Anthraquinone compounds and their uses

Abstract

The present invention relates to a compound comprising a substituted or unsubstituted anthraquinone, or a salt or isomer thereof, for use in treating a disorder caused by or associated with dysfunctional ion channel activity. The invention finds utility in the treatment of disorders associated with smooth muscle tone and contraction, such as but not limited to partial hypertension; myocardial infarction; faecal incontinence; constipation; gastro oesophageal reflux; impaired gastrointestinal passage; urinary incontinence; erectile dysfunction; and asthma.

Claims

1. A method for treating urinary incontinence, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound having the general formula (I): ##STR00049## wherein in (I); (i) R.sub.1-R.sub.4 and R.sub.7 are each a hydrogen atom; (ii) R.sub.5 is an amine; (iii) R.sub.8 is a secondary amine, and (iv) R.sub.6 is a carboxyl group; or a pharmaceutically acceptable salt, hydrate, or isomer thereof.

2. The method of claim 1, wherein the compound has the general formula (IE): ##STR00050## wherein in (IE); (i) R.sub.1 and R.sub.5 are each independently selected from the group consisting of: (a) a hydrogen atom; (b) a substituent selected from the group consisting of a halide and an oxygen atom; (c) a short chain alkyl, alkenyl, or alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (d) a short chain alkoxyl, alkenoxyl, or alkynoxyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; and (e) a short chain halo-alkyl, halo-alkenyl, or halo-alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (ii) R.sub.2 and R.sub.4 are each independently selected from the group consisting of: (a) a hydrogen atom; (b) a substituent selected from the group consisting of a halide, an oxygen atom, and an amine (c) a short chain halo-alkyl, halo-alkenyl, or halo-alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (d) a sulfonate or carboxyl group; (e) a short chain alkyl, alkenyl, or alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (f) a short chain alkoxyl, alkenoxyl, or alkynoxyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (g) a nitrile group; (h) a tetrazole; and (i) a hydroxyl group; and (iii) R.sub.3 is selected from the group consisting of: (a) a hydrogen atom; (b) a short chain halo-alkyl, halo-alkenyl, or halo-alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; (c) a substituent selected from the group consisting of a halide, an oxygen atom, and an amine; (d) a short chain alkyl, alkenyl, or alkynyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic (e) a short chain alkoxyl, alkenoxyl, or alkynoxyl group, which can be branched or unbranched, substituted or unsubstituted, linear or cyclic; and (f) a nitrile; or a pharmaceutically acceptable salt, hydrate, or isomer thereof.

3. The method of claim 2, wherein R.sub.1 and R.sub.5 are each independently selected from the group consisting of: a hydrogen atom; (ii) fluoride; (iii) a polycyclic group selected from the group consisting of a cycloalkane, cycloalkene, and cycloalkyne; (iv) a methoxyl group; and (v) a trifluoromethyl group.

4. The method of claim 2, wherein R.sub.2 and R.sub.4 are each independently selected from the group consisting of: (i) a hydrogen atom; (ii) fluoride; (iii) chloride; (iv) a trifluoromethyl group; (v) a trifluoromethoxy group (OCF.sub.3); (vi) a methyl group; (vii) an ethyl group; (viii) an isopropyl group; (ix) a tert-butyl group; (x) a cyclopropyl group; (xi) a nitrile group; (xii) a methoxyl group; (xiii) a ethoxyl group; (xiv) an isopropoxyl group; (xv) an amine, optionally a primary amine; (xvi) a polycyclic group selected from the group consisting of cycloalkane, cycloalkene, and cycloalkyne; (xvii) a benzyl group; (xviii) a tetrazole; and (xix) a hydroxyl group.

5. The method of claim 2, wherein R.sub.3 is selected from the group consisting of: (i) a hydrogen atom; (ii) a trifluoromethyl group; (iii) fluoride; (iv) chloride; (v) a benzyl group; (vi) a methyl group; (vii) a methoxyl group; (viii) an amine; and (ix) a nitrile.

6. The method of claim 2, wherein R.sub.2 is a trifluoromethyl group, and each of R.sub.1, and R.sub.3-R.sub.5 is a hydrogen atom.

7. A method for treating urinary incontinence in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of 1-amino-9,10-dioxo-4-(3-(trifluoromethyl)phenylamino)-9,10-dihydroanthracene-2-carboxylic acid, or a pharmaceutically acceptable salt or hydrate thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings specific embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

(2) FIGS. 1A-1E illustrate graphs depicting current recordings using inside-out patch clamp technique from isolated smooth muscle cells of a rabbit bladder bathed in Ca.sup.2+ solution. FIGS. 1A-1C depict current recordings over a range of applied voltages from 100 mV to 100 mV at 100 nM Ca.sup.2+, 1 M Ca.sup.2+, and 10 M Ca.sup.2+, respectively. FIG. 1D depicts a graph summarizing the results of FIGS. 1A-1C. FIG. 1E illustrates a typical example of an experiment in which single channel recordings were obtained in the presence of 1 M Ca.sup.2+ using a voltage ramp protocol before application of a BK channel blocker. FIG. 1F illustrates an experiment in which single channel recordings were obtained in the presence of 1 M Ca.sup.2+ using a voltage ramp protocol after application of Penitrem A (100 nM). FIGS. 1E and 1F demonstrate that addition of Penitrem A abolishes the single channel openings.

(3) FIG. 2 illustrates graphs depicting current recordings using the perforated patch configuration of the whole cell patch clamp technique with Acid Blue 25. The Control panel depicts a family of outward currents elicited in response to the voltage protocol in the lower panel. The AB25 (10 M) panel depicts the potentiating effects of Acid Blue 25 (10 M) on the outward BK current in response to the voltage protocol in the lower panel. The Wash panel depicts the effect of Acid Blue 25 (10 M) can be washed out, returning the currents to control levels.

(4) FIG. 3 illustrates a graph depicting the effects of SR-5-6 on spontaneous transient outward currents (STOCs). Isolated smooth muscle cells from rabbit bladder were held under voltage clamps at 30 mV and fired spontaneously active transient outward currents, as a result of activation of BK currents. Application of 10 M of SR-5-6 increased the amplitude of STOCs.

(5) FIG. 4 illustrates a graph depicting the dose response of SR-5-6 at a single voltage step as observed through perforated patch clamp recordings from isolated smooth muscle cells from a rabbit bladder. The graph demonstrates that the activation of BK channels by SR-5-6 occurs in a dose-dependent manner when the cell is held at 60 mV and then stepped to 0 mV for 500 msec.

(6) FIG. 5 illustrates a graph depicting the effect of SR-5-6 on L-type calcium channels as observed through perforated patch clamp recordings from isolated smooth muscle cells from a rabbit bladder. Isolated cells were held at 60 mV and stepped to 0 mV for 500 msec. A Cs.sup.+ solution was added and the measured inward L-type calcium current was reduced in amplitude in the presence of SR-5-6 in a dose-dependent manner.

(7) FIG. 6 illustrates a graph depicting the effect of SR-5-6 on BK single channel recordings using inside-out patch clamp technique from isolated smooth muscle cells of a rabbit bladder bathed in Ca.sup.2+ solution (200 nM). The patches were held at a potential of 100 mV and the voltage ramps were applied from 100 mV to +100 mV over 2 seconds. The upper trace depicts the current measurement in the presence of SR-5-6 (10 M) while the lower trace depicts the control measurement. The graph demonstrates that BK channels activate at lower potential in the presence of SR-5-6.

(8) FIGS. 7A-7D illustrate graphs depicting the effect of SR-5-6 on the opening of BK channels. FIG. 7A depicts a control trace while FIG. 7B depicts the effect of SR-5-6 (10 M) on BK channel activity. FIG. 7C illustrates a graph depicting the effects of SR-5-6 on an inside out patch over a range of concentrations. FIG. 7D illustrates a graph depicting the calculated V.sub.1/2 (the voltage wherein half of the channels in the patch are activated) derived from the data presented in FIG. 7C. These results show that the effects of SR-5-6 on the open probability and V.sub.1/2 for BK channels are dose-dependent.

(9) FIGS. 8A-8B illustrate graphs depicting the effect of SR-5-69 on the opening of BK channels. FIG. 8A illustrates a graph depicting the effect of SR-5-69 on BK channel openings over a range of concentrations. FIG. 8B illustrates a graph depicting the calculated V.sub.1/2 (the voltage wherein half of the channels in the patch are activated) derived from the data presented in FIG. 8A. These results show that the effects of SR-5-69 on the open probability and V.sub.1/2 for BK channels are dose-dependent.

(10) FIGS. 9A-9D illustrate graphs depicting the effect of the compounds of the invention on BK channels in cells other than bladder smooth muscle cells. FIG. 9A depicts the effect of SR-5-6 on BK channels expressed in Human Embryonic Kidney (HEK) cells transfected to express the pore forming BK subunit. FIG. 9B illustrates activation curves obtained from rabbit bladder smooth muscle cells in response to increases in Ca.sup.2+ concentration (100 nM, 1 M and 10 M Ca.sup.2+), wherein the native BK channels were more sensitive to Ca.sup.2+ compared to the BK alpha subunits shown in FIG. 9A. FIG. 9C illustrates a graph depicting a comparison of the Ca.sup.2+ sensitivity between smooth muscle cells (black circles), HEK cells expressing the BK subunit (white squares) and HEK cells co-expressing the BK.sub.1 subunit (grey circles) The results show that HEK cells expressing both BK and BK.sub.1 subunits show similar Ca.sup.2+ dependence as native bladder smooth muscle cells. FIG. 9D illustrates a graph summarizing the mean V.sub.1/2 caused by application of 10 m SR-5-6 in smooth muscle cells, HEK cells expressing the BK.sub.1 subunits and HEK cells only expressing the BK subunit.

EXAMPLES

(11) All experiments were carried out at 361 C., and for the excised patch single channel recordings, the total Ca.sup.2+ concentration required to give the free Ca.sup.2+ stated in the text was calculated using Chelator software: (http://www dot organphy dot science dot ru dot nl/chelator/Chelmain dot html).

(12) All experiments were approved by the Dundalk Institute of Technology Animal Care and Use Committee. Tissues were obtained from male and female New Zealand white rabbits immediately after they had been killed by lethal injection of pentobarbitone. The urinary bladder and most proximal 1.5 cm of the urethra was removed and placed in Krebs solution. Strips of bladder tissue, 0.5 cm in width were dissected, cut into 1 mm.sup.3 pieces and stored in Ca.sup.2+-free Hanks' solution for 30 min prior to cell dispersal. Tissue pieces were incubated in dispersal medium containing (per 5 ml) of Ca.sup.2+-free Hanks' solution: 15 mg collagenase (Sigma type 1A), 1 mg protease (Sigma type XXIV), 10 mg bovine serum albumin (Sigma) and 10 mg trypsin inhibitor (Sigma) for 10-15 min at 37 C. Tissue was then transferred to Ca.sup.2+-free Hanks' solution and stirred for a further 10-15 min to release single smooth muscle cells. These were plated in Petri dishes containing 100 M Ca.sup.2+ Hanks' solution and stored at 4 C. for use within 8 h. During experiments, the dish containing the cells was continuously perfused with Hanks' solution at 361 C. Additionally the cell under study was continuously superfused by means of a custom built close delivery system with a pipette of tip diameter 200 m placed approximately 300 m from the cell. The high K.sup.+ solution in the close delivery system could be switched to a drug-containing solution with a dead space time of less than 5 s.

(13) For whole cell recordings pipettes were pulled from borosilicate glass capillary tubing (1.5 mm outer diameter, 1.17 mm inner diameter; Clark Medical Instruments) to a tip of diameter approximately 1-1.5 m and resistance of 2-4 Mohms. For single channel recordings, pipettes were pulled from borosilicate glass capillary tubing (1.5 mm outer diameter, 0.8 mm inner diameter; Clark Medical Instruments) and fire polished before use. Voltage clamp commands were delivered via an Axopatch 1D or Axon 200B patch clamp amplifiers (Axon Instruments) and membrane currents were recorded by a 12 bit AD/DA converter (Axodata 1200 or Labmaster, Scientific Solutions) interfaced to an Intel computer running pCLAMP software.

(14) Single Channel Bath Solutions: For Free Ca.sup.2+ Less than 300 nM:

(15) All values in (mM): KCl, 140. Glucose, 10, EGTA 1 and HEPES, 10. For free Ca.sup.2+ greater than 300 nM: All values in (mM): KCl, 140. Glucose, 10, H-EDTA 1 and HEPES, 10. The same solution composition is used in the bath as in the patch pipette for these experiments except that the pipette solution contained 100 nM Ca.sup.2+.

(16) In single channel experiments voltage commands were applied using pClamp ramped potentials. This allowed more efficient measurement of slope conductance and channel activation than conventional step depolarisations. Activation curves were calculated by averaging current responses to 15 potential ramps and dividing each data point of the averaged current by the single channel amplitude at that holding potential, after leakage current correction. The rate of change of the applied ramp potentials were sufficiently slow (100 mVs.sup.1) so that the activation curves were not distorted by the time constants of activation or deactivation. This analysis provides a continuous recording of the number of open channels multiplied by the open probability (NPo) over the entire voltage range. To obtain values for the steepness of the voltage-dependent activation and half-maximal activation voltage, activation curves were fitted with Boltzman functions of the form:
NPo=n/{1+exp[K(VV.sub.1/2)]}
where N is the number of channels in the patch, n is the maximal NPo level, K.sup.1 is the steepness of the voltage-dependent activation (change in potential necessary to cause an e-fold increase in activation) and V.sub.1/2 is the voltage at which there is half-maximal activation. Again, all of the experiments were carried out at 361 C.

Example 1: Single Channel Recordings Using Inside-Out Patch Clamp Technique from Isolated Smooth Muscle Cells of the Rabbit Bladder

(17) When voltage ramps were applied to inside out patches and the cytosolic face of the patch was bathed in solutions containing 100 nM Ca.sup.2+, brief single channel openings were only observed at potentials positive to +50 mV. Referring to FIG. 1A, 3 distinct single channel openings which each carried a maximum current of 30 pA at +100 mV were apparent in this single sweep. These large single channel currents are a hallmark of large conductance K.sup.+ (BK) channels. When the Ca.sup.2+ concentration at the cytosolic face of the patch was increased to 1 M (FIG. 1B), the number of channels opening increased and the channels activated at more negative potentials. Increasing the Ca.sup.2+ concentration further to 10 M as shown in FIG. 1C further enhanced the single channel activity so that up to 4 distinct single channel openings could be observed at potentials negative to 100 mV. Note that the current is inward at voltages negative to 0 mV (reversal potential for K.sup.+) and is outward at potentials positive to this. FIG. 1D shows summary data from these experiments, and demonstrates that the voltage at which half of the channels in the patch were maximally activated V.sub.1/2 (mV) shifted negatively with increasing concentrations of Ca.sup.2+. In general, a 10 fold change in Ca.sup.2+ shifted the V.sub.1/2 by 100 mV consistent with the idea that these channels comprise the and 1 subunits of the BK channel. Having established that these channels had a large conductance, were both voltage and calcium sensitive and the currents reversed at the K+ equilibrium potential (0 mV) we next tested the effect of the selective BK channel blocker Penitrem A in a separate series of experiments. FIG. 1E shows a typical example of an experiment in which single channel recordings were obtained in the presence of 1 M Ca.sup.2+ using a voltage ramp protocol (upper panel) before (lower panel) application of the blocker. In this example, the BK channels activated at negative potentials (50 mV) reversed at 0 mV and were macimally activated at positive potentials. FIG. 1F shows the recording from same patch of membrane after application of the selective BK channel blocker Penitrem A (100 nM). As FIGS. 1E and 1F suggest, Penitrem A abolished the single channel openings.

(18) These data characterize the current under investigation, and are consistent with the characteristics that would be expected from a large conductance BK channel, showing that the current being activated is the current elicited by opening of BK channels.

Example 2: Isolated Cells Using Perforated Patch with Sodium 1-Amino-9,10-Dioxo-4-(Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate. (Acid Blue 25)

(19) Isolated smooth muscle cells are dispersed as described in Example 1. Currents were recorded using the perforated patch configuration of the whole cell patch clamp technique (Rae et al., 1991). The cell membrane was perforated using the antibiotic amphotericin B (600 g.Math.ml.sup.1). Other methods including the preparation of patch pipettes and recording of currents are as described in Example 1.

(20) K Perforated Patch Solution:

(21) All values in (mM): KCl, 132.96. MgCl.sub.2.6H.sub.20, 1. EGTA, 0.5 and HEPES, 10. Standard Hank's solution is used to bathe the cells in these experiments. Isolated smooth muscle cells from rabbit urethra we held at 60 mV. The test protocol involved stepping from 60 mV to 80 mV for 500 msecs, and then stepping the voltage up in +10 mV increments to +50 mV before returning to the holding potential of 60 mV. This is diagrammatically shown in FIG. 2.

(22) The Control panel in FIG. 2 shows a family of outward currents recorded using the perforated patch configuration and elicited under in response to the voltage protocol shown in the lower panel. These noisy outward currents are consistent with currents being carried through large conductance BK channels. The centre panel of FIG. 2 shows the potentiating effects of Acid Blue 25 (10 M) on the outward BK current in response to the same voltage steps. The right hand panel shows the effect of Acid Blue 25 can be washed out returning the currents to control levels. Acid Blue 25 (10 M) enhances the amplitude of the BK current compared to control, an effect that is reversible upon wash-out.

Example 3: Sodium 1-Amino-9,10-Dioxo-4-(3-(Trifluoromethyl)Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate (SR-5-6) Potentiates STOCs (Spontaneous Transient Outward Currents) Recorded Using the Perforated Patch Clamp Technique from Isolated Smooth Muscle Cells from the Rabbit Bladder

(23) Using the perforated patch clamp techniques described in Example 2, the effects of the compounds of the present invention were observed on spontaneous transient outward currents (STOCs). In this experiment, isolated smooth muscle cells from rabbit bladder were held under voltage clamp at 30 mV and fired spontaneously active transient outward currents, as a result of activation of BK currents.

(24) As seen in FIG. 3, application of 10 M SR-5-6 increased the amplitude of STOCs, recorded using perforated patch clamp techniques. This effect was reversible on wash-out. These data indicate that SR-5-6 activates BK channels in spontaneously active smooth muscle cells when the cells are held at 30 mV.

Example 4: Dose-Response of Sodium 1-Amino-9,10-Dioxo-4-(3-(Trifluoromethyl)Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate (SR-5-6) at Single Voltage StepPerforated Patch Clamp Recordings from Isolated Smooth Muscle Cells from the Rabbit Bladder

(25) Using the perforated patch clamp techniques described in Example 2, isolated smooth muscle cells from the rabbit bladder were held at 60 mV and stepped to 0 mV for 500 msec to elicit the BK current.

(26) As seen in FIG. 4, the outward current elicited during perforated patch recordings using this protocol is potentiated in a dose dependent manner, using the compound sodium 1-amino-9,10-dioxo-4-(3-(trifluoromethyl)phenylamino)-9,10-dihydroanthracene-2-sulfonate (SR-5-6). These data demonstrate that the activation of BK channels by SR-5-6 occurs in a dose-dependent manner when the cell is exposed to the above protocol.

Example 5: Effects of Sodium 1-Amino-9,10-Dioxo-4-(3-(Trifluoromethyl)Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate (SR-5-6) on L-Type Calcium ChannelPerforated Patch Clamp Recordings from Isolated Smooth Muscle Cells of the Rabbit Bladder, Using Cs+ in the Pipette Solution to Block BK Channels

(27) Using methods described above herein, the patch pipette solution was altered to allow for measurement of inward L-type calcium currents which although present in earlier studies are not visible due to the overwhelming size of the outward BK current. By replacing K.sup.+ with Cs.sup.+ ions, the outward current is now blocked and a prominent inward current carried by Calcium is present. This is the L-type calcium ion channel currents allowing calcium into the cells.

(28) Pipette Solution:

(29) All values in (mM): CsCl, 132.96. MgCl.sub.2.6H.sub.20, 1. EGTA, 0.5 and HEPES, 10.

(30) With reference to FIG. 5, isolated smooth muscle cells from the rabbit bladder were held at 60 mV and stepped to 0 mV for 500 ms. Pipette solutions contained Cs.sup.+ to block outward K.sup.+ currents. An inward L-type calcium current measured using perforated patch clamp technique, is reduced in amplitude in the presence of SR-5-6 in a dose-dependent manner. As the BK channels are known to be activated both by a change in voltage but also by an increase in intracellular Ca.sup.2+ ions, it is important to rule out a mechanism of action whereby SR-5-6 was activating BK channels by increasing the influx of Ca.sup.2+ through activation of voltage gated Ca.sup.2+ channels and subsequent Ca.sup.2+ activation of BK channels. This experiment shows that the increase in outward BK current with SR-5-6 observed previously (FIGS. 3-4) is not due to activation of L-type calcium currents and subsequent influx of calcium, rather that there is a modest inhibitory effect on influx of Ca.sup.2+ through these channels.

Example 6: Ramp Protocol Showing Sodium 1-Amino-9,10-Dioxo-4-(3-(Trifluoromethyl)Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate (SR-5-6) EffectsSingle Channel Recordings Using Inside-Out Patch Clamp Technique from Isolated Smooth Muscle Cells of the Rabbit Bladder

(31) Using the single channel inside-out patch clamp technique described in Example 1, recordings of BK single channels in patches of membrane from isolated smooth muscle cells from rabbit bladder, were obtained using the inside out patch clamp configuration. The patches were held at a potential of 100 mV and voltage ramps were then applied from 100 mV up to +100 mV over 2 s.

(32) As seen in FIG. 6, when Ca.sup.2+ was buffered to 200 nM, the BK channels began to activate at potentials positive to +50 mV. When the same voltage ramp was reapplied in the presence of 10 M SR5-6 the threshold voltage for activation of the channels was shifted 100 mV in the hyperpolarising direction.

(33) The results of this experiment show that the current evoked in response to the voltage ramp protocol shifted the activation potential to a much more negative potential. This is consistent with the idea that this compound activates BK channels since having a greater population of BK channels open would cause activation of an outward BK current at more negative potentials in the physiologically relevant range of potentials (i.e., negative to 0 mV). In addition, the amount of current elicited throughout the duration of the ramp is larger in the presence of SR-5-6 than in control conditions. Finally, since these data were collected from an inside-out patch, a situation where the compound is applied to the inside of the cell membrane, it seems probable that the compound is activating the BK channel on this small patch of membrane from the inside surface of the cell. Consequently one can conclude that SR-5-6 activates BK channels in an isolated patch of membrane, by directly opening the ion channel from the inside surface of the cell. However, given the data from whole cell recordings we can also conclude that the compound can activate BK channels when presented on exterior surface (outside) of the cell.

Example 7: Cumulative Data Showing Sodium 1-Amino-9,10-Dioxo-4-(3-(Trifluoromethyl)Phenylamino)-9,10-Dihydroanthracene-2-Sulfonate (SR-5-6) on RampsSingle Channel Recordings Using Inside-Out Patch Clamp Technique from Isolated Smooth Muscle Cells of the Rabbit Bladder

(34) Using the single channel inside out patch clamp technique, described in Example 1, voltage ramps were used to observe openings of BK channels, as described in Example 6.

(35) Referring to FIG. 7A shows the control trace using the ramp protocol as described above herein. FIG. 7B shows the effect of 10 M SR-5-6 on the channel activity, as seen previously. FIG. 7C shows a typical example of the effects of SR-5-6 on an inside out patch. To obtain these data, the average single channel currents were obtained from 15 voltage ramp sweeps under control conditions and then in the presence of each concentration of the drug. The mean currents were corrected for driving force by dividing the current by the single channel amplitude at each potential. Consequently, activation curves similar to those shown in FIG. 7C can be obtained. When these data are fitted with a Boltzmann relationship, the voltage at which half of the channels in the patch are activated can be calculated (V.sub.1/2). These data show that the effects of SR-5-6 are dose-dependent and shift the activation of the channels towards more physiologically relevant membrane potentials. FIG. 7D shows a summary plot of five experiments in which the mean activation V.sub.1/2 of the channels was plotted under control conditions (200 nM Ca.sup.2+) and in the presence of increasing concentrations of SR-5-6. The error bars show the standard error of the mean for each data point.

(36) These data show conclusively that the effects of SR-5-6 on the open probability and V.sub.1/2 for BK channels are dose-dependent, which is a critical factor in the development of drug candidates.

Example 8: Comparing Structure Function Relationships Using Single Channel Recordings of Inside-Out Patches from Isolated Smooth Muscle Cells of the Rabbit Bladder

(37) To compare the effect of a variety of chemical substitutions on the BK channels, experiments were carried out using voltage ramps applied to inside out patches which were bathed with either 188 nM free Ca.sup.2+ or 100 nM free Ca.sup.2+ on their cytosolic face. Patches were held at 100 mV and ramped through to +100 mV and each sweep was repeated 15 times. The same protocol was repeated after the patch was incubated in 1 M Ca.sup.2+. The patch was then returned to 100 nM Ca.sup.2+ containing solutions and 10 M of the drug of interest was applied. After a maximal effect was observed, the voltage ramps were reapplied to the patch in the presence of the drug. Data from each series of voltage ramp was then averaged. These mean currents were corrected for driving force by dividing the current by the single channel amplitude at each potential. When these data were fitted with a Boltzmann relationship, the voltage at which half of the channels in the patch were activated could be calculated (V.sub.1/2). The observed shift in activation (V.sub.1/2) under control conditions and in the presence of each drug was obtained by subtracting the V.sub.1/2 in control and the V.sub.1/2 in drug. Table 1 and Table 2 show a summary data series of experiments in which the average V.sub.1/2 of each molecule is compared.

(38) TABLE-US-00001 TABLE 1 The effect of applying 10 M of compounds on V.sub.1/2 according to a first aspect of the present invention V.sub.1/2 (mV) in Compound 100 nm Ca.sup.2+ SR-5-18 23.2 SR-5-14 24.2 SR-5-8 24.24 SR-5-15 28.4 Acid Blue 25 51.0 SR-5-26 53.5 SR-5-12 54.1 Acid Blue 62 54.37 SR-5-32 61.1 SR-5-37 77.8 SR-5-28 83.2 SR-5-34 84.0 SR-5-31 87.1 SR-5-6 90.9 SR-5-40 97.8 SR-5-44 145.4

(39) TABLE-US-00002 TABLE 2 The effect compounds on V.sub.1/2 of activation. Compound V.sub.1/2 (mV) in (Concentration) 100 nm Ca.sup.2+ SR-5-53 (10 M) 44.1 SR-5-63 (10 M) 113.1 SR-5-64 (10 M) 87.6 SR-5-65 (10 M) 116.2 SR-5-68 (10 M) 120.0 SR-5-69 (1 M) 101.6 SR-5-76 (10 M) 94.9 SR-5-88 (10 M) 98.8 SR-5-94 (10 M) 60.0 SR-5-72 (10 M) 103.8 SR-5-96 (10 M) 84.8 SR-5-97 (10 M) 78.5 SR-5-98 (10 M) 120.0 SR-5-99 (10 M) 83.4 SR-5-66 (10 M) 53.4

Example 9

(40) Using the single channel inside out patch clamp technique described in Example 1, voltage ramps were used to observe openings of BK channels, as described in Example 6.

(41) Referring to FIG. 8A shows the control trace (black) obtained using the ramp protocol as described above herein. To obtain these data, the average single channel currents were obtained from voltage ramp sweeps under control conditions and then in the presence of each concentration of the drug. The mean currents were corrected for driving force by dividing the current by the single channel amplitude at each potential. Consequently, activation curves similar to those shown in FIG. 7C can be obtained. When these data are fitted with a Boltzmann relationship, the voltage at which half of the channels in the patch are activated can be calculated (V.sub.1/2). These data show that the effects of SR-5-69 are dose-dependent and shift the activation of the channels towards more physiologically relevant membrane potentials. Application of SR-5-69 (30 nM) produced a very small shift in the activation V.sub.1/2 in this example. However, increasing the concentration of SR-5-69 to 100 nM, 300 nM and 1 M caused a concentration dependent shift in the activation curve in the hyperpolarising direction, such that at the highest concentration used (1 M), SR-5-69 shifted the V.sub.1/2 in excess of 100 mV, to approximately +5 mV. FIG. 8B-shows a summary graph in which the mean V.sub.1/2 was plotted against each concentration of the drug for n=4-6 patches containing BK channels. The error bars show the standard error of the mean for each data point.

(42) When these data were fitted with the Langmuir equation, the fit yielded a mean EC.sub.50 of 95 nM, consistent with the idea that this molecule was a potent and efficacious opener of BK channels.

Example 10: BK Openers Stimulate BK Channels Expressed in HEK Cells

(43) To demonstrate the effect of the compounds of the present invention on BK channels in cells other than bladder smooth muscle, the effects of SR-5-6 on BK channels expressed in Human Embryonic Kidney (HEK) cells were examined using the same technique and protocols described above. As FIG. 9A suggests, large currents could be recorded from HEK cells transfected to express the pore forming BK subunit. These currents were activated by depolarisation and by increasing the Ca.sup.2+ concentration at the cytosolic face of the channel ([Ca.sup.2+]i. As expected, the currents recorded from BK subunit expressing HEK cells were less sensitive to [Ca.sup.2+]i than the channels in native smooth muscle cells (FIG. 9A & FIG. 9C). Consequently, higher [Ca.sup.2+]l shifted the V.sub.1/2 of BK channels recorded from bladder smooth muscle more negatively than those recorded from HEK cells, expressing only the BK subunit. This difference in Ca.sup.2+ sensitivity between BK channels recorded in smooth muscle cells (FIG. 9C, black circles) and HEK cells expressing the BK subunit (FIG. 9C, white squares) has been well established and is due to the presence of the regulatory BK.sub.1 subunit in smooth muscle cells. Consequently we examined the effect of co-expressing the BK subunit with the BK.sub.1 subunit in HEK cells. As the grey circles in FIG. 9C show, the Ca.sup.2+ sensitivity of the HEK cells expressing BK.sub.1 subunits, was practically identical to that of the channels recorded from native bladder smooth muscle cells.

(44) FIG. 9B illustrates activation curves obtained from rabbit bladder smooth muscle cells in response to increases in Ca.sup.2+ concentration (100 nM, 1 M and 10 M Ca.sup.2+), wherein the native BK channels were more sensitive to Ca.sup.2+ compared to the BK alpha subunits shown in FIG. 9A.

(45) Having established these two cell lines, the effects of 10 M SR-5-6 on HEK cells expressing either BK subunits alone or co-expressing BK.sub.1 subunits were examined and its effects with those on native bladder smooth muscle cells were compared. FIG. 9D shows a summary barchart in which the mean shift in activation V.sub.1/2 (delta V.sub.1/2) caused by application of 10 M SR-5-6, was compared in native bladder smooth muscle cells (black bar), HEK cells expressing the BK.sub.1 subunits (hashed bar) and HEK cells expressing the BK subunit alone (white bar). The vertical lines represent the SEM and the numbers in parentheses represent the number of experiments per group. As the data suggests, SR-5-6 shifted the activation V.sub.1/2 of channels recorded from all three cell types, but it was less effective at shifting the V.sub.1/2 in cells expressing only the BK subunits (white bar). These data suggest that SR-5-6 can activate BK channels when they are expressed in HEK cells and is consistent with the idea that this molecule can activate BK channels, irrespective of what cell type they are present in.

(46) Conclusion

(47) Basilen blue had been characterized as a moderately potent BK channel activator when applied to the inside of the membrane of smooth muscle cell, shifting the activation voltage for the BK channel into the negative range (K. D. Cotton et al.).

(48) It has been demonstrated herein that a series of truncated derivatives of basilen blue can be synthesized; and the effects each thereof have been investigated on the inside out patches on rabbit smooth muscle cells. It has been shown that the commercially available dye, Acid Blue 25, synthesized from bromaminic acid, shifted the V.sub.1/2 by 56 mV. However, Bromaminic acid was also tested; and it strongly decreased the activity on BK channel, in fact, it showed an inhibitory effect.

(49) Screening of different analogues of anilinoanthraquinone, each having a different substituent on the benzene ring (D ring) of the Acid Blue 25 was also undertaken. In the present invention, the introduction of the hydrophobic substituent either in the ortho-position of the benzene ring (D ring), for example, ethyl [SR-5-96], isopropyl [SR-5-98] or in the meta-position, for example, trifluoromethyl [SR-5-6], isopropyl [SR-5-63], benzyl [SR-5-68] and tert-butyl [SR-5-76] produced BK channel agonists. However, the incorporation of hydrophobic substituent at para-position in D ring, for example, ethyl [SR-5-97], isopropy [SR-5-99], benzyl [SR-5-37] furnished BK channel openers.

(50) The replacement of the benzene ring (D ring) in SR-5-64 by indane led to similar potent compound like SR-5-6. Moreover, when the benzene ring was replaced by -tetralin [SR-5-69] or -naphthalene [SR-5-72], the dramatic increase in the activity was observed. SR-5-69 (1 M) and SR-5-72 (1 M) shifted the V.sub.1/2 by 110 mV and 93 mV respectively. However, when the benzene ring was substituted with -naphthalene ring [SR-5-66], the potency was dropped substantially. But, the replacement of the benzene ring with -tetralin ring [SR-5-65] provided a potent BK channel activator.

(51) On the other hand, introducing a polar substituent in the meta-position of the benzene ring, for example, SO.sub.3H [SR-5-18] decreases the activity dramatically.

(52) Without being bound by theory, the above examples suggest that a hydrophobic group particularly at the ortho- and meta-position of the phenyl ring is desirable for BK channel activation. However, the activity was not altered by substituting the sulfonate group in the C ring of SR-5-6 with its isostere carboxylate group [SR-5-88]. But, replacement of the sulfonate moiety by a H-atom at C-ring in Acid Blue 25 (1-amino-9,10-dioxo-4-(phenylamino)-9,10-dihydroanthracene-2-sulfonate) completely abolished the activity, indicating the importance of a negatively charged group at the C-2 position.

(53) These observations suggest that an appropriate hydrophobic region in the benzene ring (D ring) of anilinoanthraquinone and the sulfonate or carboxylate functionality of ring C are desirable for BK channel-opening activity (Scheme 1). The present SAR study based on V.sub.1/2 data showed that the Acid Blue 25 structure is a new scaffold for BK channel openers.

(54) ##STR00007##

(55) Accordingly, the present invention provides compounds useful as ion channel modulators that specifically and potently open BK channels in rabbit bladder smooth muscle cells and HEK cells transfected to allow production of BK channels. These compounds shift the activation of the BK channels in a hyperpolarising direction, towards physiological potentials.

(56) Materials & Methods

(57) General Details

(58) .sup.1H-NMR and .sup.13C-NMR data were collected at 300 K using a Bruker AMX 400 MHz NMR spectrometer at 400 MHz (.sup.1H), or 100 MHz (.sup.13C), respectively. Residual DMSO ( 2.50) was used as internal references for .sup.1H NMR spectra. The data reported as chemical shift (.sub.H ppm), relative integral, multiplicity (s=singlet, br=broad, d=doublet, t=triplet, q=quartet, m=multiplet), coupling constant (J Hz), and assignment. Solvent peak for DMSO ( 39.7) was used as internal reference for .sup.13C NMR spectra. High Resolution Mass Spectra (HRMS) was recorded by the School of Chemistry and Chemical Biology, University College Dublin using a Micromass/Waters LCT instrument. Microwave reactions were carried out using a CEM Focused Microwave Synthesis type Discover apparatus. Reactions were monitored by thin layer chromatography (TLC), which was performed on aluminum sheets pre-coated with Silica gel 60 F.sub.254 (Merck). Column-chromatography separations were performed using Merck Kieselgel 60 (0.040-0.063 mm). The columns were usually eluted with various combinations of ethyl acetate-methanol mixtures. All reagents were obtained from commercial sources and used as received.

(59) Synthetic Procedure of Ullmann Coupling Reaction

(60) ##STR00008##

(61) Procedure A:

(62) Bromaminic acid sodium salt (0.2 g, 0.495 mmol), the suitable aniline derivative (0.99 mmol), copper powder (31 mg, 0.495 mmol) and buffer solution of Na.sub.2HPO.sub.4 (0.2 M, 3 mL) and NaH.sub.2PO.sub.4 (0.12 M, 5 mL) was mixed in a 35 mL microwave reaction vial. Reaction vial was capped and irradiated with microwave reactor (70 W-80 W) for 5-20 minutes at 100-120 C. Reaction mixture was cooled to room temperature and filtered. Filtrate was extracted with ethyl acetate (40 mL3) and washed with water. Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was loaded on silica gel column and eluted with 2%-4% methanol in ethyl acetate mixture to furnish the corresponding pure anilinoanthraquinone derivative as a blue solid.

(63) TABLE-US-00003 TABLE 3 Isolated yields of material judged homogeneous by TLC and NMR. Compound Yield (%).sup.a Acid Blue 25 SR-5-6 75 SR-5-11 43 SR-5-12 83 SR-5-13 30 SR-5-14 36 SR-5-15 51 SR-5-18 25 SR-5-23 41 SR-5-26 82 SR-5-28 65 SR-5-31 79 SR-5-34 76 SR-5-37 68 SR-5-40 79 SR-5-44 75 SR-5-46 62 SR-5-47 44 SR-5-48 62 SR-5-53 65 SR-5-61 70 SR-5-63 75 SR-5-64 66 SR-5-65 68 SR-5-66 48 SR-5-68 72 SR-5-69 76 SR-5-76 68 SR-5-88 55 SR-5-91 47 SR-5-94 53 SR-5-72 67 SR-5-96 64 SR-5-97 78 SR-5-98 69 SR-5-99 75 .sup.aYields reported here are isolated yields of material judged homogeneous by TLC and NMR.

(64) Procedure B:

(65) Bromaminic acid sodium salt (0.2 g, 0.495 mmol), sodium carbonate (42 mg, 0.396 mmol), copper sulfate (16 mg, 0.1 mmol) and the suitable aniline or amine derivative were mixed in 5 mL water. Reaction mixture was stirred at 65 C. for 1 h and then refluxed gently at 105 C. for 5 h. The reaction mixture was then cooled to room temperature and filtered. Filtrate was extracted with ethyl acetate (40 mL3) and washed with water. Combined organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was loaded on silica gel column and eluted with 3% methanol in ethyl acetate to obtain the corresponding anilinoanthraquinone derivative as a blue powder.

(66) ##STR00009##

(67) TABLE-US-00004 TABLE 4 isolated yields of material judged homogeneous by TLC and NMR n Compound Yield (%).sup.a 1 SR-5-10 39 2 SR-5-20 40 3 SR-5-8 47 4 Acid blue 62 5 SR-5-32 .sup.49.sup.b .sup.aYields reported here are isolated yields of material judged homogeneous by TLC and NMR. .sup.bReaction was performed following the procedure B.

(68) SR-5-6: sodium 1-amino-9,10-dioxo-4-(3-(trifluoromethyl)phenylamino)-9,10-dihydroanthracene-2-sulfonate.

(69) ##STR00010##

(70) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(71) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.80 (s, 1H), 10.02 (brs, 1H), 8.24 (dd, J=7.2, 13.2 Hz, 2H), 8.04 (s, 1H), 7.85 (m, 2H), 7.65-7.56 (m, 3H), 7.48 (d, J=7.6 Hz, 1H).

(72) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 183.2, 182.1, 144.6, 142.2 (2C), 140.8, 138.8, 134.0, 133.4, 133.3, 132.9, 130.7 (20), 126.0, 125.9, 125.6, 122.8, 119.9, 118.4, 113.1, 109.6.

(73) HRMS (ES): m/z for C.sub.21H.sub.12N.sub.2O.sub.5F.sub.3S [MNa.sup.+], calcd. 461.0419. found 461.0440.

(74) SR-5-8: sodium 1-amino-4-(cyclopentylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(75) ##STR00011##

(76) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(77) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.86 (d, J=6.8 Hz, 1H), 10.13 (brs, 1H), 8.27-8.23 (m, 2H), 7.82 (s, 1H), 7.81-7.79 (m, 2H), 4.14-4.10 (m, 1H), 2.14-2.07 (m, 2H), 1.80-1.68 (m, 6H).

(78) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 181.5, 180.7, 144.7, 143.4, 143.0, 133.9 (2C), 132.5, 132.4, 125.8, 125.7, 121.6, 109.0, 108.7, 53.3, 33.6 (2C), 23.6 (2C).

(79) HRMS (ES): m/z for C.sub.16H.sub.18N.sub.2O.sub.6NaS [M+H.sup.+], calcd. 409.0834. found 409.0850.

(80) SR-5-10: sodium 1-amino-4-(cyclopropylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(81) ##STR00012##

(82) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(83) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.51 (d, J=2.4 Hz, 1H), 10.09 (brs, 1H), 8.27-8.24 (m, 1H), 8.22-8.20 (m, 1H), 8.17 (s, 1H), 7.82-7.80 (m, 2H), 2.80-2.67 (m, 1H), 0.94-0.90 (m, 2H), 0.65-0.62 (m, 2H).

(84) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 181.7, 181.4, 145.8, 143.4, 143.2, 133.9, 133.7, 132.6 (20), 125.9, 125.7, 122.1, 109.2, 109.1, 24.1, 7.63 (2C).

(85) HRMS (ES): m/z for C.sub.17H.sub.13N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 403.0341. found 403.0359.

(86) SR-5-11: sodium 1-amino-4-(2,6-difluorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(87) ##STR00013##

(88) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(89) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.52 (s, 1H), 9.98 (brs, 1H), 8.30-8.27 (m, 2H), 7.89-7.86 (m, 2H), 7.43-7.30 (m, 4H).

(90) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 183.5, 182.1, 158.91-156.45 (d, J=246.0 Hz), 158.87-156.40 (d, J=247.0 Hz), 144.1, 142.7, 140.6, 134.1, 133.5, 133.3, 132.9, 127.4, 126.1, 126.0, 121.8, 115.91-115.75 (d, J=16.0 Hz), 112.6, 112.4, 111.5, 109.2.

(91) HRMS (ES): m/z for C.sub.20H.sub.11N.sub.2O.sub.6F.sub.2S [MNa.sup.+], calcd. 429.0357. found 429.0348.

(92) SR-5-12: sodium 1-amino-4-(2-methoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(93) ##STR00014##

(94) R.sub.f: 0.3 (20% methanol in ethyl acetate).

(95) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.94 (s, 1H), 10.12 (brs, 1H), 8.30-8.27 (m, 2H), 7.97 (s, 1H), 7.88-7.83 (m, 2H), 7.30 (d, J=7.2 Hz, 1H), 7.20-7.17 (m, 2H), 7.05-7.01 (m, 1H), 3.88 (s, 3H).

(96) SR-5-13: sodium 4-(3,5-bis(trifluoromethyl)phenylamino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(97) ##STR00015##

(98) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(99) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.39 (s, 1H), 9.87 (brs, 1H), 8.21-8.14 (m, 2H), 7.98 (s, 1H), 7.85-7.78 (m, 4H), 7.62 (s, 1H).

(100) HRMS (ES): m/z for C.sub.22H.sub.11N.sub.2O.sub.6F.sub.6S [MNa.sup.+], calcd. 529.0293. found 529.0292.

(101) SR-5-14: sodium 1-amino-4-(3,5-difluorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(102) ##STR00016##

(103) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(104) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.55 (s, 1H), 9.98 (brs, 1H), 8.28-8.22 (m, 2H), 8.08 (s, 1H), 7.91-7.84 (m, 2H), 7.49 (brs, 1H), 7.00 (dd, J=2.4, 9.4 Hz, 2H), 6.96-6.90 (m, 1H).

(105) HRMS (ES): m/z for C.sub.20H.sub.11N.sub.2O.sub.6F.sub.2S [MNa.sup.+], calcd. 429.0357. found 429.0370.

(106) SR-5-15: sodium 1-amino-4-(3-fluorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(107) ##STR00017##

(108) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(109) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.83 (s, 1H), 10.05 (brs, 1H), 8.28-8.23 (m, 2H), 8.06 (s, 1H), 7.89-7.82 (m, 2H), 7.49-7.43 (m, 1H), 7.17-7.11 (m, 2H), 6.99 (dt, J=2.4, 8.2 Hz, 1H).

(110) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 183.0, 182.0, 164.05-161.63 (d, J=242.0 Hz), 144.5, 142.3, 141.6, 139.2, 134.1, 133.4, 133.3, 132.9, 131.2, 126.1, 126.0, 123.0, 118.1, 112.6, 110.52-110.31 (d, J=21.0 Hz), 109.4, 109.0.

(111) HRMS (ES): m/z for C.sub.20H.sub.12N.sub.2O.sub.6FS [MNa.sup.+], calcd. 411.0451. found 411.0464.

(112) SR-5-18: sodium 1-amino-9,10-dioxo-4-(3-sulfophenylamino)-9,10-dihydroanthracene-2-sulfonate.

(113) ##STR00018##

(114) R.sub.f: 0.2 (25% methanol in ethyl acetate).

(115) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.05 (s, 1H), 10.11 (brs, 1H), 8.30-8.28 (m, 2H), 8.00 (s, 1H), 7.88-7.85 (m, 2H), 7.46-7.39 (m, 3H), 7.25 (td, J=2.4, 7.1 Hz, 1H).

(116) SR-5-20: sodium 1-amino-4-(cyclobutylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(117) ##STR00019##

(118) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(119) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.74 (d, J=6.0 Hz, 1H), 10.10 (brs, 1H), 8.27-8.23 (m, 2H), 7.83-7.80 (m, 2H), 7.64 (s, 1H), 7.41 (brs, 1H), 4.22 (m, 1H), 2.49-2.46 (m, 2H), 2.06-1.96 (m, 2H), 1.90-1.83 (m, 2H).

(120) HRMS (ES): m/z for C.sub.18H.sub.16N.sub.2O.sub.6S [MNa.sup.+], calcd. 371.0702. found 371.0692.

(121) SR-5-23: sodium 1-amino-4-(3-fluoro-5-(trifluoromethyl)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(122) ##STR00020##

(123) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(124) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.51 (s, 1H), 9.96 (brs, 1H), 8.25 (dd, J=3.2, 15.6 Hz, 2H), 8.06 (s, 1H), 7.95-7.84 (m, 2H), 7.47 (s, 1H), 7.42 (d, J=10.8 Hz, 1H), 7.32 (d, J=8.0 Hz, 1H).

(125) HRMS (ES): m/z for C.sub.21H.sub.1N.sub.2O.sub.5F.sub.4S [MNa.sup.+], calcd. 479.0325. found 479.0344.

(126) SR-5-26: sodium 4-(m-toluidino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(127) ##STR00021##

(128) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(129) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.05 (s, 1H), 10.12 (brs, 1H), 8.27 (dt, J=2.4, 6.8 Hz, 2H), 8.04 (s, 1H), 7.87-7.82 (m, 2H), 7.54 (brs, 1H), 7.34 (t, J=7.6 Hz, 1H), 7.11 (s, 1H), 7.07 (dd, J=7.6, 24.4 Hz, 2H), 2.35 (s, 3H).

(130) SR-5-28: sodium 1-amino-4-(4-fluoro-3-(trifluoromethyl)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(131) ##STR00022##

(132) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(133) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.73 (s, 1H), 9.99 (brs, 1H), 8.21 (dd, J=7.2, 15.2 Hz, 2H), 7.91 (s, 1H), 7.86-7.80 (m, 2H), 7.67-7.63 (m, 2H), 7.57 (t, J=10.0 Hz, 1H).

(134) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 183.0, 182.0, 156.37-153.87 (d, J=250.0 Hz), 144.4, 142.3, 139.5, 136.7, 136.6, 134.0, 133.3 (20), 132.9, 129.0, 126.0, 125.9, 122.4, 121.3, 121.2, 118.50-118.28 (d, J=22.0 Hz), 112.7, 109.5.

(135) HRMS (ES): m/z for C.sub.21H.sub.11N.sub.2O.sub.5F.sub.4Na.sub.2S [M+Na.sup.+], calcd. 525.0120. found 525.0107.

(136) SR-5-31: sodium 1-amino-4-(3-ethylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(137) ##STR00023##

(138) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(139) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.07 (s, 1H), 10.13 (brs, 1H), 8.31-8.23 (m, 2H), 8.08 (s, 1H), 7.87-7.81 (m, 2H), 7.36 (t, J=7.6 Hz, 1H), 7.14 (s, 1H), 7.08 (dd, J=8.0, 20.2 Hz, 2H), 2.64 (ABq, J=7.6 Hz, 2H), 1.22 (t, J=7.6 Hz, 3H).

(140) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 182.3, 181.8, 145.6, 144.3, 142.6, 140.9, 139.1, 134.1, 133.5, 133.1, 132.7, 129.5, 126.0, 125.9, 124.0, 122.8, 122.5, 120.3, 111.3, 109.1, 28.1, 15.5.

(141) HRMS (ES): m/z for C.sub.22H.sub.17N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 467.0654. found 467.0666.

(142) SR-5-32: sodium 1-amino-4-(cycloheptylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(143) ##STR00024##

(144) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(145) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 10.95 (d, J=7.6 Hz, 1H), 10.18 (brs, 1H), 8.27-8.24 (m, 2H), 7.81-7.79 (m, 2H), 7.75 (s, 1H), 3.91-3.86 (m, 1H), 2.02-1.96 (m, 2H), 1.70-1.59 (m, 10H).

(146) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 181.4, 180.6, 144.2, 143.5, 143.0, 134.0, 133.9, 132.4, 132.3, 125.8, 125.7, 121.4, 109.1, 108.7, 52.1, 34.7 (2C), 27.6 (2C), 23.5 (2C).

(147) HRMS (ES): m/z for C.sub.21H.sub.21N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 459.0967. found 459.0970.

(148) SR-5-34: sodium 1-amino-9,10-dioxo-4-(3-(trifluoromethoxy)phenylamino)-9,10-dihydroanthracene-2-sulfonate.

(149) ##STR00025##

(150) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(151) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.78 (s, 1H), 10.02 (brs, 1H), 8.27-8.22 (m, 2H), 8.07 (s, 1H), 7.89-7.82 (m, 2H), 7.54 (t, J=8.0 Hz, 1H), 7.31-7.28 (m, 2H), 7.12 (d, J=8.4 Hz, 1H).

(152) .sup.13C NMR (100 MHz, DMSO-d.sub.6): 183.2, 182.1, 149.3, 144.6, 142.2, 141.7, 138.7, 134.0, 133.4, 133.3, 132.9, 131.2 (20), 126.0 (20), 123.0, 120.6, 115.6, 114.3, 113.1, 109.6.

(153) HRMS (ES): m/z for C.sub.21H.sub.12N.sub.2O.sub.6F.sub.3Na.sub.2S [M+Na.sup.+], calcd. 523.0164. found 523.0179.

(154) SR-5-37: sodium 1-amino-4-(4-benzylphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(155) ##STR00026##

(156) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(157) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.07 (s, 1H), 10.11 (brs, 1H), 8.29-8.26 (m, 2H), 8.00 (s, 1H), 7.88-7.81 (m, 2H), 7.34-7.20 (m, 9H), 3.98 (s, 2H).

(158) HRMS (ES): m/z for C.sub.27H.sub.19N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 529.0810. found 529.0812.

(159) SR-5-40: sodium 1-amino-4-(4-chloro-3-(trifluoromethyl)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(160) ##STR00027##

(161) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(162) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.61 (s, 1H), 9.96 (brs, 1H), 8.24-8.17 (m, 2H), 8.00 (s, 1H), 7.87-7.80 (m, 2H), 7.72 (s, 1H), 7.71 (d, J=12.0 Hz, 1H), 7.54 (dd, J=2.8, 8.8 Hz, 1H).

(163) HRMS (ES): m/z for C.sub.21H.sub.11N.sub.2O.sub.6F.sub.3Na.sub.2SCl [M+Na.sup.+], calcd. 540.9825. found 540.9824.

(164) SR-5-44: sodium 1-amino-4-(4-methyl-3-(trifluoromethyl)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(165) ##STR00028##

(166) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(167) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.85 (s, 1H), 10.05 (brs, 1H), 8.25-8.20 (m, 2H), 7.97 (s, 1H), 7.86-7.80 (m, 2H), 7.55 (s, 1H), 7.49-7.44 (m, 2H), 2.45 (s, 3H).

(168) HRMS (ES): m/z for C.sub.22H.sub.14N.sub.2O.sub.6F.sub.3Na.sub.2S [M+Na.sup.+], calcd. 521.0371. found 521.0382.

(169) SR-5-46: sodium 1-amino-4-(3-chlorophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(170) ##STR00029##

(171) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(172) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.79 (s, 1H), 10.04 (brs, 1H), 8.28-8.23 (m, 2H), 8.03 (s, 1H), 7.89-7.83 (m, 2H), 7.45 (t, J=8.0 Hz, 1H), 7.36 (t, J=2.0 Hz, 1H), 7.26-7.20 (m, 2H).

(173) SR-5-47: sodium 1-amino-4-(3-cyanophenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate.

(174) ##STR00030##

(175) R.sub.f: 0.3 (20% methanol in ethyl acetate).

(176) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.72 (s, 1H), 10.01 (brs, 1H), 8.28-8.23 (m, 2H), 7.99 (s, 1H), 7.90-7.83 (m, 2H), 7.74 (s, 1H), 7.62-7.56 (m, 3H).

(177) HRMS (ES): m/z for C.sub.21H.sub.12N.sub.3O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 464.0293. found 464.0280.

(178) SR-5-48: sodium 1-amino-4-((3-methoxy-5-(trifluoromethyl)phenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(179) ##STR00031##

(180) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(181) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.74 (s, 1H), 10.01 (brs, 1H), 8.29-8.24 (m, 2H), 8.09 (s, 1H), 7.91-7.84 (m, 2H), 7.50 (brs, 1H), 7.18 (s, 1H), 7.14 (s, 1H), 6.98 (s, 1H), 3.86 (s, 3H).

(182) HRMS (ES): m/z for C22H14N2O6F3Na2S [M+Na+], calcd. 537.0320. found 537.0342.

(183) SR-5-53: sodium 1-amino-4-((3-hydroxyphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(184) ##STR00032##

(185) R.sub.f: 0.3 (20% methanol in ethyl acetate).

(186) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.99 (s, 1H), 10.12 (brs, 1H), 9.68 (s, 1H), 8.28-8.24 (m, 2H), 8.08 (s, 1H), 7.87-7.81 (m, 2H), 7.52 (brs, 1H), 7.24 (t, J=8.0 Hz, 1H), 6.72-6.62 (m, 3H).

(187) SR-5-61: sodium 1-amino-4-(2-methoxy-5-(trifluoromethyl)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(188) ##STR00033##

(189) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(190) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.82 (s, 1H), 10.04 (brs, 1H), 8.28-8.25 (m, 2H), 7.98 (s, 1H), 7.89-7.84 (m, 2H), 7.60 (d, J=1.6 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.34 (d, J=8.8 Hz, 1H), 3.97 (s, 3H).

(191) HRMS (ES): m/z for C.sub.22H.sub.14N.sub.2O.sub.6F.sub.3Na.sub.2S [M+Na.sup.+], calcd. 537.0320. found 537.0344.

(192) SR-5-63: sodium 1-amino-4-((3-isopropylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(193) ##STR00034##

(194) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(195) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.10 (s, 1H), 10.13 (brs, 1H), 8.31-8.25 (m, 2H), 8.10 (s, 1H), 7.88-7.82 (m, 2H), 7.54 (brs, 1H), 7.37 (t, J=8.0 Hz, 1H), 7.18 (s, 1H), 7.10 (t, J=7.6 Hz, 2H), 2.96-2.89 (m, 1H), 1.25 (d, J=6.8 Hz, 6H).

(196) HRMS (ES): m/z for C.sub.23H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 481.0810. found 481.0816.

(197) SR-5-64: sodium 1-amino-4-((2,3-dihydro-1H-inden-5-yl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(198) ##STR00035##

(199) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(200) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.10 (s, 1H), 10.15 (brs, 1H), 8.30-8.26 (m, 2H), 7.96 (s, 1H), 7.88-7.81 (m, 2H), 7.50 (brs, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.15 (s, 1H), 7.04 (dd, J=2.0, 8.0 Hz, 1H), 2.92-2.88 (m, 4H), 2.11-2.03 (m, 2H).

(201) HRMS (ES): m/z for C.sub.23H.sub.17N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 479.0654. found 479.0677.

(202) SR-5-65: sodium 1-amino-9,10-dioxo-4-((5,6,7,8-tetrahydronaphthalen-1-yl)amino)-9,10-dihydroanthracene-2-sulfonate

(203) ##STR00036##

(204) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(205) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.03 (s, 1H), 10.15 (brs, 1H), 8.31-8.28 (m, 2H), 7.88-7.83 (m, 2H), 7.83 (s, 1H), 7.50 (brs, 1H), 7.19 (t, J=7.6 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 7.0 (d, J=7.2 Hz, 1H), 2.80 (t, J=5.6 Hz, 2H), 2.68 (t, J=5.6 Hz, 2H), 1.81-1.74 (m, 4H).

(206) HRMS (ES): m/z for C.sub.24H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 493.0810. found 493.0797.

(207) SR-5-68: sodium 1-amino-4-((3-benzylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(208) ##STR00037##

(209) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(210) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.05 (s, 1H), 10.12 (brs, 1H), 8.30-8.26 (m, 2H), 8.07 (s, 1H), 7.87-7.84 (m, 2H), 7.51 (brs, 1H), 7.36 (t, J=7.6 Hz, 1H), 7.32 (s, 2H), 7.31 (s, 2H), 7.21-7.17 (m, 2H), 7.12 (d, J=8.0 Hz, 1H), 7.05 (d, J=7.6 Hz, 1H), 3.98 (s, 2H).

(211) HRMS (ES): m/z for C.sub.27H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 529.0810. found 529.0834.

(212) SR-5-69: sodium 1-amino-9,10-dioxo-4-((5,6,7,8-tetrahydronaphthalen-2-yl)amino)-9,10-dihydroanthracene-2-sulfonate

(213) ##STR00038##

(214) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(215) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.07 (s, 1H), 10.15 (brs, 1H), 8.30-8.27 (m, 2H), 7.97 (s, 1H), 7.88-7.82 (m, 2H), 7.49 (brs, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.02-6.99 (m, 2H), 2.75 (brs, 4H), 1.77 (t, J=2.8 Hz, 4H).

(216) HRMS (ES): m/z for C.sub.24H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 493.0810. found 493.0821.

(217) SR-5-76: sodium 1-amino-4-((3-(tert-butyl)phenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(218) ##STR00039##

(219) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(220) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.12 (s, 1H), 10.14 (brs, 1H), 8.30-8.27 (m, 2H), 8.11 (s, 1H), 7.88-7.83 (m, 2H), 7.52 (brs, 1H), 7.38 (t, J=8.0 Hz, 1H), 7.32 (t, J=1.6 Hz, 1H), 7.24 (d, J=7.6 Hz, 1H), 7.10 (dd, J=1.6, 7.6 Hz, 1H), 1.32 (s, 9H).

(221) HRMS (ES): m/z for C.sub.24H.sub.21N.sub.2O.sub.5Na.sub.2S [M+Na.sup.+], calcd. 495.0967. found 495.0944.

(222) SR-5-88: 1-amino-9,10-dioxo-4-((3-(trifluoromethyl)phenyl)amino)-9,10-dihydroanthracene-2-carboxylic acid

(223) ##STR00040##

(224) R.sub.f: 0.5 (20% methanol in ethyl acetate).

(225) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 11.76 (s, 1H), 10.22 (brs, 2H), 8.33 (s, 1H), 8.28-8.22 (m, 2H), 7.88-7.81 (m, 2H), 7.64-7.59 (m, 3H), 7.44 (d, J=7.2 Hz, 1H).

(226) HRMS (ES): m/z for C.sub.22H.sub.14N.sub.2O.sub.4F.sub.3 [M+H.sup.+], calcd. 427.0906. found 427.0917.

(227) SR-5-91: sodium 4-((9H-fluoren-2-yl)amino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(228) ##STR00041##

(229) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(230) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.19 (s, 1H), 10.15 (brs, 1H), 8.32-8.29 (m, 2H), 8.08 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.90 (d, J=7.2 Hz, 1H), 7.88-7.86 (m, 2H), 7.60 (d, J=7.6 Hz, 1H), 7.52 (s, 1H), 7.40 (t, J=7.2 Hz, 1H), 7.34-7.29 (m, 2H), 3.97 (s, 2H).

(231) HRMS (ES): m/z for C.sub.27H.sub.17N.sub.2O.sub.6S [MNa.sup.+], calcd. 481.0858. found 481.0863.

(232) SR-5-94: sodium 4-([1,1-biphenyl]-3-ylamino)-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(233) ##STR00042##

(234) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(235) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.11 (s, 1H), 10.12 (brs, 1H), 8.31-8.28 (m, 2H), 8.18 (s, 1H), 7.90-7.84 (m, 2H), 7.75-7.73 (m, 2H), 7.58-7.47 (m, 5H), 7.42-7.38 (m, 1H), 7.29 (d, J=8.4 Hz, 1H).

(236) HRMS (ES): m/z for C.sub.26H.sub.17N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 515.0654. found 515.0648.

(237) SR-5-72: sodium 1-amino-4-((naphthalen-2-yl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(238) ##STR00043##

(239) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(240) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.18 (s, 1H), 10.13 (brs, 1H), 8.32-8.29 (m, 2H), 8.11 (s, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.91-7.85 (m, 3H), 7.78 (d, J=2.0 Hz, 1H), 7.55-7.46 (m, 3H).

(241) SR-5-96: sodium 1-amino-4-((2-ethylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(242) ##STR00044##

(243) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(244) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.13 (s, 1H), 10.16 (brs, 1H), 8.31-8.29 (m, 2H), 7.88-7.82 (m, 2H), 7.78 (s, 1H), 7.41 (d, J=7.6 Hz, 1H), 7.34-7.23 (m, 3H), 2.67 (ABq, J=7.2 Hz, 2H), 1.22 (t, J=7.2 Hz, 3H).

(245) HRMS (ES): m/z for C.sub.22H.sub.17N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 467.0654. found 467.0613.

(246) SR-5-97: sodium 1-amino-4-((4-ethylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(247) ##STR00045##

(248) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(249) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.09 (s, 1H), 10.17 (brs, 1H), 8.30-8.26 (m, 2H), 8.00 (s, 1H), 7.88-7.82 (m, 2H), 7.49 (brs, 1H), 7.30 (d, J=8.4 Hz, 2H), 7.21 (d, J=8.8 Hz, 2H), 2.65 (ABq, J=8.0 Hz, 2H), 1.23 (t, J=8.0 Hz, 3H).

(250) HRMS (ES): m/z for C.sub.22H.sub.17N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 467.0654. found 467.0662.

(251) SR-5-98: sodium 1-amino-4((2-isopropylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(252) ##STR00046##

(253) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(254) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.16 (s, 1H), 10.13 (brs, 1H), 8.31-8.29 (m, 2H), 7.89-7.83 (m, 2H), 7.73 (s, 1H), 7.49-7.46 (m, 1H), 7.32-7.24 (m, 3H), 3.23-3.16 (m, 1H), 1.25 (d, J=7.2 Hz, 6H).

(255) HRMS (ES): m/z for C.sub.23H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 481.0810. found 481.0798.

(256) SR-5-99: sodium 1-amino-4-((4-isopropylphenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(257) ##STR00047##

(258) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(259) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.11 (s, 1H), 10.15 (brs, 1H), 8.30-8.27 (m, 2H), 8.02 (s, 1H), 7.88-7.83 (m, 2H), 7.51 (brs, 1H), 7.34 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.8 Hz, 2H), 2.97-2.91 (m, 1H), 1.25 (d, J=6.4 Hz, 6H).

(260) HRMS (ES): m/z for C.sub.23H.sub.16N.sub.2O.sub.6Na.sub.2S [M+Na.sup.+], calcd. 481.0810. found 481.0827.

(261) SR-5-66: sodium 1-amino-4-((naphthalen-1-ylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

(262) ##STR00048##

(263) R.sub.f: 0.4 (20% methanol in ethyl acetate).

(264) .sup.1H NMR (400 MHz, DMSO-d.sub.6): 12.54 (s, 1H), 10.17 (brs, 1H), 8.35-8.31 (m, 2H), 8.10-8.04 (m, 2H), 7.91-7.86 (m, 3H), 7.82 (s, 1H), 7.65-7.60 (m, 3H), 7.52 (d, J=6.8 Hz, 1H).