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High-Affinity Inhibitors of Dihydrofolate Reductase: Antimicrobial and Anticancer Activities of 7,8-Dialkyl-1,3-diaminopyrrolo[3,2-f]quinazolines with Small Molecular Size Lee F. Kuyper,*,† David P. Baccanari,† Michael L. Jones,† Robert N. Hunter,† Robert L. Tansik,† Suzanne S. Joyner,† Christine M. Boytos,† Sharon K. Rudolph,† Vince Knick,† H. Robert Wilson,† J. Marc Caddell,† Henry S. Friedman,‡ John C. W. Comley,§ and Jeremy N. Stables§ Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K., and Duke University Medical Center, Durham, North Carolina 27710 Received July 13, 1995X
A series of 7,8-dialkylpyrrolo[3,2-f]quinazolines were prepared as inhibitors of dihydrofolate reductase (DHFR). On the basis of an apparent inverse relationship between compound size and antifungal activity, the compounds were designed to be relatively small and compact. Inhibitor design was aided by a GRID analysis of the three-dimensional structure of Candida albicans DHFR, which suggested that relatively small, branched alkyl groups at the 7- and 8-positions of the pyrroloquinazoline ring system would provide optimal interactions with a hydrophobic region of the protein. The compounds were potent inhibitors of fungal and human DHFR, with Ki values as low as 7.1 and 0.1 pM, respectively, and were highly active against C. albicans and an array of tumor cell lines. In contrast to known lipophilic inhibitors of DHFR such as trimetrexate and piritrexim, members of this series of pyrroloquinazolines were not susceptible to P-glycoprotein-mediated multidrug resistance and also showed significant distribution into lung and brain tissue. The compounds were active in lung and brain tumor models and displayed in vivo activity against Pneumocystis carinii and C. albicans. Introduction The enzyme dihydrofolate reductase (DHFR) is a known target for drug action.1,2 Inhibitors of DHFR have proven useful in the treatment of cancer,3-6 bacterial infections,7 malaria,8 and Pneumocystis carinii pneumonia (PCP).9,10 Efforts to develop new therapies based on DHFR inhibition continue. One area of recent focus is the search for inhibitors of DHFR from various opportunistic organisms such as the protozoan parasite Toxoplasma gondii 11-13 and the fungus Candida albicans.14 The importance of opportunistic diseases has risen considerably in recent years, owing to the large increase in the population of immunocompromised patients associated with the AIDS (acquired immune deficiency syndrome) epidemic, organ transplantation, and cancer chemotherapy.15-17 Therapeutic intervention is limited by the dearth of safe and effective antiparasitic and antifungal agents.17 Another area of recent interest is the development of novel lipophilic DHFR inhibitors for neoplastic disease.4 Although methotrexate (MTX, 1) is effective against acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma,3,18 the hydrophilic nature of MTX restricts its distribution to various body tissues, such as lung and brain,18 and prevents entry into cells by diffusion. Thus, the antitumor activity of MTX relies on the active transport of the drug into cancer cells,19,20 and resistance to MTX can arise from impairment of this transport mechanism.21
The lipophilic inhibitors metoprine (DDMP, 2), piritrexim (PTX, 3), and trimetrexate (TMX, 4) were designed to avoid the limitations of MTX. DDMP, for example, concentrates in the brain relative to plasma and was evaluated as a potential treatment for brain tumors.22 Both PTX and TMX are active against transport-impaired MTX-resistant cell lines and show tissue distribution profiles significantly different from that of MTX.23,24 In addition, compounds PTX and TMX also exhibit clinical activity against P. carinii, an opportunistic fungal organism afflicting many AIDS patients.25-28 As in many other fungal and bacterial species, folates are synthesized de novo by P. carinii, and the organism lacks the ability to take up folates from its environment. Thus, PTX and TMX can be given in combination with folinic acid (leucovorin), which is taken up only by the host cells and counteracts the antifolate toxicity of the drugs.9,10
* To whom correspondence should be addressed. Current address: Glaxo Wellcome Inc., Five Moore Dr., Research Triangle Park, NC 27709. † Wellcome Research Laboratories, NC. ‡ Duke University Medical Center. § Wellcome Research Laboratories, U.K. X Abstract published in Advance ACS Abstracts, January 15, 1996.
0022-2623/96/1839-0892$12.00/0
© 1996 American Chemical Society
High-Affinity Inhibitors of Dihydrofolate Reductase
Scheme 1a
Journal of Medicinal Chemistry, 1996, Vol. 39, No. 4 893
Scheme 2a
a Reagents and conditions: (a) NaN(CN) , 1-octanol, reflux, 13 2 h; (b) isopentyl tosylate, NaH, DMF.
This paper describes a series of lipophilic DHFR inhibitors that showed antimicrobial and anticancer activities and potentially advantageous tissue distribution properties. The compounds arose from an effort to identify DHFR inhibitors with useful activity against the fungus C. albicans. Initial attempts to identify lead compounds from our corporate collection of DHFR inhibitors were disappointing. Although a number of compounds showed significant inhibition of C. albicans DHFR (e.g., PTX and TMX), few displayed activity against C. albicans in vitro. Inconsistencies between the level of enzyme inhibition and antimicrobial activity were observed with other classes of inhibitors as well. As one working hypothesis, we speculated that antimicrobial activity might be inversely related to the molecular size of the inhibitor. That assumption led us to design a series of 7,8-dialkylpyrrolo[3,2-f]quinazolines 5 that were relatively small and compact but showed high affinity for DHFR. Inhibitor design was based on the three-dimensional structure of C. albicans DHFR and aided by the computer program GRID.29 The resulting compounds were potent inhibitors of bacterial, fungal, protozoal, and human cells. Compared to PTX, TMX, and MTX, the new compounds displayed (1) substantially greater activity against human and fungal DHFR, (2) more effective growth inhibition of a number of tumor and fungal cell lines, (3) activity against cells with P-glycoprotein-mediated multidrug resistance, (4) superior tissue distribution properties for lung and brain tumor applications, (5) activity in murine models for brain and lung tumors, and (6) superior in vivo activity against P. carinii and C. albicans.
Chemistry The preparation of 1,3-diamino-7H-pyrrolo[3,2-f]quinazolines was originally reported by Ledig30 and involved a thermally induced condensation of 5-aminoindole and dicyanamide. 7-Substituted analogues were prepared by alkylation of the parent pyrroloquinazoline or the intermediate indole. Our initial preparation of compound 5i, for example, employed analogous procedures, alkylating with isopentyl tosylate as illustrated in Scheme 1. However, the low yields of the alkylation reactions and the need to explore a variety of 7,8-dialkylated analogues prompted us to develop alternative procedures. We also devised improved conditions for effecting the cyclization using boron trifluoride etherate.31 Scheme 2 outlines procedures employed in our improved synthesis of compounds 5. The key 2,3-dialkyl5-nitroindole intermediates 9 (Table 1) were obtained by one of two methods: (1) N-alkylation of 2-alkylindoles
a Reagents and conditions: (a) H NR, DMSO, 50 °C; (b) ketone, 2 NaBH3CN, HCl, CH3OH; (c) ICl; (d) alkyne, CuI, Et3N, DMF, Pd(PPh3)2Cl2; (e) CuI, DMF, reflux; (f) ROTs, NaH, DMF; (g) HNO3, H2SO4; (h) H2, Pd/C; (i) NaN(CN)2, DMF; (j) refluxing diglyme or BF3‚etherate, DME.
11 using sodium hydride in DMF and the desired alkyl tosylate, followed by regioselective nitration, or (2) copper-catalyzed cyclization of 2-alkynyl-4-nitroanilines 8 (Table 2). The latter compounds were derived from the 4-nitro-N-alkylanilines 6 (Table 2), which were prepared from 4-nitroaniline via reductive alkylation32,33 or from 4-fluoronitrobenzene by aromatic nucleophilic substitution with the appropriate alkylamine.34,35 Iodination of the N-alkylanilines 6 was effected with ICl in methanol.36,37 Introduction of the alkynyl moiety employed palladium catalysis,38 and cyclization of the alkynylanilines 8 to the indoles 9 was performed with copper iodide.39,40 Conversion of the nitroindoles 9 to the aminoindoles 13 was performed in high yield using catalytic hydrogenation. Construction of the diaminopyrimidine ring followed our previously reported procedures for the preparation of the parent ring system.31 The aminoindoles 13 were condensed with dicyanamide to give the cyanoguanidine derivatives 14 (Table 3), and ring closure to the target compounds 5 (Table 4) was carried out thermally or with boron trifluoride etherate. Synthesis of tert-butylsubstituted analogues 5f,l,p,r was incompatible with the procedure employing BF3‚etherate but was effected successfully using thermal conditions. Results and Discussion Inhibitor Design. Hundreds of inhibitors of DHFR have been reported during the past several decades,41 and most of the compounds that show high affinity for DHFR appear to make use of the large hydrophobic cleft observed in X-ray crystal structures of the enzyme from various species.2,42-44 For example, PTX, a potential treatment for PCP,10 binds to P. carinii DHFR with its dimethoxybenzyl group positioned within the large hydrophobic cavity of that enzyme.45 As shown in Figure 1, the benzyl group of the inhibitor is surrounded by the side chains of Leu-25, Ile-33, Phe-36, Ile-65, Pro66, Phe-69, and Leu-72. Of particular pertinence to the work described here is a series of 7-substituted-pyrrolo[3,2-f]quinazolines
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Table 1. Substituted Indolesa
mp (°C)
yieldb (%)
Me Me H H Et
128-129 127-128 151-152 134-136 84-85
79 38 73 86 42
Me i-Pr n-Pr Et Me Et Me t-Bu Et
i-Pr Et Et t-Bu C(Me)2Et CH(Me)Et CHEt2 i-Pr CHEt2
156-158 70-72 67-68 121-123 66-68 63-64 69-71 125-126 oil
63 45 65 66 74 81 31 95 92
NO2 NO2 NO2 H H H H
n-Bu n-Pr i-Pr i-Pr n-Pr Et Et
t-Bu CHEt2 CHEt2 H H Me Et
90-91 55-56 oil 72-73 oil oil oil
71 98 92 69 81 60 46
12h 12j 12k 12o 13c 13d 13f 13g 13h
H H H H NH2 NH2 NH2 NH2 NH2
Me i-Pr n-Pr Me Me Et t-Bu Et Me
i-Pr Et Et CHEt2 Me Me H Et i-Pr
oil oil oil oil >250 220-221 >250 175-176 230-231
26 78 90 50 92 100 100 93 100
13j 13k 13o
NH2 i-Pr NH2 n-Pr NH2 Me
Et Et CHEt2
230-231 185-186 175-176
96 98 96
compd
X
R′
9c 9d 9e 9f 9g
NO2 NO2 NO2 NO2 NO2
Me Et i-Pr t-Bu Et
9h 9j 9k 9l 9m 9n 9o 9p 9q
NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2
9r 9s 9t 11e 11k 12d 12g
R
formula C10H10N2O2 C11H12N2O2 C11H12N2O2 C12H14N2O2 C12H14N2O2‚ 0.1H2O C12H14N2O2 C13H16N2O2 C13H16N2O2 C14H18N2O2 C14H18N2O2 C14H18N2O2 C14H18N2O2 C15H20N2O2 C15H20N2O‚ 0.1H2O C16H22N2O2 C16H22N2O2 C16H22N2O2 C11H13N C11H13N C11H13N C12H15N‚ 0.2hexane C12H15N C13H17N C13H17N C14H19N C10H12N2‚HCl C11H14N2‚HCl C12H16N2‚HCl C12H16N2‚HCl C12H16N2‚ 0.25EtOH‚ 0.2HCl C13H18N2‚HCl C13H18N2‚HCl C14H20N2‚HCl
a All compounds were characterized by 1H NMR, mass spectra, and elemental analyses. Other compounds in these series were not purified for analytical purposes and were characterized only by 1H NMR spectra. b Yields were not optimized.
Kuyper et al.
first reported by Ledig.30 Those compounds are inhibitors of mammalian and bacterial DHFR46 and show in vitro activity against the C. albicans organism.47 Molecular modeling suggested that this class of inhibitor binds to DHFR with the 7-substituent occupying the large hydrophobic cleft in a manner similar to the dimethoxybenzyl group of PTX; those compounds with larger 7-substituents generally displayed higher affinity for C. albicans DHFR. For example, compound 5u was comparable to PTX and TMX in its inhibition of the fungal enzyme (see Table 5). However, the inhibitors with the larger substituents were relatively ineffective inhibitors of C. albicans growth. Thus the challenge was to reduce inhibitor size and yet retain tight enzyme binding, and the design problem was to identify small substituents for the pyrroloquinazoline heterocycle that would impart high affinity for DHFR. The relatively potent activity reported for 7-(cyclopropylmethyl)-2,4diaminopyrrolo[3,2-f]quinazoline suggested that small alkyl substituents can impart high affinity for DHFR46 and effective inhibition of C. albicans cell growth.47 Further exploration of such small alkyl substituents was aided by the use of GRID, a computer program that evaluates the interaction between a protein active site and a variety of probe functions.29 A crystal structure of the C. albicans holoenzyme was furnished by Whitlow and co-workers,48 and a methyl group probe was used in GRID to analyze the binding site. As illustrated in Figure 2, regions of favorable interaction energy for a methyl group, formed by Met-25, Ile-33, Phe-36, Met54, Ile-62, and Leu-69, were adjacent to the 7- and 8-positions of enzyme-bound pyrroloquinazoline. The area next to the 8-position was relatively small and centered in the plane of the inhibitor’s ring system. Small, branched alkyl groups at the 8-position appeared to take good advantage of that site. On the other hand, the 7-position of the pyrroloquinazoline was positioned at the edge of the large hydrophobic cavity of the protein, and favorable binding locations for the methyl probe were found close to the parent inhibitor but above and below the plane of the pyrroloquinazoline ring system. Substituents such as the 3-pentyl group that would adopt conformations that are perpendicular to the pyrroloquinazoline ring system, as shown in Figure 2, were required to position methyl groups into regions
Figure 1. Stereoview of the active site region of the X-ray crystal structure of the P. carinii DHFR-NADPH-piritrexim complex. The protein backbone is represented as a dark pink tube, and and atoms of selected residues are shown. Atoms of piritrexim and NADPH are colored by atom type: carbon, white; nitrogen, cyan; oxygen, red; and phosphorus, orange.
High-Affinity Inhibitors of Dihydrofolate Reductase
Journal of Medicinal Chemistry, 1996, Vol. 39, No. 4 895
Table 2. Substituted Anilinesa
compd
X
Y
R
mp (°C)
yieldb (%)
formula
6h 6l 6m 6n 6o 7h 7l 7m 7n 7o 8l 8m 8n 8o 8q 8r 10e 10k
NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 H H
H H H H H I I I I I -CtC-Et -CtC-Me -CtC-Et -CtC-Me -CtC-Et -CtC-Bu -CtC-i-Pr -CtC-n-Pr
i-Pr t-Bu C(Me)2Et CH(Me)Et CHEt2 i-Pr t-Bu C(Me)2Et CH(Me)Et CHEt2 t-Bu C(Me)2Et CH(Me)Et CHEt2 CHEt2 t-Bu H H
82-84 68-69 oil oil oil 52-53 115-116 oil oil oil 131-132 oil oil oil oil oil oil oil
92 84 67 89 83 67 71 81 76 71 89 95 98 85 99 90 94 91
C9H12N2O2 C10H14N2O2 C11H16N2O2 C10H14N2O2 C11H16N2O2 C9H11N2O2I C10H13N2O2I C11H15N2O2I C10H13N2O2I C11H15N2O2I C14H18N2O2 C14H18N2O2 C14H18N2O2 C14H18N2O2‚0.2H2O C15H20N2O2 C16H22N2O2 C11H13N C11H13N
a All compounds were characterized by 1H NMR, mass spectra, and elemental analyses. Other compounds in these series were not purified for analytical purposes and were characterized only by 1H NMR spectra. b Yields were not optimized.
Table 3. N-Cyano-N′-(1,2-dialkylindol-5-yl)guanidinesa
compd
R′
R
mp (°C)
yield (%)b
formula
14c 14i 14k 14n 14o 14q 14r
Me H n-Pr Et Me Et n-Bu
Me CHEt2 Et CH(Me)Et CHEt2 CHEt2 t-Bu
215-216 68-69 195-196 135-137 151-152 145-147 127-128
89 47 86 57 97 37 67
C12H13N5 C15H19N5 C15H19N5 C16H21N5 C16H21N5 C17H23N5 C18H25N5
a All compounds were characterized by elemental analyses, 1H NMR, and mass spectra. Other compounds in this series were not purified for analytical purposes and were characterized only by 1H NMR spectra. b Yield was not optimized.
indicated by the GRID analysis. On the basis of this simple analysis, the series of 7,8-dialkylpyrrolo[3,2-f]quinazolines listed in Table 4 were prepared. In Vitro Biological Activities. The series of 7,8dialkylpyrrolo[3,2-f]quinazolines 5a-u were evaluated as inhibitors of C. albicans and human DHFR and for growth inhibition of fungal and human cells. As shown in Table 5, several members of the series displayed exceptional affinity for DHFR and potent inhibition of cell growth. DHFR Ki values were as low as 7.1 pM against C. albicans DHFR and 0.1 pM for the human enzyme. Compound concentrations effective against whole cells ranged as low as 1 ng/mL for C. albicans and 0.57 nM for the human colon tumor cell line HCT8. Potent activity was also observed against bacteria. For example, compound 5o inhibited Escherichia coli with a minimum inhibitory concentration (MIC) of 0.1 ng/mL. In this series of pyrroloquinazolines, only the parent compound 5a and its 7-methyl derivative 5b were less active than PTX and TMX as inhibitors of C. albicans DHFR. Activity against the fungal enzyme increased
substantially with additional alkyl substitution at either the 7- or 8-position. The compound with the lowest measured Ki value (5m, Ki 7.1 pM) was 3200-fold more active than its parent compound 5a and more than 260fold more active than PTX and TMX. Alkyl substitution at the 8-position of the pyrroloquinazoline ring system enhanced activity significantly. An 8-methyl group provided a 12-fold increase in affinity for the fungal enzyme (5c versus 5b), and isopropyl substitution increased activity more than 140-fold (5e versus 5a). Tertiary butyl and n-propyl groups also fit into the fungal enzyme binding pocket (compounds 5f,k,p,s), but, as suggested by the molecular modeling experiments, the binding pocket imposed a substituent size limit at the 8-position. The n-butyl group appeared to encounter those limits; compound 5r was significantly less active than the corresponding ethyl-substituted analogue 5l. The enzyme active site imposed fewer restrictions on size for substituents at the 7-position of the pyrroloquinazoline ring system, as exemplified by the trimethoxybenzyl group of compound 5u (and also the large substituents of PTX and TMX). However, the GRID analysis suggested that smaller 7-substituents with appropriate shape could contribute significantly to binding. This was verified by compound 5i; its 3-pentyl group provided a contribution to enzyme affinity (Ki 0.22 nM) equivalent to that of the much larger trimethoxybenzyl group of compound 5u (Ki 0.23 nM). A combination of small, compact alkyl substituents at the 7- and 8-positions provided inhibitors with exceptional affinity for the fungal enzyme (compounds 5l-q,s,t). The effects of 7- and 8-alkyl substituents on affinity for human DHFR were similar to those observed for the fungal enzyme. The most active pyrroloquinazoline inhibitors, compounds 5l-q,s,t, displayed Ki values in the sub-picomolar range and were significantly more active than PTX and TMX. For reference purposes, the most potent pyrroloquinazolines were also 1 order of magnitude more active than MTX (Ki 1.3 pM), the bench-mark hydrophilic anticancer agent.3 No evidence for substitution patterns that might selectively favor
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Kuyper et al.
Table 4. 7,8-Disubstituted-1,3-diaminopyrrolo[3,2-f]quinazolinesa
compd
R′
R
mp (°C)
yield (%)b
formula
5ac 5bc 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5uc
H H Me Et i-Pr t-Bu Et Me H i-Pr n-Pr Et Me Et Me t-Bu Et n-Bu n-Pr i-Pr H
H Me Me Me H H Et i-Pr CHEt2 Et Et t-Bu C(Me)2Et CH(Me)Et CHEt2 i-Pr CHEt2 t-Bu CHEt2 CHEt2 3,4,5-trimethoxybenzyl
264-266 248-250 303-305 >250 >250 >250 227-229 150-160 197-198 217-219 239-241 200-202 254-256 199-200 200-201 286-287 155-157 233-234 121-122 243-244 242-243
64 18 38 57 20 21 36 42 47 20 41 65 28 37 33 65 60 23 64 30 46
C10H9N5 C11H11N5‚0.6H2O C12H13N5‚0.5H2O C13H15N5 C13H15N5‚0.2EtOAc‚0.3H2O C14H17N5‚0.5H2O C14H17N5‚0.95H2O C14H17N5‚0.7H2O C15H19N5‚0.9H2O C15H19N5 C15H19N5‚0.1H2O C16H21N5 C16H21N5 C16H21N5‚0.7H2O C16H21H5‚0.25H2O C17H23N5‚0.2H2O C17H23N5‚H2O C18H25N5‚TFA C18H25N5 C18H25N5‚0.5H2O C20H21N5O3‚0.5H2O
a All compounds were characterized by 1H NMR, mass spectra, and elemental analyses. b Yield was not optimized. c Originally reported in ref 30.
Table 5. Biological Data for 7,8-Disubstituted-1,3-diaminopyrrolo[3,2-f]quinazolines
compd PTX TMX 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u a
R′
H H Me Et i-Pr t-Bu Et Me H i-Pr n-Pr Et Me Et Me t-Bu Et n-Bu n-Pr i-Pr H
R
MW
C. albicans DHFR Ki (nM)a
human DHFR Ki (pM)a
C. albicans MIC (µg/mL)
H Me Me Me H H Et i-Pr CHEt2 Et Et t-Bu C(Me)2Et CH(Me)Et CHEt2 i-Pr CHEt2 t-Bu CHEt2 CHEt2 3,4,5-trimethoxybenzyl
325 369 199 213 227 241 241 255 255 255 269 269 269 283 283 283 283 297 297 311 311 311 379
1.9 1.9 23 25 2.0 1.3 0.16 0.12* 0.33 0.22 0.22 0.33 0.65
