The present invention relates to an amide prodrug of 2-deoxy-2,2-difluorocylidinc
and pharmaceutical composition thereof.
BACKGROUND OF THE INVENTION
The present invention relates to a novel prodrug of gemcitabine, which is capable
of being given orally, traversing the intestinal tract substantially intact into the portal
bloodstream with less gastrointestinal toxicity and better bioavailability than with the
parent drug, and maintaining the efficacy of the parent drug at lower doses.
Gemcitabine hydrochloride (2',2'-difluoro-2'-deoxycytidine hydrochloride) is an
anti-tumor agent, with known antiviral action, that is currently produced and marketed as
Gemzar®; a lyophilised, powder formulation for treatment of various cancers. Gemzar®.
a process for making it and methods for using it are described in US Patent No. 5,464,
826 and US Patent No 4, 808, 614. Gemzar® is currently approved for the treatment of
pancreatic cancer, breast cancer and non-small cell lung cancer (NSCLC) and is being
evaluated for ovarian cancer. In addition Gemzar® may be used in the treatment of HCV
as well as a modulator of immune function (see US Patent No 6,555,518). Gemzar® is
currently administered by intravenous infusion at a dose of approximately 1000 to 1250
mg/m2 over 30 minutes, once weekly for up to 7 weeks followed by a week of rest from
treatment.
The use of gemcitabine orally may be limited by its poor oral bioavailability
which is the result of first pass metabolism. Shipley LA. Et. al., "Metabolism and
disposition of gemcitabine, and oncolytic deoxycytidine analog, in mice, rats, and dogs".
Drug Metabolism & Disposition. 20(6):849-5S, 1992. In addition, when dosed orally,
gemcitabine is implicated in causing adverse dose-limiting intestinal lesions characterized
by moderate-to-marked loss of mucosal epithelium (atrophic enteropathy) throughout the
entire length of the intestinal tract in mice given a single oral (gavage) gemcitabine dose
of 167,333, or 500 mg/kg. Horton ND et.al., "Toxicity of single-dose oral gemcitabine
in mice", American Association for Cancer Research, Poster Presentation, Orlando, FL,
March 27-31, 2004. Comparable exposures via intravenous dosing in previous mouse
studies did not result in death or gastrointestinal toxicity.
Methods for making prodrug and sustained released formulations of gemcitabine
are well known in the art. Examples of such prodrugs and sustained released

formulations can be found in WO 04/0412303 "Gemcitabine Prodrugs, Pharmaceutical
Compositions and Uses Thereof, Gallop et. al.; WO 98/32762 "Gemcitabine
Derivatives," Myhren, Finn, et al.; WO 02/09768 "Therapeutic polyesters and
polyamides," Uhrich, Kathryn E.; WO 02/76476 "Prodrugs of anticancer agents based on
substituted aromatic acids," Greenwald, Richard B., et al.; WO 02/65988, "Terminally-
branched polymeric linkers and polymeric conjugates as prodrug," Choe, Yun Hwang, et
al.
Gemcitabine amide derivatives have been described in the art as useful
intermediates in the synthesis of gemcitabine, see e.g. Britton, et al., U.S. Pat. No.
5,420,266 and Grindey, et al., U.S. Pat. No. 5,464,826. and also useful as prodrug
moieties for the administration of gemcitabine, see e.g. Gallop, et al., WO 04/041203.
There continues to be a need for a prodrug of gemcitabine that will allow for oral
delivery, will pass through the intestinal tract intact without substantial degradation and
deliver gemcitabine to the afflicted area with acceptable safety and efficacy.
Surprisingly, we have discovered a superior, novel amide derivative of
gemcitabine, which traverses the enterocyte substantially intact; is hydrolyzed to
gemcitabine without significant accumulation of deoxydifluorouridine (dFdU) the
predominant gemcitabine metabolite in the liver, has less toxicity than oral gemcitabine,
and maintains appropriate efficacy and safety profiles when administered orally.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a compound of Formula I

Another aspect of the invention provides for novel pharmaceutical compositions,
comprising a compound of formula I and one or more pharmaceutically acceptable
excipients.

The present invention also provides for the use of the compound of formula I for
the treatment of susceptible neoplasms in a mammal in need of such treatment.
The present invention provides for the use of the compound of formula 1 for the
treatment of susceptible viral infections in a mammal in need of such treatment.
The present invention also provides for the use of a compound of formula 1 for the
manufacture of a medicament for the treatment of susceptible neoplasms or viral
infections.
In addition, the present invention also provides a method for preparing the
compound of formula I.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the terms have the meanings indicated.
The term "mammal" is taken to mean any of various warm-blooded vertebrate
animals of the class Mammalia, most preferably humans, characterized by a covering of
hair on the skin and, in the female, milk-producing mammary glands for nourishing the
young.
The term "pharmaceutically acceptable excipient" refers to a pharmaceutical ly
acceptable formulation carrier, solution, or additive to enhance the formulation
characteristics. Such excipients must be compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof and are well known to the skilled
artisan, see e.g. Remingtons Pharmaceutical Sciences, 19th Edition, Mack Publishing
Company, 1995.
The term "co-crystal" means a physical association of two or more molecules
which owe their stability through non-covalent interaction. One or more components of
diis molecular complex provide a stable framework in the crystalline lattice. In certain
instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or
solvates, see e.g. "Crystal Engineering of the Composition of Pharmaceutical Phases. Do
Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?" Almarasson,
O., et. al., The Royal Society of Chemistry, 1889-1896,2004. Preferred co-crystals
include p-toluenesulfonic acid and benzenesulfonic acid.
The term 'pharmaceutically acceptable co-crystal" means one that is compatible
with the other ingredients of the formulation and not deleterious to the recipient thereof.

The term "susceptible neoplasm" refers to an abnormal growth of tissue in
mammals capable of being treated by the oral administration of the compound of formula
I. As this prodrug will hydrolyze to gemcitabine, the administration of the prodrug is
expected to have a broad spectrum of activity against a wide variety of tumor types, both
solid and non-solid. Preferably, susceptible neoplasms include T-cell lymphoma, soft
tissue sarcoma, pancreatic cancer, breast cancer, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, non-small cell lung cancer, ovarian cancer and bladder cancer.
The compounds of the invention are useful in the treatment of viral infections,
particularly HCV.
The term "therapeutically effective amount" means the amount of
compound/composition that will elicit the desired biologic or medicinal response of a
tissue, system or mammal that is being sought by the researcher, physician or clinician.
Gemcitabine contains three derivatisable functional groups, the 3' and 5'hydroxyl
groups and the N4-amino group. The compound of formula I can be prepared by using
suitable protecting groups to block the 3' and 5' hydroxyl functions followed by acylation
of the N4-amino group. Typical protecting groups are well know and appreciated in the
art Protecting Groups in Organic Synthesis, 3rd edition Theodora Greene, Peter Wuts
(Wiley-Interscience) 1999. Acylation of the N4-amino group may be accomplished by
reaction with an acid chloride or anhydride or by reaction with a carboxcylic acid in the
presence of a coupling reagent, such as N,N-dicyclohexylcarbodiimide (DCC), N-ethyl-
N'-(3-dimethylaminopropyl)-carbodiimide (EDC), 1,1-carbonyldiimidazole (CDI) or
other similar reagents well known to one skilled in the art of organic chemistry.
Alternatively, the compound of formula I may be prepared without the use of protecting
groups. In this case, mixtures of mono-, di- and tri-adducts will be formed and the
desired product may be separated from the mixture.
The following examples further illustrate the synthesis of compounds of the
invention. All starting materials and reagents are well known and appreciated in the art
and readily available or prepared by methods described herein. A process for preparing
gemcitabine (2',2'-difluoro-2'-deoxycytidine), for example, is disclosed in US Patent No.
4,808,614.

Example 1
1 -(2,2-difluoro-2-deoxy-β-D-ribofuranosyl)-4-(2-propyl-1 -oxopentyI)aminopyrimidin-2-
one

Dissolve 2',2,-difluoro-2'-deoxycytidine (10.0 g, 38.0 mmol) in anhydrous
pyridine (100 mL) and cool to 0°C while stirring under nitrogen. Add
chlorotrimethylsilane (24.0 mL, 190.0 mmol) dropwise, maintaining an internal
temperature < 5°C. Continue stirring at 0°C for 2 hours. In a separate flask, dissolve 2-
propylpentanoic acid (6.0 g, 41.8 mmol) in anhydrous acetonitrile (100 mL). Add 1,1-
carbonyldiimidazole (6.8 g, 41.8 mmol) in small portions over 30 minutes and stir for 2
hours. Add this acetonitrile solution dropwise to the pyridine solution at 0°C and allow
the reaction to come to ambient temperature. Heat the reaction at 45°C overnight then
cool to 30-35°C and add 100 mL absolute ethanol and heat at 45°C for 30 minutes. Add
50 mL water and heat at 45°C for 5 hours then cool to ambient temperature and
concentrate in vacuo. Partition the crude residue between ethyl acetate and water.
Acidify to pH ~2 with phosphoric acid and separate the organic layer. Back extract the
aqueous layer with additional ethyl acetate. Combine the organic solutions and wash with
saturated sodium bicarbonate solution and saturated sodium chloride solution, dry over
magnesium sulfate and concentrate in vacuo. Purify by silica gel chromatography (120 g)
eluting with a gradient of 30% to 60% ethyl acetate in methylene chloride. Isolate desired
product as a white crushable foam (11.2 g, 77% yield).
MS (ES): m/z 390.3 = [M+H]+
MS (ES): m/z 388.3 = [M-H]+
1HNMR (400 MHz, DMSO-d6) δ 0.83 (t, 6H), 1.15-1.36 (m, 6H), 1.46-1.55 (m, 2H), 2.60
(dddd, 1H, J=14.4, 9.6, 5.6, 5.6 Hz), (ddd, 1H, J=12.6, 6.2, 3.6 Hz), 3,77-3.81 (m, 1H),
3,87 (dt, 1H, J=8.4,3.0 Hz), 4.12-4.22 (m, 1H), 5.27 (t, 1H, J=5.6 Hz), 6.15 (t, 1H, J=7.4
Hz), 6.29 (d, 1H, J=6.4 Hz), 7.31 (d, 1H, J=7.2 Hz), 8.23 (d, 1H, J=8.0 Hz), 11.03 (s, 1H).

Example 2
l-(2,2-difluoro-2-deoxy-β-D-ribofuranosyl)-4-(2-propyl-l-oxopentyl)aminopyrimidin-2-
one p-toluenesulfonic acid hydrate co-crystal (2:1:1)
Dissolve 0.709 g (1.82 mmol) of the compound of Example 1 in 9 mL methanol.
In a separate flask, prepare a 0.25 M aqueous stock solution of p-toluenesulfonic acid.
Add 3.6 mL (0.9 mmol) of the aqueous solution dropwise. Add ~5 mL water and allow
the mixture to stir at room temperature (~30 min) until precipitation occurs. Collect
precipitated solid by vacuum filtration and allow to air dry.
Analytical analysis of co-crystal:
Prepare solutions of known concentration of p-toluenesulfonic acid, gemcitabine and the
compound of Example 1. Analyze a sample of the p-toluenesulfonic acid co-crystal of
Example 1 to determine component composition.
For a 2:1:1 ratio of N-(2-propyl-pentanoyl)]-2',2,-difluoro-2'-deoxy-cytidine /p-
toluenesulfonic acid/water, % toluenesulfonic acid determined:
Theory 17.8% p-toluenesulfonic acid
Found 19.1 % p-toluenesulfonic acid
HPLC:
Column: Waters Atlantis dC18, 3.0 µm, 4.6 i.d. x 150 mm
Column temp. 50°C
UV wavelength: 248 nm
1. Mobile phase
A. 5/95 acetonitrile/water + 0.1% TFA
B. 50/50 acetonitrile/water + 0.1 % TFA
2. Gradient
Time Solvent
(min) %A %B
0 100 0


Example 3
l-(2,2-difluoro-2-deoxy-β-D-ribofuranosyl)-4-(2-propyl-1-oxopentyl)aminopyrimidin-2-
one benzensulfonic acid co-crystal (1:1)
To 5mL of ethyl acetate, 550 mg of the compound of Example 1 is added. The
mixture is heated to approximately 55 °C. A one molar equivalence of benzensulfonic
acid delivered as a stock solution is added. Additional ~10mL of ethyl acetate is added
with sonication, as needed, to break up the precipitate. The suspension is allowed to cool
to room temperature and isolated by vacuum filtration. The isolated solid is allowed to
air dry. MP: 171 °C
The compound of Formula I and solvates thereof are orally available and are
normally administered orally, and so oral administration is preferred.
The pharmaceutical compositions are prepared in a manner well known in the
pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material
that can serve as a vehicle or medium for the active ingredient. Suitable carriers or
excipients are well known in the art. The pharmaceutical composition may be adapted for
oral, inhalation, parenteral, or topical use and may be administered to the patient in the
form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, or the
like. The compounds of the present invention may be administered orally, for example,
with an inert diluent or capsules or compressed into tablets. For the purpose of oral
therapeutic administration, the compounds may be incorporated with excipients and used
in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing
gums and the like. These preparations preferably contain at least 1% of the compound of
the present invention, the active ingredient, but may be varied depending upon the
particular form and may conveniently be between 1% to about 90% of the weight of the
unit. The amount of the compound present in compositions is such that a suitable dosage
will be obtained. Preferred compositions and preparations of the present invention may
be determined by methods well known to the skilled artisan.
The pharmaceutical composition of present invention comprising a compound such as
herein described and a pharmaceutically acceptable excipient showed surprising effects.

The tablets, pills, capsules, troches, and the like may also contain one or more of
the following adjuvants: binders such as povidone, hydroxypropyl cellulose,
microcrystalline cellulose, or gelatin; excipients or diluents such as: starch, lactose,
microcrystalline cellulose or dicalcium phosphate, disintegrating agents such as:
croscarmellose, crospovidone, sodium starch glycolate, corn starch and the like;
lubricants such as: magnesium stearate, steric acid, talc or hydrogenated vegetable oil;
glidants such as colloidal silicon dioxide; wetting agents such as: sodium lauryl sulfate
and polysorbate 80; and sweetening agents such as: sucrose, aspartame or saccharin may
be added or a flavoring agent such as: peppermint, methyl salicylate or orange flavoring.
When the dosage unit form is a capsule, it may contain, in addition to materials of the
above type, a liquid carrier such as polyethylene glycol or fatty oil. Other dosage unit
forms may contain other various materials that modify the physical form of the dosage
unit, for example, as coatings. Preferably the dosage form is enteric coated.
Thus, tablets or pills may be coated with sugar, hydroxypropyl methylcellulose,
polymethacrylates, or other coating agents. Syrups may contain, in addition to the present
compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings
and flavors. Materials used in preparing these various compositions should be
pharmaceutically pure and non-toxic in the amounts used.
The compounds of Formula I are generally effective over a wide dosage range.
For example, dosages per day, in single or divided doses, normally fall within the
range of about about 15 mg/day to about 200 mg/day, more preferably about 85
mg/day. In some instances dosage levels below the lower limit of the aforesaid range
may be more than adequate, while in other cases still larger doses may be employed
without causing any harmful side effect, and therefore the above dosage range is not
intended to limit the scope of the invention in any way. It will be understood that the
amount of the compound actually administered will be determined by a physician, in
the light of the relevant circumstances, including the condition to be treated, the
chosen route of administration, the actual compound or compounds administered, the
age, weight, and response of the individual patient, and the severity of the patient's
symptoms.

pH Chemical Stability Assay
The pH chemical stability is assessed using a semi-automated HPLC technique.
Samples of compound of formula I are prepared at 100 mcg/mL in five buffers
representing the pH range throughout the gastrointestinal tract (pHl -pH8). Samples are
loaded onto an HPLC autosampler incubated at 40°C. Samples are repeatedly injected on
the HPLC at hourly intervals for up to 24 hours with an HPLC column that separates a
compound of formula I from gemcitabine. The peak area of the compound of formula I is
monitored by UV detection over time and are compared to initial peak areas to determine
stability.
Less than 25% of the compound of Example 1 degraded to gemcitabine at pH
range 1 to 8 after four hours.
Pharmacokinetic Assays
Mouse Pharmacokinetics
The pharmacokinetic profiles of gemcitabine and a compound of formula I, are
assessed in male CD-1 mice following oral administration at doses selected to contain
approximately 10 mg/kg of gemcitabine. Designated animals (n = 3/time
point/compound) are sacrificed at 0.08,0.25,0.5,1,2, and 6 hours after dosing, and
systemic blood samples are collected into EDTA-treated tubes containing
tetrahydrouridine (0.5mM final concentration in blood) to inhibit further metabolism of
gemcitabine. An additional 3 animals are sacrificed at 0.08 hours for collection of hepatic
portal blood. Plasma is isolated by centrifugation and frozen prior to analysis. Plasma
concentrations of gemcitabine and prodrugs are determined by LC/MS/MS analysis.
Pharmacokinetic parameters are calculated using WinNonlin software (Pharsight Corp.,
Mountain View, CA). Pharmacokinetic parameters of each prodrug are compared to
those determined from administration of oral gemcitabine hydrochloride in a similar
study design.
The compound of Example 1 was extensively hydrolyzed in vivo to release
gemcitabine when administered orally to CD-1 mice. The plasma exposure to
gemcitabine was increased in CD-1 mice when the compound of Example 1 was
administered, compared to direct oral administration of gemcitabine hydrochloride. The

absorption of the intact prodrug is verified by relatively high concentrations of the
compound of Examplel in the hepatic portal plasma at 0.08 hours after oral dosing.
Monkey Pharmacokinetics Assay
The pharmacokinetic profiles of gemcitabine and a compound of formula 1, are
assessed in cynomolgus monkeys following oral and intravenous administration in a
crossover design. Compounds are administered at doses selected to contain
approximately 10 mg/kg of gemcitabine. Blood samples are collected into EDTA-treated
tubes containing tetrahydrouridine (0.5mM final concentration in blood) at designated
intervals for up to 48 hours. Animals are pretreated with ranitidine (intravenous, 5 mg/kg)
in the oral dosing periods. Plasma is isolated by centrifugation and frozen prior to
analysis. Plasma concentrations of gemcitabine and a compound of formula 1 are
determined by LC/MS/MS analysis. Pharmacokinetic parameters are determined using
WinNonlin software (Pharsight Corp., Mountain View, CA).
The compound of Example 1 was extensively hydrolyeed in vivo to release
gemcitabine when administered intravenously to cynomolgus monkeys. The oral
exposure to gemcitabine was increased in cynomolgus monkeys approximately 5-fold
when a compound of Example 1 was administered, compared to direct oral administration
of gemcitabine hydrochloride.
Hydrolysis Assays
Small Intestine Homogenate Assay
To determine the stability of a compound of formula I to enzymatic hydrolysis in
the intestine, crude homogenates of small intestinal epithelial cells are prepared from
sections of the upper small intestine from CD-I mice, beagle dogs, cynomolgus monkeys,
and humans. Mouse and dog homogenates are prepared from freshly collected tissue,
while monkey and human homogenates are prepared from previously frozen tissues.
Cells are gently scraped from intestinal segments, pooled, and homogenized in 50 mM
acetate buffer using Polytron (PT-10-85). Protein concentrations are determined by
standard spectrophotometric techniques. The prepared homogenates are store at -70°C
before using.

The hydrolytic rates of a compound of formula I in small intestinal homogenates
(SIH) is determined by incubating a compound of formula I (100 uM) with SIH (2.5 - 5
mg/mL of total protein) in acetate at pH 7.5 for up to 6 hours. Concentrations of
gemcitabine released via hydrolysis is determined by LC/MS analysis after the reaction is
quenched with acetonitrile. Hydrolysis rates are calculated at 30 minutes in screening
experiments and from the slope of the linear portion of the hydrolysis vs. time curves in
characterization studies of a compound of formula I.
The compound of Example 1 exhibited a slow rate of hydrolysis in the small
intestine homogenate assay. The hydrolysis was slowest in monkey and human
homogenates, with less than 3% of the total compound converted to gemcitabine in a 6
hour incubation.
Liver S9 Hydrolysis Assay
Hydrolysis of a compound of formula 1 by liver enzymes is determined by liver
hydrolysis assay. Liver homogenates are prepared from CD-I mouse, beagle dog,
cynomolgus monkey, and human livers. Liver tissues are cut into small pieces using
scissors, then, homogenized in 50 mM acetate buffer using Polytron (PT-10-85) for 1
min. Post-mitochondrial (S9) fractions are prepared from each by ultracentrifugation at
9,000 x g at 4°C for 10 min. Mouse, dog and monkey liver S9 fractions are prepared
from freshly collected tissues, while human liver S9 are prepared from previously frozen
tissue. After centrifugation, the supernatant is collected and protein concentrations are
determined by standard spectrophotometric techniques. The prepared S9 fractions are
store at -70°C before using.
The hydrolytic rates of a compound of formula I in liver S9 is determined by
incubating compound (10 uM) with S9 (2 mg/mL of total protein) in phosphate buffered
saline at pH 8.0 for up to 6 hours. Concentrations of gemcitabine released via hydrolysis
are determined by LC/MS analysis after the reaction is quenched with acetonitrile.
Hydrolysis rates are calculated at 30 minutes in screening experiments and from the slope
of the linear portion of the hydrolysis vs. time curves in characterization studies of a
compound of formul a I.

The compound of Example 1 was hydrolyzed in liver S9 of all species described.
The hydrolysis was most rapid in monkey and human homogenates, with approximately
35% of the total compound converted to gemcitabine in a 6 hour incubation.
Toxicology Assays
4-Day Mouse Screen
To evaluate the toxicity produced by orally administered compound of formula I
when administered daily to female CD-a mice for 4 days, the gastrointestinal toxicity
profiles of a compound of formula I is compared with the historical results for
gastrointestinal toxicity when gemcitabine is administered orally at 8 mg/kg in a 4-day
mouse study.
Female CD-I mice 5-8 weeks of age are dosed with a compound of formula I by
oral gavage. A dose level is chosen to approximate a molar equivalent of 8-mg/kg
gemcitabine. A dose volume of 10 mL/kg is used and doses are administered once daily
on 4 consecutive days. Necropsy is performed approximately 5-8 hours following the 4th
dose.
Clinical signs, clinical chemistry, gross pathology, and limited histopathology
(ileum, jejunum, and liver) are evaluated. There was a significant reduction in the
severity of atrophic intestinal changes or enteropathy observed after dosing mice with an
equivalent dose of the compound of Example 1 versus gemcitabine HC1.
14 Day Mouse Study
A 14-Day Mouse Study is conducted to evaluate whether a compound of formula
I produces adverse effects in mice following 14 days of oral gavage dosing and to
determine the plasma concentrations of a compound of formula I and its metabolites
gemcitabine HC1 and deoxydifluorouridine after 1 or 14 doses.
Male and female CD-I mice 9-12 weeks of age are dosed with a compound of
formula I rug by oral gavage. A range of doses is selected in an effort to determine a

maximum tolerated dose and dose limiting toxicity. A dose volume of 10 mL/kg is used
and doses are administered once daily.
Clinical signs, body weight, food consumption, hematology, clinical chemistry,
plasma concentrations of a compound of formula 1 and metabolites gemcitabine HC1 and
deoxydifluorouridine, and pathology (including gross pathology, organ weights, and
histopathology) are evaluated.
On a molar equivalent basis, the compound of Example 1 is associated with less
enteropathy than gemcitabine HCl while resulting in approximately twice the systemic
exposure to gemcitabine HCL.
7-Day Dog Study
To evaluate the toxicity profile of a compound of formula I when administered to
Beagle dogs for 7 days, and to determine the plasma concentrations of a compound of
formula I and metabolites gemcitabine HCl and deoxydifluorouridine after 1 or 7 doses, a
7-day dog study is conducted.
Male and female beagle dogs 6-48 months of age are dosed orally via capsule with
a compound of formula 1. A range of doses is selected in an effort to determine a
maximum tolerated dose and dose limiting toxicity. A dose volume of 1 mL/kg is used
and doses aree administered once daily.
Clinical signs, body weight, food consumption, body temperature, hematology
(including coagulation), clinical chemistry, urinalysis, plasma concentrations of the
compound of Example I and its metabolites gemcitabine HCl and deoxydifluorouridine,
and pathology (including gross pathology, organ weights, and histopathology) were
evaluated. Hematotoxicity and the other toxicities, including GI toxicity, are consistent
with what has been previously described for parenteral gemcitabine. Therefore, none of
these toxicities were unique to the oral dosing route.
In Vivo Assay
HCT-116 colon tumor cells are grown in vitro under standard tissue culture
conditions, harvested, washed, and 5 x 106 cells (1:1 suspension in Matrigel,

Collaborative Biomedical Products, Inc) are injected subcutaneously in the rear flank of
female nude mice (Charles River, CD1 nu/nu, 24-27 g, irradiated with 450 Rads within
24 h of implantation). Tumors are allowed to grow to -100 mm3 before initiation of
therapy. Vehicle control, a compound of formula I or gemcitabine-HCl at various dose
levels are administered to the mice by oral gavage (10 ml/kg volume) at the times
indicated in the individual experiments. Compounds are administered either daily for
fourteen days, every other day for a total of seven doses, or every third day for a total of
four doses. . For the every day dosing schedule, a compound of formula I is formulated
in 100 mM sodium phosphate buffer, pH 6.0, and is formulated in 1% sodium
carboxymethylcellulose, 0.5% sodium lauryl sulfate, 0.05% Antifoam 150 and 0.085%
povidone for the every other day and every third day dosing schedules. Gemcitabine-HCl
is prepared in physiological saline for oral administration. Tumor size is determined by
caliper measurements and tumor volume (mm ) is estimated from the formula
0.536, where / is the larger and w is the smaller of perpendicular diameters. All data
(tumor measurements and animal weights) are captured twice-weekly beginning with the
start of therapy and analyzed using a computer based tumor measurement system.
The antitumor efficacy observed with the compound of Example 1 was
comparable to that obtained with an equivalent dose of gemcitabine-HCl. However,
treatment with the compound of Example 1 resulted in less overall toxicity compared to
animals that received an equivalent amount of gemcitabine-HCl.

WE CLAIM:
1. An amide of compound 2-deoxy-2,2-difluorocytidine of the formula

2. A compound as claimed in claim 1, wherein the compound is 1-(2,2-difluoro-2-
deoxy-β-D-ribofuranosyl)-4-(2-propyl-1-oxopentyl)aminopyrimidin-2-one p-
toluenesulfonic acid co-crystal.
3. A compound as claimed in Claim 2, wherein compound is 1-(2,2-difluoro-2-
deoxy-(3-D-ribofuranosyl)-4-(2-propyl-1-oxopentyl)aminopyrimidin-2-one p-
toluenesulfonic acid hydrate co-crystal (2:1:1).
4. A pharmaceutical composition comprising a compound as claimed in Claim 1
to 3 as an active ingredient along with a pharmaceutically acceptable excipient,
wherein the active ingredient is present in an amount ranging from 1% to 90% by
weight of the composition.
5. A pharmaceutical composition as claimed in Claim 4, wherein the composition
is enteric coated.
6. A compound and/or a pharmaceutical composition substantially as herein
described with reference to the foregoing examples.

The present invention relates to novel amide of compound 2-deoxy-2,2-
difluorocytidine of the formula and pharmaceutical composition thereof.