(23) with water then regenerates fluorene(ll).

2.2 SYNTHETIC METHODS

The number of substituted derivatives of fluorene(ll) v;hich can be synthesised by direct substitution is limited by the pattern of reactivity shown by the hydrocarbon. Such syntheses will be considered in detail in Section 2.4 dealing with chemical properties; generally only 2-, 7- and 9- substituted fluorenes are readily obtainable by direct substitution.

13 Fev7 general syntheses of fluorene(ll) have been reported in the literature but fortunately there are a variety of methods available for making derivatives of fluorenone(18); reduction of these derivatives to the corresponding fluorenes(ll) is usually straightforward. The 22 hydrocarbon is best obtained from coal tar where it occurs to the 25 extent of 1*6%. It has been obtained by pyrolysis of acetylene (24), 26 27 substituted diphenylmethanes (25) or 2 - methyldiphenyls (26). Catalytic cyclodehydrogenation of diphenylmethane(25), dicyclohexylmethane(27) or 28

dicyclohexylketone(28) with platinum-charcoal at 300" also gives fluorene

(11). These reactions are suunaarised in Scheme 7,

HC=CH A Red Heat (24) r:::' (26)

Scheme 7

Friedel-Crafts reactions have been used with several different starting materials to make fluorene derivatives. The first published method involved biphenyl(29)and dichloromethane(Scheme 8)

Al CI * CH2CI2

(29)

Scheme 8 The method was later extended to give a bis-substituted-9-phenylfluorene(30) from benzal chloride and 3,3'-bis-dimethylaminobiphenyl (31,Scheme 9). 14 CHCl.

AlCI

Scheme 9 Fluorene-9-carboxylic acid(l) can be prepared from benzene and ethyl trichloroacetate(Scheme 10) or alternatively from benzilic acid(32) by cyclodehydration with aluminium chloride .

i)AlCl

* CCl3C02Et

Scheme 10 Similarly, benzoyl cyanide and benzene react to give 9-cyanofluorene(6) (Scheme 11); replacing benzene by toluene gives a methyl substituted 9-cyanofluorene(6).

AICI 3 > ^^^.^^^ Scheme 11

Crystalline orthophosphoric acid will dehydrate triphenyl carbinol(33) to give 9-phenylfluorene(34) in high yields^°(Scheme 12) Treatment of 2,2'-dibromodiphenyl(35) and dibromomethane in ether.

CPh^OH

(33) Scheme 12 31 with sodium will also produce fluorene (Scheme 13)

15 • ^CHoBro — ^ ^ ^ ether

(35)

Scheme 13

Finally, tetrahydrofluorene(36) has been syntheslsed from indene(37) 32 by a diene addition of butadiene (Scheme 14).

I6hrs.

. Scheme 14

Indirect syntheses via substituted fluorenones are much more versatile. The ketones are usually reduced to fluorenes with zinc, by boiling with phosphorus and hydriodic acid, or by heating to a high temperature with hydrazine hydrate. The following three methods are of particular interest because they give substituted fluorenones(18) where the substituents will be in known positions. These are:- a) Heating the calcium salt of diphenic acids(38) (Scheme 15)

CQ2-

(38)

Scheme 13

16 b) A Pschorr type synthesis, involving the diazotisation and subsequent cyclisation with copper powder of 2-aminobenzophenones(39) or alternatively o-aminodiphenylinethaneB (^0) (Scheme 16).

•^^^.^^.^^ o A

1) N^NOz/Ha 2) Cu. poujd^er 2) Cu powder

Scheme 16 c) Dehydration of 2-carboxybiphenyls(41) with concentrated sulphuric 34 acid (Scheme 17). This method is noted for the good yields obtainable; a disadvantage however, is that

0 HzS04 A

U1)

Scheme 17 if a second eubetltucnt is in the 3' position(i.e. in the second ring) two products(42 & 43) are obtained(e.g. Scheme 18).

(^1)

Scheme 18 A reliable method for making flucrenones involves the use of phenanthraquinone derivatives. Phenanthranuinone(16), which shows some structual similarity to aliphatic <=<-diketones, will undergo a benzilic acid type of rearrangement when warmed with aqueous alkali to give 9- hydroxyfluorene-9-carboxylic acid^^(Scheme 19). This ©^-hydroxy acid

lot KOH-fair \0l KOH

Scheme 19

is easily oxidised with loss of carbon dioxide to fluorenone with aqueous alkali at 100° in the presence of air. Alternatively alkaline permanganate will convert the quinone directly to fluorenone. o-Halogenobenzophenones(44) will eliminate halogen acid at high temperatures to form fluorenones (e.g. Scheme 20). This method, however has several disadvantages.

^^.^.^^^ HBr

Scheme 20 viz.the yields are small, the intermediates difficult to prepare, and because of the high temperatures required, rearrangements can occur. Fluoranthenes(45) can be oxidised to derivatives of fluorenone-1-carboxylic 37 acid (42) together with some fluoranthenequinone(46) (Scheme 21)

Scheme 21 18 Other syntheses of interest include the preparation of 3-hydroxyfluorene-2-carboxylic acid(47) from ethyl 2-(l* ,3*-dioxo-2*-indanylraEthentjL) acetoacetate^^(48)(Scheme 22) and the

CO^Et CH=C

Scheme 22 preparation of fluorene derivatives from o<-indanone(49) via the Mannich 39 reaction (Scheme 23)

* HCl- W\{^ ^ J^gg > ^^J[^^CH2-M1^ ^

CH3I

ci-

CH— 0 f4 Scheme 23 14 40 Fluorene with C in the 9-position has been synthesised

2'3 STRUCTURE

The structure of fluorene is as shown(ll). X-ray crystallographic analysis of fluorene has established that the three rings of the hydrocarbon are coplanar and that the two benzenoid rings are non-colinear, being inclined at angles of 78** to the plane of sijr»imphrj^(Y, fig.l ).

figures to left figures on right refer refer to bond angles to bond lengths^ jn pico metres(m, x 10 ) 1183 figure 1 l40t

19 2,4 PROPERTIES 42 Fluorene has a dipole moment of 0"62 Debye which is assumed to act along the plane of syrametry(Y fig.l) towards the ring and is due to the presence of the methylene group in position -9. The hydrogen atoms in the 9-position can be removed by strong bases, thus fluorene 43 can be regarded as an acidic hydrocarbon. The pKa of fluorene in dijnethylsulphoxide/ethanol is 21-0. In this respecti fluorene is similar to indene(50) and cyclopentadiene(51). The methylene group hydrogen atoms are progressively less acidic in the series cyclopentadiene, indene, fluorene

(50) (51) This reduction in acidity in the series is presumably due to the increasing +1 inductive effect of the benzenoid rings which reduces the inherent polarisation of the methylene C-H bonds. In each case, the extra stabilisation energy available by formation of the quasiaromatic carbanion(which is associated with 6 delocalised iT electrons) is the driving force in the ionisation process(e.g. fluorene Scheme 24).

Scheme 24

Fluorene crystallises from alcohol in colourless leaflets(mp 116") It distils at 293* - 295"* and sublimes easily. It is very soluble in carbon disulphide, ether and benzene, and insoluble* in water and liquid ammonia. Fluorene will form molecular complexes with a variety of reagents including dinitrobenzene, trinitroxylene, picric acid and tetranitronaphthalene.

20 Fluorene exhibits a wide variety of reactions, in some ways analogous to biphenyl(29) and diphenylmethane(25) in the aromatic character shown by its benzenoid rings, and similar to cyclopentadiene(51) and indene (50) in its behaviour as a weak acid.

2.4.1 OXIDATION

Fluorene can be oxidised to fluorenone(18) with sodium dichromate 44 in acetic acid . Potassium permanganate will oxidise it further to phthalic acid(52). Potassium hypochlorite will oxidise 2-acetylfluorene to fluorenone-2-carboxylic acid(Scheme 25).

(52)

This simultaneous oxidation of the methylene group to a carbonyl group is unusual; under comparable conditions fluorene itself is unchanged.

Scheme 25 45 With oxygen, fluorene forms a hydroperoxide (53). This peroxide is also thought to be the initial product in the oxidation of the 9-fluorenyl carbanion^^.

•^^^.^..^^—^ (531

Further oxidation of the hydroperoxide(53) gives fluorenone(18) which can itself be oxidised by hydrogen peroxide to fluorenone peroxide(54)

21 This peroxide rearranges when treated with acetic anhydride and sulphuric acid, producing the lactone of 2-hydroxydiphenyl-2'-carboxylic acid(55) (Scheme 26).

(CH3CO) 0 H2sq;

(55) Scheme 26

Ozonolysis of fluorene gives homophthalic acid(56)

CH2CO2H

(56) CO2H

Oxidation by removal of the hydrogen atoms at the 9-position of fluorene can occur in two steps to give first the colourless *J,9'-bCfluorcujl (57) and then the orange-red 9,9'-tiftttorttn»jlidenc 47,48,49^2gj^ Both these compounds can be obtained by heating fluorene with lead dioxide. 9,9'-6L-HucrcnijLticne ^^(58) is also obtained by heating fluorene with bromirie,

chlorine or sulphur, and by the action of methanolic potassium hydroxide on 9-bromofluorene or copper powder on ^^'^-Alti^Vorft-fVue^ene.

22 2.4.2 HYDROGENATION

Fluorene can be hydrogenated with hydriodic acid and red phosphorus to give decahydro- and perhydrofluorenes^^. The same compounds 52 are also obtained by catalytic hydrogenation under pressure , Catalytic, hydrogenation with Raney nickel requires temperatures of 200^C and pressures of 150 atmospheres; the product is a mixture of three isomers of perhydrofluorene . Recent interest in perhydrofluorenes has been due to the fact that they rearrange when treated with anhydrous aluminium bromide at 0°C to give alkyl adamantanes^^.

A hexahydrofluorene has been extracted with benzene from a *fat' coal, and at red heat this compound undergoes dehydrogenation to fluorene^^. The fact that hydrogen is given off at high temperature led to the idea that aromatj.c hydrocarbons in cpal tar are formed in the distillation and are not the constituents of coal. In fact the low temperature carbonisation of coal gives a mixture of aliphatic and alicyclic hydrocarbons. The aromatic rings of fluorene are sensitive to the action of sodium in liquid ammonia forming different hydrofluorenes depending on the conditions.

2.4.3. ELECTROPHILIC SUBSTITUTION IN THE BENZENOID RINGS

In electrophilic substitution reactions, fluorene is attacked principally at the 2-position, but there is often some 4-isomer produced as well. In most reactions a further substituent enters at the 7-position and a mixture of 2- and 2,7-disubstituted fluorenes results which is often difficult to separate. This mixture will also contain traces of 2,5-isomer derived from the 4-isomer. The electrophilic substituent deactivates its ring to further substitution giving rise to 2,7- and 2,5-isomers in preference to any 2,4-material(e.g. further nitration of 4-nitrofluorene(Scheme 27).

HNQ

(59)

Scheme 27

23 2-Nitrofluorene is obtained by direct nitration with nitric acid in acetic acid. ' Acetyl nitrate at 30* gives a mixture of 2-nitrofluorene plus about 15% of the 4-nitro compound^^; these isomers 58 can be separated by chromatography . Further nitration of fluorene gives 59 60 61 a mixture of 2,5- and 2,7 dinitrofluorenes. * * 2-Nitrofluorene can be reduced with iron powder and hydrochloric acid, or palladium on charcoal 56 62 and hydrazine hydrate to give 2-aminofluorene; * this compound and its 2-acetyl derivative are known to be potent carcinogens. Nitration of 2-aminof luorene gives mostly the 3-nitro compound plus some of the expected 63 2-amino-7-nitro-fluorene . However, nitration of the 2-aminofluorenone gives the 7-nitro derivative exclusively. Chlorination of fluorene gives a mixture of 2-chlorofluorene plus some of the 4-isomer and some multisubstituted products. These isomers are difficult to separate. 2-Chlorofluorene has been claimed to have been produced in high yields by treatment of fluorene with sulphuryl chloride^^. However, several workers have reported that the yields claimed are not reproducible . Another disputed claim, is that pure 2-chloi-ofluorene can be prepared by treatment with chlorine gas, in chloroform solution at 0**, 59 in the presence of iodine or antimony pentachloride as catalyst . Further chlorination yields 2,7-dichlorofluorene and 2,4,7-trichlorofluorene^^'^^. The best way of obtaining pure 2-chlorofluorene is from 2-aminofluorene using the Sandraeyer reaction^^. This method has been used to confirm the orientation of the chlorofluorenes which were isolated from reactions involving direct chlorination. Bromination of fluorene gives the usual mixture of 2- and 2,7-disubstituted products^^'^^ plus a tribromofluorene^^. Substitution with chlorine or bromine beyond the mono-stage gives 2,7- dihalogeno derivatives; which can be isolated in the pure form by recrystall- Isation.

Fluorene can be sulphonated with chlorosulphonic acid in chloroform solution at 0*; the product is the 2-sulphonic acid ' . The same compound is obtained using sulphuric acid in acetic acid^^. Fluorene - 2,7-disulphonic acid^^ is formed when fluorene is heated in sulphuric acid at 100".

24 Friedel Crafts alkylation reactions are temperature dependent. At slightly elevated temperatures the reactions give 2-alkyl derivatives, but at higher termperatures, multisubstituted products arise and isomer- isations occur. Friedel Crafts acylation reactions occur at the 2-position 72 With acyl halides, a mixture of 2- and 2,7-disubstituted products are fomed; acyl anhydrides are preferred for making pure 2-substituted 73 products. The structures of the ketones produced have been proven by using the Beckmann rearrangement of the corresponding oximes to give derivatives of known amino compounds. Thus 2,7-dibenzoylfluorene dioxime 74 undergoes rearrangement to 2,7-dibenzoylaminofluorene. This compound was also prepared directly by benzoylating 2,7-diamino fluorene. Chloromethylation of fluorene with formaldehyde and hydrogen chloride gives 2-chloromethyl£luorene, mercurisation also occurs at the 2-position. '

9-substituents have very little directing effect on the position of ring substitution, for example, 9-carboxy, 9-bromo, and 9-acetylamino fluorenes are all nitrated at the 2-position. Also 9-fluorenyltriraethyl- ammonium bromide, prepared from 9-bromofluorene and-triethylaraine,is nitrated at the 2- and 7-positions,This lack of influence on the position of new entrant groups is in marked contrast to the influence of nuclear sutstituents on the reactivity of the 9-position.

Electron-donating substituents are normally expected to activate benzene rings in the ortho/para positions. This is not the case with fluorene, 2-acetaraido fluorene undergoes halogenation, and 2-hydroxyfluorene is nitrated, in the 3-position. However, 2,4-toluenesulphonamido- 49 fluorene when nitrated gives a mixture of the 1- and 3-nitro derivatives.

H-S02-Ph-Me

With a substituent in position 3-, further substitution is always at the 2-position since this is the moet reactive.

25 Electron withdrawing groups deactivate a benzene ring and consequently fluorene derivatives possessing such substituents undergo further substitution in the second ring at position 7-(cf. Naphthalene, biphenyl, diphenylmethane). For example, 2-nitrofluorene can be chlorinated to give 7-chloro-2-nitrofluorene. The exception is bromine, which under suitable conditions will attack the 9-position.

Fluorene derivatives with electron donating substituents in the 2- and/or 7- position usually undergo further substitution in the 3- or 6- positions.

Some work has been done on the electronic charge distribution in 78 the aromatic rings of fluorene. The first attempt by Pullman used the LCAO method which gave the results shown in Figure II. This clearly does not explain the preference of fluorene for undergoing 2-substitutions.

voox 0039 I-000

0-998 0 039

l-OOl OI?9

Fig.II Fig.Ill

79 Fukui, using a method called 'frontier electron density' obtained results which correlate well with experimental results. Fig.III.

2.4.4 Substitution at the 9-Position

The acidity of the 9-position, as explained earlier (see page 20) is due to the aromatic chacter of the resultant carbanion. The 9-methylene group is attacked by oxidising agents and free radicals, and will also 21 undergo ionic substitution. Oxidation has been dealt with earlier (page 21 ). In carbon tetrachloride solution under a strong mercury arc lamp or in direct sunlight, fluorene is brominated by molecular 80 bromine to give 9-bromofluorene and 9,9-dibromofluorene- The latter compound is an intense skin irritant which produces very severe dermatitis. 81 9-bromofluorene is also obtained from fluoren-9-ol by classical methods, 82 but it is best prepared by bromination of fluorene using N-bromosuccinimide 83 or tetrabromophenol. 9,9-Dichlorofluorene is obtained by treating fluorene 26 84 with phosphorus oxychloride and phosphorus pentachloride. It can also be obtained from fluorene and carbon tetrachloride in the presence of benzyltrimethylammonium hydroxide and light.9,9-Di- fluorofluorene can be prepared from 9,9-dichlorofluorene and hydrogen fluoride using mercuric chloride as a catalyst, but is unstable.

Ionic substitutions at the 9-position are numerous and usually involve the 9-fluorenide ion acting as a nucleophile. They occur under alkaline conditions, when the 9-fluorenide anion produced can then react with electrophiles such as halogen derivatives, carbonyl compounds or carbon dioxide.

Benzyl chloride will react with fluorene in the presence of potassium hydroxide, to give 9,9-dibenzylfluorene. In alcoholic solution, fluorene condenses with aromatic aldehydes to give 9- arylidenefluor^nes. Using benzaldehyde, 9-ben2ylidenefluorevie (61) is produced (scheme 28). r^uDk

Scheme 28

Similarly, aliphatic aldehydes will also condense with fluorene in the presence of potassium ethoxide in xylene solution yielding 9-alkylidene derivatives.^^

One interesting condensation of fluorene is that with acrylonitrile, in the presence of catalytic amounts of benzyl• trimethylammonium hydroxide, to yield bis-9,9-(2'-cyanoethyl) fluorene^^ (62) (scheme 29). CNCHoCHo CHXHXN

Triton B CHACHCN

^^^.^^^ Scheme 29

Other condensations include that between fluorene and ethyl oxalate, in the presence of sodium, efhbxide. Hydrolysis of the product ester gives 9-fluorenylglyoxylic acid^^ (63). A similar condensation occurs between fluorene and ethyl formate yielding 9-formylfluorene^^(64) Both (63) and (64) exhibit keto-enol tautomerism due to the lability of the 9-hydrogen atom. (63)

Electron-withdrawing groups in the 9-position and in the aromatic rings increase the acidity of the 9-hydrogen atom. Both 2-nitro and 2-chlorofluorene are more acidic than fluorene and will undergo condensation react.ions more readily. One of the most acidic hydrocarbons known is a fluorene derivative, fluoradene (65) (also known as indeno [l,2,3-jk] fLuorene) which is sufficiently acidic to dissolve in dilute sodium hydroxide solution; its pKa was found to be 11.

(65)

One condensation reaction of fluorene is rather unusual, Fluorene in the presence of potassium hydroxide will condense v;ith acetone to give 4-(9*-fluorenyl)-4-methylpentan-2-one (scheme 30). When treated with hydrogen bromide in acetic acid, this last compound gives 1,1,3- trimethyl-l,10b-dihydrofluoranthQne which then can be aromatised to 1,3- dimethyl-fluoranChene^^(66) (CH3)2(

• {CH3)2CO

HBr CH^CO^

Se 300

Scheme 30 Fluorene reacts with maleic anhydride at the 9-carbon atom 92 after 40 hours at 210** to give 9-fluorenylsuccinic anhydride (67).

CHyCO^

H-CO"

(67)

Fluorene will undergo metallation reactions at the 9-position. 24 For example, when melted with potassium hydroxide or heated with metals 93 24 such as lithium or sodium , the appropriate 9-fluorenyl metal derivative is formed. Alternatively, organolithium compounds or metal amides may be used to achieve metallation. The metal derivatives can be used for many syntheses. They will react with carbon dioxide, to give the 9-carboxylic acid(scheme 31); with alkyl halides to give 9-alkylfluorenes;with acyl Iialidet to give ketones; and with carbonyl compounds to give alcohols. 9-Fluorenyl magnesium bromide can be prepared in excellent yields by heating fluorene

COols) Scheme 31 ^ , .^i. ^1. 1 . u .J 94,95 9-Fluorenyl lithium is in xylene with ethyl magnesium bromide. readily obtained from fluorene and n-butyl-lithium. These compounds, however, do not behave similarly. 9-Fluorenyl magnesium bromide adds to -unsaturated ketones by 1:2 addition, whereas 9-fluorenyl lithium 95 reacts by 1:4 addition

As mentioned previously(page27 )^ aldehydes will condense with fluorene in the presence of sodium ethoxide to give dibenzofulvene deriva• tives. Using modified conditions it is possible to isolate the intermediate 96 alcohols. For example,fluorene in dimethyl sulphoxide will condense with paraformaldehyde in the presence of sodium ethoxide as catalyst to give HOQHo H2OH

(21) 9,9-bishydroxyinethylfluorene(21). Such reactions are enhanced by electron withdrav/ing groups in the 9-position.

A novel method of alkylating fluorene in the 9-position involves heating fluorene with the appropriate alcohol at 230** under pressure in the presence of alkali metals. The mechanism is complex but the yields 97 are very good. (Scheme 32) /^LJD HR RCH20Nn RCH20H-^RCH0

RCH^ONg ^ — RCHO

RCHoOH RCH^NQ

Scheme 32

Some miscellaneous reactions of fluorene include that with diazoacetic ester to give a compound'^hic'- on hydrolysis and decarboxylation 98 suffers ring enlargement to form 1:2 benzazulene (68) (Scheme 33). When fluorenone is treated with methyl magnesium iodide, 9-methylfluoren- 9-ol is obtained.

CHN2Cn2Et > H-C02Et Comlert&aUon. I

C02H • Pd-C . Scheme 33

which on dehydration by a number of methods gives unstable 9-methylene- fluorene or dibenzofulvene. The product can be isolated in crystalline form after bromination followed by de-bromination in the dark, with zinc The fulvene can also be obtained from 9-fluorenylmethylurethane by distillation vjith lime. 30 9-Hethylfluoren-9-ol can be dehydrated by boiling with glacial acetic acid, tthen the resulting solution is hydrogenated with Adams catalyst, 9-methylfluorene is obtained.

9-Phenylfluorene(34) can be obtained by a variety of condensation reactions. It is formed when triphenyIchlororaethane is heated, and when triphenylmethane is heated with potassium. Other methods involve dehydrating triphenyl carbinol by heating with phosphoric acid; treating 9-hydroxyfluorene(69)with phosphorus pentoxide in benzene(Scheme 34) or

Ph CCl Ph CH

HCI/CH3CO2H HPQ3 Ph3C0H

Scheme 34

treating 9-chlorofluoreneC70) with aluminium chloride in benzene solution. It can also be made by reducing 9-phenylfluoren-9-ol(71) with zinc and 99 hydrogen chloride/acetic acid. 9-Phenylfluoren-9-ol, an analogue of triphenyIcarbinol can be obtained from fluorenone by the action of phenyl magnesium bromide or by oxidation of 9-phenylfluorene with sodium dichromate in acetic acid. The action of phosphorus pentachloride, acetyl chloride or gaseous hydrogen chloride on 9-phenylfluoren -9-ol gives 9-phenyl-9- chlorofluorene, which like trtphenylchloromethane is characterised by the mobility of its chlorine atom. When heated with copper powder in benzene solution, 9-phenyl-9-chlorofluorene gives 9,9'-diphenyl-9,9'-difluorenyl which is analogous in structure to hexaphenylethane but is considerably more stable. 9-Phenyl-9-chlorofluorene absorbs oxygen in solution to give bis-9-phenylfluorenyl peroxide.

31 9-Formylfluorene, as mentioned earlier (page 27), can be obtained from the reaction between fluorene and ethyl formate in the 89 presence of potassium ethoxide. The oxime of 9-formylfluorene is readily converted by thionyl chloride into 9-cyanofluorene, which on hydrolysis, gives fluorene-9-carboxylic acid. This acid can also be obtained from 9-KjirftijFUnn-enp-9.car()oxjlic acid by reduction with hydriodic acid and phosphorusj or by the action of aluminium chloride in benzene on benzilic acid; or alternatively from benzyl chloride and aluminium chloride in benzene solution by isomerisation. Fluorene-9-carboxylic acid is readily obtained from 9-fluorenyl magnesium bromide or from 9-fluorenyl- 95 lithium by treatment with carbon dioxide. The methyl ester can 100 similarly be prepared from 9-fluorenyl lithium and dimethyl carbonate . The ester is a useful starting material for the preparation of other fluorene derivatives. It forms a 9-sodio derivative which readily reacts with alkyl halides; subsequent decarboxylation of the products gives 9-alkylfluorenes. Solutions of the salts of fluorene-9-carboxylic acid decompose on standing to give fluorene, and in the presence of air, .,101 fluorenone.

32 3.DISCUSSION

3.1 CHEMISTRY OF 9,9-BISHYDROXYMETHYLFLUORENE

In 1970 Hanson^^ became interested in the preparation of 9,9-bishydroxymethylfluorene (21) from fluorene(ll) and paraformadehyde. . ' Subsequently, in collaboration with Fisons Limited, Agrochemicals Division, it was discovered that this compound exhibited plant growth regulating 13 9 activity similar to that of the morphactin chlorfluorecol methyl (9). Although the level of activity was lower than that of the morphactin,

•s^-"— (85) R j^=R2=H ,R2=R^=CH20 • CO -NH • Ph (86) Rj^=R2=H,R^/R^ =-CH20-SO-OCH2- (9) R =Cl,R„=H,R^=0H,R,=CO^6e ^1 2 3 4 \2 (87) Rl=R2=H,R^=R^=CH20•SO2•CH^

(11) R. ~Rrt "R r\ ""R /H 12 3 4 (88) Rj^=R2=H ,R^=R^=CH20 • SO2 ' C^H^Me (21) R j^=R2=H, R^=R^=CH20H (89) R^=R2=H,R2=CH^,R^=CH20H (76) ^1~^2~^'^3^^4~ ~ (90) R^=R2=H,R^=CH20H,R^=C02H (77) R^=R2=R3=H,R^=CH20H (93) R^=N02,R2=H,R^=R^=CH20H <:79) R =R-=H,R„=R =CH.0'C0-Me 1 2 3 4 2 (94) Rj^=Cl,R2=H,R^=R^=CH20H (80) R^=R2=H,R^=R^=CH20*C0*CH2C1 (95) R^=Br,R2=H,R^=R^=CH20H (81) R =R„=H,R,=R =CH^0*C0 Et 1 2 3 4 2 (97) Rj^=Cl,R2=H,R^=CH20H,R^=C02Me (82) R j^=R2=H ,R^=R^=CH20 • CO • nPr (98) R^=H,R2=N02,R3=R^=CH20H (83) R^=R2=H,R^=R^=CH20•CO'NH-Me (99) R^=H,R2=C02Me,R3=R^=CH20H (84) R =R-=H,R-=R =CH-0*CO*NH«Et 1 2 3 4 2

it was considered worthwhile to explore the structure-activity relationships in a series of hydroxymethylated fluorenes prepared in the same way as(21) from a variety of substituted fluorenes.

As mentioned earlier(Chapter 2, page 27 ) fluorene will condense with aldehydes in the presence of basic catalysts to form dibenzofulvenes(72) (Scheme 35). It is generally accepted that reactions of active methylene compounds with carbonyl groups proceed through an 'aldol' type of intermediate though no alcohols of structure(73)

33 (Scheme 36) have been isolated from the condensation reactions of 96 fluorene until recently .

. RCHO £M '^^^^^ ^ (721

Scheme 35

Alcohols of type(73) have usually been prepared by indirect methods.- While the formation of dibenzofulvenes from aromatic aldehydes is relatively easy, difficulties are encountered when aliphatic aldehydes are used as these usually undergo selfcondensation under the influence of the basic catalyst. HOCHR RCHOH

HOH /RCHO

COO

Scheme 36 96 Ghera & Sprinzak reported that fluorene will condense readily with aldehydes and some ketones in pyridine solution in the presence of benzyltrimethylammonium hydroxide(Scheme 36). The high reactivity of fluorene under these conditions, presumably due to the presence of an appreciable concentration of carbanion allows low enough'temperatures to be employed to avoid dehydration of the alcohol 3A formed, which can thus be isolated in substantial yield. In the preparation of these alcohols(73) the isolation of pure product was easier when fluorene was used in excess, presumably, because further reaction of the alcohol with aldehyde, to form glycol(74), is minimised 96 under these conditions (Scheme 36). In some cases, the product was accompanied by a considerable amount of l,l-bis-(9-fluorenyl)-alkane(75) which apparently results from the reversible addition of fluorene to the dibenzofulvene(72) produced by dehydration of the alcohol(73). By using an excess of aldehyde, fluorene could be made to react with two 96 molecules of aldehyde to form the hitherto unknown, 9,9-bis-l*-hydroxy- alkylfluorenes(74)(Scheme 36). While usually the yi-elds were poor, the reaction of fluorene with paraformaldehyde was remarkably easy, affording 9,9-bishydroxymethylfluorene(21) in 30% yield together with a resinous substance. The resin possibly results from the polymerisation of the diben2ofulvene(76) the latter being produced by dehydration of the intermediate 9-hydroxymethylfluorene(77).

Fluorene will react similarly with a number of ketones to give the corresponding tertiary alcohols. Unlike the reaction with aldehydes, the reversible formation of these alcohols is usually not 96 complicated by side reactions . For example, with acetone(Scheme 37) fluorene gives 9-(2'-hydroxy-2'-propyl)-fluorene(78).

H0Q(CH3)2

Triton B + (CH3)2CO S..^^

Scheme 37

In 1967, Wesslen^^ reported an improved method of making 9,9-bishydroxymethylfluorene(21) using dimethylsulphoxide as solvent, sodium ethoxide as catalyst, and a reaction time of only three minutes at 0**C; the yield was' 70%(Scheme 38).

35 This result suggests that the abstration of hydrogen from fluorene and from the Intermediate 9-hydroxyraethylfluorene(77) by the poorly

HOQHo CH2OH

DNaOEt HCHO 2)HCl ^^^^^..^^— (aq) (21)

Scheme 38 solvated ethoxide ions, proceeds very rapidly. The existence of this method meant that it should be possible to obtain moderately large quantities ( lOOg) of 9,9-bishydroxymethylfluorene(21) with• out difficulty. Similarly, it should be possible to perform analogous reactions using ring substituted fluorenes and obtain ring substituted 9,9-bishydroxyraethylfluorenes in sufficient quantity («^10g) for biological screening tests,

• • Initially therefore, work v/as directed towards obtaining moderately large quantities of 9,9-bishydroxymethylfluorene so that the structual features in the molecule which are essential for biological activity could be identified. However, the freezing point of dimethylsulphoxide is 18** and it was found impossible to keep the reactants in solution at temperatures below 15®. At this temperature, Wesslen's method produced the diol(21) in lower yields than those claimed. It was found that the reaction time was critical. Any increase in the time beyond 2*5 minutes markedly reduced the yield. The use of a nitrogen atmosphere to prevent oxidation of the carbanion produced in the reaction did not appear to have any effect on the yield. In general, the high yield clamed by Wesslen could not be reproduced on a large scale.

The most obvious features in the molecule of 9,9-bishydroxy• methylf luorene which might lead to biological activities are the hydroxyl groups. A number of compounds, described below, were therefore synthesised with the object of investigating the role of these groups in biological activity, 36 3.2 HYDROXYL MODIFICATIONS 3.2.1 Esters

A series of bisesters were synthesised containing acyl groups ranging from formyl to n-butyryl. It was hoped that the greater lipophilic character of the esters, associated with increasing chain length in the acyl group, would increase the ease of penetration of the compound into the plant leaves v/here the esters.might be slowly hydrolysed to yield the diol, BisacetyK79) bischloroacetyl(80) bisprop-

ionyl(81), and bis-n-butyryl esters(82) were prepared using the corres• ponding acid chlorides ^ as acylating agents. In chloroform solution the yields were generally very good, in the range 80 - 93% of theoretical The bisformyl ester was prepared from the diol and p-toluenesulphonyl-

chloride-dimethylformamide complex in dimethylformamide in 357o yield. 104 Attempts to prepare the bistrifluoroacetyl ester using trifluoroacetic anhydride were unsuccessful. Despite the use of pyridine as catalyst and molecular sieve to prevent possible hydrolysis of any product, only 9,9-bishydroxyraethyl fluorene(507o recovery of pure material identified by infra-red spectroscopy and melting point) was isolated from the reaction mixture.

A cyclic carbonate could not be isolated after treating the

diol(21) with phosgene^^^^ though a small quantity of an impure carbonyl containing substance was obtained(i.r. spectra and t.l.c.) along with unreacted glycol(21), but was insufficient to purify.

In view of the fact that raaay carbamate esters exhibit herbicidal activity, a number of such compounds were prepared from the diol. Treat• ment of 9,9-bishydroxymethylfluorene in dry pyridine with methyliethyl-; phenyl-, and chlorophenyl isocyanates gave respectively the bismethyl- (83), bisethyl-(84), bisphenyl-(85), and bis-3'-chlorophenyl carbamate esters. The yields were.in the region of 507,.

3.2.2. Chloromethyl derivatives

Since many active herbicides contain one or more chlorine atoms per molecule it was thought worthwhile to attempt to substitute chlorine atoms for the two hydroxyl groups in 9,9-bishydroxymethylfluorene.

37 Heating the diol with concentrated hydrochloric acid and anhydrous zinc chloride gave no reaction; the use of anhydrous hydrogen chloride gas and anhydrous zinc chloride in dioxan at 102* gave rise to a black tar which could not be successfully purified. Extraction of the tar with diethyl ether enabled unchanged diol(insoluble in ether) to be isolated by filtration. Gas liquid chromatography of the filtrate Indicated a mixture containing one major component. The infra-red spectrum of this fraction showed the presence of a carbonyl group. It was thought that peroxides present as impurities in the dioxan may have initiated oxidation of the mixture. Evaporation of the ether solution gave an oil which could not be purified.

The use of phosphorus pentachloride in refluxing carbon tetrachloride solution gave a substance which was purified by column chromatography ar.d then recrystallised to constant melting point. This organic compound was found to contain phosphorus and chlorine but proved impossible to analyse satisfactorily for phosphorus. Mass spectroscopy indicated the material had a molecular weight of »5i3 but its structure remains undetermined..

When the diol, in solution in chloroform, was treated v/ith thionyl chloride and pyridine at 0°C, the cyclic sulphite 9,9-sulphinyl- dioxyraethylfluorene(86) was produced, in 75% yield. The sulphite slowly lost sulphur dioxide in air. The parent diol could be regenerated by hydrolysis.

(86) 105 Bissinger & Kung have studied the use of thionyl chloride to chlorinate alcohols and their work suggested that the cyclic sulphite might react with anhydrous hydrogen chloride to give the corresponding bischloro compound. However, this method gave the parent diol, apparently due to moisture present giving rise to hydrolysis.

38 Mild chlorinating agents such as those often used in carbo• hydrate chemistry were then investigated. Triphenyl phosphine in carbon tetrachloride gave a complex mixture of products. Methanesulphonyl chloride usually forms mesyl esters with alcohols but it has been used 107 to chlorinate carbohydrates However, when the diol(21) was treated with reagent, the corresponding bismethanesulphonyl ester(87) was isolated as expected. Fieser and Fieser^^^^ reported that tolune-4-sulphonyl ejkeri of primary alcohols could be converted to the corresponding chlorides by reaction with pyridine hydrochloride. The bistoluene-4-sulphonyl ester of 9,9-bishydroxymethylfluorene(88) was therefore prepared. Treatment of this compound with pyridine hydrochloride in dimethyl formamide at 25** or 100" gave no product; starting material could be recovered in S5 100% yield in both cases. The failure to convert the hydroxyl groups to chloride groups was unexpected. Model building reveals no clear evidence of steric hindrance to the hydroxyl groups and the lack of reactivity remains to be explained.

3.2.3.Miscellaneous Reactions

Finally in this section of work, the effects of removal of one hydroxyl group and also of changing the environment of the hydroxyl groups slightly were investigated. To this end, 9-hydroxymethyl-9- methylf luorene(89) , 9-hydroxymethylfluorene-9-carboxylic acid^^(90), I 96 its methyl ester, and 9,9-bis-l-hydroxyethylfluorene (91) were synthesised. The monohydroxymethyl compound(89) was obtained in 70% 97 yield by treating 9-methylfluorene and paraformaldehyde with sodium

9^3 9^3 HOCH CHOH

ethoxide solution using hexamethylphosphoric triamide as solvent. 9-hydroxymethylfluorene-9-carboxyl-ic acid(90) was prepared by the method 14 of Stevens and Winch ; its methyl ester was synthesised independently by condensation of methylfluorene-9-carboxylate with paraformadehyde in acetonile solution at ,2** using aqueous Triton B as base. Although these compounds bear a close resemblance to 'flurecol* they do not appear to 39 be mentioned in any patents relating to the morphactins. The bis- 96 hydroxyethyl compound (91) was prepared from fluorene and acetaldehyde in dimethylformamide solution using sodium ethoxide as base; the yield obtained(12%) was somewhat better than that reported by Ghera and Spinzak who used pyridine as solvent.

3.3 RING SUBSTITUTED DERIVATIVES OF 9,9-BISHYDROXYMETHYLFLUORENE

3.3.1 2-Nitro-9,9-Bishvdroxymethylfluorene(93)

Some ring substituted products of 9,9-bishydroxymethylfluorene were prepared in order to investigate the effect of ring substituents general, and more specifically of chlorine(since this substituent occurs in chlorflurecol) on activity.

2-Nitrofluorene was prepared by the method of Diels^^. Thin layer chromomatography showed this material to be contaminated 57 58 with both fluorene and 4-aitrofluorene; ' it was therefore purified by chromatography on a column of basic alumina using 60-80** petrol for elution. Initial attempts to bis-hydroxymethylate this compound at the 9-position gave rise to several problems. The use of the method previously employed for fluorene produced a mixture, as shown by thin layer chromatography, of what appeared to be unreacted 2-nitrofluorene and a high melting resinous product which could not be purified. In view of this, production of the desired product by direct nitration of 9,9-bishydroxymethylfluorene was investigated. Nitration of the did using concentrated nitric acid in glacial acetic acid at 50" gave a complex mixture of products as indicated by thin layer chroma- 0 tography. Infra-red analysis of the oily product showed carbonyl groups to be present which suggested that acetylation was occurring This was supported by the similar Rf values to the acetyl derivative. The hydroxyl groups of the diol were therefore protected by acetylation; subsequent nitration of the bis-ester with nitric acid/sulphuric acid in glacial acetic acid at 60** gave the desired nitro-compound in low yield. This method was improved by using acetyl nitrate at 10* to give the nitro-compound in 79%-yield. Removal of the acetyl groups by hydrolysis then gave the required nitro-diol in satisfactory yield.

40 Further attempts were made to hydroxymethylate 2-nitrofluorene. It was established that the desired product could be obtained in very low yield from 2-nitrofluorene by reaction with paraformaldehyde in dimethylsulphoxide solution with sodium ethoxide as base at 25**. The yield was improved to 30% by using dimethylformamide as solvent and a reaction temperature of 10". However, the results were not reproducible. Thin layer chromatography of the reaction mixture showed that six products, together with much unreacted 2-nitrof luorene were present. Using chromatographicollij pure 2-nitrofluorene in hexamethylphosphoric triamide, a small scale (^ Ig. of 2-nitrofluorene) condensation gave the nitrodiol in 60% yield. However, using once recrystallised material and a larger scale('55 lOg) , very low yields were obtained.

The reaction mixture was examined by electron paramagnetic resonance spectroscopy whereupon it was established that a stable radical species existed in the 2-nitrofluorene solution in the various solvents containing ethoxide ions prior to the addition of paraformalde• hyde. It was deduced from the spectra that this species was probably the 2-nitrofluorenone radical anion(92). The species found was very stable and was unaffected by air. Subsequently, it was discovered that the nitrodiol could be prepared on a reasonably large scale at 0** using acetonitrile as solvent and aqueous 'Triton B' as base;

(92)

The yield was of the order of 70% and was reproducible. It seems probable that acetonitrile stabilises the anion at the expense of the radical anion.

41 3.3.2 Halogen Substituents

2-Chloro-9,9 bishydroxymethylfluorene(94)

The first route to 2-chloro-9,9-bishydroxymethylfluorene which was investigated involved initial chlorination of fluorene followed by hydroxymethylation. However, the preparation of 2-chlorofluorene by treatment of fluorene with sulphurylchloride as reported by Streitwieser^^ could not be accomplished. Closer examination of the literature showed that chlorination with chlorine produced some 4-chlorofluorene and 2,7-dichlorofluorene besides the 2-derivative and that the mixture could not be separated easily. The desired 2-chloro' compound was therefore prepared from the corresponding 2-nitro derivative. The nitro diol was reduced using hydrazine and palladised charcoal or hydrogen and palladised charcoal. The 2- amino-,9,9-bishydroxymethylf luorene thus produced was difficult to . purify and was found to deteriorate during storage. It was therefore converted to the crystalline hydrochloride salt in which form it could be handled without difficultvj. The pure hydrochloride salt was diazot- ised with nitrous acid and converted to the 2-chloro-9,9-bxshydroxy- methylfluorene, using the Sandmeyer reaction, in 89% yield,

2-Bromo-9>9-Bishydroxymethylfluorene(95)

Hydroxymethylation of 2-bromofluorene, prepared by direct 59 68 bromination of fluorene, ' was accomplished smoothly in 25% yield using dimethylformamide as solvent at 2®.

3.3.3 Acetyl Substituents

2,7-diacetyl-9,9-bishydroxymethylfluorene(96) •

2-Acetyl-9,9-bishydroxymethylfluorene could not be prepared. 2-Acetylfluorene in solution in acetonitrile apparently formed a radical anion on treatment with sodium ethoxide in a manner analogous to 2- nitrofluorene. This was confirmed by electron paramagnetic resonance. Hoii7ever, 2,7-diacetylfluorene, isolated as a by product in the Friedel Crafts acylation of fluorene with acetic anhydride to produce the 2-acetylfluorene, reacted with paraformaldehyde in dimethylsulphoxide to give 2,7-diacetyl-9,9rbishydroxymethylfluorene

42 H20H CHXO OCH ^^.^^^ (96)

3. 3. 44'Substituted Derivatives

9,9-bishydroxymethyl-4-nitrofluorerie(98)

4-Nitrofluorene was isolated by column chroraotography from crude 2-nitrofluorene prepared by the action of acetyl nitrate on fluorene. This material was treated with paraformaldehyde in acetonltrile with 'Triton B* as base at 0<* yielding 47% of 9,9-bishydroxymethyl-4-nitrofluorene.

Methyl 9,9-Bishydroxymethylfluorcne-4-carboxylate(99)

This compound was prepared from methyl fluorene-4-carboxylate using dimethylformamide as solvent and sodium ethoxide as base, as described for the 4-nitro compound.

3.4 METHYL 2-CHL0R0-9-HYDROXYMETHYLFLU0RENE-9-CARBOyYLATE(97)

Methyl 2-chlorofluorene-9-carboxylate, prepared by the method 108 of Sieglitz and Troester , reacted with paraformaldehyde in acetonitrile with 'Triton B* as base at 2° to give methyl 2-chloro-9-hydroxymethylfluorene-9-carboxylate in 457o yield.

3.5 BIOLOGICAT. SCREENING OF 9,9-BISHYDROXYMETHYLFLUORENE DERIVATIVES

A summary of the compounds tested and the screens used is shoxr.i in table 1 (page 44). The screening results are displayed in tables 2-5 (Appendix 1 page 75).

In the herbicide tests,Merck's 'chlorflurecol methyl'(9) was used as a comparipon. 9,9-Bishydroxymethylfluorene(21) showed slight herbicidal activity in the preliminary(Phase 1) post-emergent screen and moderate activity in the pre-emergent screen. Esterification to improve penetration into the plant leaves by increasing the lipophilic character (compounds 79-82) reduced the activity in the post-emergent test.

43 COMPOUND SCREENS USED ACTIVITY methyl 2-chloro-9-hydroxyfluorene-9- carboxylate(9) (chlorflurecol methyl) H H 9,9-bishydroxymethylfluorene(21) H,I.F,N,C H,N 9,9-bisacetyoxymethylfluorene(79) H,I,F,A H 9,9-bischloroacetoxymethylfluorene(80) H,I,F,A,I,C H,N 9,9-bispropionyloxymethylfluorene(81) H,F H 9,9-bisbutyryloxymethylfluorene(82) H,I,F,N,A,C H 9,9-bismethylcarbaraoyloxyTnethylfluorene(83) H,I,F H 9,9-bisethylcarbamoyloxymethylfluorene(84) H,I,F H 9,9-bisphenylcarbamoyloxymethylfluorene(85) H,I,F,N,A,C H 9.9-sulphinyldioxymethylfluorene(86) H.I,F,N,A H 9,9-bismethylsulphonyloxymethylfluorene(87) H,I,F H 9,9-bistoluene-4-sulphonyloxymethylfluorene(88) H,I,F.V H 9-hydroxyraethyl-9-methylfluorene(89) H H 9-hydroxymethylfluorene-9-carboxylic acid(90) H,I,F H 9,9-bishydroxymethyl-2-nitrofluorene(93) H,I,F.A.C H 9,9-bishydroxymethyl-2-chlorofluorene(94) H H 9,9-bishydroxymethyl-2-bromofluorene(95) H H methyl 2-chloro-9-hydroxymethylfluorene-9- carboxylate(97) H H methyl-9,9-bi.shydroxymethylf luorene-4- carboxylate(99) H,I,F H

KEY: H = Herbicide I = Insecticide F = Fungicide N , Nematicide A = Anthelmintic C - Coccidiostat V = Vlrlcide

Table 1 Biological Screening of Fluorene Derivatives

44 In the pre-emergent test the results were similar but the bisacetoxymethyl compound(79) was only slightly less active than (21). The carbamate esters (S3-85) showed greatly reduced activity; in the pre-emergent test they were virtually inactive. The sulphur containing esters (86-88) also showed a definite decrease in activity. The hydroxyl groups thus appear to be essential for herbicidal activity. The miscellaneous 9-substituted compounds(89,90) confirmed this hypothesis. Removal of one hydroxyl group in 9-hydroxymethyl-9- methylfluorene(89) reduced its activity in both tests compared with the dipl(21). Oxidation of one hydroxymethyl group, as in 9-hydrox- methylfluorene-9-carboxylic acid(90), improved the herbicidal activity; the hydroxymethylcarboxylic acid(90) was more active than the diol(21). This improvement no doubt stemmed from the increased similarity to 'chlorflurecol'(9) which was still the most active compound. Ring substitution in positions 2 and 4(compounds 93, 94, 95, 99) made little difference to the activity in either emergent tests. Howeyer, methyl 2-chloro-9-hydroxymethylfluorene-9-carboxylate(97) whose structure is closest to that of the morphactins showed a much higher activity in both tests compared with the diol(21). Compared to 'chlorflurecol methyl' (9) it was still very slightly less active.

Of the compounds screened,. only thrce(21, 90 and 97) were considered sufficiently promising to warrant more exhaustive Phase 2 tests. In these tests methyl 2-chloro-9-hydroxymethylfluorene-9- carboxylate performed well enough to justify further interest. In view of its similarity to 'chlorfluorecol' further investigation depends on confirmation that it is patent free and whether there is any commercial advantage in its use.

The other Phase 1 screens tried, when sufficient material was available, were insecticide, fungicide, nemnticide, anthelmintic, coccidioFtat and viricide. In none of these tests was any significant aetivity noted except with 9,9-bishydroxymethylfluorene(21) and 9,9-bischloroacetox>TTiethylfluorene(80). The diol(21) showed slight to moderate activity as a soil ncmatlcide at the highest application rate of 250ppm. At lower rates it was not sufficiently active to warrant closer examination. Moderate phytotoxicity was noted dovm to 62ppm., as might be expected. The chloroacetoxymethyl derivative(80) also showed

45 some nematicide activity. For example, it exhibited moderate control of root-knot eelworm at 125ppm (score 5) and at 62ppm(score 3). In neither cose was this considered sufficient to justify Phase II tests.

46 4. CONCLUSION

9,9-Bishydroxymethylfluorene derivatives can be prepared in reasonable yields on a small scale by condensation of fluorene derivatives with paraformaldehyde, in ionising solvents such as dimethyl sulphoxide, using strong base. However, in some cases, particularly with 2-nitrofluorene and 2-acetylfluorene, the reaction was complicated by the formation of a radical species which effectively stopped any hydroxymethylation. These free radical species were deduced to be the corresponding fluorenone radical anions because of their ease of formation, stability, and the pattern of their e.p.r, spectr^- They are apparently produced by autoxidation of the carbanion of the fluorene derivative.

A suitable system which does not stabilise these radical anions appears to be acetonitrile, with aqueous benzyltrimethylammoniura hydroxide as base, purged with nitrogen gas to remove dissolved oxygen. In this-system, for example, reproducible yield? of 607« biFhydroxyraethyl-2-nitrofluorene were obtained on a lOg. scale. It would be interesting to investigate some of the other reactions in this solvent, particularly those that gave lower yields on scale up.

With 9-monorubstituted fluorenes, condensation with paraformaldehyde proceeded smoothly with none of the problems found with ring-substituted fluorenes.

The hydroxyl of the hydroxymethyl groups were generally quite reactive. For example, they were easily esterified. However, substitution by chlorine was found to be impossible with the conventional reagents used. This lack of reactivity was not obviously due to steric hindrance and remains to be explained.

Direct ring-substitution of the diol was not considered to be the best method of obtaining ring-substituted hydroxymethyl compounds because of the expected reactivity of the hydroxyl groups towards a variety of reagents. This problem could be avoided by protecting the hydroxyl groups (e.g. in the nitration of the diol with nitric/acetic acids, the hydroxyl groups were esterified).

47 As regards biological activity, the Merck product 'chlorflurecol methyl' was the most active compound examined. The screening results indicate that only 9,9-bishydroxymethylfluorene (21), 9-hydroxymethylfluorene- 9-carboxylic acid(90), and methyl 2-chloro-9-hydroxymethylfluorene-9- carboxylate(97) are sufficiently active to warrant Stage II testing.

It thus appears that a 9-hydroxymethyl group, a 9-carboxylic acid group, and a 2-chloro group all contribute to the herbicidal activity. In particular, the 9-carboxyl group is essential for a compound to exhibit high activity. Possibly, the mode of action of the diol 9,9-biEhydroxymethylfluorene(21) involves oxidation of a hydroxymethyl group to produce an intermediate morphactin-like substance.

Since none of the active compounds prepared in this study are obviously cheaper to produce than 'chlorflurecol', it is unlikely that they will be used commercially. Whilst the methyl 2-chloro compound(-97) appears to be patent free, because of its similarity to *chlorflurecol', any commercial interest will depend on advantages arising from its use..

48 5. EXPERIMENTAL 5.1 GENERAL PROCEDURES

The materials and reagents used were as received from B.D.H. Lunited unless otherwise stated. During many of the reactions, water was excluded from the system by means of a calcium chloride guard-tube. Organic solutions were dried using sodium sulphate and evaporated under reduced pressure using a rotary evaporator. The purity of product was determined by thin layer chromatography using the ascending technique with 0»25mm. layers of silica gel (Kieselgel G.F. 2541 Merck)activated at 100®. The components were located by ultra-violet light at 254nm. Brockmann Grade 1 Alumina was used for column chromatography. Melting points were determined in capillary tubes using an 'Electrothermal' apparatus and are uncorrected.

Elemental analysis facilities were provided by Fisons Limited, Agrochemicals Division. Infra-red spectra were recorded from potassium bromide discs using a Pye-Unicam SP200 or a Perkin Elmer 357 spectro• photometer. Ultra-violet/visible spectra were recorded with a Perkin Elmer 402 spectrophotometer and ethanol as solvent. Nuclear magnetic resonance spectra were obtained using a Perkin Elmer RIO (60 MHz) or a Perkin Elmer R32(100 MHz) spectrophotometer for solutions r2 "1 in Hg dimethyl sulphoxide with tetramethylsilane as internal standard Mass spectra were recorded using a Perkin Elmer-Hitachi instrument, at Exeter University.

Dry ethanol was prepared according to the method of Lund and Bjerrum as described by Vogel^^^^. Sodium ethoxide solution in dry ethanol was prepared according to Vogel and stored at 0" in darkness,

5.2 HYDROXYL MODIFICATIONS

1, 9,9-Bishydroxymethylfluorene^^'^^(21)

Paraformaldehyde(32g;l'07 mol) was suspended in dimethyl- 3 sulphoxide(400cm ), cooled to 15**, and stirred vigorously, A solution of sodium ethoxide in dry ethanol^^^^(24cm^;2*85 mol.dm was added to the paraformaldehyde suspension followed immediately by a suspension of fluorene(64g;0'39 mol., recrystallised from ethanol,mp.114-116, lit.116°^^^), in more_dimethylsulphoxide(400cra^)at 15°.

49 The temperature rose to 19**. After 2'5 rain, the reaction was stopped -3 3 by adding concentrated hydrochloric acid(ll*6 mol.dm ;24cm ) and 3 pouring the mixture into water(1600cra ), The resulting emulsion was saturated with sodium chloride( 280g) and the product extracted using ethyl acetate(2 x 750cra ), The combined ethyl acetate fractions were washed with water(3 x lOOOcm*^), dried, filtered, and the filtrate evaporated to dryness. The resulting solid was V7ashed with diethyl ether to give a product haveing a m.p. of 143-145*. This material was further purified by recrystallising it twice from benzene. The colourless needles of compound(21) thus obtained(60g:69%) had m.p. 144-146%(lit^^,145-5-146;5«). Found: °^/e 226;calc. for C^^^^^O^^ 226. V max. 3300(-OH),1070,1030(C - 0, C - C) 770,750cra"^(C-C aromatic). X max. 209(52,500) ;266nm( 6 25,800). X 6-1, 4H, singlet, (CH^); 5-7, 2H, singlet (OH); 2-3, 8H, multiplet,(ArH).

2, 9,9 Bisacetoxymethylfluorene

9,9-Bishydroxymethylfluorene(9'lg;0*04 mol) was dissolved in chloroform(250cm^) by warming and acetyl chloride( 13cm'^; 14'4g; 0*18 mol) was added dropwise with stirring. Pyridine(17cm^ ;14.4g; 0*21 mol) was also added, with caution). The solution was heated under reflux for 7 hours under dry conditions, then cooled, washed 3 with water(3 x 200cra ), dried and filtered. The filtrate was evaporated to dryness giving an oily product which was crystallised, from ethanol/vjater to give white crystals of 9,9-bisacetoxymethyIfluorene (ll*Og: 88%) having a m.p. 102-104**. Found: "^/e 310;C,73-85;.H,6'05; ^19^18^4 ^^^"^^^s C,73-5;H,5*81%. S) max. 1735(C=0), 1215 (C-O), 1040(C-C), 765,735^'"'^(C-C aromatic). X max. 210, (35 ,600) ;257nm (e 16,500)16,500).. T 7*9, 6H, singlet(CH^) ;5-6 ,4H, singlet(CH2) ;2-3 ,811, multiplet(ArH)

3. 9,9-BischloroacetoxymethyIfluorene

9,9-Bishydroxymethylfluorene(4"Og;0'02mol.) was dissolved in ethyl acetatedOOcra"^) by warming. After cooling to room temperature, chloroacetylchloride(4"5cm ,6'3g,0'06 mol) was added dropwise with stirring. The solution was heated under reflux for 2 hr. under dry conditions, then cooled and evaporated to dryness.

50 The oily product was redissolved in ether(SOcm*^) , set aside to crystallise, and the crystals isolated by filtration, and recrystallised twice from petrol(80-100" fraction). The white crystals of 9,9-Bischloro- acetoxymethyIfluorene (2-2g;34%) had m.p.106-107°. Found: C, 60-30; H,4-25;C1,18-40;C,^H, .Cl.O, requires C,60'2;R,4-22;C1 18^727o. max. ly lo ^ 4 1745(C=0),1170(C-0),990(C-C),760,730 cm (C-C aromatic). L5'9,4H, singlet(CH2);5-5,4H,singlet(ai2)2-3, 8H, multiplet(ArH).

4. 9,9-Bispropionyloxymethylfluorene

9,9-Bishydroxymethylfluorene(ll'3g;0*05 mol) was dissolved 3 • 3 in chloroform(250cra ) by warming propionyl chloride(20cm ;21'2g;0"23 mol) added dropvjise with stirring. The mixture was heated under reflux for 30 min. under dry conditions, then cooled, washed with water(3 x 200cm ), dried and filtered. The filtrate was evaporated to dryness leaving an oily product which slowly crystallised. The solid product was recrystallised twice from ethanol to give v/hite crystals of 9,9-bispropionyloxymcthyl- fluorene (14'lg;837o) having a m.p. 83 -84^ Found: C,74*95;H,6-85; Sl"22^4 ^^^"i^^s C,74-6;H,6-51%. V max. 1730(C=0).1175(C-0),755, 730 cm' (C-C aromatic).

5. 9,9-Bis-n-butyryloxvmethvlfluorene

9,9-Bishydroxymethylfluorene(9'Og;0'04 mol) was dissolved in chloroform(200cm'b by warming and n-butyryl chloride(17cm^;17.Ig;0,16mol) was added dropwise with stirring. Pyridine( 16cm"^; 16'Og;0'20 mol) was also added cautiously. The solution was heated under reflux for 7 hr. under dry conditions, then cooled, washed with water(3 x 200cm ), dried and filtered. The filtrate was evaporated to dryness giving an oily product which slov;ly recrystallised to give white crystals of pure 9.9-bis-n-butyryloxymethylfluorene (14'0g;95%) having a m.p. 74-77*. The melting point was not raised by further recrystallisation from methanol. Found: C,75•20:H,7-35;C^.,H^-0, requires C,75*5;H,7'11%. 2.S ZD 4 max. 1735(C=0),1155(C-0),765,733cm"^(C-C.aromatic).

51 6. 9a9-Bisformyloxymethylfluorene

Toluene-4-sulphonylchloride(3'lg;0'02 mol) was dissolved in dimethylformamide^*^'^^(25cm'*) and the solution heated to 100° for OOhr. 9,9-Bishydro::yraethylfluorene(l*0g;0*004ml.) was added and the mixture kept at 100" for 2 hr. The solution was then poured into waterdOOcm"^) 3 and extracted with ethyl acetate (2 x lOOcm ). The organic solution was washed with water(3 x 100cm ), dried and filtered. The filtrate was evaporated to dryness, leaving a colourless oily product which solidified upon re-evaporation with ethanol. The product was washed with ether and then recrystallised from petrol(80-100** fraction) to give white crystals of 9 3 9-bisformyloxyme thy If luorene (O'4g;357o) having a m.p. 99-101". Further purification by twice recrystallising from ethanol/water gave material with a m.p. 102-103". Found: °^/e 282; C,72'60;H,4-95;C^yH^^0^ requires C,72'4;H,4*97%. V max. 1710(C=0), II70(C-0),765,735cm"^(C-C aromatic).. X max. 209,(21,800);266nm (6 9,900). T; 5*4,4H, singlet(CH^) 2-4,8n,raultiplet(ArH) , 1-8,2H, singlet(HCO^) ^/e 282.(89) ,222(42) , 194(85) , 178(100) , 177(40) , 167(46) ,

165(39%); metastable peaks at 171 and 143.

7. Attempted Preparation of 9,9-Bistrifluoroacetoxymethylfluorene^^^

9,9-Bishydroxymethylfluorene(l*0g;0'004 mol) and pyridine (3cm^;3'Og;0*04 mol) were dissolved in chloroform(25cm'^)and molecular sieve (2"0g;type 4A) was added, followed by trifluoroacetic anhydride (5*Og:0*02 ml) dropwise with stirring. The mixture was heated under reflux for 1 hr. under dry conditions, then cooled, washed with water (3 X 20cm'^), dried and filtered. The filtrate was evaporated to dryness leaving an oily residue which was crystallised from methanol/water to give 9,9-bishydroxymethylfluorene (0'4g. 40%),m.p.143 - 145"(lit^^, 145-5 - 146-5"). V max. 3300(OH), 1065, 1025(C-0,C-C) 765, 735cm'"^ (C-C aromatic).

52 8. Attempted Preparation of 9,9-Carbonyldioxvmethvlfluorene

9,9-Bishydroxymethylfluorene(l'Cg;0'004 mol) was dissolved 3 in chloroform(50cm ) by warming under dry conditions. The reaction vessel was fitted with a dry-ice condenser and phosgene gas was bubbled through the mixture for 0'3hr. The solution was then set aside for 24 hr. at room temperature and finally evaporated to dryness. The 3 acidic oily product was redissolved in ethyl acetate(50cm ), washed with water(2 x 50cm ), dried over anhydrous potassium carbonate, and filtered. Evaporation of the filtrate produced an oil which :crystallised when treated with carbon tstrachloride/petrol. The small quantity of impure product isolated(m.p. 187'*{softens)) was insufficient for identification or further purification but its infra-red spectrum; max.,1740(C=0),1255,(C-0),730cm"^(C-C aromatic), was indicative of a carbonate.

9. 9i9-Bismethylcarbamoyloxymethylfluorene

9,9-Bishydroxymethylfluorene(ll*3g;0'13 mol) was dissolved in dry pyridine(100cm ) and isocyanatomethane(7-5g;0*13mol) was added.

The solution V7as then heated under reflux for 2 hours under dry conditions, 3 cooled and poured into waterdOOcra ). The organic material was extracted with ethyl acetate (4 x lOOcm^), washed with water(2 x 200cm^), dried and filtered. The filtrate was evaporated to dryness and the oily product re-dissolved in ether and left to crystallise. The solid product was isolated by filtration and further purified by recrystallisation from benzene/petrol to give white microcrystals of 9,9-bis'methylcarbamoyloxy- methylf luorene (2-4g;14%),m.p.l78-180*'. Found: °^/e 340,C,67-30;H,6-05; N,8-50;C^^H2qN20^ requires C,67-l;H,5-88,N,8-247o. max. 3350(NH); 1690(0=0, amide I band); 1550,1270(N-H,C-N, amide II,III bands);1145(C-0); 735cm ^,(C-C aromatic, secondary amide IV band). X max. 208,(45,500); 257nm( e 21,500). T 7*4,6H,doublet(CH^,J==4Hz); 5-7,4H,singlet(CH2); 2-0,2H, broad singlet(NH);2-3,8H, multiplet(ArH). °^/e 340(6) 195(8),179(20),178(100),177(8),165(8),58(17),57(9%); metastable peaks at 143 & 94.

53 10. 9,9-Bisethylcarbamoyloxymethylfluorene

9,9-Bishydroxymethylfluorene(3.5g;0.02mol) vjas dissolved in dry pyridine(40cm^) and isocyanatoethane(2•5g;0"03mol) was added. The mixture was heated under reflux for 2hr. under dry conditions, cooled and evaporated to dryness. The solid product was washed with ether and recryst allised from ethyl acetate/petrol(40-60**) to give white microcrystals of pure 9,9-bisethylcarbamoyloxymethylfluorene (3*lg);55%), m.p.211-215". Found: C,68-75;H,6*65;N,7*80;C2^H2^N20^ requires C,68-5;H,6*52;N,7*61%.

max.3400(NH);1690(C=0,amide I band);1540,1250(N-H,C-N,amide II,III bands); I030(C-0);740cm ^(C-C aromatic, secondary amide IV band)

11. 9,9-Bisphenylcarbamoyloxymethylfluorene

9,9-Bishydroxymethylfluorene(4*0g;0'02 raol) was dissolved in 3 3 dry pyridine(15cm ) and isocyanatobenzene(4cm ;4'4g;0*04mol) was added. The mixture was heated under reflux for l*5hr. under dry conditions, cooled and evaporated to dryness. The solid product was recrystallised from chloroform to give white microcrystals of chromatographically pure 9.9-bisphenylcarbamoyloxymethylfluorene(5'5g;69%) of m.p.227-229". Found:C,74*7;H,5*00;N,6-30;C2gH2^N20^ requires C,75*O.H,5*15;H,6-05%.

Rp(ethyl acetate)0*74. ^ max.3380(NH);1690(C=0,amide I band;1535,1220

(NH,C-N,amide II,III bands);1060(C-0);740cm ^(C-C aromatic, secondary ami XV band). T5-5, 4H,singlet(CH.);2'4,20H,complex multiplet(ArH,NH,Ph-H).

12. 9 ,9-Bis-3'-chlorophenylearbamoyloxymethylfluorene

9,9-Bishydroxymethylfluorene(l'5g,0"01mol)was dissolved in 2 chloroform(25cm ) by warming and molecular sieve(2'Og;type4A) was added. The mixture was allowed to stand at 25** for 0*5hr. under dry conditions, isocyanato-3-chlorobenzene(7.2g^0.05raol)was added and the solution was heated under reflux for 5 hours. It was then cooled, and the solid product which precipitated was isolated by filtration, washed with petrol and recrystallised from methanol/water to give white microcrystals of chromatographically pure 9,9-bis-3-chlorophenylcarbamoyloxymethylfluorene (2-5g;677o),m.p.l53-155**. Found: C,65-05;H,3-90;CI, 13-45;N,5-30; ^2s\2^^'^2^i^ C,65,3;H,4.13;C1.13.3;:;,5,26%.

54 Rp(chloroform)0*68. >) max.3350(NH);1695(0=0,amide I band);1590,

1540(NH,CN,amide II bands);1275,1225(NH,CN,amide III bands);1060(C-0); 760 cm (C-C aromatic, secondary amide IV band).

13. 9,9-Sulphinyldioxymethylfluorene

9,9-Bishydrox>Tnethylfluorene(22'6g;0'10 mol) was dissolved in chloroform(450 cm ) by warming and redistilled thionyl chloride(8 13'lg;0'll mol) was added drcpwise with stirring under dry conditions. The solution was then kept at 25* for 48 hr., washed v/ith water(3 x 250cra ) containing sodium hydrogen carbonate,dried and filtered. The filtrate was evaporated to dryness and the solid product recrystallised twice from carbon tetrachloride to give cream coloured crystals of 9,9-sulphinyl- dioxyroethylfluorene (20-0g;73%) with m.p.161-162*. Found: ^/e 272;C, 66*05;H,4'60;S,11*50:C^^H^2*^2S requires C,66*2;H,4-41: S^l*77%. Rp(carbon tetrachloride)0'52. ^ max. 1450,1180(S = 0 sulphite); 990,930(S-0,C-0); 740 cm ^(C-C aromatic) (cp.1,2-sulphinyldioxyethane, max.1200,1020, 930 cra'^).

14. Hydrolysis of 9j9-Sulphinyldioxymethylfluorene

9,9-SulPhinyldioxymethylfluorene(2'0g;0*01 mol) was dissolved in dioxan(50 cm ) and sodium hydroxide(0'8g;0'02 mol) in aqueous ethanol (10 cm ,70% ethanol) was added. The mixture was heated under reflux for 2 hr., cooled, evaporated to dryness and redissolved in ethyl acetate 3 3 (100 cm ), The organic solution was washed with water(3 x 100 cm ) dried, filtered, and evaporated to dryness. The solid product was recrystallised from benzene to give colourless needles of 9,9-bishydroxymethyIfluorene (l*5g;887o), m.p.l44-146*;( mixed m.p. with authentic 9,9-bishydroxymethylfluorene 143-145*. ^ max,3300(OH);; 1060,1020(C-0,C-C); 760,735 cm'^ (C-C aromatic).

15. Attempted Preparation of 9-chloromethyl-9-hydroxymethylfluorene

9,9-Sulphinyldioxymethylfluorene(l'0g;0'004 mol) was dissolved in dioxan(50 cm ) and heated under reflux for 3 hours under dry conditions whilst dry hydrogen chloride gas was bubbled through the solution. The solvent was removed by evaporation and the product isolated by extraction with ethyl acetate(100 cm^).

55 The organic solution was washed with water( 3 x 100 cm*^), dried, filtered and evaporated to dryness. The solid product was identified by infra• red spectroscopy as unchanged 9,9-sulphinyldioxymethylfluorene (l»Og; 100%). The identity of the product was confirmed by recrystallising it twice from carbon tetrachloride, the chromatographically pure material had a m.p. of 161-163°. RF(carbon tetrachloride)0'52. ^ max.1450, 1180(S=0 sulphite):990,930(8-0,C-0) 735 cm'"^(C-C aromatic)

16. Attempted Preparation of 9,9-Bischloromethylfluorene a) with phosphorus pentachloride

9,9-Bishydroxymethylfluorene(l*0g;0'004 mol) was dissolved in carbon tetrachloride(25 cm*^) by warming and phosphorus pentachloride (2*0g;,0'01 mol) was added. The mixture v/as heated under reflux for 3hr. under dry conditions, cooled, washed with aqueous sodium bicarbonate (3 x 20 cm''), dried and filtered. The filtrate was evaporated to dryness and the solid product recrystallised from benzene/petrol to give material(0*2g) of melting point 185-186** which contained phosphorus and chlorine. Found: ^/e 513,C,70-35;H,4-85;C1,6*85;P,-.C^^H^^^^^a requires °^/e 263;C,68'44;H,4-56;C1,27"0%. Rp(carbon tetrachloride) 0*02(impurity);0'70(compound). ^ max. 1300(P=0);1040,1030(C-0,C-C, P-0-alkyl),730 cm''^(C-C aromatic,C-Cl) . °^/e 513(20) ,226(24) , 192( 191(78),179(18),178(100),177(10%), metastable peak at 161. b) with hydrogen chloride

9,9-Bishydroxymethylfluorene(2'0g;0*01 mol) was dissolved in dioxan(50 cm^) and anhydrous zinc chloride(5•2g;0"04 mol) added. The mixture was heated under reflux for 1*5 hr, under dry conditions whilst dry hydrogen chloride gas was bubbled through it. The solvent was removed by evaporation and the product isolated by extraction with ethyl acetate(50 cm^). The organic solution was washed with water (3 X 25 cm ), dried, filtered and evaporated to dryness. The oily product was extracted with diethyl ether to remove any diol but it could not be purified further by crystallisation. Gas chromatography of the ether soluble fraction indicated a mixture. G.L.C.,SE30 column, 160** major component 7-5 minutes, two minor components, ">) max,3500(0H); 1715(C=0);1450(C-C);1130(C-0);1030(C-C);765 ,735 cm""*-(C-C aromatic).

56 17. 9,9-Bismethylsulphonyloxymethylfluorene

9,9-Bishydroxymethylfluorene(ll»3g;0-05 mol.) was dissolved 3 3 in pyridinedOO cm ) and methanesulphonyl chloride was added(17»2 cm ; 25*3g;0*22 mol) dropwise with stirring. The mixture was heated under reflux for 0*5 hr. under dry conditions, poured into water(300 cm^) 3 and extracted with chloroform(2 x 200 cm ). The chloroform solution was washed with water(2 x 200 cm ) dried, filtered and evaporated to dryness. The oily product was recrystallised 3 times from methanol to give colourless needles of pure 9,9-bismethylsulphonyloxymethylfluorene (6'9g;36%)m.p. 163 - 164",decomp.l67". Found: C,-;H, 5-00,S,16-55;C„H,oO^S^ requires C,53-4;H,4*72;S,16-75%. V max. 1350, 1/ iO D ^ 1170(S=0 covalent sulphonate);950 cm' (S-0,C-0), 6'9,6H, singlet (CH^);5'4,4H,singlet(CH2);2-2,8H,multiplet(ArIi),

18. 9,9-Bistoluene-4-sulphonyloxymethylfluorene

9,9-Bishydroxymethylfluorene(A-8g;0-02 mol) was dissolved 3 in pyridine(125 cm ) and toluene-4-sulphonyl chloride added(16-lg;0-08 mol) with stirring. The mixture was heated under reflux for 2 hr, under dry conditions, poured into water(500 cm"^) and extracted with ethyl acetate(2 x 200 cm"^). The ethyl acetate solution was washed with water 2 (3 X 200 cm ) dried, filtered and evaporated to dryness. The solid product was recrystallised twice from ethanol to give colourless needles of 9,9-bistoluene-4'-sulphonyloxymethylfluorene(7'8g;69%),m.p,138-139"> decomp. 171". Found:C,65-0;H,4-95;S,ll-85:C^„H^,0.S^ requires 0,65*2, H,4-87,S,12*00%. S) max.1360,1175(5=0 covalent sulphonate),970 (S-0,C-0),865 cm'^(CH aromatic, para-disubstitution).

Q7a 19. 9-Methylfluorene

A high pressure autoclave was charged with a solution of sodium(10-2g;0-44 mol) and recrystallised fluorene(50g;0*30 mol) in absolute methanol(200 cm"^). The apparatus was then sealed, stirred and heated to 230* for 16 hr. It was then cooled and dismantled. Ethyl acetate(500 cm^) v/as added and the solution was washed with water (2 x 300 dn"^), dried, decolourised with charcoal and evaporated to dryness. The solid product was recrystallised twice from methanol to give colourless crystals of 9-methylfluorene(42-6g;79%) of melting point

57 44-46''(lit.^^^ 46-47**). V max. 1445(CH;765,735 cm"^(C-C aromatic), ^ 8-5, 3H,doublet,(CH^J=7Hz);6-0,lH,quadruplet(CH,J=7Hz);2-4,8H, multiplet (ArH).

20. 9-Hydroxymethyl-9-methyIfluorene a) 9-Methylfluorene(l'0g;0*01 mol) was dissolved in hexamethylphosphorictriamide(25 cm"^) and stirred at 2** while nitrogen gas was bubbled through for 0-5 hr. Parafonnaldehyde(0*23g,0*01 mol.) was suspended'in more hexamethylphosphorictriamide(20 cm^) and sodium ethoxide in dry ethanol(0'6 cm ,2»85 raol-dm ) was added. This mixture was then added slowly to the solution of methylfluorene. After 2*3 min,, the reaction was stopped by adding concentrated hydrochloric acid 3-3 3 (0'6 cm ;11'6 raol .dm ) and pouring the mixture into water(250 cm ). The product was extracted into ethyl acetate(2 x 50 cra"^) washed with water(3 x 50 cm"^), dried and filtered. The filtrate was evaporated to dryness and the oily product was recrystallised from petroKlOO-120** fraction) to give pure 9-hydroxymethyl-9-methylfluorene(l'Og;87yo) , m.p.146-150*. Found °^/e 210;C,85*6,H,6-90:C^^ H^^ 0^ requires °^/e 210;

C,85-7;H,6-67%. max .3400(OH);1450(CH);1060,1040(C-0,C-C);735 cra'^ (C-C aromatic) .t8-5,3H,singlet(CH^);6-3,2H.singlet(CH2);5-8,lH,broad

singlet(OH);2-4,8H,raultiplet(ArH). °^/e 210(22),180(46),179(100),178 (36),165(16%).

b) 9-Methylfluorene(10*8g;0*06 mol)was dissolved in dimethylformamide(140 cm^) and stirred at 2" while nitrogen gas was bubbled through for 0'5hr. Paraformaldehyde(2'5g.0'08 mol.) was suspended in more diinethylformamide(140 cm^) and sodium ethoxide in dry ethanol 3 3 {2'5 cm ; 2*85 mol.dm ) was added. This mixture was then added slowly to the methylfluorene solution. After I'Smin. the reaction was stopped by adding concentrated hydrochloric acid(2'5 cm'^;ll*6 mol. -3 3 dm ) and pouring the mixture into waterdOOO cm ). The product was extracted with ethyl acetate(2 x 200 cm^) washed with water( 3 x 100 cm^) dried and filtered. The filtrate was evaporated to dryness and the product recrystallised from benzene to give 9-hydroxymethyl-9-methylfluorene(10*8g;85%),m.p.146-149.

58 21. Benzilic Acid^^^

Sodium hydroxide(58*0g;1'44 mol) and sodium bromate(13*2g; 0*09 mol) were dissolved in water(100 cm^) and ben2oin(53'0g;0'25 mol) was added slowly with stirring. The mixture was then heated to 85* for 5 hr. As the reaction proceeded, the mixture thickened and more water(90 cm^) was added portionwise, as appropriate. When a test sample was completely water soluble, the mixture was diluted with water(600 cm ) and kept at 25* for 12 hr. The mixture was filtered to remove impurities and dilute sulphuric acid(160 cm"^ of 3:1/water:acid) was added to the filtrate to a point short of the liberation of bromine. The precipitate was isolated by filtration, washed with water, and dried to give benzilic acid(52»9g;937o) with m.p. 148-150*. This was purified by recrystallisation from benzene to give product of m.p. 150-151*(lit]'^^150°).

97c 22. Fluorene-9-carboxylic acid Benzilic acid(45"6g;0'2 mol) was suspended in anhydrous thiophene free benzene(700 cm"^) with stirring under dry conditions until a crystalline mass resulted. Anhydrous aluminium chloride(80*Og;0*6 mol) was added and the stirred mixture was heated under reflux for 3 hr. The solution was cooled and decomposed by the cautious addition of small pieces of ice followed by cold water(400 cm ) and then concentrated hydrochloric acid(200 cm^;ll*6 moL dm ^). Benzene was removed by evaporation and the product isolated by filtration. The lumps of product were crushed and extracted twice with boiling aqueous sodium carbonate 3 -3 (2 X 300 cm ;0*94 mol dm ). The mixture was filtered, cooled, and the filtrate was acidified with cold concentrated hydrochloric acid. The solid product was isolated by filtration, washed with water until the filtrate was neutral, dried, and recrystallised from acetic acid to give - cream crystals of fluorene-9-carboxylic acid(23•5g;567o),m.p. 230-232* (lit.^^^ 230-231*).

14 23. 9-Hydroxymethylfluorene-9-carboxylic acid Fluorene-9-carboxylic acid(17*2;0'08 mol) was mixed with 3 3-3 methanol(41 cm ) and aqueous sodium hydroxide(410 cm ;2'5 mol , dm ) 3 -3 followed by formaldehyde solution(205 cm );13*3 mol .dm ). The mixture was kept at 25* for 96 hr. then cooled to 0*, first neutralised and then acidified with cold concentrated hydrochloric acid(ll»6 mol .dm ).

59 The solid product which separated v;as isolated by filtration, washed with water until the filtrate was neutral, dried and recrystallised from benzene to give crude 9-hydroxymethylfluorene-9-carboxylic acid (ll"6g;597o) with m.p. 156-160*. A further four recrystallisations, from chloroform gave pure material(6•2g;31%) , m.p. 157-5-158*5**(lit. 157- 158*). Found: °^^e 240;C,75-15;H,5*00;calculated for (^i^\2'^3 ^/e 240'

C,75*0;H,5*00% . T 6'l,2H,singlet(CH2);2-4,9H,multiplet(ArH, H bonded OH).

°^*"/e 240(2)210(35) ,166(52) ,165(100) ,164(31) ,163(23) ,29(19%) , metastable peak at 131.

24. Methyl Fluorene-9-carboxylate^^''^

Fluorene-9-carboxylic acid(21*0g;0*l raol) was suspended in dichloromethane(200 cm"') and methanol(9*6g;0'3 mol) was added followed by concentrated sulphuric acid(l'5 cm^;10*0 mol. dm ^). The mixture was heated under reflux for 10 hr., cooled, washed with aqueous carbonate 3-3 (2 X 100 cm ;0'94 mol.dm ), dried and filtered. The filtrate was evaporated to dryness and the product recrystallised from petrol (40-60* fraction) to give methyl fluorene-9-carboxylate(16*Og;71%) with m.p. 64-5-65'5*(lit.^^^® 64-65*). X max. 209(36,800);265nm ( e 18,500).

25. Methyl 9- Hydroxymethylfluoren3-9-carboxylate

Methyl fluorene-9-carboxylate(1'Og;0*004 mol^ was dissolved in acetonitile(25 cm^) and cooled to 2* whilst nitrogen gas was bubbled through for 0*3 hr. Paraformaldehyde(0'2g;0'004 molO was suspended in 3 3 more acetonitriledO cm ) and aqueous benzyltrimethylammonium hydroxide(0.5cm , 2.6mol.dra-^) added. The suspension was then added slowly to the methyl fluorene-9-carboxylate solution. After 2 min., the reaction was stopped by adding concentrated hydrochloric acid(0'5 cm ,11-6 mol. dm ), the solution was filtered, poured into water, and kept at 25* for 12 hr. The product was isolated by filtration, washed, dried and recrystallised three times from petrol(60-80* fraction) to give methyl 9-hydroxymethyl fluorene-9-carboxylate(0-2g;18%) with m.p. 119-120*. Found: C,75-58:H, 5-85;C,^H,,0^ requires C,75-6;H,5-51%. ^ max.3840(OH);1725(C=0) ;1450 lb J (CH);1235(C-0);735 cm (C-C aromatic) A max.210(29,500);267nm(6 15,000). T 6-4,3H,singlet(CH^);6-l,2H,doublet (ai2,j=9 Hz);4-6,1H, triplet(0H,J=9 Hz); 2*4,8H,multiplet(Ar-H).

60 26. 9,9'Bis-l' - Hydrox>ethylfluorene^^

Fluorene(2*0g;0*01 mol) was dissolved in dimethylformamide (40 cm ) and cooled to -15" whilst nitrogen gas was bubbled through for 0*5 hr. Acetaldehyde(2 cm ;l*6g;0*04 mol) was dissolved in more dijnethylformamide(40 cm ) and cooled to -15". Sodium ethoxide in dry ethanold cm ;2'85 mol. dm ) was added to the fluorene solution followed by the acetaldehyde solution. More sodium ethoxide solution (3 cm ; 2*85 mol. dm ) was added as the colour of the solution faded until no more colour was produced. The reaction was stopped by pouring the mixture into dilute hydrochloric acid(500 cm ) and the product was extracted with ethyl acetate(2 x 200 cm*^). The ethyl acetate solution was washed with water(3 x 100 cm^), dried, and filtered. The filtrate was evaporated to dryness and the oily product crystallised from benzene to give colourless crystals of 9,9-bis-1'-hydroxyethylfluorene (0*36g;12%) with m.p. 155-157"(lit.157-158"). Further recrystallisation from benzene gave material of m.p. 157-158". max.3380(0H),1450(CH), 1130,1085,1050(C-0,C-C) ;755,735 cm'"^(C-C aromatic). X 5 • 13,4H,multiplet (0H,CH,J=7 Hz);2-4,8H,multiplet(ArH).°*^/Hz);2-4,8H,multip e 254(1)^93(18) ,192(100) ,191 (21),166(24).165(31),43(13%) .

5*3 RING SUBSTITUTED DERIVATIVES

27. 9,9'Bisacetoxymethyl-2-nitrofluorene

9,9-Bisacetoxymethylfluorene(12*4g;0*04 mol) was dissolved 3 in acetic anhydride(150 cm ) and cooled to 10". Concentrated nitric acid(5 cm^;ll'l mol.dm ^) was added dropwise with stirring over 0*5 hr at 10". The mixture was kept at 10* for a further hr.then poured into ice/water(1000 cm^) and stirred until all the acetic anhydride had hydrolysed. The product was isolated by filtration, dried and recrystallised from ethanol to give yellow crystals of 9,9-bisacetoxymethyl-2-nitrofluorene (11•2g;79%)/n.p.115-116". A further recrystal• lisation from petrol(80-100° fractipn)gave material with m.p. 113-119". Found: ^/e 355;C,64*10,H.4'85;N,4-05;C^gH^^NO^ requires ^/e 355;C, 64*3;H,4-78;N,3-94%. V max.l730(C=0);1520,1335(N02);1245(C-0);1040 cm'^(C-C). X max.207(31,100) ;256(11,200) ;325nm( e 13,600). T8-1,6H, singlet(CH2);5'5,4H singlet(CH2);2-0,7H,raultiplet(ArH). °^/e 355(10); 224(24);223(80);177(8);43(100%),

61 ft") 28- 2-Nitrofluorene

Recrystallised Fluorene(30g;0*18 mol)v;a5 suspended in glacial acetic acid(250 cm ) and warmed to 50* when concentrated nitric acid(40 cm'^;ll"l mol .dm ^) was added dropwise with stirring over 0*3 hr. The temperature was raised to 85** and held there for 5 mln.; the solution was then cooled. The solid product was isolated by filtration, washed with cold glacial actio acid, water then dried, and recrystallised from ethanol to give pure 2-nitrofluorene(31'0g; 907o),m.p.l61-162«(lit. ^^157«). ^ max. 1525(N02) ,1340(NO2) .750 cra'^

(C-C aromatic). X max.207(28,500) ;233(9,500) ;330 nm( e 20,800).

29. 9>9'Bishydroxymethyl-2-nitrofluorene

a) 9,9-Bisacetoxymethyl-2-nitro£luorene(2'0gi0'006 mol) was dissolved in acetone(50 cm^) and dilute hydrochloride acid(20 cm^, 1-4 mol.dm ^) was added. The solution was heated under reflux for 6*5 hr. poured into water and the product isolated by filtration. The material was recrystallised from benzene to give pale yellow crystals of 9,9-bishydroxymethyl-2-nitrGfluorene (0-95g;587o),m.p. 167-169". Found "^/e 271;C,66•7,H,4-85;N,5-00;C,^H,^NO, requires m+ 15"13 4 /e 271;C,66-5,H,4-80;N,5-1770. N max.3340(OH) , 1520(N02) 1340(N02)

1025(C-0,C-C),740 cm'"^(C-C aromatic). X max.207(21,200) ;235(5,600) ; 332 nm(e 11,500). °*^/e 271(6),224(22),223(100),194(20) , 193(26),177 (20),166(18),165(48%),metastable peak at 141 b) 2-Nitrofluorene(l'0g;0'005 mol)previously purified by column chromatography, was dissolved in hexamethylphosphorictriamide(30 cm*^) and stirred at 3** vjhilst nitrogen gas was bubbled through for 0*5 hr. Paraformaldehyde(0-5g;0'015 moL) was suspended in more hexamethylphos- 3 3 phorictriamide(10 cm )and sodium ethoxide in dry ethanol(l an ,2*85 nol. dm •^)was added. The suspension was added to the 2-nitrofluorene solution and the mixture stirred for 2*3 minutes. Concentrated hydro• chloric acidd cra^;ll*6 mol. dm ^) was added and the mixture poured into water(250 cm^). The product was extracted with ethyl acetate(100 cm*^), washed with water(3 x 100 cm*^), dried, filtered and evaporated to dryness The solid product was recrystallised from benzene to give pure 9,9-bishydroxyraethyl-2-nitrofluorene (l.Og; <707o, variable)with m.p. 169-170".

62 c) 2-Nitrofluorene(8*0g:0-04 moLJwas suspended in acetonitrile (240 cm"^) and stirred at whilst nitrogen gas was bubbled through for 0*5 hr. Paraformaldehyde(4'0g;0'12 moljwas suspended in more acetonitriledO cm )and an aqueous solution of benzyltrimethylammonium hydroxide(2 cm ,2*6 mol.dm )was added. This suspension was added slowly to the 2-nitrofluorene suspension. After 2*5 min.,concentrated hydro• chloric acid(2 cm"^,ll'6 mol.dm ^) was added and the mixture poured into 3 3 water(2000 cm ). The product was extracted with ethyl acetate(500 cm ), washed with water(3 x 250 era ),dried, and evaporated to dryness. The solid product was recrystallised from benzene to give 9^g-bishydroxymethyl-2-nitrof luorene (8'0g;717o) , m.p. 167-168**.

30. 2-Amino-9,9-bishydroxymethyIfluorene

a) 9,9-Bishydroxymethyl-2-nitrofluorene(4*Og;0'015 mol)was dissolved in ethanol(50 cm^)and palladised charcoal(0'02g;10% palladium) added follo^^ed by hydrazine hydrate(0'5 cm'^;0"08 moL) . The mixture was heated under reflux for 6 hr. and the samples were tested at intervals for the presence of nitro-groups using titanium trichloride solution. More hydrazine hydrate(l'0 cm ;0'16 mol.)and palladised charcoal(0"02g)were added after 2 hrs. The mixture was cooled, filtered and evaporated to dryness. The solid product was washed with chloroform and dried, giving 2-amino-939-bishydroxymethylfluorene (2'7g;74%),in.p. 216-218". Further recrystallisation from benzene gave material with m,p.218-220**. Found: °^e 241;C,74'5;H,6-35,N,6-00;C^^H^^N02 requires '"^ 241; C, 74-7 ,H,6' 23 ,

N,5'81%. ^ max.35O0(NH2),34OO(OH)162O(NH2),146O(OH),1035,1025(C-0,C-N),

745 cm'^(C-C aromatic). X max.210(27,300)293 nm(e 20,100). "^/e 241(97), 210(23^194(20),193(100)182(34),181(34),180(49),165(34%). b) 9,9-bishydroxymethyl-2-nitrofluorene(4*0g;0*015 mol) was dissolved in ethanoKlOO cm'')by v/arraingand palladised charcoal(0-02g; 10% palladium)added. The mixture was purged with nitrogen gas and then hydrogen gas was bubbled through slowly for 5 hr. The solution was then filtered and the filtrate evaporated to dryness. The solid product was washed with chloroform to give 2-amino-9,9-bishydroxymethylfluorene (3'0g;86%)with m.p.208-212**. Further recrystallisation from benzene gave material with m.p.218-220**.

63 31. 2-Amino-9,9-bishydroxymethylfluorene Toluene-4'-sulphonate

2-Amino-9,9-bishydroxymethylfluorene(2'7g;0*01 mol)was dissolved in ethylacetace(100 cm^). To this solution was added an excess of a solution of toluene-4-sulphonic acid in ethyl acetate. The white precipitate was isolated by filtration,dried and recrystallised from methanol/ethyl acetate to give colourless plates of 2-ainino-9,9- bishydroxymethylfluorene toluene-4*-sulphonate (l'0g;24%),m,p.231-233*. Found: C,63• 75;H,5•75;N,3-20;S»7-80:requires C,64-0;H,5-58;

N,3'39;S,7-75%).

32. 2-Amino-9,9-bishydroxymethylfluorene Hydrochloride

2-Ainino-9,9-bishydroxymethylfluorene(15*3g;0*O6 mol)was dissolved in methanol(200 cm"^) and cooled toO*. Hydrogen chloride gas (17'lg;0'47 moDwas then bubbled through the solution for 5 mins. The solution was decolourised with activated charcoal and the filtrate evaporated to dryness. The product was recrystallised three times from methanol/ethyl acetate to give 2-amino-9a9-bishydroxymethyIfluorene hydrochloride (5-6g;32%) ,ra.p. 251** decomp. Found: C,64-5;H,5'85;C1, 12*85;N,5'10;C^3Hj^^Cl NO^ requires C,64'9;H,5• 77 ,C1,12-8,N,5'04%.

33. 2-Chloro-9,9-bishydroxymethylfluorene

2-Amino-9,9-bishydroxymethylfluorene hydrochloride(6•Og;0*022 3 "3 moDwas dissolved in dilute hydrochloric acid(34 cm ;1*4 mol.dm ) and 3 -3 cooled to 5^. Aqueous sodium nitrate(30 cm ;0*83 mol.dm )was added dropwise with stirring. The solution was tested with starch/iodine paper and found to be positive. Cuprous chloride(2*9g;0*022 mol) was dissolved in concentrated hydrochloric acid(12 cm ;11'6 mol.dm )and cooled below 5**. To this solution was added the cold diazonium salt solution, dropwise with stirring, maintaining the temperature below 10**. The mixture was kept beloT.7 lO** for 0*5hr. then allowed to warm to 25®. The product was extracted with ethyl acetate(200 cm^)washed with dilute hydrochloric acid(100 cm ), water, dilute sodium hydroxide (100 cm ), water, then dried, filtered and the filtrate evaporated to dryness. The solid product was dissolved in ethanol and treated twice with activated charcoal;then the solution was re-evaporated.

64 The product was recrystallised from benzene Co give 2-chloro-9 bishydroxymethylfluorene (5*0g;89%) ,ra.p. 162-163**. Found: ™^/e262; C,69-5;H,4-75;C1,13'30;C^3 H^^ CI 0^ requires "^/e 262;C,69-1;H,

4*99;C1.13'6%. V max. 3330(OH),1295 cm"^(OH,C-0). X max.209(39,600); 272(26,000),305 nm.(e 8,400). T 6'2, 4H,singlet,CH^;5•1, 2H,triplet (OH, disappears on D^O addition;J = 6 Hz);2-4.7H,multiplet(Ar-H).

"^/e 262(5),260(16),214(34),213(16),212(100),194(27),166(23),165(57), 163(14%),metastable peaks at 136, 143 and 148.

34. 2-Bromofluorene^^

Recrystallised fluorene(35*Og,0'21 mol.) was dissolved in chloroform(300 cm ) and cooled to 2**. Bromine(37*2g;0*23 mol) was added over 0*5 hr. and the solution kept at 25* for 48 hr. The solution was then washed with water until the water was neutral, dried, filtered, and the filtrate evaporated to dryness. The solid product was recrystallised twice from othanol to give pure 2-bromof luorene( 16'0; 74%) ,ra. p.99-102** (lit. ^^"^ 110-111.5*).

35. 2-Bromo-9 j9-bishydroxymethylfluorene

Recrystallised 2-bromofluorene(l'0g;0'004 mol) was dissolved In dimethylformamide(25 cm ) and cooled to 2® whilst nitrogen gas was bubbled through with stirring for 0*3 hr. Paraformaldehyde(0'6g;0"02 mol.) was suspended in more dimethylformamide(5 cm^) and cooled to 2°. To this was added aqueous benzyltrimethylaramonium hydroxide(0'5 cm'',2*6 mol.dm the resulting suspension was added slowly to the bromofluorene solution. After 1 min. the reaction was stopped by adding concentrated hydrochloric. acid(0"5 cm"',11*6 mol, dm ^) and pouring the mixture into water( 100 cm*'). The product was extracted into ethyl acetate(2 x 25 cm^), washed with water(2 x 25 cm"'),dried, filtered and the filtrate evaporated to dryness. The oily product crystallised and the solid was recrystallised three times from benzene to give c:olourless crystals of 2-bromo-9,9-bishydroxymethylfluorene (0-34g;27%) ,m.p. 149-151**. Found: "^/e306;C,58-85;H,4-40,Br, 26-00,0^^ H^^BrO^ requires ^/e 306;C,59*0,H,4-26,Br,26-2%, T 6-3,4H, quadruplets(CH-,J ^„ =2Hz ,J(unknown)6 Hz);5•1,2H,triplet(0H,J=7Hz);

Z OH LHI 2-4,7H,complex multiplet(Ar-H). "^/e 306(14),258(76),256(74),195(20)194 (51),166(40),165(100).163(21%) ,metastable peaks at 216,142,122,111.

65 112 36- 2,7-DiacetyIfluorene

2,7-Diacetyl£luorene was isolated as a by-product from the preparation of 2-acetylfluorene by the Friedel Crafts reaction of acetic anhydride on fluorene. The crude 2-acetylfluorene was dissolved in carbon disulphide and the solution filtered. The residue was recrystallised twice from ethanol to give 2,7-diacetylfluorene(3*4g;5'7%). m.p.181-182* (lit.^^^ 183-184*). X max.209(29,900),325 nm(£ 35,500).

37. 2,7-Diacety1-9,9-bishydroxymethyl£luorene

2,7-Diacetylfluorene(l"0g;0'004 mol) was dissolved in hexamethylphosphoric triamide(30 cm^)and cooled to 3*. The solution was stirred and nitrogen gas was bubbled through for 0'3 hr. Paraformaldehyde(0'33g;0*01 moDwas suspended in more hexamethylphosphorictriamide(10 cm^) and sodium 3 -3 ethoxide in dry ethanol(0*5 cm ,2*85 mol. dm )was added. This suspension was added slowly to the diacetylfluorene solution. After 2*5 min. the reaction was stopped by adding hydrochloric acid(l cm^;ll*6 mol.dm ^) and pouring the mixture into water(250 cm ). The product was extracted with 3 3 ethyl acetate . (2x 50cm ) and the solution V7as washed with water(2 x 50cm ), dried, filtered and the filtrate evaporated to dryness. The solid product was recrystallised from benzene to give pale brown needles of 2,7-diacetyl- 9,9-bishydroxymethylfluorene (0-3g;24%), m.p.216-218'*. Found: °^/e 310; C,74-0;H,6-0,C^^H^gO^ requires °^/e 310;C,73'6;H,5-81. 'O max.3450(0H),

1730(C=0),1615(C-C arDraatic),1470(CH,OH)1300 cm"-^(aromatic ketone). X max. 209(27,600),32209(27,60 1 nm( € 28,600). ""*"/e 310(8) ,263(15) ,262(60) ,247(60) ,165(13) , 43(1007o).

CO 38. 4-Nitrofluorene Recrystallised fluorene(33•2g;0"2 mol)was suspended in acetic 3 3 -3 anhydride(400 cm ). Concentrated nitric acid(14 cm ;11'1 mol.dm ) was added dropwise with stirring to more acetic anhydride(400 cm )keeping the temperature below 30**. The latter solution of acetyl nitrate was then added over 0*5 hr. to the stirred solution of fluorene keeping the temperature below 30*. The mixture was kept at 25" for 48 hrs, then poured into ice/ water and stirred until all the acetic anhydride had hydrolysed. The solid product was isolated by filtration, washed with water and dried. The dry nitrofluorene was extracted with hot petrol(80-lOO* fraction, 1000 cm"^) and the petrol solution containing 4-nitrofluorene was concentrated by evaporation and added to a basic alumina chromatography column.

66 This was then eluted with petrol(60-80** fraction) and the 4-nitrofluorene was collected after any unreacted fluorene. The 4-nitrofluorene fractions were evaporated to dryness and the solid product recrystallised from petrol (40-60** fraction) to give pale yellow needles of 4-nitrof luorene(3•5g;87o) , m.p. 75-76*(lit.^® 75-77*). X max.210(28,200),233(16,400),338 nm(€ 4,800).

39. 9,9-Bishydroxymethyl-4-nitrofluorene

Recrystallised 4-nitrofluorene(0'5g;,0-002 moljwas dissolved in acetonitrile( 15 cm*^)and nitrogen gas was bubbled through for 0*3hr. The mixture was cooled to 0** and a suspension of paraformaldehyde(0*5g; 0"02 mol.) and aqueous benzyltrimethylammonium hydroxide(0.3cm ,2*6 mol. dm '^)in more acetonitrile( 10cm^)was added over 0*5 min. After 1-5 min. , the reaction was stopped by adding concentrated hydrochloric acid(0'5 cm ; -3 3 11*6 mol.dm ) and pouring the mixture into water(250 cm ). The product was extracted into ethyl acetate(2 x 100 cm^) , and the solution was washed 3 with water(4 x 100 cm ),then dried, filtered, and the filtrate evaporated to dryness. The solid product was recrystallised from benzene/petrol (80-100* fraction)to give yellow crystals of 9,9-bishydroxymethyl-4- nitrof luorene(0'3g;47%) ,m.p. 126-129*. Found: ^/e 271;C,66-6;H,4-70; N,5-30;C^^H^3NO^ requires °^/e 271;C,66-4;H,4•80;N,5"17%. \) max.3310(OH), 152O(N02),1345(NO2),106O cm"^(C-0,C-C). X max, 210(27,900),238(11,500), 340 nm. (e 3,800). t 6•1,4H.doublet(CH^=4Hz);5•1,2H,triplet(OH disappears on D2O addition,J^2= ^ Hz) ;2-3,7H,mult iplet(Ar-H) . /e 271(10) ,224(37) , 223(98),194(20U77(23),166(30),165(100),164(27),43(24%).

40. Fluorenone-4-carboxylic acid^^*^

Diphenic acid(80g;0-30 mol)cx. Koch-Light was mixed with 3 -3 concentrated sulphuric acid(30 cm ;10*0 mol.dm ) to make a thick paste and heated slowly to 130*. The solution was held at this temperature for 10 mins. then allowed to cool and poured into water(1000 cm*'). The precipitate was isolated by filtration, washed with water and recrystallised from ethanol to give golden yellow needles of fluorenone-4-carboxylic acid (57-9g;78%),m.p.225-228*(lit.'^^'' 227*).

67 114 41. Fluorene-4-carboxylic acid

Fluorene-4-carboxylic acid was prepared using the method of 114 Bergmann and Orchin but using ethylene glycol as solvent. The product was recrystallised from benzene to give fluorene-4-carboxylic acid(39'9g;85%), m.p. 195-197**(lit, 191-192**).

42. Methyl Fluorene-4-carboxylate^^^

Fluorene-4-carboxylic acid was esterified using the method of Fischer. The product was recrystallised from methanol/water to give methyl fluorene-4-carboxylate(7-2g;64%)with m.p.63-65*(lit.^^^63•5-64*5**) X max.210(32,900),265(13,000),312 nm(e 8,400).

43. Methyl 9,9-Bishydroxymethylfluorene-4-carboxylate

Methyl fluorene-4-carboxylate(l'lg;0"0O5 mol) was dissolved in dimethylformamide(25 cm^)and cooled to 2*. The solution was stirred and nitrogen gas was bubbled through for 0-3 hr. Paraformaldehyde (0'5g;0'02 molOwas suspended in more dimethylformamide(5 cm^)and.sodium ethoxide in dry ethanoKl cm ;2'85 raol.dm )was added. The suspension was added to the fluorene carboxylate solution slowly. After 1 minute the reaction was stopped by adding concentrated hydrochloric acid(l cm ; 11*6 mol.dm ^)and pouring the solution into water(250 cm^). The product was extracted into ethyl acetate(2 x 50 cm^), and the solution was washed with water(2 x 50 cra^),dried, filtered, and the filtrate evaporated to dryness. The oily product was crystallised from benzene/petrol then recrystallised from benzene to give colourless crystals of pure methyl- 9,9-bishydroxymethylf luorene-4-carboxylate (0-5g;36%) ,m.p. 128-129**. Found: ^^/e 284;C,72-05;H,5-55,C^yH^^O^ requires °*^/e 284;C,71-8;H,

5-64%. X max. 212(29,500):267(11,100);312 nm.(t 7,200). "^/e 284(2), 237(17),236(100),224(15),221,(22),205(27),177(19), 166(19), 165(63),163(17%); metastable peaks at 141 and 197.

5.4 METHYL 2-CHIjORO-9-HYDROXYMETHYLFLUORENE-9-CARBOXYLATE

44. Methyl 2-Chlorofluorene-9-carboxylate^^^

Methyl fluorene-9-carboxylate(26'0g;0'12 mol.)was dissolved in l,2-epoxypropane(200 cm'^)and stirred at -5**. Chlorine gas(ll*0g;0*16 mol.) was added slowly and the mixture stirred for 0'5hr.,and then allowed to warm to 25*. The solution was evaporated to dryness and the oily product was extracted with diethyl ether.

68 The ether solution was filtered and the filtrate evaporated to dryness and, recrystallised from cyclohexane to give crude methyl 2-chlorofluorene- 9-carboxylate(9'5g;31%) with m.p. 84-87*. Treatment with activated charcoal in methanol and further recrystallisation from petrol(60-80* fraction) 108 gave pure material(4'0g; 13%) with m.p. 90-92**(lit. 89-90*).

45. Methyl 2-Chloro-9-hydroxymethylfluorene-9-carboxylate

Methyl 2-chlorofluorene-9-carboxylate(4'4g;0'02 mol.) was dissolved in acetonitrile(100 cm )and nitrogen gas bubbled through for 0'3hr. The solution was stirred and.cooled to 2*. Paraformaldehyde(0"8g;0'03 molO was suspended in more acetonitrile(10 cm^)and aqueous benzyltrimethyl- 3 ammonium hydroxide(l'5 cm )added. This suspension was then added slowly to the methyl 2-chlorofluorene-9-carboxylate solution. After 5 minutes the reaction was stopped by adding concentrated hydrochloric acid(l'5 cm^; -3 3 11*6 mol.dm ) and the solution poured into water(500 cm ). The product 3 3 was extracted with ethyl acetate(2 x.lOO cm ),washed V7ith water(2 x 100 cm ), dried and filtered. The filtrate was evaporated to dryness and the solid product recrystallised twice from petrol(80-100** fraction) to give pale yellow crystals of pure methyl 2-chloro-9-hydroxymethylfluorene-9-carboxylate (2-2g;45%),m.p. 116-117**. Found: "'"^'/e 288;C,66-37;H,4-54;C1,12-31; C^^H^^CIO^ requires °^/e 288;C,66-5;H,4-50;Cl,12-3%, V max.3380(0H),

1730(C=q),1445(OH),1240(0-0),1080,104O(C-O,C-C),74O(C-Cl,C-C aromatic) t 6*4,4H,singlet(CH2);centre 6-2,2H,complex multiplet (CH2,J=7 Hz and 11 Hz pattern obscured by CH. at 6*4); 4*5,IH, triplet(OH,J^,- = 7 Hz, CHz disappears on D2O addition); 2*4,7H,complex multiplet(Ar-H). °* /e 288(29),260(34),258(100)226(43),223(36),201(34),200(26),199(97),198(31), 179(29),165(30),163(49%), metastable peak at 135.

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-74 APPENDIX 1

BIOLOGICAL SCREENING RESULTS

Compound Dose rate (kg,ha ^)11.2 2.8 1.4 0.7

•PMLROS PMLROSF PMLROS PMLROSF

9 **467658 454158 4432467 21 243322

86 022001

87 111001

88 121001

80 021221

79 121221

81 231001

82 111011

85 011221

83 121101

84 111001

89 111001

90 364115- 212001- 93 132102

94 120003-

95 1211011

97 3333247 3231227

98 021000-

* for key see p.77

TABLE 2 : PHASE I P0ST-E>1ERGENT HERBICIDAL SCREENING RESULTS

75 Compound Dose rate (kg.ha"^) 56 11.2

* FWLROMz PMLROMz

9 **888887 788887

21 888774 -

86 000000 -

87 000000 -

88 000000 -

80 575223 223110

79 797866 466322

81 335222 -

82 555655 -

85 • 000000 -

83 222020 -

84 000000 -

89 386784 023200

90 ' ' - 488753 93 221112 - 94 - 454222

95 - . 476431

97 - 788875

99 - 132101

•* for key see p.77

TABLE 3 : PHASE I PRE-PIERGENT HERBICIDAL SCREENING RESULTS

76 Compound Dose Rate (kg.ha"^) 5.6 2.8

*C M Co T F Ca W B Wo Bl By Cr R C M Co T F Ca W B Wo Bl By Cr R

9 887988779999-

21 835 68 7300 4 0 0 635654 100300-

82 6745432225122

87 875 886 656 8 4 7 -

TABLE 4 ; PHASE 2 PRE-E>1ERGENT SOIL INCORPORATION TESTS

2.8 1.4

*C M Co T F Ca W B Wo Bl By Cr R C M Co T F Ca W B Wo Bl By Cr R

9 856 886 646 8 3 7 - 755 784 22 3 73 4 -

21 233 532 - - - 133 421 - . - - - - -

82 5214321113111

97 755784223734-

TABLE 5 : PHASE 2 PRE-E^tERGENT SOIL SURFACE SPRAY APPLICATION TESTS

P m Peas C - Chickweed Wo « Wild oat

M Mustard Co Cotton Bl S3 Blackgrass

L Linseed T ts Tomato By « Barnyardgrass

R c Ryegrass F a Fathen Cr B Crabgrass

0 B Oats Ca a Carrot

S S3 Sugarbeet W m Wheat •

F sa French bean B «B Barley

** Scores from 0-10 refer to tenths of the sample killed (see Appendix 2 p.8o)

77 APPENDIX 2

HERBICIDE SCREENING METHODS AS USED BY FISONS LIMITED

Routine Phase I tests

Ail plants are grown under the artificial environment of

controlled temperature rooms. Light is provided by banks of 5 foot,

80 watt 'Ecko* white semi-reflector fluorescent tubes giving a l4 hour

light period in 24, controlled by a time clock. The air temperature is

maintained at 20*^0 - 1° with a 'Frigidaire* cooling system. Individual

plant containers are inspected daily and top watered where necessary.

Rapid growth rates facilitate experiments of 3 weeks duration from sowing

to assessment, only phase II experiments are normally left longer.

Species : Peas - Pisum sativum cultivar Onward (Post-emergent) Mustard - Sinapis alba

Linseed - Linum usitatissi mum

Ryegrass - Lolium perenne cultivar VTesterwolthe

Sugarbeet - Beta vulgaris cultivar Shai-pe's Klein E

Oats - Avena sativa cultivar Condor

French bean- Phaseolus vulgaris cultivar Canadian Wonder

(Pre-emergent) Maize - Zea mays cultivar White Horse Tooth replaces French beans are omitted. Sugarbeet;

All species are grown in John Innes I potting compost. French beans are sown 3 per 3| inch B.E.F. growers pot and thinned to 2 healthy plants immediately prior to spraying. The remaining species are sown in

7 X 3 X Zi inch Prestige 'Ovenglo* loaf tins of anodised aluminium. The seed is sown in 2 row, 1 along each side of the pan, each species in a separate pan pre-emergent but post-emergent they are sown in pairs - Peas with Oats, Mustard with Ryegrass and Linseed with Sugarbeet. Larger seeded species are counted out - Peas 8, Oats 10, Maize 6 and Sugarbeet 15 (clusters) per row, smaller species are measured out by volume giving approximately

43 Mustard, 45 Linseed and 6? Ryegrass.

78 Post-emergent technique - Candidate herbicides are normally

applied at rates equivalent to 11.2 kg in 900 l/ha using the laboratory

turntable sprayer. If the chemical is in short supply it is applied at

the micro rate of 2.8 kg in 450 l/ha. Formulation in order of preference

is as a water solution, sodium salt or hydrochloride, aqueous suspension

or aqueous acetone solution. Lissapol NX is added as a wetter to all but

the acetone formulation.

French beans are sprayed 7 and the remainder l4 days after

sowing with assessment l4 & 7 days later respectively. Damage is

visually assessed on a scale 0 B no damage, 100 c complete kill and

normally represents the mean of two assessors' opinions, plus if

necessary weighings or other measurements.

Pre-emergent - To economise in chemical usage, avoid solvent

phytotoxicity and facilitate formulation, compounds are formulated as a

dust by grinding in a mortar and pestle with attaclay and sand, then

incorporating into the potting compost immediately pre-sowing. The

initial dosage rate is 56 kg/ha but micro it is only 11.2 kg/ha on half

the number of plants - 3 pans, 2 species/pan. Assessment is 3 weeks

after sowing as with post-emergent.

Phase II tests

Active compounds are retested at lower rates either on the same

species or an extended range including weeds, mono and/or dicotyledons depending on initial activity. In post-emergent tests erraticaly germinating weeds are selectively transplanted to provide uniform populations. Compounds active pre-craergent at 2.8 kg/ha are retested at the same rate but as liquid formulations sprayed onto the soil surface instead of incorporated.

79 Additional phase II species include -

Chickweed Stellaria media

Mayweed Matricaria and Tripleurospermum spp

Cleavers Galium aparine

Redshank Polygonum persicaria

Fathen Chenopodium album

Cotton Gossypium sp.

Tomatoes Lycopersicon esculentum cultivar Moneymaker

Carrots Daucus carota spp. sativas cultivar

Chantenay Wheat Triticum aestivum cultivar Jufy Barley Hordeum vulgare cultivar Proctor Wild oat Avena fatua Blackgrass Alopecurus myosuroides

Bamyardgrass Echinochloa crus galli

Crabgrass Digitaria sanguinalis

80 Key to Herbicide Data Tables

Phase I

Post-emergent Tests

1. Species :

PEA = Pi sum sativum - pea

MST = Sinapis alba - mustard

LND c Linum usitatissimum - linseed

RY/ = Lolium sp. - ryegrass

BU* r= Fagopyrum esculentum - buckwheat (alternative species to ryegrass in earlier screening tests - if used the activity value is followed by an asterisk *)

SGR = Beta vulgaris - sugarbeet

OT/ e Avena sativa - cultivated oat

BA* B= Hordeum vulgare - barley (alternative to oat in earlier screening tests - indicated by an asterisk *)

All plants possess 1-2 true leaves at time of spraying

2. Rates : the dosage rates used in kg/ha are indicated above the groups

of species, i.e. 11.2, 5-6, 2.8, 1.4, 0.7- The compounds are normally

applied in 90O ]/ha at the 11.2 kg/ha rate the remainder are applied in

450 1/ha. (11.2 kg/ha = 10 lb/acre: 900 l/ha « 80 gal/acre.)

3. Activity levels

Seven days after application of the compound to the young growing

plants listed in 1. above an assessment of damage made to each species and a

score value allocated where O = no damage - i.e. compound inactive - to 100 : complete kill. These activity scores are shown for each plant species for all dosage rates tested. In addition the mean score for all six species is given. The individual and mean score values are indicated by the following figures:-

81 0 s: score value was 0 - i»e. totally inactive

1 a herbicidal score is between 1 and 9

2 « ^» " " " lO and 24

3 « " ti It It 25 -and 34

4 « " " " 35 and 49

5 « " " " " 50 and 64

6 « " " " 65 and 74

7 « " " " " 75 and 84

8 B " " " " 85 and 94

9 c: " " " 94 - i.e.. highly active

N.B. value followed by * indicates that the figure quoted refers to the alternative species.

82 Phase I

Pre-emergent Tests

1. Species :

PEA c Pi sum sativum - pea

MST 8 Sinapis alba - mustard

LND S5 Linum usitatissimum - linseed

MZE c 2ea mays - maize

OT/ n Avena sativa - oat

BA* R Hordeum vulgare - barley (alternative species to oat in earlier screening tests - indicated by an*)

RYE «5 Lolium sp. - ryegrass

2. Rates : the dosage rates used kg/ha are indicated above the groups

of species, i.e. 561 11.2, 5*6, 2.8, 1.4 and 0.7* The compounds are

normally thoroughly mixed in the soil prior to sowing the seeds.

3. , Activity levels

Twenty one days after planting the plants are assessed for the

pre-emergent activity on a 0 to lOO score - as in post-emergent tests.

These activity scores for each species are abbreviated as in the post-

emergent tests as follows:-

0 K score value « 0 - i.e. totally inactive.

1 R score value is between 1 and 9

2 R " " " " 10 and 24

3 « " " " " 25 and 34

4 R " " " 35 and 49

5 <= " " " " 50 and 64

6 = " " " " 65 and 74

7 = " " " " 75 and 84

8 = " " " " 85 and 94 9 «= " - " " >94

N.B. value followed by * indicates that it refers to the alternative species.

. 83 — Phase II

Pre- and Post-emergent tests

Post-emergent

Species CHICKWD = Chickweed

REDSHNK m Redshank

CORN-M B Com marigold

W-OAT » Wild oat

BLCK-GR s Blackgrass

BARN-GR B Bamyardgrass

CRAB-GR = Crabgrass

The remainder of the species have not been abbreviated and

activity levels are as in phase I tests.

Pre-emergent soil mix and surface application tests

Abbreviations as in post-emergent tests above.

Mode of Action

In addition to recording the herbicidal scores for each species

at the range of dosage rates used a brief indication of the mode of

action is given on the right-hand column of the phase I print-outs under

the heading of "Symptoms".

These qualitative descriptions of the mode of action are given as a single letter code as follows:-.

Post-emergent activity

S R indicated >'2k% contact damage or scorch occurred.

G cz indicated >24% growth inhibition occurred.

N B indicates a necrotic effect observed. The presence of this

symbol implies a systemic effect.

F = indicates that shoot formative effects observed, e.g. leaf

cockling, epinasty, apical deformity, vestigial or abnormal

leaf shapes ("hormone effects").

84 C = indicates that a marked change in the plant's pigmentation

vas observed - e.g. chlorosis (as with amino triazole) or

reddening of foliage. The yellowing sympton which-is a k- precursor of necrosis is not recorded. ^- • - A » indicates abscission interpreted as detachment of leaves

before or during assessment for whatever reason - scorch, y

necrosis or formation of an abscission layer, ^ .

T S3 indicates >2496 growth stimulation occurred or a marked

increase in lateral bud development or tillering.

Stimulation of lateral buds resulting from a removal of

the apical meristem by scorch etc. is not recorded.

W n indicates cuticular abnormalities observed, e.g. wax

reduction or glossy foliage (as with TCA).

R R indicates a visible root response whether stimulation,

inhibition, deformity, discolouration or any other effect.

Pre-emergent activity

I R Indicates >24% germination inhibition observed.

The other symbols are identical in meaning to those in the post-emergent tests except that the 'S' symbol does not appear.

All the above symbols are not related in the phase I print-outs to any particular species and they may refer to any one or all. Simple inspection of the herbicidal index will often enable a symptom to be related to an individual species.