Note: Descriptions are shown in the official language in which they were submitted.
w~ ss/22s40 2 1 8 2 5 ~ 3 ~ 49~
-1-
S ENANTIOMEI?ICAIl.Y PURE (+)-LIAROZOLE
,
10 This invention relates to novel I ;. . - . ;. Ally pure - . of formula a) useful in
treating disorders which are I -- A- t- ; ~- A by an increased ~ and!or abnormal
1' '' of normal, 1~ ;r or neoplastic epithelial cells. These ~ .
are ~ / useful in the field of ~ ' Oy . Also disclosed are ~ .
containing said novel compounds as well as methods of using the mentioned
15 to treat the mentioned disorders. lrhe ~A~UI~ - y rr~mrolln~1c of for[nula a) are
useful forthe " -- rA ~-ofamedicine fortreatingl-,.AI~ ,,-I;..,, disorders. Furtherthe
present invention provides methods of preparing the present novel _ r
The novel ~r~ o ~-~c subject to t~le present invention are the d~.AIl~ - - y isomer of
20 the compound liarozole and the 1~l ~- . - -- . ;. ~lly acceptable acid addition salts thereo
Liarozole is a racemic mixture, i.e a mixture of its optical isomers, and is specifically
mentioned as compound 28 in EP 0,371,559. Said patent application mentions the use
of I ' like lia~zole in the treatment of epithelial disorders. EP-0,200,744
25 describes the use of compounds lilce liarowle for inhibiting or lowering androgen
formation. Whereas EP-0,371,559 and EP-0,260,744 recognize that, . ' like
liarowle have ~ -v ~ is~meric forms, no example of an - .1 - - . , -lI y pure
form is given of liarozole.
Chemically liaro201e is (-)-5-[3~ ' yl]-lH-imidazol-l-ylmetbyl]-l~-benz-
imidawle, and is Trp~C~ by f~rmula (1). As can be seen ~om the chemical
structure, liarowle has one stereo~enic center (indicated with an asterisk in formula (I)).
H
N ~ N
The subject of this invention is the~ 1 ;"" , ;~ ~ll~y pure ~AkUII - y isomer or(+)-isomer of liarowle. Said isomer will hereinafter be referred to as (+)-liarowle.
w0 9~22s40 2 1 8 2 5 8 3
Many organic compounds exist in optically active forms, i.e. they have the ability to
rotate the plane of plane-polaTized light. In describing an optically active compound, the
prefixes D and L or R and S are used to denote the absolute ~ ;.... of the
molecule about its chiral center(s). The prefixes (+) and (-) or d and I are employed to
5 designate the sign of rotation of plane-polarized light by the compound, vith (-) or I
meaning that the compound is 1~ y and with (+) or d meaning that the
compound is ~' y. For a given chemical structure the optically active isomers
having an opposite sign of optical rotation are called; Said . are
identical except that they are mirror images of one another. A l: l -mixture of such
10 ; is called a racemic mixture.
St,, ~, ~ h . - - .~ ~1 pUTity is of importance in the field Of 1 ' ? ' since the respective
may have a different potency or may have a different activity. The
enantiomer of a benehcial isomer may even be deleterious rather that simply inert
15 Several examples of such differences are known in the art.
The term "~ lly pure" as used herein means that the product contains at least
90% by weight of one enantiomer and 10% by weight or less of the other .
In the most preferred ~... l~l - .l the term " - ~ - ;- ^lly pure" means that the
20 ~ contains at least 99% by weight of one enantiomer and 1% or less of the
other;
It should be noted that optical rotation of chemical substances is dependent upon
' parameters. The values shown in the ~ paTt 1~- - ' - --1- - are
25 specific rohations and the ~ conditions such as r l~ Ih the wavelength of
the plane polarized light used, the solvent as well as the I of the sample are
indicated in the ~ al way. The optical rotation may vaTy (it may even change
sign!) when for instance an acid addition salt is formed. When reference is made to the
y isomer of barozole or (+)-liarozole then the sign of the optical rotation of
30 the base form is intended under the given ~,A~J~ ' ' I conditions shown ~
It should also be noted that when a chemical reaction does not involve the
then the absolute ~" of said - L . . ~ ' '~' . remains the same, although the
optical rohation of the compound which results from said chemical reaction may be
35 different or even have an opposite sign. Hence, in order to avoid corlfusion, the
withthesameabsolute~....ri~,..,.l;....ofthe asthedesired
enantiomer of the final product will be designated with the prefix (B) before the reference
number.
wo ss/22s40 1 8 2 5 8 3 r~
The ~ aa eptable acid addition sals as rrtentioned ~ dbU ~ _ are meant to
a3mprise the i' r - l~y activl non-toxic acid addition salt forms which the compounds
of f~mula (I) are able to fonn. The latter can w.. ., '~, be obtained by treating tbe base
form with such appropriate acids as inorganic acids, for example, hydrohalic acids, e.g.
h.~,' ' ' " h.~u~lulllic and the like; sulfuric acid; nitric acid; phosphoric acid and the
like; or organic acids, for exampl~, aaetic, propanoic, ~ IIUA~ 2~ UA~1 .
2 . ~ '~ IJI~r `. . (Z)-2-L ' (E)-2-butene-
dioic, 2 h~LUA~' ' 2,3 dil~ UA~' " 2-hydn3xy-1,2,3-l ~.,u --
boxylic, ' '' ~,; ' '' ~ I '' ~ il 'l ''
r ', 2 ~.~dIUA~ J;U~ ~amino-2-l,rLuA.~u~,.l~l., and the like acids.
Conversely the salt folm can be a~nverted by treatment with aLcali into the free base folm.
The term addition salt also complises the hydrates and sûlvent addition forms which the
of formula (I) are able to fcnn. Examples of such fonms are e.g. hydrates,
alcoholates and the like.
Prefened ~ y aaceplable acids are ll~dlu~,;lulic acid and (E)-2-butenedioic
acid.
General preparation of structures lncluding liarozole have been ~At~ described in
EP-0,371,559 and EP-0,260,74~1.
r 1~ pure(+)-Lar3zolemaybepreparedbyreactingan~ ypure
' diamine of formula ~B)-(~I) with formic acid or a fumctional delivative
thereof.
1~ ~ H
Cl ~B)-~) Cl (+) ~I~
Said functional derivative of ftic acid is meant to aomprise the halide, anhydride,
amide and ester, including the orl~to and imino ester fo~Tn thereo Also
30 ' ' ' or an acid addition salt thereof can be used as cycl~zing agent.
The general neaction aonditions, ~vork-up procedures and w...~,.Aiulldl isolation
techniques fr)r calrgi3g out the a~ove and following reactions are desclibed in the prior
art. When more specific conditions are n quired they are mentioned I
wo 95/22s40 2 1 8 2 5 8 3 ~ ;r, ~
The ~ , pure ' diamine of formula (B)-al) may be prepared by
reducmg an ' of formula (B)-(lll) by a standard nitro to-amine reduction
reaction.
~N02 ~ I ~NH2
Cl
(B)4m) Cl (B)-(Il)
The desired enantiomer of the ' of formula (B)-(m) can be prepared by
fractional ~,lyDL~li~.wll of a racemic mixture of the ' of formula (m) with an
- lly pure chirdl acid. Preferred chirdl acid for the above fractional
0 ~ L~ LLiull is7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-." ~ r.",;, acid(i.e.
10-- ~ ~ lf~ r acid).
Appropriate solvents for carrying out said fractional ~I,yDL~lL~LiOll are water, ketones,
e.g. 2-propane, 2-butanone; alcohols, e.g. methanol, ethanol, 2-propanol. Mixtures of
15 ketones and water are very suitable for the above fractional ~ DL~IIi~Li~JPreferdbly a
mixture of 2-propanone and water is used.
The ratio of water/2-propanone by volume may vary from 1/10 to 1/2 Preferred range
of said ratio is 1/5 to 1/3.
The fractional cryct~ 7~ nc are suitably carried out below room ~ , preferably
below 5C
It was also found that the subsequent reaction step can be calried out without any
appreciable .,.~ - -:
Al ~.Iy the (+)-isomer of the compound of formula (I) may be prepared by
cyclizing an ~ of formula (B)-aV) following procedures as described above
for the cycli_ation of " of formula (B)-(ll) and ~t r ~ the thus
obtained of formula (B)-(V). In formulas (B)-(IV) and (B)-(V) R
represents Cl 6alkyl, wherein Cl 6alkyl means a straight or br~mch chained saturated
ll rdlu~ lJol~ radicals having 1 to 6 carbon atoms such as, for example, methyl, ethyl,
propyl, butyl, pentyl, hexyl. Preferably R is methyl.
WO 95122540 2 1 8 2 5 8 3 ~ "~ ,~ 3 ~"
.
~ ~N
Cl (B)-(IV) ~N H (B)-(V)
/>
(+)-(1)
The of formula (B)-(IV) may he prepared by reacting an ' of
formula (B)-(VI) v~ith a reagent of formula (VII), alkylating the thus formed thiourea
5 derivative of formula (B)-(Vm) ~llhcl~ql-^ntly cycli~ing the " of formula
(B)-(~), and reducing the nitro gl oup of the ' (B)-(X). In the formulas
(VII), (B)-(~rm), (B)-(IX) and (B )-(X) R represents C~ 6alkyl as defined l~ ab~ S /OR
NH-C--NH--CH2-CH
NH2 ~ NH2 S~N-CH2-CH(OR)2 ¦ ~--NH2 ~OR
~3CH~!1--No2 (Vll) ~CH~I--NO2
Cl (B)-(VI) OR Cl (B)-(V
NH-C=N-CH2 CH rN
N]~2 OR ~ ~SR
~CH ~NC2 ~CH~'NO2
C~ (B)-(IX) Cl (B)-~
~ (B)-(IV)
The; '1~1 pure ' of formula (B)-~VI) can he prepared by art-
known resolution techniques, e.g. by ~ .r using chiral stationary phases or
by forrning ~ nmro~m~lc such as forming an amide vith am
y pure chiral acid, e.g. c~ ~,J~Ay~ acid (mandelic acid), or
15 by forming -l~ - ;- salt forms using, l~y pure chiral acid
Liarozole has retinoid mimetic effects in vivo and in vitro. This means that thecompound is thought to inhibit retinoic acid (RA) cataholism, so that increased retinoic
acid ~A) levels lead to I ? RA effects at the tissue or cell level. Liarozole has
WO95112540 2 1 82583 ~ 7~
also been shown to be a potent inhibitor of androgen II;UD,~ ~ Preclinical andebnicAI studies are ongoing showing the utility of liarozole in the field of oncology and
.. . .
0~ -
5 U~ AYCA~ Y it has been found that (+)-liarozole shows increased retinoic mimetic
activity when eompared with raeemie liarozole or with the . - lly pure
l~vvl~ y isomer of liarozole, hereinafter referred to as (-)-liarozole. More in
par~icular, ( l ) ' ' is a stronger inhibitor of the retinoie acid, . ~ ' in human
skin epidermis and human tongue squamous earcinoma oells (SCC25). Moreover, the
10 increased ~,rf~l;v~ oo of (+)-liarozole as a retinoie mimetie, especially in the field of
d~ll~dtvloO~ ~ ean be evideneed by the test "Induetion of Pinnal Epidermal II~y~lyl~D~ in
Hairless Mioe". The effeet of retinoic acid at the level of normal human ~ Li.lo~yt~,D is
also more potentiated by (+)-liarozole. r~ ~L~ IIV~ it has been found from toxicity
tests that, ~ the use of (+)-liarozole is more suitable, when compared to
15 (-)-liarozole, for the ' of a ~ for treating ~ ;. .., disorders.
The increased retinoic mimetie activity of (+)-liarozole is desc~ibed in more detail in the
l part hereinafter. From the above, it ean be eonceived that by ' g
an effeetive amount of (+)-liarozole it is possible to aceomplish a more "targeted"
k:. l therapy. A more "targeted" ~' ~ O l therapy means that by using
20 the (+~isomer of liarozole, the eompound is used which has a higher retinoic mimetie
aetivity.
The use of (+)-liarozole and its l - 'ly aeceptable acid addition salts in the
method of the present invention is based on their useful property to delay the eatabolism
25 of retinoids, such as, all-~,u,~ ._t;IIV;I, aeid, 13-eis-retinoie aeid and their derivatives.
The latter results in more sust~uned / higher tissue ~ of retinoids and
improved eontrol of ~ and growth of various eell types. This action of
(+~liarozole is also called retinoic mimetic activity because A~ , (+)-liarozolecauses the same effeet as if retinoid would be _ ' ~ As such, (+)-liarowle ean
30 be used to eontrol the rate of growth and ~ of normal, ~ ;r and
neopl-Dstic epithelial cells.
(+)-Liarozole and its y~ lly acceptable aeid addition salts is therefore useful in
a method of treating disorders which are ~ by an increased l....l: F.. ,~
35 and!or abnommal ' of epithelial eells. (+)-Liarozole shows aetivity on eells
of which the growth and ~ ) is not Dul~D~Lidll~ mediated by or insensitive to
the aetions of androgens or estrogens, in partieular on eells of which the growth and
"'' - is sensitive to the aetions of retinoids. Special uses inelude the ability to
... . . . . ... .... . . . _ . . . . _ _ _ _
WO 95122540 ~ I 8 2 5 8 3 P~ l/~ 5'~ ~ ~S
cur~ IUUUl ' a variety of disorders of ~ I such as, for example, rosacea,
alcne, psoriasis, ichthyosis, warts, callosites, acanthosis nigricans, lichen planus, corneal
epithelial sbrasion, geographic tongue, Fox-Fordyce disease, ~ ,f. .u skin
conditions, such as, actinic keratoses, and keloids,,, ' ~ylic 11.~ ' Darif~s disease, pityriasis rubra pilaris, congenital i.,ll~uD;rul,ll c~ hlu~lll_,
palmatis et plantan s, mdasma, llyl 1 " (+)-Liaro~ole and its
acceptable acid addition salts is useful for the ' of a medicine
for treating I disorde}s.
10 In general it is ~ . ' ' that an effective amount to treat disorders which are
L . .~ fl by am excessive 1~ andlf r abnormal 1;.'f . . ,~ .,. of tissues,
would be from 0.001 mg/kg to 20 mglkg body weight and m~ re preferably from 0.01mg/kg to 10 mg/kg body weight.
15 The compounds of formula (I) usf d in the method of the invention are most preferably
applied in the form of appropriate ~ . As appropriate ~ there may
be cited all, , usually employed for sysLemically or topically ~
drugs. To prepare the ~ of this mvention, am effective
amount of the particular compound, optionally in acid-addition salt form, as the active
20 imgredient is combined in mtimate admixture with a 1~ y acceptable carrier,
which canrier may take a wide vatiey of fotms depending on the fonm of preparation
desired for: ' - ` These IJI~f~ ' . , - are desirable in unitary
dosage fortn suitable, y_ ~ul ly, for ~ orally, rectally, ~ , or
by parenteral injection. For example, in preparing the ~ .. 1~ .~ I;n.~ in oral dosage fotm,
25 any of the usual ~1~. ' rlledia may be employf d such as, for example, water,glycols, oils, alcohols and the liloe in the case of oral ~iquid 1~ IC such as
, syrups, elixirs and solutions; or solid cantiers such as starches, sugars,
kaolin, lubricants, binders, ' - ~ g agents and the like m the case of powders,
pills, capsules, and tablets. Becallse of their ease in All -- ~ - - tablets and capsules
30 represents the most adv _l~b~D oral dosage unit form, m which case solid
~1.~, - ' cattiers are obviously employed. For pnteral ~ the catrier
will usually comprise sterile water, at least in large patt, though other in~Aif-ntA, for
example, to aid solubility, may be included. Injectable solutions, for example, may be
prepared in which the cartier corAprises saline solution, g~ucose solution or a mixture of
35 saline and glucose solution. Injef.table , may also be prepared in which caA,e
appropriate hquid cattiens, susper~ding agents and the like may be employed. Also
included are solid form l r~ whiCh are intended to be conven~ed, shortly beffJre
use, to liquid form ~ iU..D. In the ~ - r ' suitable for I
2 1 82583
WO 9S/22540 P~ llr 1 ~5.'1
-8-
the carrier optionally comprises a penetration enhancing agent and/or a
suitable wetting agent, optionally combmed with suitable aclditives of any nature in minor
rn~innc, which additives do not introduce a significant deleterious effect on the skin.
As appropriate ~ 1l for topical application there may be cited all, I
5 usually employed for topically ~ ' ~ dtugs, e.g., creams, gellies, dtessings,
shampoos, tinctures, pastes, ointments, salves, powders, liquid or semi-liquid
and the like. Application of said . may be by aerosol e.g. with a
propellent such as nitrogen carbon dioxide, a freon, or without a propellent such as a
pump spray, drops, lotions, or a semisolid such as a thiclcened C' ~ ~ '1'''- ' ;~ . which can
10 be applied by a swab. In parlicular ~ semisold c~ ;nA c such as salves,
creams, pastes, gdlies, ointments and the like will ~,u~ tly be used.
It is especially a.l~ . ~ to forrnulate the ~ .. .li.. ~ l.l ~.. , ~r .. ;. ~l
C ~ in dosage unit for[n for ease of - ' nn and uniformity of dosage.
15 Dosage unit form as used in the ~ ri ~ and claims herein refers to physicallydiscreate units suitable as unit~ry dosages, each unit containing a L ' ~ ' quantity
of active indeM calculated to produce the desired therapeutic effect in association with
the required ~ ' ' carrier. Examples of such closage unit forms are tablets
~mcluding scored or coated tablets), capsules, pills, powders packets, wafers, injectable
20 solutions or . . I~- -r r ~ v~ r~c and the like, and segr~gated
multiples thereof.
Other ~ are l~lclJal~liull~ of the cosmetic type, such as toilet waters, packs,
lotions, skin milks or milky lotions. Said ~ICIJa aLiull~ contain, besides the active5 ingredient. - - r usually employed in such 1~ Examples of such
are oils, fats, waxes, surfactants, I thickening agents,
1;~ viscosity stabilizers, chelating agents, buffers, ~ a~ perfumes,
dyestuffs, lower alkanols, and the like. If clesired, further ingredients may beiIII,VI~ ' ' in the cnTnrnr;~inn~ e.g. y agents~ onliho~
3û (~ r vitamins, sunscreens, antibiotics, or other anti-acne agents.
In a further aspect of the invention there ate provided particular 1~ I or
cosmetical ~ which cprise an inert carrier, an effective amoumt of
(+)-liarowle or an acid addition salt form thereof and an effective amount of a retinoic
35 acid, a derivative thereof, in particular retinol, or a - ~ l . . lly isomeric form
thereo
It can be '~ ' that the retinoic acids and (+)-liarozole act in a synergistic
manner. Indeed, the combined effect of both substances is greater than the sum of their
W095122.540 2 ~ 82583 I_l~r~ 1~4~S
_9
respecive effects when ' 1 separately. The above desc~ibed retinoic æid
containing ~ - aTe particularly useful for treating acne or for retarding the
effects of aging of the skin and gelleraOy improve the quality of the skin, yol~uLul.y
human fæial skin. A y~ or cosmetical . . containing retinoic æid
5 or a daivative thereof as the activc ingredient in intimate admixture with a
' ' O "~, acceptable ca~ia can be prepared according to C;U..V~ iù~
~ techniques, such as those known for topical application of retinoic acid
and its da ivatives opionaOy in ad~nixture with ~- 1-~ or derivatives tha eof
known in the art. Preferred, . - for topical application are in form of a cream,ointment or lotion comprising from 0.001 to QS% (yGlLiuu~ y from 0.01 to 0.1%) all-
trans-retinoic æid, 13-cis-retinoic æid or a daivative thereof, in y~uli~uku . " 1, and
from 0.1 to 5% of a (+)-liarozole ar a ~ æceptable acid addition salt
thereof, in a semi-solid or liquid diluent or car~ier.
These preferred ~ shollld preferably be non-irritating and as far as possible
15 ~hey should be odorless and non toxic. For Cul.vl in applying to the skin, the
- usuaOy contain, besicles water or an organic solvent, several of cert~tin
organic emollients, emulsifiers fol the aqueous andlor non aqueous phases of the
, wetting agents yl~ d ~ and agents that facilitate the penetraion and
l.,lll~iOI~ ~ of the ætive agents in the skin.
20 In use, the retinoic æid containing c~ . . of the invenion are applicd topicaOy to
the area to be treated or protected, at regular intervals, as needcd, generaOy about 7 to
about 21 times per week. The duration of the treatment wiO depend upon the nature and
severity of the condition to be trea~ed as weO as the frecluency of application of the
~""'1"'` ''"'
F . ' part
A. Preyaration of the 't
Example I
a) A1 h uo. ~J - mixtureof(_)-~[(3-ul-luu~ imidazol-l-ylmethyl]-2-
~ .L (the preparatiol~ of which is described in EP-371,559) (500 g) in
2-propanone (2000 ml) and water (100 ml) was stirred at 22C. (-)-(lR)-7,7-dimethyl-
2-oxo-bicyclo[2.2.1]heptane-1- ' '~ acid (353.2 g) was added and the
mixture became 1~ ,. -- u~ ~ after 10 minutes. The mixture was first stirred for 18
hours at 20C and then for 3 hour; at 0-5C The precipitate was filtercd off, washed
with 2-1~lu~lul.~ 95/5 (lSO ml) and dried, yidding 308.9 g (36.2%) of product.
A sample (306.7 g) was partitioned between .' ' ' -' (SOO ml) and water (750
ml). Ammonium hydroxide (100 ml) was added. This mixture was stirred for 15
2l 82583
w0 95/22540 r~ ls~ ~
-10-
minutes. The aqueous layer was separated and extracted twice with ~' -' ' '
(250 ml each time). The separated organic layer was washed with water (250 ml), dried,
filtered and the solven~ was ~v . i yielding 179.7 g of (-)-(B) 4 [(3- ' ' ulJh~imidazol-l-ylmethyl]-2- ' , mp. 89.8C; [~]D = -19.80 (c = 0.5% in
methanol) (interm. 1).
b) A mixture of ' (1)(179.7 g) in methanol (656 ml) and a solution of
ammonia in methanol (32.7 ml) was h~ll, O I at 20-25 C with platinum on
activated carbon (13.1 g) as a catalyst in the presence of thiophene (0.27 g). After
uptake of hydrogen (3 eq.) the catalyst was filtered off and washed with 2-propanol (30
ml). A solution of hJLu~,hlul;~, acid in 2-propanol (sæ ml) was added to the filtrate at
<30C The mixture was stirred fo} 3 hours at 20 C then for 3 hours at 0-5 C The
resulting precipitate was slowly filtered off, washed with methanol (100 ml) and dried
(50 C), yielding 185.60 g (83.2%) (+)-(B)-4-[(3- h' , ' yl)-lH-imidazol-l-yl-
methyl]-1,2-' ' ~ u IllvliJu, mp. 172.5C; [r~]D = +23.73
(c = 1% in methanol) (interm. 2).
E~a~ple 2
a) A mixture of (4-amino-3 uyll~.l.yl) (3- ' ' . ' Jl) ' (50 g), formamide
(375 ml) and formic acid (63 ml) was strrred and refluxed for 17 hours. After cooling,
the mixture was poured on ice. The precipitate was filtered offand drie(i yielding 55 g
(99.4%) of (_)-N-[(4-amino-3-nitrophenyl) (3-~ luluyll~,~lyl)methyl]f ' (interm.3).
b) A mixture of ' (3) (SQ7 g), llydlu~,llluli~, acid 6N (350 ml) and
2-propanol (70 ml) was stirred and refluxed for 17 hours. The yellow precipitate was
filtered off and dried in vacuo, yielding 51 g (97.8%) of (_)-4-amino-c~-(3-chloro-
phenyl)-3- , ' ' ' J.' ' ' ', mp. 263C (interm. 4).
c) To a solution of ' (4) (43 g) in i ' yvlv~u~u. (400 ml) at rcom
was added succesively N.N-v;.,.l.Y' ' (13-8 g) and (R)-(-)-~-
hyLu,.yl, ~ ~;; acid (20.8 g). Then a solution of 1-lly~u~yh...,, .1. ;i, ,..1~
' .yl' (22.2 g) in ~Il~I.yLuLI~l (200 ml) was added. After complete addition a
solution of ~,~'-diuy11uh~"~yl~ 1;;.; lr (33.9 g) in di~.lllul~ ' (300 ml) was
introduced to the mixture. After stirring for 2 hours at room i r ' ~E ~
di~,y~,lull~,~.y- was filtered off. The filtrate was washed with a solution of potassium
carbonate (10%) and the organic layer was dried to give a mixture of .1: '~h .. . ' - ~ ` (60g)
35 (fraction 1). The same experiment with ' (4) (16 g) as starting material
resulted in a yield of 26 g of a mixture of 1 ~ (fraction 2). Fraction 1 and 2
woss~s40 2 ~ 8 2 5 8 3
were combined and purified by H~'LC (eluent: CH2C12/ethyl acetate 90:10), yielding
30g (32.3%) of ~i)-(R,B)-;~-[(4-amino-3 . ' .~1)(3~Llulu~h~ l)methyl]-~-
y~ (interml. 5).d) A mixture of (5) t30 g), 1.), ' acid 12N (300 ml) and l-propanol
5 (100 ml) was stured and refluxed for 17 hûurs and poured on ice. The mixture was
extracted with ethyl acetate. The ~Iqueous phase was basified with: hydroxide
and extracted with ' ' ' ' - The ~' ' ' ' extracts were dried, filtered
and~ 1, yielding7.3 g (36.0%) of (+)-(B)-4-amino-~-(3-. 1,1 ....1.' .~1)-3-
r, ~ ' (interm. o~.
e) A mixture of ' (6) (7.3 g), 2-i~vtlliuu.~. ~1,1-dil.. villv/~yvulallv (4.8 g)and methanol (75 ml) was stirred ;md refluxed for 2 hours. The mixture was evaporated
to an oily residue, yielding 11 g (~ 00%) of (+)-(B)-l~-[(4-ammo-3 , ' yl)(3-
~.lllulu~ yl)methyl]-N'-(2,2-di..lvlllu~vlllyl)thiourea (interm. 7).
f) A mixture of ' (7) ( ~1 g),; Yl~ r (2 ml) and potassium carbonate
(4.97 g) was stirred at room i 1 G for 48 hours. The solvent was evaporated and
the residue was taken off with ~lirlll ' and washed with water. The organic
layer was dried, filtcred and evaporated, yielding 11.4 g of (+)-(S)-methyl (B)-~-
[(4-arnino-3-nitrophenyl)(3~1.1u.v~11v.l.~l)mcthyl]-_'-(2,2 1' ' ~vtllyl)carbam- ' ' as an oily residue (intcrm. 8).
g) To - (8) (11.4 g) at 0C was added sulfuric acid (lOOml) (precooled to
5C). The mixture was stirred at 5C until complete dissolution and then was warmed to
room t ~ After stirring ior 2 hours, the solution was poured on icc and basificdwith ammonium hydroxide. The ~aqueous solution was extracted with ethyl acetate. The
organic layer was dried, filtered auld v~ r ~ The residue was purified by columncl~ y (eluent: CH2C12!'CH30H 98:2). The eluent of the desirGd fraction was
vv - r ' 1, yielding 3.7 g (38.0%) of (+)-(B)~[(3-vlllvlu~)hv~1)[2-(methylthio)-l~-
imidazol-l-yl]methyl]-2 ' (interm.9).
h) A mixture of " (9) (6.2 g), Raney rlickd (6 g) and methanol (100 ml) was
11~.' ~ ' for 2 hours at 2 bar ;und at room . After the calculated amount
3û of hydrogen was taken up, the cat~lyst was filterGd of The filtrate, (+)-(B)-4-[(3-
vllu~u~ 1)[2-(methylthio)-l~-imidazol-l-yl]methyl]-1.2-t-~ -- " (int~rm.
10), was used for the next step.
i) A mixture of ' (10) (5.7 g), ' ~ (5.2 g) and
methanol (100 ml) was stirred anc'~ refluxed for 3 hours. The reaction mixture was
evapclrated and the residue was taloen off in .1:. 1.1. ,. . - -- ;~ - - . and washed with sodium
hydrvgen carbonate (10%). The clrganic layer was dried, filtered and vv r ' ~ The
oily residue was purified by column 1 l- ' ~ y (eluent: CH2C12/CH30H 95:5).
woss/22s40 ~ l 8 ~583 r~
The eluent of the desired fraction was ~.r 1~ yielding 4.9 g (83.7%) of
(+)-(B)-5-t(3-'' .,' yl)[2-(methylthio)-l~I-imida
(interrn. 11).
B. Preparation of the final compounds
E~;amyle 3
A mixture of ' (2) (185 g) in water (512 ml) was sttrred at 20 C
Hyl' ' ' acid (289 ml) was added. Formic acid (85%) (61.17 ml) was added and
this mixture was heated to 55C The reaction mixture was stirred for 3 hours at 55 C
and then cooled to 20C Dh,lllul~ ' (1223 ml) was added. Ammonium
hydroxide (730 ml) was added dropwise at < 25C The separated organic layer was
washed with water (500 ml), dried, filtered and the solvent was c~, i yielding
152.88 g (108.5%) of product. A sample was dried (18 hours at 55 C), yielding 3.18 g
of (+)-(B)-5-[(3-chlorophenyl)-1~-imidazol-1-ylmethyll-lH-I,. . 1~ . ,1. mp.
113.7C; [a]D = +43.46 (c = 1% in methanol) (comp. 1).
~xamp1e 4 ~ =
A mixture of ' (11) (4.9 g), Raney nickel (2 g) and ethanol (lOOml) was
stirred and refluxed for 5 days, while every day an additional aTnount of Raney nickel (2
g) was added. The catalyst was filtered off and rinsed with L.,l.lu.~ ' The
filtrate was evaporated and the residue was purified twice by column ~ ' ~ , ' .y
(silica gel; CH2C12/CH30H 95:5; CH2C12/CH30H/NH40H 80:20:3). The eluent of the
desired fraction was evapr~rated and the residue was converted into the ll.y~lluclllv~i~ salt
in 2-propanol and ethanol. The salt was Iccly~l~lli~1 from 2-butanone, yielding 1.8 g
(37.2%) of (+)-(B)-5-[(3-~ Jl)(l_-imirlazol-l-yl)methyl]-lH-~ '
~ J~ ., mp. 212.1C; [a]D = +42.43 (c = 1% in ethanol) (comp. 2)
xample 5
Compound (1) (149.7 g) was dissolved in 2-butanone (2424 ml). A mixture of
h.y~ ' ' ' acid in 2-propanol (82.6 ml) in 2-butanone (727 ml) was added over a 2
hour period at 20 C. The reaction mixture was stirred for 16 hours at 20 C. The
precipitate was filtered off, washed with 2-butanone (242 ml) and dried (vacuum; 80C);
yielding 147.5 g (99.3%) of (+)-(B)-5-[(3-clllululJll~,.l.yl)-l~-imidazol-l-ylmethyl]-1~-
---;~1~ lr l~u~lOh~ , mp. 214.5C; [a]20 = +36.20 (c = 1% in methanol)
(comp. 2).
~ WC95n2s40 21 82583 r_",l ~/~e.~
-13-
Example 6
A mixture of compound (1) (0.72 g) in ethanol (5.1 ml; ~' d) was stirred at 20 C
until it became h ~ - ~c -J - (E~ A - ;- acid (0.54 g) was added. The mixture
was stirTed for 18 hours at 20 C and dhen cooled 0-5 C and 1~ resulted. MvTe
~ ' edhanol (2 ml) was adlled And the mixture was stirred for 2 hours at 20 C.
The precipitate was filtered off, w;lshed with edlanol (3 ml; I' I) and dried
(vacuum; 50 C), yidding 0.26 g ~23.4%) (B)-5-[(3~1~1v~u~ yl)-lH-imidazol- l-yl-medhyl]-lH-~ ' '~ (E)-2-1, -~ f~ (2:3).edlanolate (2:1); mp. 111.2C
(comp. 3).
c rl .A. ., . - . .l~ ;. _l Examples
FxAml?lP 7: Retinoic acid metabolism in human ton~ue squamous carcinoma cells.
Human tongue squamous carcinoma cells (SCC~5) were seeded in 6-well plates and
~Town for 4 days at 37 C The nledium used consisted of a 1 :I mixture of Hans' F12
and Dulbecco's modified Eagle's medium ~ l ' ' with ll~u~u~ vll~ and fetAI
calf serum. After 4 days of growth, the meAium was replaced by a I .~ t~, serum
free medium and the confluent ce]ls were further incubated for 3 days. The medium was
refreshed 16 hours before dhe ons(~t of dhe PYrPnmPnf To measure fhe effect of dhe test
compound on retinoic acid ' '~ test compound and/or 2 111 DMSO were added to
dho rr~edia before dhe reaction was initiated by the addition of 1 ~LCi [11,12-3H]-retinoic
acid. After 3 hours of incubation 'at 37 C the medium plus the cells were extracted and
[11.12-3Hl-retinoicacidwasanalyzedbyHPLCasdescribedbyVanWauweetal.,J.
Pharrnacol. Exp. Ther. (1992), 2t,1: 773-779. The results of dhis test showed dhat
compound No. 2, i.e. dhe HCI salt of (+)-liarozole, inhibited dhe IIJ~UAYI-IiOII of
retinoic acid in human tongue squamous carcinoma cells widh an ICso value of 1.0 IIM,
dhe HCI salt of rAcemic liarozole ~lad an Ic50 value of 2.9 IIM and dhe HCI salt of
(-)-lia~ozole was almost inactive.
Example 8: Induction of Pinnal Epidermal Hyperplasia in Hairless Mice
One of dhe cutAAneous effects of retinoids is dheir potent ability to induce epidermal
1~ in v~vo (CoMer, Models in d~ tvlu~;r 1987, Vol. 3 KaTger, Basel, 1987,
p. 23-28). Therefore, liarozole and bodh its - ~ v ~ lly isomers were compared
widh all-rra~-retinoic acid for the~r ability to induce epidermal hyperplasia in hairless
mice.
Female hairless mice, weighing 25-30 g, were treated orally and once daily for 14
~ , days widh either plac~.bo (PEG 200), all-~..., acid or dhe test
compound. On day 15, dhe animals were killed and eAAr tissue was collected from which
wo 9~/12540 2 1 8 2 5 8 3 r~
-14-
2 ~Lm thick sections were prepared for Illvll)llulog;~l analysis. The thickness of the total
viable epidermis was measured and the epidermis Qf placebo treated mice consisted of a
thin epithelium. In contrast, the epidermis of animals treated with RA or a testcompound was ;.y~ Jl~Lic. The results are presented in Table 1.
~1
Compound dose (mgtkg) % increase versus placebo
all-~ra~ retinoic acid 5 156
HCI salt of racemic liarozole 10 48
HCI salt of (+)-liarozole (Comp. No. 2) 10 49
HCI salt of (-)-liarozole 10 21
HCI salt of racemic liarozole 20 123
HCI salt of (+)-liarozole (Comp. NQ 2) 20 93
HCI salt of (-)-liarozole 20 10
r l 9 Tu~i~o~ test.
10 Dogs were ' ' daily and for one month an oral dose of the test ~ r ~ .
compound No. 2 and the HCI salt of (-)-liarozole. At a dose of 10 mgtkglday, the. ~ .. l ,~: " .~ m most dog tissues for compound No. 2 were at least a tenfold lower than
for the HCI salt of (-)-liarQr.ole.
15 D. Conlrosition Examples
The following r~ exemplify typical ~ suitable for
systemic or topical ' to animal and human subjects in accordance with the
present inYention.
"Active ingredient" (A.I.) as used throughout these examples relates to a compound of
20 formula (1) or a l,l . , . . ",, ~ ;, ~11 y acceptable acid addition salt thereof.
F ' 10: Olal dro~ls
500 g of the A.I. was dissolved in 0.51 of 2-llJ~U~y~l~, acid and 1.51 of the
~ ,...yL,I.~ glycol at 60~80~C After cooling to 30~40C there were added 351 of
25 ~ul~ glycol and the mixture was stirred well. Then there was added a solution of
1750 g of sodium saccha~in in 2.51 of purified water and while stirring there were added
251 of cocoa flavor and I~UI,~,II-JI~ , glycol q.s. to a volume of 501, providing an oral
drop solution comprising 10 mgtml of A.L The resulting solution was filled into suitable
containers.
~ wo ss/22s40 2 1 8 2 5 ~ 3 ~ ;5
-15-
Example 11: Oral solution
9 g of methyl 4-lly~v~ y~ ~ nd I g of propyl 4~ u~y' were dissolved
in 41 of boiling purified water. In 31 of this solution were dissolved first 10 g of
5 2,3 .~ LVA~ acid and thereafter 20 g of the A.I. The latter solution was
combined with the remaining part ~Df the former solution and 121 1,2,3-~ 1 ' and
31 of sorbitol 70% solution were added thereto. 40 g of sodium saccharin were
dissolved in QS I of water and 2 ml of raspberry and 2 ml of gooseberry essence were
added. The latter solution was corllbined witb the former, water was added q.s. to a
10 volume of 201 providing an oral sl)lution comprising 5 mg of the A.l. per
(5 ml). The resulting solution was filled in suitable containers.
Example 12: Capsules
20 g of the A.I., 6 g sodium lauryl sulfate, 56 g starch, 56 g lactose, 0.8 g colloidal
15 silicon dioxide, and 1.2 g ~, stearate were vigotously stirred together. The
resulting mixture was 1 . '~ filled into 1000 suitable hardened gelatin capsules,
each comprising 20 mg of the Al.
FY^~I~ 13: Film-coated tablets
pr~r"~h~m ~.f ~
A mixture of 100 g of the A.L, 57() g lactose and 200 g starch was mixed well and
thereafter humidifled with a solution of 5 g sodium dodecyl sulfate and 10 g
IJuly v .yl~ ulidvllc (Kollidon-K 90 (!9) in about 200 ml of water. The wet powder
mixture was sieved, dlied and siev~d again. Then there was added 100 g y~dllil.~,
cellulose (Avicel ~)) and 15 g ]l~ vegetable oil (SterDtex ~D). The whole was
mixed well and ~,UI~ Cd into tablets, giving 10.000 tablets, each comprising 10 mg
of the active ingredient.
.GQahn~
To a solution of 10 g methyl cellull~se (Methocel 60 HG (1~)) in 75 ml of ' '
ethanol there was added a solution of 5 g of ethyl cellulose (Ethocel 22 cps (!~) in 150 ml
of Jicllvl~ ' Then there we.re added 75 ml of di~,Llv~ull~.,L~ , and 2.5 ml
1,2,3-1 l~ . ' 10 g of pvl~Lll.~ , glycol was rnolte n and dissolved in 75 ml ofd;~,lllulv~n~,;llOllc. The latter soluticln was added to the former and then there were added
2.5 g of magnesium, l~ , 5 g of l~ulyv .r~ uLdu~lc and 30 ml of
' color suspension (Opaspray K-1-2109 ~9) and the whole was hr ~ '
The tablet cores were coated with the thus obtained mixture in a coating apparatus.
woss/22s40 21~2583 r~ c~ o
r 14 Injectable solution
1.8 g methyl 1 ~ VAy, and Q2 g propyl 4-lly~v~ . were dissolved in
about 051 of boihng water for injection. Afhsr cooling to about 50C there were added
while stirring 4 g lactic acid, Q05 g propylene glycol and 4 g of the A.L The solution
5 was ccoled to room i , amd ~ t I with water for mjection q.s. ad 1 1
volume, giving a solution of 4 mg/ml of A.L The solution was sterilized by filtration
(U.S.P. XVII p. 811) amd filled in sherile containers.
F ' 15: S~,~v~
3 g A.l. was dissolved in a solution of 3 g 2,3-dihydroxy-L - ' acid in 25 ml
PVIJ~ glycol 400. 12 G surfactant (SPAN (~) and ~ ;ly~,i~ (Witepsol 555 ~)
q.s. ad 300 g were molten together. The lather mixture was mixed well with the former
solution. The thus obtained mixture was poDd mto moulds at a h ~l. . of 37~38C
to form 100 ~ each containing 30 mg of the active ingredient.
F ' 16: 2~o cream
75 mg stearyl alcohol, 2 mg cetyl alcohol, 20 mg sorbitan and 10 mg
isopropyl myristate are imtroduced into a doublewall jacketed vessel and heated until the
mixtD has completely molten. This mixture is added to a separately prepared mixture of
purified water, 20v mg propylene glycol and 15 mg ~JVIJ ' 60 having a t. '1- '- ~` "
of 70 to 75C while using a I - -- , for liquids. The resulting emulsion is aUowed
to cc~ol to below 25C while '~, mixing. A solution of 20 mg A.I., I mg
PVIJ ' 80 and purified water and a solution of 2 mg sodium sulfite anhydrvus in
purified water are next added to the emulsion while c~ y mixing. The cream, I g
25 of the A.I. is 1,,~,,,. - 1 and filled into suitable tubes.
Example 17: 2% topical ~el
To a solution of 200 mg IIYdIVAY~IUIJYI ~-1y~10rl~Ai~ . m purified water is added 20 mg
of A.l. while stirring. Ilydlv~ lllvli~ acid is added urltil cornplete dissolution amd then
30 sodium hydroxide is added until pH 6Ø This solution is added to a dispersion of 10 mg
PJ in 50 mg propylene glycol while mixing. While mixing slowly, the
mixture is heated to 50C and allowed to cool to about 35C whereupon 50 mg ethyl
alcohol 95% (v/v) is added. The rest of the purified water q.s. ad I g is added and the
mixture is mixed to h~
F ' 18: 2% tspical cream
To a solution of 200 mg IIY~UAY~I~U~YI ~-.,y, ' ' in purified waher is added 20 mg
wo 9sn2s40 2 1 8 2 5 ~ 3 I~
-17-
of A.L while stirring. Il~ ' ' acid is added until complete dissolution and nextsodium hydroxide is added until l~H 6Ø While stirring, 50 mg glycerol and 35 mg
~1.~ ' 60 are added and the mixture is heated to 70C The resulting mixture is
added to a mixture of 100 mg mirleral oil, 20 mg stearyl alcohol, 20 mg cetyl alcohol, 20
mg glycerol and 15 mg sorbate 60 having a . of 70C while
mixing slowly. After cooling do~n to below 25C, the rest of the purified water q.s. ad
I g is added and the mixture is miixed to I
r ~ 19: 2% li~osome '( ' -
A mixttlre of 2 g A.I. microfine, 70 g I ' , ' '~,1 choline, 5 g cholesterol and 10 g
ethyl alcohol is stilred and heated at 55-60C until complete dissolution and is added to a
solution of 0.2 g methyl paraben, 0.02 g propyl pardben, 0.15 g disodium edetate and
0.3 g so~ium chloride in purified water while l~ 0.15 g Hydlw-yyluyyl-
ulùs~, in purified water ad 100 g is added and the mixing is continued until
swelling is cotnplete.
F ' 20: 2% li~osome r,."..~ ,u.l
A mixture of 10 g yllu~ylldtidyl clloline and I g cholesterol in 7.5 g ethyl alcohol is
stirred and heated at 40C until cclmplete ~icc~ ti~n 2 g A.I. microfine is dissolved in
20 purified water by mixing while heating at 40C. The alcoholic solution is adcled slowly to
the aqueous soluion while llv.llo~_l.,Lillg during 10 r~inutes. 1.5 g IIylllu~yluy~l-
hjl~llulu~ in pulified water is adcled while mixing until swelling is complete. The
resulting solution is adjusted to p~H 5.0 with sodium hydroxide I N and diluted with the
rest of the purified water ad 100 g.
