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IRREVERSIBLE ENZYME INHIBITORS. CLXVII
January 1970
Irreversible Enzyme Inhibitors. CLXVII. Thymidine Phosphorylase. X.3 On The Nature and Dimensions of the Hydrophobic Bonding Region. I1 's2
B. R. BAKERASD STEPHES E. HOPKIXS~ Department of Chemistry, University of California at Santa Barbara, Santa Barbara, California 93106
Received August 28, 1969 6-(l-Naphthylmethylamino)uracil(1) and three of its derivatives substituted on the naphthalene ring with 7-chloro (Z), 6,i-dichloro (3), and 6,i-dimethyl (4) were synthesized for evaluation as inhibitors of Escherichia coli B thymidine phosphorylase. The appropriate a-tetralone (12) was condensed with triethyl phosphonoacetate, then dehydrogenated to the naphthalene-1-acetic esters (14) with o-chloroanil. Reaction with hydrazine gave the crystalline hydrazides (15) which were degraded with NOCl to the corresponding substituted 1-iiaphthylmethylamines (17); the latter were condensed with 6-chlorouracil to give the inhibitors 1-4. These four compounds were excellent inhibitors of thymidine phosphorylase being complexed 1900-5800-fold better to the bacterial enzyme than the substrate, 5-fluoro-2'-deoxyuridine (FUDR); the best inhibition was observed with 2.
The dimensions of the hydrophobic bonding region of Escherichia coli B thymidine phosphorylase were previously determined with 29 derivatives of 6-anilinouracil and 6-benzylaminouracil where substituents were placed on the benzene ring; from these studies emerged the map of the hydrophobic bonding region shown in Figure l . 3 One of the predictions from this map was that 6-(cu-naphthylmethylamino)uracil (1) should be an excellent inhibitor by hydrophobic complexing with areas C and D. Whether position 14 or 17 was hydro-
1, K 1 = R 2 = H 2, Ri = H, Rp= C1
3, R,
R2= C1
4, Ri = R2 = CH3
phobic was less certain, although it appeared that one of these two positions was hydrophobic; that' position 14 was hydrophobic, but position 17 was not', has now been established by synthesis and evaluation of 2-4. The results are the subject of this paper.5 Enzyme Results.-Insertion of t'he D ring (Figure 1) on 6-benzylaminouracil ( 5 ) resulted in 1 which gave 30fold bet'ter binding (Table I) ; this increment in binding is the same order when the D ring is inserted on 6anilinouracil (6) t o give 7, thus verifying that the D ring complexes to a flat hydrophobic area.3 Insertion of 7-chloro group (2) on 1 gave a further threefold increment in binding indicating that position 14 (1:igure 1) is hydrophobic. The corresponding 6,7dichloro (3) and 6,'i-dimethyl (4) derivatives were no more effective than 2, indicating that' position 17 wa,s not a hydrophobic area. 6-(7-Chloro-l-naphthyl(1) This work was supported in part b y Grant C.l-08695 from the Kational Cancer Institute, U. S. Public Health Service. (2) For the previous paper of this series see B. R . Baker and pi. 11. J. Vermeulen, J . M e d . C h e m . , 13, E2 (1970). (3) For the previous paper on this enzb-me see B. R. Baker and W,Rzeszotarski, i b i d , , 11, 639 (1968). (4) NSF trainee on Grant KO.GZ-796. ( 5 ) For the possible chemotherapeutic utility of a tissue-specific blockade of this enzyme see (a) B. R . Baker, J . M e d . Chem., 10, 297 (1967), paper of this series: and (h) I?. R. Baker, "Design of Active-Site-Directed Irreversible Enzyme Inhibitors," John Kiley B; Sons, New York, N. Y., B
EO.
TABLE I
IXHIBITIO OFNTHYMIDINE ~,~ PHOSPHORYLASE BY
-
H +If concn for
No.
R
50% inhihn
(~Sl/[Il)o,sc
1 2 3 4
l-Saphthyl-CH20.21 1900 7-C1- 1-napht hyl-CHa0.079 .5800 6,7-C12-naphthy1-CH20.11 3700 6,i-lIe2-naph thyl-CH20.10 4000 6.2d 65 J CsHjCH2 40d 10 6 CsH: 7 2-Kapht hyl 2.2d 180 8 2,3-C12CsHa 0.33 1100 a The technical assistance of YIaureen Baker and Julie Leseman is acknowledged. * Thymidine phosphorylase was a 45-90c7, ( X H ~ ) ~ Sfraction OI from E. coli B prepared and a s a y e d with 400 p J I 2'-deoxy-5-fluorouridine (FUDR) in al.seiiate-succiiiate buffer (pH 5.9) containing lOyo DlISO as previonJy described.5 Ratio of 400 p M FUDR to concentratioii of inhibitor giving 50yo inhibition. Data from ref 3.
methy1)aminouracil (2) is the most pori-erful inhibitor of E. coli B thymidine phosphorylase yet observed; 2 was complexed to the enzyme 5800-fold more effectively than the substrate, FUDR. -4striking species difference in the hydrophobic binding area between the enzyme from E . colz B and Walker 256 rat tumor has been observed; both 1 and 8 were 1100-fold less effective on the tumor enzyme than the bacterial enzyme.6 Contrariwise, 5-benzyluracil was a n excellent inhibitor of the Walker 256 and rat liver enzymes with an ([SI/[ I ] ) 0 5 ratio of 60, but wa9 300fold less effective on the E . colz B enzyme; such species differences in binding to a hydrophobic bonding region adjacent to the active site have been previously observed with dihydrofolate reductase.' Chemistry.-The 6-( I-naphthylmethy1)aminouracils (1-4) 1% ere synthesized by condensation of 6-chlorouracil 11ith the appropriate 1-naphthylniethylamines (17) ; 3 the parent 1-naphthylmethylanliIie was commercially available, but its derivatives (17) were synthesized by the sequence shown in Scheme I. The (6) B. R Baker and
J. L. Kelley, manuscript in preparation. ( 7 ) B. R. Baker, J . M e d . Chem., 10, 912 (19671, paper S C V I I of this series. (E) 4 Paul and D. Sen, Indian J. Chem., 2, 212 (1964).
'29
12
CH,COOEt
CHCOOEt
14
13
15
CH,NHCOOCHJ,,H-
17
16
K,= C1; R, = H b, K l = R,=CI c , Rl=R,=CHj d. R, = CH,,; K, = H a,
aroylpropionic acids (10) - I 1 were reduced wit,li NZH? :md KOH in vthylcrte glycol1' to 11. Cyclization of 11 E. 13. liarnett ani1 F. C;. Sander., .I. Chem. Soc.. U 4 ( I 110) 8 . Rkraug and E.ScliwamLerrer. d i r n . . 461, 135 (1928). S t w k , 11. 1'. I?rundage, and L. Fletcher, J . .in!.Ctieni. S n c . , 1 5 , I117 ( l 9 , 5 3 ) . (12) !liiang-5linlori. t h z d . , 68, 2-18: j l 0 4 S ) . :$I)
January 1970
6-FLVOROTETRACYCLINES
(tip 60-110'); B, 5 : s C6H,-HOAc. All analytical samples gave combustion values within 0.4Yc of theory. y . ( 3,4-Dichlorophenyl)butyric Acid (11b).-Huang-hlirilon rediiction'2 of 10b (prepared by ref 11 at 100') gave 83% of crude product, bp 140-144' (0.1 mm), mp 48-64', that was suitable fur the next step. Three recrystallizations from EtOH-H,O gave white crystals, mp 62-65". Anal. (CloHloC1,On) C, H. 6,7-Dichloro-l-tetralone(12b).-To 184 g of polyphosphoric acid was added lS.4 g of Pz05followed by 18.4 (78 mmoles) of l l b . The mixtiire was stirred a t 100" for 30 min, then cooled to 50' mid diliited with 500 nil of ice water. The product was collected 0 1 1 a filter, washed with water, and recrystallized from EtOH-H20; yield 11.9 g (69%) of white crystals, mp 104-10S0, tic in solvent A. d n a l . (CloH&l?O) C, H, C1. Synthesized in the .*ame way were 12d [bp 77" (0.1 nim), m p 3.7' (lit.9 nip 3 5 O ) ] , 12a (nip 93-93", lit.18 nip 94")) and 12c [hp 84-00' (O.05 mm), nip 44-46'], Anal. ( C ~ P H ~C, ~ O€1.) 6,7-Dichloronaphthyl-l-acethydrazide(15b) (Method A).-To a stirred siiq~eiisioiiof 3.7 g (92 mmoles) of 60% siispension of N a l l io niiileral oil in '200 ml of dimethoxyethane (previously di,ied with nicdecdar -ieves) protected from moisture was added l S . 4 g (44 mmoles) iif triethyl phosphonoacetate. After being stirred at ambient temperatiire for 43 min, H? evoliitioii was complete. .I solutioii of 12 g (56 mmoles) of 12b in 12 nil of diiiiethoxyethaiie x a s added, then the mixture was refluxed for 24 hr. The cooled reactioii mixtiire was diluted with 400 ml of HuO and extracted twice with CHC1,. The combiiied extracts were washed with H L O , dried with lIgP04, and evaporated Zn t~acito. L)i.tillntioii gave 13.1 g (8x7;) of 13b, bp 727-130" (0.07 nim), a colorless oil; tlc in solvent A showed one major, one minor, and t w o trace spot.?. The ir and nmr spertra were in agreement with this striictiire. A >.oliitioii (Jf 14.9 g (61 ninioles) of o-chloranil and 15.7 g (56 nimolec) of 13b in 700 nil of PhLle was refluxed for 4 hr; duriiig thi.. time the wlutioii tiirned red, then orange. The solution was evapoi,ated in ruri(o and the residue dissolved in 700 ml of CHCL. (IS) J. v. Braun, A . Rohmer. H. Junpman, F. Zobel, L. Rrauns, 0. Stuckensclimidt, and J. Reutter, A n n . , 461, 1 (1926).
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The solution was washed successively with three 400-ml portions of 0.1 1V KOH and two 150-ml portions of H?O, then evaporated i n z)acuo leaving crude 14b with the proper iiv, ir, and nmr specti a. -4mixture of 1 he crude 14b, 350 ml of 8.55NnH4.H20, and 110 ml of EtOH w8.s refluxed for 4 hr, then evaporated t o a small voliime in uacuo. The solid was collected 011 a filter and washed extensively wit11 HiO. The criide product was stirred with 1.5 1. of 0.3 LY HC1 rind 1 1. of LIeOH iintil soliition was essentially complete. The filtered soliition was carefiilly made slightly bacic with 5% XaHC03. The solid wah collected on a filter, wached with HnO, then recrystallized from hIeOII-H,O with the aid of charcoal; yield 3..76 g (24'3), mp 198' on a KoflerHeizbank since a melting point taken i n the usual fashion showed no definite nielting point diie t o detompoyition. Pee Table I1 for additional data and other compoiinds prepared in this way. 6,7-Dichloro-l-naphthylmethylamineHydrobromide (17b) (Method B).--A solution of 1.49 g (2.2 mmoles) of CsHjCH2OH in 270 ml of CsH6was dried with moleciilai, sieves, then 2.93 g (11 mmoles) of 15b was added. The stirred siispension was satiirated with K0C1. After beiiig stirred 30 min at ambient temperatiire ( N ? evolved), the soliition was refluxed 4.5 hr, then evaporated in vacuo. To the residiial criide 16b dissolved in 20 ml of HOA4ctwas added a soliitioii of 4.Sg of I1Br gas in 40 ml of HOAc; CO, evoliitioii was complete in 15 min and the product begaii t o separate. The niixtu1,e was diliited Tvith several volumes of Et,O, then the prodiict was collected O I I a filter and washed als, mp 2M-284 dec, with Et,O; yield, 2.1x g (6.5%) of >ample was recrystalsuitable for the next htep. For anal) lized from EtOH-Et,O; see Table I1 for additional data and other conipoiiiids prepared by this method. 6-(6,7-Dicldoro-l -naphthylmethylamino)uracil ( 3 )(Method C). --.4mixtiire of 2.10 g (6,s nimoles) of 17b, 0.564 g (6.8 mmoles) of KaAc, 0 . i 5 g ( 5 . 2 mmoles) o 6-chl0roiiraci1,~and 78 ml of H I 0 was refliixed with stirring for 48 hr. The hot solution was filtered and the prodiict washed with H?O; yield 0.82 g (45Yc). Recrystallization from UlIF gave 0.22 g (12%) of product, mp >360"; tlc in solvent €3 showed one vpot. See Table I1 for additional data and other compoiiiids pi'epai,ed by this method.
6- Fluorotetracyclines' PANAYOTA BITHA,JOSEPH J. HLAVKA, A N D JARIES H. BOOTHE Lederle Laboratories, A Diz)isionof American Cyanamid Company, Pearl Riwr, iVew York 10565 Received July 3, 1969 The reaction of lla-hslotetracycliiies with liquid HF results in the replacement of 6-OH with F. When 1la-chloro-6-demethyltetracycline is treated with HF, the two possible 6-F stereoisomers were isolated. A tentative assignment of stereochemistry is given. When either 7,l la-dichloro-6-demethyltetracycline or 7 , l ladichlorotetracycline is treated with HF, only the more stable 6a-F isomers were isolated. In the latter caye some previou-ly reported 6-methylene derivative wab also isolated.
The lability of the 6-OH in the tetracycline molecule to both acid? and base degradation3 has thwarted past efforts at successful replacement of this group with other than H.4 Recently we have found that reaction of (1) A preliminary report of this work was given a t the First Sortheast Regional Meeting of tlie .\merican Chwnical Society, Boston, AIass., Oct 1968. (2) C. R . Stephens, L. 11. Conorer, R.Pasternack, F. A . Hochstein, W. T. hIoreland, P. P. Regna. F. J. Pilgrim. K . J. Hrunings, and R . €3. Woodward, J . A m . Ckem. Soc., 76, 3568 (1954); C. \T-aller. B. L. Hutchings, C. F. Wolf, A . A . Goldman, R. Broschard. and J. H. Williams, ibid., 74, 4981 (1952). Acid treatment of tetracycline results in a ready trans elimination of the 6-hydroxy group t o yield anhydrotetracycline.
OH
6
6,
OH
1la-chloro-6-demethyltetracycliiie (1) in liquid HF yielded a mixture of two 6-fluor0 stereoisomers, 2a,b (Chart I). Dehydrochlorination of either of these stereoisomers afforded the same 5a(6)-anhydro-Gfluoro-G-demethyltetracycline (3). An nmr of this (3) I3ase treatment of tetracycline yields isotetracycline.
(4) (a) J . R. D. RIcCormick. E. R . Jensen, P. 4. RIiller, and A . P. Doerschuk, J . Am. Chem. Soc., 82, 3381 (1960); (b) C. R . Stephens e t . al.. ibad., BO, 6324 (1958).
