Yes, the effectiveness of azithromycin may be reduced if you did not take the right dose. In your case, you took a smaller initial dose (250 mg), followed by 750 mg. The concern with this has to do with pharmacokinetic profile of azithromycin.
Unlike many antibiotics, azithromycin has a very long half-life (around 68 hours) and lasts for a long time in the body. It is commonly dosed by using a "loading dose", to achieve high concentrations in the body which are maintained for a long period of time due to the long duration of action of the drug.
Aside from the 1 gram dose (4-250 mg tablets) you were prescribed (commonly used for various STDs), azithromycin is also commonly prescribed as a 5 day course for other infections, such as bronchitis. When a 5 days course is prescribed, the first dose is often the "loading dose" (500 mg), followed by a 4 day maintenance dose (250 mg). The loading dose is used to achieve high enough concentrations in the body of the drug and the 4 day maintenance dose maintains those high concentrations.
The concern in your situation, by taking a lower than intended dose, is that drug concentrations may be too low to eliminate your chlamydia infection, even though you made up the intended total dose later in the day. A comparison, although not completely analogous, would be taking one ibuprofen all at once, versus splitting the pill into fourths, and taking that throughout the day. You will be ingesting the same total daily dose, but taking one whole tablet at once will have better analgesic effects than taking 1/4 of a dose 4 times during that day.
Now, the 5 hour difference between your doses may not make a huge difference since azithromycin lasts so long, but it would be prudent to reach out to your doctor and see what they recommend. They may re-prescribe the medication for you, just to be safe.