Compounds for inhibition of ceramide-mediated signal transduction

Novel, heterocyclic compounds having at least one ring nitrogen, disclosed side chains and, in some embodiments, an oxygen ortho to the ring nitrogen inhibit inflammatory responses associated with TNF-.alpha. and fibroblast proliferation in vivo and in vitro. The compounds of the invention neither appreciably inhibit the activity of cAMP phosphodiesterase nor the hydrolysis of phosphatidic acid, and are neither cytotoxic nor cytostatic. Preferred compounds of the invention are esters. Methods for the use of the novel compounds to inhibit ceramide-mediated intracellular responses in stimuli in vivo (particularly TN-.alpha.) are also described. The methods are expected to be of use in reducing inflammatory responses (for example, after angioplasty), in limiting fibrosis (for example, of the liver in cirrhosis), in inhibiting cell senescence, cell apoptosis and UV induced cutaneous immune suppression. Compounds having enhanced water solubility are also described.

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The invention relates to compounds effective in modulating cellular
 responses stimulated by ceramide-mediated signal transduction, in
 particular in response to stimulus by the cytokine tumor necrosis factor
 .alpha. (TNF-.alpha.). More specifically, it relates to compounds which
 inhibit the development of conditions associated with cell stimulus
 through the ceramide-mediated signal transduction pathway.
 2. History of the Prior Art
 The sphingomyelin pathway is a cellular signal transduction pathway that is
 believed to be involved in mediating cellular responses to several
 cytokines (including TNF-.alpha. and IL-I.beta.) and growth factors (e.g.,
 platelet derived growth factor and fibroblast growth factor) (see, e.g.,
 Dressier, et al., Science, 259:1715-1718, 1992; and, Jacobs and Kester,
 Amer.J.Physiol., 265: 740-747, 1993). It is believed that interaction of
 such molecules with cell surface receptors triggers activation of a plasma
 membrane sphingomyelinase. Sphingomyelinase in turn catalyzes the
 hydrolysis of sphingomyelin to ceramide and phosphocholine. Ceramide is
 believed to act as a second messenger through activation of a
 proline-directed, serine/threonine kinase (ceramide-activated protein
 kinase or "CaPK"). Ceramide also interacts with MAP kinase and protein
 kinase C zeta (see, e.g., Rivas, et al., Blood, 83:2191-2197, 1993) and
 with a serine/threonine protein phosphatase (see, Hannun, et al., TIBS,
 20:73-77, 1995).
 Recent investigation has provided evidence that the sphingomyelin pathway
 may mediate cellular senescence and apoptosis (programmed cell death) in
 response to TNF-.alpha. (see, e.g., Jayadev, et al., J.Biol.Chem.,
 270:2047-2052, 1994; and, Dbaibo, et al., J.Biol.Chem., 268:17762-17766,
 1993) and radiation (Haimovitz-Friedman, et al., J.Exp.Med., 180:525-535,
 1994). In this respect, ceramide has been presumed to mimic the effects of
 TNF-.alpha. on intracellular processes.
 SUMMARY OF THE INVENTION
 The invention is directed toward the development and use of compounds to
 inhibit cellular responses to ceramide metabolites of the sphingomyelin
 signal transduction pathway, such as inflammation, fibrosis, ultraviolet
 light induced cutaneous immune suppression, cell senescence and apoptosis.
 In one aspect, the invention consists of novel compounds comprised of
 heterocyclic molecules with biologically active side chains (the
 "compounds of the invention"). Purine, pteridine, thiadiazolopyrimidine,
 quinalozine and isoquinolone based compounds are included in the
 invention. Particularly preferred among these compounds are those which
 have enhanced water solubility; e.g., morpholinoethyl esters of the
 compounds of the invention.
 The compounds of the invention do not inhibit the activity of cAMP
 phosphodiesterase and therefore do not pose the risk of side-effects
 associated with other TNF-.alpha. inhibitors (e.g., pentoxifylline), such
 as sleeplessness and anxiety. Indeed, surprisingly, one of the more potent
 TNF-.alpha. activity inhibitors among the compounds of the invention
 (compound 37) had the least inhibitory effect on phosphodiesterase type
 IV, the predominant phosphodiesterase isoenzyme in monocytes and
 neutrophils. This is due to the fact that compounds such as 37 do not have
 a methylxanthine structure. Many common phosphodiesterase inhibitors (such
 as theophylline, theobromine, and caffeine) are methylxanthine compounds.
 Moreover, all of the compounds of the invention inhibit apoptosis and
 retard cellular responses to TNF-.alpha. in vitro and in vivo with greater
 potency than pentoxifylline. Unexpectedly, the potency of at least the
 non-isoquinolone compounds appears to be dependent in part on the presence
 of ring nitrogens (other than the pyrimidine nitrogens), suggesting that
 binding to the target receptor responsible for inhibition of the activity
 of TNF-.alpha. observed is also regulated to some extent by the presence
 of such ring nitrogens. Further, the effects of all of the compounds
 appear to be totally unrelated to phosphodiesterase inhibition. This is
 particularly interesting given that increases in cAMP levels in cells can
 induce apoptosis in B cells (see, e.g., L.o slashed.mo, et al.,
 J.Immunol., 154:1634-1643, 1995).
 Recently, studies have shown (see, e.g. Verheij, et al., Nature, 380:75-79,
 1996) that ceramide initiates apoptosis through the stress-activated
 protein kinase (SAPK/JNK) pathway. Thus, cells exposed to stresses such as
 ionizing radiation, hydrogen peroxide, UV-C radiation, heat shock, and
 TNF-.alpha., or to C-2 ceramide, acquired biochemical and morphological
 features typical of apoptosis. Indeed, these stresses have been shown to
 increase cellular levels of ceramide. Moreover, the role of one of the
 stress-activated protein kinases, c-jun (referred to as Jun kinase or JNK)
 in apoptosis was confirmed following stress and C-2 ceramide exposure. In
 this regard, compounds of the invention, particularly the isoquinolines,
 have shown activity as inhibitors of Jun kinase activation induced by
 anisomycin (a non-specific protein synthesis inhibitor) in MOLT-4 human
 lymphoblastoid cells or by LPS in RAW mouse macrophages. Therefore, a
 possible mechanism of activity of these compounds is the inhibition of one
 arm of the stress response pathway involving a Jun kinase in response to
 stress or to ceramide.
 Another aspect of the invention consists of methods for the use of the
 novel compounds in inhibiting ceramide-activated cellular responses to
 stimuli, in particular stimuli for cell senescence and apoptosis. This
 aspect of the invention has potential therapeutic significance in the
 treatment of cell death associated conditions such as stroke, cardiac
 ischemia, nerve damage and Alzheimer's disease.
 Another aspect of the invention consists of methods which exploit the
 ability of the compounds of the invention to absorb UV radiation. This
 aspect of the invention has potential therapeutic significance in the
 treatment and prevention of radiation dermatoses, including those
 associated with therapeutic regimes for treatment of cancer.
 The compounds of the invention are expected to be particularly useful in
 reducing the effects of aging in skin as well as the onset and progression
 of radiation dermatitis.
 In another aspect, the invention features a compound having the formula:
 ##STR1##
 where
 R.sub.1 is a terminally substituted normal alkyl having from 1 to 7 carbon
 atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a
 terminally substituted ether having from 2 to 6 carbon atoms, a terminally
 substituted secondary amine having from 2 to 6 carbon atoms, or
 substituted aryl having less than 8 carbons, where said terminal group is
 NH.sub.2, substituted amino, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2,
 NNO(OH), SO.sub.2 NH.sub.2, PO(OH)NH.sub.2, SO.sub.2 R or COOR, where R is
 H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4
 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group
 has from 1 to 4 carbon atoms and the amino is NH.sub.2 or a substituted
 amino where the substituents on the amino have 1 to 6 carbon atoms, one of
 which can be replaced by an oxygen atom or nitrogen atom;
 Z is C, CH, or N;
 R.sub.2 is an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or
 aralkyl having less than 7 carbon atoms when Z is C, R.sub.2 is a halogen,
 NO, amino, or substituted amino when Z is CH, or R.sub.2 is H, an alkyl, a
 cyclic alkyl, a heterocyclic alkyl, alkenyl, or aralkyl having less than 7
 carbon atoms when Z is N;
 A is CO when Z is N, or CR.sub.5 when Z is C or CH, where R.sub.5 is H, an
 alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl or aralkyl
 having less than 7 carbon atoms, OH, or an O-alkyl having from 1 to 5
 carbon atoms and there is a double bond between Z and A when Z is C and a
 single bond between Z and A when Z is CH;
 Y.sub.1 is N, NR.sub.6, or CR.sub.6, where R.sub.6 is H, NO, an amino, a
 substituted amino, an alkyl, a cyclic alkyl, a heterocyclic alkyl,
 alkenyl, or aralkyl having less than 7 carbon atoms;
 Y.sub.2 is N or CH; and
 X is S when Y.sub.1 and Y.sub.2 are N, CR.sub.7 when Y.sub.1 is NR.sub.6
 where R.sub.7 is H, OH, SH, Br, Cl, or I, or
 .dbd.C(R.sub.3)--C(R.sub.4).dbd. when Y.sub.1 is N, CH or CR.sub.6 and
 Y.sub.2 is N or CH, where each of R.sub.3 and R.sub.4, independently, is
 H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, aryl,
 alkylcarboxyl, or aralkyl having less than 7 carbon atoms, SH, OH or an
 O-alkyl having from 1 to 5 carbon atoms,
 or a salt thereof.
 In another aspect, the invention features a compound having the formula:
 ##STR2##
 where Q is a halogen or substituted amino, R.sub.2 is a halogen, NO, an
 amino, or a substituted amino, R.sub.3 is SH or OH, R.sub.4 is SH or OH,
 R.sub.5 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl,
 aryl or aralkyl having less than 7 carbon atoms, OH, or an O-alkyl having
 from 1 to 5 carbon atoms, and R.sub.6 is NO, an amino, or a substituted
 amino,
 or a salt thereof.
 In preferred embodiments, the compound has the formula:
 ##STR3##
 Preferably, R.sub.1 is a terminally substituted normal alkyl having from 1
 to 7 carbon atoms, where said terminal group and is NH.sub.2, substituted
 amino, or COOR, where R is H, an alkyl having from 1 to 4 carbon atoms, or
 an alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the
 amino is a substituted amino where the substituents on the amino have 1 to
 6 carbon atoms, one of which can be replaced by an oxygen atom or nitrogen
 atom; R.sub.2, when present, is an alkyl having less than 7 carbon atoms
 (i.e., Me, or --(CH.sub.2).sub.2 CH.sub.3); each of R.sub.3 and R.sub.4,
 independently, is H, SH, OH, an alkyl, aryl, alkylcarboxyl, or aralkyl
 having less than 7 carbon atoms, or an O-alkyl having from 1 to 5 carbon
 atoms; and R.sub.6 is NO, an amino, or a substituted amino (i.e., NHR, or
 NR.sub.2, where R is H, an alkyl having from 1 to 4 carbon atoms, an
 alkenyl having from 1 to 4 carbon atoms, tetrazolyl, benzyl, an
 alkylamino, where the alkyl group has from 1 to 4 carbon atoms and the
 amino is NH.sub.2 or a substituted amino where the substituents on the
 amino have 1 to 6 carbon atoms, one of which can be replaced by an oxygen
 atom or nitrogen atom, aryl, or substituted aryl, where the substituted
 aryl has one, two, or three substituents including halogens or alkyls
 having 1 to 4 carbons). More preferably, R.sub.1 is a terminally
 substituted normal alkyl having from 1 to 4 carbon atoms where said
 terminal group is COOR where R is an N-morpholinoalkyl group (i.e., an
 N-morpholinoethyl group), and each of R.sub.3 and R.sub.4, independently,
 is an O-alkyl having from 1 to 5 carbon atoms.
 Most preferably, R.sub.1 is --(CH.sub.2).sub.3 COOR where R is an
 N-morpholinoethyl group, and R.sub.3 and R.sub.4 each are H or methoxy
 groups, thus providing the compounds with enhanced water solubility.
 In other preferred embodiments, the compound has the formula:
 ##STR4##
 where
 R.sub.1 is a terminally substituted normal alkyl having from 1 to 7 carbon
 atoms, a terminally substituted alkenyl having from 2 to 7 carbon atoms, a
 terminally substituted ether having from 2 to 6 carbon atoms, a terminally
 substituted secondary amine having from 2 to 6 carbon atoms, or
 substituted aryl having less than 8 carbons, where said terminal group is
 NH.sub.2, substituted amino, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2,
 NNO(OH), SO.sub.2 NH.sub.2, PO(OH)NH.sub.2, SO.sub.2 R or COOR, where R is
 H, an alkyl having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4
 carbon atoms, tetrazolyl, benzyl, or an alkylamino, where the alkyl group
 has from 1 to 4 carbon atoms and the amino is NH.sub.2 or a substituted
 amino where the substituents on the amino have 1 to 6 carbon atoms, one of
 which can be replaced by an oxygen atom or nitrogen atom;
 R.sub.2 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or
 aralkyl having less than 7 carbon atoms;
 Y.sub.2 is N or CH;
 R.sub.6 is H, an alkyl, a cyclic alkyl, a heterocyclic alkyl, alkenyl, or
 aralkyl having less than 7 carbon atoms; and
 R.sub.7 is H, OH, SH, Br, Cl, or I,
 or a salt thereof.
 Further advantages and embodiments of the invention included therein will
 become apparent from the following disclosure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 I. Compounds of the Invention
 The compounds of the invention generally comprise purines, pteridines,
 thiadiazolopyrimidines and quinazolines prepared according to the schemes
 described below. For reference, the techniques used in synthesizing the
 compounds are adaptations of the well-known Traube Synthesis protocol
 (Lister, "Purines" (Wiley-Interscience, 1971), at p. 220), beginning with
 4,5-diaminopyrimidines; to wit: (1) for the purines in general, see Brown,
 "The Chemistry of Heterocyclic Compounds: Fused Pyrimidines", Part II, The
 Purines, 1971), at pp. 31-90; (2) for the 9-dieazapurines in particular,
 see Fox, et al., J. Org. Chem., 43:2536, 1978; (3) for the pteridines,
 see, for a description of the standard Timmis reaction, Nishigaki, et al.,
 Heterocycles, 15:757-759, 1981; Timmis, Nature, 164:13 9, 1949, (or other
 standard Traube-like protocols for preparing pteridines by ring closure of
 diaminopyfimidines using a two carbon reagent); and, (4) for the
 pyrimidines, see, Schrage and Hitchings, J. Org. Chem., 16:207, 1951.
 Compounds of the invention, intermediates and compounds tested for
 comparison of activity to the compounds of the invention are identified in
 the discussion below by the numbers assigned to each compound in Table 1.
 TABLE I
 Physicochemical Data for all New Compounds and TNF-.alpha.
 Inhibition Data
 Comp Type mp(.degree. C.)
 formula analyses.sup.a TNF.alpha.IC.sub.50.sup.b
 1 I ##STR5## 102-103
 85
 2 I ##STR6## 208-210
 &gt;200
 4 I ##STR7## 162-165 C.sub.11
 H.sub.14 N.sub.4 O.sub.4 C, H, N 200
 5 I ##STR8## 76-78 C.sub.12
 H.sub.16 N.sub.4 O.sub.4 HRMS 10
 6 I ##STR9## 73-75 C.sub.13
 H.sub.18 N.sub.4 O.sub.4 C, H, N 6
 7 I ##STR10## 84-85 C.sub.14
 H.sub.20 N.sub.4 O.sub.4 C, H, N 11
 8 I ##STR11## 158-160 C.sub.16
 H.sub.16 N.sub.4 O.sub.4 C, H, N &gt;200
 10 I ##STR12## 101-102 C.sub.13
 H.sub.17 BrN.sub.4 O.sub.4 C, H, N 60
 11 I ##STR13## 218-219 C.sub.16
 H.sub.15 BrN.sub.4 O.sub.4 C, H, N &gt;200
 12 I ##STR14## 202-206 C.sub.13
 H.sub.18 N.sub.4 O.sub.4 S C, H, N 100
 13 I ##STR15## &gt;293 dec C.sub.17
 H.sub.18 N.sub.4 O.sub.4 S C, H, N &gt;200
 20 V ##STR16## &gt;218 dec C.sub.12
 H.sub.20 N.sub.4 O.sub.3 C, H, N NH.sup.c
 24 I ##STR17## &gt;295 dec C.sub.12
 H.sub.18 N.sub.4 O.sub.3 C, H, N 50
 25 I ##STR18## &gt;320
 &gt;200
 31 I ##STR19## oil C.sub.15
 H.sub.22 N.sub.4 O.sub.4 C, H, N 5
 33 V ##STR20## &gt;270 dec C.sub.5
 H.sub.8 N.sub.4 O.sub.2 C, H, N ND
 34 II ##STR21## 287-289 C.sub.7
 H.sub.6 N.sub.4 O.sub.2 C, H, N ND
 35 II ##STR22## 187-189 C.sub.9
 H.sub.10 N.sub.4 O.sub.4 C, H, N ND
 36 II ##STR23## 92-92 C.sub.13
 H.sub.16 N.sub.4 O.sub.4 C, H, N 12
 36a II ##STR24## 199-202 C.sub.11
 H.sub.12 N.sub.4 O.sub.4 C, H, N &gt;200
 37 II ##STR25## 53-55 C.sub.15
 H.sub.20 N.sub.4 O.sub.4 C, H, N 5
 37a II ##STR26## 97-99 C.sub.13
 H.sub.16 N.sub.4 O.sub.4 C, H, N &gt;200
 38 II ##STR27## 118-119 C.sub.12
 H.sub.12 N.sub.4 O.sub.4 HRMS 25
 39 II ##STR28## 92-95 C.sub.17
 H.sub.22 N.sub.4 O.sub.6 C, H, N ND
 40 II ##STR29## 218-222 C.sub.11
 H.sub.14 N.sub.4 O.sub.2 C, H, N ND
 41 II ##STR30## 66-68 C.sub.17
 H.sub.24 N.sub.4 O.sub.4 C, H, N 130
 41a II ##STR31## 161-162 C.sub.15
 H.sub.20 N.sub.4 O.sub.4 C, H, N ND
 42 II ##STR32## &gt;307 dec C.sub.13
 H.sub.10 N.sub.4 O.sub.4 N/A ND
 43a II ##STR33## N/A N/A
 N/A N/A
 43b II ##STR34## N/A N/A
 N/A N/A
 43c II ##STR35## N/A N/A
 N/A N/A
 43d II ##STR36## N/A N/A
 N/A N/A
 43e II ##STR37## N/A N/A
 HRMS N/A
 43 II ##STR38## 99-100 C.sub.19
 H.sub.20 N.sub.4 O.sub.4 HRMS &gt;200
 44 III ##STR39## 73-75 C.sub.12
 H.sub.18 N.sub.4 O.sub.2 S C, H, N 75
 45 III ##STR40## 210-213 C.sub.5
 H.sub.4 N.sub.4 O.sub.2 S C, H, N ND
 46 III ##STR41## 142-144 C.sub.7
 H.sub.8 N.sub.4 O.sub.2 S C, H, N ND
 47 III ##STR42## 45-47 C.sub.11
 H.sub.14 N.sub.4 O.sub.4 S C, H, N 25
 47a III ##STR43## 121-122 C.sub.9
 H.sub.10 N.sub.4 O.sub.4 S HRMS &gt;200
 48 III ##STR44## oil C.sub.13
 H.sub.18 N.sub.2 O.sub.3 C, H, N 18
 51 IV ##STR45## oil C.sub.14
 H.sub.20 N.sub.4 O.sub.3 C, H, N.sup.f ND
 52 IV ##STR46## oil C.sub.15
 H.sub.18 N.sub.2 O.sub.4 C, H, N.sup.g 55
 52a IV ##STR47## 163-165 C.sub.13
 H.sub.14 N.sub.2 O.sub.4 C, H, N &gt;200
 .sup.a All compounds analyzed for C, H, N or by exact mass high resolution
 mass spectrometry; results were within .+-.0.4% of theoretical values.
 .sup.b Concentration of compound in .mu.M which inhibited the product of
 TNF.alpha. by 50% of control.
 .sup.c ND = no determined.
 .sup.d C: calc'd, 58.61; found 59.22.
 .sup.e H: calc'd, 4.13; found 3.70.
 .sup.f N: calc'd, 10.25; found 9.77.
 .sup.g C: calc'd, 59.62; found 60.61.
 Compound types are: I = purines; II = pteridines: III =
 thiadiazolopyrimidines; IV = quinazolines; and, V = isoquinolones.
 "N/A" means data not available (but can be obtained through conventional
 analysis techniques).
 B. Purine synthesis.
 The purines of the invention have the general formula (I):
 ##STR48##
 where Z is N or CH;
 R.sub.1 is (CH.sub.2).sub.n A, where:
 A is NH.sub.2, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2, NNO(OH), SO.sub.2
 NH.sub.2, PO(OH)NH.sub.2, SO.sub.2 R, or COOR where R is H, an alkyl
 having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon
 atoms, tetrazolyl or benzyl;
 n is any number of atoms from 1 to 7 having saturated and/or unsaturated
 carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom
 in place of a carbon atom to form, respectively, ether or amino linkages;
 and, preferably, R.sub.1 is a .omega.-carboxyalkyl, .omega.-carboxyalkenyl,
 or .omega.-carboxyaryl having from 1 to 8 carbon atoms, wherein the
 aromatic group further has as a substituent A (as defined above);
 R.sub.2 is H, an alkyl (including aliphatic and alicyclic, and
 heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a
 .omega.-hydroxyalkyl having from 1 to 7 carbon atoms;
 R.sub.3 is the same as R.sub.2 ; and
 X is H, any halogen, OH, SH, OR', or SR', where R' is an alkyl, alkenyl,
 phenyl or benzyl having from 1 to 4 carbon atoms.
 These compounds are synthesized per the below synthesis scheme (as
 described in further detail in the Examples).
 ##STR49##
 Generally, theobromine was used as the starting material under conditions
 to ensure N-1 alkylation took place in lieu of O-6 alkylation. Compounds 4
 through 8, 10 and 11 (Table I) were prepared by this method. Compounds 10
 and 11 in particular were prepared by a variation of the alkylation method
 in which theobromine was first brominated to give 8-bromotheobromine
 (compound 9), then alkylated. The 8-bromo substituent was also displaced
 by NaSH to yield the corresponding 8-thioxo derivatives, compounds 12 and
 13.
 ##STR50##
 This method is essentially based on the Traube purine synthesis protocol
 referred to supra. The method was used to prepare 1,3,8-trisubstituted
 xanthines bearing no alkyl group at the N-7 position. In this procedure,
 the N-1 substituted pyrimidine was alkylated at position N-3. Formation of
 the purine ring was complete by nitrosation, reduction of the nitroso to
 the amine by catalytic hydrogenation, then ring closure using urea or
 potassium ethyl xanthate to provide compounds 24 and 25 (respectively,
 8-oxo and 8-thioxo derivatives). A detailed description of this protocol
 is provided in the Examples.
 ##STR51##
 This method was utilized to prepare the N-3 propylpurines. The starting
 material used was n-propyl urea condensed with ethyl cyanoacetate in the
 presence of sodium ethoxide to yield the 6-amino-1-propylpyrimidinedione
 in moderate yield. Commercially available 3-n-propylxanthine could also be
 used as the starting material.
 Ring closure was accomplished as described in method A, except that
 diethoxymethyl acetate was used as the source of carbon in the ring
 closure step. Sequential alkylations were then performed using alkyl
 halides to yield the final compound 31 (ethyl
 4-(2,3,6,7-tetrahydro-2,6-dioxo-7-methyl-3-n-propyl-1H-purin-1-yl)butonoic
 acid). A detailed description of this protocol is provided in the
 Examples.
 C. Pteridine synthesis.
 The pteridines of the invention have the general formula (II):
 ##STR52##
 R.sub.1 is (CH.sub.2).sub.n A, where:
 A is NH.sub.2, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2, NNO(OH), SO.sub.2
 NH.sub.2, PO(OH) NH.sub.2, SO.sub.2 R, or COOR where R is H, an alkyl
 having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon
 atoms, tetrazolyl or benzyl;
 n is any number of atoms from 1 to 7 having saturated and/or unsaturated
 carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom
 in place of a carbon atom to form, respectively, ether or amino linkages;
 and, preferably, R.sub.1 is a .omega.-carboxyalkyl, .omega.-carboxyalkenyl,
 or .omega.-carboxyaryl having from 1 to 8 carbon atoms, wherein the
 aromatic group further has as a substituent A (as defined above);
 R.sub.2 is H, an alkyl (including aliphatic and alicyclic, and
 heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a
 .omega.-hydroxyalkyl having from 1 to 7 carbon atoms;
 R.sub.4 is the same as R.sub.2, OH or an O-alkyl having from 1 to 5 carbon
 atoms;
 R.sub.5 is the same as R.sub.2, OH or an O-alkyl having from 1 to 5 carbon
 atoms; and,
 Z is N or CH.
 These compounds are synthesized per the below synthesis scheme (which is
 described in further detail in the Examples.)
 ##STR53##
 Synthesis of the pteridines was based on orthodiaminopyrimidines as
 precursors. Ring closure fo the orthodiamines (compounds 33 and 28) was
 accomplished with a two carbon source (e.g., glyoxal) to produce compounds
 34 and 35 (N-1 substituted pteridines). Alkylation at N-3 as described
 with respect to Method A produced the desired pteridines (compounds
 36-38). Further, use of 3,4-hexanedione in the ring closure step produced
 a more lipophilic derivative (compound 41; 6,7-diethyl pteridine).
 Condensation of compound 22 with dimethylacetylene dicarboxylate formed
 compound 39 (1,3-dialkylpteridine), while treatment of compound 27
 phenethyl amine followed by alkylation provided compound 43 (6-phenyl
 dialkyl pteridine). Both of the latter protocols utilized a Timmis
 reaction to produce the desired products. A detailed description of these
 protocols is provided in the Examples.
 D. Thiadiazolopyrimidine synthesis.
 The thiadiazolopyrimidines of the invention have the general formula (III):
 ##STR54##
 R.sub.1 is (CH.sub.2).sub.n A, where:
 A is NH.sub.2, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2, NNO(OH), SO.sub.2
 NH.sub.2, PO(OH)NH.sub.2, SO.sub.2 R, or COOR where R is H, an alkyl
 having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon
 atoms, tetrazolyl or benzyl;
 n is any number of atoms from 1 to 7 having saturated and/or unsaturated
 carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom
 in place of a carbon atom to form, respectively, ether or amino linkages;
 and
 and, preferably, R.sub.1 is a .omega.-carboxyalkyl, .omega.-carboxyalkenyl,
 or .omega.-carboxyaryl having from 1 to 8 carbon atoms, wherein the
 aromatic group further has as a substituent A (as defined above); and
 R.sub.2 is H, an alkyl (including aliphatic and alicyclic, and
 heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a
 .omega.-hydroxyalkyl having from 1 to 7 carbon atoms.
 These compounds are synthesized per the below synthesis scheme (as
 described in further detail in the Examples).
 ##STR55##
 Synthesis of the pyrimidines was based on orthodiaminopyrimidines as
 precursors. Ring closure of the orthodiamines was accomplished by
 treatment with thionyl chloride in the presence of pyridine. Alkylation of
 these intermediates produced compounds 47 and 48 (disubstituted
 pyrimidines). A detailed description of this protocol is provided in the
 Examples.
 E. Isoquinolone Synthesis.
 The isoquinolones of the inventions have the general formula (IV):
 ##STR56##
 R.sub.2 is (CH.sub.2).sub.n A, where:
 A is NH.sub.2, acyloxy, SO.sub.3 H, PO.sub.4 H.sub.2, NNO(OH), SO.sub.2
 NH.sub.2, PO(OH)NH.sub.2, SO.sub.2 R, or COOR where R is H, an alkyl
 having from 1 to 4 carbon atoms, an alkenyl having from 1 to 4 carbon
 atoms, tetrazolyl or benzyl;
 n is any number of atoms from 1 to 7 having saturated and/or unsaturated
 carbon to carbon bonds, which atoms may include an oxygen or nitrogen atom
 in place of a carbon atom to form, respectively, ether or amino linkages;
 and, preferably, R.sub.2 is a .omega.-carboxyalkyl, .omega.-carboxyalkenyl,
 or .omega.-carboxyaryl having from 1 to 8 carbon atoms, wherein the
 aromatic group further has as a substituent A (as defined above); and
 R.sub.3 is H, an alkyl (including aliphatic and alicyclic, and
 heteroalicyclic forms), alkenyl, aralkyl having 1 to 7 carbon atoms or a
 .omega.-hydroxyalkyl having from 1 to 7 carbon atoms;
 R.sub.4 is H, OH, NH.sub.2 or O-alkyl having from 1-7 carbon atoms;
 R.sub.5 is H, OH, NO, NO.sub.2, NH.sub.2 an O-alkyl having from 1-4 carbon
 atoms, or X where;
 X is H, any halogen, OH, SH, OR', or SR', where R' is an alkyl, alkenyl,
 phenyl or benzyl having from 1 to 4 carbon atoms; and,
 R.sub.7 is H, OH, NO, NO.sub.2, NH.sub.2 an O-alkyl having from 1-7 carbon
 atoms, or X where:
 X is H, any halogen, OH, SH, OR', or SR', where R' is an alkyl, alkenyl,
 phenyl or benzyl having from 1 to 7 carbon atoms.
 These compounds were synthesized by purchasing isoquinolines from Aldrich
 Chemical (see, FIG. 14) and adding side chains to the ring structure as
 described above and in the Examples with respect to the purine, pteridine
 and thiadiazolopyrimidine compounds of the invention. Only the
 6,7-dimethoxy-1(2H)-isoquinoline compound (Aldrich #S52,626-6) had any
 inhibitory effect on TNF-.alpha. production prior to addition of the side
 chains described above (see also, Example 7).
 F. Quinazoline Synthesis.
 The quinazolines of the invention are synthesized according to the
 following scheme and are represented in structure by Compound 52:
 ##STR57##
 The starting material for this protocol was N-methyl isatoic anhydride. A
 detailed description of this protocol is provided in the examples.
 G. Increasing the water solubility of Compounds.
 The carboxylic ester and acid derivatives of the compounds described above
 can be converted to substituted carboxylic esters, where the substituent
 of the ester increases the water solubility of the compounds. Particular
 substituents that increase the water solubility of the compounds
 effectively are aminoalkyl esters. The amino group of the aminoalkyl ester
 preferably is a secondary or tertiary amino group. The amino substituents
 have between 1 and 6 carbon atoms, one of which can be replaced by an
 oxygen atom or nitrogen atom. The preferred aminoalkyl is a tertiary amine
 and the amino substituents are preferably are ether groups. The most
 preferred aminoalkyl is N-morpholinoethyl. The carboxylic ester and
 carboxylic acid derivatives can be converted to the morpholinoethyl ester
 form by the method shown in Scheme V.
 ##STR58##
 Scheme V
 II. Methods for Use of the Inventive Compounds
 The compounds of Formulas I through IV may be administered to a mammalian
 host to retard cellular responses associated with TNF-.alpha. and IL-1
 production and activation of the ceramide-mediated signal transduction
 pathway. In particular, the compounds of the invention may be administered
 to a mammal to retard activation of ceramide-dependent intracellular
 biochemical pathways in target cells without inhibiting the activity of
 phosphodiesterase in the target cells or affecting the levels of
 diacylglycerol therein. As exemplified herein, the methods of the
 invention are expected to be of particular use in providing protection
 against inflammation and excessive formation of fibrotic tissue by
 reducing the production of TNF-.alpha. by stimulated monocytes. As further
 exemplified herein, the compounds of the invention (particularly the
 isoquinolines and pteridines) are also expected to be efficacious in
 providing protection against cell senescence or cell apoptosis, such as
 occurs as a result of trauma (e.g., radiation dermatitis) and aging (e.g.,
 of the skin and other organs). In this context, the phrase "providing
 protection against" means clinically significant inhibition of cellular
 responses to stimuli (signals) whose transmission to the cell is
 facilitated in whole or in part by the ceramidemediated, sphingomyelin
 signal transduction pathway.
 For purposes of this disclosure, therefore, inflammation, fibroblast
 proliferation, cell senescence and cell apoptosis in response to
 ceramide-mediated signal transduction through the sphingomyelin pathway
 will be considered to be "ceramide associated" conditions. Those of
 ordinary skill in the art will be familiar with, or can readily ascertain,
 the identity and clinical signs of specific ceramide associated
 conditions, and can identify clinical signs of improvement therein (such
 as reductions in serum levels of TNF-.alpha. and improvement in clinical
 health) in addition to those exemplified herein.
 For administration, the compounds of the invention will preferably be
 formulated in a pharmaceutically acceptable carrier. Such carriers include
 sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
 Examples of non-aqueous solvents are propylene glycol, polyethylene
 glycol, vegetable oils such as olive oil, and injectable organic esters
 such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
 solutions, emulsions or suspensions, including saline and buffered media.
 Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
 dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous
 vehicles include fluid and nutrient replenishers, electrolyte replenishers
 (such as those based on Ringer's dextrose), and the like. Preservatives
 and other additives may also be present such as, for example,
 antimicrobials, antioxidants, chelating agents, and inert gases and the
 like.
 Exemplary of solid carriers are lactose, terra alba, sucrose, talc,
 gelatin, agar, pectin, acacia, magnesium stearate, stearic acid,
 microcrystalline cellulose, polymer hydrogels and the like. Similarly, the
 carrier or diluent may include any time delay material well known to the
 art, such as glyceryl monostearate or glyceryl distearate alone or with a
 wax, microcapsules, microspheres, liposomes, and hydrogels. For further
 reference, those of skill in the art may wish to consult the standard
 reference Remington's Pharmaceutical Sciences (which is incorporated
 herein by reference to illustrate knowledge in the art concerning suitable
 pharmaceutical carriers).
 A wide variety of pharmaceutical forms can be employed. Thus, when using a
 solid carrier the preparation can be tableted, placed in a hard gelatin
 capsule in powder or pellet form, or in the form of a troche, lozenge or
 suppository. When using a liquid carrier the preparation can be in the
 form of a liquid, such as an ampule, or as an aqueous or nonaqueous liquid
 suspension. Topical administration via timed release skin patches is also
 a suitable pharmaceutical form.
 Dosages of the compounds of the invention will vary depending on the age,
 weight and presenting condition of the host to be treated, as well as the
 potency of the particular compound administered. Such variables will
 readily be accounted for by those of ordinary skill in the clinical art.
 In particular, dosages will be adjusted upward or downward for each
 recipient based on the severity of the condition to be treated and
 accessibility of the target cells to the pharmaceutical formulations of
 the invention. Where possible, it will be preferable to administer the
 pharmaceutical formulations of the invention locally at the site of the
 target cells; e.g., into inflamed skin or by infusion to another organ of
 the host. Thus, dosages will also vary depending on the route of
 administration and the extent to which the formulations of the invention
 are expected to reach target cells before dilution or clearance of the
 formulation. Preferred routes of administration are by topical
 administration, local injection or parenteral infusion, although oral and
 intravascular routes may also be utilized.
 Generally, however, based on experience with other inhibitors of
 intracellular responses to external stimuli (such as pentoxifylline) and
 the data provided herein, good results can be expected to be achieved in
 an adult host of about 60 kg body weight in a dosage range of about 500 to
 about 4,000 mg/day, preferably between about 1,000 and about 3,500 mg/day
 (i.e., a "therapeutically effective dosage"). These dosages may be
 combined with other conventional pharmaceutical therapies for inflammation
 and fibrosis; e.g., administration of non-steroidal anti-inflammatory
 medications.
 The compounds of the invention vary in potency. A summary of the potency of
 each compound (expressed as a percentage of inhibition of intracellular
 responses to LPS, namely, the production of TNF-.alpha., where responses
 to pure LPS=100% and are measured as the concentration of the inventive
 compound needed to inhibit TNF-.alpha. production by 50%) is provided in
 Table 1, above. Those of ordinary skill in the art will recognize that
 lesser or greater dosages of the compounds of the invention may be
 required depending on the potency of the particular compound being
 administered.
 III. Methods for Identification of Therapeutically Effective Analogues of
 the Compounds of the Invention
 Those of ordinary skill in the art will be familiar with means to develop
 analogues to the compounds specifically described herein which, although
 not structurally identical thereto, possess the same biological activity.
 Such compounds are within the scope of the invention and may be identified
 according to the protocols described below and in the Examples.
 Though exposure of cells to the compounds of the invention under controlled
 conditions, the responsiveness of cells to inflammatory agents and
 intracellular mechanisms therefor can be investigated. This information
 will not only better elucidate the intracellular pathways responsible for
 cellular responses to particular stimuli, but will also aid in the
 identification of anti-inflammatory and anti-fibrosis therapeutic
 compounds.
 To identify and select therapeutic compounds for use in treating
 ceramide-associated conditions such as inflammation and fibrosis, cells
 (or intracellular components such as microsomes) which have not been
 exposed to an inflammatory or fibroblast proliferation inducing agent
 (e.g., LPS, TNF-.alpha., IL-1, PDGF) are exposed to such an agent and the
 candidate therapeutic compound. Specifically, a control group of cells is
 incubated with a known amount of the inflammatory or fibroblast
 proliferation inducing agent. Treatment groups of cells are exposed to the
 same amount of inflammatory or fibroblast proliferation inducing agent as
 well as aliquots of the candidate therapeutic compound. Inflammatory
 responses or fibroblast proliferation in each group are detected by
 conventional means known to those of skill in the art (such as the assay
 steps described in the examples) and compared.
 To identify and select therapeutic compounds for use in treating
 ceramide-associated conditions of cell senescence and apoptosis, cells (or
 intracellular components such as microsomes) which have not been exposed
 to a senescence or apoptosis inducing agent (e.g., cytokines such as
 TNF-.alpha. and exogenous stimuli such as heat, radiation and chemical
 agents), are exposed to such an agent and to the candidate therapeutic
 compound. Inhibition of senescence or apoptosis is measured as a function
 of cell growth. Those of ordinary skill in the art will be familiar with
 techniques for obtaining such measurements, examples of which are provided
 below.
 "Therapeutically effective compounds" will be those which, when
 administered according to the invention and sound medical practices,
 provide cells with protection against ceramide-associated conditions
 compared to control values for cellular reactions to a ceramide-associated
 condition inducing agent.
 The invention having been fully described, examples illustrating its
 practice are set forth below. These examples should not, however, be
 considered to limit the scope of the invention, which is defined by the
 appended claims.
 In the examples, the abbreviation "min." refers to minutes, "hrs" and "h"
 refer to hours, and measurement units (such as "ml") are referred to by
 standard abbreviations. "mp" refers to melting point.
 EXAMPLE 1
 INHIBITION OF CELL SENESCENCE AFTER SERUM DEPRIVATION IN SERUM-DEPENDENT
 CELLS
 Many cell types are dependent upon serum factors for growth. Thus,
 deprivation of such cells of serum provides a model for assessment of
 compounds to modulate cell responses to intracellular ceramide-mediated
 signal transduction. In particular, withdrawal of serum from
 serum-dependent cell cultures produces increased intracellular levels of
 endogenous ceramide and may also increase intracellular levels of
 endogenous diacyl glycerol (see, e.g., Jayadev, et al., J.Biol.Chem.,
 270:2047-2052, 1995).
 To evaluate the inhibitory effect of the compounds of the invention on
 ceramide-associated conditions in vitro, the serum withdrawal model was
 used. Specifically, 3T3 fibroblast cells were seeded in 96 well microtiter
 plates in DMEM in the presence of 10% fetal bovine serum. The cells were
 incubated to 90% confluence.
 The medium was removed, the cells washed and reincubated in serum-free
 DMEM. Compound no. 37 and cell permeable ceramide were added to the wells
 at concentrations of, respectively, 0, 4, 40 or 400 .mu.M compound no. 37
 and 0, 5 or 10 .mu.M of ceramide. After 24 hrs. incubation, 0.5 .mu.Ci of
 [.sup.3 H] thymidine was added to each well for 2 hrs. DNA synthesis in
 the tested cell population was assessed by conventional techniques for
 detection of [.sup.3 H] thymidine incorporation. The results of this assay
 are indicated in FIG. 1 and establish the cell senescence inhibitory
 efficacy of the inventive compounds (as represented by compound no. 37).
 EXAMPLE 2
 INHIBITION OF CELL APOPTOSIS AFTER CD95 STIMULATION
 Engagement of cell surface receptor CD95 (also known as Fas/Apo-1 antigen)
 triggers cell apoptosis. DX2 is a functional anti-FAS (CD95) antibody
 which will, on binding of CD95, activate the Smase catalysis of
 sphingomyelin hydrolysis and production of ceramide (see, re DX2, Cifone,
 et al., J.Exp.Med, 177:1547-1552, 1993, the disclosure of id which is
 incorporated herein by reference for use in accessing the DX2 antibody).
 Thus, binding of CD95 is a model for induction of apoptosis via the
 sphingomyelin signal transduction pathway.
 To assess the inhibitory effect of the compounds of the invention on
 ceramide-mediated cell apoptosis, human T lymphoblasts (Jurkat) were
 suspended at 2.times.10.sup.6 cells per ml in RPMI-1640 supplemented with
 insulin, transferrin, selenium and glutamine. After incubation for 2 hrs.
 at room temperature with either compound no. 37, compound no. 6,
 pentoxifylline or a control compound (Ro-1724), 25 ng/ml of anti-FAS
 antibody was added to each suspension. After another 2 hrs., cell
 apoptosis was measured as a function of the number of cells (counted by
 hemocytometer) that excluded the vital dye erythrosin B. The results of
 the experiment are indicated in FIG. 2 and establish the apoptosis
 inhibitory efficacy of the compounds of the invention (as represented by
 compounds nos. 6 and 37, particularly the latter).
 To assess the inhibitory effect of the compounds of the invention on death
 of human lymphocytes, human peripheral blood lymphocytes were isolated
 from normal human blood and depleted of monocytes by adherence to a
 plastic substrate. Lymphocytes were then cultured in RPMI-1640 medium with
 10% autologous plasma at an initial concentration of 2.times.10.sup.6
 cells per ml. Aliquots of the cell samples were divided and one half of
 the samples were incubated with either compound 37 or compound 1L-49 (an
 isoquinolone, the structure of which is shown in Example 7) for four days.
 The remaining half of the samples were allowed to rest for four days. Cell
 viability after four days was determined by erythrosin B dye exclusion in
 a hemocytometer.
 As shown in FIG. 13, at increasing concentrations, the compounds of the
 invention (as represented by compounds 37 and 1L-49) protected the cell
 sample population from death by up to 100% as compared to the survival
 rate of untreated lymphocytes.
 EXAMPLE 3
 INHIBITION OF THE ACTIVITY OF CERAMIDE ACTIVATED PROTEIN KINASE
 Ceramide-activated protein kinase (CaPK) is a 97 kDa protein which is
 exclusively membrane-bound and is believed to serve a role in the
 sphingomyelin signal transduction pathway. In particular, CaPK is believed
 to mediate phosphorylation of a peptide derived from the amino acid
 sequence surrounding Thr.sup.669 of the epidermal growth factor receptor
 (i.e., amino acids 663-681). This site is also recognized by the
 mitogen-activated kinase MAP (also known as a family of extracellular
 signal-regulated kinases). Thus, the effect of the compounds of the
 invention on CaPK activity in cells is indicative of the effect that the
 compounds exert on signal transduction in the sphingomyelin pathway.
 To that end, Jurkat cells were suspended at 2.times.10.sup.6 cells per ml
 in RPMI-1640 medium as described in Example 2. After incubation for 2
 hrs., either compound 37, 20 .mu.M of ceramide or 25 ng/ml of anti-FAS
 antibody DX2 were added to each suspension and incubated for 15 mins.
 After centrifugation and washing, the cells were separately homogenized in
 a dounce homogenizer.
 Ceramide kinase levels in each test sample were assayed as described by
 Liu, et al., J.Biol.Chem., 269:3047-3052, 1994 (the disclosure of which is
 incorporated herein for reference and use in assaying ceramide kinase).
 Briefly, the membrane fraction was isolated from each test sample of
 treated cell homogenate by ultracentrifugation and run on a 10% PAGE gel.
 The gel was washed with guanidine-HCL, and renatured in HEPES buffer. Then
 [.sup.32 P] -ATP was added to the gel and left there for 10 mins.
 Thereafter, the gel was extensively washed with 5% TCA. Autophosphorylated
 kinase was detected by autoradiography. The results of this assay are
 indicated in FIG. 3, and establish the CaPK inhibitory efficacy of the
 compounds of the invention (as represented by compound 37).
 EXAMPLE 4
 ABSORBANCE OF UVB RADIATION BY THE COMPOUNDS OF THE INVENTION
 Radiation (particularly in the UVB wavelength) is a major cause of skin
 damage (including apoptosis) in humans. As indicated elsewhere above, the
 sphingomyelin signal transduction pathway is believed to be involved in at
 least the early stages of development of radiation induced dermatoses
 (including radiation dermatitis, sunburn and UVB induced immune
 suppression from radiation damage to Langerhans cells in the skin--see,
 e.g., Haimovitz-Friedman, et al., J.Exp.Med., 180:525-535, 1994 (cellular
 responses to ionizing radiation); and, Kurimoto and Streilein, J.Immunol.,
 145:3072-3078, 1992 (cutnaceous immune suppression from UVB exposure)).
 Thus, a compound which will inhibit cell responses to stimulus of the
 sphingomyelin signal transduction pathway by radiation and can be
 administered topically at the site of exposure would be of great benefit
 in retarding the damage associated with radiation exposure (e.g., through
 exposure to sunlight or radiation).
 To assess the radiation absorbing abilities of the compounds of the
 invention, the ultraviolet spectra of compounds of the invention (nos. 6
 and 37, alone, in combination and as 8-oxo derivatives) were evaluated and
 compared to those of a commercially available sunscreen additive (PABA)
 and isoquinoline. The spectra were identified using a KONTRON analytical
 instrument. As indicated in FIG. 4, the compounds of the invention (as
 represented by compounds nos. 6 and 37) absorbed through most of the UVB
 region, indicating efficacy in absorbing radiation. Surprisingly, a
 mixture of compound nos. 6 and 37 Proved to absorb throughout the UVB
 region. Thus, given the somewhat greater absorbance characteristics of
 compound 37 vis-a-vis compound 6, it can be reasonably expected that
 mixtures of the two in ratios of 1:1 or greater (favoring compound 37)
 will have substantial synergistic efficacy in absorbing radiation and
 retarding its effects on cells.
 EXAMPLE 5
 INHIBITION OF TNF-.alpha. PRODUCTION BY THE COMPOUNDS OF THE INVENTION
 As shown in FIG. 5(a), compounds of the invention having N-1 chain lengths
 from 2-5 carbons are especially useful in inhibiting TNF-.alpha.
 production in vitro, while N-1 chain lengths of about 4 carbons (with a
 terminal ester) appear to be optimal in this respect (as compared to a
 control compound; FIG. 5(b)). Further, the esterified compounds were
 significantly more effective inhibitors of TNF-.alpha. production than
 their carboxylic counterparts. These data were obtained as follows:
 Peripheral blood mononuclear cells were isolated from normal human blood on
 Hypaque-Ficoll density gradients. A portion of the isolated cells were
 further purified by adherence to gelatin coated flasks.
 100 .mu.l aliquots of monocytes were placed onto 96 well microtiter plates
 at a density of 5.times.10.sup.5 cells/ml in RPMI-1640 medium containing
 10% fetal bovine serum. After incubation for 24 hrs., various
 concentrations of the test compounds (FIG. 5) were added to the plated
 cells in a volume of 100 .mu.l and incubated for 1 hr. After incubation, 1
 .mu.g/ml of LPS was added to each well.
 18 hrs. after exposure of the plated cells to LPS, 100 .mu.l of medium was
 collected from each well and assayed (by ELISA) for release of
 TNF-.alpha., using recombinant human TNF as a standard. The sensitivity of
 the assay ranged from 10-100 pg/ml.
 EXAMPLE 6
 RELATIVELY LOW INHIBITION OF PHOSPHODIESTERASE IV ACTIVITY BY COMPOUNDS OF
 THE INVENTION
 As shown in FIG. 6, there appears to be little correlation between the
 efficacy of the compounds of the invention in inhibiting the activity of
 phosphodiesterase and inhibiting activity of TNF-.alpha.. For example, the
 most active pteridine compound in inhibiting TNF-.alpha. production in
 vitro (#37) was a very poor inhibitor of phosphodiesterase IV, even at
 micromolar concentrations.
 These data confirm that the compounds of the invention do not target
 phosphodiesterase to control TNF-.alpha. production. The data were
 obtained as follows:
 The reaction was started with the addition of PDE and incubated at
 37.degree. C. for 10 minutes, then terminated by boiling for 2 minutes.
 500 .mu.l of 0.1 M HEPES/0.1 M NaCl (pH 8.5) was added to each tube, then
 the reaction mixture was applied to a boronate column. Unreacted CaMP was
 washed off with Hepes/NaCl and the reaction mixture eluted with acetic
 acid. Recovery was determined with the [.sup.14 C]-AMP.
 EXAMPLE 7
 IN VIVO AND IN VITRO LEUKOPENIA IN RESPONSE TO LPS AND INHIBITION OF SAME
 BY THE COMPOUNDS OF THE INVENTION
 As shown in FIGS. 7 through 9 and Table 1, the compounds of the invention
 effectively reduce cellular response to LPS, a known inducer of
 TNF-.alpha. production. In the presence of ceramide, the inhibitory
 activity of the compounds of the invention on LPS induced leukopenia (a
 phenomenon dependent on TNF-.alpha. induced surface expression of the
 P-selection class of adhesion molecules) was enhanced (FIG. 7). However,
 the inhibitory activity of the compounds of the invention was essentially
 unaffected by diacylglycerol (FIG. 8), indicating that the mode of action
 of the compounds of the invention are not dependent on hydrolysis of
 phosphatidic acid. These data were obtained as follows:
 The leukopenia inhibitory capacity of the test compounds was determined by
 intraperitoneal administration of 0.5 .mu.g of LPS in saline to ICR female
 mice (age 6-8 weeks; weight 19-23 g). One hour before receiving the LPS,
 the mice received the test compound by intraperitoneal injection at a dose
 of 50 mg/kg (in isotonic saline). Two hours after injection of LPS, 200
 .mu.l of blood was collected from each mouse into a heparinized tube and
 the total count of nucleated cells determined in a hemocytometer (FIG. 7
 and 9).
 Calcium independent protein kinase activity was measured, using a 1% triton
 X-100 extract of Jurkat cells (5.times.10.sup.8 /ml). The reaction mixture
 consisted of 20 mM Tris HCl pH 7.5, 20 MM MgCl.sub.2, 20 .mu.M ATP
 containing 200,000 cpm [.gamma.32P] ATP, and 50 .mu.M Myelin Basic
 Protein. The extract was pre-incubated with A) compound 37 B) compound 37
 with or without 10 .mu.M ceramide C) ceramide or D) dihydro ceramide for
 15 minutes, followed by addition of substrate and ATP, and incubation at
 30.degree. C. for 5 minutes. The total count of nucleated cells was
 measured in a hemocytometer (FIG. 9). The same protocol was followed to
 obtain the results shown in FIG. 8 (with the addition of diacyl glycerol
 to some of the test mixtures).
 An isoquinoline compound of the invention was also tested in vitro for its
 inhibitory efficacy with respect to LPS induced TNF-.alpha. production in
 human cells. The structure of the compound, 1I-49, is described below:
 Human macrophages were cultured in 96 well microtiter plates and incubated
 with LPS. Aliquots of the stimulated cells were then incubated with,
 respectively, 0.1, 1, 10, 100 or 1000 .mu.M of 1I-49, compound 37 and a
 commercially available isoquinoline (6,7-dimethoxy-1(2H)-isoquinoline from
 Aldrich Chemical; labelled S52-626-6 in FIG. 10) which, like the compounds
 of the invention, has an oxygen ortho to a ring nitrogen but, unlike the
 compounds of the invention, lacks a side chain substituent as described
 above (1L-49 is representative of the isoquinolone compounds of the
 invention having the side chain substituents described elsewhere above).
 The inhibitory efficacy of each compound was measured as a function of
 TNF-.alpha. reduction in pg/ml. The results of the experiment are
 indicated in FIG. 10 and establish that the compounds of the invention
 (represented by 1I-49 and compound 37) have inhibitory efficacy with
 respect to reduction in LPS induced TNF-.alpha. production by human cells.
 Other isoquinolines tested (FIG. 14) did not exert inhibitory activity in
 the absence of the side chain substituents added according to the
 invention.
 EXAMPLE 8
 FIBROBLAST PROLIFERATION IN RESPONSE TO LPS AND INHIBITION OF SAME BY THE
 COMPOUNDS OF THE INVENTION
 As shown in FIG. 11, PDGF induced fibroblast proliferation was selectively
 inhibited by the compounds of the invention. In addition, the compounds
 were shown not to be cytostatic or cytotoxic, insofar as they did not
 alter EGF-triggered mitogenesis in the cells tested (FIG. 12).
 These data were obtained as follows:
 Mouse fibroblast line 3T3 cells (American Type Culture Collection #CCL 92)
 were seeded into 96 well plates in complete medium and allowed to grow to
 confluence. The medium was then replaced with medium-free serum and the
 cells incubated for 24 hrs.
 The test compounds were then incubated with the cells for 1 hr before
 addition of 5 ng/ml human PDGF or EGF was added to each well. After
 another 24 hrs, 1 .mu.Ci of [.sup.3 H]-thymidine was added to each well. 4
 hrs later the cells were harvested onto glass fiber filters and the
 cellular incorporation of [.sup.3 H]-thymidine was measured by liquid
 scintillation counting (FIGS. 11 and 12).
 EXAMPLE 9
 SYNTHESIS OF COMPOUNDS 2, 4-8 AND 10-13
 General Alkylation Procedure for Compounds 4-8, 10, 11 (Method A):
 Theobromine or 8-bromotheobromine (2 mmol) was combined with anhydrous
 K.sub.2 CO.sub.3 (2.5 mmol) and dry DMF (15 mL) and the mixture was
 brought to 75.degree. C. The appropriate alkyl halide (2.5 mmol) w as
 added and the mixture was stirred at 75.degree. C. for 2-18 h. The
 reaction mixture was cooled, poured into water (125 mL) and extracted with
 ethyl acetate (2.times.75 mL). The organic layer was dried over magnesium
 sulfate and evaorated to yield a colorless oil or white solid which was
 triturated with ethyl ether. The resulting solid, often analytically pure,
 may be purified further if desired by crystallization from a small amount
 of ethanol. Yields 58-89%. Compounds 15-17, 31, 36-38, 41, 43, 47, and 48
 (described below) were prepared by this same procedure only using the
 appropriate precursors in place of theobromine.
 General Thiation Procedure for Compounds 12 and 13:
 The 8-bromoxanthine 10 or 11 (0.25 mmol) was suspended in anhydrous ethanol
 (10 mL) and heated to reflux. NaSH. (H.sub.2 O).sub.x (2.5 mmol) was added
 and the mixture became clear, green almost immediately. The mixture was
 stirred under reflux for 30 min, cooled and evaporated onto silica gel.
 Flash column chromatography using 5-7% MeOH in CH.sub.2 Cl.sub.2 provided
 a 63% and 75% yield of 12 and 13, respectively as white solids. Note:
 Compound 13 was found by .sup.1 H NMR to be the ethyl ester due to
 transesterification under the reaction conditions.
 .sup.1 H NMR spectra and elemental analyses or exact mass data were
 consistent with the assigned structures (see, Table 2 following Example
 9).
 EXAMPLE 10
 SYNTHESIS OF COMPOUNDS 24, 25, 31 AND INTERMEDIATES
 General Procedure for C-Nitrosation of Pyrimidines (Compounds 18-20, 27,
 and 32):
 The pyrimidine (15 mmol) was suspended in N HCl (30 mL) and an aqueous
 solution of sodium nitrite (20 mmol in 10 mL) was dripped in with stirring
 over 10 min. The suspension went from off-white to purple almost
 immediately. Stirring was continued for 1 h, pH adjusted to 5 with ammonia
 water and the purple solid product collected to provide 75-90% yield after
 drying. The characteristic lack of the C-5 proton in the .sup.1 H NMR was
 evident for each compound (Table 2).
 General Procedure for the Reduction of 5-Nitroso to 5-Amino Pyrimidines
 (Compounds 21-23, 28, and 33):
 The 5-nitrosopyrimidine (15 mmol) was suspended in water (50 mL) and heated
 to 80-90.degree. C. With stirring, sodium hydrosulfite (45 mmol) was added
 in portions over 5 min. The color quickly changed from purple to light
 green and stirring was continued an additional 10 min. The mixture was
 cooled in ice and filtered. The filtered solid was washed with cold water,
 EtOH and Et.sub.2 O to provide the orthodiamine in 70-88% yield as a tan
 to pale green solid.
 Synthesis of 1-n-Hexyl-3-methyluric acid intermediate (24):
 The nitrosopyrimidine 19 (270 mg, 1.06 mmol) was dissolved in ethanol (20
 mL) with warming and palladium on carbon (75 mg, 10%) was added under
 argon. Hydrogenation was performed at room temperature and 15 psi for 2 h,
 filtered to remove catalyst and evaporated to dryness. The residue was
 combined with urea (600 mg, 10 mmol) and heated neat on the hot plate with
 stirring. The temperature reached 140.degree. C. which produced a clear
 melt and was maintained for about 10 min. with additional urea added (1
 g). Upon cooling the melt solidified and was dissolved in n NaOH (25 mL)
 and boiled with decolorizing carbon for 10 min., filtered and acidified to
 pH 3-4 while hot. The resulting precipitate was collected after cooling
 and washed with water and dried to yield 160 mg (57%) of 24 as an
 off-white solid with the following characteristics: mp&gt;290.degree. C.
 dec. .sup.1 H NMR (500 MHZ, DMSO-d.sub.6) .delta. 11.80 and 10.73 (2s, 2H,
 N-7 H, N-9 H), 3.78 (t, 2H, N--CH.sub.2), 3.30 (s, 3H, N--CH.sub.3, under
 H.sub.2 O signal), 1.48 (m, 2H, 2'CH.sub.2), 1.24 (m, 6H, 3', 4', 5'
 CH.sub.2), 0.85 (t, 3H, CH.sub.3). Analysis: C.sub.12 H.sub.18 N.sub.4
 O.sub.3 (C, H, N; Table 2).
 Synthesis of 3-Methyl-8-thiouric acid (25) intermediate:
 The pyrimidinediamine 33 (100 mg, 0.63 mmol) was combined with potassium
 ethyl xanthate (810 mg, 5 mmol) and DMF (10 mL) and heated at 100.degree.
 C. The suspension became green almost immediately and reaction was
 complete after 30 min. by TLC. After a total reaction time of 1 h, the
 mixture was cooled, filtered and washed with Et.sub.2 O, dried to yield an
 off-white solid (310 mg) which presumably contained the unreacted
 potassium ethyl xanthate and the potassium salt of the desired product.
 The solid was suspended in water (5 mL) and heated to dissolve. Glacial
 acetic acid was added to pH 5 and a vigorous effervescence was noted. A
 white solid formed which was filtered warm and washed with water, then
 ethanol and dried to yield 99 mg (79%) of the title compound. .sup.1 H NMR
 (DMSO-d.sub.6) .delta. 13.40, 12.92 and 11.80 (3br s, 3H, NHs), 3.28 (s,
 3H, CH.sub.3). Analysis: C.sub.6 H.sub.6 N.sub.4 O.sub.2 S (C, H, N; Table
 2).
 Synthesis of 3-n-Propylxanthine (29) intermediate:
 The pyrimidinediamine 28 (750 mg) was combined with diethoxymethyl acetate
 (7 mL) and heated at 80.degree. C. for 2 h. The mixture was evaporated to
 dryness and water (5 mL) was added and the mixture heated for 20 min. to
 near boiling. The resulting solution was then allowed to evaporate slowly
 to yield off-white crystals. Yield 680 mg (86%); mp 282-284.degree. C.,
 Lit..sup.15 291-292.degree. C.
 EXAMPLE 11
 SYNTHESIS OF COMPOUNDS 36-39, 41 AND 43 AND INTERMEDIATES
 General Procedure for Ring Closure of Pyrimidinediamines to Pteridines:
 The orthodiamine 28 or 33 (2 mmol) was suspended in water (20 mL) and
 heated to above 70.degree. C. before a solution of glyoxal-sodium
 bisulfite addition product (10 mmol in 25 mL water) was added with
 stirring. The pale green suspension slowly became light amber and clear.
 After heating 5 min TLC indicated reaction was complete. The mixture was
 cooled and extracted with ethyl acetate (5.times.40 mL), dried over
 MgSO.sub.4 and evaporated to yield the 1-methyl (34) or
 1-n-propylpteridine (35) in 71 and 78%, respectively. .sup.1 H NMR showed
 the appearance of two aromatic signals at about 8.74 and 8.55 as doublets
 (J=2.5 Hz) for both compounds.
 Synthesis of 6,7-Diethyl-1-methylpteridine-2,4-dione (40) intermediate:
 Compound 33 (200 mg, 1.27 mmol) was suspended in acetonitrile (5 mL) and
 3,4-hexanedione (185 .mu.L, 1.52 mmol) was added. The mixture was heated
 at 70.degree. C. for 15 min with minimal product formation due to
 insolubility of 33. Therefore DMF (3 mL) and water (3 mL) were added and
 the temperature was raised to 100.degree. C. After 90 min total reaction
 time the mixture was cooled and poured into water (100 mL) and extracted
 with ethyl acetate (3.times.75 mL). The organic layer was dried over
 MgSO.sub.4 and evaporated to provide the colorless crystalline product.
 Yield 240 mg (81%); mp 218-222.degree. C.; .sup.1 H NMR (DMSO-d.sub.6)
 .delta. 11.78 (br s, 1H, NH), 3.46 (s, 3H, NCH.sub.3), 2.95 and 2.93 (2q,
 4H, 2CH.sub.2 of ethyls), 1.28 and 1.23 (2t, 6H, 2CH.sub.3 of ethyls).
 Analysis: C.sub.11 H.sub.14 N.sub.4 O.sub.2 (C, H, N; Table 2).
 Synthesis of 1-Methyl-6-phenylpteridine-2,4-dione (42) intermediate:
 The nitrosopyrimidine 32 (220 mg, 1.28 mmol) was mixed thoroughly with
 phenethyl amine hydrochloride (1.5 g, 9.5 mmol) and heated in an open
 beaker on the hot plate. After a few minutes at about 160.degree. C. the
 purple reaction mixture fused to a brown paste. TLC indicated many
 products so sulfolane (1 mL) was added and heat was continued for 15 min.
 The reaction mixture was heated in water (10 mL) and then diluted 50 mL in
 water and extracted with ethyl acetate (2.times.50 mL), the organic layer
 dried over MgSO.sub.4 and then concentrated. The residue was flash
 chromatographed on silica gel using 4% MeOH in CH.sub.2 Cl.sub.2. Yield 75
 mg (23%) of 42 as a pale yellow-orange solid. mp&gt;307.degree. C. dec.;
 .sup.1 H NMR (500 MHZ, DMSO-d.sub.6) .delta. 11.95 (br s, 1H, NH), 9.37
 (s, 1H, C-7 H), 8.17 (m, 2H, 2',6' phenyl), 7.55 (m, 3H, 3',4',5' Phenyl),
 3.51 (s, 3H, NCH.sub.3). Anal. C.sub.13 H.sub.10 N.sub.4 O.sub.2 (C, H,
 N).
 EXAMPLE 12
 SYNTHESIS OF COMPOUNDS 44, 47 AND 48
 General Method for Ring Closure of Pyrimidines to Thiadiazolo-pyrimidines
 (Compounds 44-46):
 The orthodiamine 23, 27, or 32 (2.3 mmol) was suspended in dry acetonitrile
 (5 mL) and dry pyridine (1.5 mL) was added. Thionyl chloride (1 mL, 13.7
 mmol) was added quickly and the mixture, which became clear and darkened,
 was heated at 60.degree. C. for 10 min. The mixture was then cooled and
 poured into n HCl (40 mL) with stirring. The resulting yellow solution was
 extracted with ethyl acetate (3.times.40 mL), dried over MgSO.sub.4 and
 evaporated to yield a pale yellow solid which was triturated with ether.
 Yield 65-74%.
 Alkylation of these intermediates yielded the disubstituted products 47 and
 48.
 EXAMPLE 13
 SYNTHESIS OF COMPOUNDS 50 AND 52
 Ethyl 4-[(2-methylamino)benzoyl]aminobutanoate (51):
 A mixture of N-methylisatoic anhydride (3.5 g, 19.8 mmol) was combined with
 4-aminobutyric acid (2.5 g, 24.3 mmol) in dry DMF (50 mL) and heated at
 100.degree. C. for 2 h. TLC indicated reaction to be complete and the DMF
 was removed in vacuo. The residue was used directly for esterification
 which was accomplished by dissolving the residue in 100% ethanol (50 mL)
 and adding chlorotrimethyl silane (2.5 mL, 20 mmol). The mixture was
 heated at 65.degree. C. for 6 h and then evaporated to yield a brown
 syrup. Crude yield 87% from isatoic anhydride. A small sample was purified
 for characterization and biological testing by preparative TLC using 7%
 MeOH in CH.sub.2 Cl.sub.2. The remainder of the material was used directly
 for preparation of compound 52. Analysis: C.sub.14 H.sub.20 N.sub.2
 O.sub.3 (C, H, N; Table 2).
 Ethyl 1-Methyl-1,4-dihydro-2,4-dioxo-3(2H)-quinazolinebutanoate (52):
 The residue from 51 was combined with ethyl chloroformate (10 mL) and
 heated at 90.degree. C. for 1 h. The mixture was cooled and poured into
 saturated aqueous sodium bicarbonate (50 mL) with stirring and after 10
 min extracted with ethyl acetate (2.times.75 mL). The organic layer was
 dried over MgSO.sub.4 and evaporated to yield a brown syrup. The crude
 product was flash chromatographed on silica using 3% MeOH in CH.sub.2
 Cl.sub.2 to yield g (%) of 52 as a thick oil. .sup.1 H NMR (500 MHZ,
 DMSO-d.sub.6) .delta. 7.27-7.42 (2m, 4H, C-5,6,7,8), 4.04 (t, 2H, CH.sub.2
 of ethyl), 3.88 (m, 2H, NCH.sub.2), 3.11 (s, 3H, NCH.sub.3), 2.33 (t, 2H,
 2'CH.sub.2), 1.71 (m, 2H, 3'CH.sub.2). Analysis: C.sub.15 H.sub.18 N.sub.2
 O.sub.4 (C, H, N; Table 2).
 TABLE 2
 ANALYSIS OF SELECTED INVENTIVE COMPOUNDS AND
 INTERMEDIATES (Combustion Elemental Analysis)
 4 Calc'd for C, 49.62; H, 5.30; N, 21.04.
 C.sub.11 H.sub.14 N.sub.4 O.sub.4:
 Found: C, 49.54; H, 5.31; N, 21.12.
 5 Calc'd for HRMS 281.124980
 C.sub.12 H.sub.16 N.sub.4 O.sub.4 + H.sup.+ :
 Found: 281.123300
 6 Calc'd for C, 53.05; H, 6.16; N, 19.04.
 C.sub.13 H.sub.18 N.sub.4 O.sub.4 :
 Found: C, 52.79; H, 6.00; N, 18.99.
 7 Calc'd for C, 54.54; H, 6.54; N, 18.17.
 C.sub.14 H.sub.20 N.sub.4 O.sub.4 :
 Found: C, 54.47; H, 6.42; N, 18.16.
 8 Calc'd for C, 57.29; H, 5.52; N, 17.33.
 C.sub.16 H.sub.16 N.sub.4 O.sub.4.2/3 DMF:
 Found: C, 57.17; H, 5.51; N, 17.35.
 10 Calc'd for C, 41.84; H, 4.59; N, 15.01.
 C.sub.13 H.sub.17 BrN.sub.4 O.sub.4 :
 Found: C, 41.83; H, 4.43; N, 14.99.
 11 Calc'd for C, 47.19; H, 3.71; N, 13.76.
 C.sub.16 H.sub.15 BrN.sub.4 O.sub.4 :
 Found: C, 47.02; H, 3.68; N, 13.63.
 12 Calc'd for C, 47.84; H, 5.56; N, 17.17.
 C.sub.13 H.sub.18 N.sub.4 O.sub.4 S:
 Found: C, 47.98; H, 5.44; N, 16.99.
 13 Calc'd for C, 54.53; H, 4.85; N, 14.96.
 C.sub.17 H.sub.18 N.sub.4 O.sub.4 S:
 Found: C, 54.41; H, 4.66; N, 14.72.
 20 Calc'd for C, 53.72; H, 7.51; N, 20.88.
 C.sub.12 H.sub.20 N.sub.4 O.sub.3 :
 Found: C, 54.00; H, 7.47; N, 20.65.
 4 Calc'd for C, 54.12; H, 6.81; N, 21.04.
 C.sub.12 H.sub.18 N.sub.4 O.sub.3 :
 Found: C, 54.23; H, 6.76; N, 21.04.
 31 Calc'd for C, 55.89; H, 6.88; N, 17.38.
 C.sub.15 H.sub.22 N.sub.4 O.sub.4 :
 Found: C, 55.67; H, 6.94; N, 17.22.
 33 Calc'd for C, 38.46; H, 5.16; N, 35.88.
 C.sub.5 H.sub.8 N.sub.4 O.sub.4 :
 Found: C, 38.22; H, 5.13; N, 35.84.
 34 Calc'd for C, 47.19; H, 3.39; N, 31.45.
 C.sub.7 H.sub.6 N.sub.4 O.sub.2 :
 Found: C, 47.01; H, 3.18; N, 31.25.
 35 Calc'd for C, 52.42; H, 4.89; N, 27.17.
 C.sub.9 H.sub.10 N.sub.4 O.sub.2 :
 Found: C, 52.20; H, 4.74; N, 27.22.
 36 Calc'd for C, 53.42; H, 5.52; N, 19.17.
 C.sub.13 H.sub.16 N.sub.4 O.sub.4 :
 Found: C, 53.39; H, 5.43; N, 19.17.
 36a Calc'd for C, 50.00; H, 4.58; N, 21.20.
 C.sub.11 H.sub.12 N.sub.4 O.sub.4 :
 Found: C, 49.92; H, 4.47; N, 21.26.
 37 Calc'd for C, 56.24; H, 6.29; N, 17.49.
 C.sub.15 H.sub.20 N.sub.4 O.sub.4 :
 Found: C, 56.04; H, 6.12; N, 17.42.
 37a Calc'd for C, 50.91; H, 5.78; N, 18.27.
 C.sub.13 H.sub.16 N.sub.4 O.sub.4.4/5H.sub.2 O:
 Found: C, 50.64; H, 5.81; N, 18.23.
 38 Calc'd for HRMS 277.093680
 C.sub.12 H.sub.18 N.sub.4 O.sub.4 + H.sup.+ :
 Found: 277.093800
 39 Calc'd for C, 53.96; H, 5.86; N, 14.81.
 C.sub.17 H.sub.22 N.sub.4 O.sub.6 :
 Found: C, 53.60; H, 5.73; N, 14.04.
 40 Calc'd for C, 55.34; H, 6.12; N, 23.47.
 C.sub.11 H.sub.14 N.sub.4 O.sub.4.1/4H.sub.2 O:
 Found: C, 55.37; H, 6.02; N, 23.43.
 41 Calc'd for C, 58.61; H, 6.94; H, 16.08.
 C.sub.17 H.sub.24 N.sub.4 O.sub.4 :
 Found C, 59.22; H, 7.08; N, 15.69.
 41a Calc'd for C, 56.24; H, 6.29; N, 17.49.
 C.sub.15 H.sub.20 N.sub.4 O.sub.4 :
 Found: C, 56.41; H, 6.27; N, 17.28.
 42 Calc'd for C, 60.00; H, 4.13; N, 21.53.
 C.sub.13 H.sub.10 N.sub.4 O.sub.4.1/3H.sub.2 O:
 Found: C, 59.78; H, 3.70; N, 21.14.
 43 Calc'd for HRMS 369.156280
 C.sub.19 H.sub.20 N.sub.4 O.sub.4 + H.sup.+ :
 Found: 369.154800
 44 Calad'd for HRMS 283.122873
 C.sub.12 H.sub.18 N.sub.4 O.sub.2 S + H.sup.+ :
 Found: 283.121300
 45 Calc'd for C, 32.61; H, 2.19; N, 30.42.
 C.sub.5 H.sub.4 N.sub.4 O.sub.2 S:
 Found: C, 32.65; H, 2.20; N, 30.29.
 46 Calc'd for C, 39.62; H, 3.80; N, 26.40.
 C.sub.7 H.sub.8 N.sub.4 O.sub.2 S:
 Found: C, 39.84; H, 3.60; N, 26.02.
 47 Calc'd for C, 44.29; H, 4.73; N, 18.78.
 C.sub.11 H.sub.14 N.sub.4 O.sub.4 S:
 Found: C, 44.57; H, 4.67; N, 18.80.
 47a Calc'd for HRMS 271.050102
 C.sub.9 H.sub.10 N.sub.4 O.sub.4 S + H.sup.+ :
 Found: 271.050600
 48 Calc'd for C, 47.84; H, 5.56; N, 17.17.
 C.sub.13 H.sub.18 N.sub.4 O.sub.4 :
 Found: C, 47.97; H, 5.66; N, 17.08.
 51 Calc'd for C, 61.52; H, 7.74; N, 10.25.
 C.sub.14 H.sub.20 N.sub.2 O.sub.3.1/2H.sub.2 O:
 Found: C, 61.54; H, 7.44; N, 9.77.
 52 Calc'd for C, 59.62; H, 6.88; N, 8.69.
 C.sub.15 H.sub.18 N.sub.2 O.sub.4 CH.sub.3 OH:
 Found: C, 60.61; H, 6.84; N, 8.58.
 52a Calc'd for C, 59.54; H, 5.38; N, 10.68.
 C.sub.13 H.sub.14 N.sub.2 O.sub.4 :
 Found: C, 59.55; H, 5.32; N, 10.59.
 EXAMPLE 14
 SYNTHESIS OF HIGHLY WATER SOLUBLE DERIVATIVES
 Acid Hydrolysis of Compound 53.
 Compound 53 (1.63 g, 5.1 mmol) (prepared according to Example 9 from the
 parent isoquinolone) was combined with n HCl (30 mL) and was heated at
 boiling for 75 min. The mixture was cooled and the resulting solid which
 formed was filtered, washed with cold water, and dried to yield 1.5 g
 (quantitative conversion) of the free carboxylic acid of suitable purity
 for esterification (as judged by TLC).
 Esterification to form morpholinoethyl ester Compound 54.
 The carboxylic acid obtained above in this Example (1.0 g, 3.4 mmol) was
 dissolved in dichloromethane (25 mL) with warming and then thionyl
 chloride (1 mL, 13.7 mmol) was added followed by 3 drops of DMF. After a
 few minutes a white solid precipitated (acid chloride intermediate), but
 the reaction mixture was allowed to stir at room temperature overnight.
 The mixture was evaporated to remove excess thionyl chloride and the
 residue was suspended in dry acetonitrile (25 mL). To this mixture was
 added morpholinoethanol (1.24 mL, 10.2 mmol) and heated at 80.degree. C.
 for 5 minutes. The solid suspension became clear almost immediately and
 after cooling, the reaction mixture was evaporated onto silica gel and
 loaded on a flash silica gel column and eluted with
 methanol--dichloromethane 5/95. The fractions of pure product were pooled
 and evaporated to yield an off-white residue which was dissolved in
 isopropyl alcohol (5 mL) and concentrated HCl (1 mL) was added. Upon
 concentration in vacuo, an off-white solid formed. Yield after drying 0.93
 g (62%), mp 90.degree. C. Proton NMR confirms structure assignment as the
 morpholino ethyl ester of Compound 53: .sup.1 H NMR (DMSO-d.sub.6, 500
 MHZ) .delta. 7.56 and 7.15 (2s, 2H, C-5 and C-8 aromatics), 7.35 and 6.55
 (2d, 2H, C-3 and C-4 aromatics), 4.37 (m, 2H, C-1' of ester), 3.92 (m, 2H,
 C-4 of acyl), 3.86 and 3.84 (2s, 6H, OMe's), 3.95, 3.45, 3.10 (3m, 8H,
 morpholino and C-2'), 2.38 (t, 2H, C-2 of acyl), 1.92 (m, 2H, C-3 of
 acyl).
 The invention having been fully described, modifications thereof may be
 apparent to those of ordinary skill in the art. Such modifications are
 within the scope of the invention as defined by the appended claims.