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Letters in Drug Design & Discovery, 2004, 1, 78-83

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Two Identical Twin Nitrogen Mustard Agents that Express Rapid Alkylation Activity at Physiological pH 7.4 and 37o C Ronald Bartzatt* and Laura Donigan University of Nebraska, College of Arts & Sciences, Chemistry Department, Medicinal Chemistry Department, Durham Science Center, 6001 Dodge Street, Omaha, Nebraska 68182 USA Received June 11, 2003: Accepted July 7, 2003

Abstract: Two nitrogen mustard (N-mustard ) agents were synthesized utilizing ethylene diamine and hexane diamine as the parent compounds. These N-mustard agents were solids at 25o C and stable while stored dry at -10o C. The two N-mustards assumed the configuration of identical twin drugs which when placed in aqueous solution were highly reactive. Both N-mustards were soluble in aqueous NaHCO3 buffer and expressed alkylation activity directed towards a nucleophilic primary amine target (4-chloroaniline) at blood pH 7.4 and 37o C. Utilizing the fluorescent probe fluorescamine, which is highly specific for primary amines, the quantity of unreacted nucleophilic 4-chloroaniline remaining after a known time period was determined. This enabled the calculation of rate constants and the determination of rate equation for alkylation to be first-order for N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine and zero-order for N,N,N’,N’tetrakis(2-chloroethyl)hexane-1,6-diamine. Molecular property descriptors such as Log P, parachor, molar volume, molar refractivity, dipole, molecular volume & area, and polar surface area were calculated for comparison. Zero violations of the Rule of 5 indicates these two mustard agents will have good bioavailability and good bioactivity.

Keywords: nitrogen mustard, identical twin drug, alkylation. INTRODUCTION Alkylating agents are the oldest class of anticancer drugs and are utilized to treat leukemia and solid tumors. Nitrogen mustard(N-mustards) are cytotoxic, small molecules, highly reactive, and bond covalently to electron rich nucleophilic sites of large or small biomolecules. The most frequent site of attachment on DNA is N-7 site of guanine but adducts are formed at the O-6 and N–1 sites of guanine [1]. Other sites include the N–7, N-3, and N–1of adenine; N-3 of cytosine; and O-4 of thymidine. Mechlorethamine, melphalan, and chlorambucil are N-mustards utilized clinically [2]. Alkylating agents are proliferative specific and cell cycle nonspecific [2]. Three major types of alkylating agents are N-mustards, ethylene imines (aziridines), and methane sulfonic acid esters [2]. Reaction mechanisms are unimolecular nucleophilic substitution (SN1) or bimolecular nucleophilic substitution reaction (SN2) [2]. Aromatic Nmustards react via SN1 and form a carbonium ion while aliphatic N-mustards form an ethylene immonium ion (highly strained three member ring) which converts to a reactive carbonium ion [2]. The carbonium ion is highly reactive and reacts with water.

carboxyl groups (-C(O)OH), mercapto (-SH), amino(-NH2), phosphate (-PO3H2), and hydroxyl groups (-OH) [2]. Crosslinking bases of DNA halts replication or transcription [3]. N-mustards alkylate amino acids and proteins, and induce an enol tautomer of guanine (normally keto) which base pairs with thymine causing miscoding [3]. Cytotoxic compounds are shown to be highly effective [4] and are useful in treatment of severe dermatology diseases, applied after transplantation [5], and treat small cell lung cancer [6]. Using antibody linked liposomes as carriers increases specificity of cytotoxic activity [7,8] and when used with nucleoside analogs they increase patient survival times and observed remission [9]. MATERIALS AND METHODS Reagents and Supplies All reagents were obtained from Aldrich Chemical Company, P.O.Box 355, Milwaukee, WI 53201. Spectronic 21D spectrophotometer (1 cm glass cuvettes) and Galaxy Series FTIR were utilized for visible and infrared spectra, respectively.

N-mustards are bifunctional alkylators which can crosslink bases of the DNA helix [1]. N-mustards react with

Computer Software

*Address correspondence to this author at the University of Nebraska, College of Arts & Sciences, Chemistry Department, Medicinal Chemistry Department, Durham Science Center, 6001 Dodge Street, Omaha, Nebraska 68182 USA; Tel: 402 554-3612; Fax: 402 554-3888; E-mail: [email protected]

Molecular modeling was accomplished with SPARTAN modeling software (Wavefunction, Irvine, California USA) and ChemSketch (90 Adelaide Street West, Toronto M5H 3V9, Canada). Values of partition coefficients were determined by various software: Interactive Analysis (6 Reuben Duren Way, Bedford MA 01730); Actelion (Gewerbestrasse 16, 4123 Allschwil, Switzerland); Syracuse

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© 2004 Bentham Science Publishers Ltd.

Two Identical Twin Nitrogen Mustard Agents

Letters in Drug Design & Discovery, 2004, Vol. 1, No. 1

Research Corp. (999 18th Street, Suite 1975, Denver, CO 80202); DRAGON (Milano Chemometrics and QSAR Research Group, Dept. Of Environmental Sciences, University of Milano-Bicocca, 1-20126 Milano, Italy). Remaining molecular descriptors were determined by ChemSketch (see above) and Molinspiration (Liscie udolie 2, SK-84104 Bratislava, Slovak Republic). Ionized fragmentation pattern calculated by MASS SPEC (Trinity, Plymouth NH 03264). Synthesis of Nitrogen Mustard Agents The diamine reactant (ethylene diamine: 2.25 g, 0.0374 moles or1,6-hexane diamine: 2.50 g, 0.0215 moles)was dissolved into 30 mL of acetonitrile previously dried over molecular sieves. Add 33 mL of 1,2-dichloroethane and up to 20 mL of triethylamine (a proton sink). Reflux mildly for one to two hours. Distill out excess 1,2-dichloroethane adding back dried acetonitrile to maintain liquid volume. Reduce volume to < 15 mL, precipitate N-mustard agents over ice/water or overnight at -10o C. Recrystallizing fromCH 3 CN increases purity but reduces yield. Keep Nmustard agents dry and stored in a desicator at -10o C. N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine has composition C(38.7%), H(6.5%), Cl(45.73%), N(9.03%), m.p. of ~106o C, and > 70% yield (> 8 g grams product from 2.25 g of parent). N,N,N’,N’-tetrakis(2chloroethyl)hexane-1,6-diamine has composition C(45.9%), MOLECULAR STRUCTURES OF NITROGEN MUSTARD AGENTS

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H(7.71%), Cl(38.7%), N(7.65%), m.p. of ~142o C, yields of > 5 g from 2.50 grams of parent. Check purity by spotting products and parent compounds on silica T.L.C. plates, resolving with CH2 Cl 2 , and observing spots after exposure to fuming iodine vapors. Lack ofparent compound in product lanes indicate high purity. N-mustard structures are shown in Figure 1 with carbons numbered for C-13 assignments (ppm): N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine: 1) 55.9; 2) 52.3; 3) 52.3; 4) 44.7; 5) 55.9; 6) 44.7; 7) 55.9; 8) 44.7; 9) 55.9; 10) 44.7. Molecular fragmentation after ionization (m/e): C10H20N2Cl4 (310.04), C 10H 20N 2Cl3 (274.1), C8H 16N 2Cl3 (246.04), C6H 12N 2Cl3 (186.013). C-13 for N,N,N’,N’-tetrakis(2chloroethyl)hexane-1,6-diamine (ppm): 1) 52.6; 2) 30.0; 3) 27.7; 4) 27.7; 5) 30.0; 6) 56.2; 7) 52.6; 8) 44.7; 9) 52.6; 10) 44.5; 11) 44.7; 12) 56.2; 13) 44.7; 14) 56.2. Molecular fragmentation after ionization (m/e): parent C14H 28N 2Cl4 (366.10), C14 H 28 N 2 Cl 3 (331.1), C10 H 24 N 2 Cl 3 (303.1), C10H20N2Cl2 (240.1). Alkylation Chemical Reactions All reactions were carried out in a working concentration of 0.10 molar sodium bicarbonate at pH 7.4 (pH of human blood) and 37o C. In test solutions > 600 micrograms of 4chloroaniline was dissolved and 16 to 30 milligrams of Nmustard agent added (totalvolume > 800 microliters). Add N-mustard agent, remove 60 µL at known time intervals, add 60 microliters of fluorescamine (2 mg/mL in ethanol), and measure absorbance at 400 nanometers wavelength. RESULTS AND DISCUSSION

Cl Cl

9 N

10 1 Cl

5

2

6

Nitrogen Mus tard Group

N

3

8 Cl

7

4

COMPOUND 1

Cl 13 11 Cl

14 2

N 12

1

3

7

6

4 5

N 9

COMPOUND 2

Cl 8

10 Cl

Fig. (1). Molecular structures of nitrogen mustard agents show the identical twin drug configuration. The nitrogen mustard group is designated in top structure by broken circle. Compound 1 is N,N,N',N'-tetrakis(2-chloroethyl)ethane-1,2diamine with SMILES designation as ClCCN(CCN(CCCl)CCCl)CCCl. Compound 2 is N,N,N',N'tetrakis(2-chloroethyl)hexane-1,6-diamine with SMILES designation as ClCCN(CCCCCCN(CCCl)CCCl)CCCl.

Nitrogen mustard compounds are an important member of the clinical anticancer drug regimen for lymphomas, leukemias, and myelomas. There is a high correlation between cytotoxicity and the extent of DNA alkylation. Alkylating agents cause DNA damage but differences can be observed among them based upon: 1) Electrophilic reactivity; 2) Reactive intermediates; 3) Pharmacokinetic properties; 4) Antitumor activity; and 5) Toxicity. The nonalkylating regions of their structure plays an important role in these observed differences. Molecular structures of the N–mustard agents are shown in Fig. (1). They are identical twin drugs for having two N– mustard groups of equal size and located similarly in a bisected half. There are four sites per molecule which can alkylate a nucleophilic atom on DNA or protein. Both Nmustard agents are solids at room temperature, must be kept dry to avoid degradation, highly reactive, and stored at -10o C. Infrared spectra in dried dimethyl sulfoxide showed the diagnostic -C-Cl stretch at 800 to 600cm-1 and -CH2 -Cl wagging at 1300 to 1230 cm-1. Utilizing simple aliphatic diamine parent compounds produces mustard products having no functional groups that incur steric interference during alkylation reactions. Having two sets of mustard groups (-N(CH2CH2Cl)2) and four sites of potential covalent bonding to DNA per molecule enhances the cytotoxic effects. Synthesis of these two agents utilizes well known chemistry of amines and alkyl halides. The products are

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Bartzatt and Donigan

GENERAL SYNTHESIS OF NITROGEN MUSTARD AGENTS Cl

H 2N Cl

Cl

Cl

MILD REFLUX

N HCl

SOLVENT ACETONITRILE

NH2

N

Cl

Cl

REACTI ON OF N-MUSTARD AGENT WI TH NUCLEOPHILIC PRIMARY AMINE Cl

Cl NH2 N

NH

Cl

Cl 37o C pH 7.4

N

N HCl

Cl Cl

N

Cl

Cl

NITROGEN MUSTARD AGENT

Cl

Fig. (2). Major step of synthesis is the reaction of ethylene diamine with 1,2-dichloroethane that is accomplished under mild refluxing conditions. Reaction products will have four 2-chloroethyl substituents which constitute the alkylating region. The mustard agents will attack the nucleophilic primary amine group of 4-chloroaniline (see rectangle) resulting in products having secondary amine groups (see oval). There are four sites of potential alkylation action for each mustard agent (three designated by inset arrows).

isolable and sufficiently stable for manipulation. All organic solvents utilized must be dry and stored over molecular sieves to maintain dryness. Moisture can induce significant degradation. The alkyl halide 1,2-dichloroethane alkylates the primary amine groups of the diamines and forms the very reactive nitrogen mustard moiety (see Fig. 2). Reaction with a nucleophilic primary amine group (see rectangle inset of Fig. 2 ) is shown with a single alkylation event. Additional sites of reaction with DNA bases are indicated on the product molecule by inset arrows. Alkylation products may have secondary (Fig. 2) or tertiary (for DNA) amine groups. Properties such as Log P (partition coefficient) are important and play an significant role in the pharmacokinetics and pharmacodynamics of a drug. The level of a drug’s solubility in the cellmembrane lipid bylayer can be estimated by Log P determination. Corresponding interactions of lipophilicity are hydrophobic non-aqueous activities. Partition coefficient values calculated by various methods Compound 1 and 2 are presented in Table 1. All values are positive which indicate a lipophilic characteristic.Calculations of LogKowLog P assumes only neutral species exist in the organic and aqueous layer of the octanol/water partitioning. Values for Clog P are a result of the summation of contributions made by fragments of the molecular structure. Compound 2 is significantly more lipophilic (more positive Log P values) and more soluble in lipid by-layers than Compound 1. Compound 2 is less water soluble than Compound 1. Statistical analysis of the Log P

values for Compound 1 produces a mean of 2.59, median of 2.38, and standard deviation of 0.338. Similarly for Compound 2 the mean is 4.41, median is 4.2, and standard deviation of 0.396. The addition of four methylenes (-CH2-) to Compound 1 results in the more lipid soluble sibling Compound 2. Table 1.

Comparison

of

Partition

Coefficients

for

1Compound 1 and 2Compound 2 Partition Coefficient

1Compound 1

2Compound 2

MLog P

3.12

4.11

miLog P

2.37

4.10

iaLog P

2.38

4.98

LogKowLog P

2.72

4.68

actCLog P

2.34

4.20

1N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine 2N,N,N’,N’-tetrakis(2-chloroethyl)hexane-1,6-diamine MLog P= 1. Moriguchi method by DRAGON; miLog P= Molinspiration method; iaLog P= Interactive Analysis; LogKowLog P= Syracuse Research Corp.; actCLog P= Actelion Ltd. method.

Polar surface area (PSA) (see Table 2) is a molecular descriptor which has been shown to be an accurate predictor of intestinal absorption, drug absorption, blood-brain barrier penetration, cell membrane permeability, and drug transport [10-15]. The mean Log P value for Compound 1 is 2.59, which makes this cytotoxic agent suitable for crossing the



blood-brain barrier [11, 12, 15]. The PSA values determined for Compound 1 and Compound 2 are 6.48 A2 and 6.48 A2, respectively. Previous studies have shown that these values of PSA indicate that more than 95% of drug present in the intestinal tract will be absorbed [15]. The molecular surface area calculated by SPARTAN is only 1.89% of the total surface area of Compound 1 is polar and 98.11% of molecular surface as nonpolar. Similarly for Compound 2, 1.49% of the surface is polar and 98.51% being nonpolar. Polarizability and parameters important for estimating van der Waals interactions (molar refractivity and parachor) are presented in Table 2. Molar refractivity is molar volume corrected by the refractive index and represents size and polarizability of a fragment or molecule.Refractive index is dominated by molecular weight and density. Larger steric effects are observed with larger molecular weight, however larger density values correlate with smaller steric effects. Previous studies have shown that properties such as formula weight, Clog P, H bond acceptors, and H bond donors can predict important pharmacokinetic activities [16]. Problems of absorption occur when two or more of the following parameters are violated (Rule of 5): 1) Formula weight < 500; 2) Clog P < 5.0; 3) Less than 5 H bond donors; and 4) Less than 10 H bond acceptors. Both Compound 1 and 2 have zero violations of Rule of 5 (see Table 2). This indicates that both Compound 1 and 2 will have good bioavailability and good bioactivity [16]. The values of PSA for Compound 1 and 2 indicate good penetration of the blood-brain barrier [17]. Table 2.

Molecular Descriptors of Nitrogen Mustard Agents

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from 340 nm to 520 nm. An absorbance peak appears at 400 nm (inset arrow) and is utilized to determine the amounts of unreacted primary amine target (4-chloroaniline) in kinetic studies evaluating alkylation efficiency. Physiological conditions of pH 7.4 (pH of human blood) and 37o C (body temperature) were applied in aqueous mixtures buffered by sodium bicarbonate. Aliquots of the reaction mixtures were removed at known time periods to monitor the amount of unreacted 4-chloroaniline still present. Collection of absorbance data versus time permits calculation of rate equations and rate constants. The alkylation of 4chloroaniline proceeded rapidly with Compounds 1 and 2, which went to completion in less than one hour. This rapid rate of alkylation is a beneficial attribute for the clinical treatment of aggressive tumors near the skin surface. REACTION OF FLUORESCAMINE WITH PRIMARY AMINE GROUP O O

O R

NH2

O

FLUORESCAMINE O OH COOH N R

Descriptor

4Compound 1

5Compound 2

1Formula Weight

310.09

366.20

1Molar Refractivity (CM3)

75.2

93.7

1Parachor (CM3)

635.1

794.2

1Index of Refraction

1.504

1.498

1Polarizability (CM3)

29.81E-24

37.16E-24

2Dipole (Debye)

3.419

1.502

2 Surface Area (A2 )

343.60

435.3

2Volume (A3)

324.11

408.3

3 Polar Surface Area (A2 )

6.48

6.48

3Violations of Rule Of 5

0

0

Intestinal Absorption

>95%

>95%

1Determined by ChemSketch. 2Determined by SPARTAN modeling. 3Determined by Molinspiration. 4 N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine. 5 N,N,N’,N’-tetrakis(2-chloroethyl)hexane-1,6-diamine.

Fluorescamine is a fluorescent probe which reacts with primary amine groups with high specificity. The derivative formed is highly fluorescent and has a significant absorbance peak in the visible range. Fig. (3) shows the formation of the fluorescent derivative after the reaction with a primary amine compound. The reaction results in the formation of a tertiary amine group within the derivative. Fig. (3) also shows the absorbance spectra of the fluorescent derivative

FLUORESCENT DERIVATIVE

Fig. (3). To follow the extent of alkylation reaction, aliquotsof the reaction mixture are removed at known time intervals and fluorescamine added to determine the remaining amounts of primary amine reactant remaining. The chemical reaction of fluorescamine with a primary amine group is shown here, forming a fluorescent product having a tertiary amine group. The Fluorescent derivative formed absorbs in the visible spectra from wavelength 340 nm to 460 nm. A strong absorbance peak occurs at 400 nm (see inset arrow) which is utilized to determine quantity of unreacted 4-chloroaniline.

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Bartzatt and Donigan

Fig. (4). Absorbance (A) at 400 nm is plotted as Ln(A) vs. Time (minutes) for the first-order plot of reaction of N,N,N’,N’-tetrakis(2chloroethyl)ethane-1,2-diamine with 4-chloroaniline, which gives -k1 (slope= -k1 ). Second plot shows (A) vs. Time (minutes) for zero-order kinetics of N,N,N’,N’-tetrakis(2-chloroethyl)- hexane-1,6-diamine reaction with 4-chloroaniline (slope = -k0).

Absorbance data (A) collected for alkylation reactions of Compound 1 was plotted Ln (A) versus time in minutes (Fig. 4) to obtain a good fit for a first-order rate equation: Rate = k1[Nu], where k1 is first-order rate constant and [Nu] is the nucleophile reacting with the nitrogen mustard agent. Table 3.

The numerical value of k1 is calculated from the slope of theline obtained from the plot, slope = -k 1 (k1 is 0.0707/minute for Compound 1). Similarly, it was found that a zero-order rate equation was a good fit for the rapid alkylation reaction of Compound 2 (Fig. 4, lower half). The

Comparison of 1Compound 1 and 2Compound 2 with Mechlorethamine and Cyclophosphamide Nitrogen Mustard Agent Property

Mechlorethamine

Cyclophosphamide

Compound 1

Compound 2

Half Life, t1/2

< 10 min

6.5 hours

25 min

36 min

miLog P

1.113

1.407

2.366

4.102

Polar Surface Area (PSA)

3.238

41.57

6.476

6.476

Number of O and N

1

4

2

2

Number of -OH and –NH

0

1

0

0

Violations of Rule of 5

0

0

0

0

1N,N,N’,N’-tetrakis(2-chloroethyl)ethane-1,2-diamine 2N,N,N’,N’-tetrakis(2-chloroethyl)hexane-1,6-diamine


plot of absorbance dataversus time in minutes showed a good fit line having slope = -k 0 = -0.00217 mole/(Liter● minute). The rate equation appears as follows: Rate = k0 ,(k0 is 0.00217 mole/(Liter● minute). There is no variable for the nucleophile. Zero-order reactions are indicated when the rate of reaction is a constant and independent of the concentration of reactants. When the limiting reactant (the nucleophile) is completely consumed the reaction stops. Compounds 1 and 2 were at concentrations in excess over the nucleophile 4chloroaniline. Integration of the rate equation produces the following relationship: [A] = -k0(Time) + [A]0 , where [A]0 is the y-axis intercept and absorbance at time zero. Compounds 1 and 2 show zero violations of Rule of 5 similarly to two clinical N-mustard drugs mechlorethamine and cyclophosphamide (Table 3). In addition, PSA values of all agents in Table 3 indicate high absorbance in the intestinal tract (> 95% for Compounds 1 and 2). The PSA and mean Log P value for Compound 1 indicate effective penetration of the blood-brain barrier, an advantage over methlorethamine and cyclophosphamide. Compounds 1 and 2 have half-lives of 25 minutes and 36 minutes, respectively, which is between those of mechorethamine and cyclophosphamide. Numbers of oxygens, nitrogens, H donors, and H acceptors are comparable to mechlorethamine and cyclophosphamide. Compounds 1 and 2 show comparable properties to N-mustards that are utilized clinically. ACKNOWLEGDEMENTS This study was financed by the College of Arts & Sciences and Chemistry Department, University of Nebraska, Omaha, NE, USA.


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