Note: Descriptions are shown in the official language in which they were submitted.
2~o~3~
wo95/29672 r~ l"J_~3v5~47
HALOMETHYL AMIDES AS IL-1~ PROTEASE INHIBITORS
~ BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a series of novel non-peptides which exhibit selective in
vitro and in vivo inhibition of interleukin-1~ converting enzyme, to cu",,uo~iLiolls
10 containing the novel non-peptides and to methods for therapeutic utility. More
particularly, the interleukin 1~ converting enzyme inhibitors described in this invention
comprise novel a-halomethyl amides and o~-halomethyl sulfonamides which possess
particular utility in the treatment of il1~1d~ dLuly and immune-based diseases of lun~,
central nervous system, and connective tissues.
Rer,~orted De~/eloD, I ,e~
Interleukin-1,~ (IL-1,~) protease (also known as interleukin-1~ converting enzyme
or ICE) is the enzyme ,e,spor,siLle for processing of the L;~IU~;C~A'Y inactiYe 31 kD
20 precursor IL-1~ to the Liologic~"y active 17 kD form (Kostura, M.J.; Tocci, M.J.;
Limjuco, G.; Chin, J.; Cameron, P.; Hillman, A.G.; Chartrain, N.A.; Schmidt, J.A., ~Q~,
Nat Acad. Sci.. (1989), ~, 5227-5231 and Black, R.A.; Kronheim, S.R.; Sleath, P.R.,
FEBS Let.. (1989), ~, 386-391). In addition to acting as one of the body's earlyresponses to injury and infection, IL-1,~ has also been proposed to act as a mediator of
25 a wide variety of diseases, inc!uding rheumatoid arthritis, osteoarthritis, ill~ldlllllldluly
bowel disease, sepsis, acute and chronic myelogenous leukemia and o~leuuolu:,is
(Dinarello, C.A.; Wolff, S.M., New En~l. J. Merl. (1993), ~2~, 1û6). A naturallyoccurring IL-1,B receptor antagonist has been used to de",on~l,dl~ the intermediacy of
IL-1~ in a number of human diseases and animal models (Hannum, C.H.; Wilcox, C.J.;
30 Arend, W.P.; Joslin, G.G.; Dripps, D.J.; Heimdal, P.L.; Armes, L.G.; Sommer, A.;
Eisenberg, S.P.; Thompson, R.C., ~, (1990), 343, 336-340; Eisenberg, S.P.;
Evans, R.J.; Arend, W.P.; Verderber, E.; Brewer, M.T.; Hannum, C.H.; Thompson,
R.C., Nature (1990), 343, 341-346; Ohlsson, K.; Bjork, P.; Bergenfeldt, M.; Hageman,
R.; Thompson, R.C., Nature, (1990), 348, 550-552; Wakabayashi, G., E~B, (1991),
--1--
2~8~36
WO 95/29672 PCT/IJS95/0~347
338-343; Pacifici, R.; et al. Proc. Natl. ~r ~r~. Sci. (1989), ~fi, Z398-2402 and
Yamamoto, I.; et al. Cr~ncer Rsh (198~ 242-4246). The specific role of IL-l~ in
~ ldlllflldlion and immunomodulation is supported by the recent observation that the
cowpox virus employs an inhibitor of ICE to suppress the i,,~lc,,,,,,d~ory response of its
host (Ray, C.A. et al, ~11, (1992), ~2, 597-604).
In summary, the utility of ICE inhibitors in modifying certain IL-1 mediated
disease states has been suggested and de",onal,dled in vivo by several workers in the
field. The following review of the current state of the art in ICE research further
10 supports such utility of ICE inhibitors:
1 ) WO 9309135, published 11 May 1993, teaches that peptide-based aspartic acid
arylacyloxy-and aryoxymethyl ketones are potent inhibitors of ICE in vitro. These
compounds also specifically inhibited ICE in the whole cell (in vivo) by their ability to
15 inhibit the formation of mature IL-1 B in whole cells. These ICE inhibitors also
clell,ol~L~dled utility in reducing feverand i"~ldll"l,d~k,n/swellin3 in rats.
2) Patients with Lyme disease sometimes develop Lyme arthritis. B. burgdorferi,
the causative agent of Lyme disease, is a potent inducer of IL-1 synthesis by
20 mononuclear cells. Miller et al. (Miller, L.C.; Lynch, E.A. Isa, S.; Logan, J.W.;
Dinarello, C.A.; and Steere, A.C., "Balance of synovial fluid IL-1 B and IL-1 Receptor
Anta~onist and Recovery from Lyme arthritis", L~ (1993) 341; 146-148) showed
that in patients who recovered quickly from Lyme Arthritis, the balance in synovial fluid
of IL-1-beta and IL-1 ra was in favor of IL-ra. When the balance was shifted in favor of
25 IL-1 B, it took si~ iCdr Illy longer for the disease to resolve. The conclusion was that the
excess IL-1 ra blocked the effects of the IL-113 in the patients studied.
3) IL-1 is present in affected tissues in ulcerative colitis in humans. In animal
models of the disease, IL-1~ levels correlate with disease severity. In the model,
30 ad"li"i~lldlion of lL-lra reduced tissue necrosis and the number of ir~ldlllllldL.~ry cells
in the colon.
See, Cominelli, F.; Nast, C.C.; Clark, B.D.; Schindler, R., Llerena, R.; Eysselein, V.E.;
Thompson, R.C.; and Dinarello, C.A.; "Interleukin-1 Gene Expression, Synthesis, and
2-- = . =. = .=
'2~8go~6
W095/29672 r._~ .c~.~47
Effect of Specific IL-1 Receptor Blockade in Rabbit ~mmune Complex Colitis" L5~n.
Im,e~ r~ ns (1990) Vol. 86, pp, 972-980.
4) IL-1 ra supresses joint swelling in the PG-APS model of arthritis in rats
See Schwab, J.H.; Anderle, S.K.; Brown, R.R.; Dalldorf, F.G. and Thompson, R.C.,"Pro- and Anti-l"~l~r"",~Lury Roles of Interelukin-1 in Recurrence of Bacterial Cell Wall-
lnduced Arthritis in Rats". Infect. Immun. (199~ ) ~L; 4436-4442.
5) IL-1 ra shows eflicacy in an small open-label human Rheumatoid Arthritis trial.
See, Lebsack, M.E.; Paul, C.C.; Bloedow, C.C.; Burch, F.X.; Sack, M.A.; Chase, W.,
and Catalano, M.A. "Subcutaneous IL-1 Receptor Antagonist in Patients with
Rheumatoid Arthritis", Arth. Rheum. (1991) 34; 545.
6) IL-1 appears to be an autocrine growth factor for the proliferation of chronic
myelogenous leukemia cells. Both IL-1ra and slL-1R inhibit colony growth in cells
removed from leukemia patients.
See, Estrov, Z.; Kurzrock, R.; Wetzler, M.; Kantarjian, H.; Blake, M.; Harris, D.;
Gutterman, J.U.; and Talpa2, M., "Supression of Chronic Myelogenous Leukemia
Colony Growth by Interleukin-1 (IL-1 ) Receptor Antagonist and Soluble IL-1 Receptors:
2 o a Novel Application for Inhibitors of IL-1 Activity". ~ (1991 ) ~; 1476-1484.
7) As in 6) above, but for acute myelogenous leukemia rather than chronic
myelogenous leukemia.
See, Estrov, Z.; Kurzrock, R.; Estey, E.; Wetzler, M.; Ferrajoli, A.; Harris, D.; Blake, M.;
25 Guttermann, J.U.; and Talpaz, M. "Inhibition of Acute 1\1y~1~gel,ous Leukemia Blast
Proliferation by Interleukin-1 (IL-1) Receptor Antagonist and Soluble IL-1 Receptors".
(1992)~ g79; 1938-1945.
An effective therapy has yet to be fully developed CrJIIIIII~ for the treatment
30 of IL-1~ mediated i"lld"""dlury diseases. Consequently, there is a need for
therapeutic agents effective in the treatment and prevention of these diseases.
--3--
~8~U36
WO 9~i/29672 r~ .' 17 ~
~IIARY ;)F THE lNVFNTlON
According to tha present invention, there is provided a compound of the formula
5 (A) or a pharmaceutically ~r~ept~hla salt thereof:
1~2
R3--N-Y--Rl (A)
wherein:
10 Y = CO or SO2;
R1 = independently selected from alkyl, haloalkyl and alkoxyalkyl;
R2 = H, alkyl, (CH2)-alkenyl, aralkyl, h~lerua,~ yl, carboxyalkyl, cyanoalkyl, aryl,
15 heteroaryl; and
R3 = H, alkyl, (CH2)-alkenyl, aralkyl, heteroaralkyl, aryl, heteraryl;
"Alkyl" is defined as a saturated aliphatic hydrocarbon which may be either straight - or
20 branched chain. Preferred groups have no more than 12 carbon atoms and may be methyl, ethyl, and structural isomers of propyl, butyl, up to dodecyl.
"Haloalkyl" is defined as an alkyl radical ~llhctitllted by one or more halogen (F, Cl, Br,
I). Fûr example chloromethyl, di.;l,l~ru,,,~Ll,yl, fluoromethyl, difluoromethyl,25 fluuluLil,lùrun,ethyl.
"Alkoxyalkyl" is defined as an alkyl radical .s~lhstitlltad by an alkoxy group. For example
methoxymethyl.
30 "Aryl" is defined as a phenyl on naphthyl ring which may be url~llh~titlltQd or substituted
wherein one or more of the hydrogen atoms has been replaced by the same or different
substituents including halo, alkyl, aryl, nitro, cyanû, amino, alkylacylamino, hydroxyl,
alkoxy, haloalkyl.
--4--
~89~
WO 95129672 ~ 47
"Halo" means iodo, bromo, chloro, fluoro.
- "Carboxyalkyl" means an alkyl radical .cllhctitllt~d by a carboxyl group. For example,
5 carboxymethyl.
"Aralkyl" means an alkyl radical Cllhctit~lt~d with an aryl ring. For example benzyl, 4-
chlorobenzyl.
10 "Heteroaryl" means pyridyl, thienyl or furanyl and structural isomers thereof.
le~udldlkyl" means an alkyl radical substituted by an heteroaryl ring. For example
2-thienyl ethyl.
15 "Alkenyl" is defined as an alkyl yroup containing one or more sites of unsaturation. For
example, ethenyl, ethynl, 1-butenyl, 2-butynyl, 1,3-hexadienyl.
"Cyanoalkyl" means an alkyl radical substituted by a cyano group. For example, cyano
ethyl.
The present invention also concerns the pl1al",aceutical C~""~OSi~;OII and
method of treatment of IL-I~ protease mediated disease states or disorders in a
mammal in need of such treatment comprising the d~",i"i~lldlion of IL-l~ protease
inhibitors of formula (A) as the active agent. These disease states and disorders
25 include: infectious diseases, such as meningitis and salj.i"yiiis, septic shock,
respiratory diseases; inflammatory conditions, such as arthritis, ,IIOldllyiLi~, colitis,
~llc~.hali;i~, endocerolitis, hepatitis, pancreatitis and reperfusion injury, immune-based
diseases, such as ~,y~ ensi~ ity; auto-immune diseases, such as multiple sclerosis;
bone diseases; and certain tumors and leukemias.
The present invention has particular utility in the modulation of ~,uces~i"g of IL-
1~ for the treatment of rheumatoid arthritis. Levels of IL-1,B are known to be elevated in
the synovial fluid of patients with the disease. Additionally, IL-I~ stimulates the
synthesis of enzymes believed to be invoived in i"~ld"""dlion, such as collagenase and
--5--
Zlg903~
WO 95/29672 T ~ ~ 7
PuA2, and produces joint destruction which is very similar to rheumatoid arthritis
following intra-articular injection in animals.
~,~.` i' ' '
In the practice of this invention an effective amount of a compound of the
5 invention or a pharmaceutical cul"posilion thereof is administered to the subject in
need of, or desiring, such treatment. These compounds or c~",~o~ilions may be
a.l",i"i~I~r~d by any of a variety of routes depe,ldi"g upon the specific Qnd use,
including orally, parenterally (including subcutaneous, intraarticular, intramuscular and
intravenous a.l",ini~,dlion), rectally, buccally (including sublingually), l,d,~sdel",dlly or
10 i"~,di,a~a',y. The most suitable route in any given case will depend upon the use, the
particular active ingredient, and the subject involved. The compound or composition
may also be a~",il,i~ d by means of cor,l,~''c~ release, depot implant or injectable
formulations as described more fully herein.
In general, for the uses as described in the instant invention, it is expedient to
administer the active ingredient in amounts between about 0.1 and 100 mg/kg bodyweight, most preferably from about 0.1 to 30 mg/kg body weight for human therapy, the
active ingredient will be ~",i"i~lel~d preferably in the range of from about 0.1 to about
20-50 mglkg/day. This a~ ,i"i~IIdIiol, may be ac~o"l~ l,ed by a single d.dlllilli~
20 by distribution over several ~, F' ns or by slow reiease in order to achieve the most
effective results. When administered as a single dose, a~l"i"i:,I,dIion will most
prefenably be in the range of from about 0.1 to mg/kg to about 10 mg/kg.
The exact dose and regimen for ~dlllilli~IIdIioll of these compounds and
25 co",i o~iliuns will necessarily be depellde,,I upon the needs of the individual subject
being treated, the type of treatment, and the degree of affliction or need. In general,
parenteral administration requires lower dosage than other methods of administration
which are more dependent upon di~so"~liun.
A further aspect of the present invention relates to phdll"aceutical compositions
co",~,ri~i"g as an active ingredient a compound of the present invention in admixture
with a pharmaceutically ~ pt~hle, non-toxic carrier. As ",e"liuned above, such
c"",l,o~iiiuns may be prepared for use for parenteral (subcutaneous, intraarticular,
intramuscular or intravenous) a-i"li"i~l,dlion, particularly in the form of liquid solutions
2ls~n36
WO 95/29672 P~ 7
or suspensions; for oral or buccal dd",i"i~l,dlion, particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders, nasal drops or aerosois.
., When a.ll"i"i~el~d orally (or rectally) the compounds will usually be formulated
5 into a unit dosage form such as a tablet, capsule, suppository or cachet. Suchformulations typically include a solid, semi-solid or liquid carrier or diluent. Exemplary
diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, aginates,
lld~audlllll, gelatin, syrup, methylcellulose, polyoxyethylene sorbitar monolaurate,
10 methyl hydroxybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
The compositions may be prepared by any of the methods well-known in the
pharmaceutical art, for example as described in Re,,,i,,utù,)'~ Pl,~""~lltical Sciences.
17th edition, Mack Publishing Company, Easton, PA, 1985. Formulations for parenteral
a~",il,i~lldLiun may contain as common excipients sterile water or saline, alkylene
glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils
of vegetable origin, hydrogenated naphthalenes and the like. Examples of vehicles for
parenteral ad~ dli~n include water, aqueous vehicles such as saline, Ringer's
solution, dextrose solution, and Hank's solution and nonaqueous vehicles such as fixed
20 oils (such as corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl
myristate. Steri~e saline is a preferred v~hicle and the compounds are sufficiently water
soluble to be made up as a solution for all foreseeable needs. The vehicle may contain
minor amounts of additives such as substances that enhance solubility, isotonicity, and
chemical stability, e.g., dlllioxidd"ls, buffers, and preservatives. For oral
25 ddlllilli~LIdliùll, the formula can be enhanced by the addition of bile salts and also by
the addition of ac~lca~,ili,les (Am. J. Physiol. 251:332 (1986)). Formulations for nasal
ad",i"i~l,dlil,ll may be solid and contain as excipients, for example, lactose or dextran,
or may be aqueous or oily solutions for ddlllilli~lldlioll in the form of nasal drops or
metered spray. For buccal a-llllillialldliOIl typical excipients include susars, calcium
30 stearate, magnesium stearate, pr~eldti, lal~d starch, and the like.
When formulated for nasal a.l~"illi~l,dlion the dbsolyLiùll across the nasal
mucous ",e",L,rdn~ is enhanced by surfactant acids, such as for example, glycocholic
acid, cholic acid, taurocholic acid, ethocholic acid, desoxycholic acid,
--7--
~18~36
WO 95/29672 P~ ~ 47
cl,el~odesu,~ycholic acid, dehydrocholic acid, glycodeoxy-cholic acid, and the like (See,
B.H. Vickery, "LHRH and its Analogs-Col,I,dceplion and Therapeutic Applications", Pt.
2, B.H. Vickery and J.S. Nester, Eds., MTP Press, La~ncaster, UK, 1987).
DETAILED Dt:jCP~ ION OF THE INVENTION
The compounds of this invention were prepared by using the general synthetic
methods described in the Schemes below.
0 In Scheme 1, the desired amine (Formula 1) was either purchased c~ e~;i&lll
or prepared by reductive amination of an aldehyde (Formula 2) and an amine (Formula
3), and then acylated or sulfonylated with an appropriate acid or sulfonyl chloride. This
afforded compounds of the type in Formula 4.
Alternatively (Scheme ll), direct alkylation of an acylated amine (Formula 5) was
performed to give differentially N,N-rlisllhstitllt,od amides of the type in Formula 6. The
alkylation reaction proceeds nicely using potassium t-butoxide as a base and
tetrahydrofuran as a solvent
Methods for the preparation of acid chlorides, sulfonyl chlorides, reductive
amination and alkylation of amines are well known in the art. See "Advanced Organic
Chemistry", J. March, eds. McGraw-Hill Book Co., Second Edition, 1977.
--8--
WO 9~i/29672 ~18 9 0 3 g r~ 7
Scheme I
R3-NH2 + R2- CHO
- (formula 2) (formula 3)
NaBH(OAc)3
R NH + Cl Y R base R N Y R
12 R2
(formula 1) (formula 4)
Scheme II rR2
R3-N-Y-RI (1) KOtBu R3-N-Y-RI
(formula 5) (2) W-cH2R2 (formula 6)
WO 9~i/29672 ~ ~ 8 :~ 0 3 6 PCTII~S95/05347
Exsmple 1
Preparation of N-Allyl-N-(2.4-dichlorobenzyl) chlo~ ,ide (?)
,,~ + H ~N / ~--N-- ~
Cll
+ Cl~----Q J~N~
Cl oJ c
Part A: To 1 9 of 2 4-dichiorobe"~dl.le~,yde in 6 mL of 1 2-di~ u~ll,d"e was added
428 uL of allyl amine 28û uL of acetic acid and 1.8 g of NaBH(OAc)3 in the givenorder. After 3û minutes the reaction mixture was diluted with ulllo~u~u,,,, and saturated
aqueous NaHC03. The layers were separated and the organic layer was dried
(MgS04) and c~ncer,l~dL~d in vacuo affording a colorless oil. Flash ~ lUllldlUUldUlly
(15% EtOAc-hexane) afforded 476 mg (38%) of 1 as a colorless oil.
Part B: 476 mg of 1 was dissolved in 5 mL of methylene chloride and 3û6 uL of Et3N
was added. The reaction mixture was cooled to 0C and 175 uL of ul~luruac~lyl
chloride was added and the mixture was stirred for 2 h. The reaction was then diluted
with .:I,loru,ur,,, and washed lwice with water. The combined organic layers were dried
(MgS04) and concenl,dled in vacuo provided a white solid. Flash ulliurlldluyldu~ly
(15% EtOAc-hexane) afforded 50û mg of ? as a white solid:
Low Resolution Mass Spec. mlz (relative intensity):
292 (M+H; 1ûO) 256 (76) 174 (12) 159 (34) 146 (5)
--10--
WO 9S/29672 Z ~ 8 g 0 3 6 r~ 17
Example 2
Pl~pa,dliorl of N-Ben7yl-N-~.4-di.,l,lolubel,,yl) cl,lû~ PL"r,lide (4).
,
~NH+ Cl~a ~ HN)~ ci
Cll
3 + Ph~Br Cl~\~ ~h Cl
Part A: 100 9 ûf 2,4-dichlorobenzylamine was dissûlved into 600 mL ûf CH2CI2 andthe reaction mixture was cooled to 0~C. Next, 89 mL of Et3N was added fûllowed by
the dropwise addition of 50 mL of cl,lorudc~lyl chloride. The reaction mixture was
stirred for 24 h. The reaction was then washed twice with H20, dried (MgSO4) andcollcenLldLt:d in vacuo affording a solid which was triturated with 10% hexane-EtOAc
affording 131 9 (97%) of 3 as a pure white solid.
Part B: 500 mg of 3 was dissolved in 2 mL of THF and 2 mL (1û equiv) of benzyl
15 bromide was added. Next, 276 mg of potassium t-butoxide in 7 mL of THF was added
dropwise to the reaction mixture which was then stirred for 30 minutes and finally was
concentrated in vacuo. The residue was dissolved in chlûroform and washed twice with
H20, dried (MgS04) and collcer,lldl~d in vacuo aflording a yellow oil. Flash
1111UlllULU~U,ld,Uhy (15% EfOAc-hexane) aflorded 500 m3 (73%) of 4 as a white solid.
20 Low Resolution Mass Spec. m/z (relative intensity):
356 (M+H; 11),196 (4), 91 (100)
Using the methods described in Examples 1 and 2, the following were also prepared:
--11--
WO 95129672 2 ~ 8 9 0 3 6 1 ~ J~ ~
Ex~mple 3
N-~2.4~ lolv~:"~yl?-N;methyl ~ Groac~a~ e
Low Resolution Mass Spec. mlz (relatlvè intensity):
266 (M+H; 16), 232 (53), 214 (19),188 (100),173 (16).
Exsmple 4
N-i~en~yl-N-(3-~l,lv-uber,~/l) cl~'oruac~t.,;,,~d~
Low Resolution Mass Spec. m/z (relative intensity):
308 (M+H; 36), 216 (5), 182 (17), 106 (15), 91 (100)
i_xample 5
N-Ben_yl-N-(2.5- i;cl~loroi~e"~ cl,'or.,ac~ .l, ' 'e
Low Resolution Mass Spec. mlz (relative intensity):
342 (M+H; 58), 306 (15),182 (32),106 (23), 91 (100).
Example 6
N-(4-CI~lvrvb~ vacet6~ 'e
25 Low Resolution Mass Spec. mlz (relative intensity):
308 (M+H; 22), 274 (13), 230 (11), 125 (90), 91 (100).
Example 7
3 0 N-~en7yi-N-(3.4-~icl ~lol ob~"~ !c l oac~ta", -' e
Low Resolution Mass Spec. mlz (relative intensity):
342 (M+H; 20), 106 (20), 91 (100).
--12-- =
2~8g~36
WO 95/29672 r~ 7
E~ample 8
~ Btr,c~l N (2-chlcr~,ber~yl) ~,I.loroate~ " le
.. Low Resolution Mass Spec. m/z (relative intensity~:
308 (M+H; 50), 272 (14), 182 (15), 125 (10), 106 (22), 91 (100).
E~ample g
N-Benzyl-N-(2.3-di~ ob~,.cyl) cll'or~ ,. 'e
Low Resoiution Mass Spec. m/z (reiative intensity):
342 (M+H; 36), 306 (15),182 (13),106 (17), 91 (100).
s Example 10
N-Cyanoethyl-N-(2.4-d;_l1'or~b~ yl) "le~ ,Ar..t~ " le
1H NMR (CDCI3) ~ 7.44-7.06 (m, 3H, Ar), 4.70 (s, 2H, (OCH2-O) 4.23 and 4.11 (twosinglets, 2H (rotamers), ArCH2-N) 3.63 and 3.55 (two triplets, 2H (rotamers) J = 6.53
Hz each, N-CH2-CH2) 2.68 and 2.63 (two triplets, 2H, J = 6.53Hz each (rotamers)
CH2-CN)
Example 11
N-Cyanomethyl-N-(2.4-.Ji~.l,lol~.~e~,~yl) tl\:ol~",t:ll"~ un .. i~'
1H NMR (CDCI3) â 7.55-7.28 (m, 3H, Ar), 4.72 (s, 2H, S02CH2-CI) 4.58 (s, 2H,
ArCH2N) 3.70 (t, 2H, J = 7.02Hz, N-CH2CH2) 2.61 (t, 2H, J = 7.21 Hz, CH2-CN).
Example 12
N-Cy -.~ot~ l N ~2.4-di.,l ~l GI sb~ yl) PI ~C ' ~ " d e
Low Resolution Mass Spec. m/z (relative intensity):
285 (M+H; 72), 249 (100),188 (7), 159 (9),109 (6).
' 35
--13--
~3g~3~i
W0 95/~9672 r ~ 17
Example 13
N-Cyanoethyl-N-(2.4-dichlJ,v~er~yl) fluG~ iJe
Low Resolution Mass Spec. m/z (relative intensity):
289 (M+H; 100), 253 (40), 159 (20).
Example 14
N-(2.4-Diclllorol~en~yl)-N-~(3-l~henyl)propyl~ rvc.celc~",:~e
Low Resolution Mass Spec. m/z (relative intensity):
370 (M+H; 62), 336 (53), 302 (23),185 (40),159 (69),125 (31), 93 (100).
Example 15
~(N-CI~ oact ~yl)-N-(2.4-dicl~lvrvbel,~yl)l ~Iycine
Low Resolution Mass Spec. m/z (relative intensity):
31 1 (M+H;100), 274 (46), 232 (16), 159 (1 1 ), 115 (5).
Example 16
N-(2.4-Dicl~ vb~ tl)-N-~(2-thienyl)ethyl] cl.lar-,~c.it.,.l, 'e
Low Resolution Mass Spec. m/z (relative intensity):
364 (M+H; 100), 326 (7), 266 (12),159 (29),110 (56).
Exampie 17
N-(2.4-D;chl~vber,~l)-N-r(2-thienyl)methyll tlllervac~al~ e
1H NMR (CDCI3) ~ 7.49-6.98 (m, 6H, Ar), 4.78 and 4.66 (two singlets, 2H (rotamers)
Ar-CH2-N), 4.75 ~s, 2H, COCH2-CI), 4.28 and 4.12 (two singlets, 2H, N- H2-thiophene)
--14--
WO 9~129672 ~18 9 0 ~ ~ r~ ,3,r.~ 7
Example 18
N-(3-Chlolvl;orl~yl) clllcr
Low Resolution Mass Spec. m/z (relative intensity):
218 (M+H; 80), 182 (77), 153 (11), 141 (16), 125 (100), 106 (42).
Example 1 9
N-(2.3-r :~hlGIv~e.,~yll ~,I,lor~.ac~,t~ '6
Low Resolution Mass Spec. m/z (relative intensity):
254 (M+H; 59), 216 (100),159 (74),106 (42).
Example 20
N~ r~ichlorobenzyll ~ lo
Low Resolution Mass Spec. m/z (relative intensity):
254 (M+H; 95), 216 (100),159 (95),141 (13),106 (89).
Example 21
N-(2.4-Diclll~rvbe"~yl) chlol~ ,.;Je
25 Low Resolution Mass Spec. m/z (relative intensity):
252 (M+H; 38), 217 (20), 185 (43.6), 159 (25), 132 (19), 110 (20), 93 (100), 75 (32).
Example 22
N-r(2.4-Dichloro~henyl)ethyll cl,l~l-,a~ ", :'e
Low Resolution Mass Spec. m/z (relative intensity):
266 (M+H; 23), 232 (11), 185 (56), 139 (9),170 (13), 93 (100), 75 (24).
-15-
~8~13~
W0 95/29r~72 ~ r ~ 47
Compounds of the present invention were tested for IL-I~ protease inhibition
activity according to the following protocols:
In Vitro ,
Partially purified IL-1~ protease is stored at -80C, thawed on ice, and
preincubated for 10 minutes at 37DC with 2.5 mM dillliu~ ul in a buffer solutioncontaining 10 mM Tris-HCI (pH 8.0) and 25% (v/w) glycerol. Inhibitors are prepared as
stock solutions in dimethyl sulfoxide (DMSO). The protease is preincubated with
inhibitor in a volume of 20 ,uL in a 1.5 mL polypropylene ",iu,u.id,ll,iluge tube for 15
minutes at 37C. The volume of compound added to the assay is adjusted to yield a
DMSO ~un~r~ ,dliùll in the preincubation of <15% (v/v). The enzyme assay is theninitiated by the addition of substrate (TRITC-AYVHDAPVRS-NH2) (SECI I.D. No. 1 ) to
yield a final concentration of 67 ,uM in a final volume of 30 ,uL. The reaction are carried
out for 60 minutes at 37C in the dark and are It,r",i"dL~d by the addition of 10 mL of
10% trifluoroacetic acid (TFA). Following the addition of 115 ,uL of 0.1% TFA, the
samples are analyzed by high pressure liquid ~ rUllld~U_ld,ulrly using a reverse phase
(C18) column and elution with an ac~lurlil,ile/water/TFA gradient. Substrate andproduct are monitored by their dU5~l l,a,lc~ at 550 nm and elute at 4.2 and 5.2 minutes,
2 o respectively.
The compound in example 1- poccpss~c IL-1~ protease inhibition (ICsO = <1.0
uM).
25 In Vivo
In vivo inhibition (IC~o~ was d~lel",il,ed as follows:
Human monocytes were isolated from hepd,i"i~rd leukopheresis units obtained
30 through Biological Specialty Corporation ~Lansdale, PA). Monocytes were purified by
Ficoll-Hupaque (Pharmacia Fine Chemicals, ri y~ NJ) gradient centrifugation
and more than 95% pure monocyte populations obtained by centrifugal elutriation. The
assay was performed on duplicate samples of freshly isolated human monocytes,
cultured in suspension at 37C and rotated gently in conical bottom polypropylene
--16--
218~36
wo ssl2s6n r~l,u~ 17
tubes (Sardstedt Inc., Princeton, NJ). Human monocytes at a concenl~d~ion of 5 x 106
cells/mL were resuspended in 1 mL of RPMI 1640 (a common tissue buffer from M.A.Bioproducts, Walkersville, MD) containing 1% fetal calf senum (FCS) (HyClone, Logan,
~, UT) and 50 ,ug/mL gentamycin (Gibco, Grand Island, NY). The cells were treated either
5 with a compound of the invention (i.e. test compound) or with a non-inhibitor (control
compound, typically 0.03% DMSO) for 15 minutes and then activated with 0.01% fixed
Staphylococcus aureus (The Enzyme Center, Malden, MA) for 1 hour. The cells werethen centrifuged and resuspended in 1 mL of cysteine, methionine-free RPMI mediacontainin~ 1% dialyzed FCS (Hyclone). The cells were pretreated with a test
lO compound or control compound for 15 minutes after which 0.01% fixed S. aureus plus
100 uCi Tran 35-S label (ICN, Irvine, CA) was added and the cells incubated at 37C
for 1 hour. After incubation, cells were centrifuged, washed once in phosphate buffer
saline and resuspended in 1 mL RPMI containing 1% fetal calf serum. The cells were
again pretreated with a test or control compound for 15 minutes and then 0.01% S.
15 aureus for 2 hours. At the end of the incubation, cells were centrifuged and supernates
saved for immunoprecipitation. Cells were washed once in phOalJhdl~ buffer saline
and then Iysed in RIPA, a continuous cell media buffer containing 2 mM
phenylmethylsulfonyl fluoride, 10 mM jorlo~r~Z~l~, 1 ,ug/mL pepstatin A, 1 ,ug/mL
leupeptin and 0.5 TIU aprotinin.
For the immul,oprt:~ipi~d~;ons, an equal volume of 1% dry milk in RIPA buffer
plus 50 ,uL of resuspended protein A sephdlc,se CL-4B (Plla""a~,ia, Piscataway, New
York) was added to supernates and 1 mL of 4% dry milk containing protein A
sepharose CL-4B to cell Iysates and samples rotated for 30 minutes at 4C. Beads25 were then centrifuged down, samples lldn~ d to fresh tubes and incubated
overnight with 40 ,ug rabbit anti-human IL-lp polyclonal antibody (Genzyme,
Cambridge, MA). The IL~ proteins were then prt~ ,ildled with 70 ,uL protein A
sepharose, resuspended in 60 ,uL SDS sample buflerand run on 15% SGD-PAGE gels.
Autolddio~d,olly was performed on dried gels and the amount of Id~ioa~tiJ;.y (counts
30 per minute, cpm) quantitated using a Betascope 603 analyzer.
--17--
fi g~6
WO 95/29672 PCT/US95/05347
Dsta Analysis
In the monocyte pulse chase assay, each test parameter was run in duplicate.
Data was collected from the Beta Scope using a personal computer, then transferred to
5 the VAX system for calculation of mean ~p~n and standard deviation of the mean.
When test compounds were evaluated, the percent inhibition of rQlease of mature IL-l~
was calculated as follows:
100 x [1 - (cells treated with stimuli + test compound -
unstimulated cells)/(cells treated~with stimuli + control compound-
unstimulated cells)]
These % inhibition values were then used to calculate IC50 value for each
compound. Since the human monocyte pulse chase assay uses primary cells from
15 different donors, each test compound was run in 2-3 separate experiments, using
monocytes from 2-3 different donors.
The compound in Example 1 had in vivo IC~ of <10 I~M.
--18--
W095/29672 2~89û36 r~l,u~ 47
SEQUENCE 1ISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Dolle, Roland E.
Rinker, James M-
(ii) TITLE OF INVENTION: Halomethyl Amides As IL--1~ Protease Tnh;h;~rR
(iii) NUMBER OF SEQUENCES: 1
(iv CO~ESPONDENCE ADDRESS:
~A :DD~ESSEE: SterLing Winthrop Inc.
~B TH3ET: 9 Great Valley Parkway
~CI T~ET: P.O. Box 3026
D I CI rY klalvern
E "T.,TE: PA
F CO JNTRY: USA
IG ZIP: 19355
(v) COMPUTER READABLE FOPM:
(A) MEDIUM TYPE: Diskette, 3.5 inch, 2.0 MB storage
(B~ COMPUTER: Apple Macintosh
(C) OPEFATING SYSTEM: Macintosh 7.1
(D) SOFTWME: Microso~t Word 5.1a
(vi) CURRENT APPL
(A) APPLICATION NUMBER To bé Assigned
(B) FILING DATE: 4/29/9~
(C) CLASSIFICATION: 1811
(viii) ATTORNEY/AGENT INFORMATION:
~A) NAME: Doreen M. Wells
(B) REGIsTRArrIoN NUMBER: 34, 278
(iX) TT.T~r~-MMT~NTr~TION INFORMATION:
(A) TELEPHONE: (610) 889-8684
(B) TELEFAX: (610) 889-6364
(2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHMACTERISTICS
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: -1
(D ) OTHER INFORMAT ION: / l abe l= TRITC
/note= "TRITC is tetramehylrh~ m;n~ isothiocyanate".
(ix) FEATURE:
(A) NAME/~EY: Modl~ied-site
(B) LOCATION: 11
(D) OTHER INFORMATION. /label= Xaa
/note= "Xaa is NH2 " -
xi) SEQUENCE DESCRIPTION: SEQ ID NO
Ala Tyr Val His Asp Ala Pro Val Arg Ser
1 s lo
--19--
